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Sample records for morphogenetic protein bmp-4

  1. Expression of human bone morphogenetic protein (BMP-2 and BMP-4 genes in transgenic bovine fibroblasts Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4 em fibroblastos bovinos transgênicos

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    C. Oleskovicz

    2004-08-01

    Full Text Available cDNAs dos genes bone morphogenetic protein-2 (BMP-2 e bone morphogenetic protein-4 (BMP-4 foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV. Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

  2. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

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    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  3. Comparison of newly developed anti-bone morphogenetic protein 4 llama-derived antibodies with commercially available BMP4 inhibitors.

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    Calpe, Silvia; Correia, Ana C P; Sancho-Serra, Maria Del Carmen; Krishnadath, Kausilia K

    2016-01-01

    Due to improved understanding of the role of bone morphogenetic protein 4 (BMP4) in an increasing number of diseases, the development of selective inhibitors of BMP4 is an attractive therapeutic option. The currently available BMP4 inhibitors are not suitable as therapeutics because of their low specificity and low effectiveness. Here, we compared newly generated anti-BMP4 llama-derived antibodies (VHHs) with 3 different types of commercially available BMP4 inhibitors, natural antagonists, small molecule BMPR inhibitors and conventional anti-BMP4 monoclonal antibodies. We found that the anti-BMP4 VHHs were as effective as the natural antagonist or small molecule inhibitors, but had higher specificity. We also showed that commercial anti-BMP4 antibodies were inferior in terms of both specificity and effectiveness. These findings might result from the fact that the VHHs C4C4 and C8C8 target a small region within the BMPR1 epitope of BMP4, whereas the commercial antibodies target other areas of the BMP4 molecule. Our results show that the newly developed anti-BMP4 VHHs are promising antibodies with better specificity and effectivity for inhibition of BMP4, making them an attractive tool for research and for therapeutic applications.

  4. Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

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    Di Pasquale, Elisa; Brivanlou, Ali H.

    2009-01-01

    Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

  5. Bone Morphogenetic Protein (BMP-4 and BMP-7 regulate differentially Transforming Growth Factor (TGF-β1 in normal human lung fibroblasts (NHLF

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    Lloyd Clare M

    2010-06-01

    Full Text Available Abstract Background Airway remodelling is thought to be under the control of a complex group of molecules belonging to the Transforming Growth Factor (TGF-superfamily. The Bone Morphogenetic Proteins (BMPs belong to this family and have been shown to regulate fibrosis in kidney and liver diseases. However, the role of BMPs in lung remodelling remains unclear. BMPs may regulate tissue remodelling in asthma by controlling TGF-β-induced profibrotic functions in lung fibroblasts. Methods Cell cultures were exposed to TGF-β1 alone or in the presence of BMP-4 or BMP-7; control cultures were exposed to medium only. Cell proliferation was assessed by quantification of the incorporation of [3H]-thymidine. The expression of the mRNA encoding collagen type I and IV, tenascin C and fibronectin in normal human lung fibroblasts (NHLF was determined by real-time quantitative PCR and the main results were confirmed by ELISA. Cell differentiation was determined by the analysis of the expression of α-smooth muscle actin (α-SMA by western blot and immunohistochemistry. The effect on matrix metalloproteinase (MMP activity was assessed by zymography. Results We have demonstrated TGF-β1 induced upregulation of mRNAs encoding the extracellular matrix proteins, tenascin C, fibronectin and collagen type I and IV when compared to unstimulated NHLF, and confirmed these results at the protein level. BMP-4, but not BMP-7, reduced TGF-β1-induced extracellular matrix protein production. TGF-β1 induced an increase in the activity of the pro-form of MMP-2 which was inhibited by BMP-7 but not BMP-4. Both BMP-4 and BMP-7 downregulated TGF-β1-induced MMP-13 release compared to untreated and TGF-β1-treated cells. TGF-β1 also induced a myofibroblast-like transformation which was partially inhibited by BMP-7 but not BMP-4. Conclusions Our study suggests that some regulatory properties of BMP-7 may be tissue or cell type specific and unveil a potential regulatory role for

  6. Genetic variation in bone morphogenetic protein (BMP) and colon and rectal cancer

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    Slattery, Martha L.; Lundgreen, Abbie; Herrick, Jennifer S.; Kadlubar, Susan; Caan, Bette J.; Potter, John D.; Wolff, Roger K.

    2011-01-01

    Bone morphogenetic proteins (BMP) are part of the TGF-β-signaling pathway; genetic variation in these genes may be involved in colorectal cancer. In this study we evaluated the association between genetic variation in BMP1 (11 tagSNPs), BMP2 (5 tagSNPs), BMP4 (3 tagSNPs), BMPR1A (9 tagSNPs), BMPR1B (21 tagSNPs), BMPR2 (11 tagSNPs), and GDF10 (7 tagSNPs) with risk of colon and rectal cancer and tumor molecular phenotype. We used data from population-based case-control studies (colon cancer n=1574 cases, 1970 controls; rectal cancer n=791 cases, 999 controls). We observed that genetic variation in BMPR1A, BMPR1B, BMPR2, BMP2, and BMP4 was associated with risk of developing colon cancer, with 20 to 30% increased risk for most high-risk genotypes. A summary of high-risk genotypes showed over a twofold increase in colon cancer risk at the upper risk category (OR 2.49 95% CI 1.95, 3.18). BMPR2, BMPR1B, BMP2, and GDF10 were associated with rectal cancer. BMPR2 rs2228545 was associated with an almost twofold increased risk of rectal cancer. The risk associated with the highest category of the summary score for rectal cancer was 2.97 (95% CI 1.87, 4.72). Genes in the BMP-signaling pathway were consistently associated with CIMP+ status in combination with both KRAS-mutated and MSI tumors. BMP genes interacted statistically significantly with other genes in the TGF-β-signaling pathway, including TGFβ1, TGFβR1, Smad 3, Smad 4, and Smad 7. Our data support a role for genetic variation in BMP-related genes in the etiology of colon and rectal cancer. One possible mechanism is via the TGF-β-signaling pathway. PMID:21387313

  7. Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression.

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    Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

    2016-05-05

    Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2(f/f);Bmp4(f/f)ameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling.

  8. Comparative expression analyses of bone morphogenetic protein 4 (BMP4) expressions in muscles of tilapia and common carp indicate that BMP4 plays a role in the intermuscular bone distribution in a dose-dependent manner.

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    Su, Shengyan; Dong, Zaijie

    2018-01-01

    Intermuscular bones in fish negatively influence both meet processing and attractiveness to consumers. Tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio) are both major farmed fish species globally, but whereas the former does not possess intermuscular bones, the latter does. Therefore, these two species might present a good model to study the genetic control of distribution of intermuscular bones in fish. Bone morphogenetic protein 4 (BMP4) gene is associated with tissue ossification and bone regeneration in mammals, but in fish its role in ossification remains understudied. To study the relationship between BMP4 and bone distribution in fish, we determined the expression of BMP4 in muscle tissues of common carp and tilapia on transcriptional and translational levels. As the gene has been merely predicted in silico from the genome of common carp, we have cloned and characterized it. The gene (GenBank: HQ446455) contains one intron and two exons, which encode a 400-amino acid protein with high homology to other known BMP4 protein sequences. Phylogenetic analysis showed that common carp clustered within the Cypriniformes clade (zebrafish was the closest ortholog) and tilapia within the Percomorpha clade. Using microCT scanning, we confirmed that intermuscular bones could be observed only in common carp (none in tilapia), but only in dorsal and caudal muscles (none in the ventral muscle). Expression levels of BMP4 in the muscles of common carp were in agreement with this observation both on transcriptional (qPCR) and translational (immunohistochemistry) level: higher in dorsal and caudal muscles, and lower in the ventral muscle. In tilapia, expression of BMP4 gene was also detectable in all three muscles, but expression levels in all three muscles were comparable to the one observed in the ventral muscle of carp, i.e., very low. Therefore, among the six studied muscles, the expression of BMP4 was high only in the two that possess intermuscular bones

  9. Bone Morphogenetic Proteins 2/4 Are Upregulated during the Early Development of Vascular Calcification in Chronic Kidney Disease

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    Xiao Wei

    2018-01-01

    Full Text Available Vascular calcification is a main cause of increased cardiovascular morbidity and mortality in chronic kidney disease (CKD patients. This study aimed to investigate the role of the bone morphogenetic protein (BMP signaling pathway in the early development of vascular calcification in CKD. A CKD vascular calcification rat model was established by providing rats with a 1.8% high-phosphorus diet and an intragastric administration of 2.5% adenine suspension. The kidney and aortic pathologies were analyzed. Blood biochemical indicators, serum BMP-2 and BMP-4 levels, and aortic calcium content were determined. The expression levels of BMP-2, BMP-4, bone morphogenetic protein receptor-IA (BMPR-IA, and matrix Gla protein (MGP in aorta were examined by quantitative real-time polymerase chain reaction and immunohistochemistry. Compared with the normal control (Nor rats, the CKD rats exhibited a significantly decreased body weight and an increased kidney weight as well as abnormal renal function and calcium-phosphorus metabolism. Aortic von Kossa and Alizarin red staining showed massive granular deposition and formation of calcified nodules in aorta at 8 weeks. The aortic calcium content was significantly increased, which was positively correlated with the serum BMP-2 (r=0.929; P<0.01 and serum BMP-4 (r=0.702; P<0.01 levels in CKD rats. The rat aortic BMP-2 mRNA level in the CKD rats was persistently increased, and the BMP-4 mRNA level was prominently increased at the 4th week, declining thereafter. Strong staining of BMP-2, BMP-4, BMPR-IA, and MGP proteins was observed in the tunica media of the aorta from the 4th week after model induction. In conclusion, activation of the BMP signaling pathway is involved in the early development of vascular calcification in CKD. Therefore, elevated serum BMP-2 and BMP-4 levels may serve as serum markers for CKD vascular calcification.

  10. Bone Morphogenetic Protein (BMP-7 expression is decreased in human hypertensive nephrosclerosis

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    Cohen Clemens D

    2010-11-01

    Full Text Available Abstract Background Bone Morphogenetic Protein (BMP-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. Methods BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8 expression was quantified in proximal tubular cells (HK-2. Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-β induced epithelial-to-mesenchymal transition (EMT, expression of TGF-β receptor type I (TGF-βRI and phosphorylated Smad 2 (pSmad 2 as well as on TNF-α induced apoptosis of proximal tubular cells. Results BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml were able to reverse TNF-α-induced apoptosis and TGF-β-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-βRI. In addition, BMP-7 was able to reverse TGF-β-induced phosphorylation of Smad 2. Conclusions The findings suggest a protective role for BMP-7 by counteracting the TGF-β and TNF-α-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease.

  11. Nuclear variants of bone morphogenetic proteins

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    Meinhart Christopher A

    2010-03-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. Results In all three proteins, a bipartite nuclear localization signal (NLS was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5 containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. Conclusions The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.

  12. The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

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    Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

    2000-12-01

    In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

  13. Nanostructured hydroxyapatite surfaces-mediated adsorption alters recognition of BMP receptor IA and bioactivity of bone morphogenetic protein-2.

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    Huang, Baolin; Yuan, Yuan; Ding, Sai; Li, Jianbo; Ren, Jie; Feng, Bo; Li, Tong; Gu, Yuantong; Liu, Changsheng

    2015-11-01

    Highly efficient loading of bone morphogenetic protein-2 (BMP-2) onto carriers with desirable performance is still a major challenge in the field of bone regeneration. Till now, the nanoscaled surface-induced changes of the structure and bioactivity of BMP-2 remains poorly understood. Here, the effect of nanoscaled surface on the adsorption and bioactivity of BMP-2 was investigated with a series of hydroxyapatite surfaces (HAPs): HAP crystal-coated surface (HAP), HAP crystal-coated polished surface (HAP-Pol), and sintered HAP crystal-coated surface (HAP-Sin). The adsorption dynamics of recombinant human BMP-2 (rhBMP-2) and the accessibility of the binding epitopes of adsorbed rhBMP-2 for BMP receptors (BMPRs) were examined by a quartz crystal microbalance with dissipation. Moreover, the bioactivity of adsorbed rhBMP-2 and the BMP-induced Smad signaling were investigated with C2C12 model cells. A noticeably high mass-uptake of rhBMP-2 and enhanced recognition of BMPR-IA to adsorbed rhBMP-2 were found on the HAP-Pol surface. For the rhBMP-2-adsorbed HAPs, both ALP activity and Smad signaling increased in the order of HAP-Sinuses of rhBMP-2 in clinical applications and arouse broad interests among researchers in the fields of nano-biotechnology, biomaterials and bone tissue engineering. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  14. Colloid, adhesive and release properties of nanoparticular ternary complexes between cationic and anionic polysaccharides and basic proteins like bone morphogenetic protein BMP-2.

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    Petzold, R; Vehlow, D; Urban, B; Grab, A L; Cavalcanti-Adam, E A; Alt, V; Müller, M

    2017-03-01

    Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

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    Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

    2012-04-01

    To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Bone morphogenetic protein 4 is overexpressed in and promotes migration and invasion of drug-resistant cancer cells.

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    Zhou, Kairui; Shi, Xiaoli; Huo, Jinling; Liu, Weihua; Yang, Dongxiao; Yang, Tengjiao; Qin, Tiantian; Wang, Cong

    2017-08-01

    Drug resistance and metastasis significantly hinder chemotherapy and worsen prognoses in cancer. Bone morphogenetic protein 4 (BMP4) belongs to the TGF-β superfamily, has broad biological activities in cell proliferation and cartilage differentiation and is also able to induce migration and invasion. Herein, we investigated the role of BMP4 in the regulation of metastasis in paclitaxel-resistant human esophageal carcinoma EC109 cells (EC109/Taxol) and docetaxel-resistant human gastric cancer MGC803 cells (MGC/Doc). In these drug-resistant cell lines, we found the cell motility was enhanced and BMP4 was up-regulated relative to their respective parental cell lines. Consistent with in vitro assays, migration potential and BMP4 expression were increased in EC109/Taxol nude mice. Furthermore, to address whether BMP4 was required to enhance the metastatic in EC109/Taxol cells, the pharmacological inhibitor of BMP signaling dorsomorphin was used; meanwhile, we found that the migration and invasion abilities were inhibited. Moreover, the canonical Smad signaling pathway was investigated. Overall, our studies demonstrated that BMP4 participates in the regulation of invasion and migration by EC109/Taxol cells, and inhibition of BMP4 may be a novel strategy to interfere with metastasis in cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Recombinant human bone morphogenetic protein induces bone formation

    International Nuclear Information System (INIS)

    Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

    1990-01-01

    The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

  18. Bone morphogenetic protein 4 inhibits insulin secretion from rodent beta cells through regulation of calbindin1 expression and reduced voltage-dependent calcium currents

    DEFF Research Database (Denmark)

    Christensen, Gitte L.; Jacobsen, Maria L. B.; Wendt, Anna

    2015-01-01

    AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive loss of pancreatic beta cell mass and function. Therefore, it is of therapeutic interest to identify factors with the potential to improve beta cell proliferation and insulin secretion. Bone morphogenetic protein 4 (BMP4) expression...

  19. Bone morphogenetic protein signalling in colorectal cancer

    NARCIS (Netherlands)

    Hardwick, James C.; Kodach, Liudmila L.; Offerhaus, G. Johan; van den Brink, Gijs R.

    2008-01-01

    Much of the current understanding of colorectal cancer stems from the study of rare, inherited colorectal cancer syndromes. Mutations in the bone morphogenetic protein (BMP) pathway have been found in juvenile polyposis, an inherited polyposis syndrome that predisposes to colorectal cancer. The

  20. Cyst-Like Osteolytic Formations in Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Augmented Sheep Spinal Fusion.

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    Pan, Hsin Chuan; Lee, Soonchul; Ting, Kang; Shen, Jia; Wang, Chenchao; Nguyen, Alan; Berthiaume, Emily A; Zara, Janette N; Turner, A Simon; Seim, Howard B; Kwak, Jin Hee; Zhang, Xinli; Soo, Chia

    2017-07-01

    Multiple case reports using recombinant human bone morphogenetic protein-2 (rhBMP-2) have reported complications. However, the local adverse effects of rhBMP-2 application are not well documented. In this report we show that, in addition to promoting lumbar spinal fusion through potent osteogenic effects, rhBMP-2 augmentation promotes local cyst-like osteolytic formations in sheep trabecular bones that have undergone anterior lumbar interbody fusion. Three months after operation, conventional computed tomography showed that the trabecular bones of the rhBMP-2 application groups could fuse, whereas no fusion was observed in the control group. Micro-computed tomography analysis revealed that the core implant area's bone volume fraction and bone mineral density increased proportionately with rhBMP-2 dose. Multiple cyst-like bone voids were observed in peri-implant areas when using rhBMP-2 applications, and these sites showed significant bone mineral density decreases in relation to the unaffected regions. Biomechanically, these areas decreased in strength by 32% in comparison with noncystic areas. Histologically, rhBMP-2-affected void sites had an increased amount of fatty marrow, thinner trabecular bones, and significantly more adiponectin- and cathepsin K-positive cells. Despite promoting successful fusion, rhBMP-2 use in clinical applications may result in local adverse structural alterations and compromised biomechanical changes to the bone. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. Expression of the bone morphogenetic protein-2 (BMP2 in the human cumulus cells as a biomarker of oocytes and embryo quality

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    Sirin B Demiray

    2017-01-01

    Full Text Available Background: The members of the transforming growth factor-B superfamily, as the bone morphogenetic proteins (BMPs subfamily and anti-Müllerian hormone (AMH, play a role during follicular development, and the bone morphogenetic protein-2 (BMP2, AMH, and THY1 are expressed in ovaries. Aim: This study was designed to define whether or not the expressions of these proteins in human cumulus cells (CCs can be used as predictors of the oocyte and embryo competence. Settings and Design: The study included nine female patients who were diagnosed as idiopathic infertility, aged 25–33 years (median 30 years and underwent Assisted Reproductive Technologies. Materials and Methods: The CCs from 60 oocyte–cumulus complexes obtained from the nine patients were evaluated with immunofluorescence staining in respect of BMPs, AMH and THY1 markers. The CCs surrounding the same oocytes were evaluated separately according to the oocyte and embryo quality. Statistical Analysis: Quantitative data were statistically analyzed for differences using the two-sided Mann–Whitney U test (P < 0.05. Results and Conclusions: Significant differences in immunofluorescence staining were observed in oocyte quality and embryo quality for the BMP2 only (P < 0.05. No significant differences were observed for AMH or CD90/THY1. Conclusion: These results demonstrated that there is a significant difference in the expression of BMP2 in the CCs of good quality oocytes and subsequently a good embryo.

  2. The Effects of Irradiation and Calcium-deficient Diet on the Expression of Bone Morphogenetic Protein-2/4 during Early Tooth Development

    International Nuclear Information System (INIS)

    Park, Dai Hee; Hwang, Eui Hwan; Lee, Sang Rae

    2000-01-01

    To investigate the expression of bone morphogenetic protein (BMP)-2/4 during early tooth development after irradiation and calcium-deficient diet. The pregnant three-week-old Sprague-Dawley rats were used for the study. The control group was non-irradiation/normal diet group (Group 1), and the experimental groups were irradiation/normal diet group (Group 2) and irradiation/calcium-diet group (Group 3). The abdomen of the rats at the 9th day of pregnancy were irradiated with single dose of 350 cGy. The rat pups were sacrificed at embryonic 18 days, 3 days and 14 days after delivery and the maxillae tooth germs were taken. The tissue sections of specimen were stained immunohistochemically with anti-BMP-2/4 antibody. At embryo-18 days, immunoreacivity for BMP-2/4 of the Group 1 was modetate in stratum intermedium of dental organ and weak in dental papilla and dental follicle, but that of Group 2 was weak in cell layer of dental organ, and no immunoreacivity was shown in dental papilla and dental follice of Group 2 and in all tissue components of the Group 3. At postnatal-3 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in cell layer of dental organ, odontoblasts and developing alveolar bone, but that of Group of 2 and Group 3 was weak in odontoblasts and developing alveolar bone. At postnatal-14 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in newly formed cementum, alveolar bone and odontoblasts, but that of Group 2 was weaker than that of Group 1. In the Group 3, tooth forming cell layer showed weak immunoreactivity, but other cell layers showed no immunoreactivity. The expression of bone morphogenetic protein (BMP)-2/4 during early tooth development was disturbed after irradiation and calcium-deficient diet.

  3. The Effects of Irradiation and Calcium-deficient Diet on the Expression of Bone Morphogenetic Protein-2/4 during Early Tooth Development

    Energy Technology Data Exchange (ETDEWEB)

    Park, Dai Hee; Hwang, Eui Hwan; Lee, Sang Rae [Dept. of Oral and Maxillofacial Radiology, College of Dentistry, Kyunghee University, Seoul (Korea, Republic of)

    2000-09-15

    To investigate the expression of bone morphogenetic protein (BMP)-2/4 during early tooth development after irradiation and calcium-deficient diet. The pregnant three-week-old Sprague-Dawley rats were used for the study. The control group was non-irradiation/normal diet group (Group 1), and the experimental groups were irradiation/normal diet group (Group 2) and irradiation/calcium-diet group (Group 3). The abdomen of the rats at the 9th day of pregnancy were irradiated with single dose of 350 cGy. The rat pups were sacrificed at embryonic 18 days, 3 days and 14 days after delivery and the maxillae tooth germs were taken. The tissue sections of specimen were stained immunohistochemically with anti-BMP-2/4 antibody. At embryo-18 days, immunoreacivity for BMP-2/4 of the Group 1 was modetate in stratum intermedium of dental organ and weak in dental papilla and dental follicle, but that of Group 2 was weak in cell layer of dental organ, and no immunoreacivity was shown in dental papilla and dental follice of Group 2 and in all tissue components of the Group 3. At postnatal-3 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in cell layer of dental organ, odontoblasts and developing alveolar bone, but that of Group of 2 and Group 3 was weak in odontoblasts and developing alveolar bone. At postnatal-14 days, immunoreacivity for BMP-2/4 of the Group 1 was strong in newly formed cementum, alveolar bone and odontoblasts, but that of Group 2 was weaker than that of Group 1. In the Group 3, tooth forming cell layer showed weak immunoreactivity, but other cell layers showed no immunoreactivity. The expression of bone morphogenetic protein (BMP)-2/4 during early tooth development was disturbed after irradiation and calcium-deficient diet.

  4. Bone morphogenetic protein 4 induces differentiation of colorectal cancer stem cells and increases their response to chemotherapy in mice.

    Science.gov (United States)

    Lombardo, Ylenia; Scopelliti, Alessandro; Cammareri, Patrizia; Todaro, Matilde; Iovino, Flora; Ricci-Vitiani, Lucia; Gulotta, Gaspare; Dieli, Francesco; de Maria, Ruggero; Stassi, Giorgio

    2011-01-01

    The limited clinical response observed in many patients with colorectal cancer may be related to the presence of chemoresistant colorectal cancer stem cells (CRC-SCs). Bone morphogenetic protein 4 (BMP4) promotes the differentiation of normal colonic stem cells. We investigated whether BMP4 might be used to induce differentiation of CRC-SCs and for therapeutic purposes. CRC-SCs were isolated from 25 tumor samples based on expression of CD133 or using a selection culture medium. BMP4 expression and activity on CRC-SCs were evaluated in vitro; progeny of the stem cells were evaluated by immunofluorescence, immunoblot, and flow cytometry analyses. The potential therapeutic effect of BMP4 was assessed in immunocompromised mice after injection of CRC-SCs that responded to chemotherapy (n = 4) or that did not (n = 2). CRC-SCs did not express BMP4 whereas differentiated cells did. Recombinant BMP4 promoted differentiation and apoptosis of CRC-SCs in 12 of 15 independent experiments; this effect did not depend on Small Mothers against decapentaplegic (Smad)4 expression level or microsatellite stability. BMP4 activated the canonical and noncanonical BMP signaling pathways, including phosphoInositide 3-kinase (PI3K) and PKB (protein kinase B)/AKT. Mutations in PI3K or loss of Phosphatase and Tensin homolog (PTEN) in Smad4-defective tumors made CRC-SCs unresponsive to BMP4. Administration of BMP4 to immunocompromised mice with tumors that arose from CRC-SCs increased the antitumor effects of 5-fluorouracil and oxaliplatin. BMP4 promotes terminal differentiation, apoptosis, and chemosensitization of CRC-SCs in tumors that do not have simultaneous mutations in Smad4 and constitutive activation of PI3K. BMP4 might be developed as a therapeutic agent against cancer stem cells in advanced colorectal tumors. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  5. Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

    Science.gov (United States)

    Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

    2018-02-01

    Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

  6. NOX4 mediates BMP4-induced upregulation of TRPC1 and 6 protein expressions in distal pulmonary arterial smooth muscle cells.

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    Qian Jiang

    Full Text Available Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4 mediated, elevated expression of canonical transient receptor potential (TRPC largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs. In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.We employed recombinant human BMP4 (rhBMP4 to determine the effects of BMP4 on NADPH oxidase 4 (NOX4 and reactive oxygen species (ROS production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4 and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca2+ determination in PASMCs.In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13 %, and the mean ROS level was (123.65±1.62 % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca2+]i and SOCE. Furthermore, compared with control group (0.21±0.001, the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001 (P<0.01. However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H2O2 100 µM, 24 h rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca2+]i and store-operated calcium entry (SOCE, suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.

  7. Role of Bone Morphogenetic Protein 7 (BMP7 in the Modulation of Corneal Stromal and Epithelial Cell Functions

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    Bhavani S. Kowtharapu

    2018-05-01

    Full Text Available In the cornea, healing of the wounded avascular surface is an intricate process comprising the involvement of epithelial, stromal and neuronal cell interactions. These interactions result to the release of various growth factors that play prominent roles during corneal wound healing response. Bone morphogenetic proteins (BMPs are unique multi-functional potent growth factors of the transforming growth factor-beta (TGF-β superfamily. Treatment of corneal epithelial cells with substance P and nerve growth factor resulted to an increase in the expression of BMP7 mRNA. Since BMP7 is known to modulate the process of corneal wound healing, in this present study, we investigated the influence of exogenous rhBMP7 on human corneal epithelial cell and stromal cell (SFs function. To obtain a high-fidelity expression profiling of activated biomarkers and pathways, transcriptome-wide gene-level expression profiling of epithelial cells in the presence of BMP7 was performed. Gene ontology analysis shows BMP7 stimulation activated TGF-β signaling and cell cycle pathways, whereas biological processes related to cell cycle, microtubule and intermediate filament cytoskeleton organization were significantly impacted in corneal epithelial cells. Scratch wound healing assay showed increased motility and migration of BMP7 treated epithelial cells. BMP7 stimulation studies show activation of MAPK cascade proteins in epithelial cells and SFs. Similarly, a difference in the expression of claudin, Zink finger E-box-binding homeobox 1 was observed along with phosphorylation levels of cofilin in epithelial cells. Stimulation of SFs with BMP7 activated them with increased expression of α-smooth muscle actin. In addition, an elevated phosphorylation of epidermal growth factor receptor following BMP7 stimulation was also observed both in corneal epithelial cells and SFs. Based on our transcriptome analysis data on epithelial cells and the results obtained in SFs, we

  8. In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

    Science.gov (United States)

    Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

    2015-03-01

    Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

  9. Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation

    NARCIS (Netherlands)

    Croes, M.; Kruyt, M. C.; Groen, W. M.; Van Dorenmalen, K. M.A.; Dhert, W. J.A.; Öner, F. C.; Alblas, J.

    2018-01-01

    Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is

  10. Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.

    Science.gov (United States)

    Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Nash, Gerard B; Mallat, Ziad; Chilvers, Edwin R; Upton, Paul D; Morrell, Nicholas W

    2017-08-18

    Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Thyroid hormone-induced hypertrophy in mesenchymal stem cell chondrogenesis is mediated by bone morphogenetic protein-4.

    Science.gov (United States)

    Karl, Alexandra; Olbrich, Norman; Pfeifer, Christian; Berner, Arne; Zellner, Johannes; Kujat, Richard; Angele, Peter; Nerlich, Michael; Mueller, Michael B

    2014-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair.

  12. Persistent expression of BMP-4 in embryonic chick adrenal cortical cells and its role in chromaffin cell development

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    Halbach Oliver

    2008-10-01

    Full Text Available Abstract Background Adrenal chromaffin cells and sympathetic neurons both originate from the neural crest, yet signals that trigger chromaffin development remain elusive. Bone morphogenetic proteins (BMPs emanating from the dorsal aorta are important signals for the induction of a sympathoadrenal catecholaminergic cell fate. Results We report here that BMP-4 is also expressed by adrenal cortical cells throughout chick embryonic development, suggesting a putative role in chromaffin cell development. Moreover, bone morphogenetic protein receptor IA is expressed by both cortical and chromaffin cells. Inhibiting BMP-4 with noggin prevents the increase in the number of tyrosine hydroxylase positive cells in adrenal explants without affecting cell proliferation. Hence, adrenal BMP-4 is likely to induce tyrosine hydroxylase in sympathoadrenal progenitors. To investigate whether persistent BMP-4 exposure is able to induce chromaffin traits in sympathetic ganglia, we locally grafted BMP-4 overexpressing cells next to sympathetic ganglia. Embryonic day 8 chick sympathetic ganglia, in addition to principal neurons, contain about 25% chromaffin-like cells. Ectopic BMP-4 did not increase this proportion, yet numbers and sizes of 'chromaffin' granules were significantly increased. Conclusion BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.

  13. Role of Bone Morphogenetic Proteins-7 (BMP-7 in the Renal Improvement Effect of DangGui (Angelica sinensis in Type-1 Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Ching-Hua Yeh

    2011-01-01

    Full Text Available Hyperglycemia induced reactive oxygen species (ROS generation is believed as major factors leading to diabetic nephropathy (DN. DangGui (Angelica sinensis is mentioned to show renal protective effect in combination with other herbs. Bone morphogenetic proteins-7 (BMP-7 is produced merit in protection of DN. The role of BMP-7 in DangGui-induced renal improvement is not clear. The present study investigated the effects of DangGui on renal functions, BMP-7 expression and the levels of ROS in streptozotocin (STZ-induced diabetic rats and high glucose-exposed rat mesangial cells (RMCs. After 1- or 4-week treatment, DangGui improved renal functions and increased renal BMP-7 expression in diabetic rats. The BMP-7 expression in RMCs was reduced by high glucose treatment and this could be reversed by DangGui. Moreover, RMCs exposed to high glucose were expired by BMP-7 RNAi transfection but those cells remained alive by scramble transfection. Thus, we employed regular RMCs to knock down BMP-7 with RNAi and we found that DangGui increased BMP-7 expression in these RMCs. Direct activation of BMP-7 expression by DangGui could be considered. The results of DPPH assay, DHE stain and lucigenin assay indicated that DangGui could inhibit high glucose-induced ROS in RMCs. These results suggest that DangGui has an ability to improve renal functions in STZ-diabetic rats through increasing endogenous BMP-7 expression and decreasing oxidative stress in kidney. The present study suggest that DangGui could be applied to improve renal functions in diabetic disorders.

  14. Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis

    DEFF Research Database (Denmark)

    Zhang, Hongbin; Schulz, Tim J; Espinoza, Daniel O

    2010-01-01

    Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin...... BMP7's suppressive effect on pref-1 transcription. Together, these data suggest cross talk between the insulin and BMP signaling systems by which BMP7 can rescue brown adipogenesis in cells with insulin resistance....

  15. Quantitative analysis of bone morphogenetic protein 15 (BMP15 and growth differentiation factor 9 (GDF9 gene expression in calf and adult bovine ovaries

    Directory of Open Access Journals (Sweden)

    Hayashi Ken-go

    2011-03-01

    Full Text Available Abstract Background It has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15 and growth and differentiation factor 9 (GDF9 play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes. Methods Bovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR and in situ hybridization. Immunohistochemical analysis was also performed. Results The expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry. Conclusions Our result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

  16. BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

    Science.gov (United States)

    Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

    2015-11-01

    Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

  17. Expression of bone morphogenetic proteins and Msx genes during root formation.

    Science.gov (United States)

    Yamashiro, T; Tummers, M; Thesleff, I

    2003-03-01

    Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

  18. Simvastatin enhances bone morphogenetic protein receptor type II expression

    International Nuclear Information System (INIS)

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

    2006-01-01

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

  19. Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia.

    Science.gov (United States)

    Barlow, A J; Francis-West, P H

    1997-01-01

    The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The signals that control facial outgrowth and patterning are largely unknown. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. We show that expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 and that ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. We correlate this activation of gene expression with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of shh and bifurcation of the palatine bone. We also show that application of BMP-2 can increase cell proliferation of the mandibular primordia. Our data suggest that BMP-2 and BMP-4 are part of a signalling cascade that controls outgrowth and patterning of the facial primordia.

  20. Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos

    DEFF Research Database (Denmark)

    Dunworth, William P; Cardona-Costa, Jose; Bozkulak, Esra Cagavi

    2014-01-01

    : Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. METHODS AND RESULTS: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2...... signaling in zebrafish embryos and mouse embryonic stem cell-derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox...

  1. Bone morphogenetic protein-7 promotes chondrogenesis in human amniotic epithelial cells.

    Science.gov (United States)

    Zhou, Junjie; Yu, Guangrong; Cao, Chengfu; Pang, Jinhui; Chen, Xianqi

    2011-06-01

    Bone morphogenetic proteins (BMPs) play important roles at multiple stages of chondrogenesis. This study was undertaken to investigate the potential role of bone morphogenetic protein-7 (BMP-7) in the differentiation of chondrocytes using tissue engineering techniques. The impact of BMP-7 on human amniotic epithelial cells (hAECs) was tested. The hAECs were treated either with recombinant human BMP-7 cDNA or with transforming growth factor beta 1 (TGF-β1) as a positive control for three weeks in vitro. Cartilaginous differentiation and proliferation were assayed by quantitative RT-PCR, histology, and in situ hybridization. Our results were such that hAECs treated with either BMP-7 or TGF-β1 expressed cartilage markers (aggrecan, Sox9, CEP-68, and type II and X collagens) within three weeks. Compared with a control vector, BMP-7 induced a decrease in type I collagen expression, while the transcription of the cartilage-specific type II collagen remained stable. In induction experiments, BMP-7 transgenic hAECs exhibited the largest amount of matrix synthesis. In conclusion, these data indicate that BMP-7 plays an important role in inducing the production of cartilage by hAECs in vitro. Cartilage differentiation and matrix maturation can be promoted by BMPs in a cartilage engineering paradigm. These properties make BMPs promising tools in the engineering of cartilaginous joint bio-prostheses and as candidate biological agents or genes for cartilage stabilisation.

  2. Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

    Science.gov (United States)

    Calpe, Silvia; Wagner, Koen; El Khattabi, Mohamed; Rutten, Lucy; Zimberlin, Cheryl; Dolk, Edward; Verrips, C Theo; Medema, Jan Paul; Spits, Hergen; Krishnadath, Kausilia K

    2015-11-01

    Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies. ©2015 American Association for Cancer Research.

  3. Bone Morphogenetic Protein Coating on Titanium Implant Surface: a Systematic Review

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    Haim Haimov

    2017-06-01

    Full Text Available Objectives: The purpose of the study is to systematically review the osseointegration process improvement by bone morphogenetic protein coating on titanium implant surface. Material and Methods: An electronic literature search was conducted through the MEDLINE (PubMed and EMBASE databases. The search was restricted for articles published during the last 10 years from October 2006 to September 2016 and articles were limited to English language. Results: A total of 41 articles were reviewed, and 8 of the most relevant articles that are suitable to the criteria were selected. Articles were analysed regarding concentration of bone morphogenetic protein (BMP, delivery systems, adverse reactions and the influence of the BMP on the bone and peri-implant surface in vivo. Finally, the present data included 340 implants and 236 models. Conclusions: It’s clearly shown from most of the examined studies that bone morphogenetic protein increases bone regeneration. Further studies should be done in order to induce and sustain bone formation activity. Osteogenic agent should be gradually liberated and not rapidly released with priority to three-dimension reservoir (incorporated titanium implant surface in order to avoid following severe side effects: inflammation, bleeding, haematoma, oedema, erythema, and graft failure.

  4. Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-β2 in Human TM Cells.

    Science.gov (United States)

    Mody, Avani A; Wordinger, Robert J; Clark, Abbot F

    2017-02-01

    Increased expression of TGF-β2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-β2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-β2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-β2 induction of ECM proteins in TM cells. Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-β2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-β2-induced expression of fibronectin and PAI-1 in TM cells (P protein 4 induced ID1 and ID3 expression suppresses TGF-β2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-β2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

  5. Regulation of bone morphogenetic proteins in early embryonic development

    Science.gov (United States)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  6. Survey of the Effectiveness of Internet Information on Patient Education for Bone Morphogenetic Protein.

    Science.gov (United States)

    Huang, Meng; Briceño, Valentina; Lam, Sandi K; Luerssen, Thomas G; Jea, Andrew

    2016-03-01

    In light of recent reports of potential short- and long-term complications of bone morphogenetic protein (BMP) and increasing "off-label" use among spine surgeons, we wished to analyze online information on BMP and its controversial uses, as patients frequently search the Internet for medical information, even though the quality and accuracy of available information are highly variable. Between December 2014 and January 2015, we conducted a Google search to identify the 50 most accessed websites providing BMP information using the search phrase "bone morphogenetic protein." Websites were classified based on authorship. Each website was examined for the provision of appropriate patient inclusion and exclusion criteria, surgical and nonsurgical treatment alternatives, purported benefits, disclosure of common and potential complications, peer-reviewed literature citations, and discussion of off-label use. Two percent of websites were authored by private medical groups, 2% by academic medical groups, 10% by insurance companies, 16% by biomedical industries, 4% by news sources, 0% by lawyers, and 66% by others. Sixty-two percent referenced peer-reviewed literature. Benefits and complications were reported in 44% and 26% of websites, respectively. Surgical and nonsurgical treatment alternatives were mentioned in 16% and 4% of websites, respectively. Discussion of off-label BMP use occurred in 18% of websites. Our study showed the ineffectiveness of the Internet in reporting quality information on BMP use. We found that websites authored by insurance companies provide an acceptable foundation for patient education. This, however, cannot replace the need for a thorough dialogue between doctor and patient about risks, benefits, and indications. Copyright © 2016. Published by Elsevier Inc.

  7. Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

    Science.gov (United States)

    Meyers, Emily A; Gobeske, Kevin T; Bond, Allison M; Jarrett, Jennifer C; Peng, Chian-Yu; Kessler, John A

    2016-02-01

    Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Role of bone morphogenetic protein-7 in renal fibrosis

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    Rui Xi eLi

    2015-04-01

    Full Text Available Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET. BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application.

  9. Role of bone morphogenetic protein-7 in renal fibrosis

    Science.gov (United States)

    Li, Rui Xi; Yiu, Wai Han; Tang, Sydney C. W.

    2015-01-01

    Renal fibrosis is final common pathway of end stage renal disease. Irrespective of the primary cause, renal fibrogenesis is a dynamic process which involves a large network of cellular and molecular interaction, including pro-inflammatory cell infiltration and activation, matrix-producing cell accumulation and activation, and secretion of profibrogenic factors that modulate extracellular matrix (ECM) formation and cell-cell interaction. Bone morphogenetic protein-7 is a protein of the TGF-β super family and increasingly regarded as a counteracting molecule against TGF-β. A large variety of evidence shows an anti-fibrotic role of BMP-7 in chronic kidney disease, and this effect is largely mediated via counterbalancing the profibrotic effect of TGF-β. Besides, BMP-7 reduced ECM formation by inactivating matrix-producing cells and promoting mesenchymal-to-epithelial transition (MET). BMP-7 also increased ECM degradation. Despite these observations, the anti-fibrotic effect of BMP-7 is still controversial such that fine regulation of BMP-7 expression in vivo might be a great challenge for its ultimate clinical application. PMID:25954203

  10. Bone morphogenetic protein-2 (BMP-2 and transforming growth factor-β1 (TGF-β1 alter connexin 43 phosphorylation in MC3T3-E1 Cells

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    Rudkin George H

    2001-07-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs and transforming growth factor-βs (TGF-βs are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC in MC3T3-E1 cells. Connexin 43 (Cx43 has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC. Results Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-β1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. Conclusions BMP-2 and TGF-β1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-β1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

  11. Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products.

    Science.gov (United States)

    Bae, Hyun W; Zhao, Li; Kanim, Linda E A; Wong, Pamela; Delamarter, Rick B; Dawson, Edgar G

    2006-05-20

    Enzyme-linked immunosorbent assay was used to detect bone morphogenetic proteins (BMPs) 2, 4, and 7 in 9 commercially available ("off the shelf") demineralized bone matrix (DBM) product formulations using 3 different manufacturer's production lots of each DBM formulation. To evaluate and compare the quantity of BMPs among several different DBM formulations (inter-product variability), as well as examine the variability of these proteins in different production lots within the same DBM formulation (intra-product variability). DBMs are commonly used to augment available bone graft in spinal fusion procedures. Surgeons are presented with an ever-increasing variety of commercially available human DBMs from which to choose. Yet, there is limited information on a specific DBM product's osteoinductive efficacy, potency, and constancy. There were protein extracts from each DBM sample separately dialyzed 4 times against distilled water at 4 degrees C for 48 hours. The amount of BMP-2, BMP-4, and BMP-7 was determined using enzyme-linked immunosorbent assay. RESULTS.: The concentrations of detected BMP-2 and BMP-7 were low for all DBM formulations, only nanograms of BMP were extracted from each gram of DBM (20.2-120.6 ng BMP-2/g DBM product; 54.2-226.8 ng BMP-7/g DBM). The variability of BMP concentrations among different lots of the same DBM formulation, intra-product variability, was higher than the variability of concentrations among different DBM formulations, inter-product variability (coefficient of variation range BMP-2 [16.34% to 76.01%], P DBMs are low, in the order of 1 x 10(-9) g of BMP/g of DBM. There is higher variability in concentration of BMPs among 3 different lots of the same DBM formulation than among different DBM formulations. This variability questions DBM products' reliability and, possibly, efficacy in providing consistent osteoinduction.

  12. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

    NARCIS (Netherlands)

    Xu, Cui-Ping; Ji, Wen-Min; van den Brink, Gijs R.; Peppelenbosch, Maikel P.

    2006-01-01

    To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. Fifty-four adult male Wistar rats were randomly

  13. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

    NARCIS (Netherlands)

    Xu, Cui-Ping; Ji, Wen-Min; van den Brink, Gijs R.; Peppelenbosch, Maikel P.

    2006-01-01

    AIM: To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. METHODS: Fifty-four adult male Wistar rats

  14. Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

    Science.gov (United States)

    Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

    2017-01-01

    In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

  15. Maxillary anterior ridge augmentation with recombinant human bone morphogenetic protein 2.

    Science.gov (United States)

    Edmunds, Ryan K; Mealey, Brian L; Mills, Michael P; Thoma, Daniel S; Schoolfield, John; Cochran, David L; Mellonig, Jim

    2014-01-01

    No human studies exist on the use of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an absorbable collagen sponge (ACS) as a sole graft material for lateral ridge augmentation in large ridge defect sites. This series evaluates the treatment outcome of maxillary anterior lateral ridge augmentation with rhBMP-2/ACS. Twenty patients were treated with rhBMP-2/ACS and fixation screws for space maintenance. Cone beam volumetric tomography measurements were used to determine gain in ridge width, and a bone core biopsy was obtained. The mean horizontal ridge gain was 1.2 mm across sites, and every site gained width.

  16. Comparison of Cell Viability and Embryoid Body Size of Two Embryonic Stem Cell Lines After Different Exposure Times to Bone Morphogenetic Protein 4

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    Nehleh Zarei Fard

    2015-03-01

    Full Text Available Background: Activation of bone morphogenetic protein 4 (BMP4 signaling pathway in embryonic stem (ES cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB, size, and cavitation of ES cells. Methods: Embryonic stem cells (R1 and B1 lines were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4 or after 48 h (+BMP4 of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P<0.05 was considered as significant. Results: In EB culture protocol, cell number significantly decreased in +BMP4 culture condition with greater cavity size compared to the ++BMP4 condition at day 5 (P=0.009. In contrast, in monolayer culture system, there was no significant difference in the cell number between all groups (P=0.91. Conclusion: The results suggest that short-term exposure of BMP4 is required to promote cavitation in EBs according to lower cell number in +BMP4 condition. Different cell lines showed different behavior in cavitation formation.

  17. Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP Signaling and Inflammatory StimuliSummary

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    Wei Ye

    Full Text Available Background & Aims: Gastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5 cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5+ve cells during inflammation. Methods: The H+/K+-adenosine triphosphatase β-subunit promoter was used to express the BMP inhibitor noggin (Nog in the stomach (H+/K+-Nog mice. Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1aflox/flox mice. Lgr5/GFP+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom. Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H+/K+-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction. Results: Lgr5-Cre;Bmpr1aflox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP+ve cells. Infection of Lgr5-Cre;Bmpr1aflox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea simplicifolia lectin II and Ulex europaeus agglutinin 1, and

  18. Bone morphogenetic protein-2 and bone therapy: successes and pitfalls.

    Science.gov (United States)

    Poon, Bonnie; Kha, Tram; Tran, Sally; Dass, Crispin R

    2016-02-01

    Bone morphogenetic proteins (BMPs), more specifically BMP-2, are being increasingly used in orthopaedic surgery due to advanced research into osteoinductive factors that may enhance and improve bone therapy. There are many areas in therapy that BMP-2 is being applied to, including dental treatment, open tibial fractures, cancer and spinal surgery. Within these areas of treatment, there are many reports of successes and pitfalls. This review explores the use of BMP-2 and its successes, pitfalls and future prospects in bone therapy. The PubMed database was consulted to compile this review. With successes in therapy, there were descriptions of a more rapid healing time with no signs of rejection or infection attributed to BMP-2 treatment. Pitfalls included BMP-2 'off-label' use, which lead to various adverse effects. Our search highlighted that optimising treatment with BMP-2 is a direction that many researchers are exploring, with areas of current research interest including concentration and dose of BMP-2, carrier type and delivery. © 2015 Royal Pharmaceutical Society.

  19. Form-deprivation myopia induces decreased expression of bone morphogenetic protein-2, 5 in guinea pig sclera

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    Qing Wang

    2015-02-01

    Full Text Available AIM: To identify the presence of various bone morphogenetic proteins (BMPs and their receptors in normal sclera of human, rat and guinea pigs, and to determine whether their expression changed with form-deprivation myopia (FDM in guinea pig sclera. METHODS: The expression of BMPs and BMP receptors were detected using reverse transcription polymerase chain reaction (RT-PCR and immunofluorescence. Two-week-old guinea pigs were monocularly form-deprived with a translucent lens. After fourteen days induction of FDM, total RNA was isolated and subjected to RT-PCR to examine the changes of BMPs and BMP receptors in tissues from the posterior sclera. Western blotting analysis was used to investigate their changes in protein levels. RESULTS: Human sclera expressed mRNAs for BMP-2, -4, -5, -7, -RIA, -RIB and BMP-RII. Conversely, rat sclera only expressed mRNA for BMP-7 and BMP-RIB, while the expression of BMPs and BMP receptors in guinea pigs were similar to that of humans. Human sclera also expresses BMP-2, -4, -5,-7 in protein level. Fourteen days after the induction of myopia, significant decreased expressions for BMP-2 and BMP-5 in the posterior sclera of FDM-affected eyes (PCONCLUSION: Various BMPs were expressed in human and guinea pig sclera. In the posterior sclera, expressions of BMP-2 and BMP-5 significantly decreased in FDM eyes. This finding indicates that various BMPs as components of the scleral cytokines regulating tissue homeostasis and provide evidence that alterations in the expression of BMP-2 and BMP-5 are associated with sclera remodeling during myopia induction.

  20. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    Science.gov (United States)

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Recombinant human bone morphogenetic protein 2 in augmentation procedures: case reports.

    Science.gov (United States)

    Luiz, Jaques; Padovan, Luis Eduardo Marques; Claudino, Marcela

    2014-01-01

    To successfully rehabilitate edentulous patients using endosseous implants, there must be enough available bone. Several techniques have been proposed for augmentation of sites with insufficient bone volume. Although autogenous bone has long been considered the gold standard for such procedures, the limited availability of graft material and a high morbidity rate are potential disadvantages of this type of graft. An alternative is to use recombinant human bone morphogenetic protein 2 (rhBMP-2), which is able to support bone regeneration in the oral environment. These cases demonstrate the applicability of rhBMP-2 in maxillary sinus elevation and augmentation procedures in the maxilla to enable dental implant placement. The use of rhBMP-2 in alveolar augmentation procedures had several clinical benefits for these patients.

  2. Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

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    Mehdi Forouzandeh-Moghadam

    2009-01-01

    Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

  3. The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

    International Nuclear Information System (INIS)

    Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

    2008-01-01

    Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation

  4. BMP suppresses PTEN expression via RAS/ERK signaling.

    Science.gov (United States)

    Beck, Stayce E; Carethers, John M

    2007-08-01

    Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

  5. Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

    Science.gov (United States)

    Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

    2016-04-01

    Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

  6. Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation

    NARCIS (Netherlands)

    Wang, J.; Zheng, Y.; Zhao, J.; Liu, T.; Gao, L.; Gu, Z.; Wu, G.

    2012-01-01

    Objectives To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer. Material and Methods Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created

  7. Bone morphogenetic protein use in spine surgery-complications and outcomes: a systematic review.

    Science.gov (United States)

    Faundez, Antonio; Tournier, Clément; Garcia, Matthieu; Aunoble, Stéphane; Le Huec, Jean-Charles

    2016-06-01

    Because of significant complications related to the use of autologous bone grafts in spinal fusion surgery, bone substitutes and growth factors such as bone morphogenetic protein (BMP) have been developed. One of them, recombinant human (rh) BMP-2, has been approved by the Food and Drug Administration (FDA) for use under precise conditions. However, rhBMP-2-related side effects have been reported, used in FDA-approved procedures, but also in off-label use.A systematic review of clinical data was conducted to analyse the rhBMP-2-related adverse events (AEs), in order to assess their prevalence and the associated surgery practices. Medline search with keywords "bone morphogenetic protein 2", "lumbar spine", "anterolateral interbody fusion" (ALIF) and the filter "clinical trial". FDA published reports were also included. Study assessment was made by authors (experienced spine surgeons), based on quality of study designs and level of evidence. Extensive review of randomised controlled trials (RCTs) and controlled series published up to the present point, reveal no evidence of a significant increase of AEs related to rhBMP-2 use during ALIF surgeries, provided that it is used following FDA guidelines. Two additional RCTs performed with rhBMP-2 in combination with allogenic bone dowels reported increased bone remodelling in BMP-treated patients. This AE was transient and had no consequence on the clinical outcome of the patients. No other BMP-related AEs were reported in these studies. This literature review confirms that the use of rhBMP-2 following FDA-approved recommendations (i.e. one-level ALIF surgery with an LT-cage) is safe. The rate of complications is low and the AEs had been identified by the FDA during the pre-marketing clinical trials. The clinical efficiency of rhBMP-2 is equal or superior to that of allogenic or autologous bone graft in respect to fusion rate, low back pain disability, patient satisfaction and rate of re-operations. For all other off

  8. Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein-4 increases oocyte diameter, but impairs subsequent developmental competence.

    Science.gov (United States)

    Yang, Yinghua; Kanno, Chihiro; Sakaguchi, Kenichiro; Yanagawa, Yojiro; Katagiri, Seiji; Nagano, Masashi

    2017-11-01

    Bone morphogenetic protein-4 (BMP-4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo-grown oocytes. We herein investigated whether an extended IVG culture with BMP-4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte-granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP-4 (10 ng/mL), while a 12 day culture with BMP-4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP-4 (83.2%), but was significantly lower without BMP-4 (58.9%) than the control (83.0%). Prolong-cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP-4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP-4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP-4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes. © 2017 Japanese Society of Animal Science.

  9. Estrogens increase expression of bone morphogenetic protein 8b in brown adipose tissue of mice

    NARCIS (Netherlands)

    A. Grefhorst (Aldo); J.C. van den Beukel (Anneke); A.F. van Houten (A.); J. Steenbergen (Jacobie); J.A. Visser (Jenny); A.P.N. Themmen (Axel)

    2015-01-01

    textabstractBackground: In mammals, white adipose tissue (WAT) stores fat and brown adipose tissue (BAT) dissipates fat to produce heat. Several studies showed that females have more active BAT. Members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families are expressed

  10. Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart.

    Science.gov (United States)

    Abdelwahid, E; Rice, D; Pelliniemi, L J; Jokinen, E

    2001-07-01

    The bone morphogenetic proteins BMP-2 and BMP-4 and the homeobox gene MSX-2 are required for normal development of many embryonic tissues. To elucidate their possible roles during the remodeling of the tubular heart into a fully septated four-chambered heart, we have localized the mRNA of Bmp-2, Bmp-4, Msx-2 and apoptotic cells in the developing mouse heart from embryonic day (E)11 to E17. mRNA was localized by in situ hybridization, and apoptotic cells by TUNEL (TDT-mediated dUTP-biotin nick end-labeling) as well as by transmission electron microscopy. By analyzing adjacent serial sections, we demonstrated that the expression of Msx-2 and Bmp-2 strikingly overlapped in the atrioventricular canal myocardium, in the atrioventricular junctional myocardium, and in the maturing myocardium of the atrioventricular valves. Bmp-4 was expressed in the outflow tract myocardium and in the endocardial cushion of the outflow tract ridges from E12 to E14. Msx-2 appeared in the mesenchyme of the atrioventricular endocardial cushion from E11 to E14, while Bmp-2 and Bmp-4 were detected between E11 and E14. Apoptotic cells were also detected in the mesenchyme of the endocardial cushion between E12 and E14. Our results suggest that BMP-2 and MSX-2 are tightly linked to the formation of the atrioventricular junction and valves and that BMP-4 is involved in the development of the outflow tract myocardium and of the endocardial cushion. In addition, BMP-2, BMP-4 and MSX-2 and apoptosis seem to be associated with differentiation of the endocardial cushion.

  11. Regulation of Oligodendrocyte Progenitor Cell Maturation by PPARδ: Effects on Bone Morphogenetic Proteins

    Directory of Open Access Journals (Sweden)

    Maria Vittoria Simonini

    2009-12-01

    Full Text Available In EAE (experimental autoimmune encephalomyelitis, agonists of PPARs (peroxisome proliferator-activated receptors provide clinical benefit and reduce damage. In contrast with PPARγ, agonists of PPARδ are more effective when given at later stages of EAE and increase myelin gene expression, suggesting effects on OL (oligodendrocyte maturation. In the present study we examined effects of the PPARδ agonist GW0742 on OPCs (OL progenitor cells, and tested whether the effects involve modulation of BMPs (bone morphogenetic proteins. We show that effects of GW0742 are mediated through PPARδ since no amelioration of EAE clinical scores was observed in PPARδ-null mice. In OPCs derived from E13 mice (where E is embryonic day, GW0742, but not the PPARγ agonist pioglitazone, increased the number of myelin-producing OLs. This was due to activation of PPARδ since process formation was reduced in PPARδ-null compared with wild-type OPCs. In both OPCs and enriched astrocyte cultures, GW0742 increased noggin protein expression; however, noggin mRNA was only increased in astrocytes. In contrast, GW0742 reduced BMP2 and BMP4 mRNA levels in OPCs, with lesser effects in astrocytes. These findings demonstrate that PPARδ plays a role in OPC maturation, mediated, in part, by regulation of BMP and BMP antagonists.

  12. Bone morphogenetic proteins: from structure to clinical use

    Directory of Open Access Journals (Sweden)

    Granjeiro J.M.

    2005-01-01

    Full Text Available Bone morphogenetic proteins (BMPs are multi-functional growth factors belonging to the transforming growth factor ß superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

  13. Photobiomodulation of mesenchymal stem cells encapsulated in an injectable rhBMP4-loaded hydrogel directs hard tissue bioengineering.

    Science.gov (United States)

    Diniz, Ivana M A; Carreira, Ana C O; Sipert, Carla R; Uehara, Cindi M; Moreira, Maria S N; Freire, Laila; Pelissari, Cibele; Kossugue, Patrícia M; de Araújo, Daniele R; Sogayar, Mari C; Marques, Márcia M

    2018-06-01

    Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic ® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic ® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering. © 2017 Wiley Periodicals, Inc.

  14. Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Ian P M Tomlinson

    2011-06-01

    Full Text Available Genome-wide association studies (GWAS have identified 14 tagging single nucleotide polymorphisms (tagSNPs that are associated with the risk of colorectal cancer (CRC, and several of these tagSNPs are near bone morphogenetic protein (BMP pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3, BMP4 (14q22.2, and BMP2 (20p12.3 using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10 and BMP2 (rs4813802, P = 4.65×10(-11. Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8 and rs11632715 (P = 2.30×10(-10. As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.

  15. Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

    Science.gov (United States)

    Rico-Llanos, Gustavo A; Becerra, Jose; Visser, Rick

    2017-07-01

    Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017. © 2017 Wiley Periodicals, Inc.

  16. DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

    Science.gov (United States)

    Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2009-06-01

    Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

  17. Activation of the PI3K/Akt pathway mediates bone morphogenetic protein 2-induced invasion of pancreatic cancer cells Panc-1.

    Science.gov (United States)

    Chen, Xiong; Liao, Jie; Lu, YeBin; Duan, XiaoHui; Sun, WeiJia

    2011-06-01

    Bone morphogenetic proteins (BMPs) signaling has an emerging role in pancreatic cancer. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in pancreatic cancer. In our studies, we have focused on bone morphogenetic protein 2(BMP-2) because it induces an epithelial to mesenchymal transition (EMT) and accelerates invasion in the human pancreatic cancer cell line Panc-1. It has been reported that the phosphatidylinositol 3-kinase (PI3K)/Akt pathway mediates invasion of gastric and colon cancer cells, which is unrevealed in pancreatic cancer cells. The objective of our study was to investigate whether BMP-2 mediated invasion might pass through the PI3K/Akt pathway. Our results show that expression of phosphorylation of Akt was increased by treatment with BMP-2, but not Noggin, a BMP-2 antagonist. Then pretreatment of Panc-1 cells with LY294002, an inhibitor of the PI3K/AKT pathway, significantly inhibited BMP-2-induced EMT and invasiveness. The data suggest that BMP-2 accelerates invasion of panc-1 cells via the PI3K/AKT pathway in panc-1 cells, which gives clues to searching new therapy targets in advanced pancreatic cancer.

  18. BMP4 Expression Following Stem Cells from Human Exfoliated Deciduous and Carbonate Apatite Transplantation on Rattus norvegicus

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    Tania Saskianti

    2018-04-01

    Full Text Available Background: Alveolar bone defects in children still have a high incidence. Conventional bone graft technique that has been used as a defect therapy is still not effective, so new techniques with tissue engineering approach are needed. Bone Morphogenetic Protein 4 (BMP4 as one of the indicators of osteogenic differentiation has not been widely studied, especially in the transplantation with combination of Stem Cells from Human Exfoliated Deciduous (SHED and carbonate apatite. Aim and Objectives: This research aimed to determine the expression of BMP4 after SHED and carbonate apatite transplantation on Rattus norvegicus. Material and Methods: The combinations of SHED and carbonate apatite were transplanted on alveolar bone defects of 4 rats (Rattus norvegicus as the treatment groups and another 4 rats were transplanted with carbonate apatite as the control groups. After 21 days, staining with Hematoxylin Eosin (HE and Immunohistochemistry (IHC BMP4 was performed. Results: BMP4 expression in the treatment groups was significantly higher when compared to the control groups. Discussion: Carbonate apatite has low crystallization rate and high osteoconductivity that produce more osteoblasts and increased BMP4 expression. Conclusion: The transplantation of SHED and carbonate apatite increased BMP4 expression as an indicator of osteogenic differentiation in rats.

  19. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

    International Nuclear Information System (INIS)

    Shimada, Koichi; Ikeda, Kyoko; Ito, Koichi

    2009-01-01

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-α stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-α-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-α and BMP signaling pathways.

  20. Microparticle-Mediated Delivery of BMP4 for Generation of Meiosis-Competent Germ Cells from Embryonic Stem Cells.

    Science.gov (United States)

    Esfandiari, Fereshteh; Ashtiani, Mohammad Kazemi; Sharifi-Tabar, Mehdi; Saber, Maryam; Daemi, Hamed; Ghanian, Mohammad Hossein; Shahverdi, Abdolhossein; Baharvand, Hossein

    2017-03-01

    Producing meiosis-competent germ cells (GCs) from embryonic stem cells (ESCs) is essential for developing advanced therapies for infertility. Here, a novel approach is presented for generation of GCs from ESCs. In this regard, microparticles (MPs) have been developed from alginate sulfate loaded with bone morphogenetic protein 4 (BMP4). The results here show that BMP4 release from alginate sulfate MPs is significantly retarded by the sulfated groups compared to neat alginate. Then, BMP4-laden MPs are incorporated within the aggregates during differentiation of GCs from ESCs. It is observed that BMP4-laden MPs increase GC differentiation from ESCs at least twofold compared to the conventional soluble delivery method. Interestingly, following meiosis induction, Dazl, an intrinsic factor that enables GCs to enter meiosis, and two essential meiosis genes (Stra8 and Smc1b) are upregulated significantly in MP-induced aggregates compared to aggregates, which are formed by the conventional method. Together, these data show that controlled delivery of BMP4 during ESC differentiation into GC establish meiosis-competent GCs which can serve as an attractive GC source for reproductive medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Risk variants in BMP4 promoters for nonsyndromic cleft lip/palate in a Chilean population

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    Suazo José

    2011-12-01

    Full Text Available Abstract Background Bone morphogenetic protein 4 gene (BMP4 plays a key role during maxillofacial development, since orofacial clefts are observed in animals when this gene is conditionally inactivated. We recently reported the existence of association between nonsyndromic cleft lip/palate (NSCLP and BMP4 polymorphisms by detecting transmission deviations for haplotypes that include a region containing a BMP4 promoter in case-parent trios. The aim of the present study was to search for possible causal mutations within BMP4 promoters (BMP4.1 and BMP4.2. Methods We analyzed the sequence of BMP4.1 and BMP4.2 in 167 Chilean NSCLP cases and 336 controls. Results We detected three novel variants in BMP4.1 (c.-5514G > A, c.-5365C > T and c.-5049C > T which could be considered as cleft risk factors due to their absence in controls. Additionally, rs2855530 G allele (BMP4.2 carriers showed an increased risk for NSCLP restricted to males (OR = 1.52; 95% C.I. = 1.07-2.15; p = 0.019. For this same SNP the dominant genotype model showed a higher frequency of G/G+G/C and a lower frequency of C/C in cases than controls in the total sample (p = 0.03 and in the male sample (p = 0.003. Bioinformatic prediction analysis showed that all the risk variants detected in this study could create new transcription factor binding motifs. Conclusions The sex-dependent association between rs2855530 and NSCLP could indirectly be related to the differential gene expression observed between sexes in animal models. We concluded that risk variants detected herein could potentially alter BMP4 promoter activity in NSCLP. Further functional and developmental studies are necessary to support this hypothesis.

  2. Facilitated receptor-recognition and enhanced bioactivity of bone morphogenetic protein-2 on magnesium-substituted hydroxyapatite surface

    Science.gov (United States)

    Huang, Baolin; Yuan, Yuan; Li, Tong; Ding, Sai; Zhang, Wenjing; Gu, Yuantong; Liu, Changsheng

    2016-01-01

    Biomaterial surface functionalized with bone morphogenetic protein-2 (BMP-2) is a promising approach to fabricating successful orthopedic implants/scaffolds. However, the bioactivity of BMP-2 on material surfaces is still far from satisfactory and the mechanism of related protein-surface interaction remains elusive. Based on the most widely used bone-implants/scaffolds material, hydroxyapatite (HAP), we developed a matrix of magnesium-substituted HAP (Mg-HAP, 2.2 at% substitution) to address these issues. Further, we investigated the adsorption dynamics, BMPRs-recruitment, and bioactivity of recombinant human BMP-2 (rhBMP-2) on the HAP and Mg-HAP surfaces. To elucidate the mechanism, molecular dynamic simulations were performed to calculate the preferred orientations, conformation changes, and cysteine-knot stabilities of adsorbed BMP-2 molecules. The results showed that rhBMP-2 on the Mg-HAP surface exhibited greater bioactivity, evidenced by more facilitated BMPRs-recognition and higher ALP activity than on the HAP surface. Moreover, molecular simulations indicated that BMP-2 favoured distinct side-on orientations on the HAP and Mg-HAP surfaces. Intriguingly, BMP-2 on the Mg-HAP surface largely preserved the active protein structure evidenced by more stable cysteine-knots than on the HAP surface. These findings explicitly clarify the mechanism of BMP-2-HAP/Mg-HAP interactions and highlight the promising application of Mg-HAP/BMP-2 matrixes in bone regeneration implants/scaffolds. PMID:27075233

  3. Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin

    International Nuclear Information System (INIS)

    Olsen, O E; Wader, K F; Misund, K; Våtsveen, T K; Rø, T B; Mylin, A K; Turesson, I; Størdal, B F; Moen, S H; Standal, T; Waage, A; Sundan, A; Holien, T

    2014-01-01

    Multiple myeloma is a malignancy of plasma cells predominantly located in the bone marrow. A number of bone morphogenetic proteins (BMPs) induce apoptosis in myeloma cells in vitro, and with this study we add BMP-9 to the list. BMP-9 has been found in human serum at concentrations that inhibit cancer cell growth in vitro. We here show that the level of BMP-9 in serum was elevated in myeloma patients (median 176 pg/ml, range 8–809) compared with healthy controls (median 110 pg/ml, range 8–359). BMP-9 was also present in the bone marrow and was able to induce apoptosis in 4 out of 11 primary myeloma cell samples by signaling through ALK2. BMP-9-induced apoptosis in myeloma cells was associated with c-MYC downregulation. The effects of BMP-9 were counteracted by membrane-bound (CD105) or soluble endoglin present in the bone marrow microenvironment, suggesting a mechanism for how myeloma cells can evade the tumor suppressing activity of BMP-9 in multiple myeloma

  4. Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3.

    Science.gov (United States)

    Dwivedi, Prem P; Grose, Randall H; Filmus, Jorge; Hii, Charles S T; Xian, Cory J; Anderson, Peter J; Powell, Barry C

    2013-08-01

    From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

    Science.gov (United States)

    Shen, Jia; James, Aaron W.; Zhang, Xinli; Pang, Shen; Zara, Janette N.; Asatrian, Greg; Chiang, Michael; Lee, Min; Khadarian, Kevork; Nguyen, Alan; Lee, Kevin S.; Siu, Ronald K.; Tetradis, Sotirios; Ting, Kang; Soo, Chia

    2017-01-01

    The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis. PMID:26772960

  6. TNF-α Upregulates Expression of BMP-2 and BMP-3 Genes in the Rat Dental Follicle – Implications for Tooth Eruption

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    Yao, Shaomian; Prpic, Veronica; Pan, Fenghui; Wise, Gary E.

    2011-01-01

    The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha ( TNF-α) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the 1st mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-α expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), it was the objective of this study to determine 1) if the dental follicle expresses BMP-3 and 2) if TNF-α stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-α to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-α in the dental follicle and TNF-α significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-α can upregulate the expression of bone formation genes that may be needed for tooth eruption. PMID:20067418

  7. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

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    Jin H

    2014-05-01

    Full Text Available Han Jin,1 Kai Zhang,2 Chunyan Qiao,1 Anliang Yuan,1 Daowei Li,1 Liang Zhao,1 Ce Shi,1 Xiaowei Xu,1 Shilei Ni,1 Changyu Zheng,3 Xiaohua Liu,4 Bai Yang,2 Hongchen Sun11Department of Pathology, School of Stomatology, Jilin University, Changchun, People’s Republic of China; 2State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, People’s Republic of China; 3Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA; 4Department of Biomedical Sciences, Texas A&M University Baylor College of Dentistry, Dallas, TX, USAAbstract: Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2 gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al nanocomposites plus human BMP-2 complementary(cDNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI

  8. Dose reduction of bone morphogenetic protein-2 for bone regeneration using a delivery system based on lyophilization with trehalose

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    Zhang X

    2018-01-01

    Full Text Available Xiaochen Zhang,1,* Quan Yu,2,* Yan-an Wang,1 Jun Zhao2 1Department of Oral and Maxillofacial-Head and Neck Oncology, 2Department of Orthodontics, College of Stomatology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China *These authors contributed equally to this work Introduction: To induce sufficient new bone formation, high doses of bone morphogenetic protein-2 (BMP-2 are applied in regenerative medicine that often induce serious side effects. Therefore, improved treatment strategies are required. Here, we investigate whether the delivery of BMP-2 lyophilized in the presence of trehalose reduced the dose of BMP-2 required for bone regeneration. Materials and methods: A new growth factor delivery system was fabricated using BMP-2-loaded TiO2 nanotubes by lyophilization with trehalose (TiO2-Lyo-Tre-BMP-2. We measured BMP-2 release characteristics, bioactivity, and stability, and determined the effects on the osteogenic differentiation of bone marrow stromal cells in vitro. Additionally, we evaluated the ability of this formulation to regenerate new bone around implants in rat femur defects by micro-computed tomography (micro-CT, sequential fluorescent labelling, and histological analysis. Results: Compared with absorbed BMP-2-loaded TiO2 nanotubes (TiO2-BMP-2, TiO2-Lyo-Tre-BMP-2 exhibited sustained release, consistent bioactivity, and higher stability of BMP-2, and resulted in greater osteogenic differentiation of BMSCs. Eight weeks post-operation, TiO2-Lyo-Tre-BMP-2 nanotubes, with various dosages of BMP-2, regenerated larger amounts of new bone than TiO2-BMP-2 nanotubes. Conclusion: Our findings indicate that delivery of BMP-2 lyophilized with trehalose may be a promising method to reduce the dose of BMP-2 and avoid the associated side effects. Keywords: bone morphogenetic protein-2, dose reduction, delivery system, trehalose, lyophilization, TiO2 nanotubes, BMP-2, regenerative medicine, surface

  9. A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2

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    Thanyaphoo Suphannee

    2016-09-01

    Full Text Available Silicon-substituted calcium phosphate (Si-CaP was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2 was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future.

  10. Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

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    García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

    2015-09-01

    Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

  11. Delivery of bone morphogenetic protein-2 and substance P using graphene oxide for bone regeneration

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    La WG

    2014-05-01

    Full Text Available Wan-Geun La,1 Min Jin,1 Saibom Park,1,2 Hee-Hun Yoon,1 Gun-Jae Jeong,1 Suk Ho Bhang,1 Hoyoung Park,1,2 Kookheon Char,1,2 Byung-Soo Kim1,31School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea; 2The National Creative Research Initiative Center for Intelligent Hybrids, Seoul National University, Seoul, Republic of Korea; 3Institute of Bioengineering, Institute of Chemical Processes, Engineering Research Institute, Seoul National University, Seoul, Republic of KoreaAbstract: In this study, we demonstrate that graphene oxide (GO can be used for the delivery of bone morphogenetic protein-2 (BMP-2 and substance P (SP, and that this delivery promotes bone formation on titanium (Ti implants that are coated with GO. GO coating on Ti substrate enabled a sustained release of BMP-2. BMP-2 delivery using GO-coated Ti exhibited a higher alkaline phosphatase activity in bone-forming cells in vitro compared with bare Ti. SP, which is known to recruit mesenchymal stem cells (MSCs, was co-delivered using Ti or GO-coated Ti to further promote bone formation. SP induced the migration of MSCs in vitro. The dual delivery of BMP-2 and SP using GO-coated Ti showed the greatest new bone formation on Ti implanted in the mouse calvaria compared with other groups. This approach may be useful to improve osteointegration of Ti in dental or orthopedic implants.Keywords: bone morphogenetic protein-2, bone regeneration, graphene oxides, stem cell recruitment, substance P

  12. Bone morphogenetic protein-4 and transforming growth factor-beta1 mechanisms in acute valvular response to supra-physiologic hemodynamic stresses.

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    Sun, Ling; Sucosky, Philippe

    2015-06-26

    To explore ex vivo the role of bone morphogenetic protein-4 (BMP-4) and transforming growth factor-beta1 (TGF-β1) in acute valvular response to fluid shear stress (FSS) abnormalities. Porcine valve leaflets were subjected ex vivo to physiologic FSS, supra-physiologic FSS magnitude at normal frequency and supra-physiologic FSS frequency at normal magnitude for 48 h in a double-sided cone-and-plate bioreactor filled with standard culture medium. The role of BMP-4 and TGF-β1 in the valvular response was investigated by promoting or inhibiting the downstream action of those cytokines via culture medium supplementation with BMP-4 or the BMP antagonist noggin, and TGF-β1 or the TGF-β1 inhibitor SB-431542, respectively. Fresh porcine leaflets were used as controls. Each experimental group consisted of six leaflet samples. Immunostaining and immunoblotting were performed to assess endothelial activation in terms of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expressions, paracrine signaling in terms of BMP-4 and TGF-β1 expressions and extracellular matrix (ECM) remodeling in terms of cathepsin L, cathepsin S, metalloproteinases (MMP)-2 and MMP-9 expressions. Immunostained images were quantified by normalizing the intensities of positively stained regions by the number of cells in each image while immunoblots were quantified by densitometry. Regardless of the culture medium, physiologic FSS maintained valvular homeostasis. Tissue exposure to supra-physiologic FSS magnitude in standard medium stimulated paracrine signaling (TGF-β1: 467% ± 22% vs 100% ± 6% in fresh controls, BMP-4: 258% ± 22% vs 100% ± 4% in fresh controls; P 0.05). Supra-physiologic FSS frequency had no effect on endothelial activation and paracrine signaling regardless of the culture medium but TGF-β1 silencing attenuated FSS-induced ECM degradation via MMP-9 downregulation (MMP-9: 302% ± 182% vs 100% ± 42% in fresh controls; P > 0.05). Valvular tissue is sensitive

  13. Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure

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    Nusinowitz Steven

    2001-11-01

    Full Text Available Abstract Background Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP. In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. Results Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. Conclusions We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

  14. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling.

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    Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K; Zhang, Chun-Li

    2014-01-01

    Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  15. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

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    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  16. Heterologous Secretory Expression and Characterization of Dimerized Bone Morphogenetic Protein 2 in Bacillus subtilis

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    Muhammad Umair Hanif

    2017-01-01

    Full Text Available Recombinant human Bone Morphogenetic Protein 2 (rhBMP2 has important applications in the spine fusion and ortho/maxillofacial surgeries. Here we first report the secretory expression of biological active dimerized rhBMP2 from Bacillus subtilis system. The mature domain of BMP2 gene was amplified from pTz57R/BMP2 plasmid. By using pHT43 expression vector two constructs, pHT43-BMP2-M (single BMP2 gene and pHT43-BMP2-D (two BMP2 genes coupled with a linker to produce a dimer, were designed. After primary cloning (DH5α strain and sequence analysis, constructs were transformed into Bacillus subtilis for secretory expression. Expression conditions like media (2xYT and temperature (30°C were optimized. Maximum 35% and 25% secretory expression of monomer (~13 kDa and dimer (~25 kDa, respectively, were observed on SDS-PAGE in SCK6 strain. The expression and dimeric nature of rhBMP2 were confirmed by western blot and native PAGE analysis. For rhBMP2 purification, 200 ml culture supernatant was freeze dried to 10 ml and dialyzed (Tris-Cl, pH 8.5 and Fast Protein Liquid Chromatography (6 ml, Resource Q column was performed. The rhBMP2 monomer and dimer were eluted at 0.9 M and 0.6 M NaCl, respectively. The alkaline phosphatase assay of rhBMP2 (0, 50, 100, 200, and 400 ng/ml was analyzed on C2C12 cells and maximum 200 ng/ml activity was observed in dose dependent manner.

  17. Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

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    Tellier, Liane E; Miller, Tobias; McDevitt, Todd C; Temenoff, Johnna S

    2015-10-28

    Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

  18. Smad, PI3K/Akt, and Wnt-dependent signaling pathways are involved in BMP-4-induced ESC self-renewal.

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    Lee, Min Young; Lim, Hyun Woo; Lee, Sang Hun; Han, Ho Jae

    2009-08-01

    It is known that bone morphogenetic protein 4 (BMP-4) has a diverse effect on ESCs. However, its precise mechanism in mouse ESCs is not fully understood. We evaluated the effect of BMP-4 on ESC proliferation and its related signal cascades in this study. BMP-4 significantly increased the level of [(3)H]-thymidine incorporation in time- (> or =8 hours) and dose- (> or =10 ng/ml) dependent manners. Additionally, BMP-4 increased cyclin D1 and decreased p27(kip1) expression values in a time-dependent manner. The increases in BMP-4-induced [(3)H]-thymidine incorporation and cyclin D1 expression were inhibited by the BMP-4 receptor antagonist noggin. BMP-4 increased Wnt1 expression. Wnt1 expression was attenuated by Smad4 small interfering RNA (siRNA), and BMP-4-induced cyclin D1 expression was inhibited by Smad4 and Wnt1 siRNAs. BMP-4 also activated beta-catenin, which was blocked by Smad4 and Wnt1 siRNAs. In addition, BMP-4 induced Akt phosphorylation. BMP-4-induced beta-catenin activation and cyclin D1 expression were attenuated by phosphatidyl inositol 3-kinase (PI3K) siRNA and Akt inhibitor. Additionally, downregulation of Smad4, Wnt1, and PI3K expression by siRNA decreased the levels of pluripotency marker mRNAs of ESCs, including Oct4, Sox2, and FoxD3. Our results suggested that BMP-4-induced [(3)H]-thymidine incorporation was significantly attenuated by Smad4, Wnt1, and PI3K knockdown. In conclusion, BMP-4 contributed to the maintenance of cell proliferation and the pluripotent state by Smad, PI3K/Akt, and Wnt1/beta-catenin in mouse ESCs.

  19. Effects of Recombinant Human Bone Morphogenetic Protein-2 on Vertical Bone Augmentation in a Canine Model.

    Science.gov (United States)

    Hsu, Yung-Ting; Al-Hezaimi, Khalid; Galindo-Moreno, Pablo; O'Valle, Francisco; Al-Rasheed, Abdulaziz; Wang, Hom-Lay

    2017-09-01

    Vertical bone augmentation (VBA) remains unpredictable and challenging for most clinicians. This study aims to compare hard tissue outcomes of VBA, with and without recombinant human bone morphogenetic protein (rhBMP)-2, under space-making titanium mesh in a canine model. Eleven male beagle dogs were used in the study. Experimental ridge defects were created to form atrophic ridges. VBA was performed via guided bone regeneration using titanium mesh and allografts. In experimental hemimandibles, rhBMP-2/absorbable collagen sponge was well mixed with allografts prior to procedures, whereas a control buffer was applied within controls. Dogs were euthanized after a 4-month healing period. Clinical and radiographic examinations were performed to assess ridge dimensional changes. In addition, specimens were used for microcomputed tomography (micro-CT) assessment and histologic analysis. Membrane exposure was found on five of 11 (45.5%) rhBMP-2-treated sites, whereas it was found on nine of 11 (81.8%) non-rhBMP-2-treated sites. Within 4 months of healing, rhBMP-2-treated sites showed better radiographic bone density, greater defect fill, and significantly more bone gain in ridge height (P 0.05). Under light microscope, predominant lamellar patterns were found in the specimen obtained from rhBMP-2 sites. With inherent limitations of the canine model and the concern of such a demanding surgical technique, current findings suggest that the presence of rhBMP-2 in a composite graft allows an increase of vertical gain, with formation of ectopic bone over the titanium mesh in comparison with non-rhBMP-2 sites.

  20. Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

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    Xia, Yin; Babitt, Jodie L; Bouley, Richard; Zhang, Ying; Da Silva, Nicolas; Chen, Shanzhuo; Zhuang, Zhenjie; Samad, Tarek A; Brenner, Gary J; Anderson, Jennifer L; Hong, Charles C; Schneyer, Alan L; Brown, Dennis; Lin, Herbert Y

    2010-04-01

    The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

  1. Bmp 2 and bmp 7 induce odonto- and osteogenesis of human tooth germ stem cells.

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    Taşlı, P Neslihan; Aydın, Safa; Yalvaç, Mehmet Emir; Sahin, Fikrettin

    2014-03-01

    Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.

  2. Bmp signaling is at the heart of vertebrate left-right asymmetry

    NARCIS (Netherlands)

    Verhoeven, M.C.

    2009-01-01

    Bone morphogenetic protein (Bmp) signaling is vitally important in many aspects of cardiac development. These include cardiac induction and differentiation and establishing the L/R axis. In this thesis, we focus on the role of Bmp signaling in securing proper cardiac asymmetry, by (1) establishing

  3. Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

    NARCIS (Netherlands)

    Yang, X.; Walboomers, X.F.; Dolder, J. van den; Yang, F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2008-01-01

    Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and

  4. Basolateral BMP signaling in polarized epithelial cells.

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    Masao Saitoh

    Full Text Available Bone morphogenetic proteins (BMPs regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER, counteracting reduction of TER by treatment with TGF-β or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

  5. Single nucleotide polymorphism of bone morphogenetic protein 4 gene: A risk factor of non-syndromic cleft lip with or without palate

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    Sathyaprasad Savitha

    2015-01-01

    Full Text Available Background: The bone morphogenetic protein (BMP signalling pathway is crucial in a number of developmental processes and is critical in the formation of variety of craniofacial elements including cranial neural crest, facial primordium, tooth, lip and palate. It is an important mediator in regulation of lip and palate fusion, cartilage and bone formation. Aim: To study the role of mutation of BMP4 genes in the aetiology of non-syndromic cleft lip with or without palate (NSCL ± P and identify it directly from human analyses. Materials and Methods: A case-control study was done to evaluate whether BMP4T538C polymorphism, resulting in an amino acid change of Val=Ala (V152A in the polypeptide, is associated with NSCL ± P in an Indian paediatric population. Genotypes of 100 patients with NSCL ± P and 100 controls (in whom absence of CL ± P was confirmed in three generations were detected using a polymerase chain reaction-restriction fragment length polymorphism strategy. Logistic regression was performed to evaluate allele and genotype association with NSCLP. Results: Results showed significant association between homozygous CC genotype with CL ± P (odds ratio [OR]-5.59 and 95% confidence interval [CI] = 2.85-10.99. The 538C allele carriers showed an increased risk of NSCL ± P as compared with 538 T allele (OR - 4.2% CI = 2.75-6.41. Conclusion: This study suggests an association between SNP of BMP4 gene among carriers of the C allele and increased risk for NSCLP in an Indian Population. Further studies on this aspect can scale large heights in preventive strategies for NSCLP that may soon become a reality.

  6. The evaluation of lyophilized polymer matrices for administering recombinant human bone morphogenetic protein-2.

    Science.gov (United States)

    Duggirala, S S; Rodgers, J B; DeLuca, P P

    1996-07-01

    Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.

  7. The classic: Bone morphogenetic protein.

    Science.gov (United States)

    Urist, Marshall R; Strates, Basil S

    2009-12-01

    This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406.

  8. Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge.

    Science.gov (United States)

    Matsumoto, Goichi; Ueda, Takayo; Sugita, Yoshihiko; Kubo, Katsutoshi; Mizoguchi, Megumi; Kotani, Eiji; Oda, Naoki; Kawamata, Shin; Segami, Natsuki; Mori, Hajime

    2015-08-01

    Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 × 10(7) BMP-2 polyhedra, and half were treated with sponges containing 3.6 × 10(6) BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 µg of recombinant human BMP-2 (rhBMP-2) or 1.8 × 10(7) empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development. © The Author(s) 2015.

  9. BMP4 and LGL1 are Down Regulated in an Ovine Model of Congenital Diaphragmatic Hernia

    Directory of Open Access Journals (Sweden)

    Heather eEmmerton-Coughlin

    2014-11-01

    Full Text Available Background/Purpose: The molecular pathophysiology of lung hypoplasia in congenital diaphragmatic hernia (CDH remains poorly understood. The Wnt signaling pathway and downstream targets, such as bone morphogenetic proteins (BMP 4 and other factors such as late gestation lung protein 1 (LGL1, are essential to normal lung development. Nitrofen-induced hypoplastic CDH rodent lungs demonstrate down regulation of the Wnt pathway including BMP4 and reduced LGL1 expression. The aim of the current study was to examine the molecular pathophysiology associated with a surgically induced CDH in an ovine model. Methods: Left thoracotomy was performed at 80 days in 14 fetal sheep; CDH was created in 7 experimental animals. Lungs were harvested at 136 days (term=145d. Lung weight and mean terminal bronchiole density (MTBD were measured to determine the degree of pulmonary hypoplasia. Quantitative real time PCR was undertaken to analyze Wnt2, Wnt7b, BMP4 and LGL1 mRNA expression. Results: Total lung weight was decreased while MTBD was increased in the CDH group (p<0.05, confirming pulmonary hypoplasia. BMP4 and LGL1 mRNA was significantly reduced in CDH lungs (p<0.05. Wnt2 mRNA was decreased, although not significantly (p<0.06. Conclusions: For the first time, down regulation of BMP4 and Lgl1 are reported in an ovine CDH model. In contrast to other animal models, these changes are persistent to near term. These findings suggest that mechanical compression from herniated viscera may play a more important role in causing pulmonary hypoplasia in CDH, rather than a primary defect in lung organogenesis.

  10. Emerging roles of BMP9 and BMP10 in hereditary hemorrhagic telangiectasia

    Directory of Open Access Journals (Sweden)

    Emmanuelle eTillet

    2015-01-01

    Full Text Available Rendu-Osler-Weber syndrome, also known as hereditary hemorrhagic telangiectasia (HHT, is an autosomal dominant vascular disorder. Three genes are causally related to HHT: the ENG gene encoding endoglin, a co-receptor of the TGFß family (HHT1, the ACVRL1 gene encoding ALK1 (activin receptor-like kinase 1, a type I receptor of the TGFß family (HHT2, and the SMAD4 gene, encoding a transcription factor critical for this signaling pathway. Bone morphogenetic proteins (BMPs are growth factors of the TGFß family. Among them, BMP9 and BMP10 have been shown to bind directly with high affinity to ALK1 and endoglin, and BMP9 mutations have recently been linked to a vascular-anomaly syndrome that has phenotypic overlap with HHT. BMP9 and BMP10 are both circulating cytokines in blood, and the current working model is that BMP9 and BMP10 maintain a quiescent endothelial state that is dependent on the level of ALK1/endoglin activation on endothelial cells. In accordance with this model, to explain the etiology of HHT we hypothesize that a deficient BMP9/BMP10/ALK1/endoglin pathway may lead to re-activation of angiogenesis or a greater sensitivity to an angiogenic stimulus. Resulting endothelial hyperproliferation and hypermigration may lead to vasodilatation and formation of arteriovenous malformation (AVM. HHT would thus result from a defect in the angiogenic balance. This review will focus on the emerging role played by BMP9 and BMP10 in the development of this disease and the therapeutic approaches that this opens.

  11. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    International Nuclear Information System (INIS)

    Jiang Fangxu; Harrison, Leonard C.

    2005-01-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its α 6 integrin and α-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells

  12. Exaggerated inflammatory response after use of recombinant bone morphogenetic protein in recurrent unicameral bone cysts.

    Science.gov (United States)

    MacDonald, Kevin M; Swanstrom, Morgan M; McCarthy, James J; Nemeth, Blaise A; Guliani, Teresa A; Noonan, Kenneth J

    2010-03-01

    Recurrent unicameral bone cysts (UBCs) can result in significant morbidity during a child's physical and emotional development. Multiple treatment options are available and a review of the literature fails to clearly define the optimal treatment for UBCs. Recombinant bone morphogenetic protein (BMP) has been used with success in other disorders of poor bone formation. This manuscript is the first to report on the use of recombinant BMP in the treatment of UBCs. Three patients with recurrent UBCs underwent revision surgery with recombinant BMP. Radiographic and medical review was performed and is reported here. In these patients, the use of BMP failed to fully resolve their UBC; 2 patients had complete recurrence that required further surgery. In addition to poor radiographic results, all patients developed exaggerated inflammatory responses in the acute postoperative period. Each child developed clinically significant limb swelling and pain that mimicked infection. On the basis of our poor radiographic results and a paradoxical clinical result, we no longer recommend the use of recombinant BMP in the manner reported here for the treatment of recurrent UBCs. Level IV, case series.

  13. Developmental roles of the BMP1/TLD metalloproteinases.

    Science.gov (United States)

    Ge, Gaoxiang; Greenspan, Daniel S

    2006-03-01

    -beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events. Copyright 2006 Wiley-Liss, Inc.

  14. Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells.

    Science.gov (United States)

    Ogura-Ochi, Kanako; Fujisawa, Satoshi; Iwata, Nahoko; Komatsubara, Motoshi; Nishiyama, Yuki; Tsukamoto-Yamauchi, Naoko; Inagaki, Kenichi; Wada, Jun; Otsuka, Fumio

    2017-08-01

    The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease

    International Nuclear Information System (INIS)

    Tang, Jun; Song, Min; Wang, Yanyan; Fan, Xiaotang; Xu, Haiwei; Bai, Yun

    2009-01-01

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP swe /PS1 ΔE9 mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP swe /PS1 ΔE9 transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  16. Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs. pasteurization vs. allograft.

    Science.gov (United States)

    Takata, Munetomo; Sugimoto, Naotoshi; Yamamoto, Norio; Shirai, Toshiharu; Hayashi, Katsuhiro; Nishida, Hideji; Tanzawa, Yoshikazu; Kimura, Hiroaki; Miwa, Shinji; Takeuchi, Akihiko; Tsuchiya, Hiroyuki

    2011-12-01

    Insufficient bone union is the occasional complication of biomechanical reconstruction after malignant bone tumor resection using temperature treated tumor bearing bone; freezing, pasteurization, and autoclaving. Since bone morphogenetic protein (BMP) plays an important role in bone formation, we assessed the amount and activity of BMP preserved after several temperature treatments, including -196 and -73°C for 20 min, 60 and 100°C for 30 min, 60°C for 10h following -80°C for 12h as an allograft model, and 4°C as the control. The material extracted from the human femoral bone was treated, and the amount of BMP-7 was analyzed using an enzyme-linked immunosorbent assay. Then, the activity of recombinant human BMP-7 after the treatment was assessed using a bioassay with NIH3T3 cells and immunoblotting analysis to measure the amount of phospho-Smad, one of the signaling substrates that reflect the intracellular reaction of BMPs. Both experiments revealed that BMP-7 was significantly better preserved in the hypothermia groups. The percentages of the amount of BMP-7 in which the control group was set at 100% were 114%, 108%, 70%, 49%, and 53% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. The percentages of the amount of phospho-Smad were 89%, 87%, 24%, 4.9%, and 14% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. These results suggested that freezing possibly preserves osteoinductive ability than hyperthermia treatment. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure.

    Science.gov (United States)

    Morine, Kevin J; Qiao, Xiaoying; York, Sam; Natov, Peter S; Paruchuri, Vikram; Zhang, Yali; Aronovitz, Mark J; Karas, Richard H; Kapur, Navin K

    2018-02-27

    Background -Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-β1) promotes cardiac fibrosis, but also activates counter-regulatory pathways that serve to regulate TGF-β1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFβ family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFβ co-receptor that promotes TGF-β1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3 and further that neutralizing endoglin activity promotes BMP9 activity. Methods -We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We utilized the thoracic aortic constriction (TAC) induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. Results -BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates Type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and an siRNA approach. In BMP9 -/- mice subjected to TAC, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to TAC with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 compared to vehicle treated controls. Since endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to TAC induced heart failure limits collagen production, increases BMP9 protein levels, and increases

  18. Osteogenic potential of the human bone morphogenetic protein 2 gene activated nanobone putty.

    Science.gov (United States)

    Tian, Xiao-bin; Sun, Li; Yang, Shu-hua; Zhang, Yu-kun; Hu, Ru-yin; Fu, De-hao

    2008-04-20

    Nanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. Twenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation. The tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone

  19. Combined Effects of Vascular Endothelial Growth Factor and Bone Morphogenetic Protein 2 on Odonto/Osteogenic Differentiation of Human Dental Pulp Stem Cells In Vitro.

    Science.gov (United States)

    Aksel, Hacer; Huang, George T-J

    2017-06-01

    The purpose of this study was to investigate whether combined and concerted delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) enhances odonto/osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro. Various concentrations of VEGF and/or BMP-2 with or without the presence of odonto/osteogenic medium (OM) were added into DPSC cultures for 21 days. The mineral formation in cultures was evaluated using alizarin red stain (ARS). Optimal concentrations of VEGF and BMP-2 were codelivered to DPSCs for total of 21 days with the following experimental groups: (1) group 1: OM only, (2) group 2: OM + VEGF, (3) group 3: OM + BMP-2, and (4) group 4: OM + VEGF + BMP-2 (subgroup 4a: VEGF present the first 7 days, 4b: BMP-2 present the last 14 days, and 4c, both present for 21 days). Cultures were then subjected to quantitative ARS analysis or harvested for quantitative polymerase chain reaction analysis for the expression of core-binding factor alpha 1 (CBFA1), alkaline phosphatase (ALP), and dentin matrix protein 1 (DMP-1). No mineral formation was detected by ARS when VEGF and/or BMP-2 were used without OM. OM + VEGF, but not OM + BMP-2, formed more mineralization than OM (P  .05) in the expression of the 3 genes. VEGF addition in the early phase rather than a continuous presence of both VEGF and BMP-2 enhances odonto/osteogenic differentiation of DPSCs. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  20. BMP Suppresses PTEN Expression via RAS/ERK Signaling

    OpenAIRE

    Beck, Stayce E.; Carethers, John M.

    2007-01-01

    Bone morphogenetic protein (BMP), a member of the transforming growth factor β family, classically utilizes the SMAD signaling pathway for its growth suppressive effects, and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effe...

  1. Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats

    International Nuclear Information System (INIS)

    Yin Jinbo; Ma Yuxin; Yin Qing; Xu Haiwei; An Ning; Liu Shiyong; Fan Xiaotang; Yang Hui

    2007-01-01

    The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process and explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after First PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression

  2. Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

    Directory of Open Access Journals (Sweden)

    Zhiyu Liu

    2015-05-01

    Full Text Available Bone morphogenetic proteins (BMPs belong to the transforming growth factor β (TGFβ superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.

  3. Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

    Science.gov (United States)

    Siverino, Claudia; Tabisz, Barbara; Lühmann, Tessa; Meinel, Lorenz; Müller, Thomas; Walles, Heike; Nickel, Joachim

    2018-03-29

    Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

  4. Astrocytes from the contused spinal cord inhibit oligodendrocyte differentiation of adult oligodendrocyte precursor cells by increasing the expression of bone morphogenetic proteins.

    Science.gov (United States)

    Wang, Yaping; Cheng, Xiaoxin; He, Qian; Zheng, Yiyan; Kim, Dong H; Whittemore, Scott R; Cao, Qilin L

    2011-04-20

    Promotion of remyelination is an important therapeutic strategy to facilitate functional recovery after traumatic spinal cord injury (SCI). Transplantation of neural stem cells (NSCs) or oligodendrocyte precursor cells (OPCs) has been used to enhance remyelination after SCI. However, the microenvironment in the injured spinal cord is inhibitory for oligodendrocyte (OL) differentiation of NSCs or OPCs. Identifying the signaling pathways that inhibit OL differentiation in the injured spinal cord could lead to new therapeutic strategies to enhance remyelination and functional recovery after SCI. In the present study, we show that reactive astrocytes from the injured rat spinal cord or their conditioned media inhibit OL differentiation of adult OPCs with concurrent promotion of astrocyte differentiation. The expression of bone morphogenetic proteins (BMP) is dramatically increased in the reactive astrocytes and their conditioned media. Importantly, blocking BMP activity by BMP receptor antagonist, noggin, reverse the effects of active astrocytes on OPC differentiation by increasing the differentiation of OL from OPCs while decreasing the generation of astrocytes. These data indicate that the upregulated bone morphogenetic proteins in the reactive astrocytes are major factors to inhibit OL differentiation of OPCs and to promote its astrocyte differentiation. These data suggest that manipulation of BMP signaling in the endogenous or grafted NSCs or OPCs may be a useful therapeutic strategy to increase their OL differentiation and remyelination and enhance functional recovery after SCI.

  5. Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy.

    Science.gov (United States)

    Sharma, Rekha; Ahlawat, Sonika; Maitra, A; Roy, Manoranjan; Mandakmale, S; Tantia, M S

    2013-12-10

    Bone morphogenetic proteins (BMPs) are members of the TGF-β (transforming growth factor-beta) superfamily, of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus expansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4 gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective of the present study was to detect polymorphism in intronic, exonic and 3' un-translated regions of BMP4 gene in Indian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning>40%), Osmanabadi, Sangamneri (Twinning 20-30%), Sirohi and Ganjam (Twinning<10%)) differing in prolificacy and geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) was used to design primers for the amplification of a targeted region followed by direct DNA sequencing to identify the genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic region and the 3' flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutation resulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534 locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predominant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhrana breed with a genotype frequency of 0.02. A microsatellite was identified in the 3' flanking region, only 20 nucleotides downstream from the termination site of the coding region, as a short sequence with more than nineteen continuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of a non-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is the first report of a mutation in the coding

  6. Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

    Science.gov (United States)

    Muzio, Giuliana; Martinasso, Germana; Baino, Francesco; Frairia, Roberto; Vitale-Brovarone, Chiara; Canuto, Rosa A

    2014-11-01

    In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  7. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

    Science.gov (United States)

    Zara, Janette N; Siu, Ronald K; Zhang, Xinli; Shen, Jia; Ngo, Richard; Lee, Min; Li, Weiming; Chiang, Michael; Chung, Jonguk; Kwak, Jinny; Wu, Benjamin M; Ting, Kang; Soo, Chia

    2011-05-01

    The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 μg/mL, total dose 0.375 and 0.75 μg in 75 μg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 μg/mL, total dose 2.25 μg in 75 μg total volume), and a high BMP2 concentration range (150, 300, and 600 μg/mL, total dose 11.25, 22.5, and 45 μg in 75 μg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 μg/mL.

  8. Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice

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    Barlow Linda A

    2010-10-01

    Full Text Available Abstract Background Bone Morphogenetic Protein 4 (BMP4 is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae and posterior (circumvallate papilla tongue. Results BMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III. BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers

  9. Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice.

    Science.gov (United States)

    Nguyen, Ha M; Barlow, Linda A

    2010-10-13

    Bone Morphogenetic Protein 4 (BMP4) is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae) and posterior (circumvallate papilla) tongue. BMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III). BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers and BrdU birthdating. Our data suggest that

  10. Bone morphogenetic protein signaling and olig1/2 interact to regulate the differentiation and maturation of adult oligodendrocyte precursor cells.

    Science.gov (United States)

    Cheng, Xiaoxin; Wang, Yaping; He, Qian; Qiu, Mengsheng; Whittemore, Scott R; Cao, Qilin

    2007-12-01

    Promotion of remyelination is an important therapeutic strategy for the treatment of the demyelinating neurological disorders. Adult oligodendrocyte precursor cells (OPCs), which normally reside quiescently in the adult central nervous system (CNS), become activated and proliferative after demyelinating lesions. However, the extent of endogenous remyelination is limited because of the failure of adult OPCs to mature into myelinating oligodendrocytes (OLs) in the demyelinated CNS. Understanding the molecular mechanisms that regulate the differentiation of adult OPCs could lead to new therapeutic strategies to treat these disorders. In this study, we established a stable culture of adult spinal cord OPCs and developed a reliable in vitro protocol to induce their sequential differentiation. Adult OPCs expressed bone morphogenetic protein (BMP) type Ia, Ib, and II receptor subunits, which are required for BMP signal transduction. BMP2 and 4 promoted dose-dependent astrocyte differentiation of adult OPCs with concurrent suppression of OL differentiation. Treatment of OPCs with BMP2 and 4 increased ID4 expression and decreased the expression of olig1 and olig2. Overexpression of olig1 or olig2 blocked the astrocyte differentiation of adult OPCs induced by BMP2 and 4. Furthermore, overexpression of both olig1 and olig2, but not olig1 or olig2 alone, rescued OL differentiation from inhibition by BMP2 and 4. Our results demonstrated that downregulation of olig1 and olig2 is an important mechanism by which BMP2 and 4 inhibit OL differentiation of adult OPCs. These data suggest that blocking BMP signaling combined with olig1/2 overexpression could be a useful therapeutic strategy to enhance endogenous remyelination and facilitate functional recovery in CNS demyelinated disorders. Disclosure of potential conflicts of interest is found at the end of this article.

  11. Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription.

    Science.gov (United States)

    Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

    2006-12-01

    During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

  12. Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription▿

    Science.gov (United States)

    Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

    2006-01-01

    During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22α and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription. PMID:17030628

  13. Molecular mechanisms of BMP-induced bone formation: Cross-talk between BMP and NF-κB signaling pathways in osteoblastogenesis

    Directory of Open Access Journals (Sweden)

    Eijiro Jimi

    2010-02-01

    Full Text Available Osteoblasts are bone-forming cells that differentiate from mesenchymal stem cells. Differentiation processes are coordinately and dynamically controlled in the mesenchymal cells by specific signal transduction pathways. Bone morphogenetic proteins (BMPs, members of the TGF-β superfamily, induce not only bone formation in vivo, but also osteoblast differentiation of mesenchymal cells in vitro. BMP signals are transduced from plasma membrane receptors to the nucleus through both Smad-dependent and -independent pathways, and are regulated by many extracellular and intercellular proteins that interact with BMPs or components of BMP signaling pathways. To understand the molecular mechanisms underlying the role of BMPs in osteoblast differentiation, it is important to elucidate the BMP signaling transduction pathways that are active during osteoblast differentiation. In this review, we summarize the BMP signaling pathways that are known to function in osteoblast development. We also describe our recent findings regarding the molecular mechanisms underlying the cross-talk between BMP/Smad and NF-κB pathways in osteoblast differentiation.

  14. Effect of hydrophilicity of carbon nanotube arrays on the release rate and activity of recombinant human bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Han Zhaojun; Ostrikov, Kostya [Plasma Nanoscience Centre Australia (PNCA), CSIRO Materials Science and Engineering, Lindfield, New South Wales 2070 (Australia); Tan, Cher Ming; Tay, Beng Kang [School of Electrical and Electronic Engineering, Nanyang Technological University, 639798 (Singapore); Peel, Sean A F, E-mail: zhaojun.han@csiro.au [Department of Dentistry, University of Toronto, Toronto, ON, M5G 1G6 (Canada)

    2011-07-22

    Novel nanostructures such as vertically aligned carbon nanotube (CNT) arrays have received increasing interest as drug delivery carriers. In the present study, two CNT arrays with extreme surface wettabilities are fabricated and their effects on the release of recombinant human bone morphogenetic protein-2 (rhBMP-2) are investigated. It is found that the superhydrophilic arrays retained a larger amount of rhBMP-2 than the superhydrophobic ones. Further use of a poloxamer diffusion layer delayed the initial burst and resulted in a greater total amount of rhBMP-2 released from both surfaces. In addition, rhBMP-2 bound to the superhydrophilic CNT arrays remained bioactive while they denatured on the superhydrophobic surfaces. These results are related to the combined effects of rhBMP-2 molecules interacting with poloxamer and the surface, which could be essential in the development of advanced carriers with tailored surface functionalities.

  15. Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity.

    Science.gov (United States)

    Doherty, Leo F; Taylor, Hugh S

    2015-03-01

    To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus

    Science.gov (United States)

    Levet, Sandrine; Ouarné, Marie; Ciais, Delphine; Coutton, Charles; Subileau, Mariela; Mallet, Christine; Ricard, Nicolas; Bidart, Marie; Debillon, Thierry; Faravelli, Francesca; Rooryck, Caroline; Feige, Jean-Jacques; Tillet, Emmanuelle; Bailly, Sabine

    2015-01-01

    The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFβ family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure. PMID:26056270

  17. Bone morphogenetic protein-2 loaded poly(D,L-lactide-co-glycolide microspheres enhance osteogenic potential of gelatin/hydroxyapatite/β-tricalcium phosphate cryogel composite for alveolar ridge augmentation

    Directory of Open Access Journals (Sweden)

    Hao-Chieh Chang

    2017-12-01

    Full Text Available Background/Purpose: Sufficient bony support is essential to ensure the success of dental implant osseointegration. However, the reconstruction of vertical ridge deficiencies is still a major challenge for dental implants. This study introduced a novel treatment strategy by infusing poly(D,L-lactide-co-glycolide (PLGA microspheres encapsulating bone morphogenetic protein-2 (BMP-2 within a gelatin/hydroxyapatite/β-tricalcium phosphate (gelatin/HA/β-TCP cryogel composite to facilitate supra-alveolar ridge augmentation. Methods: The gelatin scaffold was crosslinked using cryogel technique, and HA/β-TCP particles were mechanically entrapped to form the gelatin/HA/β-TCP composite. Co-axial electrohydrodynamic atomization technology was used to fabricate PLGA microspheres encapsulating BMP-2. The composites of gelatin/HA/β-TCP alone, with infusion of BMP-2 solution (BMPi or microspheres (BMPm, were fixed on rat mandibles using a titanium mini-implant for 4 weeks, and the therapeutic efficiency was evaluated by micro-computed tomography, bone fluorochrome, and histology. Results: The gelatin/HA/β-TCP composite was homogenously porous, and BMP-2 was sustained release from the microspheres without initial burst release. Ridge augmentation was noted in all specimens treated with the gelatin/HA/β-TCP composite, and greater bone deposition ratio were noted in Groups BMPi and BMPm. Compared with Group BMPi, specimens in Group BMPm showed significantly greater early osteogenesis and evident osseointegration in the supra-alveolar level. Conclusion: BMP-2 loaded PLGA microspheres effectively promoted osteogenic potential of the gelatin/HA/β-TCP composite and facilitated supra-alveolar ridge augmentation in vivo. Keywords: bone morphogenetic protein-2, bone regeneration, dental implant, tissue engineering, tissue scaffolds

  18. Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

    Science.gov (United States)

    Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

    2015-09-21

    Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment.

  19. Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection.

    Science.gov (United States)

    Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu; Takano-Yamamoto, Teruko

    2015-09-01

    The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can

  20. Unique players in the BMP pathway: Small C-terminal domain phosphatases dephosphorylate Smad1 to attenuate BMP signaling

    Science.gov (United States)

    Knockaert, Marie; Sapkota, Gopal; Alarcón, Claudio; Massagué, Joan; Brivanlou, Ali H.

    2006-01-01

    Smad transcription factors are key signal transducers for the TGF-β/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-β and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes. PMID:16882717

  1. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APP{sub swe}/PS1{sub {Delta}E9} transgenic mouse model of Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jun [Department of Medical Genetics, Third Military Medical University, Chongqing 400038 (China); Department of Physiology, Third Military Medical University, Chongqing 400038 (China); Song, Min; Wang, Yanyan [Department of Medical Genetics, Third Military Medical University, Chongqing 400038 (China); Fan, Xiaotang [Department of Histology and Embryology, Third Military Medical University, Chongqing 400038 (China); Xu, Haiwei, E-mail: haiweixu2001@yahoo.com.cn [Department of Physiology, Third Military Medical University, Chongqing 400038 (China); Bai, Yun, E-mail: baiyungene@gmail.com [Department of Medical Genetics, Third Military Medical University, Chongqing 400038 (China)

    2009-07-31

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP{sub swe}/PS1{sub {Delta}E9} mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP{sub swe}/PS1{sub {Delta}E9} transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  2. Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

    Science.gov (United States)

    Addante, Annalisa; Roncero, Cesáreo; Almalé, Laura; Lazcanoiturburu, Nerea; García-Álvaro, María; Fernández, Margarita; Sanz, Julián; Hammad, Seddik; Nwosu, Zeribe C; Lee, Se-Jin; Fabregat, Isabel; Dooley, Steven; Ten Dijke, Peter; Herrera, Blanca; Sánchez, Aránzazu

    2018-05-11

    Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Effects of bone morphogenetic protein-2 on bone cells in primary culture: immunohistochemical and electronmicroscopical studies

    Energy Technology Data Exchange (ETDEWEB)

    Schmitz, I.; Prochnow, N.; Mueller, K.M. [Berufsgenossenschaftliche Kliniken Bergmannsheil, Bochum (Germany). Inst. fuer Pathologie; Wiemann, M.; Schirrmacher, K.; Bingmann, D. [Essen Univ. (Germany). Inst. fuer Physiologie; Sebald, W. [Wuerzburg Univ. (Germany). Inst. fuer Physiologische Chemie II

    2001-02-01

    Bone morphogenetic protein 2 (BMP-2), among other morphogenetic effects on non osseous tissues, promotes bone formation in vivo. Therefore, BMP-2 may accelerate the integration of osseous implants. Although the effects of BMPs on cell proliferation have been studied extensively in vivo or in cell lines, little is published about effects on bone cells in primary cultures, especially on cell differentiation. As such information is a prerequisite to understand and to control effects of BMPs on cells at the surface of implant materials, the present experiments aimed to describe effects of BMP-2 on primary cultures derived from calvarial fragments of neonatal rats. The cells were stimulated with 50 nM BMP-2 added to the nutrient medium for 3 or 6 days. Light- and electronmicroscopical studies showed that cells in the sprouting zones were larger and more often spindle shaped. Stimulated cells had more nucleoli than control cells and the endoplasmic reticulum was widened. They retained properties of typical bone cells: An immunhistochemical analysis showed that stimulated cells increased the activity of alkaline phosphatase, they secreted collagen type I and to a minor extent collagen type III. In BMP-2 treated cells the pattern of cells stained for actin, desmin and vimentin hardly changed whereas extracellular fibronectin appeared to be less cross-linked in BMP-2 treated cultures. The distribution and labeling strength of osteocalcin, a specific marker protein of bone cells did not change markedly. After exposure to BMP-2 cells tended to detach from the cover slips. Electron microscopy showed a reduced number of cell processes possibly facilitating the detachment and/or mobility. Stimulated cells contained an increased number of lamellar bodies which may reflect an increased synthesis and/or membrane turnover. Staining of non-osseous cells with anti-CD68-or anti-myeloid antibodies revealed that the small percentage of these cells regularly occurring in primary cultures

  4. Cancer risk after use of recombinant bone morphogenetic protein-2 for spinal arthrodesis.

    Science.gov (United States)

    Carragee, Eugene J; Chu, Gilbert; Rohatgi, Rajat; Hurwitz, Eric L; Weiner, Bradley K; Yoon, S Tim; Comer, Garet; Kopjar, Branko

    2013-09-04

    Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor known to have in vitro effects on the growth and invasiveness of cancer. It has been approved by the U.S. Food and Drug Administration in limited doses for single-level anterior spinal arthrodesis, but it is commonly used off-label and at high doses. The effect of rhBMP-2 on the risk of cancer has been a concern. We sought to evaluate the risk of new cancers in patients receiving high-dose rhBMP-2. We used publicly available data from a pivotal, multicenter, randomized controlled trial of patients with degenerative lumbar spine conditions who underwent a single-level instrumented posterolateral arthrodesis with either high-dose rhBMP-2 in a compression-resistant matrix (CRM) (rhBMP-2/CRM; n = 239) or autogenous bone graft (control group; n = 224). We compared the risks of new cancers in the rhBMP-2/CRM and control groups at two and five years after surgery. At two years, with 86% follow-up, there were fifteen new cancer events in eleven patients in the rhBMP-2/CRM group compared with two new cancer events in two patients in the control group treated with autogenous bone graft. The incidence rate of new cancer events per 100 person-years was 3.37 (95% confidence interval [CI], 1.89 to 5.56) in the rhBMP-2/CRM group at two years compared with 0.50 (95% CI, 0.06 to 1.80) in the control group. The incidence rate ratio was 6.75 (95% CI, 1.57 to 60.83; p = 0.0026) at two years. Calculated in terms of the number of patients with one or more cancer events two years after the surgery, the incidence rate per 100 person-years was 2.54 (95% CI, 1.27 to 4.54) in the rhBMP-2/CRM group compared with 0.50 (95% CI, 0.06 to 1.82) in the control group at two years; the incidence rate ratio was 5.04 (95% CI, 1.10 to 46.82; p = 0.0194). At five years, there was a 37% loss of follow-up, but a significantly greater incidence of cancer events was still observed in the rhBMP-2/CRM group. A high dose of 40 mg of rhBMP

  5. Bone morphogenetic protein-induced heterotopic bone formation: What have we learned from the history of a half century?

    Directory of Open Access Journals (Sweden)

    Takenobu Katagiri, PhD

    2015-05-01

    Full Text Available Bone morphogenetic protein (BMP was originally discovered by Marshall Urist a half century ago following the observation of a unique activity that induced heterotopic bone formation in skeletal muscle tissue. The molecular mechanisms underlying the induction of heterotopic bone formation in skeletal muscle by BMPs were elucidated through the purification and molecular cloning of BMPs and identification of their functional receptors and downstream effectors, as well as from genetic disorders related to BMP activity. BMPs are important regulators of not only skeletal development and regeneration but also the homeostasis of normal skeletal muscle mass. There is still much to learn about the physiology and pathology at the interface of BMPs and skeletal muscle.

  6. SMOC can act as both an antagonist and an expander of BMP signaling.

    Science.gov (United States)

    Thomas, J Terrig; Eric Dollins, D; Andrykovich, Kristin R; Chu, Tehyen; Stultz, Brian G; Hursh, Deborah A; Moos, Malcolm

    2017-03-21

    The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone ( pent ), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

  7. Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery.

    Science.gov (United States)

    Yang, Hee Seok; La, Wan-Geun; Bhang, Suk Ho; Kim, Hak-Jun; Im, Gun-Il; Lee, Haeshin; Park, Jung-Ho; Kim, Byung-Soo

    2011-07-01

    Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.

  8. High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber.

    Science.gov (United States)

    Tashiro, Shota; Le, Minh Nguyen Tuyet; Kusama, Yuta; Nakatani, Eri; Suga, Mika; Furue, Miho K; Satoh, Taku; Sugiura, Shinji; Kanamori, Toshiyuki; Ohnuma, Kiyoshi

    2018-04-19

    Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration.

    Science.gov (United States)

    Wagner, Ines; Wang, Heng; Weissert, Philipp M; Straube, Werner L; Shevchenko, Anna; Gentzel, Marc; Brito, Goncalo; Tazaki, Akira; Oliveira, Catarina; Sugiura, Takuji; Shevchenko, Andrej; Simon, András; Drechsel, David N; Tanaka, Elly M

    2017-03-27

    Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entry with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specific dominant-negative receptor expression reduced cell cycle entry in vitro and in vivo. In vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.

    Science.gov (United States)

    Iijima, H; Ito, A; Nagai, M; Tajino, J; Yamaguchi, S; Kiyan, W; Nakahata, A; Zhang, J; Wang, T; Aoyama, T; Nishitani, K; Kuroki, H

    2017-06-01

    To evaluate the dose-response relationship of exercise loading in the cartilage-subchondral bone (SB) unit in surgically-induced post-traumatic osteoarthritis (PTOA) of the knee. Destabilized medial meniscus (DMM) surgery was performed on the right knee of 12-week-old male Wistar rats, and sham surgery was performed on the contralateral knee. Four weeks after the surgery, the animals were subjected to moderate (12 m/min) or intense (21 m/min) treadmill exercises for 30 min/day, 5 days/week for 4 weeks. PTOA development in articular cartilage and SB was examined using histological and immunohistochemical analyses, micro-computed tomography (micro-CT) analysis, and biomechanical testing at 8 weeks after surgery. Gremlin-1 was injected to determine the role of bone morphogenetic protein (BMP) signaling on PTOA development following moderate exercise. Moderate exercise increased BMP-2, BMP-4, BMP-6, BMP receptor 2, pSmad-5, and inhibitor of DNA binding protein-1 expression in the superficial zone chondrocytes and suppressed cartilage degeneration, osteophyte growth, SB damage, and osteoclast-mediated SB resorption. However, intense exercise had little effect on BMP expression and even caused progression of these osteoarthritis (OA) changes. Gremlin-1 injection following moderate exercise caused progression of the PTOA development down to the level of the non-exercise DMM-operated knee. Exercise regulated cartilage-SB PTOA development in DMM-operated knees in a dose-dependent manner. Our findings shed light on the important role of BMP expression in superficial zone chondrocytes in attenuation of PTOA development following physiological exercise loading. Further studies to support a mechanism by which BMPs would be beneficial in preventing PTOA progression are warranted. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  11. BMP signaling protects telencephalic fate by repressing eye identity and its Cxcr4-dependent morphogenesis.

    Science.gov (United States)

    Bielen, Holger; Houart, Corinne

    2012-10-16

    Depletion of Wnt signaling is a major requirement for the induction of the anterior prosencephalon. However, the molecular events driving the differential regionalization of this area into eye-field and telencephalon fates are still unknown. Here we show that the BMP pathway is active in the anterior neural ectoderm during late blastula to early gastrula stage in zebrafish. Bmp2b mutants and mosaic loss-of-function experiments reveal that BMP acts as a repressor of eye-field fate through inhibition of its key transcription factor Rx3, thereby protecting the future telencephalon from acquiring eye identity. This BMP-driven mechanism initiates the establishment of the telencephalon prior to the involvement of Wnt antagonists from the anterior neural border. Furthermore, we demonstrate that Rx3 and BMP are respectively required to maintain and restrict the chemokine receptor cxcr4a, which in turn contributes to the morphogenetic separation of eye-field and telencephalic cells during early neurulation. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage

    Science.gov (United States)

    Garrison, Presley; Yue, Shanna; Hanson, Jeffrey; Baron, Jeffrey; Lui, Julian C.

    2017-01-01

    Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may

  13. Mutation of Rubie, a novel long non-coding RNA located upstream of Bmp4, causes vestibular malformation in mice.

    Directory of Open Access Journals (Sweden)

    Kristina A Roberts

    Full Text Available The vestibular apparatus of the vertebrate inner ear uses three fluid-filled semicircular canals to sense angular acceleration of the head. Malformation of these canals disrupts the sense of balance and frequently causes circling behavior in mice. The Epistatic circler (Ecl is a complex mutant derived from wildtype SWR/J and C57L/J mice. Ecl circling has been shown to result from the epistatic interaction of an SWR-derived locus on chromosome 14 and a C57L-derived locus on chromosome 4, but the causative genes have not been previously identified.We developed a mouse chromosome substitution strain (CSS-14 that carries an SWR/J chromosome 14 on a C57BL/10J genetic background and, like Ecl, exhibits circling behavior due to lateral semicircular canal malformation. We utilized CSS-14 to identify the chromosome 14 Ecl gene by positional cloning. Our candidate interval is located upstream of bone morphogenetic protein 4 (Bmp4 and contains an inner ear-specific, long non-coding RNA that we have designated Rubie (RNA upstream of Bmp4 expressed in inner ear. Rubie is spliced and polyadenylated, and is expressed in developing semicircular canals. However, we discovered that the SWR/J allele of Rubie is disrupted by an intronic endogenous retrovirus that causes aberrant splicing and premature polyadenylation of the transcript. Rubie lies in the conserved gene desert upstream of Bmp4, within a region previously shown to be important for inner ear expression of Bmp4. We found that the expression patterns of Bmp4 and Rubie are nearly identical in developing inner ears.Based on these results and previous studies showing that Bmp4 is essential for proper vestibular development, we propose that Rubie is the gene mutated in Ecl mice, that it is involved in regulating inner ear expression of Bmp4, and that aberrant Bmp4 expression contributes to the Ecl phenotype.

  14. Duplications involving a conserved regulatory element downstream of BMP2 are associated with brachydactyly type A2

    DEFF Research Database (Denmark)

    Dathe, Katarina; Kjaer, Klaus W; Brehm, Anja

    2009-01-01

    Autosomal-dominant brachydactyly type A2 (BDA2), a limb malformation characterized by hypoplastic middle phalanges of the second and fifth fingers, has been shown to be due to mutations in the Bone morphogenetic protein receptor 1B (BMPR1B) or in its ligand Growth and differentiation factor 5 (GDF5......). A linkage analysis performed in a mutation-negative family identified a novel locus for BDA2 on chromosome 20p12.3 that incorporates the gene for Bone morphogenetic protein 2 (BMP2). No point mutation was identified in BMP2, so a high-density array CGH analysis covering the critical interval...... within the identified duplication. Our results reveal an additional functional mechanism for the pathogenesis of BDA2, which is duplication of a regulatory element that affects the expression of BMP2 in the developing limb....

  15. Calcium phosphate implants coatings as carriers for BMP-2

    NARCIS (Netherlands)

    Liu, Y.; He, J.F.; Hunziker, E.B.

    2009-01-01

    The osteoconductivity of dental implants can be improved by coating them with a layer of calcium phosphate (CaP), which can be rendered osteoinductive by functionalizing it with an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2). In the present study, we wished to compare the

  16. The effect of platelet rich plasma from bone marrow aspirate with added bone morphogenetic protein-2 on the Achilles tendon-bone junction in rabbits.

    Science.gov (United States)

    Kim, Hak Jun; Nam, Hyok-Woo; Hur, Chang-Yong; Park, Misu; Yang, Hee Seok; Kim, Byung-Soo; Park, Jung-Ho

    2011-12-01

    To determine if exogenously injected bone marrow derived platelet-rich plasma (PRP) plus bone morphogenetic protein (BMP)-2 could accelerate the healing of bone-tendon junction injuries and increase the junction holding strength during the early regeneration period. A direct injury model of the bone-tendon junction was made using an Achilles tendon-calcaneus bone junction in a rabbit. In the PRP/BMP-2/fibrin group, 0.05 mL of bone marrow derived PRP and 100 ng/mL of BMP-2 both incorporated into 0.1 mL of fibrin glue were injected into Achilles tendon-calcaneus bone junctions. The effect of the intervention was tested by comparing the results of an intervention group to a control group. The results of biomechanical testing, and histological and gross analyses were compared between the 2 groups at the following time points after surgery: 2 weeks, 4 weeks, and 8 weeks. Histologic examinations showed that woven bone developed in tendon-bone junctions at 2 weeks after surgery in the PRP/BMP-2/fibrin group. Mechanical test results showed no significant difference between the PRP/BMP-2/fibrin and control groups at 2 and 4 weeks after surgery, but the mean maximal load in the PRP/BMP-2/fibrin group was significantly higher than in the control group (p rabbit model of tendon-bone junction injury.

  17. Systems biology derived source-sink mechanism of BMP gradient formation.

    Science.gov (United States)

    Zinski, Joseph; Bu, Ye; Wang, Xu; Dou, Wei; Umulis, David; Mullins, Mary C

    2017-08-09

    A morphogen gradient of Bone Morphogenetic Protein (BMP) signaling patterns the dorsoventral embryonic axis of vertebrates and invertebrates. The prevailing view in vertebrates for BMP gradient formation is through a counter-gradient of BMP antagonists, often along with ligand shuttling to generate peak signaling levels. To delineate the mechanism in zebrafish, we precisely quantified the BMP activity gradient in wild-type and mutant embryos and combined these data with a mathematical model-based computational screen to test hypotheses for gradient formation. Our analysis ruled out a BMP shuttling mechanism and a bmp transcriptionally-informed gradient mechanism. Surprisingly, rather than supporting a counter-gradient mechanism, our analyses support a fourth model, a source-sink mechanism, which relies on a restricted BMP antagonist distribution acting as a sink that drives BMP flux dorsally and gradient formation. We measured Bmp2 diffusion and found that it supports the source-sink model, suggesting a new mechanism to shape BMP gradients during development.

  18. Recombinant human bone morphogenetic protein-2 released from polyurethane-based scaffolds promotes early osteogenic differentiation of human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Kim, Jinku; Hollinger, Jeffrey O

    2012-01-01

    The purposes of this study were to determine the pharmacokinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) from a polyurethane (PUR)-based porous scaffold and to determine the biological responses of human mesenchymal stem cells (hMSCs) to the rhBMP-2 released from those scaffolds. The rhBMP-2 was incorporated into the PUR three-dimensional (3D) porous scaffolds and release profiles were determined using enzyme-linked immunosorbent assay. The bioactivity of the rhBMP-2 containing releasates was determined using hMSCs and compared with exogenous rhBMP-2. Release of rhBMP-2 from PUR-based systems was bi-phasic and characterized by an initial burst followed by a sustained release for up to 21 days. Expression of alkaline phosphatase activity by hMSCs treated with the rhBMP-2 releasates was significantly greater than the cells alone (control) throughout the time periods. Furthermore, after 14 days of culture, the hMSCs cultured with rhBMP-2 releasate had a greater amount of mineralization compared to exogenous rhBMP-2. Overall, the rhBMP-2 release from the PUR-based scaffolds was sustained for 21 days and the releasates appeared to be bioactive and promoted earlier osteogenic differentiation and mineralization of hMSCs than the exogenous rhBMP-2. (paper)

  19. Abnormal Uterine Bleeding Is Associated With Increased BMP7 Expression in Human Endometrium.

    Science.gov (United States)

    Richards, Elliott G; El-Nashar, Sherif A; Schoolmeester, John K; Keeney, Gary L; Mariani, Andrea; Hopkins, Matthew R; Dowdy, Sean C; Daftary, Gaurang S; Famuyide, Abimbola O

    2017-05-01

    Abnormal uterine bleeding (AUB), a common health concern of women, is a heterogeneous clinical entity that is traditionally categorized into organic and nonorganic causes. Despite varied pharmacologic treatments, few offer sustained efficacy, as most are empiric, unfocused, and do not directly address underlying dysregulated molecular mechanisms. Characterization of such molecular derangements affords the opportunity to develop and use novel, more successful treatments for AUB. Given its implication in other organ systems, we hypothesized that bone morphogenetic protein (BMP) expression is altered in patients with AUB and hence comprehensively investigated dysregulation of BMP signaling pathways by systematically screening 489 samples from 365 patients for differences in the expression of BMP2, 4, 6, and 7 ligands, BMPR1A and B receptors, and downstream SMAD4, 6, and 7 proteins. Expression analysis was correlated clinically with data abstracted from medical records, including bleeding history, age at procedure, ethnicity, body mass index, hormone treatment, and histological diagnosis of fibroids, polyps, adenomyosis, hyperplasia, and cancer. Expression of BMP7 ligand was significantly increased in patients with AUB (H-score: 18.0 vs 26.7; P bleeding (menorrhagia) as their specific AUB pattern demonstrated significantly higher BMP7 expression. Significantly, no differences in the expression of any other BMP ligands, receptors, or SMAD proteins were observed in this large patient cohort. However, expression of BMPR1A, BMPR1B, and SMAD4 was significantly decreased in cancer compared to benign samples. Our study demonstrates that BMP7 is a promising target for future investigation and pharmacologic treatment of AUB.

  20. Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

    Directory of Open Access Journals (Sweden)

    Ariadne Cristiane Cabral Cruz

    2012-12-01

    Full Text Available Bone morphogenetic protein type 2 (BMP-2 is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2 or absence (ASCs+OM of BMP-2. The alkaline phosphatase (ALP activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II, osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity, intermediate (osteonectin and osteocalcin, or final (calcium deposition phases, suggesting that the exogenous addition of BMP-2 did not improve

  1. Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7.

    Science.gov (United States)

    Nishio, Kensuke; Ozawa, Yasumasa; Ito, Hisanori; Kifune, Takashi; Narita, Tatsuya; Iinuma, Toshimitsu; Gionhaku, Nobuhito; Asano, Masatake

    2017-10-01

    Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells. The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay. All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

  2. A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella.

    Science.gov (United States)

    Kuo, Dian-Han; Weisblat, David A

    2011-08-09

    Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1

    Directory of Open Access Journals (Sweden)

    Takako Saito

    2017-05-01

    Full Text Available Endoglin (ENG/CD105 is an essential endothelial cell co-receptor of the transforming growth factor β (TGF-β superfamily, mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1 and involved in tumor angiogenesis and preeclampsia. Here, we present crystal structures of the ectodomain of human ENG and its complex with the ligand bone morphogenetic protein 9 (BMP9. BMP9 interacts with a hydrophobic surface of the N-terminal orphan domain of ENG, which adopts a new duplicated fold generated by circular permutation. The interface involves residues mutated in HHT1 and overlaps with the epitope of tumor-suppressing anti-ENG monoclonal TRC105. The structure of the C-terminal zona pellucida module suggests how two copies of ENG embrace homodimeric BMP9, whose binding is compatible with ligand recognition by type I but not type II receptors. These findings shed light on the molecular basis of the BMP signaling cascade, with implications for future therapeutic interventions in this fundamental pathway.

  4. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

    Science.gov (United States)

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

  5. Oxygen-glucose deprivation preconditioning protects neurons against oxygen-glucose deprivation/reperfusion induced injury via bone morphogenetic protein-7 mediated ERK, p38 and Smad signalling pathways.

    Science.gov (United States)

    Guan, Junhong; Du, Shaonan; Lv, Tao; Qu, Shengtao; Fu, Qiang; Yuan, Ye

    2016-01-01

    Bone morphogenetic protein (BMP)-7 mediated neuroprotective effect of cerebral ischemic preconditioning (IPC) has been studied in an ischemic animal model, but the underlying cellular mechanisms have not been clearly clarified. In this study, primary cortical neurons and the SH-SY5Y cell line were used to investigate the role of BMP-7 and its downstream signals in the neuroprotective effects of oxygen-glucose deprivation preconditioning (OGDPC). Immunocytochemistry was used to detect the expression of neurofilament in neurons. MTT and lactate dehydrogenase activity assays were used to measure the cytotoxicity. Western blot was used to detect the protein expression of BMP-7 and downstream signals. BMP inhibitor, mitogen-activated protein kinase inhibitors, Smad inhibitor and siRNA of Smad 1 were used to investigate the role of corresponding signalling pathways in the OGDPC. Results showed that OGDPC-induced overexpression of BMP-7 in primary cortical neurons and SH-SY5Y cells. Both of endogenous and exogenous BMP-7 could replicate the neuroprotective effects seen in OGDPC pretreatment. In addition, extracellular regulated protein kinases, p38 and Smad signalling pathway were found to be involved in the neuroprotective effects mediated by OGDPC via BMP-7. This study primarily reveals the cellular mechanisms of the neuroprotection mediated by OGDPC, and provides evidence for better understanding of this intrinsic factor against ischemia. © 2015 Wiley Publishing Asia Pty Ltd.

  6. S100A4 and BMP-2 Co-Dependently Induce Vascular Smooth Muscle Cell Migration via pERK and Chloride Intracellular Channel 4 (CLIC4)

    Science.gov (United States)

    Spiekerkoetter, Edda; Guignabert, Christophe; de Jesus Perez, Vinicio; Alastalo, Tero-Pekka; Powers, Janine M; Wang, Lingli; Lawrie, Allan; Ambartsumian, Noona; Schmidt, Ann-Marie; Berryman, Mark; Ashley, Richard H; Rabinovitch, Marlene

    2009-01-01

    Rationale S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMC), through an interaction with the receptor for advanced glycation end products (RAGE). Objective We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (R) II, observed in pulmonary arterial hypertension (PAH). Methods and Results Both S100A4/Mts1 (500ng/ml) and BMP-2 (10ng/ml) induce migration of hPASMCS in a novel co-dependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII siRNA. Phosphorylation of ERK is induced by both ligands and is required for motility by inducing MMP2 activity, but phosphoERK1/2 is blocked by anti-RAGE and not by BMPRII siRNA. In contrast, BMPRII siRNA, but not anti-RAGE, reduces expression of intracellular chloride channel 4 (CLIC4), a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA (MHCIIA), but does alter alignment of MHCIIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodiae in motile cells. Conclusions Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, i.e., cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease. PMID:19713532

  7. Protective effect of bone morphogenetic protein 6 on RPE cells injury caused by H2O2

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    Li Chen

    2016-01-01

    Full Text Available AIM:To investigate the effect of bone morphogenetic protein 6(BMP-6on cellular morphology, proliferation and apoptosis of retinal pigment epithelial cells(ARPE-19incubated in hydrogen peroxide(H2O2. METHODS:ARPE-19 cells were cultured conventionally and divided into four groups. One group was untreated as blank group, the other three groups were incubated in 75μm/L H2O2, 150ng/mLBMP-6 or75μm/L H2O2+150ng/mL BMP-6. All the groups were incubated for 3h, 6h, 9h and 12h. We tested the cell viabilitity by MTT. We used flow cytometry to test the cell cycle and cell apoptosis.RESULTS:H2O2 significantly decreased the cell activity in time-dependent manner. The activity of cells with BMP-6+H2O2 was higher H2O2 group, and the differences between the two groups at 3h and 6h were significant(P2O2, while the cells with BMP-6 were less cell detachment and apoptosis. CONCLUSION:BMP-6 has protective effects on RPE cells from oxidative stress in certain extent.

  8. Bone morphogenetic protein-15 in follicle fluid combined with age may differentiate between successful and unsuccessful poor ovarian responders

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    Wu Yan-Ting

    2012-12-01

    Full Text Available Abstract Background The counselling of poor ovarian responders about the probability of pregnancy remains a puzzle for gynaecologists. The aim of this study was to optimise the management of poor responders by investigating the role of the oocyte-derived factor bone morphogenetic protein-15 (BMP-15 combined with chronological age in the prediction of the outcome of in-vitro fertilisation-embryo transfer (IVF-ET in poor responders. Methods A retrospective study conducted in a university hospital. A total of 207 poor ovarian responders who reached the ovum pick-up stage undergoing IVF/intracytoplasmic sperm injection (ICSI with three or fewer follicles no less than 14 mm on the day of oocyte retrieval were recruited from July 1, 2008 to December 31, 2009. Another 215 coinstantaneous cycles with normal responses were selected as controls. The BMP-15 levels in the follicular fluid (FF of the 207 poor responders were analysed by western blot. Based on the FF BMP-15 level and age, poor responders were sub-divided into four groups. The main outcome measures were the FF BMP-15 level, implantation rate, pregnancy rate, and live birth rate. Results The implantation rate (24.2% vs. 15.3%, chemical pregnancy rate (40% vs. 23.7%, clinical pregnancy rate (36.5% vs. 20.4% and live birth rate (29.4% vs. 15.1% in the high BMP-15 group were significantly higher than those in the low BMP-15 group. Furthermore, poor responders aged less than or equal to 35 years with a higher FF BMP-15 level had the best implantation, pregnancy and live birth rates, which were comparable with those of normal responders. Conclusions Our study suggests a potential role of BMP-15 in the prediction of the IVF outcome. A high FF BMP-15 combined with an age less than or equal to 35 years may be used as a potential indicator for repeating IVF cycles in poor ovarian responders.

  9. BMP-2 induces EMT and breast cancer stemness through Rb and CD44

    DEFF Research Database (Denmark)

    Huang, Peide; Chen, Anan; He, Weiyi

    2017-01-01

    Bone morphogenetic protein 2 (BMP-2) has been reported to facilitate epithelial-to-mesenchymal transition (EMT) and bone metastasis in breast cancer xenograft models. To investigate the role of BMP-2 in the development of breast cancer stem cells (BCSCs), and to further elucidate the mechanisms u...... then contribute to breast cancer metastasis. These findings may be helpful for developing new strategies for the treatment and prognosis of advanced breast cancer....

  10. In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

    Science.gov (United States)

    Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T

    2003-06-13

    Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.

  11. BMP Sustains Embryonic Stem Cell Self-Renewal through Distinct Functions of Different Krüppel-like Factors.

    Science.gov (United States)

    Morikawa, Masato; Koinuma, Daizo; Mizutani, Anna; Kawasaki, Natsumi; Holmborn, Katarina; Sundqvist, Anders; Tsutsumi, Shuichi; Watabe, Tetsuro; Aburatani, Hiroyuki; Heldin, Carl-Henrik; Miyazono, Kohei

    2016-01-12

    Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Histone deacetylases control neurogenesis in embryonic brain by inhibition of BMP2/4 signaling.

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    Maya Shakèd

    Full Text Available BACKGROUND: Histone-modifying enzymes are essential for a wide variety of cellular processes dependent upon changes in gene expression. Histone deacetylases (HDACs lead to the compaction of chromatin and subsequent silencing of gene transcription, and they have recently been implicated in a diversity of functions and dysfunctions in the postnatal and adult brain including ocular dominance plasticity, memory consolidation, drug addiction, and depression. Here we investigate the role of HDACs in the generation of neurons and astrocytes in the embryonic brain. PRINCIPAL FINDINGS: As a variety of HDACs are expressed in differentiating neural progenitor cells, we have taken a pharmacological approach to inhibit multiple family members. Inhibition of class I and II HDACs in developing mouse embryos with trichostatin A resulted in a dramatic reduction in neurogenesis in the ganglionic eminences and a modest increase in neurogenesis in the cortex. An identical effect was observed upon pharmacological inhibition of HDACs in in vitro-differentiating neural precursors derived from the same brain regions. A reduction in neurogenesis in ganglionic eminence-derived neural precursors was accompanied by an increase in the production of immature astrocytes. We show that HDACs control neurogenesis by inhibition of the bone morphogenetic protein BMP2/4 signaling pathway in radial glial cells. HDACs function at the transcriptional level by inhibiting and promoting, respectively, the expression of Bmp2 and Smad7, an intracellular inhibitor of BMP signaling. Inhibition of the BMP2/4 signaling pathway restored normal levels of neurogenesis and astrogliogenesis to both ganglionic eminence- and cortex-derived cultures in which HDACs were inhibited. CONCLUSIONS: Our results demonstrate a transcriptionally-based regulation of BMP2/4 signaling by HDACs both in vivo and in vitro that is critical for neurogenesis in the ganglionic eminences and that modulates cortical

  13. Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a

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    North Kathryn N

    2009-05-01

    Full Text Available Abstract Background Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9, but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression. Methods We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes (alkaline phosphatase, osteocalcin and bone morphogenetic protein receptor-1A as measured by quantitative PCR. Results BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated Bmpr-1a expression. Chronic BMP-2 treatment resulted in peak Bmpr-1a expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of Bmpr-1a that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine BMPR-1A/Bmpr-1a. Forced myogenic gene expression in fibroblasts was associated with a significant increase in Bmpr-1a expression and a synergistic increase in the osteogenic response to BMP-2. Conclusion These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the

  14. BMP signaling regulates satellite cell-dependent postnatal muscle growth.

    Science.gov (United States)

    Stantzou, Amalia; Schirwis, Elija; Swist, Sandra; Alonso-Martin, Sonia; Polydorou, Ioanna; Zarrouki, Faouzi; Mouisel, Etienne; Beley, Cyriaque; Julien, Anaïs; Le Grand, Fabien; Garcia, Luis; Colnot, Céline; Birchmeier, Carmen; Braun, Thomas; Schuelke, Markus; Relaix, Frédéric; Amthor, Helge

    2017-08-01

    Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57 In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool. © 2017. Published by The Company of Biologists Ltd.

  15. Poly(lactic-co-glycolide) polymer constructs cross-linked with human BMP-6 and VEGF protein significantly enhance rat mandible defect repair.

    Science.gov (United States)

    Das, Anusuya; Fishero, Brian A; Christophel, J Jared; Li, Ching-Ju; Kohli, Nikita; Lin, Yong; Dighe, Abhijit S; Cui, Quanjun

    2016-04-01

    We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P(VEGF)) or BMP-6 (P(BMP-6)) or both (P(VEGF+BMP-6)) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with P(VEGF+BMP-6) scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the P(VEGF+BMP-6) group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P(VEGF), P(BMP-6) and P(VEGF+BMP-6) but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P(VEGF), P(BMP-6), or P(VEGF+BMP-6) but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on P(VEGF+BMP-6) but not on P, P(VEGF), or P(BMP-6). Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.

  16. Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm.

    Science.gov (United States)

    Zakin, Lise; Reversade, Bruno; Kuroda, Hiroki; Lyons, Karen M; De Robertis, Eddy M

    2005-05-01

    Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.

  17. Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

    Science.gov (United States)

    Bylund, Jeffery B; Trinh, Linh T; Awgulewitsch, Cassandra P; Paik, David T; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B; Kamp, Timothy J; Hatzopoulos, Antonis K

    2017-05-01

    Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

  18. Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

    Science.gov (United States)

    Bylund, Jeffery B.; Trinh, Linh T.; Awgulewitsch, Cassandra P.; Paik, David T.; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B.; Kamp, Timothy J.

    2017-01-01

    Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling. PMID:28125926

  19. Effects of simulated weightlessness on the kinase activity of MEK1 induced by bone morphogenetic protein-2 in rat osteosarcoma cells

    Science.gov (United States)

    Zhang, S.; Wang, B.; Cao, X. S.; Yang, Z.

    Objective The mRNA expression of alpha 1 chain of type I collagen COL-I alpha 1 in rat osteosarcoma ROS17 2 8 cells induced by bone morphogenetic protein-2 BMP-2 was reduced under simulated microgravity The protein kinase MEK1 of MAPK signal pathway plays an important role in the expression of COL-I alpha 1 mRNA The purpose of this study is to investigate the effects of simulated weightlessness on the activity of MEK1 induced by BMP-2 in ROS17 2 8 cells Methods ROS17 2 8 cells were cultured in 1G control and rotating clinostat simulated weightlessness for 24 h 48 h and 72 h BMP-2 500 ng ml was added into the medium 1 h before the culture ended There was a control group in which ROS17 2 8 cells were cultured in 1G condition without BMP-2 Then the total protein of cells was extracted and the expression of phosphated-ERK1 2 p-ERK1 2 protein was detected by means of Western Blotting to show the kinase activity of MEK1 Results There were no significant differences in the expression of total ERK1 2 among all groups The expression of p-ERK1 2 was unconspicuous in the control group without BMP-2 but increased significantly when BMP-2 was added P 0 01 The level of p-ERK1 2 in simulated weightlessness group was much more lower than that in 1G group in every time point P 0 01 The expression of p-ERK1 2 gradually decreased along with the time of weightlessness simulation P 0 01 Conclusions The kinase activity of MEK1 induced by BMP-2 in rat osteosarcoma cells was reduced under simulated weightlessness

  20. Does the Addition of Bone Morphogenetic Protein 2 to Platelet-Rich Fibrin Improve Healing After Treatment for Medication-Related Osteonecrosis of the Jaw?

    Science.gov (United States)

    Park, Jung-Hyun; Kim, Jin-Woo; Kim, Sun-Jong

    2017-06-01

    To investigate the effect of the addition of bone morphogenetic protein 2 (BMP-2) to leukocyte-rich and platelet-rich fibrin (L-PRF) on the treatment of medication-related osteonecrosis of the jaws (MRONJ), this study compared the healing outcome of combined use of BMP-2 and L-PRF with single use of L-PRF. Of 55 patients who were diagnosed with MRONJ, 25 were treated with L-PRF alone and 30 were treated with L-PRF and recombinant human BMP-2. For each patient, surgical sites were evaluated postoperatively at 4 and 16 weeks. Associations between the treatment method and the resolution of MRONJ were analyzed with the adjustment of patient-specific factors that may influence the treatment outcome. At 4 and 16 weeks postoperatively, patients with MRONJ who were treated with both L-PRF and BMP-2 showed favorable outcomes with complete resolution of the lesions, which was statistically significant compared with that of the therapy using L-PRF alone (P = .028). Therefore, the additional use of BMP-2 considerably improved MRONJ healing. Among patient-specific factors, the existence of a bacterial colony in the biopsy specimen was a significant factor that negatively affected disease resolution (P = .017). The combined use of BMP-2 and L-PRF leads to the early resolution of MRONJ; thus patients who need to continue antiresorptive therapy may benefit from the combined regimen. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

  1. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    International Nuclear Information System (INIS)

    Wu, Li-An; Yuan, Guohua; Yang, Guobin; Ortiz-Gonzalez, Iris; Yang, Wuchen; Cui, Yong; MacDougall, Mary; Donly, Kevin J.; Harris, Stephen; Chen, Shuo

    2009-01-01

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  2. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  3. Gremlin-1 induces BMP-independent tumor cell proliferation, migration, and invasion.

    Directory of Open Access Journals (Sweden)

    Minsoo Kim

    Full Text Available Gremlin-1, a bone morphogenetic protein (BMP antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2 expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.

  4. FoxC2 Enhances BMP7-Mediated Anabolism in Nucleus Pulposus Cells of the Intervertebral Disc

    OpenAIRE

    Wang, Zheng; Fu, Changfeng; Chen, Yong; Xu, Feng; Wang, Zhenyu; Qu, Zhigang; Liu, Yi

    2016-01-01

    Bone-morphogenetic protein-7 (BMP-7) is a growth factor that plays a major role in mediating anabolism and anti-catabolism of the intervertebral disc matrix and cell homeostasis. In osteoblasts, Forkhead box protein C2 (FoxC2) is a downstream target of BMPs and promotes cell proliferation and differentiation. However, the role FoxC2 may play in degenerative human intervertebral disc tissue and the relationship between FoxC2 and BMP-7 in nucleus pulposus (NP) cells remain to be elucidated. Thi...

  5. Dynamics of BMP signaling in limb bud mesenchyme and polydactyly.

    Science.gov (United States)

    Norrie, Jacqueline L; Lewandowski, Jordan P; Bouldin, Cortney M; Amarnath, Smita; Li, Qiang; Vokes, Martha S; Ehrlich, Lauren I R; Harfe, Brian D; Vokes, Steven A

    2014-09-15

    Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Effect of autoclave devitalization on autograft incorporation and bone morphogenetic protein of tibia in Sprague-Dawley rats

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    Anak A.G.Y. Asmara

    2014-06-01

    Full Text Available Background: Heating process with autoclave is one of limb salvage modalities that are widely used. but the results are not satisfying, due to mechanical bone fragility. However, considering this treatment modality is widely accepted in terms of financial, religion and sociocultural aspects, we conducted a on study rats treated with resection and reconstruction with autoclave heating method to assess bone healing by sequential radiology, histopathologic osteoblasts count, and bone morphogenetic protein (BMP.Methods: Thirty six Sprague-Dawley rats were divided into two groups with one group being the autoclave group and others served as control group. In both groups, the tibial diaphysis was extracted en bloc for 7 mm. All groups were kept for 8 weeks and treated under the same condition except the autoclave group, where the extracted bones were put into autoclave at 134°C for 15 minutes and refixed again with k-wire. We performed radiological examination at 5th and 8th week using Lane and Sandhu radiological score. After extraction, the tibial bones were inspected for histological pattern using Salked modified score, osteoblast quantity counting and BMP-2 values.Results: There were statistically significant diffences between control and autoclave group on radiological score at 5th (5.12 ± 1.6 g vs 3.21 ± 2.42, p = 0.023 and 8th week (6.06 ± 1.71 vs 4.29 ± 2.53, p = 0.040, histological score between groups (6.06 ± 1.14 vs 4.14 ± 1.99, p = 0.005, osteoblast count (p < 0.001, and BMP-2 expression,  respectively.Conclusion: Autoclave recycling autograft lowered the speed of graft incorporation and BMP-2 expression. Therefore, autoclave recycling autograft as a method of limb salvage surgery must be reevaluated and not considered to be applied for treatment in bone malignancy.  

  7. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

    2017-04-01

    Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

  8. Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish

    Science.gov (United States)

    Swartz, Mary E.; McCarthy, Neil; Norrie, Jacqueline L.; Eberhart, Johann K.

    2016-01-01

    The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. PMID:27122171

  9. Depot injectable biodegradable nanoparticles loaded with recombinant human bone morphogenetic protein-2: preparation, characterization, and in vivo evaluation

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    Hassan AH

    2015-07-01

    Full Text Available Ali Habiballah Hassan,1 Khaled Mohamed Hosny,2,3 Zuahir A Murshid,1 Adel Alhadlaq,4 Ahmed Alyamani,5 Ghada Naguib6 1Department of Orthodontics, Faculty of Dentistry, 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt; 4Department of Pediatric Dentistry and Orthodontics, College of Dentistry, King Saud University, Riyadh, 5Department of Oral Surgery, 6Department of Restorative Dentistry, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Objective: The aim of this study is to utilize the biocompatibility characteristics of biodegradable polymers, viz, poly lactide-co-glycolide (PLGA and polycaprolactone (PCL, to prepare sustained-release injectable nanoparticles (NPs of bone morphogenetic protein-2 (BMP-2 for the repair of alveolar bone defects in rabbits. The influence of formulation parameters on the functional characteristics of the prepared NPs was studied to develop a new noninvasive injectable recombinant human BMP-2 (rhBMP-2 containing grafting material for the repair of alveolar bone clefts.Materials and methods: BMP-2 NPs were prepared using a water-in-oil-in-water double-emulsion solvent evaporation/extraction method. The influence of molar ratio of PLGA to PCL on a suitable particle size, encapsulation efficiency, and sustained drug release was studied. Critical size alveolar defects were created in the maxilla of 24 New Zealand rabbits divided into three groups, one of them treated with 5 µg/kg of rhBMP-2 NP formulations.Results: The results found that NPs formula prepared using blend of PLGA and PCL in 4:2 (w/w ratio showed the best sustained-release pattern with lower initial burst, and showed up to 62.7% yield, 64.5% encapsulation efficiency, 127 nm size, and more than 90% in vitro release. So, this formula was selected for

  10. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

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    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z. [The Third Hospital of Hebei Medical University, The Provincial Key Laboratory for Orthopedic Biomechanics of Hebei, Shijiazhuang, Hebei Province (China)

    2015-02-13

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

  11. Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

    International Nuclear Information System (INIS)

    Ma, W.H.; Liu, Y.J.; Wang, W.; Zhang, Y.Z.

    2015-01-01

    Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation

  12. BMP4 signaling is involved in the generation of inner ear sensory epithelia

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    Wang Yucheng

    2005-08-01

    Full Text Available Abstract Background The robust expression of BMP4 in the incipient sensory organs of the inner ear suggests possible roles for this signaling protein during induction and development of auditory and vestibular sensory epithelia. Homozygous BMP4-/- animals die before the inner ear's sensory organs develop, which precludes determining the role of BMP4 in these organs with simple gene knockout experiments. Results Here we use a chicken otocyst culture system to perform quantitative studies on the development of inner ear cell types and show that hair cell and supporting cell generation is remarkably reduced when BMP signaling is blocked, either with its antagonist noggin or by using soluble BMP receptors. Conversely, we observed an increase in the number of hair cells when cultured otocysts were treated with exogenous BMP4. BMP4 treatment additionally prompted down-regulation of Pax-2 protein in proliferating sensory epithelial progenitors, leading to reduced progenitor cell proliferation. Conclusion Our results implicate BMP4 in two events during chicken inner ear sensory epithelium formation: first, in inducing the switch from proliferative sensory epithelium progenitors to differentiating epithelial cells and secondly, in promoting the differentiation of hair cells within the developing sensory epithelia.

  13. Mechanism of recombinant human bone morphogenetic protein-2 in repairing hematopoietic injury in mice exposed to γ-rays

    International Nuclear Information System (INIS)

    Liu Shuibing; Hu Peizhen; Hou Ying; Li Xubo; Tian Qiong; Shi Mei

    2009-01-01

    Objective: To investigate the mechanism of recombinant human bone morphogenetic protein-2 (rhBMP-2) in repairing hematopoietic injury in mice irradiated with γ-ray. To prepare SRY gene probe and study the effect of rhBMP-2 in repairing hematopoietic injury in mice by in situ hybridization. Methods: Twenty-two BALB/c female mice were randomly divided into the irradiated group and BMP treated group, respectively. Bone marrow cells of normal male mice were transplanted into 22 female mice post-irradiation to 8.5 Gy of 60 Co γ rays. The left femurs of the survived female mice were re-irradiated with 9 Gy 14 days later. Mice in BMP treated group were given rhBMP-2 20 mg/kg while those in control group were treated with 0.9% saline by intraperitoneal injection every day for 6 days. These mice were killed 14 days later and paraffin sections of femurs were made. The SRY gene was detected with in situ hybridization. Results: There were more positive blots in the left femurs of the mice in irradiated group than those in BMP treated group (T=155.0, P 0.05). The number of positive blots in the left femurs of the mice in BMPtreated group was significantly less than those in the right femurs of the mice in two groups (T=155.0, 55.0, P<0.05). Conclusions: No donor cell of male mice was detected in the left femurs of BMP treated group, suggesting that rhBMP-2 promoted the restoration of residuary bone marrow cells. Thus, rhBMP-2 promotes the proliferation or differentiation of residuary mesenchymal stem cells, improves hematopoietic microenvironment and accelerates the hematopoietic restoration. (authors)

  14. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring

    Science.gov (United States)

    2016-01-01

    Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers’ oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris. PMID:26928288

  15. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

    Directory of Open Access Journals (Sweden)

    Desislava Abadjieva

    Full Text Available Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP 15 and growth differentiation factor (GDF 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR. The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

  16. Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

    Science.gov (United States)

    Abadjieva, Desislava; Kistanova, Elena

    2016-01-01

    Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

  17. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

    2017-01-01

    Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

  18. 2,2'-Bis(monoacylglycero) PO4 (BMP), but Not 3,1'-BMP, increases membrane curvature stress to enhance α-tocopherol transfer protein binding to membranes.

    Science.gov (United States)

    Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D; Katsaras, John; Atkinson, Jeffrey

    2015-03-01

    Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. We examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

  19. Profile of serum alkaline phosphatase after inoculation of mononuclear cells and bone morphogenetic protein in the repair of osteochondral defects in rabbits

    Directory of Open Access Journals (Sweden)

    Luiz Augusto de Souza

    2011-12-01

    Full Text Available In this study, serum alkaline phosphatase activity was measured in response to the repair of osteochondral defects in twenty-four New Zealand rabbits. The animals were divided into three groups: a control (GC, those treated with bone marrow mononuclear cells (GCM and those that received mononuclear cells with autologous bone morphogenetic protein (BMP + GCM. After exposing the trochlear groove of the left stifle joint, a wedge-shaped segment was removed. Later, the defect was filled with an osteochondral autograft preserved in 98% glycerin. For the GC group, only the bone graft was performed. For the GCM, in addition to the graft, 2x106 seed mononuclear cells were implanted. For the GCM + BMP, the same number of cells, associated with 1μg of bone morphogenetic protein, were intraarticularly administered. The osteoblastic response was measured by analyzing the serum alkaline phosphatase on day 0 (preoperative 3, 15, 30, and 45 after surgery, and by radiographic examinations. Analysis of variance in randomized blocks, factorial and Tukey’s test (p = 0.05 were made. The overall mean GCM was superior to the other groups and the highest rates were among the 15th and 45th days postoperatively. The discrepancy in values between individuals of the same group casts doubts on the veracity of the test.

  20. Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

    International Nuclear Information System (INIS)

    Li Huiwu; Dai Kerong; Tang Tingting; Zhang Xiaoling; Yan Mengning; Lou Jueren

    2007-01-01

    In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a β-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified by BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals

  1. Twisted gastrulation, a BMP antagonist, exacerbates podocyte injury.

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    Sachiko Yamada

    Full Text Available Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7 in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1, a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.

  2. A BMP responsive transcriptional region in the chicken type X collagen gene.

    Science.gov (United States)

    Volk, S W; Luvalle, P; Leask, T; Leboy, P S

    1998-10-01

    Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5' flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4-2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness

  3. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

    Science.gov (United States)

    2014-01-01

    Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of

  4. crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans.

    Science.gov (United States)

    Fung, Wong Yan; Fat, Ko Frankie Chi; Eng, Cheah Kathryn Song; Lau, Chow King

    2007-11-01

    We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.

  5. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye; Henriksen, Z; Sørensen, O H

    2004-01-01

    . Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin...... activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels......, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly...

  6. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.

    Science.gov (United States)

    Plikus, Maksim V; Mayer, Julie Ann; de la Cruz, Damon; Baker, Ruth E; Maini, Philip K; Maxson, Robert; Chuong, Cheng-Ming

    2008-01-17

    In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug

  7. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, Gayathri; Bialorucki, Callan [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Yildirim-Ayan, Eda, E-mail: eda.yildirimayan@utoledo.edu [Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606 (United States); Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614 (United States)

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21 days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O{sub 2} plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200 ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21 days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. - Highlights: • Injectable nanofibrous scaffold with osteoprogenitor cells and BMP2 was synthesized. • PCL nanofiber concentration within collagen scaffold affected the BMP2 retention and bioactivity. • Optimal PCL concentration was identified for mechanical stability, injectability, and osteogenic activity. • Scaffolds exhibited long-term osteoinductive capacity for bone repair and regeneration.

  8. Lactoferricin enhances BMP7-stimulated anabolic pathways in intervertebral disc cells.

    Science.gov (United States)

    Ellman, Michael B; Kim, Jaesung; An, Howard S; Chen, Di; Kc, Ranjan; Li, Xin; Xiao, Guozhi; Yan, Dongyao; Suh, Joon; van Wjnen, Andre J; Wang, James H-C; Kim, Su-Gwan; Im, Hee-Jeong

    2013-07-25

    Bone-morphogenetic protein-7 (BMP7) is a well-known anabolic and anti-catabolic growth factor on intervertebral disc (IVD) matrix and cell homeostasis. Similarly, Lactoferricin B (LfcinB) has recently been shown to have pro-anabolic, anti-catabolic, anti-oxidative and/or anti-inflammatory effects in bovine disc cells in vitro. In this study, we investigated the potential benefits of using combined peptide therapy with LfcinB and BMP7 for intervertebral disc matrix repair and to understand cellular and signaling mechanisms controlled by these factors. We studied the effects of BMP7 and LfcinB as individual treatments and combined therapy on bovine nucleus pulposus (NP) cells by assessing proteoglycan (PG) accumulation and synthesis, and the gene expression of matrix protein aggrecan and transcription factor SOX-9. We also analyzed the role of Noggin, a BMP antagonist, in IVD tissue and examined its effect after stimulation with LfcinB. To understand the molecular mechanisms by which LfcinB synergizes with BMP7, we investigated the ERK-SP1 axis as a downstream intracellular signaling regulator involved in BMP7 and LfcinB-mediated activities. Treatment of bovine NP cells cultured in alginate with LfcinB plus BMP7 synergistically stimulates PG synthesis and accumulation in part by upregulation of aggrecan gene expression. The synergism results from LfcinB-mediated activation of Sp1 and SMAD signaling pathways by (i) phosphorylation of SMAD 1/5/8; (ii) downregulation of SMAD inhibitory factors [i.e., noggin and SMAD6 (inhibitory SMAD)]; and (iii) upregulation of SMAD4 (universal co-SMAD). These data indicate that LfcinB-suppression of Noggin may eliminate the negative feedback of BMP7, thereby maximizing biological activity of BMP7 and ultimately shifting homeostasis to a pro-anabolic state in disc cells. We propose that combination growth factor therapy using BMP7 and LfcinB may be beneficial for treatment of disc degeneration. Copyright © 2013 Elsevier B.V. All

  9. Synergistic effects of dimethyloxallyl glycine and recombinant human bone morphogenetic protein-2 on repair of critical-sized bone defects in rats

    Science.gov (United States)

    Qi, Xin; Liu, Yang; Ding, Zhen-Yu; Cao, Jia-Qing; Huang, Jing-Huan; Zhang, Jie-Yuan; Jia, Wei-Tao; Wang, Jing; Liu, Chang-Sheng; Li, Xiao-Lin

    2017-02-01

    In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold. The synergistic effects of DMOG and rhBMP-2 released in the composite scaffolds on osteogenic and angiogenic differentiation of hBMSCs were investigated using real-time quantitative polymerase chain reaction and western blotting. Moreover, in vivo studies were conducted to observe bone regeneration and vascular formation of critical-sized bone defects in rats using micro-computed tomography, histological analyses, Microfil® perfusion, fluorescence labeling, and immunohistochemical analysis. The results showed that DMOG and rhBMP-2 released in the MBG-PHBHHx scaffolds did exert synergistic effects on the osteogenic and angiogenic differentiation of hBMSCs. Moreover, DMOG and rhBMP-2 produced significant increases in newly-formed bone and neovascularization of calvarial bone defects in rats. It is concluded that the co-delivery strategy of both rhBMP-2 and DMOG can significantly improve the critical-sized bone regeneration.

  10. Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2.

    Directory of Open Access Journals (Sweden)

    Roche C de Guzman

    Full Text Available Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2 has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD of 1.8 × 10(-4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7 M, within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5 M. BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks, suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5, below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the

  11. E. coli-Produced BMP-2 as a Chemopreventive Strategy for Colon Cancer: A Proof-of-Concept Study

    Directory of Open Access Journals (Sweden)

    Saravanan Yuvaraj

    2012-01-01

    Full Text Available Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bone morphogenetic protein 2 (BMP-2 is an important and powerful tumour suppressor in the colon and is thus an attractive candidate protein for delivery through genetically modified bacteria. It has not been shown, however, that BMP production in the bacterial context is effective on colon cancer cells. Here we demonstrate that transforming E. coli with a cDNA encoding an ileal-derived mature human BMP-2 induces effective apoptosis in an in vitro model system for colorectal cancer, whereas the maternal organism was not effective in this respect. Furthermore, these effects were sensitive to cotreatment with the BMP inhibitor Noggin. We propose that prevention and treatment of colorectal cancer using transgenic bacteria is feasible.

  12. Matrix-immobilized BMP-2 on microcontact printed fibronectin as in vitro tool to study BMP-mediated signaling and cell migration

    Directory of Open Access Journals (Sweden)

    Kristin eHauff

    2015-05-01

    Full Text Available During development, bone morphogenetic proteins (BMPs exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo the extracellular matrix (ECM not only provides a support for adherent cells, but also presents a reservoir of growth factors (GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell transmembrane receptors, such as integrins, which convey adhesion-mediated signaling to the intracellular compartment. Integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors, in regulating cell responses to extracellular signals. To this, we present here the immobilization of BMP-2 onto cellular fibronectin (cFN, a key protein of the ECM, to investigate their impact on GF-mediated signaling and migration.Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin (NA as cross-linker. Characterization with QCM-D and ELISA confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h.To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2 we investigated short- and long-term responses of C2C12 myoblasts in comparison to soluble BMP-2 (sBMP-2 or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation into the nucleus corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after six days.We next implemented this approach to fabricate cFN micro patterned stripes by soft lithography. These stripes only allowed cell-surface interaction on the pattern due to passivation of the surface in between, thus serving as platform for studies on directed cell migration. During a 10 h-period, cells showed an increased migratory activity upon BMP-2 exposure.Thus, this versatile tool retains the GF's bioactivity and allows the presentation of ECM

  13. Acceleration of Alveolar Ridge Augmentation Using a Low Dose of Recombinant Human Bone Morphogenetic Protein-2 Loaded on a Resorbable Bioactive Ceramic.

    Science.gov (United States)

    Fahmy, Rania A; Mahmoud, Naguiba; Soliman, Samia; Nouh, Samir R; Cunningham, Larry; El-Ghannam, Ahmed

    2015-12-01

    The aim of the present study was to evaluate the effect of a porous silica-calcium phosphate composite (SCPC50) loaded with and without recombinant human bone morphogenetic protein-2 (rhBMP-2) on alveolar ridge augmentation in saddle-type defects. Micro-granules of SCPC50 resorbable bioactive ceramic were coated with rhBMP-2 10 mg and then implanted into a saddle-type defect (12 × 7 mm) in a dog mandible and covered with a collagen membrane. Control groups included defects grafted with SCPC50 granules without rhBMP-2 and un-grafted defects. Bone healing was evaluated at 8 and 16 weeks using histologic and histomorphometric techniques. The increase in bone height and total defect fill were assessed for each specimen using the ImageJ 1.46 program. The release kinetics of rhBMP-2 was determined in vitro. The height of the bone in the grafted defects and the total defect fill were statistically analyzed. SCPC50 enhanced alveolar ridge augmentation as indicated by the increased vertical bone height, bone surface area, and bone volume after 16 weeks. SCPC50-rhBMP-2 provided a sustained release profile of a low effective dose (BMP-2 4.6 ± 1.34 pg/mL per hour) during the 1- to 21-day period. The slow rate of release of rhBMP-2 from SCPC50 accelerated synchronized complete bone regeneration and graft material resorption in 8 weeks. Successful rapid reconstruction of the alveolar ridge by SCPC50 and SCPC50-rhBMP-2 occurred without any adverse excessive bone formation, inflammation, or fluid-filled voids. Results of this study suggest that SCPC50 is an effective graft material to preserve the alveolar ridge after tooth extraction. Coating SCPC50-rhBMP-2 further accelerated bone regeneration and a considerable increase in vertical bone height. These findings make SCPC50 the primary choice as a carrier for rhBMP-2. SCPC50-rhBMP-2 can serve as an alternative to autologous bone grafting. Published by Elsevier Inc.

  14. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    International Nuclear Information System (INIS)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun; Liu Guangpeng; Zhang Peng; Hou Hongliang; Tang Tingting

    2011-01-01

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  15. Interaction of Wnt Signaling with BMP/Smad Signaling during the Transition from Cell Proliferation to Myogenic Differentiation in Mouse Myoblast-Derived Cells

    Directory of Open Access Journals (Sweden)

    Kumiko Terada

    2013-01-01

    Full Text Available Background. Wnt signaling is involved in muscle formation through β-catenin-dependent or -independent pathways, but interactions with other signaling pathways including transforming growth factor β/Smad have not been precisely elucidated. Results. As Wnt4 stimulates myogenic differentiation by antagonizing myostatin (GDF8 activity, we examined the role of Wnt4 signaling during muscle differentiation in the C2C12 myoblast cell line. Among several extrinsic signaling molecules examined in a microarray analysis of C2C12 cells during the transition from cell proliferation to differentiation after mitogen deprivation, bone morphogenetic protein 4 (BMP4 expression was prominently increased. Wnt4 overexpression had similar effects on BMP4 expression. BMP4 was able to inhibit muscle differentiation when added to the culture medium. BMP4 and noggin had no effects on the cellular localization of β-catenin induced by Wnt3a; however, the BMP4-induced phosphorylation of Smad1/5/8 was enhanced by Wnt4, but not by Wnt3a. The BMP antagonist noggin effectively stimulated muscle differentiation through binding to endogenous BMPs, and the effect of noggin was enhanced by the presence of Wnt3a and Wnt4. Conclusion. These results suggest that BMP/Smad pathways are modified through Wnt signaling during the transition from progenitor cell proliferation to myogenic differentiation, although Wnt/β-catenin signaling is not modified with BMP/Smad signaling.

  16. BMP15 Prevents Cumulus Cell Apoptosis Through CCL2 and FBN1 in Porcine Ovaries

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    Bo Zhai

    2013-07-01

    Full Text Available Background: Bone morphogenetic protein-15 (BMP15 is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. Methods: In this study, flow cytometry (FCM was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi method was used to inhibit the expression of (MCP-1/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR and western blotting. Results: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1/CCL2 and fibrillin1 (FBN1 were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. Conclusion: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.

  17. Report of a newly indentified patient with mutations in BMP1 and underlying pathogenetic aspects

    DEFF Research Database (Denmark)

    Valencia, María; Caparrós-Martin, Jose A; Sirerol-Piquer, María Salomé

    2014-01-01

    Osteogenesis imperfecta is a genetic condition characterized by bone fragility and recurrent fractures, which in the large majority of patients are caused by defects in the production of type I collagen. Mutations in the gene encoding bone morphogenetic protein 1 (BMP1, also known as procollagen C......-endopeptidase) have been associated with osteogenesis imperfecta in two sib pairs. In this report, we describe an additional patient with osteogenesis imperfecta with normal bone density and a recurrent, homozygous c.34G>C mutation in BMP1. Western blot analysis of dermal fibroblasts from this patient showed...

  18. Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia.

    Science.gov (United States)

    Suzuki, Kentaro; Adachi, Yasuha; Numata, Tomokazu; Nakada, Shoko; Yanagita, Motoko; Nakagata, Naomi; Evans, Sylvia M; Graf, Daniel; Economides, Aris; Haraguchi, Ryuma; Moon, Anne M; Yamada, Gen

    2012-01-01

    Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox)) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox) conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.

  19. Reduced BMP signaling results in hindlimb fusion with lethal pelvic/urogenital organ aplasia: a new mouse model of sirenomelia.

    Directory of Open Access Journals (Sweden)

    Kentaro Suzuki

    Full Text Available Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4(flox/flox and the Isl1 (Islet1-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4(flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.

  20. Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

    Science.gov (United States)

    Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

    2009-06-01

    The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

  1. BMP9-Induced Osteogenetic Differentiation and Bone Formation of Muscle-Derived Stem Cells

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    Li Xiang

    2012-01-01

    Full Text Available Efficient osteogenetic differentiation and bone formation from muscle-derived stem cells (MDSCs should have potential clinical applications in treating nonunion fracture healing or bone defects. Here, we investigate osteogenetic differentiation ability of MDSCs induced by bone morphogenetic protein 9 (BMP9 in vitro and bone formation ability in rabbit radius defects repairing model. Rabbit's MDSCs were extracted by type I collagenase and trypsin methods, and BMP9 was introduced into MDSCs by infection with recombinant adenovirus. Effects of BMP9-induced osteogenetic differentiation of MDSCs were identified with alkaline phosphatase (ALP activity and expression of later marker. In stem-cell implantation assay, MDSCs have also shown valuable potential bone formation ability induced by BMP9 in rabbit radius defects repairing test. Taken together, our findings suggest that MDSCs are potentiated osteogenetic stem cells which can be induced by BMP9 to treat large segmental bone defects, nonunion fracture, and/or osteoporotic fracture.

  2. Effects of atomic-level nano-structured hydroxyapatite on adsorption of bone morphogenetic protein-7 and its derived peptide by computer simulation.

    Science.gov (United States)

    Wang, Qun; Wang, Menghao; Lu, Xiong; Wang, Kefeng; Fang, Liming; Ren, Fuzeng; Lu, Guoming

    2017-11-09

    Hydroxyapatite (HA) is the principal inorganic component of bones and teeth and has been widely used as a bone repair material because of its good biocompatibility and bioactivity. Understanding the interactions between proteins and HA is crucial for designing biomaterials for bone regeneration. In this study, we evaluated the effects of atomic-level nano-structured HA (110) surfaces on the adsorption of bone morphogenetic protein-7 (BMP-7) and its derived peptide (KQLNALSVLYFDD) using molecular dynamics and density functional theory methods. The results indicated that the atomic-level morphology of HA significantly affected the interaction strength between proteins and HA substrates. The interactions of BMP-7 and its derived peptide with nano-concave and nano-pillar HA surfaces were stronger than those with flat or nano-groove HA surfaces. The results also revealed that if the groove size of nano-structured HA surfaces matched that of residues in the protein or peptide, these residues were likely to spread into the grooves of the nano-groove, nano-concave, and nano-pillar HA, further strengthening the interactions. These results are helpful in better understanding the adsorption behaviors of proteins onto nano-structured HA surfaces, and provide theoretical guidance for designing novel bioceramic materials for bone regeneration and tissue engineering.

  3. Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

    Science.gov (United States)

    Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

    2009-12-01

    The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and

  4. Association of Bone Morphogenetic Protein 6 With Exocrine Gland Dysfunction in Patients With Sjögren’s Syndrome and in Mice

    Science.gov (United States)

    Yin, Hongen; Cabrera-Perez, Javier; Lai, Zhenan; Michael, Drew; Weller, Melodie; Swaim, William D.; Liu, Xibao; Catalán, Marcelo A.; Rocha, Eduardo M.; Ismail, Nevien; Afione, Sandra; Rana, Noreen A.; Di Pasquale, Giovanni; Alevizos, Ilias; Ambudkar, Indu; Illei, Gabor G.; Chiorini, John A.

    2014-01-01

    Objective Primary Sjögren’s syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. Methods To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus–mediated gene transfer to the salivary glands of C57BL/6 mice. Results A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector–treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. Conclusion In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6–induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease. PMID:23982860

  5. Bone Regeneration Using N-Methyl-2-pyrrolidone as an Enhancer for Recombinant Human Bone Morphogenetic Protein-2 in a Rabbit Sinus Augmentation Model.

    Science.gov (United States)

    Lim, Hyun-Chang; Thoma, Daniel S; Yoon, So-Ra; Cha, Jae-Kook; Lee, Jung-Seok; Jung, Ui-Won

    2017-01-01

    The aim of this study was to determine whether N-methyl-2-pyrrolidone (NMP) can decrease the dose of recombinant human bone morphogenetic protein-2 (rhBMP-2) in sinus augmentation of rabbits. In each of 15 rabbits, 2 sinuses were randomly grafted using 1 of 3 treatment modalities: (i) biphasic calcium phosphate (BCP; control), (ii) rhBMP-2-coated BCP (BMP), or (iii) rhBMP-2-coated BCP soaked in NMP solution (BMP/NMP). The rabbits were sacrificed 2 weeks postoperatively. Histologic and histomorphometric analyses were performed. Bone formation in all groups was predominantly located close to the access window and the lateral walls. Newly formed bone within the total augmented area (NB TA ) was greatest in BMP/NMP (1.94 ± 0.69 mm 2 ), followed by BMP (1.50 ± 0.72 mm 2 ) and BCP (1.28 ± 0.52 mm 2 ) ( P > 0.05). In the center of the augmentation (NB ROI_C ) and the area close to the sinus membrane (NB ROI_M ), BMP/NMP produced the largest area of NB (NB ROI_C : 0.10 ± 0.11 mm 2 ; NB ROI_M : 0.17 ± 0.08 mm 2 ); the corresponding NB values for BCP were 0.05 ± 0.05 mm 2 and 0.08 ± 0.09 mm 2 , respectively ( P > 0.05 for all comparisons). The effect of NMP on bone regeneration was inconsistent between the specimens. Adding NMP as an adjunct to rhBMP-2-coated BCP produced inconsistent effects on bone regeneration, resulting in no significant benefit compared to controls.

  6. Outcome of nonunion fractures in dogs treated with fixation, compression resistant matrix, and recombinant human bone morphogenetic protein-2.

    Science.gov (United States)

    Massie, Anna M; Kapatkin, Amy S; Fuller, Mark C; Verstraete, Frank J M; Arzi, Boaz

    2017-03-20

    To report the use of compression resistant matrix (CRM) infused with recombinant human bone morphogenetic protein (rhBMP-2) prospectively in the healing of nonunion long-bone fractures in dogs. A longitudinal cohort of dogs that were presented with nonunion fractures were classified and treated with CRM soaked with rhBMP-2 and fracture fixation. They were followed with serial radiographs and evaluated for healing times and complications according to the time frame and definitions previously established for orthopaedic clinical cases. Eleven nonunion fractures in nine dogs were included. Median healing time was 10 weeks (range: 7-20 weeks). Major perioperative complications due to bandage morbidity were encountered in two of 11 limbs and resolved. All other complications were minor. They occurred perioperatively in eight of 11 limbs. Minor follow-up complications included short-term in one of two limbs, mid-term in one of three, and long-term in four of five limbs. Nine limbs returned to full function and two limbs returned to acceptable function at the last follow-up. Nonunion fractures given a poor prognosis via standard-of-care treatment were successfully repaired using CRM with rhBMP-2 accompanying fixation. These dogs, previously at high risk of failure, returned to full or acceptable function.

  7. Lead induces chondrogenesis and alters transforming growth factor-beta and bone morphogenetic protein signaling in mesenchymal cell populations.

    Science.gov (United States)

    Zuscik, Michael J; Ma, Lin; Buckley, Taylor; Puzas, J Edward; Drissi, Hicham; Schwarz, Edward M; O'Keefe, Regis J

    2007-09-01

    It has been established that skeletal growth is stunted in lead-exposed children. Because chondrogenesis is a seminal step during skeletal development, elucidating the impact of Pb on this process is the first step toward understanding the mechanism of Pb toxicity in the skeleton. The aim of this study was to test the hypothesis that Pb alters chondrogenic commitment of mesenchymal cells and to assess the effects of Pb on various signaling pathways. We assessed the influence of Pb on chondrogenesis in murine limb bud mesenchymal cells (MSCs) using nodule formation assays and gene analyses. The effects of Pb on transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling was studied using luciferase-based reporters and Western analyses, and luciferase-based assays were used to study cyclic adenosine monophosphate response element binding protein (CREB), beta-catenin, AP-1, and nuclear factor-kappa B (NF-kappaB) signaling. We also used an ectopic bone formation assay to determine how Pb affects chondrogenesis in vivo. Pb-exposed MSCs showed enhanced basal and TGF-beta/BMP induction of chondrogenesis, evidenced by enhanced nodule formation and up-regulation of Sox-9, type 2 collagen, and aggrecan, all key markers of chondrogenesis. We observed enhanced chondrogenesis during ectopic bone formation in mice preexposed to Pb via drinking water. In MSCs, Pb enhanced TGF-beta but inhibited BMP-2 signaling, as measured by luciferase reporter assays and Western analyses of Smad phosphorylation. Although Pb had no effect on basal CREB or Wnt/beta-catenin pathway activity, it induced NFkappaB signaling and inhibited AP-1 signaling. The in vitro and in vivo induction of chondrogenesis by Pb likely involves modulation and integration of multiple signaling pathways including TGF-beta, BMP, AP-1, and NFkappaB.

  8. Strategies for delivering bone morphogenetic protein for bone healing

    Energy Technology Data Exchange (ETDEWEB)

    Begam, Howa [School of Bioscience and Engineering, Jadavpur University, Kolkata 700032 (India); Nandi, Samit Kumar, E-mail: samitnandi1967@gmail.com [Department of Veterinary Surgery, Radiology West Bengal University of Animal and Fishery Sciences, Kolkata 700037 (India); Kundu, Biswanath, E-mail: biswa_kundu@rediffmail.com [Bioceramics and Coating Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700032 (India); Chanda, Abhijit [Department of Mechanical Engineering, Jadavpur University, Kolkata 700032 (India)

    2017-01-01

    Bone morphogenetic proteins (BMPs) are the most significant growth factors that belong to the Transforming Growth Factor Beta (TGF-β) super-family. Though more than twenty members of this family have been identified so far in humans, Food and Drug Administration (FDA) approved two growth factors: BMP-2 and BMP-7 for treatments of spinal fusion and long-bone fractures with collagen carriers. Currently BMPs are clinically used in spinal fusion, oral and maxillofacial surgery and also in the repair of long bone defects. The efficiency of BMPs depends a lot on the selection of suitable carriers. At present, different types of carrier materials are used: natural and synthetic polymers, calcium phosphate and ceramic-polymer composite materials. Number of research articles has been published on the minute intricacies of the loading process and release kinetics of BMPs. Despite the significant evidence of its potential for bone healing demonstrated in animal models, future clinical investigations are needed to define dose, scaffold and route of administration. The efficacy and application of BMPs in various levels with a proper carrier and dose is yet to be established. The present article collates various aspects of success and limitation and identifies the prospects and challenges associated with the use of BMPs in orthopaedic surgery. - Highlights: • Currently BMPs are clinically used in spinal fusion, oral and maxillofacial surgery and also in repair of long bone defects. • Different types of carrier materials are used: natural, synthetic polymers, calcium phosphate and ceramic-polymer composite • Efficacy and application of BMPs in various levels with proper carrier and dose is yet to be established • Number of research articles has been published on minute intricacies of loading process and release kinetics of BMPs • Present article collates success, limitation and identifies prospects, challenges for use of BMPs in orthopaedic surgery.

  9. Overexpression of BMP3 in the developing skeleton alters endochondral bone formation resulting in spontaneous rib fractures.

    Science.gov (United States)

    Gamer, Laura W; Cox, Karen; Carlo, Joelle M; Rosen, Vicki

    2009-09-01

    Bone morphogenetic protein-3 (BMP) has been identified as a negative regulator in the skeleton as mice lacking BMP3 have increased bone mass. To further understand how BMP3 mediates bone formation, we created transgenic mice overexpressing BMP3 using the type I collagen promoter. BMP3 transgenic mice displayed spontaneous rib fractures that were first detected at E17.0. The fractures were due to defects in differentiation of the periosteum and late hypertrophic chondrocytes resulting in thinner cortical bone with decreased mineralization. As BMP3 modulates BMP and activin signaling through ActRIIB, we examined the ribs of ActRIIB receptor knockout mice and found they had defects in late chondrogenesis and mineralization similar to BMP3 transgenic mice. These data suggest that BMP3 exerts its effects in the skeleton by altering signaling through ActRIIB in chondrocytes and the periosteum, and this results in defects in bone collar formation and late hypertrophic chondrocyte maturation leading to decreased mineralization and less bone. 2009 Wiley-Liss, Inc.

  10. BMP signaling in the human fetal ovary is developmentally regulated and promotes primordial germ cell apoptosis.

    Science.gov (United States)

    Childs, Andrew J; Kinnell, Hazel L; Collins, Craig S; Hogg, Kirsten; Bayne, Rosemary A L; Green, Samira J; McNeilly, Alan S; Anderson, Richard A

    2010-08-01

    Primordial germ cells (PGCs) are the embryonic precursors of gametes in the adult organism, and their development, differentiation, and survival are regulated by a combination of growth factors collectively known as the germ cell niche. Although many candidate niche components have been identified through studies on mouse PGCs, the growth factor composition of the human PGC niche has not been studied extensively. Here we report a detailed analysis of the expression of components of the bone morphogenetic protein (BMP) signaling apparatus in the human fetal ovary, from postmigratory PGC proliferation to the onset of primordial follicle formation. We find developmentally regulated and reciprocal patterns of expression of BMP2 and BMP4 and identify germ cells to be the exclusive targets of ovarian BMP signaling. By establishing long-term cultures of human fetal ovaries in which PGCs are retained within their physiological niche, we find that BMP4 negatively regulates postmigratory PGC numbers in the human fetal ovary by promoting PGC apoptosis. Finally, we report expression of both muscle segment homeobox (MSX)1 and MSX2 in the human fetal ovary and reveal a selective upregulation of MSX2 expression in human fetal ovary in response to BMP4, suggesting this gene may act as a downstream effector of BMP-induced apoptosis in the ovary, as in other systems. These data reveal for the first time growth factor regulation of human PGC development in a physiologically relevant context and have significant implications for the development of cultures systems for the in vitro maturation of germ cells, and their derivation from pluripotent stem cells.

  11. BMP signaling mediates effects of exercise on hippocampal neurogenesis and cognition in mice.

    Directory of Open Access Journals (Sweden)

    Kevin T Gobeske

    2009-10-01

    Full Text Available Exposure to exercise or to environmental enrichment increases the generation of new neurons in the adult hippocampus and promotes certain kinds of learning and memory. While the precise role of neurogenesis in cognition has been debated intensely, comparatively few studies have addressed the mechanisms linking environmental exposures to cellular and behavioral outcomes. Here we show that bone morphogenetic protein (BMP signaling mediates the effects of exercise on neurogenesis and cognition in the adult hippocampus. Elective exercise reduces levels of hippocampal BMP signaling before and during its promotion of neurogenesis and learning. Transgenic mice with decreased BMP signaling or wild type mice infused with a BMP inhibitor both exhibit remarkable gains in hippocampal cognitive performance and neurogenesis, mirroring the effects of exercise. Conversely, transgenic mice with increased BMP signaling have diminished hippocampal neurogenesis and impaired cognition. Exercise exposure does not rescue these deficits, suggesting that reduced BMP signaling is required for environmental effects on neurogenesis and learning. Together, these observations show that BMP signaling is a fundamental mechanism linking environmental exposure with changes in cognitive function and cellular properties in the hippocampus.

  12. Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation.

    Science.gov (United States)

    Wang, Jingxiao; Zheng, Yuanna; Zhao, Juan; Liu, Tie; Gao, Lixia; Gu, Zhiyuan; Wu, Gang

    2012-01-01

    To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer. Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created surrounding SLA-treated titanium implants (3.1 mm in diameter, 10 mm in length) in minipig's calvaria. We administrated collagen sponges with adsorbed low-dose (30 ng/mm(3) ) BMP2/7 to treat the defects using BMP2, BMP7 or no BMP as controls.2, 3 and 6 weeks after implantation, we adopted micro-computer tomography to evaluate the micro-architectures of new bone using the following parameters: relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density, and structure mode index (SMI). Bone implant contact (BIC) was also revealed histologically. Consistent with 2 and 3 weeks, after 6 weeks post-operation, BMP2/7 resulted in significantly higher BV/TV (63.033 ± 2.055%) and significantly lower SMI (-4.405 ± 0.500) than BMP2 (BV/TV: 43.133 ± 2.001%; SMI: -0.086 ± 0.041) and BMP7 (BV/TV: 41.467 ± 1.850%; SMI: -0.044 ± 0.016) respectively. Significant differences were also found in Tb.N, Tb.Th and Tb.Sp at all time points. At 2 weeks, BMP2/7 resulted in significantly higher BIC than the controls. Low-dose BMP2/7 heterodimer facilitated more rapid bone regeneration in better quality in peri-implant bone defects than BMP2 and BMP7 homodimers. © 2011 John Wiley & Sons A/S.

  13. Association of bone morphogenetic protein 6 with exocrine gland dysfunction in patients with Sjögren's syndrome and in mice.

    Science.gov (United States)

    Yin, Hongen; Cabrera-Perez, Javier; Lai, Zhenan; Michael, Drew; Weller, Melodie; Swaim, William D; Liu, Xibao; Catalán, Marcelo A; Rocha, Eduardo M; Ismail, Nevien; Afione, Sandra; Rana, Noreen A; Di Pasquale, Giovanni; Alevizos, Ilias; Ambudkar, Indu; Illei, Gabor G; Chiorini, John A

    2013-12-01

    Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  14. Randomised controlled clinical trial of augmentation of the alveolar ridge using recombinant human bone morphogenetic protein 2 with hydroxyapatite and bovine-derived xenografts: comparison of changes in volume.

    Science.gov (United States)

    Nam, J W; Khureltogtokh, S; Choi, H M; Lee, A R; Park, Y B; Kim, H J

    2017-10-01

    The aim of this randomised controlled clinical trial was to assess the early efficacy of bone morphogenetic protein-2 with hydroxyapatite granules (BMP-2/hydroxyapatite) on augmentation of the alveolar ridge, by comparing changes in volume with those associated with the use of an inorganic bovine-derived xenograft (BDX). We studied 20 patients who were divided into two groups using a table of random numbers, and BMP-2/hydroxyapatite and BDX were applied accordingly. Computed tomographic (CT) images and panoramic radiographs were obtained immediately after operation and four months later. CT images were reconstructed in three dimensions to measure volumetric changes, and linear measurements were made on panoramic images. The mean (SD) absorption rates for BMP-2/hydroxyapatite and BDX were 13.2 (8.8)% and 13.8 (20.5)%, respectively. While the mean value did not differ significantly between the two materials, the SD was higher in the BDX group than in the BMP-2/hydroxyapatite group. No clinically important complications occurred in either group. We conclude that both BMP-2/hydroxyapatite and BDX were effective in augmenting the alveolar ridge, but BMP-2/hydroxyapatite seemed to be more useful in complicated bone defects. Copyright © 2017 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  15. Bone morphogenetic protein 2 (BMP2) induces growth suppression and enhances chemosensitivity of human colon cancer cells

    DEFF Research Database (Denmark)

    Vishnubalaji, Radhakrishnan; Yue, Shijun; Alfayez, Musaad

    2016-01-01

    expression were assessed using qRT-PCR. AlamarBlue assay was used to assess cell viability in vitro. In vivo experiments were conducted using SCID mice. RESULTS: Our data revealed frequent downregulation of BMP2 in primary CRC tissues. Additionally, interrogation of publically available gene expression......, suggesting that restoration of BMP2 expression could be a potential therapeutic strategy for CRC....

  16. Vascular endothelial growth factor/bone morphogenetic protein-2 bone marrow combined modification of the mesenchymal stem cells to repair the avascular necrosis of the femoral head

    Science.gov (United States)

    Ma, Xiao-Wei; Cui, Da-Ping; Zhao, De-Wei

    2015-01-01

    Vascular endothelial cell growth factor (VEGF) combined with bone morphogenetic protein (BMP) was used to repair avascular necrosis of the femoral head, which can maintain the osteogenic phenotype of seed cells, and effectively secrete VEGF and BMP-2, and effectively promote blood vessel regeneration and contribute to formation and revascularization of tissue engineered bone tissues. To observe the therapeutic effect on the treatment of avascular necrosis of the femoral head by using bone marrow mesenchymal stem cells (BMSCs) modified by VEGF-165 and BMP-2 in vitro. The models were avascular necrosis of femoral head of rabbits on right leg. There groups were single core decompression group, core decompression + BMSCs group, core decompression + VEGF-165/BMP-2 transfect BMSCs group. Necrotic bone was cleared out under arthroscope. Arthroscopic observation demonstrated that necrotic bone was cleared out in each group, and fresh blood flowed out. Histomorphology determination showed that blood vessel number and new bone area in the repair region were significantly greater at various time points following transplantation in the core decompression + VEGF-165/BMP-2 transfect BMSCs group compared with single core decompression group and core decompression + BMSCs group (P < 0.05). These suggested that VEGF-165/BMP-2 gene transfection strengthened osteogenic effects of BMSCs, elevated number and quality of new bones and accelerated the repair of osteonecrosis of the femoral head. PMID:26629044

  17. Bmp suppression in mangrove killifish embryos causes a split in the body axis.

    Directory of Open Access Journals (Sweden)

    Sulayman Mourabit

    Full Text Available Bone morphogenetic proteins (Bmp are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL movements, and anterior-posterior (AP axis formation in embryos of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.

  18. 2-N, 6-O-sulfated chitosan-assisted BMP-2 immobilization of PCL scaffolds for enhanced osteoinduction

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Lingyan [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168 (Australia); Department of Prosthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, 639 Zhizaoju Road, Shanghai 200011 (China); Yu, Yuanman [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Wang, Jing, E-mail: biomatwj@163.com [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Werkmeister, Jerome A [CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168 (Australia); McLean, Keith M, E-mail: Keith.McLean@csiro.au [CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168 (Australia); Liu, Changsheng, E-mail: liucs@ecust.edu.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China)

    2017-05-01

    The aim of this study was to develop a 2-N, 6-O-sulfated chitosan (26SCS) modified electrospun fibrous PCL scaffold for bone morphogenetic protein-2 (BMP-2) delivery to improve osteoinduction. The PCL scaffold was modified by an aminolysis reaction using ethylenediamine (ED) and 26SCS was immobilized via electrostatic interactions (PCL-N-S). Scaffolds were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. In vitro BMP-2 adsorption and release kinetics indicated that modified PCL-N-S scaffolds showed higher levels of binding of BMP-2 (about 30–100 times), moderative burst release (about one third), and prolonged releasing time compared to the unmodified PCL scaffold. The bioactivity of released BMP-2 determined by alkaline phosphatase (ALP) activity assay was maintained and improved 8– 12 times with increasing concentration of immobilized 26SCS on the scaffolds. In vitro studies demonstrated that bone marrow mesenchymal stem cells (BMSCs) attached more readily to the PCL-N-S scaffolds with increased spreading. In conclusion, 26SCS modified PCL scaffolds can be a potent system for the sustained and bioactive delivery of BMP-2. - Graphical abstract: Limited self-regenerating capacity of human body makes the reconstruction of critical size bone defect a significant challenge. Although bone morphogenetic protein-2 (BMP-2) is an important differentiation factor inducing bone regeneration, it's short half-life in vivo and potent side effect at high dosage still show lots of concerns in the clinical use. Herein, modification of electrospun PCL scaffolds was presented through immobilizing of sulfated chitosan (26SCS). The modified scaffolds effectively improve the binding capacity of BMP-2 and exhibited an enhanced bioactivity and sustained release in vitro. Thus, the use of 26SCS modified PCL scaffolds combined with BMP-2 could be a useful scaffold for tissue

  19. Role of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Ovarian Function and Their Importance in Mammalian Female Fertility — A Review

    Directory of Open Access Journals (Sweden)

    Fernanda Cavallari de Castro

    2016-08-01

    Full Text Available Growth factors play an important role during early ovarian development and folliculogenesis, since they regulate the migration of germ cells to the gonadal ridge. They also act on follicle recruitment, proliferation/atresia of granulosa cells and theca, steroidogenesis, oocyte maturation, ovulation and luteinization. Among the growth factors, the growth differentiation factor 9 (GDF9 and the bone morphogenetic protein 15 (BMP15, belong to the transforming growth factor beta (TGF-β superfamily, have been implicated as essential for follicular development. The GDF9 and BMP15 participate in the evolution of the primordial follicle to primary follicle and play an important role in the later stages of follicular development and maturation, increasing the steroidogenic acute regulatory protein expression, plasminogen activator and luteinizing hormone receptor (LHR. These factors are also involved in the interconnections between the oocyte and surrounding cumulus cells, where they regulate absorption of amino acids, glycolysis and biosynthesis of cholesterol cumulus cells. Even though the mode of action has not been fully established, in vitro observations indicate that the factors GDF9 and BMP15 stimulate the growth of ovarian follicles and proliferation of cumulus cells through the induction of mitosis in cells and granulosa and theca expression of genes linked to follicular maturation. Thus, seeking greater understanding of the action of these growth factors on the development of oocytes, the role of GDF9 and BMP15 in ovarian function is summarized in this brief review.

  20. BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

    Directory of Open Access Journals (Sweden)

    Kersten Christian

    2005-05-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs belong to the TGF-β superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells. Results The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naïve (CD19+CD27- and memory B cells (CD19+CD27+ stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM. Conclusion In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.

  1. Effects of orthopedic implants with a polycaprolactone polymer coating containing bone morphogenetic protein-2 on osseointegration in bones of sheep.

    Science.gov (United States)

    Niehaus, Andrew J; Anderson, David E; Samii, Valerie F; Weisbrode, Steven E; Johnson, Jed K; Noon, Mike S; Tomasko, David L; Lannutti, John J

    2009-11-01

    To determine elution characteristics of bone morphogenetic protein (BMP)-2 from a polycaprolactone coating applied to orthopedic implants and determine effects of this coating on osseointegration. 6 sheep. An in vitro study was conducted to determine BMP-2 elution from polycaprolactone-coated implants. An in vivo study was conducted to determine the effects on osseointegration when the polycaprolactone with BMP-2 coating was applied to bone screws. Osseointegration was assessed via radiography, measurement of peak removal torque and bone mineral density, and histomorphometric analysis. Physiologic response was assessed by measuring serum bone-specific alkaline phosphatase activity and uptake of bone markers. Mean +/- SD elution on day 1 of the in vitro study was 263 +/- 152 pg/d, which then maintained a plateau at 59.8 +/- 29.1 pg/d. Mean peak removal torque for screws coated with polycalprolactone and BMP-2 (0.91 +/- 0.65 dN x m) and screws coated with polycaprolactone alone (0.97 +/- 1.30 dN.m) did not differ significantly from that for the control screws (2.34 +/- 1.62 dN x m). Mean bone mineral densities were 0.535 +/- 0.060 g/cm(2), 0.596 +/- 0.093 g/cm(2), and 0.524 +/- 0.142 g/cm(2) for the polycaprolactone-BMP-2-coated, polycaprolactone-coated, and control screws, respectively, and did not differ significantly among groups. Histologically, bone was in closer apposition to the implant with the control screws than with either of the coated screws. BMP-2 within the polycaprolactone coating did not stimulate osteogenesis. The polycaprolactone coating appeared to cause a barrier effect that prevented formation of new bone. A longer period or use of another carrier polymer may result in increased osseointegration.

  2. DMH1, a small molecule inhibitor of BMP type i receptors, suppresses growth and invasion of lung cancer.

    Directory of Open Access Journals (Sweden)

    Jijun Hao

    Full Text Available The bone morphogenetic protein (BMP signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.

  3. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

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    Silvia Claros

    2014-06-01

    Full Text Available Transforming growth factor-beta (TGF-β is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS for 10 days in the presence of rhTGF (recombinant human TGF-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.

  4. Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2

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    Li, Chen-Shuang; Tian, Haijun; Zou, Min; Zhao, Ke-Wei; Li, Yawei; Lao, Lifeng; Brochmann, Elsa J.; Duarte, M. Eugenia L.; Daubs, Michael D.; Zhou, Yan-Heng; Murray, Samuel S.; Wang, Jeffrey C.

    2015-01-01

    The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participated in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous. - Highlights: • Spp24 effectively inhibited the in vivo tumor growth of PANC-1. • BMP-2 dramatically promoted tumor growth by promoting PANC-1 proliferation. • Spp24 abolished the tumor growth effect of BMP-2 by promoting PANC-1 apoptosis. • Spp24 may be a candidate as a therapeutic agent of pancreatic cancer.

  5. Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2

    Energy Technology Data Exchange (ETDEWEB)

    Li, Chen-Shuang [Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing (China); Tian, Haijun, E-mail: haijuntianmd@gmail.com [Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai (China); Department of Orthopaedic Surgery, University of California, Los Angeles, Los Angeles, CA (United States); Department of Surgery, Bethune School of Medics, Shijiazhuang (China); Zou, Min [Department of Orthodontics, School and Hospital of Stomatology, Xi' an Jiaotong University, Xi' an (China); Zhao, Ke-Wei [Research Service, VA Greater Los Angeles Healthcare System, North Hills, CA (United States); Li, Yawei; Lao, Lifeng [Department of Orthopaedic Surgery, University of California, Los Angeles, Los Angeles, CA (United States); Brochmann, Elsa J. [Research Service, VA Greater Los Angeles Healthcare System, North Hills, CA (United States); Geriatric Research, Education and Clinical Center, VA Greater Los Angeles Healthcare System, North Hills, CA (United States); Department of Medicine, University of California, Los Angeles, Los Angeles, CA (United States); Duarte, M. Eugenia L. [National Institute of Traumatology and Orthopaedics, Rio de Janeiro (Brazil); Daubs, Michael D. [Division of Orthopaedic Surgery, Department of Surgery, University of Nevada School of Medicine, Las Vegas, NV (United States); Zhou, Yan-Heng, E-mail: yanhengzhou@vip.163.com [Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing (China); Murray, Samuel S. [Research Service, VA Greater Los Angeles Healthcare System, North Hills, CA (United States); Geriatric Research, Education and Clinical Center, VA Greater Los Angeles Healthcare System, North Hills, CA (United States); Department of Medicine, University of California, Los Angeles, Los Angeles, CA (United States); Wang, Jeffrey C. [Department of Orthopaedic Surgery, University of Southern California, Los Angeles, CA (United States)

    2015-10-16

    The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participated in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous. - Highlights: • Spp24 effectively inhibited the in vivo tumor growth of PANC-1. • BMP-2 dramatically promoted tumor growth by promoting PANC-1 proliferation. • Spp24 abolished the tumor growth effect of BMP-2 by promoting PANC-1 apoptosis. • Spp24 may be a candidate as a therapeutic agent of pancreatic cancer.

  6. Interactions between BMP-7 and USAG-1 (uterine sensitization-associated gene-1 regulate supernumerary organ formations.

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    Honoka Kiso

    Full Text Available Bone morphogenetic proteins (BMPs are highly conserved signaling molecules that are part of the transforming growth factor (TGF-beta superfamily, and function in the patterning and morphogenesis of many organs including development of the dentition. The functions of the BMPs are controlled by certain classes of molecules that are recognized as BMP antagonists that inhibit BMP binding to their cognate receptors. In this study we tested the hypothesis that USAG-1 (uterine sensitization-associated gene-1 suppresses deciduous incisors by inhibition of BMP-7 function. We learned that USAG-1 and BMP-7 were expressed within odontogenic epithelium as well as mesenchyme during the late bud and early cap stages of tooth development. USAG-1 is a BMP antagonist, and also modulates Wnt signaling. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Using explant culture and subsequent subrenal capsule transplantation of E15 USAG-1 mutant maxillary incisor tooth primordia supplemented with BMP-7 demonstrated in USAG-1+/- as well as USAG-1-/- rescue and supernumerary tooth development. Based upon these results, we conclude that USAG-1 functions as an antagonist of BMP-7 in this model system. These results further suggest that the phenotypes of USAG-1 and BMP-7 mutant mice reported provide opportunities for regenerative medicine and dentistry.

  7. Enhanced osteogenesis of adipose derived stem cells with Noggin suppression and delivery of BMP-2.

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    Jiabing Fan

    Full Text Available Bone morphogenetic proteins (BMPs are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH, chondroitin sulfate (CS, and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.

  8. Influence of follicular fluid GDF9 and BMP15 on embryo quality.

    Science.gov (United States)

    Gode, Funda; Gulekli, Bulent; Dogan, Erbil; Korhan, Peyda; Dogan, Seda; Bige, Ozgur; Cimrin, Dilek; Atabey, Nese

    2011-06-01

    To evaluate the association between follicular fluid levels of propeptide and mature forms of growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 with subsequent oocyte and embryo quality. Prospective clinical study. University hospital. Eighty-one infertile patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). The expression levels of the propeptide and mature forms of follicular fluid GDF9 and BMP15 were determined by western blot analysis. The levels of follicular fluid hormones (FSH, E2, and P) were measured with automated chemiluminescent enzyme immunoassays. The relationships between the levels of GDF9 and BMP15, hormones, oocyte maturation, and embryo quality. Mature GDF9 levels were significantly correlated with the nuclear maturation of oocytes. The mean mature GDF9 level was 4.87±0.60 in the high-embryo-quality group and 1.45±0.81 in the low-embryo-quality group. There were no statistically significant differences in embryo quality among the patients regarding propeptide GDF9 and BMP15 expression status. There was a negative correlation between follicular fluid levels of P and the mature form of GDF9. Higher mature GDF9 levels in the follicular fluid were significantly correlated with oocyte nuclear maturation and embryo quality. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals.

    Science.gov (United States)

    Lintern, Katherine B; Guidato, Sonia; Rowe, Alison; Saldanha, José W; Itasaki, Nobue

    2009-08-21

    Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.

  10. Distinct and overlapping gene regulatory networks in BMP- and HDAC-controlled cell fate determination in the embryonic forebrain

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    Scholl Catharina

    2012-07-01

    Full Text Available Abstract Background Both bone morphogenetic proteins (BMPs and histone deacetylases (HDACs have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling. Results Using neurospheres from the embryonic striatum as an in vitro system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level. Conclusions STAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.

  11. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats.

    Directory of Open Access Journals (Sweden)

    Michelle T Poldervaart

    Full Text Available The design of bioactive three-dimensional (3D scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2 influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.

  12. Crestal Sinus Augmentation with Recombinant Human Bone Morphogenetic Protein 2: Clinical and Radiographic Outcomes of 2-Year Pilot Trial.

    Science.gov (United States)

    Kuchler, Ulrike; Rudelstorfer, Claudia M; Barth, Barbara; Tepper, Gabor; Lidinsky, Dominika; Heimel, Patrick; Watzek, Georg; Gruber, Reinhard

    Recombinant human bone morphogenetic protein 2 (rhBMP-2) together with an absorbable collagen carrier (ACS) was approved for augmentation of the maxillary sinus prior to implant placement. The original registration trial was based on a lateral window approach. Clinical outcomes of crestal sinus augmentation with rhBMP-2 have not been reported so far. An uncontrolled pilot trial in which seven patients with a residual maxillary height below 5 mm were enrolled to receive crestal sinus augmentation with rhBMP-2/ACS was conducted. Elevation of the sinus mucosa was performed by gel pressure. Primary endpoints were the gain in augmentation height and volume measured by computed tomography after 6 months. Evaluation of bone quality at the time of implant placement was based on histology. Secondary endpoints were the clinical and radiologic evaluation of the implants and patient satisfaction by visual analog scale (VAS) at the 2-year follow-up. Median gain in augmentation height was 7.2 mm (range 0.0 to 17.5 mm). Five patients gained at least 5 mm of bone height. Two patients with a perforation of the sinus mucosa failed to respond to rhBMP-2/ACS and underwent lateral window augmentation. The median gain in augmentation volume of the five patients was 781.3 mm³ (range 426.9 to 1,242.8 mm³). Biopsy specimens showed a cancellous network consisting of primary plexiform bone with little secondary lamellar bone. After 2 years, implants were in function with no signs of inflammation or peri-implant bone loss. Patients were satisfied with the esthetic outcomes and chewing function. This pilot clinical trial supports the original concept that rhBMP-2/ACS supports bone formation, also in crestal sinus augmentation, and emphasizes the relevance of the integrity of the sinus mucosa to predict the bone gain.

  13. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

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    Alexander J Neumann

    Full Text Available Articular cartilage progenitor cells (ACPCs represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs. This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2. hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1 concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase. To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs

  14. MRI of transforaminal lumbar interbody fusion: imaging appearance with and without the use of human recombinant bone morphogenetic protein-2 (rhBMP-2)

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    Fox, Michael G.; Goldberg, Judd M.; Gaskin, Cree M.; Barr, Michelle S.; Alford, Bennett [University of Virginia, Department of Radiology and Medical Imaging, Charlottesville, VA (United States); Patrie, James T. [University of Virginia, Department of Public Health Sciences, Charlottesville, VA (United States); Shen, Francis H. [University of Virginia, Department of Orthopedic Surgery, Charlottesville, VA (United States)

    2014-09-15

    To describe the vertebral endplate and intervertebral disc space MRI appearance following TLIF, with and without the use of rhBMP-2, and to determine if the appearance is concerning for discitis/osteomyelitis. After institutional review board approval, 116 TLIF assessments performed on 75 patients with rhBMP-2 were retrospectively and independently reviewed by five radiologists and compared to 73 TLIF assessments performed on 45 patients without rhBMP-2. MRIs were evaluated for endplate signal, disc space enhancement, disc space fluid, and abnormal paraspinal soft tissue. Endplate edema-like signal was reported when T1-weighted hypointensity, T2-weighted hyperintensity, and endplate enhancement were present. Subjective concern for discitis/osteomyelitis on MRI was graded on a five-point scale. Generalized estimating equation binomial regression model analysis was performed with findings correlated with rhBMP-2 use, TLIF level, graft type, and days between TLIF and MRI. The rhBMP-2 group demonstrated endplate edema-like signal (OR 5.66; 95 % CI [1.58, 20.24], p = 0.008) and disc space enhancement (OR 2.40; 95 % CI [1.20, 4.80], p = 0.013) more often after adjusting for the TLIF level, graft type, and the number of days following TLIF. Both groups had a similar temporal distribution for endplate edema-like signal but disc space enhancement peaked earlier in the rhBMP-2 group. Disc space fluid was only present in the rhBMP-2 group. Neither group demonstrated abnormal paraspinal soft tissue and discitis/osteomyelitis was not considered likely in any patient. Endplate edema-like signal and disc space enhancement were significantly more frequent and disc space enhancement developed more rapidly following TLIF when rhBMP-2 was utilized. The concern for discitis/osteomyelitis was similar and minimal in both groups. (orig.)

  15. Alveolar ridge and maxillary sinus augmentation using rhBMP-2: a systematic review.

    Science.gov (United States)

    Freitas, Rubens Moreno de; Spin-Neto, Rubens; Marcantonio Junior, Elcio; Pereira, Luís Antônio Violin Dias; Wikesjö, Ulf M E; Susin, Cristiano

    2015-01-01

    The aim of this systematic review was to evaluate clinical and safety data for recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier when used for alveolar ridge/maxillary sinus augmentation in humans. Clinical studies/case series published 1980 through June 2012 using rhBMP-2/ACS were searched. Studies meeting the following criteria were considered eligible for inclusion: >10 subjects at baseline and maxillary sinus or alveolar ridge augmentation not concomitant with implant placement. Seven of 69 publications were eligible for review. rhBMP-2/ACS yielded clinically meaningful bone formation for maxillary sinus augmentation that would allow placement of regular dental implants without consistent differences between rhBMP-2 concentrations. Nevertheless, the statistical analysis showed that sinus augmentation following autogenous bone graft was significantly greater (mean bone height: 1.6 mm, 95% CI: 0.5-2.7 mm) than for rhBMP-2/ACS (rhBMP-2 at 1.5 mg/mL). In extraction sockets, rhBMP-2/ACS maintained alveolar ridge height while enhancing alveolar ridge width. Safety reports did not represent concerns for the proposed indications. rhBMP-2/ACS appears a promising alternative to autogenous bone grafts for alveolar ridge/maxillary sinus augmentation; dose and carrier optimization may expand its efficacy, use, and clinical application. © 2013 Wiley Periodicals, Inc.

  16. BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation.

    Science.gov (United States)

    Minina, E; Wenzel, H M; Kreschel, C; Karp, S; Gaffield, W; McMahon, A P; Vortkamp, A

    2001-11-01

    During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. To analyze the relationship between the two signaling pathways, we used an organ culture system for limb explants of mouse and chick embryos. We manipulated chondrocyte differentiation by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice did not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, could not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. We further investigated the role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes and identified three functions of BMP signaling in this process. First we found that maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel. We further identified a role for BMP signaling in modulating the expression of IHH: Finally, the application of Noggin to mouse limb explants resulted in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This

  17. Comparison of the osteogenesis and fusion rates between activin A/BMP-2 chimera (AB204) and rhBMP-2 in a beagle's posterolateral lumbar spine model.

    Science.gov (United States)

    Zheng, Guang Bin; Yoon, Byung-Hak; Lee, Jae Hyup

    2017-10-01

    Activin A/BMP-2 chimera (AB204) could promote bone healing more effectively than recombinant bone morphogenetic protein 2 (rhBMP-2) with much lower dose in a rodent model, but there is no report about the effectiveness of AB204 in a large animal model. The purpose of this study was to compare the osteogenesis and fusion rate between AB204 and rhBMP-2 using biphasic calcium phosphate (BCP) as a carrier in a beagle's posterolateral lumbar fusion model. This is a randomized control animal study. Seventeen male beagle dogs were included. Bilateral posterolateral fusion was performed at the L1-L2 and L4-L5 levels. Biphasic calcium phosphate (2 cc), rhBMP-2 (50 µg)+BCP (2 cc), or AB204 (50 µg)+BCP (2 cc) were implanted into the intertransverse space randomly. X-ray was performed at 4 and 8 weeks. After 8 weeks, the animals were sacrificed, and new bone formation and fusion rate were evaluated by manual palpation, computed tomography (CT), and undecalcified histology. The AB204 group showed significantly higher fusion rate (90%) than the rhBMP-2 group (15%) or the Osteon group (6.3%) by manual palpation. On x-ray and CT assessment, fusion rate and the volume of newly formed bone were also significantly higher in AB204 group than other groups. In contrast, more osteolysis was found in rhBMP-2 group (40%) than in AB204 group (10%) on CT study. In histologic results, new bone formation was sufficient between transverse processes in AB204 group, and obvious trabeculation and bone remodeling were observed. But in rhBMP-2 group, new bone formation was less than AB204 group and osteolysis was observed between the intertransverse spaces. A low dose of AB204 with BCP as a carrier significantly promotes the fusion rate in a large animal model when compared with the rhBMP-2. These findings demonstrate that AB204 could be an alternative to rhBMP-2 to improve fusion rate. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells.

    Science.gov (United States)

    Chang, Hsun-Ming; Cheng, Jung-Chien; Klausen, Christian; Leung, Peter C K

    2013-12-01

    In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

  19. BMP2 induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK.

    Science.gov (United States)

    Liu, Jun; Ben, Qi-Wen; Yao, Wei-Yan; Zhang, Jian-Jun; Chen, Da-Fan; He, Xiang-Yi; Li, Lei; Yuan, Yao-Zong

    2012-06-01

    The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

  20. Sequential VEGF and BMP-2 releasing PLA-PEG-PLA scaffolds for bone tissue engineering: I. Design and in vitro tests.

    Science.gov (United States)

    Eğri, Sinan; Eczacıoğlu, Numan

    2017-03-01

    Biodegradable PLA-PEG-PLA block copolymers were synthesized with desired backbone structures and molecular weights using PEG20000. Rectangular scaffolds were prepared by freeze drying with or without using NaCl particles. Bone morphogenetic protein (BMP)-2 was loaded to the matrix after the scaffold formation for sustained release while vascular endothelial growth factor (VEGF) was loaded within the pores with gelatin solution. VEGF release was quite fast and almost 60% of it was released in 2 d. However, sequential - sustained released was observed for BMP-2 in the following few months. Corporation of VEGF/BMP-2 couple into the scaffolds increased the cell adhesion and proliferation. Neither significant cytotoxicity nor apoptosis/necrosis were observed.

  1. rhBMP-2 for posterolateral instrumented lumbar fusion: a multicenter prospective randomized controlled trial.

    Science.gov (United States)

    Hurlbert, R John; Alexander, David; Bailey, Stewart; Mahood, James; Abraham, Ed; McBroom, Robert; Jodoin, Alain; Fisher, Charles

    2013-12-01

    Multicenter randomized controlled trial. To evaluate the effect of recombinant human bone morphogenetic protein (rhBMP-2) on radiographical fusion rate and clinical outcome for surgical lumbar arthrodesis compared with iliac crest autograft. In many types of spinal surgery, radiographical fusion is a primary outcome equally important to clinical improvement, ensuring long-term stability and axial support. Biologic induction of bone growth has become a commonly used adjunct in obtaining this objective. We undertook this study to objectify the efficacy of rhBMP-2 compared with traditional iliac crest autograft in instrumented posterolateral lumbar fusion. Patients undergoing 1- or 2-level instrumented posterolateral lumbar fusion were randomized to receive either autograft or rhBMP-2 for their fusion construct. Clinical and radiographical outcome measures were followed for 2 to 4 years postoperatively. One hundred ninety seven patients were successfully randomized among the 8 participating institutions. Adverse events attributable to the study drug were not significantly different compared with controls. However, the control group experienced significantly more graft-site complications as might be expected. 36-Item Short Form Health Survey, Oswestry Disability Index, and leg/back pain scores were comparable between the 2 groups. After 4 years of follow-up, radiographical fusion rates remained significantly higher in patients treated with rhBMP-2 (94%) than those who received autograft (69%) (P = 0.007). The use of rhBMP-2 for instrumented posterolateral lumbar surgery significantly improves the chances of radiographical fusion compared with the use of autograft. However, there is no associated improvement in clinical outcome within a 4-year follow-up period. These results suggest that use of rhBMP-2 should be considered in cases where lumbar arthrodesis is of primary concern.

  2. Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds.

    Science.gov (United States)

    Tabisz, Barbara; Schmitz, Werner; Schmitz, Michael; Luehmann, Tessa; Heusler, Eva; Rybak, Jens-Christoph; Meinel, Lorenz; Fiebig, Juliane E; Mueller, Thomas D; Nickel, Joachim

    2017-03-13

    The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

  3. [In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

    Science.gov (United States)

    Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

    2013-03-01

    To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

  4. [Sustained release of recombinant human bone morphogenetic protein-2 combined with stromal vascular fraction cells in promoting posterolateral spinal fusion in rat model].

    Science.gov (United States)

    Yuan, Wei; Zheng, Jun; Qian, Jinyu; Zhou, Xiaoxiao; Wang, Minghui; Wang, Xiuhui

    2017-07-01

    To observe the effect of stromal vascular fraction cells (SVFs) from rat fat tissue combined with sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2) in promoting the lumbar fusion in rat model. SVFs were harvested from subcutaneous fat of bilateral inguinal region of 4-month-old rat through the collagenase I digestion. The sustained release carrier was prepared via covalent bond of the rhBMP-2 and β-tricalcium phosphate (β-TCP) by the biominetic apatite coating process. The sustained release effect was measured by BCA method. Thirty-two rats were selected to establish the posterolateral lumbar fusion model and were divided into 4 groups, 8 rats each group. The decalcified bone matrix (DBX) scaffold+PBS, DBX scaffold+rhBMP-2/β-TCP sustained release carrier, DBX scaffold+SVFs, and DBX scaffold+rhBMP-2/β-TCP sustained release carrier+SVFs were implanted in groups A, B, C, and D respectively. X-ray films, manual spine palpation, and high-resolution micro-CT were used to evaluate spinal fusion at 8 weeks after operation; bone mineral density (BMD) and bone volume fraction were analyzed; the new bone formation was evaluated by HE staining and Masson's trichrome staining, osteocalcin (OCN) was detected by immunohistochemical staining. The cumulative release amount of rhBMP-2 was about 40% at 2 weeks, indicating sustained release effect of rhBMP-2; while the control group was almost released within 2 weeks. At 8 weeks, the combination of manual spine palpation, X-ray, and micro-CT evaluation showed that group D had the strongest bone formation (100%, 8/8), followed by group B (75%, 6/8), group C (37.5%, 3/8), and group A (12.5%, 1/8). Micro-CT analysis showed BMD and bone volume fraction were significantly higher in group D than groups A, B, and C ( P cells with bone matrix deposition, and an active osteogenic process similar to the mineralization of long bones in group D. The bone formation of group B was weaker than that of group D, and

  5. Functional cardiomyocytes derived from Isl1 cardiac progenitors via Bmp4 stimulation.

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    Esra Cagavi

    Full Text Available As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of

  6. Strontium doping promotes bioactivity of rhBMP-2 upon calcium phosphate cement via elevated recognition and expression of BMPR-IA.

    Science.gov (United States)

    Huang, Baolin; Tian, Yu; Zhang, Wenjing; Ma, Yifan; Yuan, Yuan; Liu, Changsheng

    2017-11-01

    Preserving and improving osteogenic activity of bone morphogenetic protein-2 (BMP-2) upon implants remains one of the key limitations in bone regeneration. With calcium phosphate cement (CPC) as model, we have developed a series of strontium (Sr)-doped CPC (SCPC) to address this issue. The effects of fixed Sr on the bioactivity of recombinant human BMP-2 (rhBMP-2) as well as the underlying mechanism were investigated. The results suggested that the rhBMP-2-induced osteogenic activity was significantly promoted upon SCPCs, especially with a low amount of fixed Sr (SrCO 3 content IA (BMPR-IA) to rhBMP-2 and an increased expression of BMPR-IA in C2C12 model cells. As a result, the activations of BMP-induced signaling pathways were different in C2C12 cells incubated upon CPC/rhBMP-2 and SCPCs/rhBMP-2. These findings explicitly decipher the mechanism of SCPCs promoting osteogenic bioactivity of rhBMP-2 and signify the promising application of the SCPCs/rhBMP-2 matrix in bone regeneration implants. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The multifaceted effects of agmatine on functional recovery after spinal cord injury through Modulations of BMP-2/4/7 expressions in neurons and glial cells.

    Directory of Open Access Journals (Sweden)

    Yu Mi Park

    Full Text Available Presently, few treatments for spinal cord injury (SCI are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm, a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm(2 weight for 1 min at thoracic vertebra (Th 9 segment. Mice that received an intraperitoneal (i.p. injection of Agm (100 mg/kg/day within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following

  8. The Multifaceted Effects of Agmatine on Functional Recovery after Spinal Cord Injury through Modulations of BMP-2/4/7 Expressions in Neurons and Glial Cells

    Science.gov (United States)

    Park, Yu Mi; Lee, Won Taek; Bokara, Kiran Kumar; Seo, Su Kyoung; Park, Seung Hwa; Kim, Jae Hwan; Yenari, Midori A.; Park, Kyung Ah; Lee, Jong Eun

    2013-01-01

    Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm2 weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI. PMID

  9. Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Monika; Pal, Subhashis; China, Shyamsundar Pal; Porwal, Konica [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India); Dev, Kapil [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Shrivastava, Richa [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Raju, Kanumuri Siva Rama; Rashid, Mamunur [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Trivedi, Arun Kumar; Sanyal, Sabyasachi [Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Wahajuddin, Muhammad [Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Bhaduria, Smrati [Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Maurya, Rakesh [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226031 (India); Chattopadhyay, Naibedya, E-mail: n_chattopadhyay@cdri.res.in [Division of Endocrinology and Centre for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226031 (India)

    2017-02-01

    Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuated the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda-1

  10. Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos

    International Nuclear Information System (INIS)

    He Chen; Chen Xiaozhuo

    2005-01-01

    Vascular endothelial growth factor (VEGF) is a mitogen that is critically involved in vasculogenesis, angiogenesis, and hematopoiesis. However, what and how transcription factors participate in the regulation of vegf gene expression are not fully understood. Here we report the cloning and sequencing of the zebrafish vegf promoter which revealed that the promoter contains a number of bone morphogenetic protein (BMP)-activated Smad binding elements (SBE), implicating Smad1 and Smad5 in the regulation of BMP-induced expression of vegf. Electrophoretic mobility shift assays of adding recombinant Smad proteins to the SBE-containing DNA oligonucleotides that represent portions of zebrafish vegf promoter resulted in mobility shift of the oligonucleotides. These changes demonstrate potential interactions between Smad1/5 and the vegf promoter. Reporter activity assays using the wild-type or SBE-deleted vegf promoters to drive the luciferase reporter gene expression revealed that Smad1 stimulated while Smad5 repressed the vegf promoter activity in zebrafish embryos. These data indicate that the BMP/Smad signaling pathway is involved in the regulation of zebrafish vegf transcription. In addition, we demonstrate that transgenic expression of human BMP4 in zebrafish embryos induced an expansion of the posterior intermediate cell mass (ICM, also commonly called blood island), a population of cells containing endothelial and hematopoietic precursors. In the expanded ICM, vegf and VEGF receptor 2 (flk-1) were ectopically co-expressed, suggesting that an autocrine/paracrine regulation of vegf expression may exist and contribute to the BMP-induced hemangiogenic cell proliferation

  11. Expression characteristics of BMP2, BMPR-IA and Noggin in different stages of hair follicle in yak skin.

    Science.gov (United States)

    Song, Liang-Li; Cui, Yan; Yu, Si-Jiu; Liu, Peng-Gang; Liu, Jun; Yang, Xue; He, Jun-Feng; Zhang, Qian

    2018-05-01

    Bone morphogenetic protein 2 (BMP2), BMP receptor-IA (BMPR-IA), and the BMP2 antagonist Noggin are important proteins involved in regulating the hair follicle (HF) cycle in skin. In order to explore the expression profiles of BMP2, BMPR-IA, and Noggin in the HF cycle of yak skin, we collected adult yak skin in the telogen, proanagen, and midanagen phases of HFs and evaluated gene and protein expression by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. qRT-PCR and western blotting results showed that BMP2 and BMPR-IA expression levels were highest in the telogen of HFs and higher than that of Noggin in the same phase. The expression of Noggin was significantly higher in proanagen and midanagen phases of HFs than in the telogen phase, with the highest expression observed in the proanagen phase. Moreover, the expression of Noggin in the proanagen phase was significantly higher than those of BMP2 and BMPR-IA during the same phase. Immunohistochemistry results showed that BMP2, BMPR-IA, and Noggin were expressed in the skin epidermis, sweat glands, sebaceous glands, HF outer root sheath, and hair matrix. In summary, the characteristic expression profiles of BMP2, BMPR-IA, and Noggin suggested that BMP2 and BMPR-IA had inhibitory effects on the growth of HFs in yaks, whereas Noggin promoted the growth of yak HFs, mainly by affecting skin epithelial cell activity. These results provide a basis for further studies of HF development and cycle transition in yak skin. Copyright © 2017. Published by Elsevier Inc.

  12. Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

    Science.gov (United States)

    Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

    2016-03-01

    SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. © 2016 Authors; published by Portland Press Limited.

  13. Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme.

    Science.gov (United States)

    Tucker, A S; Al Khamis, A; Sharpe, P T

    1998-08-01

    Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45-58). A target gene for Bmp4 signalling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. We also show that the mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future

  14. Requirement of Xmsx-1 in the BMP-triggered ventralization of Xenopus embryos.

    Science.gov (United States)

    Yamamoto, T S; Takagi, C; Ueno, N

    2000-03-01

    Signaling triggered by polypeptide growth factors leads to the activation of their target genes. Several homeobox genes are known to be induced in response to polypeptide growth factors in early Xenopus development. In particular, Xmsx-1, an amphibian homologue of vertebrate Msx-1, is well characterized as a target gene of bone morphogenetic protein (BMP). Here, using a dominant-negative form of Xmsx-1 (VP-Xmsx-1), which is a fusion protein made with the virus-derived VP16 activation domain, we have examined whether Xmsx-1 activity is required in the endogenous ventralizing pathway. VP-Xmsx-1 induced a secondary body axis, complete with muscle and neural tissues, when overexpressed in ventral blastomeres, suggesting that Xmsx-1 activity is necessary for both mesoderm and ectoderm to be ventralized. We have also examined the epistatic relationship between Xmsx-1 and another ventralizing homeobox protein, Xvent-1, and show that Xmsx-1 is likely to be acting upstream of Xvent-1. We propose that Xmsx-1 is required in the BMP-stimulated ventralization pathway that involves the downstream activation of Xvent-1.

  15. O uso de rhBMP-2 para aumento ósseo maxilar: relato de caso clínico

    OpenAIRE

    Freitas, Rubens; Spin Neto, Rubens; Marcantonio, Claudio [UNESP; Marcantonio, Rosemary Adriana Chierici [UNESP; Pereira, Luis Antonio Violin Dias; Marcantonio Júnior, Elcio [UNESP

    2012-01-01

    The bone resorption in the anterior maxilla, due to its aesthetic importance, can be considered one of the challenges in implant dentistry. Autogenous bone graft is the most indicated bone augmentation procedure, aiming an implant supported rehabilitation.. Alternatively, some other graft procedures can be done with homogenous and xenogenous bone graft, biomaterials and different associations. Additionally to the mentioned biomaterials, the bone morphogenetic protein (BMP), specially the rhBM...

  16. Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation

    Directory of Open Access Journals (Sweden)

    Nikolina Basic-Jukic

    2016-11-01

    Full Text Available Background/Aims: Delayed graft function (DGF is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2 is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF and DGF. Methods: 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients and DGF group (15 patients. BMP-2 expression in intima media (BMP2m and endothelium (BMP2e of epigastric artery was assessed by immunohistochemistry. Results: Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001 (Pst grade expression. Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477] and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]. Conclusions: Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF.

  17. BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

    Directory of Open Access Journals (Sweden)

    Yann Gibert

    2011-01-01

    Full Text Available Hemojuvelin (Hjv, a member of the repulsive-guidance molecule (RGM family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

  18. Differing impact of the deletion of hemochromatosis-associated molecules HFE and transferrin receptor-2 on the iron phenotype of mice lacking bone morphogenetic protein 6 or hemojuvelin.

    Science.gov (United States)

    Latour, Chloé; Besson-Fournier, Céline; Meynard, Delphine; Silvestri, Laura; Gourbeyre, Ophélie; Aguilar-Martinez, Patricia; Schmidt, Paul J; Fleming, Mark D; Roth, Marie-Paule; Coppin, Hélène

    2016-01-01

    Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and β2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice. © 2015 by the American Association for the Study of Liver Diseases.

  19. Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects.

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    Markus D Schofer

    Full Text Available Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM.The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2 into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1 left unfilled, or treated with (2 bovine spongiosa, (3 PLLA scaffolds alone or (4 PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5.PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups.Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone

  20. Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.

    Science.gov (United States)

    Tatsinkam, Arnold Junior; Mulloy, Barbara; Rider, Christopher C

    2015-08-15

    Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers. © 2015 Authors; published by Portland Press Limited.

  1. Development and Assessment of a 3D-Printed Scaffold with rhBMP-2 for an Implant Surgical Guide Stent and Bone Graft Material: A Pilot Animal Study

    OpenAIRE

    Bae, Ji Cheol; Lee, Jin-Ju; Shim, Jin-Hyung; Park, Keun-Ho; Lee, Jeong-Seok; Bae, Eun-Bin; Choi, Jae-Won; Huh, Jung-Bo

    2017-01-01

    In this study, a new concept of a 3D-printed scaffold was introduced for the accurate placement of an implant and the application of a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded bone graft. This preliminary study was conducted using two adult beagles to evaluate the 3D-printed polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP)/bone decellularized extracellular matrix (bdECM) scaffold conjugated with rhBMP-2 for the simultaneous use as an implant surgical guide stent an...

  2. Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells.

    Science.gov (United States)

    Rici, Rose Eli Grassi; Alcântara, Dayane; Fratini, Paula; Wenceslau, Cristiane Valverde; Ambrósio, Carlos Eduardo; Miglino, Maria Angelica; Maria, Durvanei Augusto

    2012-02-22

    The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. We propose that rhBMP-2 has great

  3. Biphasic effects of FGF2 on odontoblast differentiation involve changes in the BMP and Wnt signaling pathways.

    Science.gov (United States)

    Sagomonyants, Karen; Mina, Mina

    2014-08-01

    Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study, we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of the components of the BMP (Bmp2, Dlx5, Msx2, Osx) and Wnt (Wnt10a, Wisp2) pathways, and decreased expression of an inhibitor of the Wnt signaling, Nkd2. Further addition of FGF2 during the differentiation/mineralization phase of growth resulted in decreased expression of components of the BMP signaling (Bmp2, Runx2, Osx) and increased expression of inhibitors of the Wnt signaling (Nkd2, Dkk3). This suggests that both BMP and Wnt pathways may be involved in mediating the effects of FGF2 on dental pulp cells.

  4. A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

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    Courtney M Tate

    Full Text Available Bone morphogenetic proteins (BMPs, members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

  5. A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

    Science.gov (United States)

    Tate, Courtney M; Mc Entire, Jacquelyn; Pallini, Roberto; Vakana, Eliza; Wyss, Lisa; Blosser, Wayne; Ricci-Vitiani, Lucia; D'Alessandris, Quintino Giorgio; Morgante, Liliana; Giannetti, Stefano; Larocca, Luigi Maria; Todaro, Matilde; Benfante, Antonina; Colorito, Maria Luisa; Stassi, Giorgio; De Maria, Ruggero; Rowlinson, Scott; Stancato, Louis

    2015-01-01

    Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

  6. Recombinant human bone morphogenetic protein 2 outcomes for maxillary sinus floor augmentation: a systematic review and meta-analysis.

    Science.gov (United States)

    Lin, Guo-Hao; Lim, Glendale; Chan, Hsun-Liang; Giannobile, William V; Wang, Hom-Lay

    2016-11-01

    To study the effect of the recombinant human bone morphogenetic protein 2 (rhBMP-2) on sinus volumetric and histometric changes after sinus floor augmentation compared to a conventional approach of non-biologic bone grafting materials. An electronic search of 4 databases (January 1990-February 2015), including PubMed/MEDLINE, EMBASE, Web of Science and Cochrane Central, and a hand search of peer-reviewed journals for relevant articles were performed. Human clinical trials with data on comparison of sinus volumetric and/or histometric outcomes with and without the use of rhBMP-2 in sinus grafting procedures, with ≥10 augmentation sites in each study group, and with a follow-up period of at least 6 months, were included. Random-effects meta-analyses were performed to analyze weighted mean difference (WMD) and confidence interval (CI) for the recorded variables according to PRISMA guidelines. Six randomized controlled trials (RCTs) were included. The results of the meta-analyses showed that the WMD of vertical bone height gain was -0.14 mm (95% CI = -1.91 to 1.62 mm, P = 0.87), the WMD of bone density was -142.42 mg/cm 3 (95% CI = -310.62-25.78 mg/cm 3 , P = 0.10), the WMD of the percentage of vital bone was -4.59% (95% CI = -11.73-2.56%, P = 0.21), and the WMD of the percentage of residual bone grafting materials was -9.90% (95% CI = -26.38-6.58%, P = 0.21). The comparison of implant survival rate presented an overall risk ratio of 1.00 (95% CI = 0.94-1.07). The two approaches (conventional bone grafting compared to BMPs) demonstrated comparable effectiveness for both clinical and histomorphometric measures. This systematic review revealed that the use of rhBMP-2 in maxillary sinus floor augmentation achieved similar clinical and histometric outcomes when compared to conventional sinus grafting procedures after a healing period of 6-9 months. However, previous studies showed the morbidity and other patient-reported outcomes were improved in

  7. Quinoline compound KM11073 enhances BMP-2-dependent osteogenic differentiation of C2C12 cells via activation of p38 signaling and exhibits in vivo bone forming activity.

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    Seung-hwa Baek

    Full Text Available Recombinant human bone morphogenetic protein (rhBMP-2 has been approved by the FDA for clinical application, but its use is limited due to high cost and a supra-physiological dose for therapeutic efficacy. Therefore, recent studies have focused on the generation of new therapeutic small molecules to induce bone formation or potentiate the osteogenic activity of BMP-2. Here, we show that [4-(7-chloroquinolin-4-yl piperazino][1-phenyl-5-(trifluoromethyl-1H-pyrazol-4-yl]methanone (KM11073 strongly enhances the BMP-2-stimulated induction of alkaline phosphatase (ALP, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. The KM11073-mediated ALP induction was inhibited by the BMP antagonist noggin, suggesting that its osteogenic activity occurs via BMP signaling. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of KM11073 accompanied by enhanced expression of BMP-2, -6, and -7 mRNA. Furthermore, the in vivo osteogenic activity of KM11073 was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its single use for bone formation. In conclusion, the combination of rhBMP-2 with osteogenic small molecules could reduce the use of expensive rhBMP-2, mitigating the undesirable side effects of its supra-physiological dose for therapeutic efficacy. Moreover, due to their inherent physical properties, small molecules could represent the next generation of regenerative medicine.

  8. Exogenous BMP7 in aortae of rats with chronic uremia ameliorates expression of profibrotic genes, but does not reverse established vascular calcification

    DEFF Research Database (Denmark)

    Gravesen, Eva; Lerche Mace, Maria; Nordholm, Anders

    2018-01-01

    Hyperphosphatemia and vascular calcification are frequent complications of chronic renal failure and bone morphogenetic protein 7 (BMP7) has been shown to protect against development of vascular calcification in uremia. The present investigation examined the potential reversibility of established...... uremic vascular calcification by treatment of uremic rats with BMP7. A control model of isogenic transplantation of a calcified aorta from uremic rats into healthy littermates examined whether normalization of the uremic environment reversed vascular calcification. Uremia and vascular calcification were...... induced in rats by 5/6 nephrectomy, high phosphate diet and alfacalcidol treatment. After 14 weeks severe vascular calcification was present and rats were allocated to BMP7, vehicle or aorta transplantation. BMP7 treatment caused a significant decrease of plasma phosphate to 1.56 ± 0.17 mmol/L vs 2.06 ± 0...

  9. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

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    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  10. Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling.

    Science.gov (United States)

    Graef, Jennifer L; Rendina-Ruedy, Elizabeth; Crockett, Erica K; Ouyang, Ping; King, Jarrod B; Cichewicz, Robert H; Lucas, Edralin A; Smith, Brenda J

    2018-05-01

    Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome.

    Science.gov (United States)

    Tanaka, Mari; Asada, Misako; Higashi, Atsuko Y; Nakamura, Jin; Oguchi, Akiko; Tomita, Mayumi; Yamada, Sachiko; Asada, Nariaki; Takase, Masayuki; Okuda, Tomohiko; Kawachi, Hiroshi; Economides, Aris N; Robertson, Elizabeth; Takahashi, Satoru; Sakurai, Takeshi; Goldschmeding, Roel; Muso, Eri; Fukatsu, Atsushi; Kita, Toru; Yanagita, Motoko

    2010-03-01

    The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization-associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3-/- mice, which model Alport syndrome. Ablation of Usag1 in Col4a3-/- mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3-/- mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.

  12. Enhancement of Tendon–Bone Healing for Anterior Cruciate Ligament (ACL Reconstruction Using Bone Marrow-Derived Mesenchymal Stem Cells Infected with BMP-2

    Directory of Open Access Journals (Sweden)

    Shiyi Chen

    2012-10-01

    Full Text Available At present, due to the growing attention focused on the issue of tendon–bone healing, we carried out an animal study of the use of genetic intervention combined with cell transplantation for the promotion of this process. Here, the efficacy of bone marrow stromal cells infected with bone morphogenetic protein-2 (BMP-2 on tendon–bone healing was determined. A eukaryotic expression vector containing the BMP-2 gene was constructed and bone marrow-derived mesenchymal stem cells (bMSCs were infected with a lentivirus. Next, we examined the viability of the infected cells and the mRNA and protein levels of BMP-2-infected bMSCs. Gastrocnemius tendons, gastrocnemius tendons wrapped by bMSCs infected with the control virus (bMSCs+Lv-Control, and gastrocnemius tendons wrapped by bMSCs infected with the recombinant BMP-2 virus (bMSCs+Lv-BMP-2 were used to reconstruct the anterior cruciate ligament (ACL in New Zealand white rabbits. Specimens from each group were harvested four and eight weeks postoperatively and evaluated using biomechanical and histological methods. The bMSCs were infected with the lentivirus at an efficiency close to 100%. The BMP-2 mRNA and protein levels in bMSCs were significantly increased after lentiviral infection. The bMSCs and BMP-2-infected bMSCs on the gastrocnemius tendon improved the biomechanical properties of the graft in the bone tunnel; specifically, bMSCs infected with BMP-2 had a positive effect on tendon–bone healing. In the four-week and eight-week groups, bMSCs+Lv-BMP-2 group exhibited significantly higher maximum loads of 29.3 ± 7.4 N and 45.5 ± 11.9 N, respectively, compared with the control group (19.9 ± 6.4 N and 21.9 ± 4.9 N (P = 0.041 and P = 0.001, respectively. In the eight-week groups, the stiffness of the bMSCs+Lv-BMP-2 group (32.5 ± 7.3 was significantly higher than that of the bMSCs+Lv-Control group (22.8 ± 7.4 or control groups (12.4 ± 6.0 (p = 0.036 and 0.001, respectively. Based on the

  13. Bone morphogenetic protein-2 functions as a negative regulator in the differentiation of myoblasts, but not as an inducer for the formations of cartilage and bone in mouse embryonic tongue

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    Suzuki Erika

    2011-07-01

    Full Text Available Abstract Background In vitro studies using the myogenic cell line C2C12 demonstrate that bone morphogenetic protein-2 (BMP-2 converts the developmental pathway of C2C12 from a myogenic cell lineage to an osteoblastic cell lineage. Further, in vivo studies using null mutation mice demonstrate that BMPs inhibit the specification of the developmental fate of myogenic progenitor cells. However, the roles of BMPs in the phases of differentiation and maturation in skeletal muscles have yet to be determined. The present study attempts to define the function of BMP-2 in the final stage of differentiation of mouse tongue myoblast. Results Recombinant BMP-2 inhibited the expressions of markers for the differentiation of skeletal muscle cells, such as myogenin, muscle creatine kinase (MCK, and fast myosin heavy chain (fMyHC, whereas BMP-2 siRNA stimulated such markers. Neither the recombinant BMP-2 nor BMP-2 siRNA altered the expressions of markers for the formation of cartilage and bone, such as osteocalcin, alkaline phosphatase (ALP, collagen II, and collagen X. Further, no formation of cartilage and bone was observed in the recombinant BMP-2-treated tongues based on Alizarin red and Alcian blue stainings. Neither recombinant BMP-2 nor BMP-2 siRNA affected the expression of inhibitor of DNA binding/differentiation 1 (Id1. The ratios of chondrogenic and osteogenic markers relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene were approximately 1000-fold lower than those of myogenic markers in the cultured tongue. Conclusions BMP-2 functions as a negative regulator for the final differentiation of tongue myoblasts, but not as an inducer for the formation of cartilage and bone in cultured tongue, probably because the genes related to myogenesis are in an activation mode, while the genes related to chondrogenesis and osteogenesis are in a silencing mode.

  14. Plasma Treated High-Density Polyethylene (HDPE Medpor Implant Immobilized with rhBMP-2 for Improving the Bone Regeneration

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    Jin-Su Lim

    2014-01-01

    Full Text Available We investigate the bone generation capacity of recombinant human bone morphogenetic protein-2 (rhBMP-2 immobilized Medpor surface through acrylic acid plasma-polymerization. Plasma-polymerization was carried out at a 20 W at an acrylic acid flow rate of 7 sccm for 5 min. The plasma-polymerized Medpor surface showed hydrophilic properties and possessed a high density of carboxyl groups. The rhBMP-2 was immobilized with covalently attached carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide. Carboxyl groups and rhBMP-2 immobilization on the Medpor surface were identified by Fourier transform infrared spectroscopy. The activity of Medpor with rhBMP-2 immobilized was examined using an alkaline phosphatase assay on MC3T3-E1 cultured Medpor. These results showed that the rhBMP-2 immobilized Medpor increased the level of MC3T3-E1 cell differentiation. These results demonstrated that plasma surface modification has the potential to immobilize rhBMP-2 on polymer implant such as Medpor and can be used for the binding of bioactive nanomolecules in bone tissue engineering.

  15. Bone marrow concentrate promotes bone regeneration with a suboptimal-dose of rhBMP-2.

    Science.gov (United States)

    Egashira, Kazuhiro; Sumita, Yoshinori; Zhong, Weijian; I, Takashi; Ohba, Seigo; Nagai, Kazuhiro; Asahina, Izumi

    2018-01-01

    Bone marrow concentrate (BMC), which is enriched in mononuclear cells (MNCs) and platelets, has recently attracted the attention of clinicians as a new optional means for bone engineering. We previously reported that the osteoinductive effect of bone morphogenetic protein-2 (BMP-2) could be enhanced synergistically by co-transplantation of peripheral blood (PB)-derived platelet-rich plasma (PRP). This study aims to investigate whether BMC can effectively promote bone formation induced by low-dose BMP-2, thereby reducing the undesirable side-effects of BMP-2, compared to PRP. Human BMC was obtained from bone marrow aspirates using an automated blood separator. The BMC was then seeded onto β-TCP granules pre-adsorbed with a suboptimal-dose (minimum concentration to induce bone formation at 2 weeks in mice) of recombinant human (rh) BMP-2. These specimens were transplanted subcutaneously to the dorsal skin of immunodeficient-mice and the induction of ectopic bone formation was assessed 2 and 4 weeks post-transplantation. Transplantations of five other groups [PB, PRP, platelet-poor plasma (PPP), bone marrow aspirate (BM), and BM-PPP] were employed as experimental controls. Then, to clarify the effects on vertical bone augmentation, specimens from the six groups were transplanted for on-lay placement on the craniums of mice. The results indicated that BMC, which contained an approximately 2.5-fold increase in the number of MNCs compared to PRP, could accelerate ectopic bone formation until 2 weeks post-transplantation. On the cranium, the BMC group promoted bone augmentation with a suboptimal-dose of rhBMP-2 compared to other groups. Particularly in the BMC specimens harvested at 4 weeks, we observed newly formed bone surrounding the TCP granules at sites far from the calvarial bone. In conclusion, the addition of BMC could reduce the amount of rhBMP-2 by one-half via its synergistic effect on early-phase osteoinduction. We propose here that BMC transplantation

  16. Sizzled controls dorso-ventral polarity by repressing cleavage of the Chordin protein.

    Science.gov (United States)

    Muraoka, Osamu; Shimizu, Takashi; Yabe, Taijiro; Nojima, Hideaki; Bae, Young-Ki; Hashimoto, Hisashi; Hibi, Masahiko

    2006-04-01

    The Bone morphogenetic protein (Bmp) signalling gradient has a major function in the formation of the dorso-ventral axis. The zebrafish ventralized mutant, ogon, encodes Secreted Frizzled (Sizzled). sizzled is ventrally expressed in a Bmp-dependent manner and is required for the suppression of Bmp signalling on the ventral side of zebrafish embryos. However, it remains unclear how Sizzled inhibits Bmp signalling and controls ventro-lateral cell fate. We found that Sizzled stabilizes Chordin, a Bmp antagonist, by binding and inhibiting the Tolloid-family metalloproteinase, Bmp1a, which cleaves and inactivates Chordin. The cysteine-rich domain of Sizzled is required for inhibition of Bmp1a activity. Loss of both Bmp1a and Tolloid-like1 (Tll1; another Tolloid-family metalloproteinase) function leads to a complete suppression and reversal of the ogon mutant phenotype. These results indicate that Sizzled represses the activities of Tolloid-family proteins, thereby creating the Chordin-Bmp activity gradient along the dorso-ventral axis. Here, we describe a previously unrecognized role for a secreted Frizzled-related protein.

  17. Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

    Science.gov (United States)

    Basic-Jukic, Nikolina; Gulin, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Katalinic, Lea; Coric, Marijana; Veda, Marija Varnai; Ivkovic, Vanja; Kes, Petar; Jelakovic, Bojan

    2016-01-01

    Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF. © 2016 The Author(s) Published by S. Karger AG, Basel.

  18. BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression.

    Science.gov (United States)

    Ren, Wei; Sun, Xiaoxiao; Wang, Ke; Feng, Honglei; Liu, Yuehong; Fei, Chang; Wan, Shaoheng; Wang, Wei; Luo, Jinyong; Shi, Qiong; Tang, Min; Zuo, Guowei; Weng, Yaguang; He, Tongchuan; Zhang, Yan

    2014-03-01

    Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.

  19. Enhanced healing of rabbit segmental radius defects with surface-coated calcium phosphate cement/bone morphogenetic protein-2 scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yi; Hou, Juan; Yin, ManLi [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Wang, Jing, E-mail: biomatwj@163.com [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Liu, ChangSheng, E-mail: csliu@sh163.net [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China)

    2014-11-01

    Large osseous defects remain a difficult clinical problem in orthopedic surgery owing to the limited effective therapeutic options, and bone morphogenetic protein-2 (BMP-2) is useful for its potent osteoinductive properties in bone regeneration. Here we build a strategy to achieve prolonged duration time and help inducting new bone formation by using water-soluble polymers as a protective film. In this study, calcium phosphate cement (CPC) scaffolds were prepared as the matrix and combined with sodium carboxymethyl cellulose (CMC-Na), hydroxypropylmethyl cellulose (HPMC), and polyvinyl alcohol (PVA) respectively to protect from the digestion of rhBMP-2. After being implanted in the mouse thigh muscles, the surface-modified composite scaffolds evidently induced ectopic bone formation. In addition, we further evaluated the in vivo effects of surface-modified scaffolds in a rabbit radius critical defect by radiography, three dimensional micro-computed tomographic (μCT) imaging, synchrotron radiation-based micro-computed tomographic (SRμCT) imaging, histological analysis, and biomechanical measurement. The HPMC-modified CPC scaffold was regarded as the best combination for segmental bone regeneration in rabbit radius. - Highlights: • A simple surface-coating method was used to fabricate composite scaffolds. • Growth factor was protected from rapid depletion via superficial coating. • Significant promotion of bone regeneration was achieved. • HPMC-modification displayed optimal effect of bone regeneration.

  20. [Association of the genetic variations of bone morphogenetic protein 7 gene with diabetes and insulin resistance in Xinjiang Uygur population].

    Science.gov (United States)

    Yan, Zhi-tao; Li, Nan-fang; Guo, Yan-ying; Yao, Xiao-guang; Wang, Hong-mei; Hu, Jun-li

    2011-06-01

    To investigate the association between the genetic variations of the functional region in bone morphogenetic protein gene (BMP7) with type 2 diabetes mellitus in Chinese Uygur individuals. A case-control study was conducted based on epidemiological investigation. A total of 717 Uygur subjects (276 males and 441 females) were selected and divided into two groups: diabetes mellitus group (n = 502, 191 males and 311 females) and control group (n = 215, 85 males and 130 females). All exons, flanking introns and the promoter regions of (BMP7) gene were sequenced in 48 Uygur diabetics. Representative variations were selected according to the minor allele frequency (MAF) and linkage disequilibrium and genotyped using the TaqMan polymerase chain reaction method in 717 Uygur individuals, a relatively isolated general population in a relatively homogeneous environment and a case-control study was conducted to test the association between the genetic variations of (BMP7) gene and type 2 diabetes mellitus. Five novel and 8 known variations in the (BMP7) gene were identified. All genotype distributions were tested for deviations from Hardy-Weinberg equilibrium (P> 0.05). There was significant difference of genotype distribution of rs6025422 between type 2 diabetes mellitus and control groups in the male population (P 0.05), but there was no difference in total and female population (P> 0.05). And the means of fasting blood glucose (FBG), fasting insulin and HOMA-index significantly decreased in individuals with AA, AG and GG genotypes of rs6025422 in male population (Ppopulation (P> 0.05). The logistic regression analysis showed that GG genotype of rs6025422 variation might be a protective factor for diabetes in male (OR= 0.637, 95% confidence interval 0.439-0.923, P< 0.05). The present study suggests that the rs6025422 polymorphism in (BMP7) gene may be associated with diabetes mellitus and insulin resistance in Uygur men.

  1. BMP6 down-regulates GDNF expression through SMAD1/5 and ERK1/2 signaling pathways in human granulosa-lutein cells.

    Science.gov (United States)

    Zhang, Xin-Yue; Chang, Hsun-Ming; Taylor, Elizabeth L; Leung, Peter C K; Liu, Rui-Zhi

    2018-05-09

    Bone morphogenetic protein 6 (BMP6) is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line-derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein cells as in vitro cell models. Our results showed that BMP6 significantly down-regulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells. Using dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF. In addition, BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced down-regulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.

  2. Drosophila Nociceptive Sensitization Requires BMP Signaling via the Canonical SMAD Pathway.

    Science.gov (United States)

    Follansbee, Taylor L; Gjelsvik, Kayla J; Brann, Courtney L; McParland, Aidan L; Longhurst, Colin A; Galko, Michael J; Ganter, Geoffrey K

    2017-08-30

    Nociceptive sensitization is a common feature in chronic pain, but its basic cellular mechanisms are only partially understood. The present study used the Drosophila melanogaster model system and a candidate gene approach to identify novel components required for modulation of an injury-induced nociceptive sensitization pathway presumably downstream of Hedgehog. This study demonstrates that RNAi silencing of a member of the Bone Morphogenetic Protein (BMP) signaling pathway, Decapentaplegic (Dpp), specifically in the Class IV multidendritic nociceptive neuron, significantly attenuated ultraviolet injury-induced sensitization. Furthermore, overexpression of Dpp in Class IV neurons was sufficient to induce thermal hypersensitivity in the absence of injury. The requirement of various BMP receptors and members of the SMAD signal transduction pathway in nociceptive sensitization was also demonstrated. The effects of BMP signaling were shown to be largely specific to the sensitization pathway and not associated with changes in nociception in the absence of injury or with changes in dendritic morphology. Thus, the results demonstrate that Dpp and its pathway play a crucial and novel role in nociceptive sensitization. Because the BMP family is so strongly conserved between vertebrates and invertebrates, it seems likely that the components analyzed in this study represent potential therapeutic targets for the treatment of chronic pain in humans. SIGNIFICANCE STATEMENT This report provides a genetic analysis of primary nociceptive neuron mechanisms that promote sensitization in response to injury. Drosophila melanogaster larvae whose primary nociceptive neurons were reduced in levels of specific components of the BMP signaling pathway, were injured and then tested for nocifensive responses to a normally subnoxious stimulus. Results suggest that nociceptive neurons use the BMP2/4 ligand, along with identified receptors and intracellular transducers to transition to a

  3. BMP9 ameliorates amyloidosis and the cholinergic defect in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Burke, Rebecca M; Norman, Timothy A; Haydar, Tarik F; Slack, Barbara E; Leeman, Susan E; Blusztajn, Jan Krzysztof; Mellott, Tiffany J

    2013-11-26

    Bone morphogenetic protein 9 (BMP9) promotes the acquisition of the cholinergic phenotype in basal forebrain cholinergic neurons (BFCN) during development and protects these neurons from cholinergic dedifferentiation following axotomy when administered in vivo. A decline in BFCN function occurs in patients with Alzheimer's disease (AD) and contributes to the AD-associated memory deficits. We infused BMP9 intracerebroventricularly for 7 d in transgenic AD model mice expressing green fluorescent protein specifically in cholinergic neurons (APP.PS1/CHGFP) and in wild-type littermate controls (WT/CHGFP). We used 5-mo-old mice, an age when the AD transgenics display early amyloid deposition and few cholinergic defects, and 10-mo-old mice, by which time these mice exhibit established disease. BMP9 infusion reduced the number of Aβ42-positive amyloid plaques in the hippocampus and cerebral cortex of 5- and 10-mo-old APP.PS1/CHGFP mice and reversed the reductions in choline acetyltransferase protein levels in the hippocampus of 10-mo-old APP.PS1/CHGFP mice. The treatment increased cholinergic fiber density in the hippocampus of both WT/CHGFP and APP.PS1/CHGFP mice at both ages. BMP9 infusion also increased hippocampal levels of neurotrophin 3, insulin-like growth factor 1, and nerve growth factor and of the nerve growth factor receptors, tyrosine kinase receptor A and p75/NGFR, irrespective of the genotype of the mice. These data show that BMP9 administration is effective in reducing the Aβ42 amyloid plaque burden, reversing cholinergic neuron abnormalities, and generating a neurotrophic milieu for BFCN in a mouse model of AD and provide evidence that the BMP9-signaling pathway may constitute a therapeutic target for AD.

  4. Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice

    Directory of Open Access Journals (Sweden)

    Yoko Hashimoto

    2014-01-01

    Full Text Available Canola oil (Can and hydrogenated soybean oil (H2-Soy are commonly used edible oils. However, in contrast to soybean oil (Soy, they shorten the survival of stroke-prone spontaneously hypertensive (SHRSP rats. It has been proposed that the adverse effects of these oils on the kidney and testis are caused at least in part by dihydro-vitamin K (VK 1 in H2-Soy and unidentified component(s in Can. Increased intake of dihydro-VK1 is associated with decreased tissue VK2 levels and bone mineral density in rats and humans, respectively. The aim of the present study was to determine the effects of these oils on bone morphogenetic protein (BMP-induced ectopic bone formation, which is promoted by VK2 deficiency, in relation to the role of VK in the γ-carboxylation of osteocalcin and matrix Gla protein. A crude extract of BMPs was implanted into a gap in the fascia of the femoral muscle in 5-week-old mice maintained on a Soy, Can, or H2-Soy diet. Newly formed bone volume, assessed by three-dimensional X-ray micro-computed tomography and three-dimensional reconstruction imaging for bone, was 4-fold greater in the Can and H2-Soy groups than in the Soy group. The plasma carboxylated osteocalcin (Gla-OC and total OC (Gla-OC plus undercarboxylated osteocalcin [Glu-OC] levels were significantly lower in the Can group than in the Soy group (p < 0.05. However, these levels did not significantly differ between the H2-Soy and Soy groups. The plasma Gla-OC/Glu-OC ratio in the Can and H2-Soy groups was significantly lower (in Can; p = 0.044 or was almost significantly lower (in H2-Soy; p = 0.053 than that in the Soy group. In conclusion, Can and H2-Soy accelerated BMP-induced bone formation in mice to a greater extent than Soy. Further research is required to evaluate whether the difference in accelerated ectopic bone formation is associated with altered levels of VK2 and VK-dependent protein(s among the three dietary groups.

  5. A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

    Science.gov (United States)

    Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

    2012-01-01

    In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

  6. Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Zhiwei; Huangfu, Changxin; Wang, Yanying; Ge, Hongwei; Yao, Yao; Zou, Ping; Wang, Guangtu [College of Science, Sichuan Agricultural University, Ya' an 625014 (China); He, Hua [Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Wenjiang, Sichuan 611130 (China); Rao, Hanbing, E-mail: rhbscu@gmail.com [College of Science, Sichuan Agricultural University, Ya' an 625014 (China)

    2015-07-01

    The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer–Emmett–Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g{sup −1}, respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG < 0) and entropy increasing (ΔS > 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. - Highlights: • A novel protein adsorption studies based on sheet, rod and whisker of HA were designed. • Kinetic and thermodynamics parameters of BMP-2 and HA bonded materials were evaluated. • Surface topographies of the HA effect BMP-2 adsorption • The HA-whisker material had excellent adsorption performance for protein enrichment. • The electrostatic interaction is responsible for the

  7. Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features

    International Nuclear Information System (INIS)

    Lu, Zhiwei; Huangfu, Changxin; Wang, Yanying; Ge, Hongwei; Yao, Yao; Zou, Ping; Wang, Guangtu; He, Hua; Rao, Hanbing

    2015-01-01

    The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer–Emmett–Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g −1 , respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG < 0) and entropy increasing (ΔS > 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. - Highlights: • A novel protein adsorption studies based on sheet, rod and whisker of HA were designed. • Kinetic and thermodynamics parameters of BMP-2 and HA bonded materials were evaluated. • Surface topographies of the HA effect BMP-2 adsorption • The HA-whisker material had excellent adsorption performance for protein enrichment. • The electrostatic interaction is responsible for the BMP-2

  8. Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilize chondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.

    Science.gov (United States)

    Legendre, Florence; Ollitrault, David; Hervieu, Magalie; Baugé, Catherine; Maneix, Laure; Goux, Didier; Chajra, Hanane; Mallein-Gerin, Frédéric; Boumediene, Karim; Galera, Philippe; Demoor, Magali

    2013-07-01

    Cartilage healing by tissue engineering is an alternative strategy to reconstitute functional tissue after trauma or age-related degeneration. However, chondrocytes, the major player in cartilage homeostasis, do not self-regenerate efficiently and lose their phenotype during osteoarthritis. This process is called dedifferentiation and also occurs during the first expansion step of autologous chondrocyte implantation (ACI). To ensure successful ACI therapy, chondrocytes must be differentiated and capable of synthesizing hyaline cartilage matrix molecules. We therefore developed a safe procedure for redifferentiating human chondrocytes by combining appropriate physicochemical factors: hypoxic conditions, collagen scaffolds, chondrogenic factors (bone morphogenetic protein-2 [BMP-2], and insulin-like growth factor I [IGF-I]) and RNA interference targeting the COL1A1 gene. Redifferentiation of dedifferentiated chondrocytes was evaluated using gene/protein analyses to identify the chondrocyte phenotypic profile. In our conditions, under BMP-2 treatment, redifferentiated and metabolically active chondrocytes synthesized a hyaline-like cartilage matrix characterized by type IIB collagen and aggrecan molecules without any sign of hypertrophy or osteogenesis. In contrast, IGF-I increased both specific and noncharacteristic markers (collagens I and X) of chondrocytes. The specific increase in COL2A1 gene expression observed in the BMP-2 treatment was shown to involve the specific enhancer region of COL2A1 that binds the trans-activators Sox9/L-Sox5/Sox6 and Sp1, which are associated with a decrease in the trans-inhibitors of COL2A1, c-Krox, and p65 subunit of NF-kappaB. Our procedure in which BMP-2 treatment under hypoxia is associated with a COL1A1 siRNA, significantly increased the differentiation index of chondrocytes, and should offer the opportunity to develop new ACI-based therapies in humans.

  9. A Comparative Analysis of Recombinant Human Bone Morphogenetic Protein-2 with a Demineralized Bone Matrix versus Iliac Crest Bone Graft for Secondary Alveolar Bone Grafts in Patients with Cleft Lip and Palate: Review of 501 Cases.

    Science.gov (United States)

    Hammoudeh, Jeffrey A; Fahradyan, Artur; Gould, Daniel J; Liang, Fan; Imahiyerobo, Thomas; Urbinelli, Leo; Nguyen, JoAnna T; Magee, William; Yen, Stephen; Urata, Mark M

    2017-08-01

    Alveolar cleft reconstruction using iliac crest bone graft is considered standard of care for children with complete cleft lip and palate at the time of mixed dentition. Harvesting bone may result in donor-site morbidity and additional operating time and length of hospitalization. Recombinant human bone morphogenetic protein (rhBMP)-2 with a demineralized bone matrix is an alternative bone source for alveolar cleft reconstruction. The authors investigated the outcomes of rhBMP-2/demineralized bone matrix versus iliac crest bone graft for alveolar cleft reconstruction by reviewing postoperative surgical complications and cleft closure. A retrospective chart review was conducted for 258 rhBMP-2/demineralized bone matrix procedures (mean follow-up, 2.9 years) and 243 iliac crest bone graft procedures (mean follow-up, 4.1 years) on 414 patients over a 12-year period. The authors compared complications, canine eruption, and alveolar cleft closure between the two groups. In the rhBMP-2/demineralized bone matrix group, one patient required prolonged intubation because of intraoperative airway swelling not thought to be caused by rhBMP-2, 36 reported facial swelling and one required outpatient steroids as treatment, and 12 had dehiscence; however, half of these complications resolved without intervention. Twenty-three of the 228 rhBMP-2/demineralized bone matrix patients and 28 of the 242 iliac crest bone graft patients required repeated surgery for alveolar cleft repair. Findings for canine tooth eruption into the cleft site through the graft were similar between the groups. The rhBMP-2/demineralized bone matrix appears to be an acceptable alternative for alveolar cleft repair. The authors found no increase in serious adverse events with the use of this material. Local complications, such as swelling and minor wound dehiscence, predominantly improved without intervention. Therapeutic, III.

  10. The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

    Science.gov (United States)

    Nakagata, Naomi; Miyagawa, Shinichi; Suzuki, Kentaro; Kitazawa, Sohei; Yamada, Gen

    2012-01-01

    Background Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. Methodology/Principal Findings To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. Conclusions/Significance This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh

  11. Nanopolymers Delivery of the Bone Morphogenetic Protein-4 Plasmid to Mesenchymal Stem Cells Promotes Articular Cartilage Repair In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Junjun Shi

    2012-01-01

    Full Text Available The clinical application of viral vectors for gene therapy is limited for biosafety consideration. In this study, to promote articular cartilage repair, poly (lactic-co glycolic acid (PLGA nanopolymers were used as non-viral vectors to transfect rabbit mesenchymal stem cells (MSCs with the pDC316-BMP4-EGFP plasmid. The cytotoxicity and transfection efficiency in vitro were acceptable measuring by CCK-8 and flow cytometry. After transfection, Chondrogenic markers (mRNA of Col2a1, Sox9, Bmp4, and Agg of experimental cells (MSCs being transfected with BMP-4 plasmid by PLGA nanopolymers were increased more than those of control cells (MSCs being transfected with naked BMP-4 plasmid alone. In vivo study, twelve rabbits (24 knees with large full thickness articular cartilage defects were randomly divided into the experimental group (MSCs being transfected with BMP-4 plasmid by PLGA nanopolymers and the control group (MSCs being transfected with naked BMP-4 plasmid. The experimental group showed better regeneration than the control group 6 and 12 weeks postoperatively. Hyaline-like cartilage formed at week 12 in the experimental group, indicating the local delivery of BMP-4 plasmid to MSCs by PLGA nanopolymers improved articular cartilage repair significantly. PLGA nanopolymers could be a promising and effective non-viral vector for gene therapy in cartilage repair.

  12. Repair of rat cranial bone defect by using bone morphogenetic protein-2-related peptide combined with microspheres composed of polylactic acid/polyglycolic acid copolymer and chitosan

    International Nuclear Information System (INIS)

    Li, Jingfeng; Jin, Lin; Zhu, Shaobo; Wang, Mingbo; Xu, Shuyun

    2015-01-01

    The effects of the transplanted bone morphogenetic protein-2 (BMP2) -related peptide P24 and rhBMP 2 combined with poly(lactic-co-glycolic acid) (PLGA)/chitosan (CS) microspheres were investigated in promoting the repair of rat cranial bone defect. Forty white rats were selected and equally divided into four groups (group A: 1 μg of rhBMP 2 /PLGA/CS composite; group B: 3 mg of P24/PLGA/CS composite; group C: 0.5 μg of rhBMP 2 + 1.5 mg of P24/PLGA/CS composite; group D: blank PLGA/CS material), and rat cranial bone defect models with a diameter of 5 mm were established. The materials were transplanted to the cranial bone defects. The animals were sacrificed on weeks 6 and 12 post-operation. Radiographic examinations (x-ray imaging and 3D CT scanning) and histological evaluations were performed. The repaired areas of cranial bone defects were measured, and the osteogenetic abilities of various materials were compared. Cranial histology, imaging, and repaired area measurements showed that the osteogenetic effects at two time points (weeks 6 and 12) in group C were better than those in groups A and B. The effects in groups A and B were similar. Group D achieved the worst repair effect of cranial bone defects, where a large number of fibrous connective tissues were observed. The PLGA/CS composite microspheres loaded with rhBMP 2 and P24 had optimal concrescence and could mutually increase their osteogenesis capability. rhBMP 2 + P24/PLGA/CS composite is a novel material for bone defect repair with stable activity to induce bone formation. (paper)

  13. Regulation of Msx-1, Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development.

    Science.gov (United States)

    Phippard, D J; Weber-Hall, S J; Sharpe, P T; Naylor, M S; Jayatalake, H; Maas, R; Woo, I; Roberts-Clark, D; Francis-West, P H; Liu, Y H; Maxson, R; Hill, R E; Dale, T C

    1996-09-01

    Expression of the Msx-1 and Msx-2 homeobox genes have been shown to be coordinately regulated with the Bmp-2 and Bmp-4 ligands in a variety of developing tissues. Here we report that transcripts from all four genes are developmentally regulated during both foetal and postnatal mammary gland development. The location and time-course of the Bmp and Msx expression point to a role for Msx and Bmp gene products in the control of epithelial-mesenchymal interactions. Expression of Msx-2, but not Msx-1, Bmp-2 or Bmp-4 was decreased following ovariectomy, while expression of the human Msx-2 homologue was regulated by 17beta-oestradiol in the MCF-7 breast cancer cell line. The regulation of Msx-2 expression by oestrogen raises the possibility that hormonal regulation of mammary development is mediated through the control of epithelial-mesenchymal interactions.

  14. Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Grassi Rici Rose

    2012-02-01

    Full Text Available Abstract Background The bone morphogenetic proteins (BMPs belong to a unique group of proteins that includes the growth factor TGF-β. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs and belong to the University of São Paulo, College of Veterinary Medicine (FMVZ-USP stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p

  15. Bone mesenchymal stem cells co-expressing VEGF and BMP-6 genes to combat avascular necrosis of the femoral head.

    Science.gov (United States)

    Liao, Hongxing; Zhong, Zhixiong; Liu, Zhanliang; Li, Liangping; Ling, Zemin; Zou, Xuenong

    2018-01-01

    The aim of the present study was to investigate the potential of bone mesenchymal stem cells (BMSCs) treated with a combination of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) genes for the treatment of avascular necrosis of the femoral head (ANFH). Rat BMSCs were isolated and purified using a density gradient centrifugation method. The purity and characteristics of the BMSCs were detected by cell surface antigens identification using flow cytometry. The experimental groups were administered with one of the following adeno-associated virus (AAV) vector constructs: AAV-green fluorescent protein (AAV-GFP), AAV-BMP-6, AAV-VEGF or AAV-VEGF-BMP-6. The expression of VEGF and BMP-6 was detected by reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA assays. The effects of VEGF and BMP-6 on BMSCs were evaluated by angiogenic and osteogenic assays. The transfected BMSCs were combined with a biomimetic synthetic scaffold poly lactide-co-glycolide (PLAGA) and they were then subcutaneously implanted into nude mice. After four weeks, the implants were analyzed with histology and subsequent immunostaining to evaluate the effects of BMSCs on blood vessel and bone formation in vivo . In the AAV-VEGF-BMP-6 group, the expression levels of VEGF and BMP-6 were significantly increased and human umbilical vein endothelial cells tube formation was significantly enhanced compared with other groups. Capillaries and bone formation in the AAV-VEGF-BMP-6 group was significantly higher compared with the other groups. The results of the present study suggest that BMSCs expressing both VEGF and BMP-6 induce an increase in blood vessels and bone formation, which provides theoretical support for ANFH gene therapy.

  16. BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

    Science.gov (United States)

    Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

    2015-01-01

    The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

  17. Xerogel Interfaced Nanofibers Stimulate Bone Regeneration Through the Activation of Integrin and Bone Morphogenetic Protein Pathways.

    Science.gov (United States)

    Lee, Yoo-Mi; Yun, Hyung-Mun; Lee, Hye-Young; Lim, Hyun-Chang; Lee, Hae-Hyoung; Kim, Hae-Won; Kim, Eun-Cheol

    2017-02-01

    A xerogel was interfaced onto biopolymer nanofibers though a core–shell electrospinning design for bone regeneration. The xerogel-interfaced biopolymer nanofibrous matrix was bioactive and highly hydrophilic, with a significant decrease in the water contact angle. The matrix showed excellent in vitro responses of primary osteoblasts in terms of adhesion, proliferation, and migration. Furthermore, the osteoblastic differentiation of cells, including alkaline phosphatase activity, mineralization, and gene expression, was significantly upregulated by the xerogel interface. In vivo animal tests in a critical-sized calvarial defect confirmed the new bone formation ability of the xerogel-surfaced nanofiber matrices. The underlying signaling mechanisms of the stimulation were implied to be integrin and bone morphogenetic protein (BMP) pathways, as demonstrated by the activation of integrin (α2β1) and downstream signaling molecules (FAK, paxillin, RhoA, MAPK, and NF-κB), as well as the BMPs and the downstream transcription factor Smad1/5/8. Taking these findings together, the xerogel-surfaced biopolymer nanofibers are proposed to be a promising scaffold candidate for bone regeneration.

  18. Trends Analysis of rhBMP2 Utilization in Single-Level Anterior Lumbar Interbody Fusion in the United States.

    Science.gov (United States)

    Lao, Lifeng; Cohen, Jeremiah R; Buser, Zorica; Brodke, Darrel S; Yoon, S Tim; Youssef, Jim A; Park, Jong-Beom; Meisel, Hans-Joerg; Wang, Jeffrey C

    2018-04-01

    Retrospective case study. To evaluate the trends and demographics of recombinant human bone morphogenetic protein 2 (rhBMP2) utilization in single-level anterior lumbar interbody fusion (ALIF) in the United States. Patients who underwent single-level ALIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database (PearlDiver Technologies, Fort Wayne, IN), a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were analyzed for each patient. A total of 921 patients were identified who underwent a single-level ALIF in this study. The average rate of single-level ALIF with rhBMP2 utilization increased (35%-48%) from 2005 to 2009, but sharply decreased to 16.7% in 2010 and 15.0% in 2011. The overall incidence of single-level ALIF without rhBMP2 (0.20 cases per 100 000 patients) was more than twice of the incidence of single-level ALIF with rhBMP2 (0.09 cases per 100 000 patients). The average rate of single-level ALIF with rhBMP2 utilization is highest in West (41.4%), followed by Midwest (33.3%), South (26.5%) and Northeast (22.2%). The highest incidence of single-level ALIF with rhBMP2 was observed in the group aged less than 65 years (compared with any other age groups, P level ALIF increased from 2006 to 2009, but decreased in 2010 and 2011. The Northeast region had the lowest incidence of rhBMP2 utilization. The group aged less than 65 years trended to have the higher incidence of single-level ALIF with rhBMP2 utilization.

  19. Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction.

    Science.gov (United States)

    James, Rebecca E; Hoover, Kendall M; Bulgari, Dinara; McLaughlin, Colleen N; Wilson, Christopher G; Wharton, Kristi A; Levitan, Edwin S; Broihier, Heather T

    2014-12-08

    Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Recombinant human bone morphogenetic protein-2 in the treatment of bone fractures

    Directory of Open Access Journals (Sweden)

    Neil Ghodadra

    2008-09-01

    Full Text Available Neil Ghodadra, Kern SinghDepartment of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, USAAbstract: Over one million fractures occur per year in the US and are associated with impaired healing increasing patient morbidity, stress, and economic costs. Despite improvements in surgical technique, internal fixation, and understanding of biologics, fracture healing is delayed or impaired in up to 4% of all fractures. Complications due to impaired fracture healing present therapeutic challenges to the orthopedic surgeon and often lead to chronic functional and psychological disability for the patient. As a result, it has become clinically desirable to augment mechanical fixation with biologic strategies in order to accelerate osteogenesis and promote successful arthrodesis. The discovery of bone morphogenic protein (BMP has been pivotal in understanding the biology of fracture healing and has been a source of intense clinical research as an adjunct to fracture treatment. Multiple in vitro and in vivo studies in animals have elucidated the complex biologic interactions between BMPs and cellular receptors and have convincingly demonstrated rhBMP-2 to be a safe, effective treatment option to enhance bone healing. Multiple clinical trials in trauma surgery have provided level 1 evidence for the use of rhBMP-2 as a safe and effective treatment of fractures. Human clinical trials have provided further insight into BMP-2 dosage, time course, carriers, and efficacy in fracture healing of tibial defects. These promising results have provided hope that a new biologic field of technology has emerged as a useful adjunct in the treatment of skeletal injuries and conditions.Keywords: bone morphogenic protein-2, bone fracture, bone healing

  1. Improved healing of transected rabbit Achilles tendon after a single injection of cartilage-derived morphogenetic protein-2.

    Science.gov (United States)

    Forslund, Carina; Aspenberg, Per

    2003-01-01

    Achilles tendon ruptures in humans might be treated more efficiently with the help of a growth factor. Cartilage-derived morphogenetic protein-2 has been shown to induce formation of tendon-like tissue. Cartilage-derived morphogenetic protein-2 has a positive effect on mechanical parameters for tendon healing in a rabbit model with Achilles tendon transection. Controlled laboratory study. The right Achilles tendon of 40 rabbits was transected without tendon suture. Cartilage-derived morphogenetic protein-2 (10 micro g) or vehicle control (acetate buffer) was injected locally 2 hours postoperatively. All tendons were tested biomechanically at 8 and 14 days, and treated tendons were histologically and radiographically evaluated at 56 days. At 14 days, both failure load and stiffness of treated tendons were increased by 35%. The treated tendons had significantly larger callus size at 8 and 14 days. Histologic and radiographic examination showed no signs of ossification in the treated tendons after 56 days. A single injection of cartilage-derived morphogenetic protein-2 led to a stronger and stiffer tendon callus than that in the controls without inducing bone formation. Similar results from a larger animal model would suggest a possible future use of cartilage-derived morphogenetic protein-2 in the treatment of human Achilles tendon ruptures.

  2. Deproteinized bovine bone functionalized with the slow delivery of BMP-2 for the repair of critical-sized bone defects in sheep.

    Science.gov (United States)

    Liu, Tie; Wu, Gang; Wismeijer, Daniel; Gu, Zhiyuan; Liu, Yuelian

    2013-09-01

    As an alternative to an autologous bone graft, deproteinized bovine bone (DBB) is widely used in the clinical dentistry. Although DBB provides an osteoconductive scaffold, it is not capable of enhancing bone regeneration because it is not osteoinductive. In order to render DBB osteoinductive, bone morphogenetic protein 2 (BMP-2) has previously been incorporated into a three dimensional reservoir (a biomimetic calcium phosphate coating) on DBB, which effectively promoted the osteogenic response by the slow delivery of BMP-2. The aim of this study was to investigate the therapeutic effectiveness of such coating on the DBB granules in repairing a large cylindrical bone defect (8 mm diameter, 13 mm depth) in sheep. Eight groups were randomly assigned to the bone defects: (i) no graft material; (ii) autologous bone; (iii) DBB only; (iv) DBB mixed with autologous bone; (v) DBB bearing adsorbed BMP-2; (vi) DBB bearing a coating but no BMP-2; (vii) DBB bearing a coating with adsorbed BMP-2; and (viii) DBB bearing a coating-incorporated depot of BMP-2. 4 and 8 weeks after implantation, samples were withdrawn for a histological and a histomorphometric analysis. Histological results confirmed the excellent biocompatibility and osteoconductivity of all the grafts tested. At 4 weeks, DBB mixed with autologous bone or functionalized with coating-incorporated BMP-2 showed more newly-formed bone than the other groups with DBB. At 8 weeks, the volume of newly-formed bone around DBB that bore a coating-incorporated depot of BMP-2 was greatest among the groups with DBB, and was comparable to the autologous bone group. The use of autologous bone and BMP-2 resulted in more bone marrow formation. Multinucleated giant cells were observed in the resorption process around DBB, whereas histomorphometric analysis revealed no significant degradation of DBB. In conclusion, it was shown that incorporating BMP-2 into the calcium phosphate coating of DBB induced strong bone formation around DBB

  3. Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

    Science.gov (United States)

    Rutkovskiy, Arkady; Sagave, Julia; Czibik, Gabor; Baysa, Anton; Zihlavnikova Enayati, Katarina; Hillestad, Vigdis; Dahl, Christen Peder; Fiane, Arnt; Gullestad, Lars; Gravning, Jørgen; Ahmed, Shakil; Attramadal, Håvard; Valen, Guro; Vaage, Jarle

    2017-09-01

    We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

  4. Early inhibitory effects of zoledronic acid in tooth extraction sockets in dogs are negated by recombinant human bone morphogenetic protein.

    Science.gov (United States)

    Gerard, David A; Carlson, Eric R; Gotcher, Jack E; Pickett, David O

    2014-01-01

    This study was conducted with 2 purposes. The first was to determine the effect of a single dose of zoledronic acid (ZA) on the healing of a tooth extraction socket in dogs. The second was to determine if placement of recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge (ACS) - INFUSE, (Medtronic, Memphis, TN) into these extraction sockets would inhibit the inhibition on bone healing and remodeling by ZA. Nine adult female beagle dogs (2 to 3 yr old) were placed into 3 groups of 3 dogs each. Group I received 15 mL of sterile saline intravenously; group II received 2.5 mg of ZA intravenously; and group III received 5 mg of ZA intravenously. Forty-five days after treatment, all dogs underwent extraction of noncontiguous right and left mandibular first molars and second premolars. In group I, the right mandibular extraction sockets had nothing placed in them, whereas the left mandibular sockets had only ACS placed in them. In groups II and III, the right mandibular sockets had rhBMP-2/ACS placed in them, whereas the left mandibular sockets had only ACS placed. All extraction sockets were surgically closed. Tetracycline was given intravenously 5 and 12 days later, and all animals were euthanized 15 days after tooth extraction. The extraction sockets and rib and femur samples were harvested immediately after euthanasia, processed, and studied microscopically. A single dose of ZA significantly inhibited healing and bone remodeling in the area of the tooth extractions. The combination of rhBMP-2/ACS appeared to over-ride some of the bone remodeling inhibition of the ZA and increased bone fill in the extraction sites, and remodeling activity in the area was noted. The effects of rhBMP-2/ACS were confined to the area of the extraction sockets because bone activity at distant sites was not influenced. A single dose of ZA administered intravenously inhibits early healing of tooth extraction sockets and bone remodeling in this animal model. The

  5. Systematic Review and Meta-Analysis of Recombinant Human Bone Morphogenetic Protein-2 in Localized Alveolar Ridge and Maxillary Sinus Augmentation.

    Science.gov (United States)

    Kelly, Mick P; Vaughn, Olushola L Akinshemoyin; Anderson, Paul A

    2016-05-01

    Recombinant human bone morphogenetic protein-2 (rhBMP-2) is approved by the Food and Drug Administration as a viable alternative to bone graft in spinal fusion and maxillary sinus lift. The research questions for meta-analysis were: Is rhBMP-2 an effective bone graft substitute in localized alveolar ridge augmentation and maxillary sinus floor augmentation? What are the potential adverse events? A search of MEDLINE from January 1980 to January 2014 using PubMed, the Cochrane Database of Systematic Reviews and Controlled Trials, CINAHL, and EMBASE was performed. Searches were performed from Medical Subject Headings. The quality of each study included was graded by Review Manager software. The primary outcome variable was bone formation measured as change in bone height on computed tomogram. A systematic review of adverse events also was performed. A random-effects model was chosen. Continuous variables were calculated using the standardized mean difference and 95% confidence intervals (CIs) comparing improvement from baseline of the experimental group with that of the control group. Change in bone height was calculated using logarithmic odds ratio. Test of significance used the Z statistic with a P value of .05. Ten studies met the criteria for systematic review; 8 studies were included in the meta-analysis. Five studies assessed localized alveolar ridge augmentation and resulted in an overall standardized mean difference of 0.56 (CI, 0.20-0.92) in favor of BMP; this result was statistically important. Three studies assessed maxillary sinus floor augmentation and resulted in an overall standardized mean difference of -0.50 (CI, -0.93 to -0.09), which was meaningfully different in favor of the control group. Adverse events were inconsistently reported, ranging from no complications to widespread adverse events. For localized alveolar ridge augmentation, this meta-analysis showed that rhBMP-2 substantially increases bone height. However, rhBMP-2 does not perform as

  6. BMP4 density gradient in disk-shaped confinement

    Science.gov (United States)

    Bozorgui, Behnaz; Teimouri, Hamid; Kolomeisky, Anatoly B.

    We present a quantitative model that explains the scaling of BMP4 gradients during gastrulation and the recent experimental observation that geometric confinement of human embryonic stem cells is sufficient to recapitulate much of germ layer patterning. Based on a assumption that BMP4 diffusion rate is much smaller than the diffusion rate of it's inhibitor molecules, our results confirm that the length-scale which defines germ layer territories does not depend on system size.

  7. Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an equine bone defect model.

    Science.gov (United States)

    Seo, Jong-Pil; Tsuzuki, Nao; Haneda, Shingo; Yamada, Kazutaka; Furuoka, Hidefumi; Tabata, Yasuhiko; Sasaki, Naoki

    2014-03-01

    Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2 × 10(5) MSCs; M-6: 2 × 10(6) MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.

  8. NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

    Science.gov (United States)

    Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

    2003-08-01

    Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

  9. Association between two polymorphisms of the bone morpho-genetic protein-2 gene with genetic susceptibility to ossification of the posterior longitudinal ligament of the cervical spine and its severity

    Institute of Scientific and Technical Information of China (English)

    WANG Hao; YANG Zhao-hui; LIU Dong-mei; WANG Ling; MENG Xiang-long; TIAN Bao-peng

    2008-01-01

    Background Ossification of the posterior longitudinal ligament (OPLL) has a strong genetic background. Previous studies have shown that bone morphogenetic protein-2 (BMP2) and BMP2 mRNA are expressed in ossifying matrix and chondrocytes adjacent to cartilaginous areas of OPLL tissues and mesenchymal cells with fibroblastic features in the immediate vicinity of the cartilaginous areas. It is suggested that BMP2 plays different roles in the different stages of development of OPLL. However, it remains unknown which factors induce ligament cells to produce BMP2.Methods OPLL patients (n=192) and non-OPLL controls (n=304) were studied. Radiographs of the cervical spine were analyzed for extent of OPLL. We investigated whether single nucleotide polymorphisms of exons 3(-726) TIC and 3(-583) A/G in the BMP2 gene are statistically associated with genetic susceptibility to OPLL in Chinese Han subjects.Results There was no statistical difference between the occurrence of exons 3(-726) TIC and 3(-583) A/G and the occurrence of OPLL in the cervical spine. However, there was a significant association between occurrence of exon 3(-726) TIC polymorphism and occurrence of OPLL in males of cases and controls in the cervical spine. In addition, no significant association was found between the exons 3(-726) TIC and 3(-583) A/G with number of ossified cervical vertebrae in OPLL patients.Conclusions Exon 3(-583) A/G polymorphism in BMP2 gene is not associated with the occurrence and the extent of OPLL in the cervical spine. Chinese Han male patients with TC and CC genotypes in exon 3(-726) T/C have genetic susceptibility to OPLL but not to more extensive OPLL in the cervical spine.

  10. Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface.

    Science.gov (United States)

    Cho, Hankyu; Tarafder, Solaiman; Fogge, Michael; Kao, Kristy; Lee, Chang H

    2016-11-01

    Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.

  11. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

    Science.gov (United States)

    Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

    2012-03-01

    Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

  12. A high concentration of recombinant human bone morphogenetic protein-2 induces low-efficacy bone regeneration in sinus augmentation: a histomorphometric analysis in rabbits.

    Science.gov (United States)

    Hong, Ji-Youn; Kim, Min-Soo; Lim, Hyun-Chang; Lee, Jung-Seok; Choi, Seong-Ho; Jung, Ui-Won

    2016-12-01

    The aim of the study was to elucidate the efficacy of bone regeneration at the early stage of healing in rabbit sinuses grafted with a biphasic calcium phosphate (BCP) carrier soaked in a high concentration of recombinant human bone morphogenetic protein-2 (rhBMP-2). Both maxillary sinuses of eight male rabbits were used. The sinus on one side (assigned randomly) was grafted with BCP loaded with rhBMP-2 (1.5 mg/ml; test group) using a soaking method, while the other was grafted with saline-soaked BCP (control group). After a 2-week healing period, the sinuses were analyzed by micro-computed tomography and histomorphometry. The total augmented area and soft tissue space were significantly larger in the test group than in the control group, whereas the opposite was true for the area of residual material and newly formed bone. Most of the new bone in the test group was localized to the Schneiderian membrane (SM), while very little bone formation was observed in the window and center regions of the sinus. New bone was distributed evenly in the control group sinuses. Within the limitations of this study, it appeared that application of a high concentration of rhBMP-2 soaked onto a BCP carrier inhibited bone regeneration from the pristine bone and increased soft tissue swelling and inflammatory response at the early healing stage of sinus augmentation, although osteoinductive potential was found along the SM. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. The Effects of Tocotrienol and Lovastatin Co-Supplementation on Bone Dynamic Histomorphometry and Bone Morphogenetic Protein-2 Expression in Rats with Estrogen Deficiency

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    Kok-Yong Chin

    2017-02-01

    Full Text Available Both tocotrienol and statins are suppressors of the mevalonate pathway. Supplementation of tocotrienol among statin users could potentially protect them against osteoporosis. This study aimed to compare the effects of tocotrienol and lovastatin co-supplementation with individual treatments on bone dynamic histomorphometric indices and bone morphogenetic protein-2 (BMP-2 gene expression in ovariectomized rats. Forty-eight female Sprague-Dawley rats were randomized equally into six groups. The baseline was sacrificed upon receipt. All other groups were ovariectomized, except for the sham group. The ovariectomized groups were administered orally daily with (1 lovastatin 11 mg/kg/day alone; (2 tocotrienol derived from annatto bean (annatto tocotrienol 60 mg/kg/day alone; (3 lovastatin 11 mg/kg/day, and annatto tocotrienol 60 mg/kg/day. The sham and ovariectomized control groups were treated with equal volume of vehicle. After eight weeks of treatment, the rats were sacrificed. Their bones were harvested for bone dynamic histomorphometry and BMP-2 gene expression. Rats supplemented with annatto tocotrienol and lovastatin concurrently demonstrated significantly lower single-labeled surface, but increased double-labeled surface, mineralizing surface, mineral apposition rate and bone formation rate compared to individual treatments (p < 0.05. There was a parallel increase in BMP-2 gene expression in the rats receiving combined treatment (p < 0.05. The combination of annatto tocotrienol and lovastatin exerted either additively or synergistically on selected bone parameters. In conclusion, tocotrienol can augment the bone formation and mineralization in rats receiving low-dose statins. Supplementation of tocotrienol in statin users can potentially protect them from osteoporosis.

  14. rhBMP-2 (ACS and CRM formulations) overcomes pseudarthrosis in a New Zealand white rabbit posterolateral fusion model.

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    Lawrence, James P; Waked, Walid; Gillon, Thomas J; White, Andrew P; Spock, Christopher R; Biswas, Debdut; Rosenberger, Patricia; Troiano, Nancy; Albert, Todd J; Grauer, Jonathan N

    2007-05-15

    The study design consisted of a New Zealand white rabbit model of pseudarthrosis repair. Study groups consisting of no graft, autograft, or recombinant human bone morphogenetic protein-2 (rhBMP-2) with absorbable collagen sponge (ACS) or compression resistant matrix (CRM) were evaluated. To evaluate the relative efficacy of bone graft materials (autograft, ACS, and CRM). rhBMP-2 has been shown to have a 100% fusion rate in a primary rabbit fusion model, even in the presence of nicotine, which is known to inhibit fusion. Seventy-two New Zealand white rabbits underwent posterolateral lumbar fusion with iliac crest autograft. To establish pseudarthroses, nicotine was administered to all animals. At 5 weeks, the spines were explored and all pseudarthroses were redecorticated and implanted with no graft, autograft, rhBMP-2/ACS, or rhBMP-2/CRM. At 10 weeks, fusions were assessed by manual palpation and histology. Eight rabbits (11%) were lost to complications. At 5 weeks, 66 (97%) had pseudarthroses. At 10 weeks, attempted pseudarthrosis repairs were fused in 1 of 16 of no graft rabbits (6%), 5 of 17 autograft rabbits (29%), and 31 of 31 rhBMP-2 rabbits (with ACS or CRM) (100%). Histologic analysis demonstrated more mature bone formation in the rhBMP-2 groups. The 2 rhBMP-2 formulations led to significantly higher fusion rates and histologic bone formation than no graft and autograft controls in this pseudarthrosis repair model.

  15. Tissue engineering for lateral ridge augmentation with recombinant human bone morphogenetic protein 2 combination therapy: a case report.

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    Mandelaris, George A; Spagnoli, Daniel B; Rosenfeld, Alan L; McKee, James; Lu, Mei

    2015-01-01

    This case report describes a tissue-engineered reconstruction with recombinant human bone morphogenetic protein 2/acellular collagen sponge (rhBMP-2/ ACS) + cancellous allograft and space maintenance via Medpor Contain mesh in the treatment of a patient requiring maxillary and mandibular horizontal ridge augmentation to enable implant placement. The patient underwent a previously unsuccessful corticocancellous bone graft at these sites. Multiple and contiguous sites in the maxilla and in the mandibular anterior, demonstrating advanced lateral ridge deficiencies, were managed using a tissue engineering approach as an alternative to autogenous bone harvesting. Four maxillary and three mandibular implants were placed 9 and 10 months, respectively, after tissue engineering reconstruction, and all were functioning successfully after 24 months of follow-up. Histomorphometric analysis of a bone core obtained at the time of the maxillary implant placement demonstrated a mean of 76.1% new vital bone formation, 22.2% marrow/cells, and 1.7% residual graft tissue. Tissue engineering for lateral ridge augmentation with combination therapy requires further research to determine predictability and limitations.

  16. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

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    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  17. Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo.

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    Jun Zhao

    Full Text Available Calcium phosphate (Ca-P scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2 loaded calcium-deficient hydroxyapatite (CDHA scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR and enzyme-linked immunosorbent assay (ELISA demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2 promoted osteogenic differentiation of bone marrow stromal cells (bMSCs significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ± 5.32% and area (40.71% ± 7.14% as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ± 0.44%, calcein: 6.08% ± 1.37% and mineral apposition rate (4.13 ± 0.62 µm/day in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17 ± 15.02 Mpa and

  18. Kinetics and thermodynamics studies on the BMP-2 adsorption onto hydroxyapatite surface with different multi-morphological features.

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    Lu, Zhiwei; Huangfu, Changxin; Wang, Yanying; Ge, Hongwei; Yao, Yao; Zou, Ping; Wang, Guangtu; He, Hua; Rao, Hanbing

    2015-01-01

    The effect of the surface topography on protein adsorption process is of great significance for designing hydroxyapatite (HA) ceramic material surfaces. In this work, three different topographies of HA materials HA-sheet, HA-rod, and HA-whisker were synthesized and testified by X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Brunauer-Emmett-Teller (BET) and a field emission scanning electron microscopy (FE-SEM). We have systematically investigated the adsorption kinetics and thermodynamics of bone morphogenetic proteins (BMP-2) on the three different topography surfaces of HA, respectively. The results showed that the maximum adsorption capacities of HA-sheet, HA-rod and HA-whisker were (219.96 ± 10.18), (247.13 ± 12.35), and (354.67 ± 17.73) μg · g(-1), respectively. Kinetic parameters, rate constants, equilibrium adsorption capacities and related correlation coefficients, for each kinetic model were calculated as well as discussed. It demonstrated that the adsorption of BMP-2 onto HA could be described by the pseudo second-order equation. Adsorption of BMP-2 onto HA followed the Langmuir isotherm. It confirmed that compared with other samples HA-whisker had more adsorption sites for its high specific surface area which could provide more opportunities for protein molecules. The adsorption processes were endothermic (ΔH > 0), spontaneous (ΔG 0). A possible adsorption mechanism has been proposed. In addition, the BMP-2 could be adsorbed to the surface which existed slight conformational changes by FT-IR. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways.

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    Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

    2014-01-01

    Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.

  20. Autologous implantation of BMP2-expressing dermal fibroblasts to improve bone mineral density and architecture in rabbit long bones.

    Science.gov (United States)

    Ishihara, Akikazu; Weisbrode, Steve E; Bertone, Alicia L

    2015-10-01

    Cell-mediated gene therapy may treat bone fragility disorders. Dermal fibroblasts (DFb) may be an alternative cell source to stem cells for orthopedic gene therapy because of their rapid cell yield and excellent plasticity with bone morphogenetic protein-2 (BMP2) gene transduction. Autologous DFb or BMP2-expressing autologous DFb were administered in twelve rabbits by two delivery routes; a transcortical intra-medullar infusion into tibiae and delayed intra-osseous injection into femoral drill defects. Both delivery methods of DFb-BMP2 resulted in a successful cell engraftment, increased bone volume, bone mineral density, improved trabecular bone microarchitecture, greater bone defect filling, external callus formation, and trabecular surface area, compared to non-transduced DFb or no cells. Cell engraftment within trabecular bone and bone marrow tissue was most efficiently achieved by intra-osseous injection of DFb-BMP2. Our results suggested that BMP2-expressing autologous DFb have enhanced efficiency of engraftment in target bones resulting in a measurable biologic response by the bone of improved bone mineral density and bone microarchitecture. These results support that autologous implantation of DFb-BMP2 warrants further study on animal models of bone fragility disorders, such as osteogenesis imperfecta and osteoporosis to potentially enhance bone quality, particularly along with other gene modification of these diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  1. Multi-protein delivery by nanodiamonds promotes bone formation.

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    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

  2. Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

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    Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

    2018-04-01

    The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

  3. Rapamycin inhibits BMP-7-induced osteogenic and lipogenic marker expressions in fetal rat calvarial cells.

    Science.gov (United States)

    Yeh, Lee-Chuan C; Ma, Xiuye; Ford, Jeffery J; Adamo, Martin L; Lee, John C

    2013-08-01

    Bone morphogenetic proteins (BMPs) promote osteoblast differentiation and bone formation in vitro and in vivo. BMPs canonically signal through Smad transcription factors, but BMPs may activate signaling pathways traditionally stimulated by growth factor tyrosine kinase receptors. Of these, the mTOR pathway has received considerable attention because BMPs activate P70S6K, a downstream effector of mTOR, suggesting that BMP-induced osteogenesis is mediated by mTOR activation. However, contradictory effects of the mTOR inhibitor rapamycin (RAPA) on bone formation have been reported. Since bone formation is thought to be inversely related to lipid accumulation and mTOR is also important for lipid synthesis, we postulated that BMP-7 may stimulate lipogenic enzyme expression in a RAPA-sensitive mechanism. To test this hypothesis, we determined the effects of RAPA on BMP-7-stimulated expression of osteogenic and lipogenic markers in cultured fetal rat calvarial cells. Our study showed that BMP-7 promoted the expression of osteogenic and lipogenic markers. The effect of BMP-7 on osteogenic markers was greater in magnitude than on lipogenic markers and was temporally more sustained. RAPA inhibited basal and BMP-7-stimulated osteogenic and lipogenic marker expression and bone nodule mineralization. The acetyl CoA carboxylase inhibitor TOFA stimulated the expression of osteoblast differentiation markers, whereas palmitate suppressed their expression. We speculate that the BMP-7-stimulated adipogenesis is part of the normal anabolic response to BMPs, but that inappropriate activation of the lipid biosynthetic pathway by mTOR could have deleterious effects on bone formation and could explain paradoxical effects of RAPA to promote bone formation. Copyright © 2013 Wiley Periodicals, Inc.

  4. Trends Analysis of rhBMP Utilization in Single-Level Posterior Lumbar Interbody Fusion in the United States.

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    Lao, Lifeng; Cohen, Jeremiah R; Buser, Zorica; Brodke, Darrel S; Youssef, Jim A; Park, Jong-Beom; Yoon, S Tim; Wang, Jeffrey C; Meisel, Hans-Joerg

    2017-10-01

    Retrospective study. Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used in spinal fusion surgery, but there is little information on rhBMP-2 utilization in single-level posterior lumbar interbody fusion (PLIF). The purpose of our study was to evaluate the trends and demographics of rhBMP-2 utilization in single-level PLIF. Patients who underwent single-level PLIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database, a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were recorded for each patient. Results were reported for each variable as the incidence of procedures identified per 100 000 patients searched in the database. A total of 2735 patients had single-level PLIF. The average rate of single-level PLIF with rhBMP-2 maintained at a relatively stable level (28% to 31%) from 2005 to 2009, but decreased in 2010 (9.9%) and 2011 (11.8%). The overall incidence of single-level PLIF without rhBMP-2 (0.68 cases per 100 000 patients) was statistically higher ( P level PLIF with rhBMP-2 (0.21 cases per 100 000 patients). The average rate of single-level PLIF with rhBMP-2 utilization was the highest in West (30.1%), followed by Midwest (26.9%), South (20.5%), and Northeast (17.8%). The highest incidence of single-level PLIF with rhBMP-2 was observed in the age group level PLIF. There was a 3-fold increase in the rate of PLIF without rhBMP-2 compared to PLIF with rhBMP-2, with both procedures being mainly done in patients less than 65 years of age.

  5. Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

    Science.gov (United States)

    McCarthy, Bryce; Yuan, Yuan; Koria, Piyush

    2016-07-08

    Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016. © 2016 American Institute of Chemical Engineers.

  6. Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

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    Guo, H.H.; Yu, C.C.; Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China); Ma, X.J. [Ningxia Medical Autonomous Region of the First People' s Hospital, Department of Orthopedic Surgery, Yinchuan (China); Yang, X.C.; Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China)

    2013-10-02

    Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

  7. Efficacy of rhBMP-2 Loaded PCL/β-TCP/bdECM Scaffold Fabricated by 3D Printing Technology on Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Eun-Bin Bae

    2018-01-01

    Full Text Available This study was undertaken to evaluate the effect of 3D printed polycaprolactone (PCL/β-tricalcium phosphate (β-TCP scaffold containing bone demineralized and decellularized extracellular matrix (bdECM and human recombinant bone morphogenetic protein-2 (rhBMP-2 on bone regeneration. Scaffolds were divided into PCL/β-TCP, PCL/β-TCP/bdECM, and PCL/β-TCP/bdECM/BMP groups. In vitro release kinetics of rhBMP-2 were determined with respect to cell proliferation and osteogenic differentiation. These three reconstructive materials were implanted into 8 mm diameter calvarial bone defect in male Sprague-Dawley rats. Animals were sacrificed four weeks after implantation for micro-CT, histologic, and histomorphometric analyses. The findings obtained were used to calculate new bone volumes (mm3 and new bone areas (%. Excellent cell bioactivity was observed in the PCL/β-TCP/bdECM and PCL/β-TCP/bdECM/BMP groups, and new bone volume and area were significantly higher in the PCL/β-TCP/bdECM/BMP group than in the other groups (p<.05. Within the limitations of this study, bdECM printed PCL/β-TCP scaffolds can reproduce microenvironment for cells and promote adhering and proliferating the cells onto scaffolds. Furthermore, in the rat calvarial defect model, the scaffold which printed rhBMP-2 loaded bdECM stably carries rhBMP-2 and enhances bone regeneration confirming the possibility of bdECM as rhBMP-2 carrier.

  8. Safety study: is there a pathologic IGF-1, PDGF and TGF-ß cytokine expression caused by adjunct BMP-7 in tibial and femoral non-union therapy?

    Directory of Open Access Journals (Sweden)

    Fischer C

    2018-04-01

    Full Text Available Christian Fischer,1 Christian Reiner,2 Gerhard Schmidmaier,1 Julian Doll,1 Christopher Child,3 Paul Alfred Grützner,4 Bahram Biglari,4 Sonja Boxriker,5 Arash Moghaddam5 1Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, HTRG – Heidelberg Trauma Research Group, Heidelberg University Hospital, Heidelberg, Germany; 2Department of Trauma and Orthopedic Surgery, Paracelsus Medical University, Nuremberg Hospital South, Nuremberg, Germany; 3Department of Trauma Surgery, University Hospital Zurich, Zurich, Switzerland; 4Trauma and Orthopedics, BG Trauma Center Ludwigshafen, Ludwigshafen, Germany; 5Center of Orthopedics, Trauma Surgery and Sport Medicine, ATORG Aschaffenburg-Alzenau, Aschaffenburg, Germany Background: In this prospective safety study, we investigated if the characteristic cytokine expression during bone regeneration is manipulated by the local application of bone morphogenetic protein-7 (BMP-7 in non-union surgery. Therefore, the levels of insulin like growth factor 1 (IGF-1, platelet-derived growth factor AB (PDGF-AB and transforming growth factor beta (TGF-β were compared between patients with the gold standard use of autologous bone graft (ABG and those with additional application of BMP-7 as part of the diamond concept.Patients and methods: Between 2009 and 2014, of the 153 patients with tibial and femoral non-unions, a matched pair analysis was performed to compare the serological cytokine expressions. Blood samples were collected preoperatively, 1, 2 and 6 weeks as well as 3 and 6 months after non-union surgery. Matching criteria were smoking status, fracture location, gender, age and body mass index (BMI. Patients in G1 (n=10 were treated with ABG and local BMP-7 while their matching partners in G2 (n=10 received ABG only. The routine clinical and radiologic follow-up was 1 year.Results: Although the IGF-1 quantification in G2 showed higher pre- and postoperative values compared to G1 (p<0.05, the courses of both

  9. Vitamin C-linker-conjugated tripeptide AHK stimulates BMP-2-induced osteogenic differentiation of mouse myoblast C2C12 cells.

    Science.gov (United States)

    Jung, Jung-Il; Park, Kyeong-Yong; Lee, Yura; Park, Mira; Kim, Jiyeon

    2018-03-15

    Vitamin C-linker-conjugated Ala-His-Lys tripeptide (Vit C-AHK) is a derivative of Vitamin C-conjugated tripeptides, which were originally developed as a component of a product for collagen synthesis enhancement or human dermal fibroblast growth. Here, we investigated the effect of Vit C-AHK on bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Vit C-AHK enhanced proliferation of C2C12 cells and induction of BMP-2-induced alkaline phosphatase, a typical marker of osteoblast differentiation. Vit C-AHK also stimulated the phosphorylation and translocation of Smad1/5/8 to the nucleus and phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2 and p38. In addition, Vit C-AHK enhanced the BMP-2-induced mRNA expression of osteoblast differentiation-related genes such as ALP, BMP-2, Osteocalcin, and Runx2. Our results suggest that Vit C-AHK exerts an enhancing effect on osteoblast proliferation and differentiation through activation of Smad1/5/8 and MAPK ERK1/2 and p38 signaling and without significant cytotoxicity. These results provide important data for the development of peptide-based bone-regenerative agents and treatment of bone-related disorders. Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  10. In vitro and in vivo evaluation of calcium phosphate composite scaffolds containing BMP-VEGF loaded PLGA microspheres for the treatment of avascular necrosis of the femoral head.

    Science.gov (United States)

    Zhang, Hao-Xuan; Zhang, Xiu-Ping; Xiao, Gui-Yong; Hou, Yong; Cheng, Lei; Si, Meng; Wang, Shuai-Shuai; Li, Yu-Hua; Nie, Lin

    2016-03-01

    Avascular necrosis of the femoral head (ANFH) is difficult to treat due to high pressure and hypoxia, and reduced levels of growth factors such as bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF). We generated a novel calcium phosphate (CPC) composite scaffold, which contains BMP-VEGF-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (BMP-VEGF-PLGA-CPC). The BMP-VEGF-loaded microspheres have an encapsulation efficiency of 89.15% for BMP, and 78.55% for VEGF. The BMP-VEGF-PLGA-CPC scaffold also demonstrated a porosity of 62% with interconnected porous structures, and pore sizes of 219 μm and compressive strength of 6.60 MPa. Additionally, bone marrow mesenchymal stem cells (BMSCs) were seeded on scaffolds in vitro. Further characterization showed that the BMP-VEGF-PLGA-CPC scaffolds were biocompatible and enhanced osteogenesis and angiogenesis in vitro. Using a rabbit model of ANFH, BMP-VEGF-PLGA-CPC scaffolds were implanted into the bone tunnels of core decompression in the femoral head for 6 and 12 weeks. Radiographic and histological analysis demonstrated that the BMP-VEGF-PLGA-CPC scaffolds exhibited good biocompatibility, and osteogenic and angiogenic activity in vivo. These results indicate that the BMP-VEGF-PLGA-CPC scaffold may improve the therapeutic effect of core decompression surgery and be used as a treatment for ANFH. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Effect of heat treatment at 600 degree C for 10 hours on human BMP

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    Izawa, H.; Hachiya, Y.; Muramatsu, K.; Narita, Y.; Kawai, T.

    1999-01-01

    Viral infection are an extremely serious in allogeneic bone transplantations. While it is essential to kill viruses such as HIV in allogeneic bone graft, the osteoinductive activity must be preserved. Heat treatment of allogeneic bone graft at 60 degree C for 10 hours is effective in killing viruses such as HIV, but it is unclear to what extent the activity of human bone morphogenetic protein (hBMP) is preserved. In this experiment crude hBMP was extracted from both heated and non-heated human bones which were decalcified by the Urist method. Gelatin capsules containing 5mg of crude hBMP were transplanted into the thigh muscles of 5 week old mice. Human bone samples heated in a water bath at 60 degree C for 10 hours and non-heated samples were each transplanted into 5 mice. At 20 days after transplantation, the heterotopic bone formation was compared by evaluation of X-ray and histologicic analysis. X-rays showed heterotopic bone formation in both heated and non-heated samples. Further, histologic analysis showed that peripheral osteoid tissue had developed into laminar bone formation and interlaminar bone marrow was observed. Heterotopic bone formation was induced by crude hBMP from heated bones in a similar way to crude hBMP from non-heated bones observed in X-ray. There was no significant difference in histologic analysis. The crude hBMP, extracted from bones which were heat-treated at 60 degree C for 10 hours induced heterotopic bone formation similar to that in non-heated bone observed by X-ray and histologic analysis at 20 days after transplantation. This demostrates that the heat-treated bone preserved osteoinduction

  12. Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

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    Kang, Myoung Hee [Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Oh, Sang Cheul [Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Lee, Hyun Joo [Department of Pathology, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kang, Han Na; Kim, Jung Lim [Graduate School of Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Kim, Jun Suk [Division of Oncology/Hematology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of); Yoo, Young A., E-mail: ydanbi@korea.ac.kr [Brain Korea 21 Program for Biomedical Science, Korea University College of Medicine, Korea University, Seoul 136-705 (Korea, Republic of)

    2011-07-15

    Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.

  13. [EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS].

    Science.gov (United States)

    Fu, Weili; Chen, Gang; Tang, Xin; Li, Qi; Ll, Jian

    2015-04-01

    To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000). Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into

  14. Wnt/β-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis

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    Aurrekoetxea, Maitane; Irastorza, Igor; García-Gallastegui, Patricia; Jiménez-Rojo, Lucia; Nakamura, Takashi; Yamada, Yoshihiko; Ibarretxe, Gaskon; Unda, Fernando J.

    2016-01-01

    Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity. Results: Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8), which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1) were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b) and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts. PMID:27066482

  15. Wnt/β-catenin regulates the activity of Epiprofin/Sp6, SHH, FGF and BMP to coordinate the stages of odontogenesis

    Directory of Open Access Journals (Sweden)

    Maitane eAurrekoetxea

    2016-03-01

    Full Text Available Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO, a specific inhibitor of GSK-3 activity. Results: Overactivatingthe Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh, Epiprofin (Epfn and Fibroblast growth factor8 (Fgf8, which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4, Fibroblast growth factor10 (Fgf10, Muscle segment homeobox 1 (Msx-1, Bone Morphogenetic protein 4 (Bmp4 and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1 were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts.

  16. 3D bioprinting of BMSC-laden methacrylamide gelatin scaffolds with CBD-BMP2-collagen microfibers.

    Science.gov (United States)

    Du, Mingchun; Chen, Bing; Meng, Qingyuan; Liu, Sumei; Zheng, Xiongfei; Zhang, Cheng; Wang, Heran; Li, Hongyi; Wang, Nuo; Dai, Jianwu

    2015-12-18

    Three-dimensional (3D) bioprinting combines biomaterials, cells and functional components into complex living tissues. Herein, we assembled function-control modules into cell-laden scaffolds using 3D bioprinting. A customized 3D printer was able to tune the microstructure of printed bone mesenchymal stem cell (BMSC)-laden methacrylamide gelatin scaffolds at the micrometer scale. For example, the pore size was adjusted to 282 ± 32 μm and 363 ± 60 μm. To match the requirements of the printing nozzle, collagen microfibers with a length of 22 ± 13 μm were prepared with a high-speed crusher. Collagen microfibers bound bone morphogenetic protein 2 (BMP2) with a collagen binding domain (CBD) as differentiation-control module, from which BMP2 was able to be controllably released. The differentiation behaviors of BMSCs in the printed scaffolds were compared in three microenvironments: samples without CBD-BMP2-collagen microfibers in the growth medium, samples without microfibers in the osteogenic medium and samples with microfibers in the growth medium. The results indicated that BMSCs showed high cell viability (>90%) during printing; CBD-BMP2-collagen microfibers induced BMSC differentiation into osteocytes within 14 days more efficiently than the osteogenic medium. Our studies suggest that these function-control modules are attractive biomaterials and have potential applications in 3D bioprinting.

  17. In silico Mechano-Chemical Model of Bone Healing for the Regeneration of Critical Defects: The Effect of BMP-2.

    Directory of Open Access Journals (Sweden)

    Frederico O Ribeiro

    Full Text Available The healing of bone defects is a challenge for both tissue engineering and modern orthopaedics. This problem has been addressed through the study of scaffold constructs combined with mechanoregulatory theories, disregarding the influence of chemical factors and their respective delivery devices. Of the chemical factors involved in the bone healing process, bone morphogenetic protein-2 (BMP-2 has been identified as one of the most powerful osteoinductive proteins. The aim of this work is to develop and validate a mechano-chemical regulatory model to study the effect of BMP-2 on the healing of large bone defects in silico. We first collected a range of quantitative experimental data from the literature concerning the effects of BMP-2 on cellular activity, specifically proliferation, migration, differentiation, maturation and extracellular matrix production. These data were then used to define a model governed by mechano-chemical stimuli to simulate the healing of large bone defects under the following conditions: natural healing, an empty hydrogel implanted in the defect and a hydrogel soaked with BMP-2 implanted in the defect. For the latter condition, successful defect healing was predicted, in agreement with previous in vivo experiments. Further in vivo comparisons showed the potential of the model, which accurately predicted bone tissue formation during healing, bone tissue distribution across the defect and the quantity of bone inside the defect. The proposed mechano-chemical model also estimated the effect of BMP-2 on cells and the evolution of healing in large bone defects. This novel in silico tool provides valuable insight for bone tissue regeneration strategies.

  18. Bone Morphogenic Protein-2 (rhBMP2)-Loaded Silk Fibroin Scaffolds to Enhance the Osteoinductivity in Bone Tissue Engineering

    Science.gov (United States)

    Du, Guang-Yu; He, Sheng-Wei; Sun, Chuan-Xiu; Mi, Li-Dong

    2017-10-01

    There is an increasing demand for formulations of silk fibroin (SF) scaffolds in biomedical applications. SF was crosslinked via glutaraldehyde with osteoinductive recombinant human bone morphogenic protein-2 (rhBMP2) of different ratios viz. (i) 3% SF with no rhBMP2 (SF), (ii) 3% SF with equal amount of rhBMP2 (SF+BMP2), and (iii) 12% SF with 3% of rhBMP2 (4SF+BMP2), and these solutions were used in electrospinning-based fabrication of nanoscaffolds for evaluating increased osteoinductive potential of SF scaffolds with rhBMP2. Stress-strain relationship suggested there is no loss in mechanical strength of fibers with addition of rhBMP2, and mechanical strength of scaffold was improved with increase in concentration of SF. rhBMP2 association increased the water retention capacity of scaffold as evident from swelling studies. Viability of hMSCs was found to be higher in conjugated scaffolds, and scaffolds do not exhibit any cytotoxicity towards guest cells. Cells were found to have higher alkaline phosphatase activity in conjugated scaffolds under in vitro and in vivo conditions which establishes the increased osteoinductivity of the novel construct. The scaffolds were found to be effective for in vivo bone formation as well.

  19. Continuity resection of the mandible after ameloblastoma - feasibility of oral rehabilitation with rhBMP-2 associated to bovine xenograft followed by implant installation.

    Science.gov (United States)

    Lustosa, Romulo Maciel; Macedo, Diogo de Vasconcelos; Iwaki, Lilian Cristina Vessoni; Tolentino, Elen de Souza; Hasse, Paulo Norberto; Marson, Giordano Bruno de Oliveira; Iwaki Filho, Liogi

    2015-10-01

    Recombinant human morphogenetic protein (rhBMP) is a graft alternative for extensive mandibular reconstruction after tumor resections. However, the feasibility of rhBMP-2 to receive osseointegrated implants and prosthetic rehabilitation has been rarely reported. This study reports on a case of an extensive solid ameloblastoma along the mandibular body. The treatment consisted of resection followed by off-label use of rhBMP type 2 associated with bovine bone xenograft. Eleven months postoperatively, the patient was prosthetically rehabilitated with dental implants, without evidence of resorption or complications. The literature on mandibular reconstructions using rhBMP and their feasibility for future osseointegrated implant placement was also reviewed. Based on the presented case, the association between rhBMP-2 and a bovine bone xenograft could be considered a feasible option for the reconstruction and rehabilitation of large mandibular defects after tumor resection. According to the literature, the use of rhBMP as a graft material is encouraging, with good clinical outcome. However, there are no long-term studies demonstrating success and survival rates of implants placed in these grafts. Future investigations will be required to ascertain the long-term survival of implants in areas grafted with rhBMP. Also, there is a lack of information regarding the prosthetic rehabilitation of these patients. Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  20. Adipose-derived stem cells and BMP-2 delivery in chitosan-based 3D constructs to enhance bone regeneration in a rat mandibular defect model.

    Science.gov (United States)

    Fan, Jiabing; Park, Hyejin; Lee, Matthew K; Bezouglaia, Olga; Fartash, Armita; Kim, Jinku; Aghaloo, Tara; Lee, Min

    2014-08-01

    Reconstructing segmental mandiblular defects remains a challenge in the clinic. Tissue engineering strategies provide an alternative option to resolve this problem. The objective of the present study was to determine the effects of adipose-derived stem cells (ASCs) and bone morphogenetic proteins-2 (BMP-2) in three-dimensional (3D) scaffolds on mandibular repair in a small animal model. Noggin expression levels in ASCs were downregulated by a lentiviral short hairpin RNA strategy to enhance ASC osteogenesis (ASCs(Nog-)). Chitosan (CH) and chondroitin sulfate (CS), natural polysaccharides, were fabricated into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP-2. The efficacy of 3D apatite-coated CH/CS scaffolds supplemented with ASCs(Nog-) and BMP-2 were evaluated in a rat critical-sized mandibular defect model. After 8 weeks postimplantation, the scaffolds treated with ASCs(Nog-) and BMP-2 significantly promoted rat mandibular regeneration as demonstrated by micro-computerized tomography, histology, and immunohistochemistry, compared with the groups treated with ASCs(Nog-) or BMP-2 alone. These results suggest that our combinatorial strategy of ASCs(Nog-)+BMP-2 in 3D apatite microenvironments can significantly promote mandibular regeneration, and these may provide a potential tissue engineering approach to repair large bony defects.

  1. Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

    Science.gov (United States)

    Wertheimer, Tobias; Velardi, Enrico; Tsai, Jennifer; Cooper, Kirsten; Xiao, Shiyun; Kloss, Christopher C.; Ottmüller, Katja J.; Mokhtari, Zeinab; Brede, Christian; deRoos, Paul; Kinsella, Sinéad; Palikuqi, Brisa; Ginsberg, Michael; Young, Lauren F.; Kreines, Fabiana; Lieberman, Sophia R.; Lazrak, Amina; Guo, Peipei; Malard, Florent; Smith, Odette M.; Shono, Yusuke; Jenq, Robert R.; Hanash, Alan M.; Nolan, Daniel J.; Butler, Jason M.; Beilhack, Andreas; Manley, Nancy R.; Rafii, Shahin; Dudakov, Jarrod A; van den Brink, Marcel RM

    2018-01-01

    The thymus is extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood and this capacity diminishes considerably with age. Here we show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration, via their production of BMP4. ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signalling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance and regeneration; and its downstream targets such as Dll4, itself a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity. PMID:29330161

  2. Influence of Mussel-Derived Bioactive BMP-2-Decorated PLA on MSC Behavior in Vitro and Verification with Osteogenicity at Ectopic Sites in Vivo.

    Science.gov (United States)

    Chen, Zhuoyue; Zhang, Zhen; Feng, Juantao; Guo, Yayuan; Yu, Yuan; Cui, Jihong; Li, Hongmin; Shang, Lijun

    2018-04-11

    Osteoinductive activity of the implant in bone healing and regeneration is still a challenging research topic. Therapeutic application of recombinant human bone morphogenetic protein-2 (BMP-2) is a promising approach to enhance osteogenesis. However, high dose and uncontrolled burst release of BMP-2 may introduce edema, bone overgrowth, cystlike bone formation, and inflammation. In this study, low-dose BMP-2 of 1 μg was used to design PLA-PD-BMP for functionalization of polylactic acid (PLA) implants via mussel-inspired polydopamine (PD) assist. For the first time, the binding property and efficiency of the PD coating with BMP-2 were directly demonstrated and analyzed using an antigen-antibody reaction. The obtained PLA-PD-BMP surface immobilized with this low BMP-2 dose can endow the implants with abilities of introducing strong stem cell adhesion and enhanced osteogenicity. Furthermore, in vivo osteoinduction of the PLA-PD-BMP-2 scaffolds was confirmed by a rat ectopic bone model, which is marked as the "gold standard" for the evidence of osteoinductive activity. The microcomputed tomography, Young's modulus, and histology analyses were also employed to demonstrate that PLA-PD-BMP grafted with 1 μg of BMP-2 can induce bone formation. Therefore, the method in this study can be used as a model system to immobilize other growth factors onto various different types of polymer substrates. The highly biomimetic mussel-derived strategy can therefore improve the clinical outcome of polymer-based medical implants in a facile, safe, and effective way.

  3. Off-label use of rhBMP-2 as bone regeneration strategies in mandibular ameloblastoma unicystic.

    Science.gov (United States)

    Silva, Henrique Celestino Lima E; Cheim, Adonai Peixoto; Moreno, Roberto; Miranda, Sérgio Luis de

    2017-01-01

    Jawbone reconstruction after tumor resection is one of the most challenging clinical tasks for maxillofacial surgeons. Osteogenic, osteoinductive, osteoconductive and non-antigenic properties of autogenous bone place this bone as the gold standard for solving problems of bone availability. However, the need for a second surgical site to harvest the bone graft increases significantly both the cost and the morbidity associated with the reconstructive procedures. Bone grafting gained an important tool with the discovery of bone morphogenetic proteins in 1960. Benefit of obtaining functional and real bone matrix without need of second surgical site seems to be the great advantage of use bone morphogenetic proteins. This study analyzed the use of rhBMP-2 in unicystic ameloblastoma of the mandible, detailing its structure, mechanisms of cell signaling and biological efficacy, in addition to present possible advantages and disadvantages of clinical use of rhBMP-2 as bone regeneration strategy. RESUMO A reconstrução óssea dos maxilares após ressecções tumorais é uma das tarefas mais difíceis para o cirurgião maxilofacial. As propriedades osteogênicas, osteoindutoras, osteocondutoras e não antigênicas do osso autógeno o colocam como o padrão-ouro para a solução de problemas de disponibilidade óssea. Entretanto a coleta do enxerto ósseo necessita de um segundo sítio cirúrgico, aumentando significativamente o custo e a morbidade associados ao procedimento reconstrutivo. A enxertia óssea ganhou uma excelente ferramenta com a descoberta das proteínas ósseas morfogenéticas na década de 1960. O benefício da obtenção de matriz óssea verdadeira e funcional, sem a necessidade de um segundo sítio cirúrgico, parece ser a grande vantagem do uso das proteínas ósseas morfogenéticas. Neste contexto, o objetivo deste estudo foi analisar a utilização da rhBMP-2 na regeneração óssea de ameloblastoma mandibular unicístico, detalhando sua estrutura, seus

  4. Bone regeneration in osteoporosis by delivery BMP-2 and PRGF from tetronic-alginate composite thermogel.

    Science.gov (United States)

    Segredo-Morales, Elisabet; García-García, Patricia; Reyes, Ricardo; Pérez-Herrero, Edgar; Delgado, Araceli; Évora, Carmen

    2018-05-30

    As the life expectancy of the world population increases, osteoporotic (OP) fracture risk increase. Therefore in the present study a novel injectable thermo-responsive hydrogel loaded with microspheres of 17β-estradiol, microspheres of bone morphogenetic protein-2 (BMP-2) and plasma rich in growth factors (PRGF) was applied locally to regenerate a calvaria critical bone defect in OP female rats. Three systems were characterized: Tetronic® 1307 (T-1307) reinforced with alginate (T-A), T-A with PRGF and T-A-PRGF with microspheres. The addition of the microspheres increased the viscosity but the temperature for the maximum viscosity did not change (22-24 °C). The drugs were released during 6 weeks in one fast phase (three days) followed by a long slow phase. In vivo evaluation was made in non-OP and OP rats treated with T-A, T-A with microspheres of 17β-estradiol (T-A-βE), T-A-βE prepared with PRGF (T-A-PRGF-βE), T-A-βE with microspheres of BMP-2 (T-A-βE-BMP-2) and the combination of the three (T-A-PRGF-βE-BMP). After 12 weeks, histological and histomorphometric analyzes showed a synergic effect due to the addition of BMP-2 to the T-A-βE formulation. The PRGF did not increased the bone repair. The new bone filling the OP defect was less mineralized than in the non-OP groups. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. An experimental setup to evaluate innovative therapy options for the enhancement of bone healing using BMP as a benchmark – a pilot study

    Directory of Open Access Journals (Sweden)

    B Preininger

    2012-04-01

    Full Text Available Critical or delayed bone healing in rat osteotomy (OT models is mostly achieved through large defects or instability. We aimed to design a rat OT model for impaired bone healing based on age, gender and parity. The outcome should be controllable through variations of the haematoma in the OT including a bone morphogenetic protein (BMP 2 guided positive control.Using external fixation to stabilise femoral a 2 mm double OT in 12 month old, female Sprague Dawley rats after a minimum of 3 litters healing was characterised following in situ haematoma formation (ISH-group, transplantation of a BMP charged autologous blood clot (BMP-group and the artificial blood clot only (ABC-group into the OT-gap. In vivo micro-computer tomography (µCT scans were performed after 2, 4 and 6 weeks. After 6 weeks specimens underwent histological analyses.In µCT examinations and histological analyses no bony bridging was observed in all but one animal in the ISH-group. In the BMP group complete bridging was achieved in all animals. The ABC-group showed less mineralised tissue formation and smaller bridging scores during the course of healing than the ISH-group.In this pilot study we introduce a model for impaired bone healing taking the major biological risk factors into account. We could show that the in situ fracture haematoma is essential for bone regeneration. Using BMP as a positive control the presented experimental setup can serve to evaluate innovative therapeutical concepts in long bone application.

  6. S113R mutation in SLC33A1 leads to neurodegeneration and augmented BMP signaling in a mouse model

    Directory of Open Access Journals (Sweden)

    Pingting Liu

    2017-01-01

    Full Text Available The S113R mutation (c.339T>G (MIM #603690.0001 in SLC33A1 (MIM #603690, an ER membrane acetyl-CoA transporter, has been previously identified in individuals with hereditary spastic paraplegia type 42 (SPG42; MIM #612539. SLC33A1 has also been shown to inhibit the bone morphogenetic protein (BMP signaling pathway in zebrafish. To better understand the function of SLC33A1, we generated and characterized Slc33a1S113R knock-in mice. Homozygous Slc33a1S113R mutant mice were embryonic lethal, whereas heterozygous Slc33a1 mutant mice (Slc33a1wt/mut exhibited behavioral abnormalities and central neurodegeneration, which is consistent with hereditary spastic paraplegia (HSP phenotypes. Importantly, we found an upregulation of BMP signaling in the nervous system and mouse embryonic fibroblasts of Slc33a1wt/mut mice. Using a sciatic nerve crush injury model in vivo and dorsal root ganglion (DRG culture in vitro we showed that injury-induced axonal regeneration in Slc33a1wt/mut mice was accelerated and mediated by upregulated BMP signaling. Exogenous addition of BMP signaling antagonist, noggin, could efficiently alleviate the accelerated injury-induced axonal regrowth. These results indicate that SLC33A1 can negatively regulate BMP signaling in mice, further supporting the notion that upregulation of BMP signaling is a common mechanism of a subset of hereditary spastic paraplegias.

  7. Gelatin-Derived Graphene–Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Yulong [Department of Orthopaedic; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Qazvini, Nader Taheri [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Zane, Kylie [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Sadati, Monirosadat [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Wei, Qiang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Liao, Junyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Fan, Jiaming [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Song, Dongzhe [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department of Conservative Dentistry and Endodontics, West China School of Stomatology, Sichuan University, Chengdu 610041, China; Liu, Jianxiang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; amp, Technology, Wuhan 430022, China; Ma, Chao [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Departments of Neurosurgery and Otolaryngology-Head; amp, Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China; Qu, Xiangyang [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Chen, Liqun [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Yu, Xinyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Zhicai [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; amp, Technology, Wuhan 430022, China; Zhao, Chen [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zeng, Zongyue [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Ruyi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Yan, Shujuan [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Wu, Tingting [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Departments of Neurosurgery and Otolaryngology-Head; amp, Neck Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430071, China; Wu, Xingye [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Shu, Yi [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Li, Yasha [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; Zhang, Wenwen [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department; Reid, Russell R. [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Department of Surgery, Section of Plastic; Lee, Michael J. [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Wolf, Jennifer Moritis [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Tirrell, Matthew [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; He, Tong-Chuan [Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, Illinois 60637, United States; Ministry of Education Key Laboratory of; de Pablo, Juan J. [Institute for Molecular Engineering, The University of Chicago, Chicago, Illinois 60637, United States; Argonne National Laboratory, Argonne, Illinois 60439, United States; Deng, Zhong-Liang [Department of Orthopaedic

    2017-05-04

    Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPAR gamma was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.

  8. Combination of BMP-2-releasing gelatin/β-TCP sponges with autologous bone marrow for bone regeneration of X-ray-irradiated rabbit ulnar defects.

    Science.gov (United States)

    Yamamoto, Masaya; Hokugo, Akishige; Takahashi, Yoshitake; Nakano, Takayoshi; Hiraoka, Masahiro; Tabata, Yasuhiko

    2015-07-01

    The objective of this study is to evaluate the feasibility of gelatin sponges incorporating β-tricalcium phosphate (β-TCP) granules (gelatin/β-TCP sponges) to enhance bone regeneration at a segmental ulnar defect of rabbits with X-ray irradiation. After X-ray irradiation of the ulnar bone, segmental critical-sized defects of 20-mm length were created, and bone morphogenetic protein-2 (BMP-2)-releasing gelatin/β-TCP sponges with or without autologous bone marrow were applied to the defects to evaluate bone regeneration. Both gelatin/β-TCP sponges containing autologous bone marrow and BMP-2-releasing sponges enhanced bone regeneration at the ulna defect to a significantly greater extent than the empty sponges (control). However, in the X-ray-irradiated bone, the bone regeneration either by autologous bone marrow or BMP-2 was inhibited. When combined with autologous bone marrow, the BMP-2 exhibited significantly high osteoinductivity, irrespective of the X-ray irradiation. The bone mineral content at the ulna defect was similar to that of the intact bone. It is concluded that the combination of bone marrow with the BMP-2-releasing gelatin/β-TCP sponge is a promising technique to induce bone regeneration at segmental bone defects after X-ray irradiation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

    Science.gov (United States)

    Shintani, Nahoko; Siebenrock, Klaus A; Hunziker, Ernst B

    2013-01-01

    Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2) induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2) and transforming growth factor beta 1 (TGF-ß1) were investigated. Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml) for 4 (or 6) weeks. FGF-2 (10 ng/ml) or TGF-ß1 (10 ng/ml) was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs) only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2), but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the

  10. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

    Directory of Open Access Journals (Sweden)

    Nahoko Shintani

    Full Text Available Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2 induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2 and transforming growth factor beta 1 (TGF-ß1 were investigated.Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml for 4 (or 6 weeks. FGF-2 (10 ng/ml or TGF-ß1 (10 ng/ml was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2, but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume.TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of clinical repair, our findings will be of importance in fine-tuning the

  11. Dual delivery of rhPDGF-BB and bone marrow mesenchymal stromal cells expressing the BMP2 gene enhance bone formation in a critical-sized defect model.

    Science.gov (United States)

    Park, Shin-Young; Kim, Kyoung-Hwa; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

    2013-11-01

    Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

  12. HIF-1α as a Regulator of BMP2-Induced Chondrogenic Differentiation, Osteogenic Differentiation, and Endochondral Ossification in Stem Cells

    Directory of Open Access Journals (Sweden)

    Nian Zhou

    2015-04-01

    Full Text Available Background/Aims: Joint cartilage defects are difficult to treat due to the limited self-repair capacities of cartilage. Cartilage tissue engineering based on stem cells and gene enhancement is a potential alternative for cartilage repair. Bone morphogenetic protein 2 (BMP2 has been shown to induce chondrogenic differentiation in mesenchymal stem cells (MSCs; however, maintaining the phenotypes of MSCs during cartilage repair since differentiation occurs along the endochondral ossification pathway. In this study, hypoxia inducible factor, or (HIF-1α, was determined to be a regulator of BMP2-induced chondrogenic differentiation, osteogenic differentiation, and endochondral bone formation. Methods: BMP2 was used to induce chondrogenic and osteogenic differentiation in stem cells and fetal limb development. After HIF-1α was added to the inducing system, any changes in the differentiation markers were assessed. Results: HIF-1α was found to potentiate BMP2-induced Sox9 and the expression of chondrogenesis by downstream markers, and inhibit Runx2 and the expression of osteogenesis by downstream markers in vitro. In subcutaneous stem cell implantation studies, HIF-1α was shown to potentiate BMP2-induced cartilage formation and inhibit endochondral ossification during ectopic bone/cartilage formation. In the fetal limb culture, HIF-1α and BMP2 synergistically promoted the expansion of the proliferating chondrocyte zone and inhibited chondrocyte hypertrophy and endochondral ossification. Conclusion: The results of this study indicated that, when combined with BMP2, HIF-1α induced MSC differentiation could become a new method of maintaining cartilage phenotypes during cartilage tissue engineering.

  13. Functionalization of deproteinized bovine bone with a coating-incorporated depot of BMP-2 renders the material efficiently osteoinductive and suppresses foreign-body reactivity.

    Science.gov (United States)

    Wu, Gang; Hunziker, Ernst B; Zheng, Yuanna; Wismeijer, Daniel; Liu, Yuelian

    2011-12-01

    The repair of critical-sized bony defects remains a challenge in the fields of implantology, maxillofacial surgery and orthopaedics. As an alternative bone-defect filler to autologous bone grafts, deproteinized bovine bone (DBB) is highly osteoconductive and clinically now widely used. However, this product suffers from the disadvantage of not being intrinsically osteoinductive. In the present study, this property was conferred by coating DBB with a layer of calcium phosphate into which bone morphogenetic protein 2 (BMP-2) was incorporated. Granules of DBB bearing a coating-incorporated depot of BMP-2--together with the appropriate controls (DBB bearing a coating but no BMP-2; uncoated DBB bearing adsorbed BMP-2; uncoated DBB bearing no BMP-2)--were implanted subcutaneously in rats. Five weeks later, the implants were withdrawn for a histomorphometric analysis of the volume densities of (i) bone, (ii) bone marrow, (iii) foreign-body giant cells and (iv) fibrous capsular tissue. Parameters (i) and (ii) were highest, whilst parameters (iii) and (iv) were lowest in association with DBB bearing a coating-incorporated depot of BMP-2. Hence, this mode of functionalization not only confers DBB with the property of osteoinductivity but also improves its biocompatibility--thus dually enhancing its clinical potential in the repair of bony defects. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. [The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

    Science.gov (United States)

    Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

    2003-09-01

    To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

  15. Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

    Science.gov (United States)

    Chen, Da-Fu; Zhou, Zhi-Yu; Dai, Xue-Jun; Gao, Man-Man; Huang, Bao-Ding; Liang, Tang-Zhao; Shi, Rui; Zou, Li-Jin; Li, Hai-Sheng; Bünger, Cody; Tian, Wei; Zou, Xue-Nong

    2014-03-01

    The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion (ALIF) model in pigs using equine bone protein extract (BPE), recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge (ACS), and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, miR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine (serotonin) receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

  16. Bone morphogenic protein: an elixir for bone grafting--a review.

    Science.gov (United States)

    Shah, Prasun; Keppler, Louis; Rutkowski, James

    2012-12-01

    Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor beta superfamily. This literature review focuses on the molecular biology of BMPs, their mechanism of action, and subsequent applications. It also discusses uses of BMPs in the fields of dentistry and orthopedics, research on methods of delivering BMPs, and their role in tissue regeneration. BMP has positive effects on bone grafts, and their calculated and timely use with other growth factors can provide extraordinary results in fractured or nonhealing bones. Use of BMP introduces new applications in the field of implantology and bone grafting. This review touches on a few unknown facts about BMP and this ever-changing field of research to improve human life.

  17. Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics

    Directory of Open Access Journals (Sweden)

    D Wulsten

    2011-02-01

    Full Text Available This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue.Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

  18. In vitro and in vivo evaluation of calcium phosphate composite scaffolds containing BMP-VEGF loaded PLGA microspheres for the treatment of avascular necrosis of the femoral head

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hao-Xuan [Department of Orthopedics, Shandong University Qilu Hospital, Jinan, Shandong (China); Zhang, Xiu-Ping [School of Public Health, Fudan University, Shanghai (China); Xiao, Gui-Yong [School of Materials Science and Engineering, Shandong University, Jinan, Shandong (China); Key Laboratory for Liquid–Solid Structural Evolution and Processing of Materials, Ministry of Education, Shandong University, Jinan, Shandong (China); Hou, Yong; Cheng, Lei; Si, Meng; Wang, Shuai-Shuai [Department of Orthopedics, Shandong University Qilu Hospital, Jinan, Shandong (China); Li, Yu-Hua, E-mail: qiluyuhua@126.com [Department of Orthopedics, Shandong University Qilu Hospital, Jinan, Shandong (China); Nie, Lin, E-mail: hoho05@126.com [Department of Orthopedics, Shandong University Qilu Hospital, Jinan, Shandong (China)

    2016-03-01

    Avascular necrosis of the femoral head (ANFH) is difficult to treat due to high pressure and hypoxia, and reduced levels of growth factors such as bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF). We generated a novel calcium phosphate (CPC) composite scaffold, which contains BMP-VEGF-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (BMP-VEGF-PLGA-CPC). The BMP-VEGF-loaded microspheres have an encapsulation efficiency of 89.15% for BMP, and 78.55% for VEGF. The BMP-VEGF-PLGA-CPC scaffold also demonstrated a porosity of 62% with interconnected porous structures, and pore sizes of 219 μm and compressive strength of 6.60 MPa. Additionally, bone marrow mesenchymal stem cells (BMSCs) were seeded on scaffolds in vitro. Further characterization showed that the BMP-VEGF-PLGA-CPC scaffolds were biocompatible and enhanced osteogenesis and angiogenesis in vitro. Using a rabbit model of ANFH, BMP-VEGF-PLGA-CPC scaffolds were implanted into the bone tunnels of core decompression in the femoral head for 6 and 12 weeks. Radiographic and histological analysis demonstrated that the BMP-VEGF-PLGA-CPC scaffolds exhibited good biocompatibility, and osteogenic and angiogenic activity in vivo. These results indicate that the BMP-VEGF-PLGA-CPC scaffold may improve the therapeutic effect of core decompression surgery and be used as a treatment for ANFH. - Highlights: • BMP-VEGF-PLGA-CPC scaffolds were biocompatible and enhanced osteogenesis and angiogenesis in vitro. • BMP-VEGF-PLGA-CPC scaffolds exhibited good biocompatibility, and osteogenic and angiogenic activity in vivo. • BMP-VEGF-PLGA-CPC scaffolds provided a new approach for the treatment of avascular necrosis of the femoral head (ANFH).

  19. In vitro and in vivo evaluation of calcium phosphate composite scaffolds containing BMP-VEGF loaded PLGA microspheres for the treatment of avascular necrosis of the femoral head

    International Nuclear Information System (INIS)

    Zhang, Hao-Xuan; Zhang, Xiu-Ping; Xiao, Gui-Yong; Hou, Yong; Cheng, Lei; Si, Meng; Wang, Shuai-Shuai; Li, Yu-Hua; Nie, Lin

    2016-01-01

    Avascular necrosis of the femoral head (ANFH) is difficult to treat due to high pressure and hypoxia, and reduced levels of growth factors such as bone morphogenetic protein (BMP), and vascular endothelial growth factor (VEGF). We generated a novel calcium phosphate (CPC) composite scaffold, which contains BMP-VEGF-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (BMP-VEGF-PLGA-CPC). The BMP-VEGF-loaded microspheres have an encapsulation efficiency of 89.15% for BMP, and 78.55% for VEGF. The BMP-VEGF-PLGA-CPC scaffold also demonstrated a porosity of 62% with interconnected porous structures, and pore sizes of 219 μm and compressive strength of 6.60 MPa. Additionally, bone marrow mesenchymal stem cells (BMSCs) were seeded on scaffolds in vitro. Further characterization showed that the BMP-VEGF-PLGA-CPC scaffolds were biocompatible and enhanced osteogenesis and angiogenesis in vitro. Using a rabbit model of ANFH, BMP-VEGF-PLGA-CPC scaffolds were implanted into the bone tunnels of core decompression in the femoral head for 6 and 12 weeks. Radiographic and histological analysis demonstrated that the BMP-VEGF-PLGA-CPC scaffolds exhibited good biocompatibility, and osteogenic and angiogenic activity in vivo. These results indicate that the BMP-VEGF-PLGA-CPC scaffold may improve the therapeutic effect of core decompression surgery and be used as a treatment for ANFH. - Highlights: • BMP-VEGF-PLGA-CPC scaffolds were biocompatible and enhanced osteogenesis and angiogenesis in vitro. • BMP-VEGF-PLGA-CPC scaffolds exhibited good biocompatibility, and osteogenic and angiogenic activity in vivo. • BMP-VEGF-PLGA-CPC scaffolds provided a new approach for the treatment of avascular necrosis of the femoral head (ANFH).

  20. Effect of Emdogain enamel matrix derivative and BMP-2 on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells.

    Science.gov (United States)

    Fawzy El-Sayed, Karim M; Dörfer, Christof; Ungefroren, Hendrick; Kassem, Neemat; Wiltfang, Jörg; Paris, Sebastian

    2014-07-01

    The objective of this study was to evaluate the effect of Emdogain (Enamel Matrix Derivative, EMD) and Bone Morphogenetic Protein-2 (BMP-2), either solely or in combination, on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells. Stem/progenitor cells were isolated from human alveolar bone proper, magnetically sorted using STRO-1 antibodies, characterized flowcytometrically for their surface markers' expression, and examined for colony formation and multilineage differentiation potential. Subsequently, cells were treated over three weeks with 100 μg/ml Emdogain (EMD-Group), or 100 ng/ml BMP-2 (BMP-Group), or a combination of 100 ng/ml BMP-2 and 100 μg/ml Emdogain (BMP/EMD-Group). Unstimulated stem/progenitor cells (MACS(+)-Group) and osteoblasts (OB-Group) served as controls. Osteogenic gene expression was analyzed using RTq-PCR after 1, 2 and 3 weeks (N = 3/group). Mineralized nodule formation was evaluated by Alizarin-Red staining. BMP and EMD up-regulated the osteogenic gene expression. The BMP Group showed significantly higher expression of Collagen-I, III, and V, Alkaline phosphatase and Osteonectin compared to MACS(+)- and OB-Group (p < 0.05; Two-way ANOVA/Bonferroni) with no mineralized nodule formation. Under in-vitro conditions, Emdogain and BMP-2 up-regulate the osteogenic gene expression of stem/progenitor cells. The combination of BMP-2 and Emdogain showed no additive effect and would not be recommended for a combined clinical stimulation. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

  1. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    International Nuclear Information System (INIS)

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-01-01

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  2. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  3. Controlled Retention of BMP-2-Derived Peptide on Nanofibers Based on Mussel-Inspired Adhesion for Bone Formation.

    Science.gov (United States)

    Lee, Jinkyu; Perikamana, Sajeesh Kumar Madhurakkat; Ahmad, Taufiq; Lee, Min Suk; Yang, Hee Seok; Kim, Do-Gyoon; Kim, Kyobum; Kwon, Bosun; Shin, Heungsoo

    2017-04-01

    Although bone morphogenetic protein-2 (BMP-2) has been frequently used to stimulate bone formation, it has several side effects to be addressed, including the difficulty in optimization of clinically relevant doses and unwanted induction of cancerous signaling processes. In this study, an osteogenic peptide (OP) derived from BMP-2 was investigated as a substitute for BMP-2. In vitro studies showed that OP was able to enhance the osteogenic differentiation and mineralization of human mesenchymal stem cells (hMSCs). The peptides were then conjugated onto biocompatible poly-ι-lactide electrospun nanofibers through polydopamine chemistry. Surface chemical analysis proved that more than 80% of the peptides were stably retained on the nanofiber surface after 8 h of polydopamine coating during at least 28 days, and the amount of peptides that was retained increased depending on the polydopamine coating time. For instance, about 65% of the peptides were retained on nanofibers after 4 h of polydopamine coating. Also, a relatively small dose of peptides could effectively induce bone formation in in vivo critical-sized defects on the calvarial bones of mice. More than 50.4% ± 16.9% of newly formed bone was filled within the defect after treatment with only 10.5 ± 0.6 μg of peptides. Moreover, these groups had similar elastic moduli and contact hardnesses with host bone. Taken together, our results suggest that polydopamine-mediated OP immobilized on nanofibers can modulate the retention of relatively short lengths of peptides, which might make this an effective therapeutic remedy to guide bone regeneration using a relatively small amount of peptides.

  4. MiR-22 Suppresses BMP7 in the Development of Cirrhosis

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    Dong Ji

    2015-06-01

    Full Text Available Background/Aims: New strategies for the prevention and treatment of cirrhosis are urgently needed for improving therapeutic outcome. A role of microRNAs (miRNAs in the pathogenesis of cirrhosis has been recently acknowledged, whereas the exact involved miRNAs as well as the associated molecular signaling pathways have not been determined. Specifically, the studies on the relationship between miR-22 and bone morphogenic protein 7 (BMP7 in the development of cirrhosis are lacking. Methods: We examined the correlation of the levels of miR-22 and bone morphogenic protein 7 (BMP7 in the liver biopsies from patients with cirrhosis. We examined overexpression or suppression of miR-22 on BMP7 in hepatocytes. We examined the binding of miR-22 to the 3'-UTR of BMP7 mRNA. Finally, in a carbon tetrachloride (CCl4-induced cirrhosis model in mice, we gave mice adeno-associated viruses carrying antisense of miR-22, and examined its effects on BMP7 levels and the hallmarks of cirrhosis. Results: The levels of miR-22 and BMP7 in the liver biopsies from patients were strongly and inversely correlated. MiR-22 inhibited BMP7 expression in hepatocytes, through directly binding the 3'-UTR of BMP7 mRNA. Expression of antisense miR-22 significantly attenuated the levels of liver fibrosis, portal hypertension and sodium retention caused by CCl4, possibly through upregulation of BMP7. Conclusions: MiR-22 promotes the development of cirrhosis through BMP7 suppression.

  5. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

    Science.gov (United States)

    Sorescu, George P.; Sykes, Michelle; Weiss, Daiana; Platt, Manu O.; Saha, Aniket; Hwang, Jinah; Boyd, Nolan; Boo, Yong C.; Vega, J. David; Taylor, W. Robert; hide

    2003-01-01

    Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

  6. Injectable perlecan domain 1-hyaluronan microgels potentiate the cartilage repair effect of BMP2 in a murine model of early osteoarthritis

    International Nuclear Information System (INIS)

    Srinivasan, Padma P; McCoy, Sarah Y; Yang Weidong; Farach-Carson, Mary C; Kirn-Safran, Catherine B; Jha, Amit K; Jia Xinqiao

    2012-01-01

    The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2-free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2-treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.

  7. A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

    Science.gov (United States)

    Guenther, Catherine A; Wang, Zhen; Li, Emma; Tran, Misha C; Logan, Catriona Y; Nusse, Roel; Pantalena-Filho, Luiz; Yang, George P; Kingsley, David M

    2015-08-01

    Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Mesenchymal Stem Cells Mitigate Cirrhosis through BMP7

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    Bing Li

    2015-01-01

    Full Text Available Background/Aims: Transplantation of mesenchymal stem cells (MSCs has therapeutic effects on various diseases, while its effect on developing cirrhosis as well as the underlying mechanism remained largely unknown. Methods: Twenty C57BL/6 mice were randomly separated into 2 groups of ten each. One group received transplantation of MSCs, while the other group received saline as control. The mice then received intraperitoneal injection of carbon tetrachloride (CCl4 twice per week for 8 weeks to develop cirrhosis. After another 4 weeks, the levels of cirrhosis in these mice were evaluated by liver fibrosis area, portal pressure, sodium balance and excretion. Transcripts of transforming growth factor β 1 (TGFβ1 and bone morphogenic protein 7 (BMP7 in the mouse livers were quantified by RT-qPCR. BMP7-depleted MSCs were prepared and applied in this model, and compared to MSCs. Results: Liver fibrosis, portal hypertension and sodium retention that were developed by CCl4, were all significantly alleviated by MSCs transplantation, which decreased TGFβ1 levels and increased BMP7 levels in the injured liver. MSCs were found to express extremely high levels of BMP7. Knockdown of BMP7 in MSCs completely abolished the protective effect of MSCs against CCl4-induced cirrhosis. Conclusions: MSCs mitigate cirrhosis through their production of BMP7 against the fibrogenic effect of TGFβ1 in the injured liver.

  9. Lack of Obvious Influence of PLLA Nanofibers on the Gene Expression of BMP-2 and VEGF during Growth and Differentiation of Human Mesenchymal Stem Cells

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    Markus D. Schofer

    2009-01-01

    Full Text Available Growth factors like bone morphogenetic protein 2 (BMP-2 and vascular endothelial growth factor (VEGF play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid (PLLA nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2–enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers.

  10. Lack of Obvious Influence of PLLA Nanofibers on the Gene Expression of BMP-2 and VEGF during Growth and Differentiation of Human Mesenchymal Stem Cells

    Science.gov (United States)

    Schofer, Markus D.; Fuchs-Winkelmann, S.; Wack, C.; Rudisile, M.; Dersch, R.; Leifeld, I.; Wendorff, J.; Greiner, A.; Paletta, J. R. J.; Boudriot, U.

    2009-01-01

    Growth factors like bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid) (PLLA) nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC) differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2–enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers. PMID:19412560

  11. Bone regeneration by gelatin hydro-gel seat containing BMP-2 and its application to canine orbital floor fracture Model

    International Nuclear Information System (INIS)

    Mochizuki, Yuichi

    2007-01-01

    Reported are preparation of the gel seat in the title (GHG) for sustained release of BMP-2 (bone morphogenetic protein-2) and its application to the fracture model of orbital floor of the dog. The chemically linked GHG was prepared from gelatin and glutaraldehyde and lyophilized. BMP-2 solution was dropped on the GHG seat to be contained there. To see the biobehavior of BMP-2 and GHG, they were labeled with 125 I and were given subcutaneously in the back of nude mice, of which remaining radioactivity was periodically measured by Aloka ARC-310B gamma counter. This experiment revealed the sustained release of BMP-2 along with degradation of the GHG. Then a complex of the GHG and bio-degradable polymer (L-lactide-ε-caprolactone) was prepared and implanted to the artificially fractured region (10 x 10 mm) of dog orbit floor, of which recovering process was evaluated by analysis of bone structure with soft X-ray (SOFRON, TRS-1005) roentgenography, histology, and micro-CT imaging (Comscantecno's Scan Xmate-A090S) for trabecular bone volume, thickness, number and separation. This experiment revealed that new bone was effectively induced to regenerate on the complex, of which structure was found similar to the normal trabecula. Thus in future, the complex can be useful for ideal treatment of the orbit floor fracture without necessity of donor. (R.T.)

  12. Spatial control of bone formation using a porous polymer scaffold co-delivering anabolic rhBMP-2 and anti-resorptive agents

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    NYC Yu

    2014-01-01

    Full Text Available Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid (PLGA scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg ± anti-resorptive agents: zoledronic acid (5 µg ZA, ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2 % HA or IkappaB kinase (IKK inhibitor (10 µg PS-1145. Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3 % TV, p < 0.01. The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1 % higher (p < 0.01. Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2 % and +52.0 %, respectively (p ≤ 0.01. Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.

  13. Bone regeneration of critical calvarial defect in goat model by PLGA/TCP/rhBMP-2 scaffolds prepared by low-temperature rapid-prototyping technology.

    Science.gov (United States)

    Yu, D; Li, Q; Mu, X; Chang, T; Xiong, Z

    2008-10-01

    Active artificial bone composed of poly lactide-co-glycolide (PLGA)/ tricalcium phosphate (TCP) was prefabricated using low-temperature rapid-prototyping technology so that the process of osteogenesis could be observed in it. PLGA and TCP were the primary materials, they were molded at low temperature, then recombinant human bone morphogenetic protein-2 (rhBMP-2) was added to form an active artificial bone. Goats with standard cranial defects were randomly divided into experimental (implants with rhBMP-2 added) and control (implants without rhBMP-2) groups, and osteogenesis was observed and evaluated by imaging and biomechanical and histological examinations. The PLGA-TCP artificial bone scaffold (90% porosity) had large and small pores of approximately 360microm and 3-5microm diameter. Preliminary and complete repair of the cranial defect in the goats occurred 12 and 24 weeks after surgery, respectively. The three-point bending strength of the repaired defects attained that of the normal cranium. In conclusion, low-temperature rapid-prototyping technology can preserve the biological activity of this scaffold material. The scaffold has a good three-dimensional structure and it becomes an active artificial bone after loading with rhBMP-2 with a modest degradation rate and excellent osteogenesis in the goat.

  14. Bases teóricas y aplicación clínica de las proteínas morfogenéticas óseas en cirugía maxilofacial Base theories and the clinical application of bone morphogenetic proteins in maxillofacial surgery

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    C.M. Ardila Medina

    2009-06-01

    Full Text Available Uno de los grandes avances en la neoformación ósea ha sido la identificación de factores de crecimiento importantes para ella como son las proteinas morfogenéticas óseas (PMO que regulan la diferenciación ósea y cartilaginosa in vivo. La depuración, clonación genética y expresión de las PMO han establecido las bases para el análisis celular y molecular del desarrollo y la regeneración ósea. El estudio genético de las PMO señala que son esenciales para la función normal animal y en la osteogénesis postfetal es importante en el desarrollo embrionario orgánico, esquelético y de los tejidos dentales y craneofaciales. La disponibilidad de las PMO proporciona retos y oportunidades para mejorar los conocimientos que regulan la regeneración ósea con el fin de optimizar los resultados en el paciente.One of the fundamental advances in bone neoformation has been the identification of important growth factors like the bone morphogenetic proteins that regulate live cartilage and bone differentiation. The cleansing, genetic cloning and expression of recombinant human bone morphogenetic proteins (BMP have laid the basis for cellular and molecular analysis of bone development and regeneration. The genetic study of the BMPs indicates that they are essential to the normal development and function of animals. BMP post-natal bone development is also very important in embryonic organic, skeletal, craniofacial and dental tissue development. The availability of BMPs provides several challenges and opportunities to improve insights into the mechanisms that regulate the regeneration of bone for optimal outcome in the patient.

  15. Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

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    Shriram Nallamshetty

    Full Text Available The effects of retinoids, the structural derivatives of vitamin A (retinol, on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA and its precursor all trans retinaldehyde (Rald, exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1, the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT demonstrated that Aldh1a1-deficient (Aldh1a1(-/- female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/- mice. In serum assays, Aldh1a1(-/- mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/- mesenchymal stem cells (MSCs expressed significantly higher levels of bone morphogenetic protein 2 (BMP2 and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/- mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/- mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

  16. Deficiency of retinaldehyde dehydrogenase 1 induces BMP2 and increases bone mass in vivo.

    Science.gov (United States)

    Nallamshetty, Shriram; Wang, Hong; Rhee, Eun-Jung; Kiefer, Florian W; Brown, Jonathan D; Lotinun, Sutada; Le, Phuong; Baron, Roland; Rosen, Clifford J; Plutzky, Jorge

    2013-01-01

    The effects of retinoids, the structural derivatives of vitamin A (retinol), on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA) and its precursor all trans retinaldehyde (Rald), exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1), the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT) demonstrated that Aldh1a1-deficient (Aldh1a1(-/-) ) female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT) mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1(-/-) mice. In serum assays, Aldh1a1(-/-) mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1(-/-) mesenchymal stem cells (MSCs) expressed significantly higher levels of bone morphogenetic protein 2 (BMP2) and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1(-/-) mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1(-/-) mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR)-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

  17. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling

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    Song eQin

    2014-04-01

    Full Text Available Neural stem cells (NSCs are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates BMP-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  18. Platelet-rich plasma stimulates osteoblastic differentiation in the presence of BMPs

    International Nuclear Information System (INIS)

    Tomoyasu, Akihiro; Higashio, Kanji; Kanomata, Kazuhiro; Goto, Masaaki; Kodaira, Kunihiko; Serizawa, Hiroko; Suda, Tatsuo; Nakamura, Atsushi; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2007-01-01

    Platelet-rich plasma (PRP) is clinically used as an autologous blood product to stimulate bone formation in vivo. In the present study, we examined the effects of PRP on proliferation and osteoblast differentiation in vitro in the presence of bone morphogenetic proteins (BMPs). PRP and its soluble fraction stimulated osteoblastic differentiation of myoblasts and osteoblastic cells in the presence of BMP-2, BMP-4, BMP-6 or BMP-7. The soluble PRP fraction stimulated osteoblastic differentiation in 3D cultures using scaffolds made of collagen or hydroxyapatite. Moreover, heparin-binding fractions obtained from serum also stimulated osteoblastic differentiation in the presence of BMP-4. These results suggested that platelets contain not only growth factors for proliferation but also novel potentiator(s) for BMP-dependent osteoblastic differentiation

  19. Dual Delivery of BMP-2 and bFGF from a New Nano-Composite Scaffold, Loaded with Vascular Stents for Large-Size Mandibular Defect Regeneration

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    Hang Zhao

    2013-06-01

    Full Text Available The aim of this study was to investigate the feasibility and advantages of the dual delivery of bone morphogenetic protein-2 (BMP-2 and basic fibroblast growth factor (bFGF from nano-composite scaffolds (PLGA/PCL/nHA loaded with vascular stents (PLCL/Col/nHA for large bone defect regeneration in rabbit mandibles. Thirty-six large bone defects were repaired in rabbits using engineering bone composed of allogeneic bone marrow mesenchymal stem cells (BMSCs, bFGF, BMP-2 and scaffolds composed of PLGA/PCL/nHA loaded with PLCL/Col/nHA. The experiments were divided into six groups: BMSCs/bFGF/BMP-2/scaffold, BMSCs/BMP-2/scaffold, BMSCs/bFGF/scaffold, BMSCs/scaffold, scaffold alone and no treatment. Sodium alginate hydrogel was used as the carrier for BMP-2 and bFGF and its features, including gelling, degradation and controlled release properties, was detected by the determination of gelation and degradation time coupled with a controlled release study of bovine serum albumin (BSA. AlamarBlue assay and alkaline phosphatase (ALP activity were used to evaluate the proliferation and osteogenic differentiation of BMSCs in different groups. X-ray and histological examinations of the samples were performed after 4 and 12 weeks post-implantation to clarify new bone formation in the mandible defects. The results verified that the use of sodium alginate hydrogel as a controlled release carrier has good sustained release ability, and the combined application of bFGF and BMP-2 could significantly promote the proliferation and osteogenic differentiation of BMSCs (p < 0.05 or p < 0.01. In addition, X-ray and histological examinations of the samples exhibited that the dual release group had significantly higher bone formation than the other groups. The above results indicate that the delivery of both growth factors could enhance new bone formation and vascularization compared with delivery of BMP-2 or bFGF alone, and may supply a promising way of repairing large

  20. BMP2 and BMP7 play antagonistic roles in feather induction

    Science.gov (United States)

    Michon, Frédéric; Forest, Loïc; Collomb, Elodie; Demongeot, Jacques; Dhouailly, Danielle

    2008-01-01

    Summary During embryonic development, feathers first appear as primordia consisting of an epidermal placode associated with a dermal condensation. In most previous studies, the BMPs have been proposed to function as inhibitors of the formation of cutaneous appendages. We showed that the function of BMPs is quite nuanced: BMP-2 and BMP-7, which are expressed in both skin components, act antagonistically and yet are both involved in the dermal condensations formation. BMP-7, the first to be expressed, is implicated in chemotaxis which leads to cell recruitment to the condensation, whereas BMP-2, which is expressed later, leads to an arrest of cell migration, likely via its modulation of EIIIA Fibronectin domain and α4-Integrin expression. We also propose a mathematical model, a reaction-diffusion system, based on cell proliferation, chemotaxis and the timing of BMP-2 and BMP-7 expression, which simulates the endogenous situation and reproduces the negative effects of excess BMP-2 or BMP-7 on feather patterning. PMID:18635609

  1. Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Chang Liu

    2018-03-01

    Full Text Available Background/Aims: Bone morphogenetic protein 15 (BMP15 and growth differentiation factor 9 (GDF9, which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. Methods: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs. Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8 analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. Results: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK

  2. Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

    International Nuclear Information System (INIS)

    Sung, Nak-Yun; Choi, Jong-il

    2015-01-01

    Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60 cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants - Highlights: • Demineralized bone matrix (DBM) was gamma-irradiated for sterilization. • Irradiated DBM had higher alkaline phosphatase and osteocalcin production. • It was reasoned the more released bone morphogenetic proteins by irradiation. • This result supports the application of radiation sterilization for bone implants

  3. AcvR1-mediated BMP signaling in second heart field is required for arterial pole development: implications for myocardial differentiation and regional identity.

    Science.gov (United States)

    Thomas, Penny S; Rajderkar, Sudha; Lane, Jamie; Mishina, Yuji; Kaartinen, Vesa

    2014-06-15

    BMP signaling plays an essential role in second heart field-derived heart and arterial trunk development, including myocardial differentiation, right ventricular growth, and interventricular, outflow tract and aortico-pulmonary septation. It is mediated by a number of different BMP ligands, and receptors, many of which are present simultaneously. The mechanisms by which they regulate morphogenetic events and degree of redundancy amongst them have still to be elucidated. We therefore assessed the role of BMP Type I receptor AcvR1 in anterior second heart field-derived cell development, and compared it with that of BmpR1a. By removing Acvr1 using the driver Mef2c[AHF]-Cre, we show that AcvR1 plays an essential role in arterial pole morphogenesis, identifying defects in outflow tract wall and cushion morphology that preceded a spectrum of septation defects from double outlet right ventricle to common arterial trunk in mutants. Its absence caused dysregulation in gene expression important for myocardial differentiation (Isl1, Fgf8) and regional identity (Tbx2, Tbx3, Tbx20, Tgfb2). Although these defects resemble to some degree those in the equivalent Bmpr1a mutant, a novel gene knock-in model in which Bmpr1a was expressed in the Acvr1 locus only partially restored septation in Acvr1 mutants. These data show that both BmpR1a and AcvR1 are needed for normal heart development, in which they play some non-redundant roles, and refine our understanding of the genetic and morphogenetic processes underlying Bmp-mediated heart development important in human congenital heart disease. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Radiographic and Histologic Evaluation of a Bone Void that Formed After Recombinant Human Bone Morphogenetic Protein-2-Mediated Sinus Graft Augmentation: A Case Report.

    Science.gov (United States)

    Kang, Hyun-Joo; Jun, Choong-Man; Yun, Jeong-Ho

    2016-01-01

    In the present case report, the authors describe radiographic and histologic observations of a bone void that formed after a sinus augmentation using a graft material that contained recombinant human bone morphogenetic protein-2 (rhBMP-2) and discuss clinical and histologic implications of their findings. Sinus augmentation was performed using a graft material comprising 1 g of hydroxyapatite/β-tricalcium phosphate, which contained 1 mg of rhBMP-2. Radiographic evaluation was conducted with panoramic radiographs and computed tomography images of the augmented maxillary sinus, which were analyzed using a three-dimensional image-reconstruction program. Histologic evaluation was also performed on a biopsy specimen obtained 6 months after the sinus augmentation. The total augmented volume increased from 1,582.2 mm(3) immediately after the sinus augmentation to 3,344.9 mm3 at 6 months after the augmentation because of the formation of a bone void. Twenty-six months after the sinus augmentation, the bone void remained but had reduced in volume, with the total augmented volume reduced to 2,551.7 mm(3). Histologically, new bone was observed to be in contact with the grafted particles, and a fatty marrow-like tissue was present in the area of the bone void. This case report shows that the bone void that had formed after sinus augmentation resolved over time and seemed to be partially replaced with new bone. Furthermore, none of the implants failed, and clinical adverse events were not observed during the follow-up period.

  5. Wnt and BMP signaling crosstalk in regulating dental stem cells: Implications in dental tissue engineering

    Directory of Open Access Journals (Sweden)

    Fugui Zhang

    2016-12-01

    Full Text Available Tooth is a complex hard tissue organ and consists of multiple cell types that are regulated by important signaling pathways such as Wnt and BMP signaling. Serious injuries and/or loss of tooth or periodontal tissues may significantly impact aesthetic appearance, essential oral functions and the quality of life. Regenerative dentistry holds great promise in treating oral/dental disorders. The past decade has witnessed a rapid expansion of our understanding of the biological features of dental stem cells, along with the signaling mechanisms governing stem cell self-renewal and differentiation. In this review, we first summarize the biological characteristics of seven types of dental stem cells, including dental pulp stem cells, stem cells from apical papilla, stem cells from human exfoliated deciduous teeth, dental follicle precursor cells, periodontal ligament stem cells, alveolar bone-derived mesenchymal stem cells (MSCs, and MSCs from gingiva. We then focus on how these stem cells are regulated by bone morphogenetic protein (BMP and/or Wnt signaling by examining the interplays between these pathways. Lastly, we analyze the current status of dental tissue engineering strategies that utilize oral/dental stem cells by harnessing the interplays between BMP and Wnt pathways. We also highlight the challenges that must be addressed before the dental stem cells may reach any clinical applications. Thus, we can expect to witness significant progresses to be made in regenerative dentistry in the coming decade.

  6. Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

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    Shepherd Trevor G

    2010-02-01

    Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and

  7. Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

    Directory of Open Access Journals (Sweden)

    David Garciadiego-Cázares

    Full Text Available The Integrin β1 family is the major receptors of the Extracellular matrix (ECM, and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA. In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β Superfamily, such as Growth differentiation factor 5 (Gdf-5 and Bone morphogenetic protein 7 (Bmp-7, play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh, Gdf-5 and α5 integrin to maintain articular cartilage and prevent

  8. Effect of bone morphogenetic protein-7 (BMP-7 on in vitro survival of caprine preantral follicles Efeito da proteína morfogenética óssea 7 (BMP-7 para a sobrevivência in vitro de folículos pré-antrais caprinos

    Directory of Open Access Journals (Sweden)

    Valdevane R. Araújo

    2010-04-01

    Full Text Available This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+ supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml. Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM. Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.O presente trabalho foi conduzido de modo a se verificar o efeito de diferentes concentrações da BMP-7 no desenvolvimento in vitro de folículos pré-antrais caprinos. Fragmentos de tecido cortical ovariano caprino foram cultivados por 1 ou 7 dias em Minimum Essential Medium (MEM+ suplementado com diferentes concentrações de BMP-7 (1, 10, 50 ou 100ng/ml. Os fragmentos não cultivados ou aqueles cultivados por 1 ou 7 dias foram processados para histologia clássica e microscopia eletrônica de transmissão (TEM, sendo avaliados parâmetros morfológicos indicativos de viabilidade, ativação e crescimento. Os resultados mostraram que o percentual de folículos morfologicamente normais diminuiu significativamente em

  9. CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

    DEFF Research Database (Denmark)

    Mönnich, Maren; Borgeskov, Louise; Breslin, Loretta

    2018-01-01

    The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends...... on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure...... but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss...

  10. TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

    Science.gov (United States)

    Murphy, Meghan K; Huey, Daniel J; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-03-01

    Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs. © 2014 AlphaMed Press.

  11. Local rhBMP-12 on an Absorbable Collagen Sponge as an Adjuvant Therapy for Rotator Cuff Repair - A Phase 1, Randomized, Standard of Care Control, Multicenter Study: Safety and Feasibility.

    Science.gov (United States)

    Greiner, Stefan; Ide, Junji; Van Noort, Arthur; Mochizuki, Yu; Ochi, Hiroshi; Marraffino, Shannon; Sridharan, Sudhakar; Rudicel, Sally; Itoi, Eiji

    2015-08-01

    Recombinant human bone morphogenetic protein-12 (rhBMP-12) has been shown to induce tendon and ligament formation in rats and to improve tendon healing; however, the safety and feasibility of implanting rhBMP-12/absorbable collagen sponge (ACS) in humans are not known. To investigate the safety and feasibility of rhBMP-12 on an ACS as an adjuvant therapy in open rotator cuff repair. Randomized controlled trial; Level of evidence, 2. This study consisted of 20 patients with full-thickness rotator cuff tears. Patients were randomized either to standard of care (SOC) treatment (open rotator cuff repair) or to receive 0.015 mg/mL rhBMP-12/ACS and SOC treatment during their open rotator cuff repair (rhBMP-12/ACS group) at a rate of 1/4 SOC/rhBMP-12/ACS. The feasibility of implanting the product and the safety of the product were evaluated during the 1-year follow-up period. The evaluation involved up to 10 postoperative visits, which included physical examinations, radiographs, computed tomography (CT) scans, magnetic resonance imaging (MRI) scans with an emphasis on heterotopic ossification (HO), pharmacokinetics, immunogenicity, laboratory evaluations, and local and systemic adverse events at specified time points. Small amounts of HO were seen on follow-up CT scans in 10 of 16 patients in the rhBMP-12/ACS group and in 2 of 3 patients in the SOC group. HO did not increase at 26 weeks and was not associated with any adverse events or unsatisfactory clinical outcomes. Pharmacokinetics demonstrated that circulating levels of rhBMP-12 were not detectable after administration. Five of 16 patients showed a postoperative immunogenic response but did not show any correlating adverse events. Complete healing of the rotator cuff was observed in 14 of 16 patients; 2 of 16 imaging results could not be analyzed because of artifacts in the rhBMP-12 group on MRI scans. In the SOC group, 1 of 4 patients showed a retear at 12 weeks after surgery. The use of rhBMP-12/ACS has been shown

  12. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Science.gov (United States)

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  13. Twisted Gastrulation as a BMP Modulator during Mammary Gland Development and Tumorigenesis

    Science.gov (United States)

    2014-05-01

    tissues ( Bonilla -Claudio et al., 2012). In WT TEBs (Fig. 8A–D) and mature ducts (Fig. 8I–L) almost all luminal cells expressed GATA-3. In contrast, Twsg1...PubMed: 12952901] Bonilla -Claudio M, Wang J, Bai Y, Klysik E, Selever J, Martin JF. Bmp signaling regulates a dosedependent transcriptional...deficient mice. Arch Oral Biol. 2006; 51:433–8. [PubMed: 16289463] Metallo CM, Ji L, de Pablo JJ, Palecek SP. Retinoic Acid and Bone Morphogenetic

  14. Cryogenic 3D printing for producing hierarchical porous and rhBMP-2-loaded Ca-P/PLLA nanocomposite scaffolds for bone tissue engineering.

    Science.gov (United States)

    Wang, Chong; Zhao, Qilong; Wang, Min

    2017-06-07

    The performance of bone tissue engineering scaffolds can be assessed through cell responses to scaffolds, including cell attachment, infiltration, morphogenesis, proliferation, differentiation, etc, which are determined or heavily influenced by the composition, structure, mechanical properties, and biological properties (e.g. osteoconductivity and osteoinductivity) of scaffolds. Although some promising 3D printing techniques such as fused deposition modeling and selective laser sintering could be employed to produce biodegradable bone tissue engineering scaffolds with customized shapes and tailored interconnected pores, effective methods for fabricating scaffolds with well-designed hierarchical porous structure (both interconnected macropores and surface micropores) and tunable osteoconductivity/osteoinductivity still need to be developed. In this investigation, a novel cryogenic 3D printing technique was investigated and developed for producing hierarchical porous and recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium phosphate (Ca-P) nanoparticle/poly(L-lactic acid) nanocomposite scaffolds, in which the Ca-P nanoparticle-incorporated scaffold layer and rhBMP-2-encapsulated scaffold layer were deposited alternatingly using different types of emulsions as printing inks. The mechanical properties of the as-printed scaffolds were comparable to those of human cancellous bone. Sustained releases of Ca 2+ ions and rhBMP-2 were achieved and the biological activity of rhBMP-2 was well-preserved. Scaffolds with a desirable hierarchical porous structure and dual delivery of Ca 2+ ions and rhBMP-2 exhibited superior performance in directing the behaviors of human bone marrow-derived mesenchymal stem cells and caused improved cell viability, attachment, proliferation, and osteogenic differentiation, which has suggested their great potential for bone tissue engineering.

  15. Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model

    Directory of Open Access Journals (Sweden)

    Boos AM

    2014-11-01

    Full Text Available Anja M Boos,1,* Annika Weigand,1,* Gloria Deschler,1 Thomas Gerber,2 Andreas Arkudas,1 Ulrich Kneser,1 Raymund E Horch,1 Justus P Beier11Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg FAU, Erlangen, 2Institute of Physics, University of Rostock, Rostock, Germany *These authors contributed equally to this work Abstract: New therapeutic strategies are required for critical size bone defects, because the gold standard of transplanting autologous bone from an unharmed area of the body often leads to several severe side effects and disadvantages for the patient. For years, tissue engineering approaches have been seeking a stable, axially vascularized transplantable bone replacement suitable for transplantation into the recipient bed with pre-existing insufficient conditions. For this reason, the arteriovenous loop model was developed and various bone substitutes have been vascularized. However, it has not been possible thus far to engineer a primary stable and axially vascularized transplantable bone substitute. For that purpose, a primary stable silica-embedded nanohydroxyapatite (HA bone substitute in combination with blood, bone marrow, expanded, or directly retransplanted mesenchymal stem cells, recombinant human bone morphogenetic protein 2 (rhBMP-2, and different carrier materials (fibrin, cell culture medium, autologous serum was tested subcutaneously for 4 or 12 weeks in the sheep model. Autologous serum lead to an early matrix change during degradation of the bone substitute and formation of new bone tissue. The best results were achieved in the group combining mesenchymal stem cells expanded with 60 µg/mL rhBMP-2 in autologous serum. Better ingrowth of fibrovascular tissue could be detected in the autologous serum group compared with the control (fibrin. Osteoclastic activity indicating an active bone remodeling process was observed after 4 weeks, particularly

  16. Macrophages support splenic erythropoiesis in 4T1 tumor-bearing mice.

    Directory of Open Access Journals (Sweden)

    Min Liu

    Full Text Available Anemia is a common complication of cancer; a role of spleen in tumor-stress erythropoiesis has been suggested. However, the molecular mechanisms involved in the splenic erythropoiesis following tumor maintenance remain poorly understood. Here we show that tumor development blocks medullar erythropoiesis by granulocyte colony-stimulating factor (G-CSF and then causes anemia in murine 4T1 breast tumor-bearing mice. Meanwhile, tumor-stress promotes splenic erythropoiesis. Splenectomy worsened tumor-induced anemia, and reduced tumor volume and tumor weight, indicating the essential role of spleen in tumor-stress erythropoiesis and tumor growth. Tumor progression of these mice led to increased amounts of bone morphogenetic protein 4 (BMP4 in spleen. The in vivo role of macrophages in splenic erythropoiesis under tumor-stress conditions was investigated. Macrophage depletion by injecting liposomal clodronate decreased the expression of BMP4, inhibited splenic erythropoiesis, aggravated the tumor-induced anemia and suppressed tumor growth. Our results provide insight that macrophages and BMP4 are positive regulators of splenic erythropoiesis in tumor pathological situations. These findings reveal that during the tumor-stress period, the microenvironment of the spleen is undergoing changes, which contributes to adopt a stress erythropoietic fate and supports the expansion and differentiation of stress erythroid progenitors, thereby replenishing red blood cells and promoting tumor growth.

  17. Surface functionalization of nanoporous alumina with bone morphogenetic protein 2 for inducing osteogenic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Song, Yuanhui; Ju, Yang; Morita, Yasuyuki; Xu, Baiyao; Song, Guanbin

    2014-01-01

    Many studies have demonstrated the possibility to regulate cellular behavior by manipulating the specific characteristics of biomaterials including the physical features and chemical properties. To investigate the synergistic effect of chemical factors and surface topography on the growth behavior of mesenchymal stem cells (MSCs), bone morphorgenic protein 2 (BMP2) was immobilized onto porous alumina substrates with different pore sizes. The BMP2-immobilized alumina substrates were characterized with scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Growth behavior and osteogenic differentiation of MSCs cultured on the different substrates were investigated. Cell adhesion and morphological changes were observed with SEM, and the results showed that the BMP2-immobilized alumina substrate was able to promote adhesion and spreading of MSCs. MTT assay and immunofluorescence staining of integrin β1 revealed that the BMP2-immobilized alumina substrates were favorable for cell growth. To evaluate the differentiation of MSCs, osteoblastic differentiation markers, such as alkaline phosphatase (ALP) activity and mineralization, were investigated. Compared with those of untreated alumina substrates, significantly higher ALP activities and mineralization were detected in cells cultured on BMP2-immobilized alumina substrates. The results suggested that surface functionalization of nanoporous alumina substrates with BMP2 was beneficial for cell growth and osteogenic differentiation. With the approach of immobilizing growth factors onto material substrates, it provided a new insight to exploit novel biofunctional materials for tissue engineering. - Highlights: • BMP2 was immobilized onto nanoporous alumina substrates with different pore sizes. • BMP2-immobilized substrates were able to promote adhesion and spreading of MSCs. • BMP2-immobilized substrates were favorable for cell growth of MSCs. • BMP2-immobilized substrates promoted osteogenic

  18. Diverse bone morphogenetic protein expression profiles and smad pathway activation in different phenotypes of experimental canine mammary tumors.

    Directory of Open Access Journals (Sweden)

    Helena Wensman

    Full Text Available BACKGROUND: BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones. CONCLUSIONS/SIGNIFICANCE: We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors.

  19. Calcium citrate: a new biomaterial that can enhance bone formation in situ

    Directory of Open Access Journals (Sweden)

    WANG Li-ming

    2012-11-01

    Full Text Available 【Abstract】 Objective: To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 (rhBMP-2 on bone regeneration in a bone defect rabbit model. Methods: Totally 30 male New Zealand white rabbits were randomly and equally divided into calcium citrate-rhBMP-2 (CC-rhBMP-2 group and rhBMP-2 only group. Two 10 mm-long and 5 mm-deep bone defects were respec-tively created in the left and right femoral condyles of the rabbits. Subsequently 5 pellets of calcium citrate (10 mg combined with rhBMP-2 (2 mg or rhBMP-2 alone were im-planted into the bone defects and compressed with cotton swab. Bone granules were obtained at 2, 4 and 6 weeks after procedure and received histological analysis. LSD t-test and a subsequent t-test were adopted for statistical analysis. Results: Histomorphometric analysis revealed newly formed bones, and calcium citrate has been absorbed in the treatment group. The percent of newly formed bone area in femoral condyle in control group and CC-rhBMP-2 group was respectively 31.73%±1.26% vs 48.21%±2.37% at 2 weeks; 43.40%±1.65% vs 57.32%±1.47% at 4 weeks, and 51.32%±7.80% vs 66.74%±4.05% at 6 weeks (P<0.05 for all. At 2 weeks, mature cancellous bone was observed to be already formed in the treatment group. Conclusion: From this study, it can be concluded that calcium citrate combined with rhBMP-2 signifcantly en-hances bone regeneration in bone defects. This synthetic gelatin matrix stimulates formation of new bone and bone marrow in the defect areas by releasing calcium ions. Key words: Bone morphogenetic protein-2; Biocompatible materials; Calcium citrate; Gelatin

  20. Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Knudsen, Kirsten Quyen Nguyen; Olesen, Ping; Ledet, Thomas

    2007-01-01

    The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformat......The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved...... in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL......) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were...

  1. Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin.

    Science.gov (United States)

    Szláma, György; Vásárhelyi, Viktor; Trexler, Mária; Patthy, László

    2016-12-01

    The NTR domain of WFIKKN1 protein has been shown to have significant affinity for the prodomain regions of promyostatin and latent myostatin but the biological significance of these interactions remained unclear. In view of its role as a myostatin antagonist, we tested the assumption that WFIKKN1 inhibits the release of myostatin from promyostatin and/or latent myostatin. WFIKKN1 was found to have no effect on processing of promyostatin by furin, the rate of cleavage of latent myostatin by BMP1, however, was significantly enhanced in the presence of WFIKKN1 and this enhancer activity was superstimulated by heparin. Unexpectedly, WFIKKN1 was also cleaved by BMP1 and our studies have shown that the KKN1 fragment generated by BMP1-cleavage of WFIKKN1 contributes most significantly to the observed enhancer activity. Analysis of a pro-TGF-β -based homology model of homodimeric latent myostatin revealed that the BMP1-cleavage sites are buried and not readily accessible to BMP1. In view of this observation, the most plausible explanation for the BMP1-enhancer activity of the KKN1 fragment is that it shifts a conformational equilibrium of latent myostatin from the closed circular structure of the homodimer to a more open form, making the cleavage sites more accessible to BMP1. On the other hand, the observation that the enhancer activity of KKN1 is superstimulated in the presence of heparin is explained by the fact KKN1, latent myostatin, and BMP1 have affinity for heparin and these interactions with heparin increase the local concentrations of the reactants thereby facilitating the action of BMP1. Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  2. Noggin and Wnt3a enable BMP4-dependent differentiation of telencephalic stem cells into GluR-agonist responsive neurons

    DEFF Research Database (Denmark)

    Andersson, Therese; Duckworth, Joshua K; Fritz, Nicolas

    2011-01-01

    levels, that in turn exerted a concentration-dependent inhibition of BMP4-mediated mesenchymal differentiation of NSCs. Instead, BMP4 exposure of NSCs induced neuronal differentiation in mesenchyme-preventing conditions, whereas treatment with recombinant noggin alone did not. Wnt signaling is known...... to be essential for the development of neurons derived from the dorsal telencephalon, and co-stimulation of NSCs with BMP4+Wnt3a resulted in a synergistic effect yielding significantly increased number of mature neurons compared to stimulation with each factor alone. Thus whereas only a subset of BMP4-induced...... neurons derived from telencephalic NSCs, responded to glutamate receptor (GluR) agonists, over 80% of BMP4+Wnt3a-induced neurons responded appropriately to GluR-agonists. Our results increase the understanding of the role for BMP4 in differentiation of telencephalic multipotent progenitors, and reveal...

  3. The cholinergic-inducing effect of BMP4 on rat's cerebral neural stem cells

    International Nuclear Information System (INIS)

    Chang Yan; Xue Yilong; Luo Yun; Tian Lei; Pan Jingkun; Cui Xin

    2004-01-01

    The cholinergic-inducing effect of BMP4 on isolated and cultivated rat's cerebral neural stem cells (NSC) was examined. NSC isolated from two months old rat's brain region like hippocampus and striatum was cultivated in a DMEM/F12 medium containing EGF and bFGF, and was identified with morphological character and nestin immunocytochemistry test. After 24 hours, cultivating the NSC with the BMP4-added medium for 7-8 days, then the microscopical change were observed, ChAT and nestin double-labelling immunocytochemistry test was done. Results showed that about 34% NSC of neuron-like character was observed by microscope in the paper. That ChAT-positive cells coexist with nestin-positive cells was found by immunocytochemistry test. There were 28% ChAT-positive cells and 38% nestin-positive cells in the study. Cholinergic neurons differentiated from NSC could be induced by adding BMP4 to the medium

  4. Bone Morphogenetic Proteins and myostatin pathways: key mediator of human sarcopenia.

    Science.gov (United States)

    Scimeca, Manuel; Piccirilli, Eleonora; Mastrangeli, Francesca; Rao, Cecilia; Feola, Maurizio; Orlandi, Augusto; Gasbarra, Elena; Bonanno, Elena; Tarantino, Umberto

    2017-02-15

    Sarcopenia, osteoporosis and osteoarthritis are the most frequent musculoskeletal disorders affecting older people. The main aim of this study was to test the hypothesis that the balance between BMPs and myostatin pathways regulates the age-related muscle degeneration in OP and OA patients. To this end, we investigated the relationship among the expression of BMP-2/4-7, myostatin and phosphorylated Smads1-5-8 and the muscle quality, evaluated in term of fibers atrophy and satellite cells activity. In this retrospective study, we collected 123 biopsies of vastus lateralis: 48 biopsies from patients who underwent hip arthroplasty for subcapital fractures of the femur (OP), 55 biopsies from patients who underwent hip arthroplasty for osteoarthritis (OA) and 20 biopsies from patients who underwent hip arthroplasty for high-energy hip fractures (CTRL). Muscle biopsies were fixed in 4% paraformaldehyde and paraffin embedded. Serial sections were used for morphometrical and immunohistochemical analysis (BMP/2/4-7, myostatin, Smads1-5-8, Pax7 and myogenin). In addition, 1 mm 3 of muscle tissue of each patient was embedded in epon for ultrastructural study. Morphometric data indicated an increase of the number of atrophic fibers in OP patients compare to OA. In line with these data, we found an high regenerative potential in muscle tissues of OA patients due to the significant amount of both Pax7 and myogenin positive satellite cells detected in OA group. In addition, our data showed the decrease of BMP2/4 and -7 expression in OP patients compared to both OA group and CTRL. Conversely, OP patients were characterized by high levels of myostatin expression. A different expression profile was also found for phosphorylated Smad1-5-8 between OP and OA patients. In particular, OP patients showed a low number of positive phosphorylated Smad1-5-8 nuclei. The identification of molecular pathways involved in the pathogenesis of sarcopenia open new prospective for the development of

  5. Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD.

    Science.gov (United States)

    White, Courtney L; Kitich, Aleksandar; Gober, James W

    2010-05-01

    In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.

  6. Development and Assessment of a 3D-Printed Scaffold with rhBMP-2 for an Implant Surgical Guide Stent and Bone Graft Material: A Pilot Animal Study

    Directory of Open Access Journals (Sweden)

    Ji Cheol Bae

    2017-12-01

    Full Text Available In this study, a new concept of a 3D-printed scaffold was introduced for the accurate placement of an implant and the application of a recombinant human bone morphogenetic protein-2 (rhBMP-2-loaded bone graft. This preliminary study was conducted using two adult beagles to evaluate the 3D-printed polycaprolactone (PCL/β-tricalcium phosphate (β-TCP/bone decellularized extracellular matrix (bdECM scaffold conjugated with rhBMP-2 for the simultaneous use as an implant surgical guide stent and bone graft material that promotes new bone growth. Teeth were extracted from the mandible of the beagle model and scanned by computed tomography (CT to fabricate a customized scaffold that would fit the bone defect. After positioning the implant guide scaffold, the implant was placed and rhBMP-2 was injected into the scaffold of the experimental group. The two beagles were sacrificed after three months. The specimen block was obtained and scanned by micro-CT. Histological analysis showed that the control and experimental groups had similar new bone volume (NBV, % but the experimental group with BMP exhibited a significantly higher bone-to-implant contact ratio (BIC, %. Within the limitations of this preliminary study, a 3D-printed scaffold conjugated with rhBMP-2 can be used simultaneously as an implant surgical guide and a bone graft in a large bone defect site. Further large-scale studies will be needed to confirm these results.

  7. Development and Assessment of a 3D-Printed Scaffold with rhBMP-2 for an Implant Surgical Guide Stent and Bone Graft Material: A Pilot Animal Study

    Science.gov (United States)

    Bae, Ji Cheol; Lee, Jin-Ju; Shim, Jin-Hyung; Park, Keun-Ho; Lee, Jeong-Seok; Bae, Eun-Bin; Choi, Jae-Won; Huh, Jung-Bo

    2017-01-01

    In this study, a new concept of a 3D-printed scaffold was introduced for the accurate placement of an implant and the application of a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded bone graft. This preliminary study was conducted using two adult beagles to evaluate the 3D-printed polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP)/bone decellularized extracellular matrix (bdECM) scaffold conjugated with rhBMP-2 for the simultaneous use as an implant surgical guide stent and bone graft material that promotes new bone growth. Teeth were extracted from the mandible of the beagle model and scanned by computed tomography (CT) to fabricate a customized scaffold that would fit the bone defect. After positioning the implant guide scaffold, the implant was placed and rhBMP-2 was injected into the scaffold of the experimental group. The two beagles were sacrificed after three months. The specimen block was obtained and scanned by micro-CT. Histological analysis showed that the control and experimental groups had similar new bone volume (NBV, %) but the experimental group with BMP exhibited a significantly higher bone-to-implant contact ratio (BIC, %). Within the limitations of this preliminary study, a 3D-printed scaffold conjugated with rhBMP-2 can be used simultaneously as an implant surgical guide and a bone graft in a large bone defect site. Further large-scale studies will be needed to confirm these results. PMID:29258172

  8. Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

    Science.gov (United States)

    Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-08-01

    Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. © 2015 Wiley Periodicals, Inc.

  9. BMP-2, hypoxia, and COL1A1/HtrA1 siRNAs favor neo-cartilage hyaline matrix formation in chondrocytes.

    Science.gov (United States)

    Ollitrault, David; Legendre, Florence; Drougard, Carole; Briand, Mélanie; Benateau, Hervé; Goux, Didier; Chajra, Hanane; Poulain, Laurent; Hartmann, Daniel; Vivien, Denis; Shridhar, Vijayalakshmi; Baldi, Alfonso; Mallein-Gerin, Frédéric; Boumediene, Karim; Demoor, Magali; Galera, Philippe

    2015-02-01

    Osteoarthritis (OA) is an irreversible pathology that causes a decrease in articular cartilage thickness, leading finally to the complete degradation of the affected joint. The low spontaneous repair capacity of cartilage prevents any restoration of the joint surface, making OA a major public health issue. Here, we developed an innovative combination of treatment conditions to improve the human chondrocyte phenotype before autologous chondrocyte implantation. First, we seeded human dedifferentiated chondrocytes into a collagen sponge as a scaffold, cultured them in hypoxia in the presence of a bone morphogenetic protein (BMP), BMP-2, and transfected them with small interfering RNAs targeting two markers overexpressed in OA dedifferentiated chondrocytes, that is, type I collagen and/or HtrA1 serine protease. This strategy significantly decreased mRNA and protein expression of type I collagen and HtrA1, and led to an improvement in the chondrocyte phenotype index of differentiation. The effectiveness of our in vitro culture process was also demonstrated in the nude mouse model in vivo after subcutaneous implantation. We, thus, provide here a new protocol able to favor human hyaline chondrocyte phenotype in primarily dedifferentiated cells, both in vitro and in vivo. Our study also offers an innovative strategy for chondrocyte redifferentiation and opens new opportunities for developing therapeutic targets.

  10. Release and Cytokine Production of BmpB from BmpB-Loaded pH-Sensitive and Mucoadhesive Thiolated Eudragit Microspheres.

    Science.gov (United States)

    Singh, Bijay; Jiang, Tao; Kim, You-Kyoung; Kang, Sang-Kee; Choi, Yun-Jaie; Cho, Chong-Su

    2015-01-01

    Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 μ. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.

  11. Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation

    International Nuclear Information System (INIS)

    Kadouchi, Ichiro; Sakamoto, Kei; Tangjiao, Liu; Murakami, Takashi; Kobayashi, Eiji; Hoshino, Yuichi; Yamaguchi, Akira

    2009-01-01

    Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

  12. Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells.

    Science.gov (United States)

    Son, Hyo-Eun; Kim, Eun-Jung; Jang, Won-Gu

    2018-01-15

    Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells. Copyright © 2017. Published by Elsevier Inc.

  13. Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Jodie M Fleming

    Full Text Available Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR. Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2 was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers.

  14. Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

    Science.gov (United States)

    Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

    2017-07-01

    Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic

  15. Effects of piezosurgery in accelerating the movement of orthodontic alveolar bone tooth of rats and the expression mechanism of BMP-2.

    Science.gov (United States)

    Han, Jinyou; He, Hong

    2016-11-01

    The aim of the study was to investigate the effects of piezosurgery in accelerating the movement of orthodontic alveolar bone tooth of rats and the expression mechanism of bone morphogenetic protein-2 (BMP-2). Adult male Wistar rats (n=30), with an age range of 14-15 weeks, and an average weight of 250±16 g were used. The animals were randomly divided into the control and observation groups. The rats in the control group were injected with 25-dihydroxyvitamin (1,25-dihydroxycholecalciferol) into their dental ligament. The rats in the observation group were placed with an orthodontic device between the first molar and central incisor in the maxillary. On the first day after animal treatment, piezosurgery stimulation was performed on the first molar in maxillary. The changes of the movement distance of the first molar and gum surface temperature on days 1, 3, 5, 7 and 14 were then compared. Immunohistochemical staining was performed to detect the expression of BMP-2 of periodontal tissue in the tension side of the first molar. Tooth movement distance in the observation group on days 5, 7 and 14 was significantly longer than that in the control group (ppiezosurgery may significantly accelerate the movement of orthodontic alveolar bone tooth of rats and be associated with an increasing BMP-2 expression.

  16. Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

    International Nuclear Information System (INIS)

    Kihara, Tasuku; Ichikawa, Saki; Yonezawa, Takayuki; Lee, Ji-Won; Akihisa, Toshihiro; Woo, Je Tae; Michi, Yasuyuki; Amagasa, Teruo; Yamaguchi, Akira

    2011-01-01

    Research highlights: → Acerogenin A stimulated osteoblast differentiation in osteogenic cells. → Acerogenin A-induced osteoblast differentiation was inhibited by noggin. → Acerogenin A increased Bmp-2, Bmp-4 and Bmp-7 mRNA expression in MC3T3-E1 cells. → Acerogenin A is a candidate agent for stimulating bone formation. -- Abstract: We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.

  17. * Calvarial Bone Regeneration Is Enhanced by Sequential Delivery of FGF-2 and BMP-2 from Layer-by-Layer Coatings with a Biomimetic Calcium Phosphate Barrier Layer.

    Science.gov (United States)

    Gronowicz, Gloria; Jacobs, Emily; Peng, Tao; Zhu, Li; Hurley, Marja; Kuhn, Liisa T

    2017-12-01

    A drug delivery coating for synthetic bone grafts has been developed to provide sequential delivery of multiple osteoinductive factors to better mimic aspects of the natural regenerative process. The coating is composed of a biomimetic calcium phosphate (bCaP) layer that is applied to a synthetic bone graft and then covered with a poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) film. Bone morphogenetic protein-2 (BMP-2) was applied before the coating process directly on the synthetic bone graft and then, bCaP-PEM was deposited followed by adsorption of fibroblast growth factor-2 (FGF-2) into the PEM layer. Cells access the FGF-2 immediately, while the bCaP-PEM temporally delays the cell access to BMP-2. In vitro studies with cells derived from mouse calvarial bones demonstrated that Sca-1 and CD-166 positive osteoblast progenitor cells proliferated in response to media dosing with FGF-2. Coated scaffolds with BMP-2 and FGF-2 were implanted in mouse calvarial bone defects and harvested at 1 and 3 weeks. After 1 week in vivo, proliferation of cells, including Sca-1+ progenitors, was observed with low dose FGF-2 and BMP-2 compared to BMP-2 alone, indicating that in vivo delivery of FGF-2 activated a similar population of cells as shown by in vitro testing. At 3 weeks, FGF-2 and BMP-2 delivery increased bone formation more than BMP-2 alone, particularly in the center of the defect, confirming that the proliferation of the Sca-1 positive osteoprogenitors by FGF-2 was associated with increased bone healing. Areas of bone mineralization were positive for double fluorochrome labeling of calcium and alkaline phosphatase staining of osteoblasts, along with increased TRAP+ osteoclasts, demonstrating active bone formation distinct from the bone-like collagen/hydroxyapatite scaffold. In conclusion, the addition of a bCaP layer to PEM delayed access to BMP-2 and allowed the FGF-2 stimulated progenitors to populate the scaffold before differentiating in

  18. Combined Inhibition of the BMP pathway and the RANK-RANKL axis in a Mixed Lytic/blastic Prostate Cancer Lesion

    Science.gov (United States)

    Virk, Mandeep S.; Alaee, Farhang; Petrigliano, Frank A.; Sugiyama, Osamu; Chatziioannou, Arion F.; Stout, David; Dougall, William C.; Lieberman, Jay R.

    2010-01-01

    The purpose of this study was to investigate the influence of combined inhibition of RANKL (receptor activator of nuclear factor kappa-B ligand) and bone morphogenetic protein (BMP) activity in a mixed lytic/blastic prostate cancer lesion in bone. Human prostate cancer cells (C4 2b) were injected into immunocompromised mice using an intratibial injection model to create mixed lytic/blastic lesions. RANK-Fc, a recombinant RANKL antagonist, was injected subcutaneously three times a week (10mg/kg) to inhibit RANKL and subsequent formation, function and survival of osteoclasts. Inhibition of BMP activity was achieved by transducing prostate cancer cells ex vivo with a retroviral vector expressing noggin (retronoggin; RN). There were three treatment groups (RANK-Fc treatment, RN treatment and combined RN and RANK-Fc treatment) and two control groups (untreated control and empty vector control for the RN treatment group). The progression of bone lesion and tumor growth was evaluated using plain radiographs, hind limb tumor size, 18F-Fluorodeoxyglucose and 18F-fluoride micro PET-CT, histology and histomorphometry. Treatment with RANK-Fc alone inhibited osteolysis and transformed a mixed lytic/blastic lesion into an osteoblastic phenotype. Treatment with RN alone inhibited the osteoblastic component in a mixed lytic/blastic lesion and resulted in formation of smaller osteolytic bone lesion with smaller soft tissue size. The animals treated with both RN and RANK-Fc demonstrated delayed development of bone lesions, inhibition of osteolysis, small soft tissue tumors and preservation of bone architecture with less tumor induced new bone formation. This study suggests that combined inhibition of the RANKL and the BMP pathway may be an effective biologic therapy to inhibit the progression of established mixed lytic/blastic prostate cancer lesions in bone. PMID:21073986

  19. Three-Dimensional Cone Beam Computed Tomography Volumetric Outcomes of rhBMP-2/Demineralized Bone Matrix versus Iliac Crest Bone Graft for Alveolar Cleft Reconstruction.

    Science.gov (United States)

    Liang, Fan; Yen, Stephen L-K; Imahiyerobo, Thomas; Sanborn, Luke; Yen, Leia; Yen, Daniel; Nazarian, Sheila; Jedrzejewski, Breanna; Urata, Mark; Hammoudeh, Jeffrey

    2017-10-01

    Recent studies indicate that recombinant human bone morphogenetic protein-2 (rhBMP-2) in a demineralized bone matrix scaffold is a comparable alternative to iliac bone autograft in the setting of secondary alveolar cleft repair. Postreconstruction occlusal radiographs demonstrate improved bone stock when rhBMP-2/demineralized bone matrix (DBM) scaffold is used but lack the capacity to evaluate bone growth in three dimensions. This study uses cone beam computed tomography to provide the first clinical evaluation of volumetric and density comparisons between these two treatment modalities. A prospective study was conducted with 31 patients and 36 repairs of the alveolar cleft over a 2-year period. Twenty-one repairs used rhBMP-2/DBM scaffold and 14 repairs used iliac bone grafting. Postoperatively, occlusal radiographs were obtained at 3 months to evaluate bone fill; cone beam computed tomographic images were obtained at 6 to 9 months to compare volumetric and density data. At 3 months, postoperative occlusal radiographs demonstrated that 67 percent of patients receiving rhBMP-2/DBM scaffold had complete bone fill of the alveolus, versus 56 percent of patients in the autologous group. In contrast, cone beam computed tomographic data showed 31.6 percent (95 percent CI, 24.2 to 38.5 percent) fill in the rhBMP-2 group compared with 32.5 percent (95 percent CI, 22.1 to 42.9 percent) in the autologous population. Density analysis demonstrated identical average values between the groups (1.38 g/cc). These data demonstrate comparable bone regrowth and density values following secondary alveolar cleft repair using rhBMP-2/DBM scaffold versus autologous iliac bone graft. Cone beam computed tomography provides a more nuanced understanding of true bone regeneration within the alveolar cleft that may contribute to the information provided by occlusal radiographs alone. Therapeutic, II.

  20. Surface Modification of Titanium with BMP-2/GDF-5 by a Heparin Linker and Its Efficacy as a Dental Implant

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    Dae Hyeok Yang

    2017-01-01

    Full Text Available In this study, we prepared human bone morphogenetic protein-2 (hBMP-2/human growth and differentiation factor-5 (hGDF-5-coated titanium (Ti disc and screw types for controlled release of the growth factors (GFs. The two growth factors were coated onto Ti with a smooth surface using their specific interaction with heparin, because they have heparin binding sites in their molecular structures. Efficacy of the two growth factor-coated Ti for enhancement of bone formation and osseointegration was compared to pristine Ti, and hBMP-2- and hGDF-5-coated Ti in vivo. The surface chemical composition, surface morphology, and wettability characteristics of the metal samples were determined by X-ray photoelectron spectroscopy (XPS, scanning electron microscopy (SEM, and contact angle measurement, respectively. The initial burst of hBMP-2, hGDF-5, and their combination, occurred within one day of the release study, resulting in 12.5%, 4.5%, and 13.5%/3.2%, and then there was a sustained, even release of these two growth factors from the coated metal for 30 days. In vitro tests revealed that MC3T3-E1 cells cultured on the two growth factor-coated Ti had a higher proliferation rate and a higher activity for alkaline phosphatase (ALP, which led to a larger amount of calcium deposition and larger expressions of type I collagen (COL 1, ALP, and osteocalcin (OCN mRNAs. In vivo animal tests using ten white New Zealand rabbits showed that the two growth factor-coated Ti enhanced bone formation and osseointegration at the interface between the implants and host bone. In addition, histological evaluation showed that bone remodeling, including bone formation by osteoblasts and bone resorption by osteoclasts, actively occurred between the two growth factor-coated Ti and host bone. Consequently, it is suggested that Ti surface modification with the combination of hBMP-2 and hGDF-5 for the two growth factor-coated Ti implants can improve the clinical properties of

  1. Bone Morphogenic Protein 4-Smad-Induced Upregulation of Platelet-Derived Growth Factor AA Impairs Endothelial Function.

    Science.gov (United States)

    Hu, Weining; Zhang, Yang; Wang, Li; Lau, Chi Wai; Xu, Jian; Luo, Jiang-Yun; Gou, Lingshan; Yao, Xiaoqiang; Chen, Zhen-Yu; Ma, Ronald Ching Wan; Tian, Xiao Yu; Huang, Yu

    2016-03-01

    Bone morphogenic protein 4 (BMP4) is an important mediator of endothelial dysfunction in cardio-metabolic diseases, whereas platelet-derived growth factors (PDGFs) are major angiogenic and proinflammatory mediator, although the functional link between these 2 factors is unknown. The present study investigated whether PDGF mediates BMP4-induced endothelial dysfunction in diabetes mellitus. We generated Ad-Bmp4 to overexpress Bmp4 and Ad-Pdgfa-shRNA to knockdown Pdgfa in mice through tail intravenous injection. SMAD4-shRNA lentivirus, SMAD1-shRNA, and SMAD5 shRNA adenovirus were used for knockdown in human and mouse endothelial cells. We found that PDGF-AA impaired endothelium-dependent vasodilation in aortas and mesenteric resistance arteries. BMP4 upregulated PDGF-AA in human and mouse endothelial cells, which was abolished by BMP4 antagonist noggin or knockdown of SMAD1/5 or SMAD4. BMP4-impared relaxation in mouse aorta was also ameliorated by PDGF-AA neutralizing antibody. Tail injection of Ad-Pdgfa-shRNA ameliorates endothelial dysfunction induced by Bmp4 overexpression (Ad-Bmp4) in vivo. Serum PDGF-AA was elevated in both diabetic patients and diabetic db/db mice compared with nondiabetic controls. Pdgfa-shRNA or Bmp4-shRNA adenovirus reduced serum PDGF-AA concentration in db/db mice. PDGF-AA neutralizing antibody or tail injection with Pdgfa-shRNA adenovirus improved endothelial function in aortas and mesenteric resistance arteries from db/db mice. The effect of PDGF-AA on endothelial function in mouse aorta was also inhibited by Ad-Pdgfra-shRNA to inhibit PDGFRα. The present study provides novel evidences to show that PDGF-AA impairs endothelium-dependent vasodilation and PDGF-AA mediates BMP4-induced adverse effect on endothelial cell function through SMAD1/5- and SMAD4-dependent mechanisms. Inhibition of PGDF-AA ameliorates vascular dysfunction in diabetic mice. © 2016 American Heart Association, Inc.

  2. Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Schultz, Robbie D; Bennett, Emily E; Ellis, E Ann; Gumienny, Tina L

    2014-01-01

    In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

  3. Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Robbie D Schultz

    Full Text Available In mammals, Bone Morphogenetic Protein (BMP pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.

  4. Effects of airborne particulate matter on alternative pre-mRNA splicing in colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Buggiano, Valeria; Petrillo, Ezequiel; Alló, Mariano; Lafaille, Celina [Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires (Argentina); Redal, María Ana [Instituto de Ciencias Básicas y Medicina Experimental, Hospital Italiano de Buenos Aires (Argentina); Alghamdi, Mansour A. [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Khoder, Mamdouh I. [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah (Saudi Arabia); Shamy, Magdy [Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah (Saudi Arabia); Muñoz, Manuel J., E-mail: mmunoz@fbmc.fcen.uba.ar [Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires (Argentina); and others

    2015-07-15

    Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5′ untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing. - Highlights: • Airborne particulate matter (PM10) affects alternative splicing in colon cells. • PM10 upregulates one of the two mRNA variants of the growth factor BMP-4. • This variant has a longer 5′ unstranslated region and introduces an upstream AUG. • By regulating BMP-4 mRNA splicing PM10 inhibits total expression of BMP-4 protein. • BMP-4 downregulation was previously reported to be associated to colon cancer.

  5. Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway.

    Science.gov (United States)

    Blackburn, Brody J; Li, Shuaizhi; Roznowski, Aaron P; Perez, Alexis R; Villarreal, Rodrigo H; Johnson, Curtis J; Hardy, Margaret; Tuckerman, Edward C; Burch, April D; Fane, Bentley A

    2017-12-15

    Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability. IMPORTANCE Genetic analyses have been

  6. Breast cancer cells obtain an osteomimetic feature via epithelial-mesenchymal transition that have undergone BMP2/RUNX2 signaling pathway induction.

    Science.gov (United States)

    Tan, Cong-Cong; Li, Gui-Xi; Tan, Li-Duan; Du, Xin; Li, Xiao-Qing; He, Rui; Wang, Qing-Shan; Feng, Yu-Mei

    2016-11-29

    Bone is one of the most common organs of breast cancer metastasis. Cancer cells that mimic osteoblasts by expressing bone matrix proteins and factors have a higher likelihood of metastasizing to bone. However, the molecular mechanisms of osteomimicry formation of cancer cells remain undefined. Herein, we identified a set of bone-related genes (BRGs) that are ectopically co-expressed in primary breast cancer tissues and determined that osteomimetic feature is obtained due to the osteoblast-like transformation of epithelial breast cancer cells that have undergone epithelial-mesenchymal transition (EMT) followed by bone morphogenetic protein-2 (BMP2) stimulation. Furthermore, we demonstrated that breast cancer cells that transformed into osteoblast-like cells with high expression of BRGs showed enhanced chemotaxis, adhesion, proliferation and multidrug resistance in an osteoblast-mimic bone microenvironment in vitro. During these processes, runt-related transcription factor 2 (RUNX2) functioned as a master mediator by suppressing or activating the transcription of BRGs that underlie the dynamic antagonism between the TGF-β/SMAD and BMP/SMAD signaling pathways in breast cancer cells. Our findings suggest a novel mechanism of osteomimicry formation that arises in primary breast tumors, which may explain the propensity of breast cancer to metastasize to the skeleton and contribute to potential strategies for predicting and targeting breast cancer bone metastasis and multidrug resistance.

  7. Improvement of ovarian response and oocyte quality of aged female by administration of bone morphogenetic protein-6 in a mouse model

    Directory of Open Access Journals (Sweden)

    Park Seung S

    2012-12-01

    Full Text Available Abstract Background Advancing female age remains a difficult problem in infertility treatment. Ovarian angiogenesis plays an important role in follicular development and the activation of ovarian angiogenesis has been emerged as a new strategy for the improvement of age-related decline of oocyte quality. BMP-6 affect gonadotropin signals in granulosa cells and it promotes normal fertility by enabling appropriate response to LH and normal oocyte quality. BMP-6 has a potential role in regulation of angiogenesis and regulates the expression of inhibitor of DNA-binding proteins (Ids. Ids involved in the control and timing of follicle selection and granulosa cells differentiation. Especially, Id-1 is well-characterized target of BMP-6 signaling. Therefore, this study investigated whether co-administration of BMP-6 during superovulation process improves ovarian response, oocyte quality and expression of Id-1 and vascular endothelial growth factor (VEGF in the ovary of aged female using a mouse model. Methods Aged C57BL/6 female mice (26–31 weeks old were superovulated by injection with 0.1 mL of 5 IU equine chorionic gonadotropin (eCG containing recombinant mouse BMP-6 at various doses (0, 0.01, 0.1, 1, and 10 ng, followed by injection with 5 IU human chorionic gonadotropin (hCG 48 h later. Then, the mice were immediately paired with an individual male. The aged control group was superovulated without BMP-6. Young mice of 6–9 weeks old were superovulated without BMP-6 as a positive control for superovulation and in vitro culture of embryos. Eighteen hours after hCG injection, zygotes were retrieved and cultured for 4 days. Both ovaries of each mouse were provided in the examination of ovarian expression of Id-1 and VEGF by reverse transcriptase-polymerase chain reaction, western blot, and immunohistochemistry. Results Administration of 0.1 ng BMP-6 significantly increased the number and blastocyst formation rate of oocytes ovulated and ovarian

  8. A computational model for BMP movement in sea urchin embryos.

    Science.gov (United States)

    van Heijster, Peter; Hardway, Heather; Kaper, Tasso J; Bradham, Cynthia A

    2014-12-21

    Bone morphogen proteins (BMPs) are distributed along a dorsal-ventral (DV) gradient in many developing embryos. The spatial distribution of this signaling ligand is critical for correct DV axis specification. In various species, BMP expression is spatially localized, and BMP gradient formation relies on BMP transport, which in turn requires interactions with the extracellular proteins Short gastrulation/Chordin (Chd) and Twisted gastrulation (Tsg). These binding interactions promote BMP movement and concomitantly inhibit BMP signaling. The protease Tolloid (Tld) cleaves Chd, which releases BMP from the complex and permits it to bind the BMP receptor and signal. In sea urchin embryos, BMP is produced in the ventral ectoderm, but signals in the dorsal ectoderm. The transport of BMP from the ventral ectoderm to the dorsal ectoderm in sea urchin embryos is not understood. Therefore, using information from a series of experiments, we adapt the mathematical model of Mizutani et al. (2005) and embed it as the reaction part of a one-dimensional reaction-diffusion model. We use it to study aspects of this transport process in sea urchin embryos. We demonstrate that the receptor-bound BMP concentration exhibits dorsally centered peaks of the same type as those observed experimentally when the ternary transport complex (Chd-Tsg-BMP) forms relatively quickly and BMP receptor binding is relatively slow. Similarly, dorsally centered peaks are created when the diffusivities of BMP, Chd, and Chd-Tsg are relatively low and that of Chd-Tsg-BMP is relatively high, and the model dynamics also suggest that Tld is a principal regulator of the system. At the end of this paper, we briefly compare the observed dynamics in the sea urchin model to a version that applies to the fly embryo, and we find that the same conditions can account for BMP transport in the two types of embryos only if Tld levels are reduced in sea urchin compared to fly. Copyright © 2014 Elsevier Ltd. All rights

  9. Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

    Science.gov (United States)

    Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

    2014-12-01

    Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (pAPC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  10. Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

    Science.gov (United States)

    Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

    2011-01-01

    MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

  11. Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.

    Science.gov (United States)

    Su, Yu-Wen; Chung, Rosa; Ruan, Chun-Sheng; Chim, Shek Man; Kuek, Vincent; Dwivedi, Prem P; Hassanshahi, Mohammadhossein; Chen, Ke-Ming; Xie, Yangli; Chen, Lin; Foster, Bruce K; Rosen, Vicki; Zhou, Xin-Fu; Xu, Jiake; Xian, Cory J

    2016-06-01

    Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016

  12. Gelatin Tight-Coated Poly(lactide-co-glycolide Scaffold Incorporating rhBMP-2 for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Juan Wang

    2015-03-01

    Full Text Available Surface coating is the simplest surface modification. However, bioactive molecules can not spread well on the commonly used polylactone-type skeletons; thus, the surface coatings of biomolecules are typically unstable due to the weak interaction between the polymer and the bioactive molecules. In this study, a special type of poly(lactide-co-glycolide (PLGA-based scaffold with a loosened skeleton was fabricated by phase separation, which allowed gelatin molecules to more readily diffuse throughout the structure. In this application, gelatin modified both the internal substrate and external surface. After cross-linking with glutaraldehyde, the surface layer gelatin was tightly bound to the diffused gelatin, thereby preventing the surface layer gelatin coating from falling off within 14 days. After gelatin modification, PLGA scaffold demonstrated enhanced hydrophilicity and improved mechanical properties (i.e., increased compression strength and elastic modulus in dry and wet states. Furthermore, a sustained release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2 was achieved in the coated scaffold. The coated scaffold also supported the in vitro attachment, proliferation, and osteogenesis of rabbit bone mesenchymal stem cells (BMSCs, indicating the bioactivity of rhBMP-2. These results collectively demonstrate that the cross-linked-gelatin-coated porous PLGA scaffold incorporating bioactive molecules is a promising candidate for bone tissue regeneration.

  13. BMP4 and FGF3 haplotypes increase the risk of tendinopathy in volleyball athletes.

    Science.gov (United States)

    Salles, José Inácio; Amaral, Marcus Vinícius; Aguiar, Diego Pinheiro; Lira, Daisy Anne; Quinelato, Valquiria; Bonato, Letícia Ladeira; Duarte, Maria Eugenia Leite; Vieira, Alexandre Rezende; Casado, Priscila Ladeira

    2015-03-01

    To investigate whether genetic variants can be correlated with tendinopathy in elite male volleyball athletes. Case-control study. Fifteen single nucleotide polymorphisms within BMP4, FGF3, FGF10, FGFR1 genes were investigated in 138 elite volleyball athletes, aged between 18 and 35 years, who undergo 4-5h of training per day: 52 with tendinopathy and 86 with no history of pain suggestive of tendinopathy in patellar, Achilles, shoulder, and hip abductors tendons. The clinical diagnostic criterion was progressive pain during training, confirmed by magnetic resonance image. Genomic DNA was obtained from saliva samples. Genetic markers were genotyped using TaqMan real-time PCR. Chi-square test compared genotypes and haplotype differences between groups. Multivariate logistic regression analyzed the significance of covariates and incidence of tendinopathy. Statistical analysis revealed participant age (p=0.005) and years of practice (p=0.004) were risk factors for tendinopathy. A significant association between BMP4 rs2761884 (p=0.03) and tendinopathy was observed. Athletes with a polymorphic genotype have 2.4 times more susceptibility to tendinopathy (OR=2.39; 95%CI=1.10-5.19). Also, association between disease and haplotype TTGGA in BMP4 (p=0.01) was observed. The FGF3 TGGTA haplotype showed a tendency of association with tendinopathy (p=0.05), and so did FGF10 rs900379. FGFR1 showed no association with disease. These findings indicate that haplotypes in BMP4 and FGF3 genes may contribute to the tendon disease process in elite volleyball athletes. Copyright © 2014 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  14. Inactivation of Bmp4 from the Tbx1 Expression Domain Causes Abnormal Pharyngeal Arch Artery and Cardiac Outflow Tract Remodeling

    Science.gov (United States)

    Nie, Xuguang; Brown, Christopher B.; Wang, Qin; Jiao, Kai

    2011-01-01

    Maldevelopment of outflow tract and aortic arch arteries is among the most common forms of human congenital heart diseases. Both Bmp4 and Tbx1 are known to play critical roles during cardiovascular development. Expression of these two genes partially overlaps in pharyngeal arch areas in mouse embryos. In this study, we applied a conditional gene inactivation approach to test the hypothesis that Bmp4 expressed from the Tbx1 expression domain plays a critical role for normal development of outflow tract and pharyngeal arch arteries. We showed that inactivation of Bmp4 from Tbx1-expressing cells leads to the spectrum of deformities resembling the cardiovascular defects observed in human DiGeorge syndrome patients. Inactivation of Bmp4 from the Tbx1 expression domain did not cause patterning defects, but affected remodeling of outflow tract and pharyngeal arch arteries. Our further examination revealed that Bmp4 is required for normal recruitment/differentiation of smooth muscle cells surrounding the PAA4 and survival of outflow tract cushion mesenchymal cells. PMID:21123999

  15. Study of the association between the BMP4 gene and congenital anomalies of the kidney and urinary tract

    Directory of Open Access Journals (Sweden)

    Geisilaine Soares dos Reis

    2014-01-01

    Conclusions: The present data suggest that Brazilian individuals with polymorphisms of the BMP4 gene have a higher risk to develop CAKUT, especially the malformations related to nephrogenesis and initial branching such as MDK and UPJO. Conversely, VUR appeared not to be related to BMP4 gene.

  16. Bone induction through controlled release of novel BMP-2-related peptide from PTMC11-F127-PTMC11 hydrogels

    International Nuclear Information System (INIS)

    Tang Shuo; Li Jingfeng; Teng Yu; Guo Xiaodong; Zhao Jingjing; Xu Shuyun; Quan Daping

    2012-01-01

    Bone morphogenetic protein 2 (BMP-2) is the most powerful osteogenic factor; its effectiveness in enhancing osteoblastic activation has been confirmed both in vitro and in vivo. We developed a novel peptide (designated P24) derived from the ‘knuckle’ epitope of BMP-2 and found it also had osteogenic bioactivity to some extent. The main objective of this study was to develop a controlled release system based on poly(trimethylene carbonate)–F127–poly(trimethylene carbonate) (PTMC 11 -F127-PTMC 11 ) hydrogels for the P24 peptide, to promote bone formation. By varying the copolymer concentrations, we demonstrated that P24/PTMC 11 -F127-PTMC 11 hydrogels were an efficient system for the sustained release of P24 over 21–35 days. The P24-loaded hydrogels elevated alkaline phosphatase activity and promoted the expression of osteocalcin mRNA in bone marrow stromal cells (BMSCs) in vitro. Radiographic and histological examination showed that P24-loaded hydrogels could induce more effective ectopic bone formation in vivo than P24-free hydrogels. These results indicate that the PTMC 11 -F127-PTMC 11 hydrogel is a suitable carrier for the controlled release of P24, and is a promising injectable biomaterial for the induction of bone regeneration. (paper)

  17. Asymmetric BMP4 signalling improves the realism of kidney organoids.

    Science.gov (United States)

    Mills, Christopher G; Lawrence, Melanie L; Munro, David A D; Elhendawi, Mona; Mullins, John J; Davies, Jamie A

    2017-11-01

    We present a strategy for increasing the anatomical realism of organoids by applying asymmetric cues to mimic spatial information that is present in natural embryonic development, and demonstrate it using mouse kidney organoids. Existing methods for making kidney organoids in mice yield developing nephrons arranged around a symmetrical collecting duct tree that has no ureter. We use transplant experiments to demonstrate plasticity in the fate choice between collecting duct and ureter, and show that an environment rich in BMP4 promotes differentiation of early collecting ducts into uroplakin-positive, unbranched, ureter-like epithelial tubules. Further, we show that application of BMP4-releasing beads in one place in an organoid can break the symmetry of the system, causing a nearby collecting duct to develop into a uroplakin-positive, broad, unbranched, ureter-like 'trunk' from one end of which true collecting duct branches radiate and induce nephron development in an arrangement similar to natural kidneys. The idea of using local symmetry-breaking cues to improve the realism of organoids may have applications to organoid systems other than the kidney.

  18. The Expression of Bone Morphogenetic Protein 2 and Matrix Metalloproteinase 2 through Retinoic Acid Receptor Beta Induced by All-Trans Retinoic Acid in Cultured ARPE-19 Cells.

    Directory of Open Access Journals (Sweden)

    Zhenya Gao

    Full Text Available All-trans retinoic acid (ATRA plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2 and matrix metalloproteinase 2 (MMP-2 and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19 cells.The effects of ATRA (concentrations from 10-9 to 10-5 mol/l on the expression of retinoic acid receptors (RARs in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10-9 to 10-5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR and enzyme-linked immunosorbent assay (ELISA. The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ.RARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10-9 to 10-5 mol/l with a maximum effect observed at 10-6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10-6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135.ATRA induced upregulation of RARβ in ARPE-19 cells and stimulated these cells to secrete BMP-2 and MMP-2.

  19. Dorsoventral patterning in hemichordates: insights into early chordate evolution.

    Directory of Open Access Journals (Sweden)

    Christopher J Lowe

    2006-09-01

    Full Text Available We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called "dorsal." On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates

  20. Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

    Science.gov (United States)

    Chang, Wing Y; Andrews, Joseph; Carter, David E; Dagnino, Lina

    2006-08-01

    E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes.

  1. Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription▿

    OpenAIRE

    Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

    2006-01-01

    During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene ex...

  2. Chronological gene expression of parathyroid hormone-related protein (PTHrP) in the stellate reticulum of the rat: implications for tooth eruption.

    Science.gov (United States)

    Yao, Shaomian; Pan, Fenghui; Wise, Gary E

    2007-03-01

    Tooth eruption is a localized event that requires the expression of certain molecules at precise times to regulate bone resorption and bone formation. Parathyroid hormone-related protein (PTHrP) may be one of those molecules. Although PTHrP is produced in the stellate reticulum (SR) of the tooth and exerts its effect on the adjacent dental follicle, its expression pattern in the SR is unknown. Thus, it was the objectives of this study to determine the chronology of expression of PTHrP, and then to determine its effect on vascular endothelial growth factor (VEGF) expression for osteoclastogenesis and on bone morphogenetic protein-2 (BMP-2) for bone growth. Laser capture microdissection and RT-PCR were used to determine the chronological expression of PTHrP in vivo. In vitro, dental follicle cells were incubated with PTHrP and RT-PCR was conducted to determine its effect on VEGF and BMP-2 gene expression. PTHrP was maximally expressed at day 7 postnatally in the SR with the level of expression still high at day 9. In vitro, PTHrP upregulated VEGF120 and VEGF164 expression after 4h of incubation with a maximum effect at 6h. PTHrP upregulated BMP-2 gene expression with a maximal effect at 2h. Because the secondary burst of osteoclastogenesis needed for eruption occurs around day 10, it is possible that PTHrP is stimulating this osteoclastogenesis by upregulating VEGF. Concurrently, the upregulation of BMP-2 by PTHrP may stimulate bone growth at the base of the bony crypt to promote eruption.

  3. Bone Morphogenetic Protein-2, but Not Mesenchymal Stromal Cells, Exert Regenerative Effects on Canine and Human Nucleus Pulposus Cells

    NARCIS (Netherlands)

    Bach, Frances C.; Miranda-Bedate, Alberto; Van Heel, Ferdi W M; Riemers, Frank M.; Müller, Margot C M E; Creemers, Laura B.; Ito, Keita; Benz, Karin; Meij, Björn P.; Tryfonidou, Marianna A.

    2017-01-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2

  4. Bone morphogenetic protein-2, but not mesenchymal stromal cells, exert regenerative effects on canine and human nucleus pulposus cells

    NARCIS (Netherlands)

    Bach, Frances; Miranda-Bedate, Alberto; van Heel, Ferdi; Riemers, Frank; Muller, Margot; Creemers, Laura; Ito, Keita; Benz, Karin; Meij, Björn; Tryfonidou, M

    2017-01-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2

  5. Bone morphogenetic protein-2, but not mesenchymal stromal cells, exert regenerative effects on Canine and human nucleus pulposus cells

    NARCIS (Netherlands)

    Bach, F.C.; Miranda-Bedate, A.; Van Heel, F.W.M.; Riemers, F.M.; Müller, M.C.M.E.; Creemers, L.B.; Ito, K.; Benz, K.; Meij, B.P.; Tryfonidou, M.A.

    2017-01-01

    Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein-2

  6. Evaluation of bioactive glass incorporated poly(caprolactone)-poly(vinyl alcohol) matrix and the effect of BMP-2 modification

    Energy Technology Data Exchange (ETDEWEB)

    Keothongkham, Khamsone [Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Charoenphandhu, Narattaphol [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10900 (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10900 (Thailand); Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170 (Thailand); Thongbunchoo, Jirawan; Suntornsaratoon, Panan; Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok 10900 (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10900 (Thailand); Tang, I-Ming [Department of Materials Science Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Pon-On, Weeraphat, E-mail: fsciwpp@ku.ac.th [Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand)

    2017-05-01

    Composite materials having mechanical and biological properties similar to those of human bones are needed for bone regeneration and repair. In the present study, composites were made by incorporating bioactive glass (BG) into polycaprolactone (PCL)-polyvinyl alcohol (PVA) (PCLPVA) matrix. Composites with different BG contents of 10, 25 and 50 wt% were fabricated by an in-situ blending method. Physicochemical properties measurements found that the composite with 50 wt% BG in the PCLPVA organic matrix exhibited the best mechanical properties (compressive strength and compressive young's modulus up to 32.26 MPa and 530.91 MPa, respectively). We investigated the effects of the BG content on cell adhesion, proliferation and osteogenic activity of UMR-106 cells grown on the scaffolds using in vitro cell culture assay. The composite scaffolds having 25 wt% BG showed a significant increase in their cell adhesion capability and a faster cell proliferation. They also exhibited cell adhesion and spreading morphology after only 5 days of culturing. For these reasons, we chose to attach the bone morphogenetic protein (BMP)-2 to this composite. The resulting composite (labeled BMP-2-loaded PCLPVABG25) showed significant improvement in the UMR-106 cells adhesion, in the enhancement in osteogenic differentiation and osteoinductivity of this composite. - Highlights: • Preparation of PCLPVABGx composite scaffolds and their physical properties. • Mechanical properties could be adjusted by controlling BG contents in PCLPVA matrix. • In vitro cell availability tests confirmed the osteoblast grow on the PCLPVABGx composite scaffolds surface. • Upon the BMP-2-loaded PCLPVABG25 scaffolds can enhance cell attachment and significantly improved osteogenicity.

  7. Prevention: Exercise

    Medline Plus

    Full Text Available ... Supplements Surgical Options Anterior Cervical Fusion Artifical Disc Replacement (ADR) Bone Graft Alternatives Bone Morphogenetic Proteins (BMP) Cervical Disc Replacement Cervical Laminoplasty Lumbar (Open) Microscopic Discectomy Percutaneous Vertebral ...

  8. Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Sahar Ansari

    2014-01-01

    Full Text Available Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR. The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

  9. An automated decision-tree approach to predicting protein interaction hot spots.

    Science.gov (United States)

    Darnell, Steven J; Page, David; Mitchell, Julie C

    2007-09-01

    Protein-protein interactions can be altered by mutating one or more "hot spots," the subset of residues that account for most of the interface's binding free energy. The identification of hot spots requires a significant experimental effort, highlighting the practical value of hot spot predictions. We present two knowledge-based models that improve the ability to predict hot spots: K-FADE uses shape specificity features calculated by the Fast Atomic Density Evaluation (FADE) program, and K-CON uses biochemical contact features. The combined K-FADE/CON (KFC) model displays better overall predictive accuracy than computational alanine scanning (Robetta-Ala). In addition, because these methods predict different subsets of known hot spots, a large and significant increase in accuracy is achieved by combining KFC and Robetta-Ala. The KFC analysis is applied to the calmodulin (CaM)/smooth muscle myosin light chain kinase (smMLCK) interface, and to the bone morphogenetic protein-2 (BMP-2)/BMP receptor-type I (BMPR-IA) interface. The results indicate a strong correlation between KFC hot spot predictions and mutations that significantly reduce the binding affinity of the interface. 2007 Wiley-Liss, Inc.

  10. Drug: D03779 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available Recombinant human bone morphogenetic protein-2 (BMP2) [HSA:650] [KO:K04662] Osteoinductive agent for use in orthopaedic surgery..., dental/craniofacial surgery, or spine surgery, as an adjunc

  11. Genetic and evolutionary analyses of the human bone morphogenetic protein receptor 2 (BMPR2 in the pathophysiology of obesity.

    Directory of Open Access Journals (Sweden)

    Dorit Schleinitz

    2011-02-01

    Full Text Available Human bone morphogenetic protein receptor 2 (BMPR2 is essential for BMP signalling and may be involved in the regulation of adipogenesis. The BMPR2 locus has been suggested as target of recent selection in human populations. We hypothesized that BMPR2 might have a role in the pathophysiology of obesity.Evolutionary analyses (dN/dS, Fst, iHS were conducted in vertebrates and human populations. BMPR2 mRNA expression was measured in 190 paired samples of visceral and subcutaneous adipose tissue. The gene was sequenced in 48 DNA samples. Nine representative single nucleotide polymorphisms (SNPs were genotyped for subsequent association studies on quantitative traits related to obesity in 1830 German Caucasians. An independent cohort of 925 Sorbs was used for replication. Finally, relation of genotypes to mRNA in fat was examined.The evolutionary analyses indicated signatures of selection on the BMPR2 locus. BMPR2 mRNA expression was significantly increased both in visceral and subcutaneous adipose tissue of 37 overweight (BMI>25 and 30 kg/m² compared with 44 lean subjects (BMI< 25 kg/m² (P<0.001. In a case-control study including lean and obese subjects, two intronic SNPs (rs6717924, rs13426118 were associated with obesity (adjusted P<0.05. Combined analyses including the initial cohort and the Sorbs confirmed a consistent effect for rs6717924 (combined P = 0.01 on obesity. Moreover, rs6717924 was associated with higher BMPR2 mRNA expression in visceral adipose tissue.Combined BMPR2 genotype-phenotype-mRNA expression data as well as evolutionary aspects suggest a role of BMPR2 in the pathophysiology of obesity.

  12. Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Duch, Mogens R.; Mygind, Tina

    2006-01-01

    -old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno...

  13. Reprogramming Antagonizes the Oncogenicity of HOXA13-Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells.

    Science.gov (United States)

    Wu, Deng-Chyang; Wang, Sophie S W; Liu, Chung-Jung; Wuputra, Kenly; Kato, Kohsuke; Lee, Yen-Liang; Lin, Ying-Chu; Tsai, Ming-Ho; Ku, Chia-Chen; Lin, Wen-Hsin; Wang, Shin-Wei; Kishikawa, Shotaro; Noguchi, Michiya; Wu, Chu-Chieh; Chen, Yi-Ting; Chai, Chee-Yin; Lin, Chen-Lung Steve; Kuo, Kung-Kai; Yang, Ya-Han; Miyoshi, Hiroyuki; Nakamura, Yukio; Saito, Shigeo; Nagata, Kyosuke; Lin, Chang-Shen; Yokoyama, Kazunari K

    2017-10-01

    Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)-homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS-like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed-lineage leukemia 1 lysine N-methyltransferase, WD repeat-containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis-element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13-mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13-HOTTIP and HOXA13-HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer-binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115-2128. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  14. Nonsyndromic cleft lip with or without cleft palate: Increased burden of rare variants within Gremlin-1, a component of the bone morphogenetic protein 4 pathway.

    Science.gov (United States)

    Al Chawa, Taofik; Ludwig, Kerstin U; Fier, Heide; Pötzsch, Bernd; Reich, Rudolf H; Schmidt, Gül; Braumann, Bert; Daratsianos, Nikolaos; Böhmer, Anne C; Schuencke, Hannah; Alblas, Margrieta; Fricker, Nadine; Hoffmann, Per; Knapp, Michael; Lange, Christoph; Nöthen, Markus M; Mangold, Elisabeth

    2014-06-01

    The genes Gremlin-1 (GREM1) and Noggin (NOG) are components of the bone morphogenetic protein 4 pathway, which has been implicated in craniofacial development. Both genes map to recently identified susceptibility loci (chromosomal region 15q13, 17q22) for nonsyndromic cleft lip with or without cleft palate (nsCL/P). The aim of the present study was to determine whether rare variants in either gene are implicated in nsCL/P etiology. The complete coding regions, untranslated regions, and splice sites of GREM1 and NOG were sequenced in 96 nsCL/P patients and 96 controls of Central European ethnicity. Three burden and four nonburden tests were performed. Statistically significant results were followed up in a second case-control sample (n = 96, respectively). For rare variants observed in cases, segregation analyses were performed. In NOG, four rare sequence variants (minor allele frequency elements. © 2014 Wiley Periodicals, Inc.

  15. Prevention: Exercise

    Medline Plus

    Full Text Available ... Testing Specialized Nerve Tests: EMG, NCV and SEEP Alternative Medicine Acupuncture Herbal Supplements Surgical Options Anterior Cervical Fusion Artifical Disc Replacement (ADR) Bone Graft Alternatives Bone Morphogenetic Proteins (BMP) Cervical Disc Replacement Cervical ...

  16. Comparison of recombinant human bone morphogenetic protein-2-infused absorbable collagen sponge, recombinant human bone morphogenetic protein-2-coated tricalcium phosphate, and platelet-rich fibrin-mixed tricalcium phosphate for sinus augmentation in rabbits

    Directory of Open Access Journals (Sweden)

    Chul-Hun Kim

    2017-09-01

    Conclusion: Our histological evaluation demonstrates that Type I ACS can be used as a carrier of rhBMP-2, and rhBMP-2+ACS showed rapid bone formation, remodeling, and calcification at Week 2 in rabbit.

  17. Prevention of early postnatal hyperalimentation protects against activation of transforming growth factor-β/bone morphogenetic protein and interleukin-6 signaling in rat lungs after intrauterine growth restriction.

    Science.gov (United States)

    Alcázar, Miguel Angel Alejandre; Dinger, Katharina; Rother, Eva; Östreicher, Iris; Vohlen, Christina; Plank, Christian; Dötsch, Jörg

    2014-12-01

    Intrauterine growth restriction (IUGR) is intimately linked with postnatal catch-up growth, leading to impaired lung structure and function. However, the impact of catch-up growth induced by early postnatal hyperalimentation (HA) on the lung has not been addressed to date. The aim of this study was to investigate whether prevention of HA subsequent to IUGR protects the lung from 1) deregulation of the transforming growth factor-β(TGF-β)/bone morphogenetic protein (BMP) pathway, 2) activation of interleukin (IL)-6 signaling, and 3) profibrotic processes. IUGR was induced in Wistar rats by isocaloric protein restriction during gestation by feeding a control (Co) or a low-protein diet with 17% or 8% casein, respectively. On postnatal day 1 (P1), litters from both groups were randomly reduced to 6 pups per dam to induce HA or adjusted to 10 pups and fed with standard diet: Co, Co with HA (Co-HA), IUGR, and IUGR with HA (IUGR-HA). Birth weights in rats after IUGR were lower than in Co rats (P < 0.05). HA during lactation led to accelerated body weight gain from P1 to P23 (Co vs. Co-HA, IUGR vs. IUGR-HA; P < 0.05). At P70, prevention of HA after IUGR protected against the following: 1) activation of both TGF-β [phosphorylated SMAD (pSMAD) 2; plasminogen activator inhibitor 1 (Pai1)] and BMP signaling [pSMAD1; inhibitor of differentiation (Id1)] compared with Co (P < 0.05) and Co or IUGR (P < 0.05) rats, respectively; 2) greater mRNA expression of interleukin (Il) 6 and Il13 (P < 0.05) as well as activation of signal transducer and activator of transcription 3 (STAT3) signaling (P < 0.05) after IUGR-HA; and 3) greater gene expression of collagen Iα1 and osteopontin (P < 0.05) and increased deposition of bronchial subepithelial connective tissue in IUGR-HA compared with Co and IUGR rats. Moreover, HA had a significant additive effect (P < 0.05) on the increased enhanced pause (indicator of airway resistance) in the IUGR group (P < 0.05) at P70. This study demonstrates

  18. Gata4 expression in lateral mesoderm is downstream of BMP4 and isactivated directly by Forkhead and GATA transcription factors through adistal enhancer element

    Energy Technology Data Exchange (ETDEWEB)

    Rojas, Anabel; De Val, Sarah; Heidt, Analeah B.; Xu, Shan-Mei; Bristow, James; Black, Brian L.

    2005-05-20

    The GATA family of zinc-finger transcription factors plays key roles in the specification and differentiation of multiple cell types during development. GATA4 is an early regulator of gene expression during the development of endoderm and mesoderm, and genetic studies in mice have demonstrated that GATA4 is required for embryonic development.Despite the importance of GATA4 in tissue specification and differentiation, the mechanisms by which Gata4 expression is activated and the transcription factor pathways upstream of GATA4 remain largely undefined. To identify transcriptional regulators of Gata4 in the mouse,we screened conserved noncoding sequences from the mouse Gata4 gene for enhancer activity in transgenic embryos. Here, we define the regulation of a distal enhancer element from Gata4 that is sufficient to direct expression throughout the lateral mesoderm, beginning at 7.5 days of mouse embryonic development. The activity of this enhancer is initially broad but eventually becomes restricted to the mesenchyme surrounding the liver. We demonstrate that the function of this enhancer in transgenic embryos is dependent upon highly conserved Forkhead and GATA transcription factor binding sites, which are bound by FOXF1 and GATA4,respectively. Furthermore, the activity of the Gata4 lateral mesoderm enhancer is attenuated by the BMP antagonist Noggin, and the enhancer is not activated in Bmp4-null embryos. Thus, these studies establish that Gata4 is a direct transcriptional target of Forkhead and GATA transcription factors in the lateral mesoderm, and demonstrate that Gata4lateral mesoderm enhancer activation requires BMP4, supporting a model in which GATA4 serves as a downstream effector of BMP signaling in the lateral mesoderm.

  19. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    NARCIS (Netherlands)

    Yang, X.; Kraan, P.M. van der; Dolder, J. van den; Walboomers, X.F.; Bian, Z.; Fan, M.; Jansen, J.A.

    2007-01-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and

  20. Intraoperative engineering of osteogenic grafts combining freshly harvested, human adipose-derived cells and physiological doses of bone morphogenetic protein-2

    Directory of Open Access Journals (Sweden)

    A Mehrkens

    2012-09-01

    Full Text Available Engineered osteogenic constructs for bone repair typically involve complex and costly processes for cell expansion. Adipose tissue includes mesenchymal precursors in large amounts, in principle allowing for an intraoperative production of osteogenic grafts and their immediate implantation. However, stromal vascular fraction (SVF cells from adipose tissue were reported to require a molecular trigger to differentiate into functional osteoblasts. The present study tested whether physiological doses of recombinant human BMP-2 (rhBMP-2 could induce freshly harvested human SVF cells to generate ectopic bone tissue. Enzymatically dissociated SVF cells from 7 healthy donors (1 x 106 or 4 x 106 were immediately embedded in a fibrin gel with or without 250 ng rhBMP-2, mixed with porous silicated calcium-phosphate granules (Actifuse®, Apatech (final construct size: 0.1 cm3 and implanted ectopically for eight weeks in nude mice. In the presence of rhBMP-2, SVF cells not only supported but directly contributed to the formation of bone ossicles, which were not observed in control cell-free, rhBMP-2 loaded implants. In vitro analysis indicated that rhBMP-2 did not involve an increase in the percentage of SVF cells recruited to the osteogenic lineage, but rather induced a stimulation of the osteoblastic differentiation of the committed progenitors. These findings confirm the feasibility of generating fully osteogenic grafts using an easily accessible autologous cell source and low amounts of rhBMP-2, in a timing compatible with an intraoperative schedule. The study warrants further investigation at an orthotopic site of implantation, where the delivery of rhBMP-2 could be bypassed thanks to the properties of the local milieu.

  1. Prevention: Exercise

    Medline Plus

    Full Text Available ... Disc Replacement (ADR) Bone Graft Alternatives Bone Morphogenetic Proteins (BMP) Cervical Disc Replacement Cervical Laminoplasty Lumbar (Open) ... Flexibility Aerobic Exercise Cervical Exercise Strength Training for the Elderly Other Back Pack Safety Pregnancy and Back ...

  2. 3-OST-7 regulates BMP-dependent cardiac contraction.

    Directory of Open Access Journals (Sweden)

    Shiela C Samson

    2013-12-01

    Full Text Available The 3-O-sulfotransferase (3-OST family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4 expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.

  3. Injectable Shear-Thinning CaSO4/FGF-18-Incorporated Chitin-PLGA Hydrogel Enhances Bone Regeneration in Mice Cranial Bone Defect Model.

    Science.gov (United States)

    Sivashanmugam, A; Charoenlarp, Pornkawee; Deepthi, S; Rajendran, Arunkumar; Nair, Shantikumar V; Iseki, Sachiko; Jayakumar, R

    2017-12-13

    For craniofacial bone regeneration, shear-thinning injectable hydrogels are favored over conventional scaffolds because of their improved defect margin adaptability, easier handling, and ability to be injected manually into deeper tissues. The most accepted method, after autografting, is the use of recombinant human bone morphogenetic protein-2 (BMP-2); however, complications such as interindividual variations, edema, and poor cost-efficiency in supraphysiological doses have been reported. The endogenous synthesis of BMP-2 is desirable, and a molecule which induces this is fibroblast growth factor-18 (FGF-18) because it can upregulate the BMP-2 expression  by supressing noggin. We developed a chitin-poly(lactide-co-glycolide) (PLGA) composite hydrogel by regeneration chemistry and then incorporated CaSO 4 and FGF-18 for this purpose. Rheologically, a 7-fold increase in the elastic modulus was observed in the CaSO 4 -incorporated chitin-PLGA hydrogels as compared to the chitin-PLGA hydrogel. Shear-thinning Herschel-Bulkley fluid nature was observed for both hydrogels. Chitin-PLGA/CaSO 4 gel showed sustained release of FGF-18. In vitro osteogenic differentiation showed an enhanced alkaline phosphatase (ALP) expression in the FGF-18-containing chitin-PLGA/CaSO 4 gel when compared to cells alone. Further, it was confirmed by studying the expression of osteogenic genes [RUNX2, ALP, BMP-2, osteocalcin (OCN), and osteopontin (OPN)], immunofluorescence staining of BMP-2, OCN, and OPN, and alizarin red S staining. Incorporation of FGF-18 in the hydrogel increased the endothelial cell migration. Further, the regeneration potential of the prepared hydrogels was tested in vivo, and longitudinal live animal μ-CT was performed. FGF-18-loaded chitin-PLGA/CaSO 4 showed early and almost complete bone healing in comparison with chitin-PLGA/CaSO 4 , chitin-PLGA/FGF-18, chitin-PLGA, and sham control systems, as confirmed by hematoxylin and eosin and osteoid tetrachrome stainings

  4. Hair follicle defects and squamous cell carcinoma formation in Smad4 conditional knockout mouse skin.

    Science.gov (United States)

    Qiao, W; Li, A G; Owens, P; Xu, X; Wang, X-J; Deng, C-X

    2006-01-12

    Smad4 is the common mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in skin development and epidermal tumorigenesis, we disrupted this gene in skin using the Cre-loxP approach. We showed that absence of Smad4 blocked hair follicle differentiation and cycling, leading to a progressive hair loss of mutant (MT) mice. MT hair follicles exhibited diminished expression of Lef1, and increased proliferative cells in the outer root sheath. Additionally, the skin of MT mice exhibited increased proliferation of basal keratinocytes and epidermal hyperplasia. Furthermore, we provide evidence that the absence of Smad4 resulted in a block of both TGFbeta and bone morphogenetic protein (BMP) signaling pathways, including p21, a well-known cyclin-dependent kinase inhibitor. Consequently, all MT mice developed spontaneous malignant skin tumors from 3 months to 13 months of age. The majority of tumors are malignant squamous cell carcinomas. A most notable finding is that tumorigenesis is accompanied by inactivation of phosphatase and tensin homolog deleted on chromosome 10 (Pten), activation of AKT, fast proliferation and nuclear accumulation of cyclin D1. These observations revealed the essential functions of Smad4-mediated signals in repressing skin tumor formation through the TGFbeta/BMP pathway, which interacts with the Pten signaling pathway.

  5. Generation of an rhBMP-2-loaded beta-tricalcium phosphate/hydrogel composite and evaluation of its efficacy on peri-implant bone formation

    International Nuclear Information System (INIS)

    Lee, Jae Hyup; Baek, Hae-Ri; Lee, Ji-Ho; Ryu, Mi Young; Seo, Jun-Hyuk; Lee, Kyung-Mee

    2014-01-01

    Dental implant insertion on a site with low bone quality or bone defect should be preceded by a bone graft or artificial bone graft insertion to heal the defect. We generated a beta-tricalcium phosphate (β-TCP) and poloxamer 407-based hydrogel composite and penetration of the β-TCP/hydrogel composite into the peri-implant area of bone was evaluated by porous bone block experiments. The maximum penetration depth for porous bone blocks and dense bone blocks were 524 μm and 464 μm, respectively. We report the in-vivo performance of a composite of β-TCP/hydrogel composite as a carrier of recombinant human bone morphogenetic protein (rhBMP-2), implanted into a rabbit tibial defect model. Three holes drilled into each tibia of eight male rabbits were (1) grafted with dental implant fixtures; (2) filled with β-TCP/hydrogel composite (containing 5 μg of rhBMP-2), followed by grafting of the dental implant fixtures. Four weeks later, bone-implant contact ratio and peri-implant bone formation were analyzed by radiography, micro-CT and histology of undecalcified specimens. The micro-CT results showed a significantly higher level of trabecular thickness and new bone and peri-implant new bone formation in the experimental treatment compared to the control treatment. Histomorphometry revealed a significantly higher bone-implant contact ratio and peri-implant bone formation with the experimental treatment. The use of β-TCP/poloxamer 407 hydrogel composite as a carrier of rhBMP-2 significantly promoted new bone formation around the dental implant fixture and it also improved the quality of the new bone formed in the tibial marrow space. (paper)

  6. Localization of Bmp-4, Shh and Wnt-5a transcripts during early mice tooth development by in situ hybridization Localização de transcritos de Bmp-4, Shh e Wnt-5a durante as fases iniciais do desenvolvimento dentário de camundongos por hibridização in situ

    Directory of Open Access Journals (Sweden)

    Fábio Daumas Nunes

    2007-06-01

    Full Text Available A comparative nonisotopic in situ hybridization (ISH analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.No presente trabalho, realizou-se uma análise comparativa não isotópica por hibridização in situ a fim de se detectar a presença de transcritos de Bmp-4, Shh e Wnt-5a durante as fases iniciais da odontogênese em camundongos, desde a iniciação até o estágio de capuz. No estágio de iniciação, observou-se expressão precoce de Bmp-4 no epitélio e no mesênquima subjacente, enquanto que a expressão de Shh ocorreu durante o estágio de capuz, na região de espessamento do revestimento epitelial odontogênico, tornando-se mais intensa na área de nó do esmalte. Os transcritos de Wnt-5a foram expressos somente no mesênquima durante os estágios de iniciação, botão e capuz, com intenso sinal na região no mesênquima na fase de botão. Estes resultados mostraram que Bmp-4, Shh e Wnt-5a são expressos desde os estágios mais precoces do desenvolvimento dentário, sugerindo que o gene Wnt-5a seja expresso em populações celulares distintas daquelas que expressam Bmp-4 e Shh.

  7. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

    International Nuclear Information System (INIS)

    Nemoto, Eiji; Ebe, Yukari; Kanaya, Sousuke; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi

    2012-01-01

    Highlights: ► Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. ► Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. ► Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. ► Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

  8. Expressional and functional analyses of transcription factors activated by BMP-4s signaling in early xenopus embryo; BMP-4 shigunaru dentatsu kiko to sono hyoteki kakunai tensha inshi ni kansuru kenkyu

    Energy Technology Data Exchange (ETDEWEB)

    Maeno, Mitsugu [Niigata University, Niigata (Japan). Faculty of Science

    1998-12-16

    The expression and physiological function of two transcription factors, GATA-2 and Xmsx-1, in amphibian embryos has been analyzed. The expression of these mRNAs in embryonic cells were firmly regulated by the BMP-4 signaling, that plays a central role in the formation of ventral tissues. The microinjection studies of GATA-2 RNA into embryonic cells suggested that this factor functions in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation in ventral area of embryo. Embryos injected with Xmsx-1 RNA, but not with GATA-2, in dorsal blastomeres exhibited a ventralized phenotype, with microcephaly and swollen abdomen. Thus, Xmsx-1 is a ventralizing agent. However, on the basis of molecular marker analyses, Xmsx-1 did not promote erythropoietic differentiation, but promoted muscle tissue formation. It has been concluded that Xmsx-1 si a target transcription factor of the BMP-4 signaling, but possesses a distinct activity on dorso-ventral patterning of mesodermal tissues. (author)

  9. GREM1, FRZB and DKK1 mRNA levels correlate with osteoarthritis and are regulated by osteoarthritis associated factors

    NARCIS (Netherlands)

    Leijten, Jeroen Christianus Hermanus; Bos, Steffan D.; Landman, Ellie; Georgi, Nicole; Jahr, Holger; Meulenbelt, Ingrid; Post, Janine Nicole; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

    2013-01-01

    Introduction: Osteoarthritis is, at least in a subset of patients, associated with hypertrophic differentiation of articular chondrocytes. Recently, we have identified the bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) signaling antagonists Gremlin 1 (GREM1),

  10. Low-level bisphenol A increases production of glial fibrillary acidic protein in differentiating astrocyte progenitor cells through excessive STAT3 and Smad1 activation

    International Nuclear Information System (INIS)

    Yamaguchi, Hideaki; Zhu, Jun; Yu, Tao; Sasaki, Kazuo; Umetsu, Hironori; Kidachi, Yumi; Ryoyama, Kazuo

    2006-01-01

    The effects of bisphenol A (BPA) on the differentiation of serum-free mouse embryo (SFME) cells, the astrocyte progenitor cells in the central nervous system, were examined. SFME cells were exposed to 10 ng/ml leukemia inhibitory factor (LIF) and 10 ng/ml bone morphogenetic protein 2 (BMP2) to increase glial fibrillary acidic protein (GFAP) expression and induce cell differentiation. Various concentrations of BPA (0.1 pg/ml-1 μg/ml) were then added to determine their effects on the cell differentiation. SFME cells were effectively differentiated by LIF and BMP2 in completely serum-free cultures. Cell proliferation following cell differentiation was not significantly affected by low-level BPA. However, GFAP expression was significantly increased in SFME cells in the presence of 1-100 pg/ml BPA. These increases were due to excessive activation of signal transducer and activator of transcription 3 (STAT3) and mothers against decapentaplegic homolog 1 (Smad1) by the low-level BPA

  11. Molecular Genetic and Gene Therapy Studies of the Musculoskeletal System

    Science.gov (United States)

    2004-10-01

    Center during the past 12 months. Both lectures were designed to educate and tutor Army project investigators in viral gene transfer technology. One...sponge char- Y, Sakamoto K, et al. Effects of recombinant human acteristics and protein pI on in vivo rhBMP pharma - bone morphogenetic protein-2 on

  12. Changes in Serum Levels of Bone Morphogenic Protein 4 and Inflammatory Cytokines after Bariatric Surgery in Severely Obese Korean Patients with Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Mee Kyoung Kim

    2013-01-01

    Full Text Available Serum bone morphogenic protein- (BMP- 4 levels are associated with human adiposity. The aim of this study was to investigate changes in serum levels of BMP-4 and inflammatory cytokines after Roux-en-Y gastric bypass (RYGB. Fifty-seven patients with type 2 diabetes underwent RYGB. Serum levels of BMP-4 and various inflammatory markers, including high-sensitivity C-reactive protein (hsCRP, free fatty acids (FFAs, and plasminogen activator inhibitor- (PAI- 1, were measured before and 12 months after RYGB. Remission was defined as glycated hemoglobin <6.5% for at least 1 year in the absence of medications. Levels of PAI-1, hsCRP, and FFAs were significantly decreased at 1 year after RYGB. BMP-4 levels were also significantly lower at 1 year after RYGB than at baseline (P=0.024. Of the 57 patients, 40 (70% had diabetes remission at 1 year after surgery (remission group. Compared with patients in the nonremission group, patients in the remission group had lower PAI-1 levels and smaller visceral fat areas at baseline. There was a difference in the change in the BMP-4 level according to remission status. Our data demonstrate a significant beneficial effect of bariatric surgery on established cardiovascular risk factors and a reduction in chronic nonspecific inflammation after surgery.

  13. The translational regulator Cup controls NMJ presynaptic terminal morphology.

    Science.gov (United States)

    Menon, Kaushiki P; Carrillo, Robert A; Zinn, Kai

    2015-07-01

    During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with two genes, EndoA and Dap160, that encode proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. A review paper on biomimetic calcium phosphate coatings

    NARCIS (Netherlands)

    Lin, X.; de Groot, K.; Wang, D.; Hu, Q.; Wismeijer, D.; Liu, Y.

    2015-01-01

    Biomimetic calcium phosphate coatings have been developed for bone regeneration and repair because of their biocompatibility, osteoconductivity, and easy preparation. They can be rendered osteoinductive by incorporating an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2), into the

  15. Activation of PKA/CREB Signaling is Involved in BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

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    Hongyu Zhang

    2015-09-01

    Full Text Available Background/Aims: BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cells (MSCs although the molecular mechanism involved is largely unknown. Here, we explored the detail role of PKA/CREB signaling in BMP9-induced osteogenic differentiation. Methods: Activation status of PKA/CREB signaling is assessed by nonradioactive assay and Western blot. Using PKA inhibitors and a dominant negative protein of CREB (A-CREB, we investigated the effect of PKA/CREB signaling on BMP9-induced osteogenic differentiation. Results: We found that BMP9 promotes PKA activity and enhances CREB phosphorylation in MSCs. BMP9 is shown to down-regulate protein kinase A inhibitor γ (PKIγ expression. We demonstrated that PKA inhibitors suppress BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs as well as late osteogenic markers osteopontin (OPN, osteocalcin (OCN and matrix mineralization. We found that PKA inhibitor reduces BMP9-induced Runx2 activation and p38 phosphorylation in MSCs. Lastly, interference of CREB function by A-CREB decreased BMP9-induced osteogenic differentiation as well. Conclusion: Our results revealed that BMP9 may activate PKA/CREB signaling in MSCs through suppression of PKIγ expression. It is noteworthy that inhibition of PKA/CREB signaling may impair BMP9-induced osteogenic differentiation of MSCs, implying that activation of PKA/CREB signaling is required for BMP9 osteoinductive activity.

  16. Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors.

    Directory of Open Access Journals (Sweden)

    Franka Klatte-Schulz

    Full Text Available Tendon bone healing of the rotator cuff is often associated with non-healing or recurrent defects, which seems to be influenced by the patient's age and sex. The present study aims to examine cellular biological characteristics of tenocyte-like cells that may contribute to this impaired rotator cuff healing. Moreover, a therapeutic approach using growth factors could possibly stimulate tendon bone healing. Therefore, our second aim was to identify patient groups who would particularly benefit from growth factor stimulation. Tenocyte-like cells isolated from supraspinatus tendons of female donors younger and older than 65 years of age were characterized with respect to different cellular biological parameters, such as cell density, cell count, marker expression, collagen-I protein synthesis, and stem cell potential. Furthermore, cells of the donor groups were stimulated with BMP-2 and BMP-7 (200 and 1000 ng/ml in 3D-culture and analyzed for cell count, marker expression and collagen-I protein synthesis. Female donors older than 65 years of age showed significantly decreased cell count and collagen-I protein synthesis compared to cells from donors younger than 65 years. Cellular biological parameters including cell count, collagen-I and -III expression, and collagen-I protein synthesis of cells from both donor groups were stimulated with BMP-2 and BMP-7. The cells from donors older than 65 years revealed a decreased stimulation potential for cell count compared to the younger group. Cells from female donors older than 65 years of age showed inferior cellular biological characteristics. This may be one reason for a weaker healing potential observed in older female patients and should be taken into consideration for tendon bone healing of the rotator cuff.

  17. A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

    Science.gov (United States)

    Bohnenpoll, Tobias; Wittern, Anna B; Mamo, Tamrat M; Weiss, Anna-Carina; Rudat, Carsten; Kleppa, Marc-Jens; Schuster-Gossler, Karin; Wojahn, Irina; Lüdtke, Timo H-W; Trowe, Mark-Oliver; Kispert, Andreas

    2017-08-01

    The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

  18. A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

    Directory of Open Access Journals (Sweden)

    Tobias Bohnenpoll

    2017-08-01

    Full Text Available The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH family of secreted proteins, Sonic hedgehog (SHH as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT.

  19. Effect of a Novel Nonviral Gene Delivery of BMP-2 on Bone Healing

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    P. Schwabe

    2012-01-01

    Full Text Available Background. Gene therapeutic drug delivery approaches have been introduced to improve the efficiency of growth factors at the site of interest. This study investigated the efficacy and safety of a new nonviral copolymer-protected gene vector (COPROG for the stimulation of bone healing. Methods. In vitro, rat osteoblasts were transfected with COPROG + luciferase plasmid or COPROG + hBMP-2 plasmid. In vivo, rat tibial fractures were intramedullary stabilized with uncoated versus COPROG+hBMP-2-plasmid-coated titanium K-wires. The tibiae were prepared for biomechanical and histological analyses at days 28 and 42 and for transfection/safety study at days 2, 4, 7, 28, and 42. Results. In vitro results showed luciferase expression until day 21, and hBMP-2-protein was measured from day 2 – day 10. In vivo, the local application of hBMP-2-plasmid showed a significantly higher maximum load after 42 days compared to that in the control. The histomorphometric analysis revealed a significantly less mineralized periosteal callus area in the BMP-2 group compared to the control at day 28. The rt-PCR showed no systemic biodistribution of luciferase RNA. Conclusion. A positive effect on fracture healing by nonviral BMP-2 plasmid application from COPROG-coated implants could be shown in this study; however, the effect of the vector may be improved with higher plasmid concentrations. Transfection showed no biodistribution to distant organs and was considered to be safe.

  20. Genetic predisposition to fracture non-union: a case control study of a preliminary single nucleotide polymorphisms analysis of the BMP pathway

    Directory of Open Access Journals (Sweden)

    Giannoudis Peter V

    2011-02-01

    Full Text Available Abstract Background Despite the known multi-factorial nature of atrophic fracture non-unions, a possible genetic predisposition for the development of this complication after long bone fractures remains unknown. This pilot study aimed to address this issue by performing a preliminary SNP analysis of specific genes known to regulate fracture healing. Methods A total of fifteen SNPs within four genes of the Bone Morphogenetic Protein (BMP pathway (BMP-2, BMP-7, NOGGIN and SMAD6 were examined, in 109 randomly selected patients with long bone fractures as a result of motor vehicle accident, fall or direct blow. There were sixty-two patients with atrophic non-union and forty-seven patients (54 fractures with uneventful fracture union. Overall SNPs frequencies were computed with respect to patient's age, gender, smoking habits, fracture-associated parameters and the use of nonsteroidal anti-inflammatory drugs (NSAIDs, and tested for their association to the impaired bone healing process, using binary logistic regression (STATA 11.1; StataCorp, Texas USA. Results Statistical analysis revealed age to be an important covariate in the development of atrophic non-union (p = 0.01, OR 1.05 [per year], and two specific genotypes (G/G genotype of the rs1372857 SNP, located on NOGGIN and T/T genotype of the rs2053423 SNP, located on SMAD6 to be associated with a greater risk of fracture non-union (p = 0.02, OR 4.56 and p = 0.04, OR 10.27, respectively, after adjustment for age. Conclusions This is the first clinical study to investigate the potential existence of genetic susceptibility to fracture non-union. Even though no concrete conclusions can be obtained from this pilot study, our results indicate the existence of a potential genetically predetermined impairment within the BMP signalling cascade, initiated after a fracture and when combined with other risk factors could synergistically increase the susceptibility of a patient to develop non-union. Further