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Sample records for bmp-2 co-dependently induce

  1. Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

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    Liao, Junyi; Hu, Ning; Zhou, Nian; Lin, Liangbo; Zhao, Chen; Yi, Shixiong; Fan, Tingxu; Bao, Wei; Liang, Xi; Chen, Hong; Xu, Wei; Chen, Cheng; Cheng, Qiang; Zeng, Yongming; Si, Weike; Yang, Zhong; Huang, Wei

    2014-01-01

    Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.

  2. Regulation of BMP2-induced intracellular calcium increases in osteoblasts.

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    Xu, Wenfeng; Liu, Bo; Liu, Xue; Chiang, Martin Y M; Li, Bo; Xu, Zichen; Liao, Xiaoling

    2016-10-01

    Although bone morphogenetic protein-2 (BMP2) is a well-characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca(2+) signaling. In this study, intracellular Ca(2+) concentration ([Ca(2+) ]i ) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3-E1 osteoblast cell line using fluorescence resonance energy transfer-based biosensors. Increase in [Ca(2+) ]i , FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF-573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2-stimulated [Ca(2+) ]i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2-induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca(2+) ]i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725-1733, 2016.

  3. PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

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    Xu, Xiaolong; Qiu, Sujun; Zhang, Yuxian; Yin, Jie; Min, Shaoxiong

    2017-03-01

    Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

  4. BMP-2 induces versican and hyaluronan that contribute to post-EMT AV cushion cell migration.

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    Kei Inai

    Full Text Available Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM components, versican and hyaluronan (HA, and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.

  5. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

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    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  6. Endocytosis contributes to BMP2-induced Smad signalling and neuronal growth.

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    Hegarty, Shane V; Sullivan, Aideen M; O'Keeffe, Gerard W

    2017-02-08

    Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons.

  7. 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.

  8. BMP2-induced inflammation can be suppressed by the osteoinductive growth factor NELL-1.

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    Shen, Jia; James, Aaron W; Zara, Janette N; Asatrian, Greg; Khadarian, Kevork; Zhang, James B; Ho, Stephanie; Kim, Hyun Ju; Ting, Kang; Soo, Chia

    2013-11-01

    Bone-morphogenetic protein 2 (BMP2) is currently the only Food and Drug Administration-approved osteoinductive growth factor used in clinical settings for bone regeneration and repair. However, the use of BMP2 is encumbered by numerous clinical complications, including postoperative inflammation and life-threatening cervical swelling. Thus, methods to prevent BMP2-induced inflammation would have far-reaching clinical implications toward improving current BMP2-based methods for bone regeneration. For the first time, we investigate the potential role of the growth factor Nel-like molecule-1 (NELL-1) in inhibiting BMP2-induced inflammation. Adult rats underwent a femoral bone onlay procedure, treated with either BMP2 protein (4 mg/mL), NELL-1 protein (4 mg/mL), or both proteins combined. Animals were evaluated at 3, 7, and 14 days postoperatively by histology, histomorphometry, immunohistochemistry, and real-time PCR for markers of inflammation (TNFα, IL6). The relative levels of TNFα and IL6 in serum were also detected by ELISA. The mechanism for NELL-1's anti-inflammatory effect was further assessed through examining inflammatory markers and generation of reactive oxygen species (ROS) in the mouse embryonic fibroblast NIH3T3 cells. BMP2 significantly induced local inflammation, including an early and pronounced polymorphonuclear cell infiltration accompanied by increased expression of TNFα and IL6. Treatment with NELL-1 alone elicited no significant inflammatory response. However, NELL-1 significantly attenuated BMP2-induced inflammation by all markers and at all timepoints. These local findings were also confirmed using systemic serum inflammatory biomarkers (TNFα, IL6). In each case, NELL-1 fully reversed BMP2-induced systemic inflammation. Lastly, our findings were recapitulated in vitro, where NELL-1 suppressed BMP2 induced expression of inflammatory markers, as well as NF-κB transcriptional activity and generation of ROS. BMP2-induced inflammation is a

  9. BMP2 induces osteoblast apoptosis in a maturation state and noggin-dependent manner.

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    Hyzy, Sharon L; Olivares-Navarrete, Rene; Schwartz, Zvi; Boyan, Barbara D

    2012-10-01

    Large doses of bone morphogenetic protein 2 (BMP2) are used clinically to induce bone formation in challenging bone defects. However, complications after treatment include swelling, ectopic bone formation, and adjacent bone resorption. While BMP2 can be effective, it is important to characterize the mechanism of the deleterious effects to optimize its use. The aim of this study was to determine the effect of BMP2 on apoptosis in osteoblast lineage cells and to determine the role of the BMP inhibitor Noggin in this process. Human mesenchymal stem cells (MSCs), immature osteoblast-like MG63 cells, and mature normal human osteoblasts (NHOst) were treated with BMP2. A model system of increased endogenous BMP signaling was created by silencing Noggin (shNOG-MG63). Finally, the BMP pathway regulating apoptosis in NHOst was examined using BMP signaling inhibitors (5Z-7-oxozeaenol, dorsomorphin, H-8). Apoptosis was characterized by caspase-3, BAX/BCL2, p53, and DNA fragmentation. BMP2 induced apoptosis in a cell-type dependent manner. While the effect was minor in MSCs, MG63 cells had modest increases and NHOst cells had robust increases apoptosis after BMP2 treatment. Apoptosis was significantly higher in shNOG-MG63 than MG63 cells. 5Z-7-oxozeaenol and dorsomorphin eliminated the BMP2-induced increase in DNA fragmentation in NHOst, suggesting roles for TAB/TAK1 and Smad signaling. These results indicate that the apoptotic effect of BMP2 is dependent on cell maturation state, inducing apoptosis in committed osteoblasts through Smad and TAB/TAK1 signaling, and is regulated by Noggin. Dose and delivery must be optimized in therapeutic applications of BMP2 to minimize complications.

  10. 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.

  11. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

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    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  12. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

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    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Kyunghwa [Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Gangwondo (Korea, Republic of); Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Jeong-Hwa, E-mail: baekjh@snu.ac.kr [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of)

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  13. Kaempferol induces chondrogenesis in ATDC5 cells through activation of ERK/BMP-2 signaling pathway.

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    Nepal, Manoj; Li, Liang; Cho, Hyoung Kwon; Park, Jong Kun; Soh, Yunjo

    2013-12-01

    Endochondral bone formation occurs when mesenchymal cells condense to differentiate into chondrocytes, the primary cell types of cartilage. The aim of the present study was to identify novel factors regulating chondrogenesis. We investigated whether kaempferol induces chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Kaempferol treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Kaempferol-treated ATDC5 cells stained more intensely with alcian blue staining than control cells, suggesting greater synthesis of matrix proteoglycans in the kaempferol-treated cells. Similarly, kaempferol induced greater activation of alkaline phosphatase activity than control cells, and it enhanced the expression of chondrogenic marker genes, such as collagen type I, collagen type X, OCN, Runx2, and Sox9. Kaempferol induced an acute activation of extracellular signal-regulated kinase (ERK) but not c-jun N-terminal kinase or p38 MAP kinase. PD98059, an inhibitor of MAPK/ERK, decreased in stained cells treated with kaempferol. Furthermore, kaempferol greatly expressed the protein and mRNA levels of BMP-2, suggesting chondrogenesis was stimulated via a BMP-2 pathway. Taken together, our results suggest that kaempferol has chondromodulating effects via an ERK/BMP-2 signaling pathway and could potentially be used as a therapeutic agent for bone growth disorders.

  14. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

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    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  15. Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage

    NARCIS (Netherlands)

    Davidson, E.N.; Vitters, E.L.; Bennink, M.B.; Lent, P.L. van; Caam, A.P. van; Blom, A.B.; Berg, W.B. van den; Loo, F.A.J. van de; Kraan, P.M. van der

    2015-01-01

    OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse

  16. 民猪BMP2基因的冷诱导研究%Expression of BMP2 in Min Pig during Cold Induced

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    张冬杰

    2010-01-01

    以75日龄民猪为试验材料,将骨形成蛋白2(BMP2)基因作为影响民猪抗寒特性的候选基因,对其在低温冷诱导后在民猪肌肉组织内的表达变化情况进行了分析.结果表明:BMP2基因在民猪冷诱导前后表达水平没有显著变化.

  17. Expression Products of Chimeric BMP2 and BMP7 Induce Osteoblast Differentiation%BMP2与BMP7嵌合表达产物可诱导成骨细胞分化

    Institute of Scientific and Technical Information of China (English)

    胡丽玲; 李晓霞; 张镜宇; 王宝利

    2009-01-01

    目的:构建骨形态发生蛋白(BMP)2与BMP7嵌合表达的分泌型基因载体pcDNA3-BMP2/7,检测表达产物的成骨诱导活性.方法:聚合酶链反应(PCR)扩增BMP2与BMP7的成熟肽编码基因,利用重叠延伸PCR以柔性肽(Gly_4Ser)_3编码序列使两者嵌合并克隆到质粒pcDNA3/sec上,转染CHO-K1细胞筛选得到稳定克隆,以其条件培养基处理鼠胚胎成纤维细胞C3H10T1/2,通过RT-PCR研究BMP2/7嵌合表达产物的活性.结果:BMP2/7嵌合表达产物可以明显提高C3H10T1/2细胞碱性磷酸酶(Alkaline phosphatase,ALP)、骨钙素(Osteocalcin,Oc)成骨细胞表型基因以及特异性转录因子Runx2 (runt-related transcription factor 2)mRNA的表达(P < 0.01).结论:制备的BMP2/7嵌合表达产物能够形成异源二聚体,诱导非骨源性细胞向成骨细胞分化.%Objective: To study the osteoinductive activity of chimeric molecule of bone morphogenetic protein(BMP)2 and BMP7 expressed in mammalian cells. Methods: Sequences encoding mature peptides of BMP2 and BMP7 were separately amplified by PCR and then linked by overlap-extension PCR with a DNA sequence encoding a flexible peptide (Gly_4Ser)_3 between them. The chimeric DNA sequence was cloned into secretory expression plasmid pcDNA3/sec and then the recombinant plasmid pcDNA3 -BMP2/7 was transfected into CHO-K1 cells. In the presence of G418,cells that stably expressed BMP2/7 were screened out. Thereafter, the conditioned culture medium of the transfected cells was collected and used to treat C3H10T1/2 cells. RT-PCR was employed to study the activity of the recombinant product in inducing osteoblast differentiation. Results: The expression products of chimeric BMP2/7 significantly enhanced the mRNA expression levels of osteoblast phenotype genes, such as alkaline phosphatase, osteocalcin and osteoblast specific transcription factor runt-related transcription factor 2 in C3H10T1/2 cells(P < 0.01). Conclusion: The chimeric expression products of BMP2

  18. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

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    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-01-01

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  19. YAP stabilizes SMAD1 and promotes BMP2-induced neocortical astrocytic differentiation.

    Science.gov (United States)

    Huang, Zhihui; Hu, Jinxia; Pan, Jinxiu; Wang, Ying; Hu, Guoqing; Zhou, Jiliang; Mei, Lin; Xiong, Wen-Cheng

    2016-07-01

    ‪YAP (yes-associated protein), a key transcriptional co-factor that is negatively regulated by the Hippo pathway, is crucial for the development and size control of multiple organs, including the liver. However, its role in the brain remains unclear. Here, we provide evidence for YAP regulation of mouse neocortical astrocytic differentiation and proliferation. YAP was undetectable in neurons, but selectively expressed in neural stem cells (NSCs) and astrocytes. YAP in NSCs was required for neocortical astrocytic differentiation, with no apparent role in self-renewal or neural differentiation. However, YAP in astrocytes was necessary for astrocytic proliferation. Yap (Yap1) knockout, Yap(nestin) conditional knockout and Yap(GFAP) conditional knockout mice displayed fewer neocortical astrocytes and impaired astrocytic proliferation and, consequently, death of neocortical neurons. Mechanistically, YAP was activated by BMP2, and the active/nuclear YAP was crucial for BMP2 induction and stabilization of SMAD1 and astrocytic differentiation. Expression of SMAD1 in YAP-deficient NSCs partially rescued the astrocytic differentiation deficit in response to BMP2. Taken together, these results identify a novel function of YAP in neocortical astrocytic differentiation and proliferation, and reveal a BMP2-YAP-SMAD1 pathway underlying astrocytic differentiation in the developing mouse neocortex.

  20. A protein kinase A (PKA)/β-catenin pathway sustains the BMP2/DLX3-induced osteogenic differentiation in dental follicle cells (DFCs).

    Science.gov (United States)

    Viale-Bouroncle, S; Klingelhöffer, C; Ettl, T; Reichert, T E; Morsczeck, C

    2015-03-01

    The directed expression of osteogenic transcription factors via a balanced activation of signaling pathways is an important prerequisite for the development of mineralized tissues. A positive-feedback loop of the BMP2-dependent SMAD signaling pathway and the DLX3 transcription factor (BMP2/DLX3 pathway) directs the osteogenic differentiation of periodontal precursor cells from the dental follicle (DFCs). However, little is known how this BMP2/DLX3 pathway interacts with other crucial signaling pathways such as the WNT/β-catenin signaling pathway. This study investigated the interaction between the BMP2/DLX3 pathway and the WNT pathway during the osteogenic differentiation of DFCs. BMP2 induced the WNT/β-catenin pathway in DFCs and phosphorylates β-catenin via protein kinase A (PKA). Moreover, only BMP2 facilitated the binding of LEF1/SMAD4/β-catenin complex to the DLX3 promoter, while an inducer of the canonical WNT pathway, WNT3A, act as an inhibitor. Although WNT3A inhibits the osteogenic differentiation of DFCs the expression of β-catenin was crucial for both the expression of DLX3 and for the osteogenic differentiation. In conclusion, while the activation of the canonical WNT pathway inhibits the osteogenic differentiation of DFCs, β-catenin sustains the BMP2/DLX3-mediated osteogenic differentiation via the activation of PKA.

  1. Uptake of nickel from 316L stainless steel into contacting osteoblastic cells and metal ion interference with BMP-2-induced alkaline phosphatase.

    Science.gov (United States)

    Mölders, Martina; Felix, Joachim; Bingmann, Dieter; Hirner, Alfred; Wiemann, Martin

    2007-11-01

    Bone cells contacting nickel (Ni)-containing implant materials may be affected by Ni species via disturbed signaling pathways involved in bone cell development. Here we analyze effects of the Ni-containing steel 316L and major metal constituents thereof on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) of MC3T3-E1 cells. While cells grew normally on 316L, cellular Ni content increased 10-fold vs. control within 4 days. With respect to the major components of 316L, Ni2+ (3-50 microM) was most inhibitory to BMP-2-induced ALP, whereas even 50 microM Fe3+, Cr3+, Mo5+, or Mn2+ had no such effect. In line with this, BMP-2-induced ALP was significantly reduced in cells on 316L. This effect was not prevented by the metal ion chelator diethylenetriaminepentaacetic acid (DTPA). Instead, DTPA abolished the stimulatory effect of BMP-2 on ALP, pointing to chelatable metal ions involved. Zn2+, as one possible candidate, antagonized the Ni2+ inhibition of BMP-2-induced ALP in both MC3T3-E1 and human bone marrow stromal cells. Results show that cells contacting 316L steel are exposed to increased concentrations of Ni which suffice to impair BMP-2-induced ALP activity. Zn2+, as a competitor of this inhibition, may help to restore normal osteoblastic function and bone development under these conditions.

  2. Activation of Bmp2-Smad1 signal and its regulation by coordinated alteration of H3K27 trimethylation in Ras-induced senescence.

    Directory of Open Access Journals (Sweden)

    Atsushi Kaneda

    2011-11-01

    Full Text Available Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF, we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence.

  3. Insights into the osteoblast precursor differentiation towards mature osteoblasts induced by continuous BMP-2 signaling

    Directory of Open Access Journals (Sweden)

    Omar F. Zouani

    2013-07-01

    Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors; and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

  4. β3 integrin-mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner.

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Picart, Catherine; Albiges-Rizo, Corinne

    2016-03-14

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin-dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2-induced Smad signaling through a Cdc42-Src-FAK-ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine.

  5. β3 integrin–mediated spreading induced by matrix-bound BMP-2 controls Smad signaling in a stiffness-independent manner

    Science.gov (United States)

    Fourel, Laure; Valat, Anne; Faurobert, Eva; Guillot, Raphael; Bourrin-Reynard, Ingrid; Ren, Kefeng; Lafanechère, Laurence; Planus, Emmanuelle; Albiges-Rizo, Corinne

    2016-01-01

    Understanding how cells integrate multiple signaling pathways to achieve specific cell differentiation is a challenging question in cell biology. We have explored the physiological presentation of BMP-2 by using a biomaterial that harbors tunable mechanical properties to promote localized BMP-2 signaling. We show that matrix-bound BMP-2 is sufficient to induce β3 integrin–dependent C2C12 cell spreading by overriding the soft signal of the biomaterial and impacting actin organization and adhesion site dynamics. In turn, αvβ3 integrin is required to mediate BMP-2induced Smad signaling through a Cdc42–Src–FAK–ILK pathway. β3 integrin regulates a multistep process to control first BMP-2 receptor activity and second the inhibitory role of GSK3 on Smad signaling. Overall, our results show that BMP receptors and β3 integrin work together to control Smad signaling and tensional homeostasis, thereby coupling cell adhesion and fate commitment, two fundamental aspects of developmental biology and regenerative medicine. PMID:26953352

  6. BMP2诱导C3H10细胞成骨分化的TGF-βⅠ型受体的体外分析%Analyses of TypeⅠTGF-β Receptors in BMP2 Induced Osteogenic Differentiation of C3H10 Cell in Vitro

    Institute of Scientific and Technical Information of China (English)

    张燕; 翁亚光; 文巍; 冯涛; 罗进勇

    2009-01-01

    筛选和分析与BMP2诱导间充质干细胞C3H10成骨分化有关的TGF-βⅠ型受体.利用显性负性突变型TGF-βⅠ型受体竞争抑制配体功能的特性,运用碱性磷酸酶定量测定、Real time PCR等方法,初步筛选出可能与BMP2诱导间充质干细胞C3H10成骨分化有关的的TGF-βⅠ型受体,随后运用RNA干扰的方法抑制相应TGF-βⅠ型受体的表达,进一步证实相关TGF-βⅠ型受体与BMP2发挥诱导成骨活性的关系.结果证实,显性负性突变的ALK3和ALK6能够抑制BMP2诱导的C3H10细胞成骨分化;RNA干扰抑制ALK3或(和)ALK6表达后,BMP2诱导C3H10细胞向成骨分化的趋势受到抑制.因此,ALK3和ALK6是与BMP2诱导C3H10细胞成骨分化有关的TGF-βⅠ型受体.%It was to analysis the typeⅠ TGF-β receptors in BMP2 induced osteogenic differentiation. The typeⅠ TGF-β receptors involved in BMP2 induced osteogenic differentiation of C3H10 cell was identified by using ALP quantitative assay and Real time PCR. The expression of BMP2 related typeⅠ TGF-β receptors was knock down by RNA interference, to confirm the relationship between osteogenic activity of BMP2 and typeⅠ TGF-β receptors. Results showed that dominant negative forms of ALK3 and ALK6 can inhibit BMP2 induced osteogenic differentiation of C3H10 cell,and ALK3/ALK6 knock-down by RNAi can inhibit BMP2 induced osteogenic differentiation of C3H10 cell. It can prove that ALK3 and ALK6 are the typeⅠ TGF-β receptors that can involve in BMP2 induced osteogenic differentiation of C3H10 cell.

  7. BMP-2 up-regulates PTEN expression and induces apoptosis of pulmonary artery smooth muscle cells under hypoxia.

    Directory of Open Access Journals (Sweden)

    Weifeng Pi

    Full Text Available AIM: To investigate the role of bone morphogenetic protein 2 (BMP-2 in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN and apoptosis of pulmonary artery smooth muscle cells (PASMCs under hypoxia. METHODS: Normal human PASMCs were cultured in growth medium (GM and treated with BMP-2 from 5-80 ng/ml under hypoxia (5% CO(2+94% N(2+1% O(2 for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR. Protein expression levels of PTEN, AKT and phosph-AKT (pAKT were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic (PTEN inhibitor and GW9662 (PPARγ antagonist were used to determine the signalling pathways. RESULTS: Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%. BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic and GW9662 (PPARγ inhibitor inhibited PTEN protein expression and recovered PASMCs proliferation rate. CONCLUSION: BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.

  8. 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

    datasets revealed significant downregulation of BMP2 in metastatic recurrent compared to non-metastatic cancer (p = 0.02). Global gene expression analysis in CRC cells over-expressing BMP2 revealed multiple dysregulated pathways mostly affecting cell cycle and DNA damage response. Concordantly, lentiviral......BACKGROUND: Molecular profiling of colorectal cancer (CRC) based on global gene expression has revealed multiple dysregulated signalling pathways associated with drug resistance and poor prognosis. However, the role of BMP2 signaling in CRC is not fully characterised. METHODS: Bioinformatics data...... 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...

  9. Retinal and choroidal expression of BMP-2 in lens-induced myopia and recovery from myopia in guinea pigs.

    Science.gov (United States)

    Li, Honghui; Wu, Juan; Cui, Dongmei; Zeng, Junwen

    2016-03-01

    The present study investigated the retinal and choroidal expression of bone morphogenetic protein-2 (BMP-2) in myopia and in myopia recovery in a guinea pig model. For this investigation, two groups of guinea pigs, lens‑induced myopia and recovery from myopia, were used, and defocused myopia was induced the guinea pigs wearing ‑4.00 D lenses on the right eyes for 3 weeks, with the left eyes serving as the contralateral. In the following week, the lenses of the guinea pigs in the recovery group were removed, and the refractive power and axial length were measured. The expression of BMP‑2 in the eyeballs was observed using immunohistochemistry and analyzed using Western blot analysis. After 3 weeks, the eyes acquired relative myopia and longer axial lengths in the two groups of guinea pigs. After 1 week without lenses in the recovery group, the myopia and axial lengths regressed. Immunofluorescence staining showed that BMP‑2 was expressed in the posterior retina, RPE, choroid and sclera. The expression of BMP‑2 decreased in the myopic retina of the guinea pigs. Following the regression of myopia in the recovery group, no difference in the expression of BMP‑2 was observed between the recovered treated eyes and the contralateral eyes. The choroidal expression level of BMP‑2 in the treated eyes showed no significant changes in either group. Therefore, BMP‑2 may be involved in the development of myopia, however, it does not have a primary role in the retinal and choroidal signals regulating scleral remodeling.

  10. BMP2 Mediates Decorin-induced Vascular Smooth Muscle Cell Calcification%BMP2参与Decorin诱导的血管平滑肌细胞钙化

    Institute of Scientific and Technical Information of China (English)

    颜建云; 周芹; 陈燕玲; 宋艳; 陆立鹤

    2014-01-01

    [目的]阐明BMP2是否参与了Decorin诱导的血管平滑肌细胞(VSMC)钙化.[方法]本研究采用人血管平滑肌细胞钙化体外模型,用腺病毒载体过表达Decorin,检测骨相关蛋白BMP2、Msx2、Osterix的表达和细胞钙化程度.观察抑制或增加BMP2信号对Decorin诱导的血管平滑肌细胞钙化和Msx2、Osterix表达的影响.[结果]Decorin能促进人血管平滑肌细胞钙化,同时上调BMP2、Msx2、Osterix的表达水平.抑制BMP2能减轻Decorin诱导的细胞钙化,而增加BMP2加速Decorin诱导的细胞钙化.[结论]Decofin通过调节BMP2促进人血管平滑肌细胞钙化.

  11. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  12. Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells.

    NARCIS (Netherlands)

    Jong, D.S. de; Steegenga, W.T.; Hendriks, J.M.; Zoelen, E.J.J. van; Olijve, W.; Dechering, K.J.

    2004-01-01

    The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type a

  13. Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells

    NARCIS (Netherlands)

    Jong, de D.S.; Steegenga, W.T.; Hendriks, J.M.A.; Zoelen, van E.J.J.; Olijve, W.; Dechering, K.J.

    2004-01-01

    The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type a

  14. The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism.

    Science.gov (United States)

    Khattab, Hany Mohamed; Ono, Mitsuaki; Sonoyama, Wataru; Oida, Yasutaka; Shinkawa, Shigehiko; Yoshioka, Yuya; Maekawa, Kenji; Tabata, Yasuhiko; Sugama, Kazushige; Sebald, Walter; Kuboki, Takuo

    2014-12-01

    Bone morphogenetic protein 2 (BMP2) is a potent osteoinductive cytokine that plays crucial roles in bone repair. However, large amounts of BMP2 are required to induce sufficient bone formation in humans possibly due to a feedback response of BMP antagonists. The engineered BMP2 variant L51P is deficient in BMP receptor type I activation but maintains affinity for BMP antagonists and can allow for the inactivation of BMP antagonists, and eventually enhance BMP2 action. As hypothesized, simultaneous addition of L51P enhanced the BMP2-induced osteogenesis. To test the ability of L51P to competitively inactivate BMP antagonists, cell binding affinity of BMP2 ligands was investigated in the presence or absence of L51P. Because the BMP antagonists were highly expressed 3 days after exogenous BMP2 stimulation, we collected supernatants from 3-day stimulated cell cultures and used as condition culture media (CM). The results showed a significant decrease in the cell binding of BMP2 ligands when cells were incubated with exogenous BMP2 and CM, whereas L51P addition competitively rescued the suppression of BMP2-to-cell binding induced by CM incubation. In a delayed experimental model, L51P was applied 3 days after exogenous BMP2 stimulation and we could observe a striking enhancement of the BMP2-induced SMAD-1/5/8 phosphorylation and luciferase activity of the Id1 promoter compared to the simultaneous addition of the two factors. These findings provide a deeper insight into the cellular and molecular mechanisms involved in the effect of L51P in suppressing the BMP antagonists and enhancing BMP activity. Additionally, these results demonstrate that L51P is a promising down regulator of BMP-induced negative feedback, which could have a significant impact in future applications of BMP2 in research and clinical settings.

  15. BMP2 induced osteogenic differentiation of human umbilical cord stem cells in a peptide-based hydrogel scaffold

    Science.gov (United States)

    Lakshmana, Shruthi M.

    Craniofacial tissue loss due to traumatic injuries and congenital defects is a major clinical problem around the world. Cleft palate is the second most common congenital malformation in the United States occurring with an incidence of 1 in 700. Some of the problems associated with this defect are feeding difficulties, speech abnormalities and dentofacial anomalies. Current treatment protocol offers repeated surgeries with extended healing time. Our long-term goal is to regenerate bone in the palatal region using tissue-engineering approaches. Bone tissue engineering utilizes osteogenic cells, osteoconductive scaffolds and osteoinductive signals. Mesenchymal stem cells derived from human umbilical cord (HUMSCs) are highly proliferative with the ability to differentiate into osteogenic precursor cells. The primary objective of the study was to characterize HUMSCs and culture them in a 3D hydrogel scaffold and investigate their osteogenic potential. PuraMatrix(TM) is an injectable 3D nanofiber scaffold capable of self-assembly when exposed to physiologic conditions. Our second objective was to investigate the effect of Bone Morphogenic Protein 2 (BMP2) in enhancing the osteogenic differentiation of HUMSCs encapsulated in PuraMatrix(TM). We isolated cells isolated from Wharton's Jelly region of the umbilical cord obtained from NDRI (New York, NY). Isolated cells satisfied the minimal criteria for mesenchymal stem cells (MSCs) as defined by International Society of Cell Therapy in terms of plastic adherence, fibroblastic phenotype, surface marker expression and osteogenic differentiation. Flow Cytometry analysis showed that cells were positive for CD73, CD90 and CD105 while negative for hematopoietic marker CD34. Alkaline phosphatase activity (ALP) of HUMSCs showed peak activity at 2 weeks (pBMP2 at doses of 50ng/ml, 100ng/ml and 200ng/ml. A significant upregulation of ALP gene in BMP2 treated cells was seen compared to HUMSCs treated in osteogenic medium (pBMP2 dose of

  16. 抑制Runx2的表达增强BMP2诱导的干细胞成软骨分化%Suppression of Runx2 Potentiates BMP2-induced Chondrogenic Differentiation

    Institute of Scientific and Technical Information of China (English)

    孙泽绪; 赵辰; 廖军义; 王琦; 徐伟; 陈诚; 黄伟

    2016-01-01

    目的:利用腺病毒介导RNA干扰抑制成骨分化关键转录调控因子Runx2(Runt-related transcription factor 2)的表达,研究其对骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)诱导间充质干细胞(mesenchymal stem cells,MSCs)成软骨分化的影响,并初步探讨相关机制.方法:利用腺病毒Ad-GFP、Ad-BMP2、Ad-SiRunx2感染C3H10T1/2细胞;共分4组:GFP组、BMP2组、BMP2+ SiRunx2组和SiRunx2组.Alcian blue染色检测各组软骨细胞基质糖胺聚糖分泌;RT-PCR检测Runx2、早期成软骨标志物(Col2a1)、蛋白聚糖(Aggrecan)及晚期成软骨标志物(Col10a1) mRNA表达水平;Western blot检测目的蛋白BMP2、Ⅱ型胶原(Col2a1)及X型胶原(Col10a1)的蛋白表达.结果:在BMP2诱导C3H10T1/2细胞成软骨分化过程中,下调Runx2的表达可以增强Col2a1、Aggrecan及软骨细胞外基质糖胺聚糖的合成,抑制Col10a1的合成.结论:下调Runx2表达可以增强BMP2诱导间充质干细胞成软骨分化能力,并抑制软骨细胞的成熟和肥大.

  17. Experimental study of the synergistic effect of Wnt11 and BMP-2 on inducing the differentiation of rat bone marrow mesenchymal stem cells into myocardium-like cells under%心肌微环境中Wnt11与BMP2协同作用促大鼠BM-MSCs分化为心肌样细胞的实验研究

    Institute of Scientific and Technical Information of China (English)

    马志峰; 董红燕; 张志峰; 袁红花; 张中明

    2011-01-01

    Objective To investigate the synergetic effect of Wnt11 and BMP2 on inducing differentiation of BM -MSCs into cardiomyocyte - like cells under the paracrine effect of myocardial micro - environment. Methods The Transwell co - culture model of BM - MSCs cells was establishment, with cardiac myocytes (CMs) together with Wnt11 plasmid -transfected 3T3 cells laid on the upper layer and BM - MSCs underneath. For the first three days, cells were cultured with 50 μg/L noggin ( BMP - inhibitor), followed by replacement with 0.5 μg/L BMP2 protein till the 14th day. According to the different inducing conditions, cells were divided into seven groups: positive control (heart group), myocardial -induction ( CM group), BMP2 - induction in myocardial micro - environment ( CM + BMP2 group), Wnt11 - induction in myocardial micro- environment (CM + Wnt11 group), Wnt11 and BMP2 co- induction (Wnt11 + BMP2 group), Wnt11 and BMP2 co- induction in myocardial micro- environment (CM + Wnt11 + BMP2 group), and negative control (BM -MSCs group). At the end of 14 days of induction culture, RT - PCR was employed to detect the expression levels of nucleic acid of the myocardium- specific transcription factors (Nkx2.5, GATA4 and Mef2c) and mature CM -specific genes (cTrtl, ANP, α - MHC and β - MHC) to evaluate the biological effect of diversified induction on the differentiation of BM -MSCs into cardiomyocyte - like cells. Results RT - PCR results revealed that in the myocardial micro - environment of paracrine the expression levels of myocardium - specific transcription factors and mature CM - specific genes in CM + Wnt11 + BMP2 group was significantly higher than the BM - MSCs group and the other co - culture groups but lower than the heart group ( P < 0. 05 ). Conclusions In cardiac paracrine micro - environment, the synergistic effect of Wnt11 and BMP - 2 can improve the efficiency of the differentiation from BM - MSCs into cardiomyocyte - like cells.%目的 探讨在心肌微

  18. Platelet-released supernatant induces osteoblastic differentiation of human mesenchymal stem cells: potential role of BMP-2

    Directory of Open Access Journals (Sweden)

    M Alini

    2010-12-01

    Full Text Available Platelet-rich preparations have recently gained popularity in maxillofacial and dental surgery, but their beneficial effect is still under debate. Furthermore, very little is known about the effect of platelet preparations at the cellular level, and the underlying mechanisms. In this study, we tested the effect of platelet-released supernatant (PRS on human mesenchymal stem cell (MSC differentiation towards an osteoblastic phenotype in vitro. Cultures of MSC were supplemented with PRS and typical osteoblastic markers were assessed at up to 28 days post-confluence. PRS showed an osteoinductive effect on MSC, as shown by an increased expression of typical osteoblastic marker genes such as collagen Ialpha1, bone sialoprotein II, BMP-2 and MMP-13, as well as by increased 45Ca2+ incorporation. Our results suggest that the effect of PRS on human MSC could be at least partially mediated by BMP-2.Activated autologous PRS could therefore provide an alternative to agents like recombinant bone growth factors by increasing osteoblastic differentiation of bone precursor cells at bone repair sites, although further studies are needed to fully support our observations.

  19. Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

    Science.gov (United States)

    Rogers, Melissa B; Shah, Tapan A; Shaikh, Nadia N

    2015-10-01

    The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an

  20. BMP-2 and titanium particles synergistically activate osteoclast formation

    Energy Technology Data Exchange (ETDEWEB)

    Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedics, Yinchuan, Ningxia Hui Autonomous Region, China, Department of Orthopedics, Affiliated Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Guo, H.H. [Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Zhang, J. [Institute of Pathology, Xi' an Jiaotong University, Xi' an Shaanxi, China, Institute of Pathology, Xi' an Jiaotong University, Xi' an Shaanxi (China); Yu, B. [Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, China, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China); Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedics, Yinchuan, Ningxia Hui Autonomous Region, China, Department of Orthopedics, Affiliated Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region (China)

    2014-05-09

    A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.

  1. Delta-like 1/fetal antigen 1(DLK1/FA1) inhibits BMP2 induced osteoblast differentiation through modulation of NFκB signaling pathway

    DEFF Research Database (Denmark)

    Qiu, Weimin; Abdallah, Basem; Kassem, Moustapha

    as assessed by reduced Alp activity and osteogenic gene expression including Alp, Col1a1, Runx2 and Bglap. In addition, DLK1/FA1 inhibited BMP signaling as demonstrated by reduced gene expression of BMP-responsive genes: Junb and Id1, reduced BMP2 induced luciferase activity in C2C12 BMP luciferase reporter....... Besides, we observed that DLK1/FA1 induced strong NFκB activity evidenced by NFκB responsive luciferase reporter assay and real-time RT-PCR analysis of NFκB target genes. The inhibitory effect of NFκB signaling on BMP signaling was confirmed by luciferase assay in C2C12 BMP luciferase reporter cells...

  2. 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

  3. Effect of grafting BMP2-derived peptide to nanoparticles on osteogenic and vasculogenic expression of stromal cells.

    Science.gov (United States)

    Mercado, Angel E; Yang, Xiaoming; He, Xuezhong; Jabbari, Esmaiel

    2014-01-01

    Bone morphogenetic protein-2 (BMP2) plays a major role in initiating the cascade of osteogenesis. However, high doses of exogenous BMP2 coupled with diffusion away from the intended site cause adverse side-effects. An alternative is to use biodegradable polymeric nanoparticles (NPs) grafted with peptides of the active domains of BMP2. NPs present a multivalent form of the peptide for stronger interaction with cell surface receptors, leading to a stronger activation of osteogenic signalling pathways. The objective of this work was to compare osteogenic activity of the BMP2 peptide (BMP2Pe), corresponding to residues 73-92 of BMP2 protein (BMP2Pr), grafted to biodegradable NPs with that of BMP2 protein (BMP2Pr). BMP2Pe was functionalized with a cysteine residue and grafted to poly(lactide fumarate) and poly(lactide-co-ethylene oxide fumarate) (PLAF/PLEOF) NPs via a thioether link. The calcium content of bone marrow stromal (BMS) cells cultured in osteogenic medium supplemented with BMP2 peptide/protein-grafted NPs (BMP2Pe-gNP and BMP2Pr-gNP) was slightly higher than other BMP2-treated groups, but all osteogenic groups showed similar levels of mineralization after 21 days. The expression pattern of master transcription factors Dlx5 and Runx2 indicated that BMP2 protein induced faster osteogenic signalling than the BMP peptide. The expression level of Osteopontin (OP), Osteocalcin (OC) and PECAM-1 in the NP-grafted BMP2 groups was significantly higher than those of ungrafted BMP2Pr and BMP2Pe groups, which may be due to a more effective presentation of the peptide/protein to cell surface receptors, thus leading to a stronger interaction of the peptide/protein with clustered cell surface receptors.

  4. HIF-1α potentiates BMP2-induced chondrogenic differentiation but inhibits osteogenic differentiation in stem cells%低氧诱导因子-1α对骨形态发生蛋白2诱导的干细胞成软骨、成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    周年; 黄伟; 廖军义; 胡宁; 陈筱蓉; 梁熙; 司维柯; 杨忠; 易世雄

    2014-01-01

    目的 探讨低氧通路中关键转录调控因子低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)对骨形态发生蛋白2(bone morpho-genetic protein 2,BMP2)诱导干细胞骨、软骨分化的影响,阐明HIF-1 α在干细胞成骨、软骨分化中的作用.方法 构建相应腺病毒AdBMP2、AdHIF-1 α、AdGFP,单独或共同感染干细胞,Western blot法检测成软骨、成骨分化关键转录调控因子Sox9、Runx2的表达,Real-time PCR法检测成软骨、成骨分化标志物COL2A1、aggrecan、COL1 A1和ALP mRNA表达,Alcian blue、ALP及Alizarin red S染色检测软骨细胞外基质及骨基质钙盐沉积情况.进行干细胞裸鼠皮下移植,观察不同处理组形成骨块的组织结构情况,探讨HIF-1α对BMP2诱导干细胞成骨、软骨分化的影响.结果 诱导分化后第1、3天,BMP2+ HIF-1α组Sox9蛋白表达明显高于BMP2单独处理组,而BMP2+ HIF-1α组Runx2蛋白表达明显低于BMP2单独处理组.诱导分化后第7、9天,BMP2+ HIF-1α组COL2A1、aggrecan mRNA相对表达明显高于BMP2单独处理组(P<0.05),而BMP2+ HIF-1α组COL1 A1、ALP mRNA相对表达明显低于BMP2单独处理组(P<0.05).Alcianblue染色发现BMP2+ HIF-1α组软骨细胞外基质分泌多于BMP2单独处理组,染色更深;ALP染色发现BMP2+ HIF-1 α组ALP的活性弱于BMP2单独处理组;茜素红染色发现BMP2+ HIF-1α组较BMP2单独处理组骨基质钙盐沉积更少;体内试验组织学观察见BMP2+ HIF-1α组软骨成分更多,骨化不明显,BMP2单独处理组软骨成分少,软骨内骨化更明显.结论 HIF-1α明显增强了BMP2诱导的干细胞成软骨分化,抑制了成骨分化及软骨内骨化,维持了软骨分化表型.

  5. Biological activity of a genetically modified BMP-2 variant with inhibitory activity

    Directory of Open Access Journals (Sweden)

    Kübler Alexander C

    2009-02-01

    Full Text Available Abstract Background Alterations of the binding epitopes of bone morphogenetic protein-2 (BMP-2 lead to a modified interaction with the ectodomains of BMP receptors. In the present study the biological effect of a BMP-2 double mutant with antagonistic activity was evaluated in vivo. Methods Equine-derived collagenous carriers were loaded with recombinant human BMP-2 (rhBMP-2 in a well-known dose to provide an osteoinductive stimulus. The study was performed in a split animal design: carriers only coupled with rhBMP-2 (control were implanted into prepared cavities of lower limb muscle of rats, specimens coupled with rhBMP-2 as well as BMP-2 double mutant were placed into the opposite limb in the same way. After 28 days the carriers were explanted, measured radiographically and characterized histologically. Results As expected, the BMP-2 loaded implants showed a typical heterotopic bone formation. The specimens coupled with both proteins showed a significant decreased bone formation in a dose dependent manner. Conclusion The antagonistic effect of a specific BMP-2 double mutant could be demonstrated in vivo. The dose dependent influence on heterotopic bone formation by preventing rhBMP-2 induced osteoinduction suggests a competitive receptor antagonism.

  6. Complexation and sequestration of BMP-2 from an ECM mimetic hyaluronan gel for improved bone formation.

    Directory of Open Access Journals (Sweden)

    Marta Kisiel

    Full Text Available Bone morphogenetic protein-2 (BMP-2 is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs dermatan sulfate (DS or heparin (HP, prior to loading it into a hyaluronic acid (HA hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.

  7. Construction of pcDNA3-BMP2 and Induced Expression in E. coli DH-5α%BMP2转基因载体的构建及在大肠杆菌DH-5α中的诱导表达

    Institute of Scientific and Technical Information of China (English)

    王国贤; 崔大祥; 刘丹平; 刘贺亮; 陈勇; 郭晏海; 刘晓虹

    2003-01-01

    BMP2基因来源于pSP6-BMP2质粒,用pcDNA3作载体,构建BMP2转基因载体并在大肠杆菌DH-5α中的诱导表达.用HindⅢ与XbaI双酶切pSP6-BMP2与pcDNA3质粒,回收BMP2基因片段与pcDNA3载体,用连接酶连接后转化JM109,提质粒后酶切鉴定;把构建好的载体转化DH-5α细菌,并诱导表达,用SDS-PAGE电泳鉴定有无表达,用WesternBlot鉴定表达蛋白.pcDNA3-BMP2载体酶切鉴定与预期片段相符,SDS-PAGE显示有BMP2蛋白表达;Western Blot证明表达蛋白有免疫原活性.成功地构建了表明pcDNA3-BMP2转基因载体并在大肠杆菌DH-5α中诱导表达了BMP蛋白.

  8. Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215.

    Science.gov (United States)

    Jung, Jae Woo; Ahn, Chihoon; Shim, Sun Young; Gray, Peter C; Kwiatkowski, Witek; Choe, Senyon

    2014-10-01

    Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LβT2 cells in which Activin A and BMP2 synergistically induce FSHβ. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LβT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LβT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LβT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHβ. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHβ induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHβ induction in LβT2 cells apparently due to its ability to block Activin A signaling.

  9. The effect of Smad6 RNA interference on BMP-2 induced osteogenic differentiation of mesenchymal stem cells%Smad6信号干扰对MSCs骨向分化的影响

    Institute of Scientific and Technical Information of China (English)

    刘猛; 董伟; 冯晓洁; 邓久鹏; 戚孟春; 李金源

    2011-01-01

    Objective To investigate the effect of Smad6 mRNA interference on bone morphogenetic protein 2 (BMP-2) induced osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). Methods The bone marrow MSCs of mice were cultured and underwent BMP-2 induced osteogenic differentiation. The cells were divided into 3 groups: the cells in group A were transfected with recombinant Smad6 RNA interference vector,which was labeled with green fluorescent protein (GFP) , and the cells in group B were transfected with control vector,and the cells in group C served as controls. The activity of alkaline phosphonate (ALP) and levels of osteocalcin were detected at five days after transfection by ALP staining and radioimmunoassay, respectively. The formation of mineralization nodus was also examined by alizarin red staining. Results The GFP was obviously expressed in MSCs after viral transfection, and viral transfection efficiency reached 98. 5%. As compared with group B,Smad6 RNA interference increased significantly ALP activity and osteocalcin levels in group A ( P < 0.01) .however,both ALP activity and osteocalcin levels in group C were significantly lower than those in the other two groups( P <0.01). The results of alizarin red staining showed that the counts of mineralization nodus in group A were significantly more than those in group B ( P <0.05) ,but no mineralization nodus was found in group C. Conclusion Smad6 mRNA interference. Can promote effectively BMP-2 induced osteogenic differentiation of MSCs,which may be a valuable method for bone regeneration for bone defect in bone tissue engineering.%目的 研究Smad6信号干扰对骨形态发生蛋白2(BMP-2)诱导的骨髓间充质干细胞(MSCs)骨向分化的促进效应.方法 培养小鼠MSCs,用BMP-2诱导骨向分化.细胞分为3组:A组细胞用携带绿色荧光蛋白(GFP)的Smad6重组RNA干扰载体转染;B组细胞用空白载体转染;C组细胞作为对照.结果 病毒转染后GFP在MSCs中有

  10. Soluble VEGFR1 reverses BMP2 inhibition of intramembranous ossification during healing of cortical bone defects.

    Science.gov (United States)

    Hu, Kai; Besschetnova, Tatiana Y; Olsen, Bjorn R

    2016-09-07

    BMP2 is widely used for promotion of bone repair and regeneration. However, bone formation induced by BMP2 is quite variable. Bone forming progenitor cells in different locations appear to respond to BMP2 in different ways, and repair outcomes can vary as a consequence of modulating effects by other factors. In this study, we have examined the effects of VEGF on BMP2-induced repair of a cortical bone defect, a 1 mm diameter drill hole, in the proximal tibia of mice. Treatment of the defect with either a bolus of PBS or soluble VEGFR1 (sVEGFR1), a decoy receptor for VEGF, had the same effects on bone formation via intramembranous ossification in the defect and cartilage formation and injured periosteum, during the healing process. In contrast, treatment with BMP2 inhibited intramembranous bone formation in the defect while it promoted cartilage and endochondral bone formation in the injured periosteum compared with mice treated with PBS or sVEGFR1. The inhibitory effect of BMP2 on bone formation was unlikely due to increased osteoclast activity and decreased invasion of blood vessels in the defect. Most importantly, co-delivery of BMP2 and sVEGFR1 reversed the inhibition of intramembranous bone formation by BMP2. Furthermore, the decreased accumulation of collagen and production of bone matrix proteins in the defect of groups with BMP2 treatment could also be prevented by co-delivery of BMP2 and sVEGFR1. Our data indicate that introducing a VEGF-binding protein, such as sVEGFR1, to reduce levels of extracellular VEGF, may enhance the effects of BMP2 on intramembranous bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  11. L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin.

    Science.gov (United States)

    Albers, Christoph E; Hofstetter, Wilhelm; Sebald, Hans-Jörg; Sebald, Walter; Siebenrock, Klaus A; Klenke, Frank M

    2012-09-01

    Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.

  12. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Chandler, Ronald L. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Fritz, David T. [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States); Mortlock, Douglas P. [Department of Molecular Physiology and Biophysics, Center for Human Genetics Research, Vanderbilt University School of Medicine, Nashville, TN (United States); Rogers, Melissa B., E-mail: rogersmb@umdnj.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry (UMDNJ), New Jersey Medical School (NJMS), Newark, NJ (United States)

    2010-02-05

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  13. Role of Smad7 in Sox9-potentiated and BMP2-induced differentiation of mouse mesenchymal stem cells into chondrocytes%Smad7在Sox9增强BMP2成软骨效应中的作用

    Institute of Scientific and Technical Information of China (English)

    赵辰; 黄伟; 梁熙; 廖军义; 周年; 胡宁; 赵智; 简长春

    2015-01-01

    目的 探讨Smad7在Sox9增强BMP2成软骨效应中的作用和机制.方法 将小鼠骨髓间充质干细胞(C3 H10T1/2)作为种子细胞,重组腺病毒Ad-BMP2和/或Ad-Sox9感染细胞,Ad-GFP感染细胞为对照.采用Real-time PCR、免疫细胞化学和Western blot分别检测感染后各组Smad7 mRNA表达水平和蛋白表达水平.采用Real-time PCR检测与Smad7相关因子MMP13与OPN mRNA的表达.结果 BMP2+Sox9组感染细胞7、11d时,Smad7 mRNA和蛋白表达水平均明显低于BMP2组(P<0.05);免疫细胞化学染色结果显示,BMP2+Sox9组Smad7染色明显弱于BMP2组;同时BMP2+Sox9组中与软骨最终成熟因子OPN与MMP13的表达均低于BMP2组(P<0.05).结论 在BMP2诱导间充质干细胞成软骨分化中,高表达的Smad7可被Sox9抑制,并抑制Smad7相关因子MMP13与OPN表达,从而解除了Smad7对BMP2成软骨的抑制作用,阻止了软骨细胞最终成熟骨化,有利于保持软骨发育与正常状态.

  14. BMP2-SMAD signaling represses the proliferation of embryonic neural stem cells through YAP.

    Science.gov (United States)

    Yao, Minghui; Wang, Yadong; Zhang, Peng; Chen, Hong; Xu, Zhiheng; Jiao, Jianwei; Yuan, Zengqiang

    2014-09-03

    Previous studies have shown that the Hippo pathway effector yes-associated protein (YAP) plays an important role in maintaining stem cell proliferation. However, the precise molecular mechanism of YAP in regulating murine embryonic neural stem cells (NSCs) remains largely unknown. Here, we show that bone morphogenetic protein-2 (BMP2) treatment inhibited the proliferation of mouse embryonic NSCs, that YAP was critical for mouse NSC proliferation, and that BMP2 treatment-induced inhibition of mouse NSC proliferation was abrogated by YAP knockdown, indicating that the YAP protein mediates the inhibitory effect of BMP2 signaling. Additionally, we found that BMP2 treatment reduced YAP nuclear translocation, YAP-TEAD interaction, and YAP-mediated transactivation. BMP2 treatment inhibited YAP/TEAD-mediated Cyclin D1 (ccnd1) expression, and knockdown of ccnd1 abrogated the BMP2-mediated inhibition of mouse NSC proliferation. Mechanistically, we found that Smad1/4, effectors of BMP2 signaling, competed with YAP for the interaction with TAED1 and inhibited YAP's cotranscriptional activity. Our data reveal mechanistic cross talk between BMP2 signaling and the Hippo-YAP pathway in murine NSC proliferation, which may be exploited as a therapeutic target in neurodegenerative diseases and aging.

  15. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

    Directory of Open Access Journals (Sweden)

    E Ferreira

    2012-07-01

    Full Text Available Transplantation of mesenchymal stem cells (MSCs with electrotransferred bone morphogenetic protein-2 (BMP-2 transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

  16. Acute BMP2 upregulation following induction of ischemic osteonecrosis in immature femoral head.

    Science.gov (United States)

    Kamiya, Nobuhiro; Shafer, Sasha; Oxendine, Ila; Mortlock, Douglas P; Chandler, Ronald L; Oxburgh, Leif; Kim, Harry K W

    2013-03-01

    Juvenile ischemic osteonecrosis of the femoral head (IOFH) is one of the most serious hip conditions causing the femoral head deformity. Little is known about BMP signaling following ischemic osteonecrosis. In this study, we found acute BMP2 upregulation in the femoral head cartilage 24h after ischemic induction using our immature pig IOFH model. Similarly, in our ischemic osteonecrosis mouse model, BMP2 expression and BMP signaling were enhanced in the articular cartilage surrounding the necrotic bone. BMP2 was increased in cartilage explants and primary chondrocytes under hypoxia (1% O(2)) compared with normoxia (21% O(2)). Addition of the hypoxia inducible factor 1 (HIF1) activator DFO significantly increased BMP2 while HIF1 silencing (siHIF1) only partially reduced BMP2, suggesting other mechanisms of BMP2 upregulation being present. Hypoxia is known to induce the production of free oxygen radicals, which are converted to hydrogen peroxide (H(2)O(2)) by superoxide dismutase 2 (SOD2). As an alternative mechanism, we investigated the effect of H(2)O(2)/SOD2 production on BMP2 upregulation. Chondrocytes produced more H(2)O(2) under hypoxia than normoxia. H(2)O(2) addition to the chondrocyte culture also significantly increased BMP2 expression. SOD2 was also dramatically increased in the ischemic pig cartilage at 24h following surgery and in primary chondrocytes/cartilage explants culture under hypoxia. SOD2 protein addition to the chondrocyte culture significantly increased BMP2. Moreover, DFO significantly increased SOD2 while HIF1 silencing only partially reduced SOD2. These results suggest that the acute BMP2 response of chondrocytes to ischemic osteonecrosis is more dominantly through the H(2)O(2) production and only partly through the HIF1 pathway.

  17. BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair.

    Science.gov (United States)

    Myers, Timothy J; Longobardi, Lara; Willcockson, Helen; Temple, Joseph D; Tagliafierro, Lidia; Ye, Ping; Li, Tieshi; Esposito, Alessandra; Moats-Staats, Billie M; Spagnoli, Anna

    2015-11-01

    The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12(+)-BMP2(+) endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2(cKO/+)) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2(cKO/+) mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2(cKO/+) mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2(cKO/cKO) endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12(+)-BMP2(+) perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12(+)-BMP2(+) to osteogenesis while departing their supportive role to angiogenesis. Our findings have far

  18. 转化生长因子β1(TGF-β1)和骨形成蛋白2(BMP2)体外联合诱导成牙本质细胞样细胞分化%Transforming Growth Factor β1 (TGFβ1) and Bone Morphogenetic Protein 2 (BMP2) Induce Odontoblast-like Cell Differentiation in vitro

    Institute of Scientific and Technical Information of China (English)

    朱奇; 樊明文; 张旗; 陈智; 边专

    2005-01-01

    目的:观察转化生长因子β1(transforming growth factor β1, TGF-β1)和骨形成蛋白2(bone morphogenetic protein 2,BMP2)联合应用对体外培养的鼠牙乳头成牙本质细胞分化的影响.方法:取17 d胎龄小鼠下颌第一磨牙牙胚,胰蛋白酶消化分离牙乳头,置半固态培养基培养6 d,半固态培养基中单独加入重组TGFβ1、BMP2,或分别与肝素联合,或TGFβ1和BMP2联合,组织学观察牙乳头的形态学变化.结果:TGF-β1或BMP2 单独加入时未见细胞极化, 但基质分泌增加.TGF-β1或BMP2 加肝素可诱导牙乳头周边细胞发生极化,并分泌胞外基质.半固态培养基中同时加入 TGF-β1和BMP2的牙乳头培养6 d后,牙乳头周边细胞出现极化和功能性分化,牙乳头周边胞外基质沉积明显,且可见牙乳头尖形态的维持及从牙乳头尖至牙乳头基底部成牙本质细胞分化梯度的维持.结论:本研究结果证实,在没有内釉上皮和完整的基底膜存在的情况下,TGF-β1和BMP2加肝素可诱导培养的牙乳头出现成牙本质细胞分化,并促进胞外基质的分泌.TGF-β1和BMP2 均能诱导成牙本质细胞的细胞学分化和分泌功能,二者联合应用可协同发挥作用增强诱导效果.

  19. BMP-2 gene-fibronectin-apatite composite layer enhances bone formation

    Directory of Open Access Journals (Sweden)

    Sogo Yu

    2011-08-01

    Full Text Available Abstract Background Safe and efficient gene transfer systems are needed for tissue engineering. We have developed an apatite composite layer including the bone morphogenetic protein-2 (BMP-2 gene and fibronectin (FB, and we evaluated its ability to induce bone formation. Methods An apatite composite layer was evaluated to determine the efficiency of gene transfer to cells cultured on it. Cells were cultured on a composite layer including the BMP-2 gene and FB, and BMP-2 gene expression, BMP-2 protein concentrations, alkaline phosphatase (ALP activity, and osteocalcin (OC concentrations were measured. A bone defect on the cranium of rats was treated with hydroxyapatite (HAP-coated ceramic buttons with the apatite composite layer including the BMP-2 gene and FB (HAP-BMP-FB. The tissue concentration of BMP-2, bone formation, and the expression levels of the BMP-2, ALP, and OC genes were all quantified. Results The apatite composite layer provided more efficient gene transfer for the cultured cells than an apatite composite layer without FB. The BMP-2 concentration was approximately 100~600 pg/mL in the cell-culture medium. Culturing the cells on the apatite composite layer for 27 days increased ALP activity and OC concentrations. In animal experiments, the tissue concentrations of BMP-2 were over 100 pg/mg in the HAP-BMP-FB group and approximately 50 pg/mg in the control groups. Eight weeks later, bone formation was more enhanced in the HAP-BMP-FB group than in the control groups. In the tissues surrounding the HAP button, the gene expression levels of ALP and OC increased. Conclusion The BMP-2 gene-FB-apatite composite layer might be useful for bone engineering.

  20. 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

    D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP...... enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce...

  1. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    2014-01-01

    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  2. Improving the osteogenic efficacy of BMP2 with mechano growth factor by regulating the signaling events in BMP pathway.

    Science.gov (United States)

    Deng, Moyuan; Liu, Peng; Xiao, Hualiang; Zhang, Yuanyuan; Wang, Yuanliang; Zhao, Jianhua; Xu, Jianzhong

    2015-09-01

    Local application of bone morphogenetic protein 2 (BMP2) is known to promote large bone defect healing and BMP2-initiated bone regeneration could be enhanced by an additional mechanical stimulation. The C-terminal 24-a.a. peptide of mechano growth factor (MGF24E), a mechanical-sensitive molecule, has been demonstrated to promote bone healing. Here, we propose a hypothesis that MGF24E could also improve the osteogenic efficacy of BMP2 by regulating the signaling events in the BMP pathway. To confirm the hypothesis, the potentials of MGF24E, BMP2 and BMP2/MGF24E combination treatments on the phosphorylation of Smad 1/5/8, the downstream osteogenesis-related gene expression and osteoblasts mineralization, are investigated with or without the blocking of Smad 5 siRNA. Furthermore, 15-mm rabbit radial bone defects were healed with the cytokine treatments and then evaluated by radiographic examination, histological assessment and immunohistochemical analysis. MGF24E could enhance the BMP2-induced Smad signaling pathway by upregulating the p-Smad protein expression and the downstream osteogenic gene expression. An amount of 5 nM BMP2 in a sub-25 nM concentration of MGF24E medium achieved a higher expression for ALP mRNA and a greater calcium mineral content compared with BMP2 alone. Nevertheless, the inhibition of the MGF24E-regulated BMP pathway could block osteogenesis induced by the dual treatment. In vivo, MGF24E treatment upregulated the endogenous BMP2 expression and the addition of MGF24E into the BMP2 treatment remarkably enhanced the bone mineral density (BMD), the radiographic scores and the histological restoration of the regenerated tissue against BMP2 treatment, suggesting a new strategy for BMP2 in bone defect healing.

  3. 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.

  4. Identification of BMP2 as an epigenetically silenced growth inhibitor in rhabdomyosarcoma.

    Science.gov (United States)

    Wolf, Sebastian; Hagl, Beate; Kappler, Roland

    2014-05-01

    Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of infancy and although therapy has improved over the years, mortality is still fairly high. The establishment of new treatments has been hampered by the limited knowledge of the molecular mechanisms driving development of RMS. One characteristic of cancer cells is aberrant DNA methylation, which could lead to silencing of tumor suppressor genes. However, only a few epigenetically silenced genes have been described in RMS so far. We performed an expression profiling analysis of three RMS cell lines that were treated with the demethylating agent 5'-aza-2'-deoxycytidine (5-Aza‑dC) facilitating re-expression of epigenetically silenced genes. This treatment induced the gene BMP2 (bone morphogenetic protein 2) throughout all cell lines. Detailed methylation analysis of CpG sites in the BMP2 promoter region by bisulfite sequencing and methylation-specific PCR revealed that a high degree of DNA methylation is causatively associated with the suppression of BMP2 in RMS cells. Consequently, treatment of the RMS cell lines with 5-Aza-dC resulted in DNA demethylation of the BMP2 promoter, most prominently in alveolar RMS. Supplementation of recombinant human BMP2 (rhBMP2) led to a reduced viability of RMS cells. Altogether, these findings suggest that suppression of BMP2 by epigenetic silencing may play a critical role in the genesis of RMS, thereby providing a rationale for the development of a new treatment strategy for RMS.

  5. Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix.

    Science.gov (United States)

    Ben-David, Dror; Srouji, Samer; Shapira-Schweitzer, Keren; Kossover, Olga; Ivanir, Eran; Kuhn, Gisela; Müller, Ralph; Seliktar, Dror; Livne, Erella

    2013-04-01

    Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 μg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects.

  6. Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro.

    Science.gov (United States)

    Lysdahl, Helle; Baatrup, Anette; Foldager, Casper Bindzus; Bünger, Cody

    2014-12-01

    Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and protein level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and protein levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided.

  7. 核心结合因子α1在BMP-2调控细胞外基质蛋白表达中的作用%Study of cbfα1 on the expression of extracellular matrix proteins in dental papilla cells induced by BMP-2 in vitro

    Institute of Scientific and Technical Information of China (English)

    余擎; 朱庆林; 孙汉堂; 田宇; 何文喜; 肖明振

    2008-01-01

    目的:探讨核心结合因子α1(cbfa1)在BMP-2调控体外培养的牙乳头细胞表达细胞外基质蛋白中的作用.方法:采用反义核酸技术,体外阻断培养的牙乳头细胞中cbfα1的表达,分别用RT-PCR、Western印迹等方法观察200ng/mL BMP-2作用6h后细胞中相关基质蛋白,碱性磷酸酶(ALP)、骨钙素(OC)、骨连蛋白(ON)、骨桥素(OPN)、骨涎蛋白(BSP)、牙本质基质蛋白1(DMP-1)以及牙本质涎磷蛋白(DSPP)的表达,采用SPSS 11.0软件包对数据进行方差分析.结果:外源性BMP-2能明显上调牙乳头细胞中ALP、OC含量以及OPN、BSP和ON的表达,当反义阻断cbfα1的表达时,ALP、OC、OPN和BSP的表达显著降低(P<0.01).结论:cbfα1参与了BMP-2调控体外培养的牙乳头细胞表达细胞外基质蛋白的信号转导过程.

  8. Sequential Treatment with SDF-1 and BMP-2 Potentiates Bone Formation in Calvarial Defects.

    Science.gov (United States)

    Hwang, Hee-Don; Lee, Jung-Tae; Koh, Jeong-Tae; Jung, Hong-Moon; Lee, Heon-Jin; Kwon, Tae-Geon

    2015-07-01

    Stromal cell-derived factor-1 (SDF-1) protein and its receptor, CXCR-4, play an important role in tissue repair and regeneration in various organs, including the bone. SDF-1 is indispensable for bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. However, SDF-1 is not needed after the osteogenic induction has been activated. Since the precise condition for the additive effects of combined DF-1 and BMP-2 in bone healing had not been fully investigated, we aimed to determine the optimal conditions for SDF-1- and BMP-2-mediated bone regeneration. We examined the in vitro osteoblastic differentiation and cell migration after sequential treatments with SDF-1 and BMP-2. Based on the in vitro additive effects of SDF-1 and BMP-2, the critical size defects of mice calvaria were treated with these cytokines in various sequences. Phosphate buffered saline (PBS)-, SDF-1-, or BMP-2-soaked collagen scaffolds were implanted into the calvarial defects (n=36). Periodic percutaneous injections of PBS or the cytokine SDF-1 and BMP-2 into the implanted scaffolds were performed on days 3 and 6, postoperatively. Six experimental groups were used according to the types and sequences of the cytokine treatments. After 28 days, the mice were euthanized and bone formation was evaluated with microcomputed tomography and histology. The molecular mechanism of the additive effect of SDF-1 and BMP-2 was evaluated by analyzing intracellular signal transduction through Smad and Erk phosphorylation. The in vitro experiments revealed that, among all the treatments, the treatment with BMP-2 after SDF-1 showed the strongest osteoblastic differentiation and enhanced cell migration. Similarly, in the animal model, the treatment with SDF-1 followed by BMP-2 treatment showed the highest degree of new bone regeneration than any other groups, including the one with continuous BMP-2 treatment. This new bone formation can be partially explained by the activation of Smad and Erk pathways

  9. Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2.

    Science.gov (United States)

    Salazar, Valerie S; Ohte, Satoshi; Capelo, Luciane P; Gamer, Laura; Rosen, Vicki

    2016-12-01

    Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1(+) osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3.

  10. Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

    Science.gov (United States)

    Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

    2017-01-01

    In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

  11. Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

    Science.gov (United States)

    Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

    2017-01-01

    In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro. PMID:28134270

  12. Periosteal BMP2 activity drives bone graft healing.

    Science.gov (United States)

    Chappuis, Vivianne; Gamer, Laura; Cox, Karen; Lowery, Jonathan W; Bosshardt, Dieter D; Rosen, Vicki

    2012-10-01

    Bone graft incorporation depends on the orchestrated activation of numerous growth factors and cytokines in both the host and the graft. Prominent in this signaling cascade is BMP2. Although BMP2 is dispensable for bone formation, it is required for the initiation of bone repair; thus understanding the cellular mechanisms underlying bone regeneration driven by BMP2 is essential for improving bone graft therapies. In the present study, we assessed the role of Bmp2 in bone graft incorporation using mice in which Bmp2 has been removed from the limb prior to skeletal formation (Bmp2(cKO)). When autograft transplantations were performed in Bmp2cKO mice, callus formation and bone healing were absent. Transplantation of either a vital wild type (WT) bone graft into a Bmp2(cKO) host or a vital Bmp2(cKO) graft into a WT host also resulted in the inhibition of bone graft incorporation. Histological analyses of these transplants show that in the absence of BMP2, periosteal progenitors remain quiescent and healing is not initiated. When we analyzed the expression of Sox9, a marker of chondrogenesis, on the graft surface, we found it significantly reduced when BMP2 was absent in either the graft itself or the host, suggesting that local BMP2 levels drive periosteal cell condensation and subsequent callus cell differentiation. The lack of integrated healing in the absence of BMP2 was not due to the inability of periosteal cells to respond to BMP2. Healing was achieved when grafts were pre-soaked in rhBMP2 protein, indicating that periosteal progenitors remain responsive in the absence of BMP2. In contrast to the requirement for BMP2 in periosteal progenitor activation in vital bone grafts, we found that bone matrix-derived BMP2 does not significantly enhance bone graft incorporation. Taken together, our data show that BMP2 signaling is not essential for the maintenance of periosteal progenitors, but is required for the activation of these progenitors and their subsequent

  13. 骨钙素在BMP2/7异源二聚体诱导种植体周围骨缺损再生中的表达%Expression of osteocalcin using rhBMP2/7 heterodimer in peri-implant bone defects compared to BMP2 homodimer and BMP7 homodimer

    Institute of Scientific and Technical Information of China (English)

    孙平; 王利民; 冯剑颖

    2011-01-01

    目的:研究骨钙素在使用BMP2/7异源二聚体促进种植体周骨缺损再生中的表达.方法:建立小型猪种植体周骨缺损模型,并使用BMP2/7异源二聚体及BMP2、BMP7同源二聚体促进骨再生,采用免疫组化方法分别在2、3、6周时检测新骨中骨钙素表达.结果:在各实验组中,骨钙素3周时表达达到最高值.在2、3、6周,BMP2/7组骨钙素表达均强于BMP2、BMP7同源二聚体组及对照组.结论:在以同样较低浓度(30ng/ml)作用于小型猪种植体周骨缺损诱导新骨形成过程中,BMP2/7异源二聚体较BMP、BMP7同源二聚体有效促进骨钙素的表达.%To delineate expression of osteocalcin induced by recombinant human bone morphogenetic protein (BMP)2/7 heterodimer in peri-implant bone defects in comparison to BMP2 and BMP7 homodimer.Methods: Identical peri-implant bone defects were created on the frontal skull of minipig. Collagen sponges with the same dose BMP2/7 heterodimer、 BMP2 homodimer 、 BMP7 homodimer 、 or BMPs were adopted to treat the freshly created implant bed. Titanium implants were centrally implanted with 4mm-flxture within bone defects. Immunohistochemical method was applied to evaluate the expression of osteocalcin (OCN) after 2, 3, and 6 weeks of implantation. Results: The OCN proteins displayed intensity peaks at 3 weeks (P<0.05) in all the groups. Immunoreactive expression of OCN of BMP2/7 group was more intensity than BMP2、 BMP7 and control group at 2, 3, and 6 weeks post-operation (P<0.05). Conclusions: Purified recombinant human BMP2/7 heterodimer can enhance the expression of OCN with significant differences over BMP2 homodimers or BMP7 homodimers with the same low-dose (30ng/ml).

  14. Improving Osteogenesis Activity on BMP-2-Immobilized PCL Fibers Modified by the γ-Ray Irradiation Technique

    Directory of Open Access Journals (Sweden)

    Young-Pil Yun

    2015-01-01

    Full Text Available The purpose of this study was to demonstrate the ability of BMP-2-immobilized polycaprolactone (PCL fibers modified using the γ-ray irradiation technique to induce the osteogenic differentiation of MG-63 cells. Poly acrylic acid (AAc was grafted onto the PCL fibers by the γ-ray irradiation technique. BMP-2 was then subsequently immobilized onto the AAc-PCL fibers (BMP-2/AAc-PCL. PCL and surface-modified PCL fibers was characterized by evaluation with a scanning electron microscope (SEM, X-ray photoelectron spectroscopy (XPS, and contact angle. The biological activity of the PCL and surface-modified PCL fibers were characterized by alkaline phosphatase (ALP activity, calcium deposition, and the mRNA expression of osteocalcin and osteopontin in MG-63 cells. Successfully grafted AAc and PCL fibers with immobilized BMP-2 were confirmed by XPS results. The results of the contact angle showed that BMP-2/AAc-PCL fibers have more hydrophilic properties in comparison to PCL fibers. The ALP activity, calcium deposition, and gene expressions of MG-63 cells grown on BMP-2/AAc-PCL fibers showed greatly induced osteogenic differentiation in comparison to the PCL fibers. In conclusion, these results demonstrated that BMP-2/AAc-PCL fibers have the potential to effectively induce the osteogenic differentiation of MG-63 cells.

  15. BMP-2 regulates the formation of oral sulcus in mouse tongue by altering the balance between TIMP-1 and MMP-13.

    Science.gov (United States)

    Fukui, Tadayoshi; Suga, Takeo; Iida, Ryo-Hei; Morito, Mitsuhiko; Luan, Xianghong; Diekwisch, Thomas G H; Nakamura, Yoshiki; Yamane, Akira

    2010-08-01

    The aim of this study is to investigate whether BMP-2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP-2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP-2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP-2 siRNA induced a 60% decrease in the expression of TIMP-1 mRNA (P sulcus in the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 220% increases in the expression of TIMP-1 mRNA and the area of the immunostaining for TIMP-1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP-2 than the vehicle. The BMP-2 siRNA induced a 60% increase in the expression of MMP-13 protein and a marked increase in the staining intensity for MMP-13 was observed in the epithelial region of the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 70% decrease in the expression of MMP-13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP-2, TIMP-1, and MMP-13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP-2 regulates the formation of oral sulcus by altering the balance between TIMP-1 and MMP-13.

  16. Designer Nodal/BMP2 Chimeras Mimic Nodal Signaling, Promote Chondrogenesis, and Reveal a BMP2-like Structure

    Science.gov (United States)

    Esquivies, Luis; Blackler, Alissa; Peran, Macarena; Rodriguez-Esteban, Concepcion; Izpisua Belmonte, Juan Carlos; Booker, Evan; Gray, Peter C.; Ahn, Chihoon; Kwiatkowski, Witek; Choe, Senyon

    2014-01-01

    Nodal, a member of the TGF-β superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries. PMID:24311780

  17. Induction of chronic pancreatitis by pancreatic duct ligation activates BMP2, apelin, and PTHrP expression in mice.

    Science.gov (United States)

    Rastellini, Cristiana; Han, Song; Bhatia, Vandanajay; Cao, Yanna; Liu, Ka; Gao, Xuxia; Ko, Tien C; Greeley, George H; Falzon, Miriam

    2015-10-01

    Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-β (TGF-β) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-β, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.

  18. An Activin A/BMP2 chimera displays bone healing properties superior to those of BMP2

    Science.gov (United States)

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C.; Ye, Sang-kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-01-01

    Recombinant Bone Morphogenetic Protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue which when mutated to the corresponding BMP2 residue resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures. PMID:24692083

  19. An activin A/BMP2 chimera, AB204, displays bone-healing properties superior to those of BMP2.

    Science.gov (United States)

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C; Ye, Sang-Kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-09-01

    Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures.

  20. Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Lin, Zhaowei; Wang, Jiang-Sheng; Lin, Lijun; Zhang, Jingwen; Liu, Yunlong; Shuai, Ming; Li, Qi

    2014-03-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are dominant seed cell sources for bone regeneration. Bone morphogenetic proteins (BMPs) initiate cartilage and bone formation in a sequential cascade. Vascular endothelial growth factor (VEGF) is an essential coordinator of extracellular matrix remodeling, angiogenesis and bone formation. In the present study, the effects of the vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP2) genes on bone regeneration were investigated by the lentivirus-mediated cotransfection of the two genes into rat bone marrow-derived MSCs. The successful co-expression of the two genes in the MSCs was confirmed using quantitative polymerase chain reaction (qPCR) and western blot analysis. The results of alizarin red and alkaline phosphatase (ALP) staining at 14 days subsequent to transfection showed that the area of staining in cells transfected with BMP2 alone was higher than that in cells transfected with BMP2 and VEGF165 or untransfected control cells, while the BMP2 + VEGF165 group showed significantly more staining than the untransfected control. This indicated that BMP2 alone exhibited a stronger effect in bone regeneration than BMP2 in combination with VEGF165. Similarly, in inducing culture medium, the ALP activity of the BMP2 + VEGF165 group was notably suppressed compared with that of the BMP2 group. The overexpression of VEGF165 inhibited BMP2-induced MSC differentiation and osteogenesis in vitro. Whether or not local VEGF gene therapy is likely to affect bone regeneration in vivo requires further investigation.

  1. BMP2 rescues deficient cell migration in Tgfbr3(-/-) epicardial cells and requires Src kinase.

    Science.gov (United States)

    Allison, Patrick; Espiritu, Daniella; Camenisch, Todd D

    2016-05-03

    During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(-/-) mouse embryos, we show that Tgfbr3(-/-) epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3(-/-) epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3(+/+) cells; BMP2 induces cellular migration to Tgfbr3(+/+) levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3(-/-) migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3(-/-) epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.

  2. Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice.

    Science.gov (United States)

    McBride-Gagyi, Sarah Howe; McKenzie, Jennifer A; Buettmann, Evan G; Gardner, Michael J; Silva, Matthew J

    2015-12-01

    Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10-24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.

  3. Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro

    DEFF Research Database (Denmark)

    Lysdahl, Helle; Baatrup, Anette; Foldager, Casper Bindzus;

    2014-01-01

    treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured...... in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of h......MSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided....

  4. Bone Regeneration using an Alpha 2 Beta 1 Integrin-Specific Hydrogel as a BMP-2 Delivery Vehicle

    Science.gov (United States)

    Shekaran, Asha; García, José R.; Clark, Amy Y.; Kavanaugh, Taylor E.; Lin, Angela S.; Guldberg, Robert E.; García, Andrés J.

    2014-01-01

    Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential. PMID:24726536

  5. Accelerated bone growth in vitro by the conjugation of BMP2 peptide with hydroxyapatite on titanium alloy.

    Science.gov (United States)

    Cai, Yanli; Wang, Xiaoyan; Poh, Chye Khoon; Tan, Hark Chuan; Soe, Min Tun; Zhang, Sam; Wang, Wilson

    2014-04-01

    Titanium alloys have been widely used in orthopedic practice due to their inherent bioactivity, however it is still insufficient to truly and reliably incorporate into living bone. In this work, polydopamine film was employed to induce the growth of hydroxyapatite (HA) on titanium alloy to enhance its osteoconductivity. Bone morphogenetic protein-2 (BMP2) peptide was absorbed into the HA particles for osteoinductivity. The precipitation of HA and the existence of BMP2 peptide were examined by X-ray diffraction, X-ray photoelectron spectroscopy and fluorescence microscopy. The dissolution of HA and the release of BMP2 peptide were monitored by measuring the concentrations of calcium ions and BMP2 peptide in phosphate buffered saline solution, respectively. The effect of BMP2 peptide incorporated into HA coating on bone growth was evaluated in vitro by cell culture tests, including cell attachment, alkaline phosphatase (ALP) activity, and gene expression. The results show that the HA particles grown on the substrate are mediated by the polydopamine film. The BMP2 peptide is distributed uniformly on HA-coated substrate and released in a sustained manner. Moreover, the conjunction of HA and BMP2 peptide increases cell adhesion, ALP activity and gene expression of osteogenic markers, which are potentially useful in the development of enhanced orthopedic medical devices.

  6. Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

    Science.gov (United States)

    Ding, H F; Liu, R; Li, B G; Lou, J R; Dai, K R; Tang, T T

    2007-11-03

    We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (pAPA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

  7. The influence of Aloe vera and xenograft XCB toward of bone morpho protein 2 BMP2 expression and amount of osteoblast of alveolar bone induced into tooth extraction sockets Cavia cobaya

    Directory of Open Access Journals (Sweden)

    Utari Kresnoadi

    2014-12-01

    Full Text Available Tooth extraction can cause inflammation leading to alveolar ridge resorption. In addition, prominent ridge has crucial role for making denture su-ccessfully. Thus, socket preservation is needed to prevent greater alveolar ridge resorption. An innovative material, a combination of Aloe vera and xe-nograft (XCB, is then considered as a biogenic stimulator that can reduce inflammation, as a result, the growth of alveolar bone is expected to be impro-ved. This research is aimed to prove whether the mixture of Aloe vera and xenograft can stimulate BMP2 and increase osteoblasts. Forty-eight Cavia co-baya animals were divided into eight groups each of which consisted of six animals. The mandibular incisors of those Cavia cobaya animals were then extracted and filled with PEG as Group Control, XCB as Group XCB, Aloe vera as Group Aloe vera, and a combination of Aloe vera +XCB as Group Aloe vera +XCB. Next, the first four groups were sacrificed seven days after extraction, and the second four groups were sacrificed 30 days after extrac-tion. And then, immunohistochemical and histopathology examinations were conducted to examine BMP2 expression and osteoblasts. Based on the re-sult known that the mixture of Aloe vera and xenograft can increase BMP2 expression and amount of osteoblasts. It can be concluded that the mixture of Aloe vera and xenograft can increase BMP2 expression and amount of osteoblast cel . It can be used as an alternative material to increase the growth of alveolar bone after extraction.

  8. 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 surroun

  9. Crosstalk of FGF-2 and BMP-2 in Osteoblastic Differentiation of Cranial Suture Cells%FGF-2与BMP-2在颅缝细胞成骨分化中的相互作用

    Institute of Scientific and Technical Information of China (English)

    姜陶然; 曹德君

    2015-01-01

    目的:探讨碱性成纤维生长因子2(FGF-2)与骨形成蛋白2(BMP-2)在颅缝细胞成骨分化中的相互作用及其机制。方法获取新生SD大鼠颅骨矢状缝及冠状缝处颅缝细胞,在培养体系中添加FGF-2,观察BMP-2表达情况。同时在培养体系中添加FGF-2及BMP-2,ALP染色、矿化染色、qPCR检测成骨标志物,观察颅缝细胞成骨分化情况。添加BMP-2抑制剂Noggin后,观察颅缝细胞成骨分化的转归。结果 FGF-2可促进BMP-2在颅缝细胞中的表达,呈浓度依赖性及时间依赖性;两者同时作用颅缝细胞可促进其晚期成骨分化,抑制其早期成骨分化。 Noggin阻断BMP-2信号通道后,FGF-2及FGF-2+BMP-2促进颅缝细胞晚期成骨分化作用均减弱。结论 BMP-2是FGF-2调控颅缝细胞晚期成骨分化不可或缺的下游因子。%Objective To explore the interaction of FGF-2 and BMP-2 in osteoblastic differentiation of calvarial suture cells. Methods Neonatal calvarial suture cells of SD rat were harvested. FGF-2 was added into cell cultures and BMP-2 expression in cranial suture cells was observed. Meanwhile, FGF-2 and BMP-2 were both added into cell cultures and the osteoblastic differentiation of cranial suture cells was observed by ALP staining, mineralized nodule staining and qPCR. Then Noggin was added to observe the changes of cells’ osteoblastic differentiation. Results BMP-2 expression increased in a time-dependent manner after the cells treated with FGF-2 and increased in a dose-dependent manner up to 50 ng/ml FGF-2, after which BMP-2 expression reached a plateau;After FGF-2 and BMP-2 co-stimulation, the expression of early marker of osteoblast differentiation (COL-1) was decreased while the expression of late markers (ALP, OC and BSP) were increased to accelerate mineralization. The natural BMP antagonist Noggin inhibited the expression of FGF2-induced OC and BSP by 1.40-fold and 1.41-fold respectively, and inhibited the

  10. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

    Directory of Open Access Journals (Sweden)

    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

  11. The expression analysis of Bmpr1a and Bmp2 during hindgut development in rat embryos with anorectal malformations.

    Science.gov (United States)

    Tang, Xiao Bing; Zhang, Jin; Wang, Wei Lin; Yuan, Zheng Wei; Bai, Yu Zuo

    2016-08-01

    The aim of this study was to determine Bmpr1a and Bmp2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Bmpr1a and Bmp2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos. The embryos were harvested by Cesarean deliveries. The expression of Bmpr1a and Bmp2 was evaluated in normal rat embryos (n=213) and ARM embryos (n=236) from GD14 to GD16. Immunohistochemical staining revealed, in normal embryos, that Bmpr1a and Bmp2 was mainly expressed on the epithelium of the urorectal septum (URS) and the cloacal membrane (CM) on GD14 and GD15. When the rectum separated from the urogenital sinus (UGS) on GD16, Bmpr1a- and Bmp2-immunolabeled cells were observed on the anorectal epithelium. In ARM embryos, the epithelium of the hindgut and URS demonstrated faint immunostaining for Bmpr1a and Bmp2. Analyses by Western blot and Real-time PCR revealed that Bmpr1a and Bmp2 protein and mRNA expression were significantly decreased in the ARM hindgut compared with normal hindgut on GD14 and GD15 (PBmp2 was noted during anorectal morphogenesis from GD14 to GD16. Therefore, downregulation of Bmpr1a and Bmp2 at the time of cloacal separation into the primitive rectum and UGS might be related to the development of ARM.

  12. Epigenetic regulation of BMP2 by 1,25-dihydroxyvitamin D3 through DNA methylation and histone modification.

    Science.gov (United States)

    Fu, Baisheng; Wang, Hongwei; Wang, Jinhua; Barouhas, Ivana; Liu, Wanqing; Shuboy, Adam; Bushinsky, David A; Zhou, Dongsheng; Favus, Murray J

    2013-01-01

    Genetic hypercalciuric stone-forming (GHS) rats have increased intestinal Ca absorption, decreased renal tubule Ca reabsorption and low bone mass, all of which are mediated at least in part by elevated tissue levels of the vitamin D receptor (VDR). Both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and bone morphogenetic protein 2 (BMP2) are critical for normal maintenance of bone metabolism and bone formation, respectively. The complex nature of bone cell regulation suggests a potential interaction of these two important regulators in GHS rats. In the present study, BMP2 expression is suppressed by the VDR-1,25(OH)2D3 complex in Bone Marrow Stromal Cells (BMSCs) from GHS and SD rat and in UMR-106 cell line. We used chromatin immunoprecipitation (ChIP) assays to identify VDR binding to only one of several potential binding sites within the BMP2 promoter regions. This negative region also mediates suppressor reporter gene activity. The molecular mechanisms underlying the down-regulation of BMP2 by 1,25(OH)2D3 were studied in vitro in BMSCs and UMR-106 cells using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA). Both DAC and TSA activate BMP2 expression in combination with 1,25(OH)2D3. Bisulfite DNA pyrosequencing reveals 1,25(OH)2D3 to completely hypermethylate a single CpG site in the same BMP2 promoter region identified by the ChIP and reporter gene assays. ChIP assays also show that 1,25(OH)2D3 can increase the repressive histone mark H3K9me2 and reduce the acetylation of histone H3 at the same BMP2 promoter region. Taken together, our results indicate that 1,25(OH)2D3 binding to VDR down-regulates BMP2 gene expression in BMSCs and osteoblast-like UMR-106 cells by binding to the BMP2 promoter region. The mechanism of this 1,25(OH)2D3-induced transcriptional repression of BMP2 involves DNA methylation and histone modification. The study provides novel evidence that 1,25(OH)2D3 represses bone

  13. Skeletal trauma generates systemic BMP2 activation that is temporally related to the mobilization of CD73+ cells.

    Science.gov (United States)

    Marsell, Richard; Steen, Brandon; Bais, Manish V; Mortlock, Douglas P; Einhorn, Thomas A; Gerstenfeld, Louis C

    2014-01-01

    The relationship between BMP2 expression and the recruitment of skeletogenic stem cells was assessed following bone marrow reaming. BMP2 expression was examined using transgenic mice in which β-galactosidase had been inserted into the coding region of BMP2. Stem cell mobilization was analyzed by FACS analysis using CD73, a marker associated with bone marrow stromal stem cells. BMP2 expression was induced in endosteal lining cells, cortical osteocytes and periosteal cells in both the reamed and in contralateral bones. BMP2 mRNA expression in the reamed bone showed an early peak within the first 24 h of reaming followed by a later peak at 7 days, while contralateral bones only showed the 7 days peak of expression. FACS analysis sorting on CD73 positive cells showed a 50% increase of these cells at 3 and 14 days in the marrow of the injured bone and a single peak at 14 days of the marrow cell population of the contralateral bone. A ∼20% increase of CD73 positive cells was seen in the peripheral blood 2 days after reaming. These data showed that traumatic bone injury caused a systemic induction of BMP2 expression and that this increase is correlated with the mobilization of CD73 positive cells.

  14. Enhanced healing of rat calvarial defects with MSCs loaded on BMP-2 releasing chitosan/alginate/hydroxyapatite scaffolds.

    Directory of Open Access Journals (Sweden)

    Xiaoning He

    Full Text Available In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2, and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy.

  15. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    Science.gov (United States)

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells.

  16. Experimental Research on Ectopic Osteogenesis of BMP2-derived Peptide P24 Combined with PLGA Copolymers

    Institute of Scientific and Technical Information of China (English)

    DUAN Zhixia; ZHENG Qixin; GUO Xiaodong; YUAN Quan; CHEN Shunguang

    2007-01-01

    To experimentally evaluate the ectopic osteogenetic capacity of synthesized BMP2-derived peptide P24 combined with poly lactic-co-glycolic acid (PLGA), Wistar rats were divided into two groups: group A, in which BMP2-derived peptide P24/PLGA complex was implanted,and group B which received simple PLGA implant. The complex was respectively implanted into the back muscles of rats. Samples were taken the 1 st, 4 th, 8 th, and the 12 th week after the implantation.Their bone formation was detected by X-ray examination, and tissue response was histologically observed. Western blotting was used for the detection of the expression of collagen Ⅰ (Col- Ⅰ ) and osteopontin (OPN). There was acute inflammation in the tissue around both types of implants at early stage. The cartilage was found around implant areas 4 weeks after the implantation of BMP2-derived peptide p24/PLGA complex, 8 weeks after the implantation, osteoblasts were found, and 12 weeks after the implantation, typical trabecular bone structure was observed. In group B, after 12 weeks, no osteoblasts were found. It is concluded that PLGA is an ideal scaffold material for bone tissue engineering. BMP2-derived peptide can start endochondral ossification and is more effective in inducing ectopic osteogenesis.

  17. 淫羊藿苷、补骨脂素、齐墩果酸、二苯乙烯苷正交配伍调控Bmp2、Smad1、4诱导BMSCs成骨分化的影响%Orthogonal Compatibility of Icariin, Psoralen, Oleanolic Acid, Stilbene Glucoside on Regulation of Bmp2, Smad1, and Smad 4 Induced Osteogenic Differentiation of BMSCs

    Institute of Scientific and Technical Information of China (English)

    高璐; 郑洪新; 陈谊敬; 宗志红; 林庶茹

    2014-01-01

    目的:观察4种补肾中药淫羊藿、补骨脂、女贞子、何首乌有效成分淫羊藿苷、补骨脂素、齐墩果酸、二苯乙烯苷正交配伍,两种非补肾中药川芎、黄芪有效成分川芎嗪、黄芪甲苷,对大鼠骨髓间充质干细胞(BMSCs)的调控作用。方法:65只SD大鼠随机分为正常对照组、阳性转化液对照组、补肾配伍组(1组、2组、3组、4组、5组、6组、7组、8组、9组)、非补肾药对照组(黄芪甲苷组、川芎嗪组)。补肾配伍组、非补肾药对照组予灌胃给药,每日1次,连续3天。正常对照组、阳性转化液对照组予等剂量生理盐水灌胃。灌胃第3天,大鼠处死后取各组含药血清培养骨髓间充质干细胞6、12、18天;ELISA法分别定量检测6、12、18天骨形态发生蛋白2(Bmp2)活性表达及含量,评价成骨细胞分化程度。用实时定量PCR法检测含药血清培养骨髓间充质干细胞第18天Bmp2、Smad1、Smad4 mRNA 表达。结果:补肾配伍药可提高体外培养骨髓间充质干细胞Bmp2的活性表达及含量,在12天达到高峰值。补肾配伍组能上调Bmp2、Smad1、Smad4的mRNA表达。结论:补肾中药有效成分配伍能促进骨髓间充质干细胞分化为成骨细胞,其机制可能与上调 Bmp2、Smad1、Smad4 mRNA 表达量及 Bmp2的活性及含量有关。%This study was aimed to observe four kinds of kidney-tonification medicine, which were Epimedium, pso-ralen, Ligustrum lucidum, Polygonum with the active ingredient of icariin, psoralen, oleanolic acid, stilbene glucoside and their orthogonal compatibility. There were two kinds of non-kidney tonification medicine, which were Chuanx-iong and astragalus with the active ingredient of TMP and astragaloside. The observation was made on the regulatory role of rat bone marrow stem cells (BMSCs). A total of 65 SD rats were randomly divided into the normal control group, positive transformed control

  18. Negative Fgf8-Bmp2 feed-back is regulated by miR-130 during early cardiac specification.

    Science.gov (United States)

    Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

    2015-10-01

    It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification.

  19. Synergistic effect ofRhBMP-2 and bFGF on ectopic osteogenesis in mice

    Institute of Scientific and Technical Information of China (English)

    Shu-Yuan Ma; Zhi-Qiang Feng; Ren-Fa Lai; Zhi-Ying Zhou; Zhong-Da Yin

    2015-01-01

    the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP-2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.

  20. High-concentration of BMP2 reduces cell proliferation and increases apoptosis via DKK1 and SOST in human primary periosteal cells.

    Science.gov (United States)

    Kim, Harry K W; Oxendine, Ila; Kamiya, Nobuhiro

    2013-05-01

    BMP2, a well-known osteoinductive agent approved by FDA, is currently being used for various off-label orthopedic applications. Recently, concerns about its efficacy for off-label use, concentration, and complications have emerged. Interestingly, there is an extremely large discrepancy in BMP2 concentration between clinical use (i.e. 1.5mg/ml) and in vitro studies (50-300 ng/ml). The purpose of this study was to determine the effects of a relatively high-concentration of BMP2 on cell proliferation and apoptosis using human primary periosteal cells as BMP2 is generally applied around the periosteum in orthopedic surgeries. We isolated periosteal cells from three independent patients. The cell proliferation assessed by MTT activity was significantly reduced by a high-concentration of BMP2 (~2000 ng/ml), while such a reduction was not observed by using a low-concentration of BMP2 (~200 ng/ml). The cell apoptosis assessed by caspase activity was significantly increased by high-concentration BMP2, while such an increase was not observed by low-concentration BMP2. We found that Wnt signaling activity was significantly reduced by high-concentration BMP2 along with a dramatic increase in DKK1 and SOST, key inhibitors of Wnt signaling in bone. The addition of DKK1 or SOST protein to the primary periosteal cells reduced MTT activity and significantly increased caspase activity. Silencing the DKK1 or SOST expression using the siRNA technique normalized cell proliferation and apoptosis in the periosteum-derived cells when exposed to a high-concentration BMP2. Taken together, these results suggest that a high-concentration BMP2 decreases human periosteal cell proliferation and induces apoptosis via the activation of Wnt inhibitors DKK1 and SOST. This study provides new insights to the effects of high BMP2 concentration on human periosteal cells and brings out the possibility of multiple effects of current BMP2 therapy on various skeletal tissues.

  1. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

    Science.gov (United States)

    Wegman, F; Bijenhof, A; Schuijff, L; Oner, F C; Dhert, W J A; Alblas, J

    2011-03-15

    Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

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

    Science.gov (United States)

    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

    2015-10-16

    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.

  3. 骨形成蛋白2重组腺病毒的构建及异位诱导成骨的研究%Construction of a recombinant adenovirus carrying human BMP2 gene and its function of inducing bone formation

    Institute of Scientific and Technical Information of China (English)

    孙大铭; 侯树勋; 付小兵

    2006-01-01

    目的构建骨形成蛋白2(BMP2)重组腺病毒,探讨其诱导成骨的作用.方法将BMP2基因克隆到转移载体pAdTrack-CMV中,在细菌BJ5183中与pAdEasy腺病毒基因组进行同源重组,得到BMP2重组腺病毒基因组,通过转染HEK293细胞,包装出重组腺病毒.然后,用其进行裸鼠异位诱导成骨.结果经过PCR及酶切鉴定,证明获得了BMP2转移质粒pAdTrack-BMP2BMP2腺病毒基因组,并包装出重组腺病毒.组织学观察显示,2周时局部大量纤维样细胞聚集,软骨细胞分化;5周时骨小梁形成,软骨细胞已退化.结论 BMP2组腺病毒的构建及其异位诱导成骨的功能为其在BMP2基因治疗中的应用奠定了良好的基础.

  4. Optimisation of BMP-2 dosage for the osseointegration of porous titanium implants in an ovine model.

    Science.gov (United States)

    Hunziker, E B; Jovanovic, J; Horner, A; Keel, M J; Lippuner, K; Shintani, N

    2016-11-18

    In clinical orthopaedics, total joint replacements and spinal fusions are routine undertakings. Many of the implicated patients suffer from osteoporosis, severe arthrosis or osteopaenia. In individuals thus afflicted, the bony bed lacks the mechanical stability that is a requisite for a firm anchorage of the implant and its functional competence. To promote the bony bondage of an implant it is necessary to induce neo-ossification by the introduction of an osteogenic agent, such as bone morphogenetic protein 2 (BMP-2). Since this growth factor is generally applied in a free form and at high dosages to maximise its osteogenicity, untoward side effects frequently ensue. We hypothesise that the administration of BMP-2 using a suitable delivery vehicle, and its gradual, low dose release therefrom in a cell-mediated manner, would avert the triggering of undesired side effects and enhance its efficacy. To test this postulate, implants of porous titanium were coated with a layer of calcium phosphate into which BMP-2 was biomimetically incorporated at dosages ranging from 0.8 to 500 µg/g of coating material (delivery system) prior to their surgical placement in the tibiae of adult sheep. The volume and the surface area of newly-formed bone were evaluated histomorphometrically after 3 and 6 weeks. The highest values were achieved using BMP-2 dosages of 20 to 100 µg/g of coating: The deposition of bone was confined to the immediate vicinity of the implant and was observed deep within the interstices of its meshwork, to the walls of which it bonded well. The findings of the study attest to the validity of our hypothesis.

  5. Material-related effects of BMP-2 delivery systems on bone regeneration.

    Science.gov (United States)

    Hernández, Antonio; Sánchez, Esther; Soriano, Isabel; Reyes, Ricardo; Delgado, Araceli; Évora, Carmen

    2012-02-01

    Material-related effects of a brushite and a PLGA controlled release system loaded with two distinct doses of bone morphogenetic protein-2 (BMP-2) (3.5 and 17.5 μg), pre-encapsulated in poly(lactic-co-glycolic acid) (PLGA), were investigated in an intramedullary femur defect model in rabbits. The systems were characterized in vitro and in vivo over 12 weeks in terms of morphology, release kinetics, porosity, molecular weight, and composition using scanning electron microscopy, mercury porosimetry, radioactivity counting, X-ray diffractometry, differential scanning calorimetry, and gel permeation chromatography. During the experimental period the investigated systems underwent significant changes in vitro as well as in vivo. It should be stressed that the two in vitro release patterns were similar, however in vivo parallel profiles were observed with a higher burst effect for BMP-2 in the PLGA system. The PLGA system degraded and disintegrated significantly faster than the brushite system, which suffered slowly progressing external erosion and, additionally, material resorption by osteoclasts in vivo. The consequences of this were reflected in the degree of bone regeneration. Although a sustained delivery of BMP-2 was achieved with both systems, the brushite construct, independent of the loaded growth factor dose, failed to consistently induce defect repair, a result attributed to its slow resorption rate. In contrast, the PLGA system resulted in complete regeneration with mature trabecular bone formation 8 weeks after implantation.

  6. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Directory of Open Access Journals (Sweden)

    Sunita Sharma

    Full Text Available 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.

  7. WNT4 acts downstream of BMP2 and functions via β-catenin signaling pathway to regulate human endometrial stromal cell differentiation.

    Science.gov (United States)

    Li, Quanxi; Kannan, Athilakshmi; Das, Amrita; Demayo, Franco J; Hornsby, Peter J; Young, Steven L; Taylor, Robert N; Bagchi, Milan K; Bagchi, Indrani C

    2013-01-01

    Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/β-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of β-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/β-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.

  8. 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 osteoind

  9. Bmp2 and Bmp4 accelerate alveolar bone development.

    Science.gov (United States)

    Ou, Mingming; Zhao, Yibing; Zhang, Fangming; Huang, Xiaofeng

    2015-06-01

    Alveolar bone remodeling is a continuous process that takes place during development and in response to various physiological and pathological stimuli. However, detailed knowledge regarding the underlying mechanisms involved in alveolar bone development is still lacking. This study aims at improving our understanding of alveolar bone formation and the role of bone morphogenetic proteins (Bmps) in this process. Mice at embryonic (E) day 13.5 to postnatal (PN) day 15.5 were selected to observe the process of alveolar bone development. Alveolar bone development was found to be morphologically observable at E14.5. Molar teeth isolated from mice at PN7.5 were pretreated with Bmp2, Bmp4, Noggin, or BSA, and grafted subcutaneously into mice. The subcutaneously implanted tooth germs formed alveolar bone indicating the role of the dental follicle in alveolar bone development. Alveolar bone formation was increased after pretreatment with Bmp2 and Bmp4, but not with Noggin. Gene expression levels in dental follicle cells from murine molars were also determined by real-time RT-PCR. The expression levels of Runx2, Bsp, and Ocn were significantly higher in dental follicle cells cultured with Bmp2 or Bmp4, and significantly lower in those cultured with Noggin when compared with that of the BSA controls. Our results suggest that the dental follicle participates in alveolar bone formation and Bmp2/4 appears to accelerate alveolar bone development.

  10. 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

  11. Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO2 nanotube layers fabricated by lyophilization following trehalose addition

    Science.gov (United States)

    Zhang, Xiaochen; Zhang, Zhiyuan; Shen, Gang; Zhao, Jun

    2016-01-01

    To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2) significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs. PMID:26869786

  13. Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO₂ nanotube layers fabricated by lyophilization following trehalose addition.

    Science.gov (United States)

    Zhang, Xiaochen; Zhang, Zhiyuan; Shen, Gang; Zhao, Jun

    2016-01-01

    To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2) significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs.

  14. Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying BMPR1B cell response.

    Science.gov (United States)

    Chapellier, Marion; Bachelard-Cascales, Elodie; Schmidt, Xenia; Clément, Flora; Treilleux, Isabelle; Delay, Emmanuel; Jammot, Alexandre; Ménétrier-Caux, Christine; Pochon, Gaëtan; Besançon, Roger; Voeltzel, Thibault; Caron de Fromentel, Claude; Caux, Christophe; Blay, Jean-Yves; Iggo, Richard; Maguer-Satta, Véronique

    2015-02-10

    Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

  15. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

    Directory of Open Access Journals (Sweden)

    Marion Chapellier

    2015-02-01

    Full Text Available Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

  16. Disequilibrium of BMP2 Levels in the Breast Stem Cell Niche Launches Epithelial Transformation by Overamplifying BMPR1B Cell Response

    Science.gov (United States)

    Chapellier, Marion; Bachelard-Cascales, Elodie; Schmidt, Xenia; Clément, Flora; Treilleux, Isabelle; Delay, Emmanuel; Jammot, Alexandre; Ménétrier-Caux, Christine; Pochon, Gaëtan; Besançon, Roger; Voeltzel, Thibault; Caron de Fromentel, Claude; Caux, Christophe; Blay, Jean-Yves; Iggo, Richard; Maguer-Satta, Véronique

    2015-01-01

    Summary Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer. PMID:25601208

  17. Research progress of BMP2 gene function during fat formation%BMP2基因在脂肪形成过程中的功能研究进展

    Institute of Scientific and Technical Information of China (English)

    何林芝; 冷丽; 李辉

    2012-01-01

    Bone morphogenetic proteins (BMPs) belong to the transforming growth factor P (TGF-p) superfamily. It's a kind of secretory protein with multiple biological functions. Researches indicates that BMP2 not only can induce the formation of bone cells, but also can promote emesenchymal stem cells to adipocytes, and plays a critical role in the formation of fat. This review summarized recent research progresses in gene structure, expression and function of BMP2.%骨形态发生蛋白2 (BMP2)属于转化生长因子β(TGF-β)超家族成员,是一种分泌性蛋白,具有多重生物学功能.BMP2基因不仅可以诱导骨细胞的形成,还可以促进间充质干细胞向脂肪细胞分化,在脂肪的形成过程中发挥着重要作用.就该基因的结构、表达及其在诱导脂肪细胞形成方面的功能等进行综述.

  18. 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.

  19. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    F Wegman

    2011-03-01

    Full Text Available Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2, which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix.

  20. 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.

  1. 重组人BMP-2修饰的β磷酸三钙/胶原材料制备及其诱导成牙性能的初步研究%PREPARATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 DECORATED β TRICALCIUM PHOSPHATE/COLLAGEN AND PRELIMINARY STUDIES ON ITS PROPERTIES OF INDUCING TOOTH FORMATION

    Institute of Scientific and Technical Information of China (English)

    张文涛; 刘建华; 王慧明; 李志勇

    2011-01-01

    性良好,可作为牙组织工程支架材料的良好选择.%Objective To explore a novel nanometer biomaterial which could induce the regeneration of tooth tissues intelligently, and to evaluate the feasibility of using this kind of biomaterial as the scaffold for tooth tissue engineering by investigating the role it plays in tooth tissue engineering.Methods The scaffold for tooth tissue engineering containing recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared by mixing nanoscale β tricalcium phosphate (β-TCP)/collagen particles.Forty-six 8-10 weeks old specific pathogen free Sprague Dawley (SD) rats, including 34 females and 12 males, weighing 250-300 g, were involved in this study.Tooth germs were removed under a stereomicroscope from the mandible of newborn SD rat, then digested and suspended.Scanning electronic microscope (SEM), adhesion rate of cells, and MTT assay were used to evaluate the effects of the scaffold on the tooth germ cells cultured in vitro.The tissue engineered tooth germ which was constructed by tooth germ cells and scaffold was transplanted under SD rat's kidney capsule as the experimental group (n=12); the tooth germ cells (cell-control group, n=12) or scaffold without cells (material-control group, n=4) were transplanted separately as control groups.Specimens were harvested to perform general and histological observations at 4 and 8 weeks after transplantation.Results β-TCP/collagen showed a loose and porous appearance with soft texture and excellent hydrophilicity.Tooth germ cells grew well and could attach to the scaffold tightly 3 days after coculture.The adhesion rates of tooth germ cells were 27.20% ± 2.37%,44.52% ± 1.87%, and 73.81% ± 4.15% when cocultured with scaffold for 4, 8, and 12 hours, respectively.MTT assay showed that the cell proliferation status of experimental group was similar to that of the control group, showing no significant difference (P >0.05).Some white calcified specimens

  2. BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects

    Directory of Open Access Journals (Sweden)

    Xiong L

    2015-01-01

    Full Text Available Long Xiong,1 Jianhua Zeng,1 Aihua Yao,2 Qiquan Tu,3 Jingtang Li,1 Liang Yan,4 Zhiming Tang1 1Department of Osteology, People’s Hospital of Jiangxi Province, Nanchang, Jiangxi, People’s Republic of China; 2School of Materials Science and Engineering, Tongji University, Shanghai, People’s Republic of China; 3Department of Osteology, People’s Hospital of Jiujiang County, Jiujiang, Jiangxi, People’s Republic of China; 4Department of Osteology, The Third Hospital of Nanchang City, Nanchang, Jiangxi, People’s Republic of China Abstract: 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 m2/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

  3. Mechanical loading increased BMP-2 expression which promoted osteogenic differentiation of tendon-derived stem cells.

    Science.gov (United States)

    Rui, Yun Feng; Lui, Pauline Po Yee; Ni, Ming; Chan, Lai Shan; Lee, Yuk Wa; Chan, Kai Ming

    2011-03-01

    This study aimed to investigate the effect of repetitive tensile loading on the expression of BMP-2 and the effect of BMP-2 on the osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro. Repetitive stretching was applied to TDSCs isolated from rat patellar tendon at 0%, 4%, and 8%, 0.5 Hz. The expression of BMP-2 was detected by Western blotting and qPCR. To study the osteogenic effects of BMP-2 on TDSCs, BMP-2 was added to the TDSC monolayer for the detection of ALP activity and calcium nodule formation in a separate experiment. TDSCs adhered, proliferated, and aligned along the direction of externally applied tensile force while they were randomly oriented in the control group. Western blotting showed increased expression of BMP-2 in 4% and 8% stretching groups but not in the control group. Up-regulation of BMP-2 mRNA was also observed in the 4% stretching group. BMP-2 increased the osteogenic differentiation of TDSCs as indicated by higher ALP cytochemical staining, ALP activity, and calcium nodule formation. Repetitive tensile loading increased the expression of BMP-2 and addition of BMP-2 enhanced osteogenic differentiation of TDSCs. Activation of BMP-2 expression in TDSCs during tendon overuse might provide a possible explanation of ectopic calcification in calcifying tendinopathy.

  4. Effect of BMP-2 and BMP-7 homodimers and a mixture of BMP-2/BMP-7 homodimers on osteoblast adhesion and growth following culture on a collagen scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Laflamme, Claude; Rouabhia, Mahmoud [Groupe de recherche en ecologie buccale, Faculte de medecine dentaire, Universite Laval, Quebec City, Quebec G1K 7P4 (Canada)], E-mail: Mahmoud.rouabhia@fmd.ulaval.ca

    2008-03-01

    In the present study, we studied the involvement of BMP-2 and BMP-7 as homodimers and as a mixture of homodimers in bone regeneration using an engineered bone model. The engineered bone model consisted of a collagen scaffold populated with osteoblasts that acted as a carrier for the BMPs. BMP-2, BMP-7 and a mixture of BMP-2/BMP-7 were used at final concentrations of 10 and 100 ng ml{sup -1}. Osteoblasts seeded onto a collagen scaffold were cultured for 24 h before being stimulated with the BMPs. Four days later, osteoblast adhesion to and growth on the scaffold were assessed. Osteocalcin, IL-6, metalloproteinase (MMP-2 and MMP-9) and protease inhibitor (TIMP-1 and TIMP-2) mRNA and protein levels were measured. Our results showed that the BMP-2, BMP-7 and a mixture of BMP-2/BMP-7 all promoted osteoblast growth on the collagen scaffold, with the mixture of BMP-2/BMP-7 enhancing the most growth. BMP-2 and the mixture of BMP-2/BMP-7 enhanced osteocalcin (an osteoblast differentiation marker) mRNA expression and protein secretion, likely via the IL-6 pathway given that IL-6 secretion was upregulated by BMP-7 and a mixture of BMP-2/BMP-7. BMPs promote extracellular matrix production by inhibiting MMP-2 mRNA and increasing TIMP-1 and TIMP-2 mRNA expressions and protein secretions. BMP-2, BMP-7 and the mixture of BMP-2/BMP-7 could promote bone regeneration via different mechanisms involving IL-6 and MMP inhibitors.

  5. Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO2 nanotube layers fabricated by lyophilization following trehalose addition

    Directory of Open Access Journals (Sweden)

    Zhang X

    2016-01-01

    Full Text Available Xiaochen Zhang,1 Zhiyuan Zhang,1 Gang Shen,2 Jun Zhao2 1Department of Oral and Maxillofacial Surgery, 2Department of Orthodontics, College of Stomatology, Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China Abstract: To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2 significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs. Keywords: osteogenesis, anti-inflammation, TiO2 nanotube layers, Lenti-BMP-2, lyophilization, trehalose 

  6. Gallic acid inhibits vascular calcification through the blockade of BMP2-Smad1/5/8 signaling pathway.

    Science.gov (United States)

    Kee, Hae Jin; Cho, Soo-Na; Kim, Gwi Ran; Choi, Sin Young; Ryu, Yuhee; Kim, In Kyeom; Hong, Young Joon; Park, Hyung Wook; Ahn, Youngkeun; Cho, Jeong Gwan; Park, Jong Chun; Jeong, Myung Ho

    2014-11-01

    Vascular calcification is associated with increased risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. Gallic acid, a natural compound found in gallnut and green tea, is known to be antifungal, antioxidant, and anticancer. Here we investigated the effect of gallic acid on vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Gallic acid inhibited inorganic phosphate-induced osteoblast differentiation markers as well as calcification phenotypes (as determined by calcium deposition, Alizarin Red, and Von Kossa staining). Knockdown of BMP2 or Noggin blocked phosphate-induced calcification. Gallic acid suppressed phosphorylation of Smad1/5/8 protein induced by inorganic phosphate. Taken together, we suggest that gallic acid acts as a novel therapeutic agent of vascular calcification by mediating BMP2-Smad1/5/8 signaling pathway.

  7. 成釉细胞瘤中bmp2基因突变的发现%Gene mutation of bmp2 in ameloblastoma

    Institute of Scientific and Technical Information of China (English)

    岳文; 杨连甲; 朱峰; 晏伟

    2000-01-01

    目的:分析成釉细胞瘤组织中bmp2成熟肽基因片段的序列,探讨bmp2基因突变存在的可能性以明确其病理机制.方法:提取肿瘤组织中的RNA,用RT-PCR方法得到bmp2成熟肽基因片段,克隆后进行序列测定和分析.结果:首次发现成釉细胞瘤组织中有bmp2基因突变:AAG→AAA,GAG→AAG,并引起相应多肽的结构改变.结论:成釉细胞瘤中存在bmp2基因突变,并有可能在病理机制中起重要作用.

  8. BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1

    Institute of Scientific and Technical Information of China (English)

    Xiaobin Song; Shaohua Liu; Xun Qu; Yingwei Hu; Xiaoying Zhang; TaoWang; FengcaiWei

    2011-01-01

    Inadequate vascularization limits the repair of bone defects,In order to improve angiogenesis and accelerate osteogenesis,the synergism of co-cultured cells with genetic modification in bone regeneration was investigated in this study.Endothelial progenitor cells (EPCs) and bone marrow stem cells (BMSCs) were transfected with the genes of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) by adenovirus,respectively.The co-cultured cells,designated as four groups including BMSC + EPC,Ad-BMP2-BMSC +EPC,BMSC + Ad-VEGF-EPC,and Ad-BMP2-BMSC + Ad-VEGF-EPC groups,were seeded on an alginate gel and then implanted into rat intramuscularly to evaluate the effects on angiogenesis and osteogenesis.Both VEGF and BMP2 could induce the overexpression of inhibitor of DNA-binding 1(Id1) gene which significantly promoted tube formation in vitro and increase the amount of blood vessels in the Ad-BMP2-BMSC + Ad-VEGF-EPC group after implantation.Nevertheless,overexpression of Id1 retarded the terminal differentiation of osteoblasts and the bone formation.Later,osteogenic gene expression at transcriptional level,calcium nodules,and alkaline phosphatase (ALP) activity showed a gradual decrease and the amount of newly formed osteogenesis area exhibited a small increase in the Ad-BMP2-BMSC + Ad-VEGF-EPC group.This finding suggests that a balanced regulation of Id1 expression in VEGF-EPCs and BMP2-BMSCs may be critical to cell-based and gene-based approaches for bone regeneration.

  9. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bone marrow mesenchymal stemcells(MSCs)are pluripotential stemcells that have the capacitytodifferentiate into chondrocytes and osteoblasts[1].Ithas been well documented that bone morphogeneticproteins(BMPs),a group of proteins belonging tothe TGF-βsuperfamily,can induce bone for mationbothin vivoandin vitroas well as promote osteo-blastic differentiation of MSC[2].HeterologousBMP2is successfully transferred to MSCs and genetherapy is employed based on repairing bony andcartilage defects,spinal fusion[3-5]....

  10. Osteogenesis and Degradation Behavior of rhBMP-2/β-Tricalcium Phosphate Porous Composite Materials

    Institute of Scientific and Technical Information of China (English)

    Weizhong YANG; Dali ZHOU; Ge LIANG; Guangfu YIN; Yun ZHANG

    2004-01-01

    Ultrafine -tricalcium phosphate (β-TCP) powders with good crystalline structure were produced by a new wet process. Throughbone tissue engineering approach, porous β-TCP ceramic was combined with recombined human bone morphogenetic proteins- 2 (rhBMP-2) to develop a novel composite material. Osteogenesis capacity of the composite was investigated intramuscularlyin rat with histological analyses and SEM examination. Pure β-TCP porous ceramic was investigated as the control. Resultsshow that the composite materials possess good biocompatibility, biodegradation and strong osteogenesis capacity throughinductive process after implantation. Material degradation began from 2 weeks post-implantation accompanying with thechanging of pore structure, with the enwrapping and separation of materials by hyperplatic mesenchymal cells and fibroblast,and with the phagocytose reaction of multinucleated giant cells. Early in 72 h, immature cartilage could be found within novelcomposite; mature lamellar bone was induced to generate after 3 weeks. With strong osteoinduction capacity and controllablebiodegradation, the novel rhBMP-2/β-TCP porous ceramic is expected to be a promising bone grafting substitute for bonetissue engineering.

  11. Direct BMP2/4 signaling through BMP receptor IA regulates fetal thymocyte progenitor homeostasis and differentiation to CD4+CD8+ double-positive cell.

    Science.gov (United States)

    Hager-Theodorides, Ariadne L; Ross, Susan E; Sahni, Hemant; Mishina, Yuji; Furmanski, Anna L; Crompton, Tessa

    2014-01-01

    BMP2/4 signaling is required for embryogenesis and involved in thymus morphogenesis and T-lineage differentiation. In vitro experiments have shown that treatment of thymus explants with exogenous BMP4 negatively regulated differentiation of early thymocyte progenitors and the transition from CD4-CD8- (DN) to CD4+CD8+ (DP). Here we show that in vivo BMP2/4 signaling is required for fetal thymocyte progenitor homeostasis and expansion, but negatively regulates differentiation from DN to DP cell. Unexpectedly, conditional deletion of BMPRIA from fetal thymocytes (using the Cre-loxP system and directing excision to hematopoietic lineage cells with the Vav promoter) demonstrated that physiological levels of BMP2/4 signaling directly to thymocytes through BMPRIA are required for normal differentiation and expansion of early fetal DN thymocytes. In contrast, the arrest in early thymocyte progenitor differentiation caused by exogenous BMP4 treatment of thymus explants is induced in part by direct signaling to thymocytes through BMPRIA, and in part by indirect signaling through non-hematopoietic cells. Analysis of the transition from fetal DN to DP cell, both by ex vivo analysis of conditional BMPRIA-deficient thymocytes and by treatment of thymus explants with the BMP4-inhibitor Noggin demonstrated that BMP2/4 signaling is a negative regulator at this stage. We showed that at this stage of fetal T-cell development BMP2/4 signals directly to thymocytes through BMPRIA.

  12. 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.

  13. BMP2-encapsulated chitosan coatings on functionalized Ti surfaces and their performance in vitro and in vivo.

    Science.gov (United States)

    Han, Lu; Lin, Hong; Lu, Xiong; Zhi, Wei; Wang, Ke-Feng; Meng, Fan-Zhi; Jiang, Ou

    2014-07-01

    Bone morphogenic protein-2 (BMP2)-encapsulated chitosan (CS) coatings were prepared to immobilize BMP2 on titanium (Ti) surfaces. The Ti substrates were functionalized through a three-step process: alkali treatment, silanization with 3-aminopropyltriethoxysilane and aldehydation with glutaraldehyde (GA). BMP2-encapsulated CS coatings (BMP2-CS) were bonded to Ti surfaces through reactions between the aldehyde groups of GA and the amine groups of CS. Direct BMP2 immobilization on aldehyde-treated Ti (BMP2-Ti) and pure CS coatings (CS-Ti) were used as controls. The release rate of BMP2-CS-Ti was half of that of BMP2-Ti at initial stage, which indicates that the CS coatings are suitable carriers for sustained BMP2 release. The osteoinductivities of BMP2-CS-Ti, BMP2-Ti, CS-Ti and pristine Ti were examined by both in vitro cell tests and in vivo experiments. Bone marrow stem cell (BMSC) culture indicated that BMP2-CS-Ti is more potent in stimulating the differentiation of the adhering BMSC than the three other groups. Rabbit femur implantation revealed the excellent osteoinductivity of BMP2-CS-coated Ti implants. These results demonstrate that the BMP2-encapsulated CS coatings are stable osteoinductive coatings that realize the sustained release of BMP2 and maintain the activity of the protein.

  14. 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.

  15. Cell saver filtering of extravasated rhBMP-2 after degenerative scoliosis reconstruction

    Directory of Open Access Journals (Sweden)

    Gabriel Liu, MBBCh, MSc, FRCS, FAMS (Orth

    2015-06-01

    Full Text Available RhBMP-2 is a bone fusion enhancer commonly used in scoliosis reconstruction surgery. It is delivered via an absorbable collagen sponge but has been known to migrate away from its delivery site. RhBMP-2 extravasation in surgical drainage has been noted during first two days post-surgery. Cell savers are widely used in scoliosis reconstruction to limit transfusion requirements and are commonly deployed in cases where rhBMP-2 is used for fusion augmentation. It is not known whether rhBMP-2 is present in salvaged blood or filtered away during cell saver recycling. Through this case series of four patients who underwent scoliosis reconstruction, we assess cell saver efficacy in filtering rhBMP-2 molecules by quantifying the amount of rhBMP-2 present in salvaged blood obtained after postoperative drainage recycling by OrthoPAT® cell saver and comparing it to rhBMP-2 leakage in postoperative drainage without cell saver recycling. We report an almost 10-fold reduction of rhBMP-2 concentration in salvaged blood obtained after cell saver recycling of postoperative drainage, suggesting cell saver effectiveness in filtering rhBMP-2 molecules.

  16. 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.

  17. BMP2/BMPR1A is linked to tumour progression in dedifferentiated liposarcomas.

    Science.gov (United States)

    O'Neill, Hannah L; Cassidy, Amy P; Harris, Olivia B; Cassidy, John W

    2016-01-01

    Bone Morphogenic Protein 2 (BMP2) is a multipurpose cytokine, important in the development of bone and cartilage, and with a role in tumour initiation and progression. BMP2 signal transduction is dependent on two distinct classes of serine/threonine kinase known as the type I and type II receptors. Although the type I receptors (BMPR1A and BMPR1B) are largely thought to have overlapping functions, we find tissue and cellular compartment specific patterns of expression, suggesting potential for distinct BMP2 signalling outcomes dependent on tissue type. Herein, we utilise large publicly available datasets from The Cancer Genome Atlas (TCGA) and Protein Atlas to define a novel role for BMP2 in the progression of dedifferentiated liposarcomas. Using disease free survival as our primary endpoint, we find that BMP2 confers poor prognosis only within the context of high BMPR1A expression. Through further annotation of the TCGA sarcoma dataset, we localise this effect to dedifferentiated liposarcomas but find overall BMP2/BMP receptor expression is equal across subsets. Finally, through gene set enrichment analysis we link the BMP2/BMPR1A axis to increased transcriptional activity of the matrisome and general extracellular matrix remodelling. Our study highlights the importance of continued research into the tumorigenic properties of BMP2 and the potential disadvantages of recombinant human BMP2 (rhBMP2) use in orthopaedic surgery. For the first time, we identify high BMP2 expression within the context of high BMPR1A expression as a biomarker of disease relapse in dedifferentiated liposarcomas.

  18. Losartan Inhibits Vascular Calcification by Suppressing the BMP2 and Runx2 Expression in Rats In Vivo.

    Science.gov (United States)

    Li, Mincai; Wu, Panfeng; Shao, Juan; Ke, Zhiqiang; Li, Dan; Wu, Jiliang

    2016-04-01

    The blockade of renin-angiotensin II system has been shown to reduce morbidity and mortality in hypertension, atherosclerosis, diabetes and chronic kidney disease. Since vascular calcification (VC) is commonly found in these diseases, the aim of this study was to examine whether or not losartan, a widely used angiotensin II receptor blockers, inhibits VC in rats in vivo. A rat model of VC was generated by treating rats with a combination of warfarin and vitamin K1. Two weeks after the treatments, the rats were treated with vehicle or without losartan (100 ng/kg/day) for 2 weeks. At the end of the experiments, aortic arteries were isolated for the examination of calcification morphology, mRNA and protein expression of BMP2 and Runx2, and osteoblast differentiation. Warfarin and vitamin K instigated vascular remodeling with calcified plaques in the aortic arteries in rats. Losartan significantly attenuated warfarin- and vitamin K-induced vascular injury and calcification. Consistently, losartan suppressed the levels of mRNA and protein expression of BMP2 and Runx2, two key factors for VC. Further, vascular calcified lesion areas expressed angiotensin II 1 receptor (AT1R). Finally, losartan treatment significantly inhibited apoptosis in vascular smooth muscle cell (VSMC) in rat arteries. We conclude that losartan suppresses VC by lowering the expression of AT1R, Runx2 and BMP2, and by inhibiting the apoptosis of VSMC in rat aortic arteries.

  19. Evaluation of a Novel HA/ZrO2-Based Porous Bioceramic Artificial Vertebral Body Combined with a rhBMP-2/Chitosan Slow-Release Hydrogel

    Science.gov (United States)

    Shi, Yihui; Quan, Renfu; Xie, Shangju; Li, Qiang; Cao, Guoping; Zhuang, Wei; Zhang, Liang; Shao, Rongxue; Yang, Disheng

    2016-01-01

    A new HA/ZrO2-based porous bioceramic artificial vertebral body (AVB), carried a recombinant human bone morphogenetic protein-2 (rhBMP-2)/chitosan slow-release hydrogel was prepared to repair vertebral bone defect in beagles. An ionic cross-linking was used to prepare the chitosan hydrogel (CS gel) as the rhBMP-2 slow-release carrier. The vertebral body defects were implanted with the rhBMP-2-loaded AVB in group A, or a non-drug-loaded AVB in group B, or autologous iliac in group C. The encapsulation rate of rhBMP-2 in rhBMP-2-loaded CS gel was 91.88±1.53%, with a drug load of 39.84±2.34 ng/mg. At 6, 12, 24 weeks postoperatively, radiography showed that the bone calluses gradually increased with time in group A, where the artificial vertebral body had completely fused with host-bone at 24 weeks after surgery. In group C, an apparent bone remodeling was occurred in the early stages, and the graft-bone and host-bone had also fused completely at 24 weeks postoperatively. In group B, fusion occurred less than in groups A and C. At 24 weeks after surgery, micro-computed tomography (Micro-CT) revealed that the volume of newly-formed bone in group A was significantly more than in group B (pbioceramic AVB carried the rhBMP-2-loaded CS gel can promote the repair of bony defect, and induce bone tissue to grow into the pores, which may replace iliac bone grafts as commonly applied in clinical practice. PMID:27400197

  20. Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells.

    Science.gov (United States)

    Liu, Danping; Hu, Liang; Zhang, Zheng; Li, Quan Ying; Wang, Guoxian

    2013-02-01

    The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (PBMP2 mRNA between group A and C (PBMP2 in group A and C was markedly higher than that in groups B, D and E (PBMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

  1. 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.

  2. 消旋聚乳酸复合rhBMP-2/hTGF-β1诱导小鼠成骨及其机制的研究%Gene expression and ectopic osteogenesis induced by recombinant human bone morphogenetic protein-2/human transforming growth factor-β1 delivered in poly-DL-lactic acid

    Institute of Scientific and Technical Information of China (English)

    王伟; 邱蔚六; 袁文化; 胡勤刚; 沈健; 陈强

    2004-01-01

    目的:评价消旋聚乳酸(PDLLA)载体材料复合重组rhBMP-2和hTGF-β1诱导小鼠体内异位成骨的能力,初步探讨其作用机制.方法:建立诱导RCI小鼠股部肌内异位成骨的18只动物模型,实验组(各6只)分别植入PDLLA/rhBMP-2/hTGF-β1和PDLLA/rhBMP-2复合材料,对照组(6只)植入PDLLA材料.采用图像处理技术、组织病理学和RT-PCR等分子生物学方法,对消旋聚乳酸载体材料复合2种细胞因子诱导小鼠体内异位成骨能力进行评价;并对其诱导异位成骨过程中,Ⅱ型胶原蛋白、骨桥蛋白和骨钙蛋白等骨基质蛋白基因表达进行检测.结果:PDLLA/rhBMP-2/hTGF-β1和PDLLA/rhBMP-2组复合材料均具有明显的诱导小鼠肌内异位成骨能力,而单纯PDLLA载体材料无诱导异位成骨能力,表现为小鼠股部复合材料植人区的相对密度指数存在显著性差异.Ⅱ胶原、骨桥蛋白和骨钙蛋白及其基因在2种细胞因子复合材料植入组中均有较高表达,而在单纯PDLLA组则无表达.结论:PDLLA复合rhBMP-2/hTGF-β1具有诱导异位成骨作用,软骨内成骨是其诱导小鼠体内异位成骨的主要方式.

  3. Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Jung-Bo Huh

    2015-07-01

    Full Text Available Anorganic bovine bone matrix (Bio-Oss® has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2 has been suggested to overcome this limitation of Bio-Oss®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter were formed in a white rabbit model and then implanted or not (controls with Bio-Oss® or BMP-2/Bio-Oss®. The Bio-Oss® and BMP-2/Bio-Oss® groups had significantly greater new bone areas (expressed as percentages of augmented areas than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss® group (16.50 ± 2.87 (n = 6 had significantly greater new bone areas than the Bio-Oss® group (9.43 ± 3.73 (n = 6 at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss® markedly enhances bone regeneration.

  4. Chondrocyte BMP2 signaling plays an essential role in bone fracture healing.

    Science.gov (United States)

    Mi, Meng; Jin, Hongting; Wang, Baoli; Yukata, Kiminori; Sheu, Tzong-Jen; Ke, Qiao Han; Tong, Peijian; Im, Hee-Jeong; Xiao, Guozhi; Chen, Di

    2013-01-10

    The specific role of endogenous Bmp2 gene in chondrocytes and in osteoblasts in fracture healing was investigated by generation and analysis of chondrocyte- and osteoblast-specific Bmp2 conditional knockout (cKO) mice. The unilateral open transverse tibial fractures were created in these Bmp2 cKO mice. Bone fracture callus samples were collected and analyzed by X-ray, micro-CT, histology analyses, biomechanical testing and gene expression assays. The results demonstrated that the lack of Bmp2 expression in chondrocytes leads to a prolonged cartilage callus formation and a delayed osteogenesis initiation and progression into mineralization phase with lower biomechanical properties. In contrast, when the Bmp2 gene was deleted in osteoblasts, the mice showed no significant difference in the fracture healing process compared to control mice. These findings suggest that endogenous BMP2 expression in chondrocytes may play an essential role in cartilage callus maturation at an early stage of fracture healing. Our studies may provide important information for clinical application of BMP2.

  5. Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis.

    Science.gov (United States)

    Koch, Philipp-Sebastian; Olsavszky, Victor; Ulbrich, Friederike; Sticht, Carsten; Demory, Alexandra; Leibing, Thomas; Henzler, Thomas; Meyer, Mathias; Zierow, Johanna; Schneider, Sven; Breitkopf-Heinlein, Katja; Gaitantzi, Haristi; Spencer-Dene, Bradley; Arnold, Bernd; Klapproth, Kay; Schledzewski, Kai; Goerdt, Sergij; Géraud, Cyrill

    2017-01-26

    Microvascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2(fl/fl) (Bmp2(LSECKO)) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

  6. Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists.

    Science.gov (United States)

    Alborzinia, Hamed; Schmidt-Glenewinkel, Hannah; Ilkavets, Iryna; Breitkopf-Heinlein, Katja; Cheng, Xinlai; Hortschansky, Peter; Dooley, Steven; Wölfl, Stefan

    2013-01-01

    Bone morphogenetic proteins (BMPs) are members of the TGFβ family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFβ receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose- and time-dependent manner, which can provide an explanation of some properties of the BMP regulatory network.

  7. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

    Science.gov (United States)

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    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 GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

  8. A carboxy terminal BMP/TGF-β binding site in secreted phosphoprotein 24 kD independently affects BMP-2 activity.

    Science.gov (United States)

    Tian, Haijun; Li, Chen-Shuang; Zhao, Ke-Wei; Wang, Jeffrey C; Duarte, M Eugenia L; David, Cynthia L; Phan, Kevin; Atti, Elisa; Brochmann, Elsa J; Murray, Samuel S

    2015-04-01

    Secreted phosphoprotein 24 kD (spp24) is a bone matrix protein isolated during attempts to identify osteogenic proteins. It is not osteogenic but performs other important roles in the regulation of bone metabolism, at least in part, by binding to and affecting the activity of members of the BMP/TGF-β family of cytokines. Spp24 exists in a number of forms that preserve the N-terminus and are truncated at the C-terminus. The hypothesized cytokine binding domain is present within the cystatin domain which is preserved in all of the N-terminal products. In this report, we describe a C-terminal fragment that is distinct from the cystatin domain and which independently binds to BMP-2 and TGF-β. This fragment inhibited BMP-2 activity in an ectopic bone forming assay. A shorter C-terminal product did not inhibit BMP-2 activity but improved bone quality induced by BMP-2 and produced increased calcium deposition outside of bone. Spp24 has been used to develop several potential therapeutic proteins. These results provide more information on the function of spp24 and provide other materials that can be exploited for clinical interventions.

  9. Biological Silicon Stimulates Collagen Type 1 and Osteocalcin Synthesis in Human Osteoblast-Like Cells Through the BMP-2/Smad/RUNX2 Signaling Pathway.

    Science.gov (United States)

    Dong, Meng; Jiao, Guangjun; Liu, Haichun; Wu, Wenliang; Li, Shangzhi; Wang, Qingshi; Xu, Daxia; Li, Xiaofeng; Liu, Huan; Chen, Yunzhen

    2016-10-01

    Silicon is essential for bone formation. A low-silicon diet leads to bone defects, and numerous animal models have demonstrated that silicon supplementation increases bone mineral density (BMD) and reduces bone fragility. However, the exact mechanism of this action has not been characterized. In this study, we aimed to determine the role of biological silicon in the induction of osteoblast differentiation and the possible underlying mechanism. We examined whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin synthesis through the bone morphogenetic protein-2 (BMP-2)/Smad1/5/runt-related transcription factor 2 (RUNX2) signaling pathway by investigating its effect in vitro at several concentrations on COL-1 and osteocalcin synthesis in human osteosarcoma cell lines (MG-63 and U2-OS). The expression of relevant proteins was detected by Western blotting following exposure to noggin, an inhibitor of BMP-2. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, phosphorylated Smad1/5 (P-Smad1/5), and RUNX2. Furthermore, rat bone mesenchymal stem cells (BMSCs) were used to determine the effect of orthosilicic acid on osteogenic differentiation. Exposure to 10 μM orthosilicic acid markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1, and osteocalcin in osteosarcoma cell lines. Enhanced ALP activity and the formation of mineralized nodules were also observed under these conditions. Furthermore, preconditioning with noggin inhibited the silicon-induced upregulation of P-Smad1/5, RUNX2, and COL-1 expression. In conclusion, the BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synthesis, and orthosilicic acid promotes the osteogenic differentiation of rat BMSCs.

  10. Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2

    Directory of Open Access Journals (Sweden)

    Vincent Thea

    2007-12-01

    Full Text Available Abstract Background Articular cartilage has little capacity for repair in vivo, however, a small number of studies have shown that, in vitro, a damage/repair response can be induced. Recent work by our group has shown that cartilage can respond to single impact load and culture by producing repair cells on the articular surface. The purpose of this study was to identify whether chondrocyte outgrowth into a 3D scaffold could be observed following single impact load and culture. The effect of bone morphogenic-2 (BMP-2 on this process was investigated. Methods Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. Cell numbers in the scaffold, on and extruding from the articular surface were quantified and the immunohistochemistry used to identify the cellular phenotype. Results Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events. Conclusion These studies demonstrate that articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response. It may be that the inhibitory effect of BMP-2 in this experimental model provides a clue to the apparent inability of articular cartilage to heal itself following damage in vivo.

  11. Experimental study of human BMP-2 on osteogenic induction in BMSCs of dogs in vitro%人BMP-2体外定向诱导犬BMSCs向成骨方向分化的实验研究

    Institute of Scientific and Technical Information of China (English)

    许蕾; 韩建国; 李家锋

    2015-01-01

    Objective:To provide seed cells for bone tissue engineering in the late establishment by establishing the cul-ture system of bone marrow mesenchymal stem cells( BMSCs)of dogs in vitro,and using human BMP-2 to make them in-duced to differentiate into osteoblasts. Methods:The extraction of BMSCs of adult beagle dogs was made,then the whole marrow adherence method and density gradient centrifugation were used to isolate and culture BMSCs in vitro,and observe the cell growth morphology everyday. The third generation BMSCs with good growth form was divided into two groups. The experimental group were cultured with adding 200ng/ml human BMP-2 containing fetal bovine serum(FBS)while the control group were cultured only with complete medium containing FBS. Then we used the detection of alkaline phosphatase staining after 3 weeks′induction,alizarin red staining and Von-Kossa staining after 4 weeks′induction to identify the differentiation of osteoblasts. Results:After 3 weeks of induction of experimental group with alkaline phosphatase,staining showed the cyto-plasm of positive expression of black particles,and it was negative in the control group;After 4 weeks of induction of experi-mental group with alizarin red staining and Von-Kossa staining showed positive expression of calcium nodules,and it was negative in the control group. All the staining results in the experimental group showed the characteristics of osteoblasts. Conclusion:BMSCs of dogs,which are extracted and cultivated in vitro,can directionally differentiate into osteoblasts under the action of human BMP-2.%目的:通过将犬骨髓间充质干细胞( bone marrow mesenchymal stem cells,BMSCs)建立体外培养体系,运用人骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)体外定向诱导分化为成骨细胞,为后期建立骨组织工程提供种子细胞。方法提取比格犬BMSCs,全骨髓贴壁法结合密度梯度离心法行体外分离培养,每日观察细

  12. Cell-mediated BMP-2 liberation promotes bone formation in a mechanically unstable implant environment.

    Science.gov (United States)

    Hägi, Tobias T; Wu, Gang; Liu, Yuelian; Hunziker, Ernst B

    2010-05-01

    The flexible alloplastic materials that are used in bone-reconstruction surgery lack the mechanical stability that is necessary for sustained bone formation, even if this process is promoted by the application of an osteogenic agent, such as BMP-2. We hypothesize that if BMP-2 is delivered gradually, in a cell-mediated manner, to the surgical site, then the scaffolding material's lack of mechanical stability becomes a matter of indifference. Flexible discs of Ethisorb were functionalized with BMP-2, which was either adsorbed directly onto the material (rapid release kinetics) or incorporated into a calcium-phosphate coating (slow release kinetics). Unstabilized and titanium-plate-stabilized samples were implanted subcutaneously in rats and retrieved up to 14 days later for a histomorphometric analysis of bone and cartilage volumes. On day 14, the bone volume associated with titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2 was 10-fold higher than that associated with their mechanically unstabilized counterparts. The bone volume associated with discs bearing a coating-incorporated depot of BMP-2 was similar in the mechanically unstabilized and titanium-plate-stabilized groups, and comparable to that associated with the titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2. Hence, if an osteogenic agent is delivered in a cell-mediated manner (via coating degradation), ossification can be promoted even within a mechanically unstable environment.

  13. 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.

  14. Effect of a Novel Nonviral Gene Delivery of BMP-2 on Bone Healing

    Directory of Open Access Journals (Sweden)

    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.

  15. Cloning of BMP-2 Gene and Its Expression Study in E. coli in Order to Produce a Recombinant Drug

    Directory of Open Access Journals (Sweden)

    N. Mohammadi

    2014-10-01

    Full Text Available Introduction & Objective: Bone morphogenetic proteins are a group of cytokines that belongs to superfamily TGF?. These proteins play an important role in evolution of many of organs and tissues through germinal period followed by amending and rebuilding of bone tissue and car-tilage. The aim of this study was to clone and expression analysis of BMP-2 gene in E. coli bacteria. Materials & Methods: In this experimental study the sequence of cDNA related to the mature peptide of human morphogenetic protein-2 (BMP-2 in E.coli was synthesized and cloned in a PET system. After sequencing, recombinant plasmid pET28a/BMP-2 was transformed into the expression host, E.coli BL21 (DE3. The transformed bacteria were cultured in LB me-dium containing kanamaycin antibiotic at 37° C for O/N. Then, induction with IPTG took place. The expression was evaluated by reverse transcriptase PCR and SDS-PAGE followed by western blotting to confirm its identity. The observed band on SDS-PSGE showed the presence of the expressed protein at the 14k Dalton segment which was confirmed by west-ern blotting technique. Results: The gene sequence was amplified by PCR. After gene and plasmid preparation, lega-tion was performed. Sequencing confirmed accuracy of cloning. Protein expression was demonstrated by RT-PCR and SDS-PAGE. Results were confirmed by western blotting. Conclusion: In this study over-expression of this recombinant protein was achieved in a pro-karyotic system. Different concentrations of inducer were applied and harvesting was per-formed in different times after induction. The best expression was detected in 4 hours after induction with a concentration of 1mM IPTG. (Sci J Hamadan Univ Med Sci 2014; 21 (3: 196-202

  16. Phenotype characterization and sequence analysis of BMP2 and BMP4 variants in two Mexican families with oligodontia.

    Science.gov (United States)

    Mu, Y; Xu, Z; Contreras, C I; McDaniel, J S; Donly, K J; Chen, S

    2012-11-28

    Both BMP2 and BMP4 are involved in tooth development. We examined phenotypes and BMP2 and BMP4 gene variations in two Mexican oligodontia families. Physical and oral examinations and panoramic radiographs were performed on affected and unaffected members in these two families. The affected members lacked six or more teeth. DNA sequencing was performed to detect BMP2 and BMP4 gene variations. Three single nucleotide polymorphisms (SNPs) in BMP2 and BMP4 genes were identified in the two families, including one synonymous and two missense SNPs: BMP2 c261A>G, pS87S, BMP2 c570A>T, pR190S, and BMP4 c455T>C, pV152A. Among the six affected patients, 67% carried "GG" or "AG" genotype in BMP2 c261A>G and four were "TT" or "AT" genotype in BMP2 c570A>T (pR190S). Polymorphism of BMP4 c455T>C resulted in amino acid changes of Val/Ala (pV152A). BMP2 c261A>G and BMP4 c455T>C affect mRNA stability. This was the first time that BMP2 and BMP4 SNPs were observed in Mexican oligodontia families.

  17. BMP2基因重组慢病毒载体质粒的构建及鉴定%Construction and identification of lentiviral vector plasmid recombined by BMP2 gene

    Institute of Scientific and Technical Information of China (English)

    林昭伟; 李奇; 林荔军; 刘云龙; 帅明; 谢小波

    2013-01-01

    目的 构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP-2/T2A/EGFP并进行鉴定.方法 从Genbank获得BMP2基因序列,结合载体上的酶切位点需要,设计上下游引物,通过PCR方法扩增目的 基因片段,利用Gateway技术 BP反应构建pDown-BMP2-T2A-EGFP,并进行阳性克隆测序,应用LR反应把pDown-BMP2-T2A-EGFP重组入慢病毒目的 载体质粒pLV.Des2d.P/neo,进行阳性克隆测序.结果 获得长度为1 191 bp的BMP2目的 基因片段,质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP经双酶切后凝胶电泳鉴定正确,测序结果与Genbank报道序列一致.结论 成功构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP.%Objective To construct and identify a lentiviral vector carrying human BMP2 gene. Methods BMP2 gene was obtained from Genbank directly. Primers were designed according to the BMP2 gene sequences reported in Genbank and the restriction sites of the vector. The BMP2 gene was amplified by polymerase chain reaction(PCR). pDown-BMP2-T2A-EGFP was constructed with the BP reaction of Gateway technology, then positive cloning was sequenced. pDown-BMP2-T2A-EGFP was inserted into destination vector plasmid pLV. Des2d. P/neo with the LR reaction of Gateway technology. The positive cloning was sequenced. Results 1 191 bp BMP2 gene fragment was obtained,pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP was identified with double digestion and sequencing, the result was completely in accordance with the BMP2 gene sequences reported in Genbank. Conclusion Recombination lentiviral vector plasmid pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP is constructed successfully.

  18. Local Application of BMP-2 Specific Plasmids in Fibrin Glue does not Promote Implant Fixation

    Directory of Open Access Journals (Sweden)

    Plank Christan

    2011-07-01

    Full Text Available Abstract Background BMP-2 is known to accelerate fracture healing and might also enhance osseointegration and implant fixation. Application of recombinant BMP-2 has a time-limited effect. Therefore, a gene transfer approach with a steady production of BMP-2 appears to be attractive. The aim of this study was to examine the effect of locally applied BMP-2 plasmids on the bone-implant integration in a non-weight bearing rabbit tibia model using a comparatively new non-viral copolymer-protected gene vector (COPROG. Methods Sixty rabbits were divided into 4 groups. All of them received nailing of both tibiae. The verum group had the nails inserted with the COPROG vector and BMP-2 plasmids using fibrin glue as a carrier. Controls were a group with fibrin glue only and a blank group. After 28 and 56 days, these three groups were sacrificed and one tibia was randomly chosen for biomechanical testing, while the other tibia underwent histomorphometrical examination. In a fourth group, a reporter-gene was incorporated in the fibrin glue instead of the BMP-2 formula to prove that transfection was successful. Results Implant fixation strength was significantly lower after 28 and 56 days in the verum group. Histomorphometry supported the findings after 28 days, showing less bone-implant contact. In the fourth group, successful transfection could be confirmed by detection of the reporter-gene in 20 of 22 tibiae. But, also systemic reporter-gene expression was found in heterotopic locations, showing an undesired spreading of the locally applied gene formula. Conclusion Our results underline the transfecting capability of this vector and support the idea that BMP-2 might diminish osseointegration. Further studies are necessary to specify the exact mechanisms and the systemic effects.

  19. Osseointegration of titanium implants by addition of recombinant bone morphogenetic protein 2 (rhBMP-2)

    Energy Technology Data Exchange (ETDEWEB)

    Lichtinger, T.K.; Mueller, R.T.; Schuermann, N.; Oldenburg, M. [Essen Univ. (Germany). Dept. of Orthopaedic Surgery; Wiemann, M. [Inst. of Physiology, Univ. of Essen (Germany); Chatzinikolaidou, M.; Jennissen, H.P. [Inst. of Physiological Chemistry, Univ. of Essen (Germany); Rumpf, H.M.

    2001-12-01

    The osseointegration of long-term implants is often incomplete such that gaps remain between the implant surface and the surrounding hard tissue. This study examines the effect of soluble recombinant human bone morphogenic protein 2 (rhBMP-2) on gap healing and osseous integration. The effect of a single, intraoperative application of soluble rhBMP-2 on the formation of new bone around titanium implants was studied. A total of 8 titanium-alloy cylinders (Ti-6Al-4V) with a plasma spray coating (TPS; 400 {mu}m thickness) were implanted into femoral condyles of mature sheep: rhBMP-2 solution (1 {mu}g) was pipetted into the 1 mm wide cleft around 4 implants; 4 further implants served as rhBMP-2-free controls. Two of these controls exhibited an additional calciumphosphate-coating. The cleft around the implants served as testing zone to study the formation of new bone by microradiographical and histological analyses. The follow-up periods were 4 and 9 weeks, respectively. A significant amount of new bone contacting the implants' surface was detected where rhBMP-2-solution had been used: In 50% a circumferential osseoinduction occurred within 4 weeks and a nearly complete osseointegration was observed after 9 weeks. In all cases bone formation was exaggerated and filled the spongiosa with compact bone. Time matched TPS-controls and controls with calciumphosphate coating showed no notable formation of new bone. The results suggest that a single administration of soluble rhBMP-2 into a bone cavity can augment bone formation and also osseointegration of titanium implants. Further investigations based on these findings are necessary to develop long-term implants (e.g. joint replacements) with rhBMP-2-biocoating for humans. (orig.)

  20. Reconstruction and expression of recombinant adenovirus co-expressing BMP2 and BMP9%双表达骨形态发生蛋白2、9重组腺病毒载体的构建和表达

    Institute of Scientific and Technical Information of China (English)

    林春阳; 陈亮; 罗进勇; 邓忠良

    2011-01-01

    Objective To reconstruct and identify the recombinant adenovirus co-expressing BMP2 and BMP9.Method The genes of BMP2 and BMP9 were amplified from AdEasy vector by PCR and sub-cloned into pASG2 vector.The co-expression shuttle plasmid pASG2-BMP2,9 was confirmed by restriction endonuclease digestion, PCR and gene sequencing, then pASG2-BMP2,9 was electro-transducted into competent AdEasier cells to acquire recombinant adenovirus plasmid.Then, the recombinant vector was transfected into HEK293 cells and high-titer recombinant adenovirus (AdBMP2,9) was gained after rounds of amplification.The expression and bone induction capacity of AdBMP2,9 was observed in C3H10 cells.Result AdBMP2,9 was constructed successfully and the virus titer was 1010 IU/mL after amplification.AdBMP2,9 could express and induce alkaline phosphatase activity in C3H10 cells.Conclusion The recombinant adenovirus co-expressing BMP2 and BMP9 was constructed successfully, which provides a useful tool for bone tissue engineering.%目的 构建双表达骨形态发生蛋白(Bone morphogenic protein,BMP)2、9腺病毒重组体并进行鉴定.方法 自单一表达的BMP2或BMP9 AdEasy质粒上扩增BMP2和BMP9片段,先后亚克隆至穿梭质粒pASG2,获得双表达穿梭质粒pASG2-BMP2、9.酶切及PCR鉴定确认、测序正确后同源重组获得双表达BMP2、BMP9腺病毒质粒,转染至HEK-293细胞中包装和扩增得到高滴度双表达BMP2、BMP9腺病毒,体外感染C3H10细胞,RT-PCR鉴定并观察其早期诱导成骨情况.结果 成功构建双表达BMP2、BMP9的腺病毒,滴度约为1010IU/mL,RT-PCR证实双表达腺病毒在C3H10细胞中表达,其感染的C3H10细胞早期碱性磷酸酶含量较单一表达的BMP2或BMP9腺病毒组增加.结论 成功构建双表达BMP2、9的重组腺病毒载体,为进一步研究BMP2和BMP9的协同成骨作用和制备高效的组织工程人工骨提供了有利的工具.

  1. BMP2/7异源二聚体调控CIZ的表达与自身活性的关系%Relationship Between the Bioactivity of BMP2/7 Heterodimers and Its Regulation of CIZ Expression

    Institute of Scientific and Technical Information of China (English)

    潘秋辉; 杨松海; 董群伟; 孙奋勇

    2007-01-01

    BMP2/7异源二聚体的活性显著高于BMP2同源二聚体,但其机制并不清楚.采用哺乳动物细胞表达的BMP2/7异源二聚体处理成骨细胞MC3T3-E1,细胞化学染色发现BMP2/7的活性显著高于BMP2,报告载体p3GC2-LUX检测发现BMP2/7能够明显上调BMP/Smad通路的活性(P<0.05).但在成骨细胞中过表达CIZ(casinteracting zinc finger protein),能够显著抑制BMP2/7上调ALP与Osteocalcin的作用,并阻断BMP2/7对BMP/Smad通路的激活.同时发现BMP蛋白能够上调CIZ的表达,但BMP2/7的作用明显低于BMP2同源二聚体.可以认为BMP2/7能够诱导CIZ的表达,但由于作用较弱,所以对自身活性的反馈抑制作用也较弱,这可能是BMP2/7有着较强生物活性的关键所在.

  2. Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance.

    Science.gov (United States)

    Porlan, Eva; Morante-Redolat, José Manuel; Marqués-Torrejón, María Ángeles; Andreu-Agulló, Celia; Carneiro, Carmen; Gómez-Ibarlucea, Esther; Soto, Atenea; Vidal, Anxo; Ferrón, Sacri R; Fariñas, Isabel

    2013-11-01

    Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.

  3. 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

  4. 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.

  5. 大鼠pEGFP-C3/BMP-2真核表达载体的构建%Construction of rat pEGFP-C3/BMP-2 recombinant eukaryotic expressing vector

    Institute of Scientific and Technical Information of China (English)

    孙欣; 曾荣; 郭伟韬; 肖启贤; 王斌; 黄云; 林颢

    2012-01-01

    目的 通过克隆大鼠的BMP2基因,构建EGFP-C3/BMP2基因的真核细胞表达载体.方法 把大鼠的基因组DNA通过PCR获得BMP2,克隆构建载体pEGFP/C3-BMP2,并将其转化到大肠杆菌里面,最后进行重组真核表达载体pEGFP-C3-BMP2的构建和鉴定,并可观察其在真核细胞中的表达.结果 以大鼠总DNA为模板扩增出1 200 bp左右的特异性条带,测序结果与Gene-Bank测序结果相比,翻译成的氨基酸序列相同并完全一致,并可在真核细胞中表达.对重组质粒pEGFP-C3/BMP2进行双酶切鉴定并测序,结果也完全一致.结论 为进一步研究利用BMP2基因修饰骨组织工程骨,促进骨折愈合再生提供实验基础.%Objective To construct a recombinant eukaryotic expressing vector pEGFP-C3/BMP-2 by using rat bone morphogenetic protein 2 (BMP-2) gene clone. Methods BMP-2 was amplified with PCR and cloned into pEGFP-C3 vector after sequencing, recombinant eukaryotic expressing vector pEGFP-C3/BMP-2 was constructed and identified by sequencing, the expression of BMP-2 in eukaryotic cells was observed and analyzed. Results The sequencing of BMP-2 gene from the rat complied with the Gene-Bank result and with the same amino acid sequence after translation. The recombinant expressing vector pEGFP-C3/BMP-2 was confirmed by double enzyme digestion and sequencing, the successful expression of BMP-2 in eukaryotic cells was observed. Conclusion For the further study BMP2 genetic modification of bone tissue engineering, and promote the regeneration of fracture healing to provide the basis.

  6. Fucoidan promotes osteoblast differentiation via JNK- and ERK-dependent BMP2-Smad 1/5/8 signaling in human mesenchymal stem cells.

    Science.gov (United States)

    Kim, Beom Su; Kang, Hyo-Jin; Park, Ji-Yun; Lee, Jun

    2015-01-09

    Fucoidan has attracted attention as a potential drug because of its biological activities, which include osteogenesis. However, the molecular mechanisms involved in the osteogenic activity of fucoidan in human alveolar bone marrow-derived mesenchymal stem cells (hABM-MSCs) remain largely unknown. We investigated the action of fucoidan on osteoblast differentiation in hABM-MSCs and its impact on signaling pathways. Its effect on proliferation was determined using the crystal violet staining assay. Osteoblast differentiation was evaluated based on alkaline phosphatase (ALP) activity and the mRNA expression of multiple osteoblast markers. Calcium accumulation was determined by Alizarin red S staining. We found that fucoidan induced hABM-MSC proliferation. It also significantly increased ALP activity, calcium accumulation and the expression of osteoblast-specific genes, such as ALP, runt-related transcription factor 2, type I collagen-α 1 and osteocalcin. Moreover, fucoidan induced the expression of bone morphogenetic protein 2 (BMP2) and stimulated the activation of extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase by increasing phosphorylation. However, the effect of fucoidan on osteogenic differentiation was inhibited by specific inhibitors of ERK (PD98059) and JNK (SP600125) but not p38 (SB203580). Fucoidan enhanced BMP2 expression and Smad 1/5/8, ERK and JNK phosphorylation. Moreover, the effect of fucoidan on osteoblast differentiation was diminished by BMP2 knockdown. These results indicate that fucoidan induces osteoblast differentiation through BMP2-Smad 1/5/8 signaling by activating ERK and JNK, elucidating the molecular basis of the osteogenic effects of fucoidan in hABM-MSCs.

  7. Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice.

    Science.gov (United States)

    Charles, Lyndon F; Woodman, Jessica L; Ueno, Daisuke; Gronowicz, Gloria; Hurley, Marja M; Kuhn, Liisa T

    2015-04-01

    There is an age-associated reduction in the bone healing activity of bone morphogenetic protein-2 (BMP-2) that is currently addressed by administering higher doses of BMP-2 in elderly patients. The unwanted medical complications from high dose BMP-2 motivated this investigation to determine whether the addition of a low dose of fibroblast growth factor 2 (FGF-2) could enhance the ability of a lower dose of BMP-2 to heal calvarial bone defects in old mice (18-20 months old). FGF-2 (5 ng) and BMP-2 (2 μg) were administered by a controlled release two-phase biomaterial scaffold placed into the bone defect. FGF-2 released more rapidly and completely in vitro than BMP-2 (40% vs 2%). In vivo, both BMP-2 and FGF-2+BMP-2 groups formed more new bone in calvarial defects than scaffold alone (p FGF-2 only groups (p FGF-2 to BMP-2 as measured by microCT, but the pattern of bone deposition was different. In old mice, but not young, there was enhanced bony fill in the central bone defect area when the BMP-2 was supplemented with FGF-2. Histological analysis of the center of the defect revealed an increased bone volume (%BV/TV (p = 0.004)) from the addition of FGF-2. These studies suggest that combining a low dose of FGF-2 with a low dose of BMP-2 has the potential to increase bone healing in old mice relative to BMP-2 alone.

  8. Long bone structure and strength depend on BMP2 from osteoblasts and osteocytes, but not vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Sarah H McBride

    Full Text Available The importance of bone morphogenetic protein 2 (BMP2 in the skeleton is well known. BMP2 is expressed in a variety of tissues during development, growth and healing. In this study we sought to better identify the role of tissue-specific BMP2 during post-natal growth and to determine if BMP2 knockout affects the ability of terminally differentiated cells to create high quality bone material. We targeted BMP2 knockout to two differentiated cell types known to express BMP2 during growth and healing, early-stage osteoblasts and their progeny (osterix promoted Cre and vascular endothelial cells (vascular-endothelial-cadherin promoted Cre. Our objectives were to assess post-natal bone growth, structure and strength. We hypothesized that removal of BMP2 from osteogenic and vascular cells (separately would result in smaller skeletons with inferior bone material properties. At 12 and 24 weeks of age the osteoblast knockout of BMP2 reduced body weight by 20%, but the vascular knockout had no effect. Analysis of bone in the tibia revealed reductions in cortical and cancellous bone size and volume in the osteoblast knockout, but not in the vascular endothelial knockout. Furthermore, forelimb strength testing revealed a 30% reduction in ultimate force at both 12 and 24 weeks in the osteoblast knockout of BMP2, but no change in the vascular endothelial knockout. Moreover, mechanical strength testing of femurs from osteoblast knockout mice demonstrated an increased Young's modulus (greater than 35% but decreased post-yield displacement (greater than 50% at both 12 and 24 weeks of age. In summary, the osteoblast knockout of BMP2 reduced bone size and altered mechanical properties at the whole-bone and material levels. Osteoblast-derived BMP2 has an important role in post-natal skeletal growth, structure and strength, while vascular endothelial-derived BMP2 does not.

  9. Long bone structure and strength depend on BMP2 from osteoblasts and osteocytes, but not vascular endothelial cells.

    Science.gov (United States)

    McBride, Sarah H; McKenzie, Jennifer A; Bedrick, Bronwyn S; Kuhlmann, Paige; Pasteris, Jill D; Rosen, Vicki; Silva, Matthew J

    2014-01-01

    The importance of bone morphogenetic protein 2 (BMP2) in the skeleton is well known. BMP2 is expressed in a variety of tissues during development, growth and healing. In this study we sought to better identify the role of tissue-specific BMP2 during post-natal growth and to determine if BMP2 knockout affects the ability of terminally differentiated cells to create high quality bone material. We targeted BMP2 knockout to two differentiated cell types known to express BMP2 during growth and healing, early-stage osteoblasts and their progeny (osterix promoted Cre) and vascular endothelial cells (vascular-endothelial-cadherin promoted Cre). Our objectives were to assess post-natal bone growth, structure and strength. We hypothesized that removal of BMP2 from osteogenic and vascular cells (separately) would result in smaller skeletons with inferior bone material properties. At 12 and 24 weeks of age the osteoblast knockout of BMP2 reduced body weight by 20%, but the vascular knockout had no effect. Analysis of bone in the tibia revealed reductions in cortical and cancellous bone size and volume in the osteoblast knockout, but not in the vascular endothelial knockout. Furthermore, forelimb strength testing revealed a 30% reduction in ultimate force at both 12 and 24 weeks in the osteoblast knockout of BMP2, but no change in the vascular endothelial knockout. Moreover, mechanical strength testing of femurs from osteoblast knockout mice demonstrated an increased Young's modulus (greater than 35%) but decreased post-yield displacement (greater than 50%) at both 12 and 24 weeks of age. In summary, the osteoblast knockout of BMP2 reduced bone size and altered mechanical properties at the whole-bone and material levels. Osteoblast-derived BMP2 has an important role in post-natal skeletal growth, structure and strength, while vascular endothelial-derived BMP2 does not.

  10. Evaluation of collagen/heparin coated TCP/HA granules for long-term delivery of BMP-2.

    Science.gov (United States)

    Hannink, Gerjon; Geutjes, Paul J; Daamen, Willeke F; Buma, Pieter

    2013-02-01

    Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. The purpose of this study was to develop a sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2). We covalently attached heparin to a cross-linked collagen type I coated tricalciumphosphate/hydroxyapatite (TCP/HA) bone substitute and subsequently loaded it with BMP-2. To systematically evaluate the contribution of each component with respect to the binding and release of BMP-2, six constructs were prepared and characterized: TCP/HA, TCP/HA with collagen (TCP/HACol), and TCP/HA with collagen and heparin (TCP/HAColHep) with and without BMP-2 (B). More BMP-2 bound to the TCP/HAColHep + B (92.9 ± 4.8 ng BMP-2/mg granule) granules as compared to the TCP/HACol + B (69.0 ± 9.6 ng BMP-2/mg granule) and TCP/HA + B granules (62.9 ± 5.4 ng BMP-2/mg granule). No difference in release pattern was found between the TCP/HA + B and TCP/HACol + B granules. Up to day 14, BMP-2 was still bound to the TCP/HAColHep + B granules, whereas most BMP had been released from TCP/HACol + B and TCP/HA + B granules at that time. After 21 days most BMP-2 also had been released from the TCP/HAColHep + B granules. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and could possibly enhance bone regeneration efficacy for the treatment of large bone defects.

  11. Heterotopic ossification following single-level anterior cervical discectomy and fusion: results from the prospective, multicenter, historically controlled trial comparing allograft to an optimized dose of rhBMP-2.

    Science.gov (United States)

    Arnold, Paul M; Anderson, Karen K; Selim, Abdulhafez; Dryer, Randall F; Kenneth Burkus, J

    2016-09-01

    range of motion, more so in the rhBMP-2/ACS group. At 24 months, HO negatively affected Neck Disability Index scores (excluding neck/arm pain scores), neurological status, and overall success in patients in the rhBMP-2/ACS group, but not in patients in the allograft group. CONCLUSIONS Implantation of rhBMP-2/ACS at 1.5 mg/ml with polyetheretherketone spacer and titanium plate is effective in inducing fusion and improving pain and function in patients undergoing ACDF for symptomatic single-level cervical degenerative disc disease. At 24 months, the rate and dimensions (length and anteroposterior diameter) of HO were higher in the rhBMP-2/ACS group. At 24 months, range of motion was reduced, with Park Grade 3 HO in both treatment groups. The impact of Park Grades 2 and 3 HO on Neck Disability Index success, neurological status, and overall success was not consistent among the treatment groups. The study data may offer a deeper understanding of HO after ACDF and may pave the way for improved device designs. Clinical trial registration no.: IDE# G060021; data compared with pooled data from control arms of IDE# G010188/NCT00642876 and IDE# G000123/NCT00437190 ( www.clinicaltrials.gov ).

  12. The Effects of rhBMP-2 Used for Spinal Fusion on Spinal Cord Pathology After Traumatic Injury

    Science.gov (United States)

    2009-07-29

    et al., 2004). In 2002, the FDA approved the use of the recombinant human BMP-2 (rhBMP-2) for treatment of discogenic pain in the lower lumbar spine...concomitant SCI (Personal communication with COL(R) Kuklo, MD). 7 Spinal column arthrodesis using rhBMP-2 with concomitant SCI As surgical...first 8 hours after injury. Therefore, in a rat model of dorsal hemisection SCI, we performed spinal arthrodesis with or without rhBMP-2 30 minutes post

  13. 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.

  14. Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

    Science.gov (United States)

    Yang, Wuchen; Guo, Dayong; Harris, Marie A; Cui, Yong; Gluhak-Heinrich, Jelica; Wu, Junjie; Chen, Xiao-Dong; Skinner, Charles; Nyman, Jeffry S; Edwards, James R; Mundy, Gregory R; Lichtler, Alex; Kream, Barbara E; Rowe, David W; Kalajzic, Ivo; David, Val; Quarles, Darryl L; Villareal, Demetri; Scott, Greg; Ray, Manas; Liu, S; Martin, James F; Mishina, Yuji; Harris, Stephen E

    2013-09-15

    We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

  15. Correlation between BMP2, TGFβ1 mRNA expression and apoptosis during healing of the mandible%骨折愈合过程中BMP2和TGFβ1 mRNA的表达与凋亡的相关性

    Institute of Scientific and Technical Information of China (English)

    司晓辉; 金岩; 杨连甲

    2001-01-01

    AIM To elucidate the correlation of bone morphogenetic protein 2 (BMP2), transforming growth factor β1 (TGFβ1) mRNA expression and apoptosis during fracture healing and its significance.METHODS An experimental model of fracture was established to detect the expression of BMP2, TGFβ1 mRNA and apoptosis in the different stages of healing by using in situ hybridization and TUNEL methods.RESULTS ①BMP2 mRNA was expressed in mesenchymal cells, osteoblasts and chondrocytes on 5~11 d whereas TGFβ1 mRNA in osteoblasts and chondrocytes on 11~14 d post-fracture; ②the apoptotic cells increased markedly on 5 d and 11 d, especially in mesenchymal cells and chondrocytes; and ③there was positive correlation between BMP2 mRNA expression and apoptosis (r=0.887, P0.05).CONCLUSION Apoptosis is a normal concomitant of fracture healing serving the purposes of controling cell number and eliminating useless cells.BMP2 may participate directly in inducing the apoptotic process.%目的 探讨骨折愈合过程中骨形成蛋白2(BMP2),转化生长因子β1(TGFβ1) mRNA的表达与凋亡发生的相关性及意义.方法 建立颌骨骨折模型,利用原位杂交方法检测骨折愈合的不同时期BMP2和TGFβ1 mRNA的表达,同时采用TUNEL法检测细胞凋亡,并进行相关性分析.结果 ①骨折愈合早期(5~11 d)的间充质细胞、成骨细胞和软骨细胞内有BMP2 mRNA表达,而TGFβ1 mRNA主要在骨折愈合后期(11~14 d)的成骨细胞和软骨细胞内表达;②细胞凋亡在骨折愈合的5 d和11 d最多,以间充质细胞和软骨细胞为主;③BMP2 mRNA的表达与凋亡具有正相关(r=0.887, P0.05).结论 骨折愈合过程伴随凋亡的发生,以控制细胞的数量和清除无用的细胞,BMP2可能直接参与了凋亡发生过程.

  16. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

    Directory of Open Access Journals (Sweden)

    Adelina A. Hernandez-Hurtado

    2016-01-01

    Full Text Available Adipose-derived mesenchymal stem cells (ADMSCs are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs. This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

  17. Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

    Science.gov (United States)

    Hernandez-Hurtado, Adelina A.; Lara-Arias, Jorge; Romero-Diaz, Viktor J.; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F.; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G.; Espinoza-Juarez, Marcela A.; Mendoza Lemus, Oscar F.

    2016-01-01

    Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology. PMID:27818692

  18. Local expression and role of BMP-2/4 in injured spinal cord.

    Science.gov (United States)

    Cui, Z S; Zhao, P; Jia, C X; Liu, H J; Qi, R; Cui, J W; Cui, J H; Peng, Q; Lin, B; Rao, Y J

    2015-08-07

    We investigated local changes in BMP-2/4 expression in rat spinal cords 1 week following injury to study the damage effects of BMP-2/4 in spinal cord injury (SCI). Sprague Dawley rats (45, 4 months old) were randomized into three groups comprising 15 rats each: a SHAM group, an SCI without noggin group (SCIO), and an SCI with noggin group (SCID). The SCIO and SCID groups were subjected to spinal cord hemisection, and motor activity was assessed using the BBB score. Expression of BMP-2/4 in each injured spinal cord section was examined by hematoxylin and eosin staining, immunohistochemistry, and western blot. There were no significant differences in BBB scores among the three groups (P > 0.05). Following hemisection, the BBB score in the SHAM group was significantly higher than in the other two groups on the 1st day after modeling (P 0.05). Seven days after modeling, the BBB score in the SHAM group was significantly higher than in the other two groups (P < 0.05), and the BBB score in the SCID group was obviously higher than in the SCIO group (P < 0.05). The expression of BMP-2/4 was highest in the SCIO group and lowest in the SHAM group (P < 0.05). SCI can cause severe impairment of motor activity in rats. Seven days after SCI, the local expression of BMP-2/4 had obviously increased; noggin can effectively inhibit the expression of BMP-2/4 and reduce impairment.

  19. Role of BMP2 in differentiation of neural stem cells from anterior subventricular zone into GABAergic neurons%BMP2在SVZa神经干细胞向GABA能神经元分化中的调控作用

    Institute of Scientific and Technical Information of China (English)

    陈锦华; 杨辉; 尹昌林; 张治元; 刘仕勇; 何家全; 高方友

    2007-01-01

    目的 研究BMP2在SVZa神经干细胞向γ-氨基丁酸(GABA)能神经元分化中的调控作用.方法 体外分离培养P0昆明小鼠室管膜下区(SVZa)神经干细胞,纯化传代培养3代后,使用不同浓度BMP2诱导SVZa神经干细胞,采用流式细胞仪检测不同浓度BMP2作用下SVZa神经干细胞分化为GABA能神经元的比例;另外利用活体荧光GFP标记GAD67特异性启动子,动态地研究BMP2在SVZa神经干细胞向GABA能神经元分化中的作用.在此基础之上,采用RT-PCR检测不同浓度BMP2作用下Mash1的表达.结果 不同浓度BMP2作用组分化为GABA能神经元的比例均高于空白对照组,10 ng/ml浓度的BMP2组比例最高;10 ng/ml浓度BMP2组,GAD67-GFP标记阳性细胞数目明显高于对照组;10 ng/ml浓度BMP2组Mash1表达高于其他组.结论 BMP2促进SVZa神经干细胞向GABA能神经元的分化;10 ng/ml浓度的BMP2显著促进Mash1的表达.

  20. BMP2基因转染犬牙髓细胞的实验研究

    Institute of Scientific and Technical Information of China (English)

    冯艳红; 刁志虹; 高毅; 李威

    2011-01-01

    目的 构建骨形态发生蛋白2(bone morphogenetic proteins 2,BMP2)绿色荧光融合蛋白pEGFP-N1-BMP2真核表达质粒,然后再用其在体外转染犬牙髓细胞,探讨BMP2基因转染对牙髓细胞BMP2基因表达的影响,及是否表达BMP2蛋白.方法 构建pEGFP-N1-BMP2真核表达质粒,采用阳离子脂质体转染法将BMP2基因转染体外培养的犬牙髓细胞,检测转染后细胞的BMP2基因表达及蛋白表达情况.结果 成功构建pEGFP-N1-BMP2真核表达质粒,对构建的BMP2真核重组质粒用XhoI、HindIII进行双酶切,其产物进行琼脂糖凝胶电泳后,在1.2kb、4.7kb可见2条特异条带;并进行全基因序列测序,报告100%符合,证明pEGFP-N1-BMP2重组质粒构建成功.转染后的细胞可见BMP2基因表达,细胞免疫组织化学检测转染后的细胞中有BMP2蛋白的表达.结论 pEGFP-N1-BMP2 真核表达质粒转染后的牙髓细胞能够表达BMP2基因,BMP2蛋白表达阳性.

  1. 骨形态发生蛋白2在骨髓间充质干细胞向肝细胞分化中的作用%Effect of BMP2 on mesenchymal stem cells differentiating into hepatocytes

    Institute of Scientific and Technical Information of China (English)

    温彪; 周述仙

    2014-01-01

    Objective To observe the effect of BMP2 on bone marrow mesenchymal stem cells (BMSCs) differentia-ting into hepatocytes .Methods BMSCs in thighbone of rats were isolated and cultured using adherent culture method . The third generation of amplification of BMSCs in vitro was made into cell climbing piece , and directional inducting hepato-cyte.BMSCs was divided into Hepatocyte growth factor (HGF) group , HGF +BMP2 group, BMP2 group and blank con-trol group depending added inducing factor .Cells were collected after culturing about 10 days.Changes in cell morphology was observed .Using enzyme-linked immunosorbent assay to culture supernatant of liver cell-specific marker alpha-fetopro-tein (AFP), albumin (ALB), Cytokeratin immunohistochemistry assay cells -18 (CK-18).Results ALB, AFP and CK-18 in HGF group and HGF +BMP2 group can be detected , and were higher than those in HGF +BMP2 group ( all P<0.05).Conclusion BMP2 can not alonely induce BMSCs differentiating into hepatocytes ,enhance the effect of HGF-in-duced hepatocyte differenting into BMSCs .%目的:观察骨形态发生蛋白2(BMP2)在骨髓间充质干细胞(BMSCs)向肝细胞分化中的作用。方法采用贴壁法分离培养大鼠股骨BMSCs ,将体外扩增的第3代BMSCs制作细胞爬片,并行肝细胞定向诱导。根据诱导因子不同分为:肝细胞生长因子( HGF)组、HGF+BMP2组、BMP2组及空白对照组。培养10 d左右收集细胞,观察各组细胞形态的变化,并采用ELISA法检测培养液上清中肝细胞特异性标志物甲胎蛋白( AFP)、白蛋白( ALB),免疫细胞化学法检测诱导分化后细胞CK-18的表达。结果 HGF组和HGF+BMP2组可检测到ALB、AFP及CK-18,且HGF+BMP2组ALB、AFP及CK-18明显高于HGF组(P均<0.05)。结论 BMP2不能单独诱导BMSCs向肝细胞分化,但能增强HGF诱导BMSCs向肝细胞分化的作用。

  2. Biodegradable chitosan nanoparticle coatings on titanium for the delivery of BMP-2.

    Science.gov (United States)

    Poth, Nils; Seiffart, Virginia; Gross, Gerhard; Menzel, Henning; Dempwolf, Wibke

    2015-01-08

    A simple method for the functionalization of a common implant material (Ti6Al4V) with biodegradable, drug loaded chitosan-tripolyphosphate (CS-TPP) nanoparticles is developed in order to enhance the osseointegration of endoprostheses after revision operations. The chitosan used has a tailored degree of acetylation which allows for a fast biodegradation by lysozyme. The degradability of chitosan is proven via viscometry. Characteristics and degradation of nanoparticles formed with TPP are analyzed using dynamic light scattering. The particle degradation via lysozyme displays a decrease in particle diameter of 40% after 4 days. Drug loading and release is investigated for the nanoparticles with bone morphogenetic protein 2 (BMP-2), using ELISA and the BRE luciferase test for quantification and bioactivity evaluation. Furthermore, nanoparticle coatings on titanium substrates are created via spray-coating and analyzed by ellipsometry, scanning electron microscopy and X-ray photoelectron spectroscopy. Drug loaded nanoparticle coatings with biologically active BMP-2 are obtained in vitro within this work. Additionally, an in vivo study in mice indicates the dose dependent induction of ectopic bone growth through CS-TPP-BMP-2 nanoparticles. These results show that biodegradable CS-TPP coatings can be utilized to present biologically active BMP-2 on common implant materials like Ti6Al4V.

  3. Biodegradable Chitosan Nanoparticle Coatings on Titanium for the Delivery of BMP-2

    Directory of Open Access Journals (Sweden)

    Nils Poth

    2015-01-01

    Full Text Available A simple method for the functionalization of a common implant material (Ti6Al4V with biodegradable, drug loaded chitosan-tripolyphosphate (CS-TPP nanoparticles is developed in order to enhance the osseointegration of endoprostheses after revision operations. The chitosan used has a tailored degree of acetylation which allows for a fast biodegradation by lysozyme. The degradability of chitosan is proven via viscometry. Characteristics and degradation of nanoparticles formed with TPP are analyzed using dynamic light scattering. The particle degradation via lysozyme displays a decrease in particle diameter of 40% after 4 days. Drug loading and release is investigated for the nanoparticles with bone morphogenetic protein 2 (BMP-2, using ELISA and the BRE luciferase test for quantification and bioactivity evaluation. Furthermore, nanoparticle coatings on titanium substrates are created via spray-coating and analyzed by ellipsometry, scanning electron microscopy and X-ray photoelectron spectroscopy. Drug loaded nanoparticle coatings with biologically active BMP-2 are obtained in vitro within this work. Additionally, an in vivo study in mice indicates the dose dependent induction of ectopic bone growth through CS-TPP-BMP-2 nanoparticles. These results show that biodegradable CS-TPP coatings can be utilized to present biologically active BMP-2 on common implant materials like Ti6Al4V.

  4. Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity.

    Science.gov (United States)

    Intini, Giuseppe; Nyman, Jeffry S

    2015-06-01

    Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1(+/-)) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2(c/c);Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1(+/-) mice were crossed with Bmp2(c/c);Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy.

  5. Effect of BMP-2 Delivery Mode on Osteogenic Differentiation of Stem Cells

    Science.gov (United States)

    Kim, Yong-Jin; Kim, Ki-Suk; Jang, Hyon-Seok; Chung, Hyung-Min

    2017-01-01

    Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy. PMID:28197209

  6. Nanotubes Functionalized with BMP2 Knuckle Peptide Improve the Osseointegration of Titanium Implants in Rabbits.

    Science.gov (United States)

    Ma, Yuanping; Zhang, Zhenting; Liu, Yiran; Li, Hongyi; Wang, Na; Liu, Wenwen; Li, Wenjun; Jin, Lingling; Wang, Jinshu; Chen, Su

    2015-02-01

    To determine the effects of surface modification on implant osseointegration in vivo, we first immobilized polydopamine onto 70-nm diameter TiO2 nanotubes as an intermediate layer, and then conjugated a 21 amino acid peptide sequence (the so-called "knuckle peptide") of bone morphogenetic protein-2 (BMP-2) onto the nanotubes created by electrochemical anodization. We inserted these implants into the tibiae of rabbits and measured the gene expression and bone formation around them. The successful fabrication of BMP-2 knuckle peptide was confirmed by contact angle measurement, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The histological features and the amount of bone fluorescence around the implants on non-decalcified sections were investigated at 3, 5, 8, and 12 weeks after implantation using traditional light and fluorescence microscopy, and the gene expression of alkaline phosphatase, osterix, collagen-I, and tartrate-resistant acid phosphatase was examined by real-time PCR at 1, 2, 3, 4, and 5 weeks after implantation. The results demonstrated a significant increase in bone-implant contact, quantity of fluorescence, and gene expression levels of the bone attached to implants with immobilized BMP-2 knuckle peptide compared with the other two control groups. In conclusion, the surface functionalization of TiO2 nanotubes with BMP-2 knuckle peptide was beneficial for osseointegration and this approach could be further developed to improve Ti-based implants for various applications.

  7. Improving the osteogenic potential of BMP-2 with hyaluronic acid hydrogel modified with integrin-specific fibronectin fragment

    NARCIS (Netherlands)

    Kisiel, M.; Martino, M.M.; Ventura, M.; Hubbell, J.A.; Hilborn, J.; Ossipov, D.A.

    2013-01-01

    While human bone morphogenetic protein-2 (rhBMP-2) is a promising growth factor for bone regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyalur

  8. Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering

    NARCIS (Netherlands)

    Kempen, Diederik H. R.; Lu, Lichun; Hefferan, Teresa E.; Creemers, Laura B.; Maran, Avudaiappan; Classic, Kelly L.; Dhert, Wouter J. A.; Yaszemski, Michael J.

    2008-01-01

    In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedd

  9. Construction and Identification of PIRES-BMP2-TGFβ3 Bicistronic Eukayotic Expression Vector%双基因真核表达载体pIRES-BMP2-TGFβ3的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    马小松; 王英振; 王昌耀; 刘金钊

    2011-01-01

    Objective: To construct a bicistronic eukayotic expression vector pIRES-BMP2-TGF. Methods: The BMP2 gene was obtained from pGEMT/BMP2 plasmid by PCR. And it was inserted into bicistronic eukaryotic expression plasmid vector pIRES. The TGFP 3 was extracted from human embryonal tissue by RT-PCR, then the gene was inserted into the plasmid pIRES-BMP2. The inserted target genes in the plasmid were detected by restriction enzyme digestion and nucleotide sequencing. Results: The direction and sequences of the new bicistronic eukaryotic expression vector pIRES-BMP2-TGFβ3 were correct. Conclusion: The bicistronic eukaryotic expression vector was successfully constructed.%目的:构建与鉴定骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体pIRES-BMP2-TGFβ3.方法:首先,用PCR方法从质粒pGEMT/BMP2中扩增出BMP2基因全长,并将其连入双基因真核表达载体pIRES,得到质粒pIRES-BMP2,其次,从人胚胎组织提取总RNA,反转录成cDNA,以反转录的cDNA为模板,PCR扩增出TGFβ3基因全长,将TGFβ3基因连入质粒pIRES-BMP2;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定.结果:酶切鏊定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确.结论:成功构建PIRES-BMP2/TGFβ3双基因真核表达载体.

  10. 颌骨牙骨质化纤维瘤中BMP-2的表达及其意义%The expression of BMP-2 in cementifying fibroma of the jaw and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    黄宏杰; 平飞云; 彭加萍

    2006-01-01

    目的观察颌骨牙骨质化纤维瘤(cementifying fibroma,CF)中骨形成蛋白-2(BMP-2)的表达,探讨其与牙骨质小体及牙骨质样物质形成的关系.方法采用原位杂交和免疫组化方法对10例CF标本进行BMP-2mRNA和BMP-2蛋白的检测.结果 10例CF标本中,9例有BMP-2mRNA表达,7例有BMP-2蛋白表达,阳性信号位于牙骨质小体及牙骨质样物质周围环绕的短梭形细胞,以及瘤样增生的纤维组织中的多数纤维母细胞和部分纤维细胞胞质中,未钙化的牙骨质基质也有BMP-2蛋白表达.结论 BMP-2在CF中的表达可能与牙骨质小体及牙骨质样物质的形成有关.

  11. CTLA4Ig depresses immune response of exogenous BMP2 transgenic MSC transplantation%CTLA4Ig抑制BMP2基因转染的MSCs诱导的免疫应答

    Institute of Scientific and Technical Information of China (English)

    张晓玲; 张超; 汤亭亭; 楼觉人; 戴尅戎

    2007-01-01

    目的 通过腺病毒介导人细胞毒T淋巴细胞相关抗原4免疫球蛋白(CTLA4Ig)及人骨形态发生蛋白2(BMP2)在骨髓间充质干细胞(MSCs)中的表达,探讨CTLA4Ig对BMP2转染的异基因MSCs诱导的免疫应答的抑制作用.方法 以CTLA4Ig和BMP2重组腺病毒转染MSCs.ELISA法检测包装的病毒感染MSCs后,CTLA4Ig及BMP2蛋白的表达;观察CTLA4Ig对混合淋巴细胞反应(MLR)的抑制作用.结果 腺病毒载体介导CTLA4Ig和BMP2体外感染的MSCs能够分泌CTLA4Ig及BMP2蛋白,且CTLA4Ig融合蛋白可以有效抑制AdBMP2基因转染的MSCs的刺激作用.结论 给予CTLA4Ig腺病毒进行基因治疗可有效的抑制AdBMP2转染的异基因MSCs引起的免疫应答,诱导MSCs移植耐受;为BMP2基因修饰的同种异体间的MSCs移植提供了实验依据.

  12. Bone formation of a porous Gelatin-Pectin-biphasic calcium phosphate composite in presence of BMP-2 and VEGF.

    Science.gov (United States)

    Amirian, Jhaleh; Linh, Nguyen Thuy Ba; Min, Young Ki; Lee, Byong-Taek

    2015-05-01

    A composite scaffold of gelatin (Gel)-pectin (Pec)-biphasic calcium phosphate (BCP) was fabricated for the successful delivery of growth factors. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) were coated on the Gel-Pec-BCP surface to investigate of effect of them on bone healing. Surface morphology was investigated by scanning electron microscopy, and BCP dispersion in the hydrogel scaffolds was measured by energy dispersive X-ray spectroscopy. The results obtained from Fourier transform infrared spectroscopy showed that BMP-2 and VEGF were successfully coated on Gel-Pec-BCP hydrogel scaffolds. MC3T3-E1 preosteoblasts were cultivated on the scaffolds to investigate the effect of BMP-2 and VEGF on cell viability and proliferation. VEGF and BMP-2 loaded on Gel-Pec-BCP scaffold facilitated increased cell spreading and proliferation compared to Gel-Pec-BCP scaffolds. In vivo, bone formation was examined using rat models. Bone formation was observed in Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds within 4 weeks, and was greatest with Gel-Pec-BCP/BMP-2 scaffolds. In vitro and in vivo results suggest that Gel-Pec-BCP/BMP-2 and Gel-Pec-BCP/VEGF scaffolds could enhance bone regeneration.

  13. 转染Ad-hBMP2的脂肪干细胞与壳聚糖/磷酸三钙复合物支架的相容性%Compatibility of Adipose-derived Stem Cells Transfected by Ad-hBMP2 Gene and CTCP Scaffold in vitro

    Institute of Scientific and Technical Information of China (English)

    方忠; 杨琴; 熊伟; 李光辉; 廖晖; 李锋; 肖骏

    2012-01-01

    Objective To observe the biological behavior of cultured adipose-derived stem cells(ADSCs)transfected by Ad-hBMP2 combined with chitosan/tricalcium phosphate(CTCP)scaffold and investigate the feasibility of the composite for cartilage tissue engineering. Methods The ADSCsQ X 106/mL) transfected with Ad-hBMP2 plasmid vector were co-cultured with the CTCP scaffold. The adhesion and proliferation of ADSCs, and the morphological changes were observed. RT-PCR, Western blot and immunohistochemistry were applied to detect the expression of Osteocalcin and collagen I in the scaffold. Results The poriferous CTCP scaffold has macro and micro poriferous structures,and the porous rate was 83%. The ADSCs transfected by Ad-hBMP2 have been successfully cultured in vitro. The induced cells adhered to the surface of the scaffold and proliferated well. The RT-PCR,Western blot and immunohistochemistry revealed that the expression of Osteocalcin and collagen I was detected after the co-culture. Conclusion The poriferous CTCP scaffold with excellent property should be a good "matrix" for ADSCs transfected by Ad-hBMP2, and could be used for bone tissue engineering.%目的 探讨转染腺病毒骨形态发生蛋白(Ad-hBMP2)基因的脂肪干细胞(ADSCs)与壳聚糖/磷酸三钙(CTCP)复合物支架的相容性,以期为ADSCs修复骨缺损寻找理想的组织工程骨支架材料.方法 将壳聚糖与磷酸三钙进行复合制成CTCP复合物材料,再将其与转染Ad-hBMP2的ADSCs(密度1×106/mL)复合培养,进行细胞复合物支架的一般与超微形态学观察,观察细胞粘附能力、增殖活力,RT-PCR及Western blot测定成骨细胞骨钙素和Ⅰ型胶原水平.结果 CTCP支架孔径200~350 μm,孔隙率83%;电镜显示转染Ad-hBMP2的ADSCs与CTCP复合物在体外培养期间支架无塌陷及形变,且其在支架上粘附、增殖良好,并能分泌细胞外基质如骨钙素和Ⅰ型胶原等;随着培养时间延长,骨的组织学特征

  14. Repair of segmental bone defects with bone marrow and BMP-2 adenovirus in the rabbit radius

    Science.gov (United States)

    Cheng, Lijia; Lu, Xiaofeng; Shi, Yujun; Li, Li; Xue, Jing; Zhang, Li; Xia, Jie; Wang, Yujia; Zhang, Xingdong; Bu, Hong

    2012-12-01

    Bone tissue engineering (BTE) is approached via implantation of autogenous mesenchymal stem cells (MSCs), marrow cells, or platelet-rich plasma, etc. To the contrary, gene therapy combining with the bone marrow (BM) has not been often reported. This study was performed to investigate whether a modified BTE method, that is, the BM and a recombinant human bone morphogenetic protein-2 adenovirus (Ad.hBMP-2) gene administering in hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramics could accelerate the healing of segmental defects in the rabbit radius. In our study, ceramics were immersed in the adenovirus overnight, and half an hour before surgery, autologous BM aspirates were thoroughly mixed with the ceramics; at the same time, a 15-mm radius defect was introduced in the bilateral forelimbs of all animals, after that, this defect was filled with the following: (1) Ad.hBMP-2 + HA/β-TCP + autologous BM (group 1); (2) HA/β-TCP + Ad.hBMP-2 (group 2); (3) HA/β-TCP alone (group 3); (4) an empty defect as a control (group 4). Histological observation and μ-CT analyses were performed on the specimens at weeks 2, 4, 8, and 12, respectively. In group 1, new bone was observed at week 4 and BM appeared at week 12, in groups 2 and 3, new bone was observed at week 8 and it was more mature at week 12, in contrast, the defect was not bridged in group 4 at week 12. The new bone area percentage in group 1 was significantly higher than that in groups 2 and 3. Our study indicated that BM combined with hBMP-2 adenovirus and porous ceramics could significantly increase the amount of newly formed bone. And this modified BTE method thus might have potentials in future clinical application.

  15. Development of Physiologically Based Pharmacokinetic Model (PBPK) of BMP2 in Mice.

    Science.gov (United States)

    Utturkar, Aditya; Paul, Bikram; Akkiraju, Hemanth; Bonor, Jeremy; Dhurjati, Prasad; Nohe, Anja

    2013-01-01

    Bone Morphogenetic protein 2 holds great promise for potential applications in the clinic. It is a potent growth factor for the use in the cervical spine surgery (FDA approved 2002) and has been marketed as "Infuse" for treating open tibial shaft fractures (FDA approved 2004). However, its use is limited by several significant side effects that maybe due to its potency and effect on different stem cell populations in the spine. BMP2 is expressed throughout the human body in several tissues and at a very high concentration in the blood. BMP receptors, especially BMP receptor type Ia, is ubiquitously expressed in most tissues. Currently, it is difficult to determine how BMP2 is physiologically distributed in mice or humans and no quantitative models are available. A Physiologically-Based Pharmaco-Kinetic (PBPK) model has been developed to determine steady-state distribution of BMP2 in mice. The multi-compartmental PBPK model represents relevant organ/tissues with physiological accuracy. The organs/tissue compartments chosen were brain, lung, heart, liver, pancreas, kidney, uterus, bone and fat. A blood compartment maintained connectivity among the various organs. Four processes characterized the change in the concentration of the protein in every compartment: blood flow in, blood flow out, protein turnover and receptor binding in the organ. The unique aspects of the model are the determination of elimination using receptor kinetics and generation using protein turnover. The model also predicts steady state concentrations of BMP2 in tissues in mice and may be used for possible scale-up of dosage regimens in humans.

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

    Directory of Open Access Journals (Sweden)

    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

  17. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    Science.gov (United States)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  18. Stiffness-dependent cellular internalization of matrix-bound BMP-2 and its relation to Smad and non-Smad signaling.

    Science.gov (United States)

    Gilde, Flora; Fourel, Laure; Guillot, Raphael; Pignot-Paintrand, Isabelle; Okada, Takaharu; Fitzpatrick, Vincent; Boudou, Thomas; Albiges-Rizo, Corinne; Picart, Catherine

    2016-12-01

    Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue.

  19. 青海藏系绵羊BMP2基因的克隆及其序列分析%BMP2 gene cloning and sequence analysis of Qinghai Tibetan Sheep

    Institute of Scientific and Technical Information of China (English)

    宋静; 杨发龙; 陈刚; 王杰; 钟金诚; 郑玉才

    2013-01-01

    从青海藏系绵羊的皮肤中提取总RNA,根据其他物种BMP2基因的保守序列设计特异性引物,采用RT-PCR技术首次扩增出藏系绵羊BMP2DNA序列,提交至GenBank,并已取得序列号HM066200.将此片段克隆到pMD18-T载体中,经菌落PCR鉴定和DNA序列测定分析验证.结果显示,该DNA序列为356 bp的部分CDS区,编码118个氨基酸.证实所克隆序列为BMP2,符合BMP2基因的特征.

  20. BMP2和TGFβ3双基因真核表达载体的构建%Construction of bicistronic eukaryotic expression plasmid containing BMP2 and TGFβ3 genes

    Institute of Scientific and Technical Information of China (English)

    马小松; 刘金钊; 王昌耀; 王英振; 刘世海; 刘相萍

    2011-01-01

    [目的]构建骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体.[方法]从人胚胎组织提取总RNA,反转录成cDNA,以pGEMT/BMP2和反转录的cDNA为模板,PCR扩增出BMP2和TGFβ3两个基因全长,将两个基因片段分别定向连入双基因真核表达载体pIRES;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定.[结果]酶切鉴定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确.[结论]成功构建pIRESBMP2/TGFβ3双基因真核表达载体.%[ Objective]To construct a bicistronic eukayotic expression plasmid consisting of BMP2 and TGFβ3 target genes.[ Method ] The DNA fragment of BMP2 and TGFβ3 genes were obtained from pGEMT/BMP2 plasmid and human embryonal tissue by PCR, respectively. They were inserted into bicistronic eukaryotic expression plasmid vector plRES. The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing. [ Result ] The direction and sequences of the new bicistronic eukaryotic expression plasmid pIRES - BMP2 -TGFβ3 were correct. [ Conclusion] The bicistronic eukaryotic expression plasmid has been constructed successfully.

  1. Parathyroid hormone stimulate osteoblast differentiation by up-regulation of BMP2 expression and function%骨形态发生蛋白2介导甲状旁腺素促进成骨细胞分化的实验研究

    Institute of Scientific and Technical Information of China (English)

    徐莹; 田野; 孟凌新

    2012-01-01

    Objective To study the important role of BMP2 in the PTH-induced osteoblast differentiation. Methods MC3T3-E1 cells were divided into 4 groups: 1) Controls; 2) PTH treatment; 3) Dorso-morphin treatment; 4) PTH + Dorsomorphin treatment. Gene and protein expression levels of BMP2, BMP2 downstream genes and osteoblastic genes and protein expressions were detected by Real-time PCR and Western blot respectively. Alkaline phosphatase (ALP) staining was performed to detect ALP activity. 12xSBE-OC luciferase activity was measured using dual luciferase reporter assays. Results The expression level of BMP2 and osteoblastic marker genes was higher in the PTH group than in controls. PTH also increases 12xSBE-OC luciferase activity significantly. In the other hand, the expression of BMP2, BMP2 downstream genes and osteoblastic genes was lower in Dorsomorphin and PTH + Dorsomorphin group, than in controls. However, the expression was similar in Dorsomorphin and PTH + Dorsomorphin groups. Conclusion PTH stimulates osteoblast differentiation by up-regulating BMP2 expression and function.%目的 探讨骨形态发生蛋白2(BMP2)在甲状旁腺素(PTH)促进成骨细胞分化过程中的重要介导作用.方法培养MC3T3-E1细胞,分为4组:1)盐水对照组;2)PTH组;3)6-[4-[2-(1-哌啶基)乙氧基]苯基]-3-(4-吡啶基)吡唑并[1,5-a]嘧啶 (Dorsomorphin) 组;4) PTH+Dorsomorphin组.Real-time PCR法和Westernblot方法检测细胞BMP2BMP2下游基因和成骨因子的表达,碱性磷酸酶(ALP)染色方法检测细胞ALP的活性;双荧光素酶报告基因检测方法检测12xSBE-OC荧光素酶的活性.结果:PTH组BMP-2、成骨因子的表达及其12xSBE-OC荧光素酶的活性,明显高于盐水对照组.Dorsomorphin组和PTH+Dorsomorphin组BMP-2BMP-2下游基因和成骨因子的表达,均明显低于盐水对照组;但其表达于两组间无明显差别.结论 BMP2介导PTH促进成骨细胞的分化,PTH可通过上调BMP2的表达,提高其功能,促进成骨细胞的成熟分化.

  2. Decreased BMP2 signal in GIT1 knockout mice slows bone healing.

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    Sheu, T J; Zhou, Wei; Fan, Jin; Zhou, Hao; Zuscik, Michael J; Xie, Chao; Yin, Guoyong; Berk, Bradford C

    2014-12-01

    Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor β (TGFβ) superfamily plays important roles and comprises TGFβs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFβs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFβ signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFβ1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFβ1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site.

  3. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla.

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    Zhang, Wen; Zhang, Xiaolei; Ling, Junqi; Wei, Xi; Jian, Yutao

    2016-05-01

    Stem cells from apical papilla (SCAP) possess clear osteo‑/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo‑/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo‑/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector‑mediated gene transfection was conducted with SCAP in order to construct blank vector‑transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene‑transfected SCAP (SCAP‑VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP‑VEGF was demonstrated to exhibit increased proliferation, and SCAP‑BMP2‑VEGF exhibited reduced proliferation during eight days observation. SCAP‑BMP2‑VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP‑BMP2‑VEGF group exhibited a significantly greater number of ALP‑positive mineralized nodules than the other

  4. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

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    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.

  5. A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.

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    Katherine L Hammond

    Full Text Available The Bone Morphogenetic Protein (BMP genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/- embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

  6. 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.

  7. Effect of BMP2-Platelet-rich Plasma-Biphasic Calcium Phosphate Scaffold on Accelerated Osteogenesis in Mastoid Obliteration.

    Science.gov (United States)

    Jang, Chul Ho; Choi, Cheol Hee; Cho, Yong Beom

    The aim of this study was to evaluate the synergistic effect of platelet-rich plasma (PRP) and recombinant human bone morphogenic protein (BMP)-2 on accelerated osteogenesis of hydroxyapatite/β-tricalcium phosphate mixture and biphasic calcium phosphate (BCP) in mastoid obliteration. To the best of our knowledge, there have been no studies reporting the enhancing effects of BCP, combined with BMP2 and PRP, on osteogenesis in mastoid obliteration. Mastoid obliteration was performed in a control group (BCP only, n=7), a group treated with BMP2 and BCP (experimental group I, n=7), and a group treated with BMP2, PRP and BCP (experimental group II, n=7). The animals were administered fluorescent bone labels for a qualitative evaluation of bone formation; oxytetracycline hydrochloride was administered at 2 weeks, calcein at 4 weeks, and alizarin red at 8 weeks. The animals were sacrificed 12 weeks post-surgery and osteogenesis was evaluated by micro-computed tomography, histological investigation, and histomorphometry. Both experimental groups showed accelerated osteogenesis compared to the control group. However, there were no statistically significant differences between experimental groups I and II. From these results, it can be concluded that BMP2 activated BCP for the enhancement of bone regeneration. However, no synergistic effect of BMP2 and PRP on the osteogenesis of BCP was observed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun [Department of Ophthalmology, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Liu Guangpeng [Key Laboratory of Tissue Engineering, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China); Zhang Peng [Center for Translational Medicine Research and Development, Shenzhen Institute of Advanced Technology, Chinese Academy of Science (China); Hou Hongliang; Tang Tingting, E-mail: drfanxianqun@126.com [Department of Orthopedics, Shanghai Ninth People' s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200011 (China)

    2011-02-15

    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.

  9. Experimental Comparison of Cranial Particulate Bone Graft, rhBMP-2, and Split Cranial Bone Graft for Inlay Cranioplasty.

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    Hassanein, Aladdin H; Couto, Rafael A; Kurek, Kyle C; Rogers, Gary F; Mulliken, John B; Greene, Arin K

    2013-05-01

    Background :  Particulate bone graft and recombinant human bone morphogenetic protein-2 (rhBMP-2) are options for inlay cranioplasty in children who have not developed a diploic space. The purpose of this study was to determine whether particulate bone graft or rhBMP-2 has superior efficacy for inlay cranioplasty and to compare these substances to split cranial bone. Methods :  A 17 mm × 17 mm critical-sized defect was made in the parietal bones of 22 rabbits and managed in four ways: Group I (no implant; n=5), Group II (particulate bone graft; n=5), Group III (rhBMP-2; n=7), and Group IV (split cranial bone graft; n=5). Animals underwent microcomputed tomography and histologic analysis 16 weeks after cranioplasty. Results :  Defects without an implant (Group I) demonstrated inferior ossification (41.4%; interquartile range [IQR], 28.9% to 42.5%) compared to those treated with particulate bone graft (Group II: 99.5%; IQR, 97.8% to 100%), rhBMP-2 (Group III: 99.6%; IQR, 99.5% to 100%), or split cranial bone (Group IV: 100%) (P inlay calvarial defect areas equally, although the thickness of bone healed with rhBMP-2 is inferior. Clinically, particulate bone graft or split cranial bone graft may be superior to rhBMP-2 for inlay cranioplasty.

  10. 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.

  11. Low-power GaAlAs laser irradiation promotes the proliferation and osteogenic differentiation of stem cells via IGF1 and BMP2.

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    Jyun-Yi Wu

    Full Text Available Low-power laser irradiation (LPLI has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells. D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs laser at dose of 0, 1, 2, or 4 J/cm(2. The lactate dehydrogenase (LDH assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm(2 significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1 and bone morphogenetic protein 2 (BMP2 signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.

  12. Does Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Use in Adult Spinal Deformity (ASD) Increase Complications and Are Complications Associated With Location of rhBMP-2 Use?: A Prospective, Multicenter Study of 279 Consecutive Patients.

    Science.gov (United States)

    Bess, Shay; Line, Breton G; Lafarge, Virginie; Schwab, Frank; Shaffrey, Christopher I; Hart, Robert A; Boachie-Adjei, Oheneba; Akbarnia, Behrooz A; Ames, Christopher P; Burton, Douglas C; Deverin, Vedat; Fu, Kai-Ming G; Gupta, Munish; Hostin, Richard; Kebaish, Khaled; Klineberg, Eric; Mundis, Gregory; O'Brien, Michael; Shelokov, Alexis; Smith, Justin S

    2013-11-18

    Study Design. Multi-center, prospective analysis of consecutive ASD patients.Objective. Evaluate complications associated with rhBMP-2 use in ASDSummary of Background Data. Off-label rhBMP-2 use is common, however under-reporting of rhBMP-2 associated complications has been recently scrutinized.Methods. ASD patients consecutively enrolled into a prospective, multicenter database, were evaluated for type and timing of acute perioperative complications. Inclusion criteria: age ≥ 18 years, ASD, spinal arthrodesis >4 levels, and ≥3 months follow-up. Patients divided into those receiving rhBMP-2 (BMP) or no rhBMP-2 (NOBMP). BMP divided into location of use: posterior (PBMP), interbody (IBMP), and interbody + posterior spine (I+PBMP). Correlations between acute perioperative complications and rhBMP-2 use including total dose, dose/level and location of use were evaluated.Results. 279 patients (mean age 57 years, mean spinal levels fused 12.0, mean follow-up 28.8 months) met inclusion criteria. BMP (n = 172; average posterior dose = 2.5 mg/level, average interbody dose = 5 mg/level) had similar age, smoking history, previous spine surgery, total spinal levels fused, estimated blood loss, and duration of hospital stay as NOBMP (n = 107; p>0.05). BMP had greater Charlson Comorbidity Index (1.9 vs. 1.2), greater scoliosis (43° vs. 38°), longer operative time (488.2 vs. 414.6 minutes), more osteotomies/patient (4.0 vs. 1.6) and greater percentage of anteroposterior fusion (APSF; 20.9% vs. 8.4%) than NOBMP, respectively (p0.05). Multivariate analysis demonstrated small to non-existent correlations between rhBMP-2 use and complications.Conclusions. RhBMP-2 use and location of rhBMP-2 use in ASD surgery, at reported doses, does not increase acute major, neurological or wound complications. Research is needed for higher rhBMP-2 dosing and long-term follow-up.

  13. BMP2-海藻酸钠-壳聚糖微球对骨折愈合影响%EFFECT OF BMP2-CHITOSAN-ALGINATE MICROCASULES ON FRACTURE HEALING: AN EXPERIMENTAL STUDY

    Institute of Scientific and Technical Information of China (English)

    赛佳明; 陈东亮; 江晓路

    2016-01-01

    目的 探讨骨形态发生蛋白2(BMP2)-海藻酸钠-壳聚糖微球的制备及其促进兔桡骨骨折愈合的作用.方法 采用脉冲电场法制备BMP2-海藻酸钠-壳聚糖微球,观测其形态、尺寸,测定BMP2载药量、包封率,并进行体外释放实验.制备新西兰兔骨缺损模型,随机分BMP2微球组、BMP2注射组、空微球组及空白对照组4组.于术后第30天行X线检查,比较各组骨折愈合情况.结果 微球具有很好的圆形形态,平均粒径为900.79μm;微球的平均包封率为(84.97±3.49)%,BMP2载药量平均为(16.34±0.48)%.体外模拟体液中,微球所载BMP2被缓释出来,在第7天时87.33%的BMP2被释放出来,在第14天时BMP2被完全释放出来.术后第30天BMP2微球组的骨折愈合情况较各对照组好,差异有显着性(H=6.87,P<0.05).结论 BMP2-海藻酸钠-壳聚糖微球具有良好的缓释效果,局部植入能促进骨折修复愈合.

  14. Uniaxial cyclic stretch promotes osteogenic differentiation and synthesis of BMP2 in the C3H10T1/2 cells with BMP2 gene variant of rs2273073 (T/G).

    Science.gov (United States)

    Li, Jia-mou; Zhang, Yao; Ren, Yuan; Liu, Bao-ge; Lin, Xin; Yang, Jiang; Zhao, Hu-cheng; Wang, Ya-jie; Song, Lei

    2014-01-01

    Ossification of the posterior longitudinal ligament of the cervical spine (OPLL) is characterized by the replacement of ligament tissues with ectopic bone formation, and this result is strongly affected by genetic and local factors. Two single nucleotide polymorphisms (SNPs) of rs2273073 (T/G) and rs235768 (A/T) of bone morphogenetic protein 2 (BMP2) gene which are associated with OPLL have been reported in our previous report. In this study, we confirmed the connection in 18 case samples analysis of BMP2 gene in OPLL patients; additionally, it was also shown from the OPLL patients with ligament tissues that enchondral ossification and expression of BMP2 were significantly higher compared with the non-OPLL patients by histological examination, immunohistochemistry and Western blotting analysis. To investigate the underlying mechanism, we studied the effect of SNPs in cell model. The C3H10T1/2 cells with different BMP2 gene variants were constructed and then subjected to uniaxial cyclic stretch (0.5 Hz, 10% stretch). In the presence of mechanical stress, the expression of BMP2 protein in C3H10T1/2 cells transfected by BMP2 (rs2273073 (T/G)) and BMP2 (rs2273073 (T/G), rs235768 (A/T)) were significantly higher than the corresponding static groups (PBMP2 gene variant of rs2273073 (T/G) could not only increase cell susceptibility to bone transformation similar to pre-OPLL change, but also increase the sensibility to mechanical stress which might play an important role during the progression of OPLL.

  15. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

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    Finnson Kenneth

    2010-02-01

    Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

  16. Treatment of osteonecrosis of femoral head with BMSCs-seeded bio-derived bone materials combined with rhBMP-2 in rabbits

    Institute of Scientific and Technical Information of China (English)

    XIAO Zeng-ming; JIANG Hua; ZHAN Xin-li; WU Zhen-guo; ZHANG Xing-lin

    2008-01-01

    Objective: To evaluate the effect of autologous bone marrow mesenchymal stem cells (BMSCs) seeded bio-derived bone materials (BBM) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in repairing defect of osteonecrosis of femoral head (ONFH). Methods: Early-stage osteonecrosis in the left hip was induced in 36 adult New Zealand white rabbits (provided by the Animal Center of Guangxi Medical University, Nanning, China) after core decompression and delivery of liquid nitrogen into the femoral head. Then the animals were divided into three groups according to the type of implants for bone repair: 12 rabbits with nothing (GroupⅠ, the blank control group), 12 with BBM combined with rhBMP-2 (Group Ⅱ), and 12 with BMSCs-seeded BBM combined with rhBMP-2 (Group Ⅲ). At 4, 8, and 12 weeks after surgery, X-ray of the femoral head of every 4 rabbits in each group was taken, and then they were killed and the femoral heads were collected at each time point, respectively. Gross observation was made on the femoral heads. After hematoxylin and eosin staining, Lane-sandhu scores of X-ray and bone densitometry were calculated and the histomorphometric measurements were made for the new bone trabeculae. Results: At 12 weeks after surgery, two femoral heads collapsed in GroupⅠ, but none in Group Ⅱor Group Ⅲ. X-ray examination showed that the femoral heads in Group Ⅰ had defect shadow or collapsed while those in Group Ⅱ had a low density and those in Group Ⅲ presented with a normal density. Histologically, the defects of femoral heads were primarily filled with no new bone but fibrous tissues in Group Ⅰ. In contrast, new bone regeneration and fibrous tissues occurred in Group Ⅱ and only new bone regeneration occurrd in Group Ⅲ. Lane-sandhu scores of X-ray, bone mineral density and rate of new bone in trabecular area in Group Ⅲ were higher signifIcantly than those of the other two groups. Conclusions: Our findings indicate a superior choice of

  17. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J.; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E.; Macdougall, Mary; Chen, Shuo

    2016-01-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2fx/fx dp) cells. iBmp2ko/ko dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2ko/ko dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmpko/ko cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  18. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-11-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages.

  19. Reciprocal repression between Fgf8 and miR-133 regulates cardiac induction through Bmp2 signaling

    Directory of Open Access Journals (Sweden)

    Carmen Lopez-Sanchez

    2015-12-01

    Full Text Available This data article contains complementary figures and results related to the research article entitled “Negative Fgf8-Bmp2 feed-back is controlled by miR-130 during early cardiac specification” [15], which reveals what specific role miR-130 plays during the cardiac induction process. This study evidenced miR-130 a putative microRNA that targets Erk1/2 (Mapk1 3′UTR- as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2. Thus, miR-130 regulates a negative Fgf8-Bmp2 feed-back loop responsible to achieve early cardiac specification. A significant aspect supporting our conclusions is given by the expression pattern of miR-130 during early cardiac specification, as well as by those results obtained after the designed experimental procedures. The data presented here reveal that miR-133 is also expressed within the precardiac areas during early cardiogenesis, pattern which is comparable to that of FGFR1, receptor involved in the Fgf8/ERK signaling pathway. Interestingly, our miR-133 overexpression experiments resulted in a decrease of Fgf8 expression, whereas we observed an increase of Bmp2 and subsequently of cardiac specific markers Nkx-2.5 and Gata4. Additionally, our loss-of-function experiments -through Fgf8 siRNA electroporation- showed an increase of miR-133 expression. Finally, after our Bmp2 experiments, we observed that miR-133 is upstream-regulated by Bmp2. All those results suggest that miR-133 also constitutes a crucial linkage in the crosstalk between Fgf8 and Bmp2 signaling by regulating the Fgf8/ERK pathway during cardiac induction.

  20. Reciprocal repression between Fgf8 and miR-133 regulates cardiac induction through Bmp2 signaling.

    Science.gov (United States)

    Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

    2015-12-01

    This data article contains complementary figures and results related to the research article entitled "Negative Fgf8-Bmp2 feed-back is controlled by miR-130 during early cardiac specification" [15], which reveals what specific role miR-130 plays during the cardiac induction process. This study evidenced miR-130 a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR- as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2. Thus, miR-130 regulates a negative Fgf8-Bmp2 feed-back loop responsible to achieve early cardiac specification. A significant aspect supporting our conclusions is given by the expression pattern of miR-130 during early cardiac specification, as well as by those results obtained after the designed experimental procedures. The data presented here reveal that miR-133 is also expressed within the precardiac areas during early cardiogenesis, pattern which is comparable to that of FGFR1, receptor involved in the Fgf8/ERK signaling pathway. Interestingly, our miR-133 overexpression experiments resulted in a decrease of Fgf8 expression, whereas we observed an increase of Bmp2 and subsequently of cardiac specific markers Nkx-2.5 and Gata4. Additionally, our loss-of-function experiments -through Fgf8 siRNA electroporation- showed an increase of miR-133 expression. Finally, after our Bmp2 experiments, we observed that miR-133 is upstream-regulated by Bmp2. All those results suggest that miR-133 also constitutes a crucial linkage in the crosstalk between Fgf8 and Bmp2 signaling by regulating the Fgf8/ERK pathway during cardiac induction.

  1. Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch

    Directory of Open Access Journals (Sweden)

    Warren O. Haggard

    2011-08-01

    Full Text Available The objective of this study was to evaluate the osteoinductivity of composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pouch model. Previous in vitro characterization demonstrated the ability of the scaffolds to promote bone regeneration and as a carrier for local delivery of BMP-2. Composite microspheres were prepared using a co-precipitation method, and scaffolds were fabricated using an acid wash to adhere beads together. To determine the in vivo osteoinductivity of the scaffolds, the following groups (n = 6 were implanted into muscle pouches created in the latissimus dorsi of Sprague Dawley rats: (A lyophilized scaffolds without rhBMP-2, (B lyophilized scaffolds with rhBMP-2, (C non-lyophilized scaffolds with rhBMP-2, and (D absorbable collagen sponge with rhBMP-2 (control. Groups B, C, and D were loaded with 4 mL of a 9.0 μg/mL solution of rhBMP-2 for 48 h. The rats were sacrificed after one month and samples were analyzed for amount of residual implant material, new bone, and osteoid. Although the experimental groups displayed minimal degradation after one month, all of the scaffolds contained small amounts of woven bone and considerable amounts of osteoid. Approximately thirty percent of the open space available for tissue ingrowth in the scaffolds contained new bone or osteoid in the process of mineralization. The ability of the composite scaffolds (with and without BMP-2 to promote ectopic bone growth in vivo was demonstrated.

  2. Effect of rhBMP-2 sustained-release nanocapsules on the ectopic osteogenesis process in Sprague-Dawley rats

    Institute of Scientific and Technical Information of China (English)

    Ren-Fa Lai; Ze-Jian Li; Zhi-Ying Zhou; Zhi-Qiang Feng; Qing-Tong Zhao

    2013-01-01

    Objective:To explore the effect of sustained-release recombinant human bone morphogenetic protein-2(rhBMP-2) on ectopic osteogenesis in the muscle pouches of rats through preparing rhBMP-2 sustained-release capsules by wrapping morphogenesis protein bones-2(BMP-2) using chitosan nanoparticles, and compositing collagen materials.Methods:Twenty fourSprague-Dawley rats were randomly divided into four groups with six rats in each group, that isGroupA (control group),GroupB(only treated with collagen),GroupC(rhBMP-2+collagen treated group) andGroupD(rhBMP-2/cs+collagen treated group).The composite materials for each group were implanted in the bilateral peroneal muscle pouches in rats.The peroneal muscles were only separated without implanting any materials in control group.Rats were sacrificed2 weeks and4 weeks post treatment and samples were cut off for general observation,MicroCT scans and histological observation.Results:General observation showed no new bone formation in GroupsA andB mice, while new bones were formed inGroupsC andD mice.Two weeks after treatmentMicroCT scans showed thatThe bone volume fraction(BVF), trabecular thickness(Tb. Th), bone mineral density(BMD) inGroupC mice were all higher than that inGroupD(P<0.05). At the fourth week, theBVF,Tb.Th andBMD were significantly higher than that at the second week(P<0.01).Conclusions:The slow-release effect of rhBMP-2/cs sustained-release capsules can significantly promote ectopic osteogenesis.Its bone formation effect is better than that of rhBMP-2 burst-release group.

  3. BMP2作用下人牙乳头细胞内Smad1 mRNA表达的变化%Smad1 mRNA expression and modulation in the human dental papilla cells treated by BMP2

    Institute of Scientific and Technical Information of China (English)

    何文喜; 牛忠英; 赵守亮; 陈健

    2001-01-01

    目的:观察人牙乳头细胞内Smad1 mRNA 的表达及在BMP2作用下,细胞内Smad1 mRNA的表达变化,探讨人牙乳头细胞内Smad1 信号途径在BMP2调控牙乳头细胞分化中的作用.方法:原代培养的人牙乳头细胞用BMP2处理后,提取总RNA,采用Northern blot法,从mRNA水平观察Smad1基因的表达及含量变化.结果:从mRNA水平观察到Smad1基因在人牙乳头细胞内的表达,但在BMP2作用6、12、24h后,Smad1 mRNA表达量无显著变化.结论:人牙乳头细胞内存在Smad1信号转导途径,牙乳头细胞内Sma d1 mRNA表达量不受BMP2调控.

  4. Divergent palate morphology in turtles and birds correlates with differences in proliferation and BMP2 expression during embryonic development.

    Science.gov (United States)

    Abramyan, John; Leung, Kelvin Jia-Mien; Richman, Joy Marion

    2014-02-01

    During embryonic development, amniotes typically form outgrowths from the medial sides of the maxillary prominences called palatal shelves or palatine processes. In mammals the shelves fuse in the midline and form a bony hard palate that completely separates the nasal and oral cavities. In birds and lizards, palatine processes develop but remain unfused, leaving a natural cleft. Adult turtles do not possess palatine processes and unlike other amniotes, the internal nares open into the oral cavity. Here we investigate craniofacial ontogeny in the turtle, Emydura subglobosa to determine whether vestigial palatine processes develop and subsequently regress, or whether development fails entirely. We found that the primary palate in turtles develops similarly to other amniotes, but secondary palate ontogeny diverges. Using histology, cellular dynamics and in situ hybridization we found no evidence of palatine process development at any time during ontogeny of the face in the turtle. Furthermore, detailed comparisons with chicken embryos (the model organism most closely related to turtles from a molecular phylogeny perspective), we identified differences in proliferation and gene expression patterns that correlate with the differences in palate morphology. We propose that, in turtles, palatine process outgrowth is never initiated due to a lack of mesenchymal bone morphogenetic protein 2 (BMP2) expression in the maxillary mesenchyme, which in turn fails to induce the relatively higher cellular proliferation required for medial tissue outgrowth. It is likely that these differences between turtles and birds arose after the divergence of the lineage leading to modern turtles.

  5. Osteogenesis differentiation of human periodontal ligament cells by CO2 laser-treatment stimulating macrophages via BMP2 signalling pathway

    Science.gov (United States)

    Hsieh, Wen-Hui; Chen, Yi-Jyun; Hung, Chi-Jr; Huang, Tsui-Hsien; Kao, Chia-Tze; Shie, Ming-You

    2014-11-01

    Immune reactions play an important role in determining the biostimulation of bone formation, either in new bone formation or inflammatory fibrous tissue encapsulation. Macrophage cell, the important effector cells in the immune reaction, which are indispensable for osteogenesis and their heterogeneity and plasticity, render macrophages a primer target for immune system modulation. However, there are very few studies about the effects of macrophage cells on laser treatment-regulated osteogenesis. In this study, we used CO2 laser as a model biostimulation to investigate the role of macrophage cells on the CO2 laser stimulated osteogenesis. Bone morphogenetic protein 2 (BMP2) was also significantly up regulated by the CO2 laser stimulation, indicating that macrophage may participate in the CO2 laser stimulated osteogenesis. Interestingly, when laser treatment macrophage-conditioned medium were applied to human periodontal ligament cells (hPDLs), the osteogenesis differentiation of hPDLs was significantly enhanced, indicating the important role of macrophages in CO2 laser-induced osteogenesis. These findings provided valuable insights into the mechanism of CO2 laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment.

  6. Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2

    DEFF Research Database (Denmark)

    Zou, Xuenong; Li, Haisheng; Chen, Li

    2004-01-01

    In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex) and recombinant human bone morphogenic...... protein-2 (rhBMP-2). Cellular proliferation was determined by 3H-thymidine incorporation into DNA at both Days 2 and 7 when BMSc was cultivated with HY at concentrations of 0, 0.5, 1.0, 2.0 and 4.0 mg/ml. HY accelerated cellular proliferation when compared with cultures in the absence of HY at both Days 2...... and 7. BMSc proliferation under the high HY concentration of 4 mg/ml was significantly higher than under the other, lower HY concentrations of 0.5, 1.0 and 2.0 mg/ml. When BMSc were cultivated under HY at concentrations of 0, 1.0 and 4.0 mg/ml and its 12 combinations with rhBMP-2 at concentrations of 0...

  7. Linkage of osteoporosis to chromosome 20p12 and association to BMP2.

    Directory of Open Access Journals (Sweden)

    Unnur Styrkarsdottir

    2003-12-01

    Full Text Available Osteoporotic fractures are a major cause of morbidity and mortality in ageing populations. Osteoporosis, defined as low bone mineral density (BMD and associated fractures, have significant genetic components that are largely unknown. Linkage analysis in a large number of extended osteoporosis families in Iceland, using a phenotype that combines osteoporotic fractures and BMD measurements, showed linkage to Chromosome 20p12.3 (multipoint allele-sharing LOD, 5.10; p value, 6.3 x 10(-7, results that are statistically significant after adjusting for the number of phenotypes tested and the genome-wide search. A follow-up association analysis using closely spaced polymorphic markers was performed. Three variants in the bone morphogenetic protein 2 (BMP2 gene, a missense polymorphism and two anonymous single nucleotide polymorphism haplotypes, were determined to be associated with osteoporosis in the Icelandic patients. The association is seen with many definitions of an osteoporotic phenotype, including osteoporotic fractures as well as low BMD, both before and after menopause. A replication study with a Danish cohort of postmenopausal women was conducted to confirm the contribution of the three identified variants. In conclusion, we find that a region on the short arm of Chromosome 20 contains a gene or genes that appear to be a major risk factor for osteoporosis and osteoporotic fractures, and our evidence supports the view that BMP2 is at least one of these genes.

  8. The Enhancement of Osteogenesis by Scaffold Based on Mineralized Recombinant Human-like Collagen Loading with rhBMP-2

    Institute of Scientific and Technical Information of China (English)

    WU Bin; ZHENG Qixin; GUO Xiaodong; WU Yongchao; WANG Yu; CUI Fuzai

    2009-01-01

    A biomimetic scaffold based on mineralized recombinant collagen,nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid)(nHA/RHLC/PLA),was prepared with recombinant human bone morphogenic protein-2(rhBMP-2)for improving the os-teoinductive property of the scaffold.The nHA/RHLC/PLA scaffolds loaded with 10μg rhBMP-2 and the unloaded scaffolds were implanted subcutaneously in the rat model.The osteogenetic capacity of these composites was evaluated by CT scan,ALP activity test and histological observation at 4 and 8 weeks after implantation.The experimental results indicated that the osteogenic capability of the scaffolds loaded with rhBMP-2 was superior to the unloaded scaffold.It was concluded that rhBMP-2 can enhance the osteoinductive property of the nHA/RHLC/PLA scaffold and the nHA/RHLC/PLA scaffold loaded with rhBMP-2 have the good potential of being used in bone tissue engineering.

  9. 腺病毒介导BMP2和BMP7基因共转染骨髓间充质干细胞表达人BMP2/7异源二聚体%The feasibility of adenoviral co-transduction of BMP2 and BMP7 for the expression of recombinant human BMP2/7 heterodimer in rat bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    高涛; 唐胜建; 牛常英; 苗春雷; 张伟; 周陆陆; 吴彩风; 刘方军

    2016-01-01

    Objective To investigate the feasibility of rat bone marrow mesenchymal stem cells (BMSCs) as the target cell of adenovirus-mediated co-transduction of BMP2 and BMP7 genes and then facilitate the expression of recombinant BMP2/7 heterodimer protein.Methods 3 adult male Fischer 344 rats of about 10 weeks of age were used for harvest and in vitro culture of rat BMSCs.Recombinant adenovirus vector carrying BMP2 or BMP7 target genes were constructed with AdMax vector system,and production of high-titer adenoviruses were packaged with HEK293T cells and then concentrated with CSCl2 density-gradient ultra-centrifugation.Rat BMSCs from passage 3 were seeded in 6-well plates at the concentration of 10 000 cells/cm2.After overnight pre-culture,BMSCs were allowed to culture in 200 μl serum-free alpha MEM containing both Ad-BMP2 and Ad-BMP7 adenovirus (100 MOI of each virus).After 7 days in vitro culture,conditioned cell culture supernatants were collected and followed by immunoprecipitation through immune protein G columns pre-loaded with mouse anti-human BMP7 antibody.The resulted protein immune-precipitates were used to assay the expression of BMP2/7 heterodimers via Western Blot and ELISA assay.As a negative control,Rat BMSCs were also genetically transduced with Ad-GFP virus at a concentration of 200 MOI.Results Our data demonstrated that recombinant adenoviruses carrying BMP2 or BMP7 target gene was successfully reconstructed,packaged,and confirmed via Western Blot assay,which as respected,presented as an unique band at 55 000 size for BMP2 or 49 000 size for BMP7.Adenovirus Ad-GFP was used to verify the integrity of recombinant virus and its transfection efficiency in rat BMSCs,which showed well cell attachment to culture plate and had no cytotoxicity.Green fluorescent protein in BMSCs was also noted eminently under fluorescent microscope.Combined transduction with AdBMP2 plus Ad-BMP7 resulted in the formation of BMP2/7 heterodimers from rat BMSCs.Analysis of

  10. Effects of rhBMP-2 on Mandibular Distraction Osteogenesis and its OPG Expression in Rabbits%rhBMP-2对兔下颌骨牵引成骨区骨保护素表达的影响

    Institute of Scientific and Technical Information of China (English)

    周蕊; 付颖; 李新

    2011-01-01

    目的:通过动物实验,研究应用外源性rhBMP-2对兔下颌骨牵引成骨区骨保护素( osteoprotegerin,OPG)的影响.方法:在48只成年大耳白兔的一侧下颌骨前部行骨切开术,分别将空白胶原、rhBMP-2 1.5 mg胶原复合物植入下颌骨切开处,用牵引器延长一侧下颌骨4 mm,稳定期第1、3、7、14天,分别处死各组动物,取牵引区新生骨痂行组织学及OPG免疫组化染色.结果:下颌牵引延长后牵引间隙均有新骨形成,应用rhBMP-2 1.5mg效果好.免疫组化染色OPG主要定位于成骨细胞的胞浆中.在同一时间内,应用rhBMP-2组较对照组有显著性差异(P<0.05).结论:动物实验表明,rhBMP-2能促进兔下颌骨牵引成骨区新骨的生成.%Objective: To investigate the effects of rhBMP- 2 on mandibular distraction osteogenesis and the expression of OPG in the period of distraction osteogenesis. Methods: Unilateral mandibular osteotomies were performed in 48 mature rabbit. rhBMP - 2 1. 5mg with the collagen carrier was implanted in the osteotomy side of mandibles. Only collagen sponge was implanted in the control group. The mandibles of 48 rabbits were lengthened by 4 mm using a distractor , The animals were killed on 1,3,7,14 days of consolidation period. The distracted calluses were harvested and processed for histological and immunohistochemistry study of OPG. Results: The regenerated bone was found in the distraction gap after mandibular lengthening. But the rhBMP-2 was better. Staining for OPG was localized in osteoblasts of the periosteal region after completion of distraction. The mandibular side treated with rhBMP-2 had greater amount of new bone formation than that treated with collagen sponge. OPG was found in the cytoplasm of osteoblasts and stained brown or dark brown by immunohistochemistry. At the same time, OPG expression indicated that there was siganificant difference between rhBMP-2 group and control group(P<0. 05). Conclusion: rhBMP -2 could accelerate

  11. Early cellular responses of BMSCs genetically modified with bFGF/BMP2 co-cultured with ligament fibroblasts in a three-dimensional model in vitro.

    Science.gov (United States)

    Li, Bin; Jha, Ramesh Kumar; Qi, Yong-Jian; Ni, Qu-Bo; Wang, Hui; Chen, Biao; Chen, Liao-Bin

    2016-11-01

    Currently, a number of strategies including the implantation of bone marrow-derived mesenchymal stem cells (BMSCs) and growth factors have been developed to regenerate the tendon-to-bone interface after performing anterior cruciate ligament reconstruction. However, the mechanisms behind the interactions of the implanted BMSCs and tendon cells remain to be elucidated. The aim of this study was to evaluate the early cellular responses of BMSCs genetically modified with basic growth factor growth factor (bFGF)/bone morphogenic protein 2 (BMP2) and ligament fibroblasts in a three-dimensional co-culture model. BMSCs and ligament fibroblasts were both isolated from male Wistar rats. The BMSCs were then transfected with an adenoviral vector carrying bFGF or BMP2. The transfected BMSCs and ligament fibroblasts both encapsulated in alginate beads were co-cultured for 6 days in three-dimensional model. On days 0, 3 and 6, cell proliferation was assayed. On day 6, the expression of several tendon-bone related markers was evaluated. In the co-culture system, bFGF and BMP2 were highly expressed at the mRNA and protein level. During the process, bFGF significantly promoted cell proliferation, as well as the expression of scleraxis (SCX) and collagen (COL) type Ⅰ (COL1) in the BMSCs; however, it markedly decreased the expression of phenotype markers in the ligament fibroblasts, including COL1 and COL3. BMP2 markedly increased the expression of alkaline phosphatase and osteocalcin in the BMSCs and ligament fibroblasts, whereas it had no obvious effect on cell proliferation and collagen synthesis in the ligament fibroblasts. The combination of bFGF and BMP2 resulted in the similarly enhanced proliferation of BMSCs and ligament fibroblasts as observed with bFGF alone; however, this combination more potently promoted osteogenic differentiation than did BMP2 alone. The findings of our study demonstrate the superiority of the combined use of growth factors in inducing

  12. 实验性骨延长区BMP2的基因表达和细胞定位%Expression and localization of BMP2 gene in the experimental bone lengthening area

    Institute of Scientific and Technical Information of China (English)

    阮默; 邹培; 李峻辉; 林月秋; 周中英; 李主一

    2006-01-01

    目的:了解骨延长过程中BMP2基因在骨延长区组织细胞的表达和定位,从分子水平探讨延长区骨修复的机制.方法:采用兔胫骨上干骺端截骨延长动物模型,24只新西兰大白兔分8个时相点取材.通过延长区组织石蜡切片,BMP2地高辛标记的mRNA探针原位杂交.结果:BMP2 mRNA阳性表达持续到延长结束后20d,表达主要定位于延长区假性生长板中的成纤维细胞样间充质细胞、成纤维细胞、骨膜生发层的骨形成细胞及骨小梁边缘的成骨细胞,并随着骨延长不同阶段而有所变化.达到预定延长长度20d以后,延长区BMP2 mRNA表达逐渐转为阴性.结论:骨延长区BMP2 mRNA的表达和细胞定位与骨折愈合不同.BMP2在延长区高水平、长时效的表达对其骨修复具有重要作用,使延长区骨组织能够持续形成,短时间内以骨再生形式修复大段延长间隙.

  13. Construction and bio-activity of the chimeric protein of BMP2-EGFP%BMP2-EGFP融合蛋白的构建及其生物活性

    Institute of Scientific and Technical Information of China (English)

    张银刚; 郭雄; 师常宏; 邹爱民; 许鹏

    2005-01-01

    目的:构建BMP2/pLEGFP重组逆转录病毒表达载体,并检测其表达的融合蛋白的生物活性.方法:用基因重组技术构建BMP2/pLEGFP重组逆转录病毒表达载体,并通过酶切和PCR鉴定.脂质体法转染COS-7细胞,用荧光显微镜检测和Western blotting分析其在COS-7细胞表达,细胞活性实验和动物体内异位成骨实验进一步检测融合蛋白的生物活性.结果:经酶切和PCR鉴定重组质粒BMP2/pLEGFP构建成功.转染COS-7细胞后,荧光显微镜和Western blotting检测均显示融合蛋白在细胞中表达;分泌的产物经细胞活性实验和动物体内异位成骨实验证实具有BMP2和EGFP的双重蛋白活性.结论:基因重组技术成功构建BMP2/pLEGFP重组体,重组体表达的融合蛋白具有GFP和BMP2的双重活性.

  14. BMP2-coprecipitated calcium phosphate granules enhance osteoinductivity of deproteinized bovine bone, and bone formation during critical-sized bone defect healing.

    Science.gov (United States)

    Liu, Tie; Zheng, Yuanna; Wu, Gang; Wismeijer, Daniel; Pathak, Janak L; Liu, Yuelian

    2017-01-31

    Most materials used clinically for filling critical-sized bone defects (CSBD), such as deproteinized bovine bone (DBB), lack osteoinductivity so that their therapeutic effects are far from satisfactory. The effect of bone morphogenic protein 2 (BMP2)-coprecipitated biomimetic calcium phosphate granules (BMP2-cop.BioCaP) on osteoinduction of DBB graft(s) during CSBD healing is still unknown. We investigated whether BMP2-cop.BioCaP affects the osteoinductivity of DBB, bone formation, and foreign body reaction during CSBD healing. DBB + BMP2-cop.BioCaP, DBB, DBB + BMP2, DBB + BioCaP, and autologous bone grafts were implanted in the CSBD of sheep. Bone formation, DBB/BioCaP degradability, foreign body reaction, and osteoinductivity of DBB were analyzed histologically and histomorphometrically at week 4 and 8. Combination of BMP2-cop.BioCaP and DBB healed CSBD as effectively as autologous bone grafts. About 95% of the BMP2-cop.BioCaP had been degraded and replaced by new bone at week 8 in the DBB + BMP2-cop.BioCaP-group. Foreign body reaction was reduced in the DBB + BMP2-cop.BioCaP-group compared to the other groups. The independent use of the BMP2-cop.BioCaP did not achieve a satisfactory bone repair. In conclusion, the BMP2-cop.BioCaP showed good degradability and biocompatibility, and enhanced osteoinductivity of DBB during CSBD healing in sheep, suggesting BMP2-cop.BioCaP as a potential osteoinducer to enhance the therapeutic effects of the graft materials in clinic.

  15. BMP2基因修饰犬脂肪源性基质细胞修复自体大段骨缺损%Repairing canine segmental bone defects using BMP2 gene modified adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    李慧武; 戴尅戎; 汤亭亭; 张晓玲; 唐坚; 孙晓江; 张双燕; 楼觉人

    2008-01-01

    Objective To evaluate osteogenetic effectiveness of porous β-tricalcium phosphate(β-TCP) ceramic mixed with human bone morphogenetic protein2 gene(Adv-hBMP2)modified adipose derived stromal cells (ADSCs) in the repair of critical-sized bone defects..Methods The ADSCs taken from the back of beagle dogs were modified by the BMP2 gene.The expression and bone-induction ability of BMP2 was identified by ELISA and ectopic bone formation in nude mice.The cells were applied to a β-tricalcium phosphate (TGP)carrier and implanted into ulnar bone defects in the canine model.18 ulnar bone defects were divided into three groups randomly and filled with granular TCP alone,granular TCP and ADSCs,or TCP and ADSCs transduced with Adv-hBMP2 respectively.All dogs were followed clinically and roentgenographically for 16 weeks and then sacrificed.Results ELISA and ectopic bone formation in nude mice showed the recombinant ADSCs could express BMP2 highly and stably.No bone defects healed after implanting granular TCP alone or granular TCP and ADSCs.In the TCP and ADSCs transduced with AdvhBMP2 group,two defects healed,four partly healed.Histological examination showed woven bone at the both end of the cortices but entirelv fibrous tissue in the middle in which defects filled with TCP alone or TCP and ADSCs.Defects filled with TCP and transduced ADSCs showed substatial new bone formation.Histomorphometry showed TCP combined with ADSCs did not significantly increase new bone area compared with TCP alone.TCP and recombinant ADSCs produced a significant increase in newly formed bone area.Conclusion ADSCs tansduced with BMP2 gene in a TCP carrier can enhance bone regeneratmn to repair the critically-sized bone defect.%目的 评价BMP2基因修饰的犬脂肪源性基质细胞(ADSCs)与β-磷酸三钙(β-TCP)复合修复自体大段骨缺损的疗效.方法 从比格犬背部脂肪组织中提取基质细胞,转染腺病毒介导的人BMP2基因(Adv-hBMP2),通过ELISA和裸鼠体内异位成骨实验鉴定BMP

  16. Comparison of rhBMP-2 versus Autogenous Iliac Crest Bone Graft for 2-Level Anterior Cervical Discectomy and Fusion for Cervical Spondylotic Myelopathy

    OpenAIRE

    2015-01-01

    Background Few studies have examined the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) in 2-level anterior cervical discectomy and fusion (ACDF). The purpose of this study was to compare the outcomes in a series of patients with CSM treated with 2-level ACDF with or without rhBMP-2. Material/Methods The retrospective study included a total of 146 patients with CSM. The rhBMP-2 group consisted of 73 patients who underwent 2-level ACDF with rhBMP-2. A total of 73 patients...

  17. Bicomponent Fibrous Scaffolds Made through Dual-source Dual-power Electrospinning: Dual Delivery of rhBMP-2 and Ca-P Nanoparticles and Enhanced Biological Performances.

    Science.gov (United States)

    Wang, Chong; Weijia Lu, William; Wang, Min

    2017-04-05

    Electrospun scaffolds incorporated with both calcium phosphates (Ca-P) and bone morphogenetic protein-2 (BMP-2) have been used for bone tissue regeneration. However, in most cases BMP-2 and Ca-P were simply mixed and loaded in a monolithic structure, risking low BMP-2 loading level, reduced BMP-2 biological activity, uncontrolled BMP-2 release and inhomogeneous Ca-P distribution. In this investigation, novel bicomponent scaffolds having evenly distributed rhBMP-2-containing fibers and Ca-P nanoparticle-containing fibers were made using an established dual-source dual-power electrospinning technique with the assistance of emulsion electrospinning and blend electrospinning. The release behaviour of rhBMP-2 and Ca(2+) ions could be separately tuned and the released rhBMP-2 retained a 68% level for biological activity. MC3T3-E1 cells showed high viability and normal morphology on scaffolds. Compared to monocomponent scaffolds, enhanced cell proliferation, alkaline phosphatase activity, cell mineralization and gene expression of osteogenic markers were achieved for bicomponent scaffolds due to the synergistic effect of rhBMP-2 and Ca-P nanoparticles. Bicomponent scaffolds with a double mass elicited further enhanced cell adhesion, spreading, proliferation and osteogenic differentiation. This article is protected by copyright. All rights reserved.

  18. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

    Science.gov (United States)

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E; Ginzburg, Yelena Z

    2016-03-01

    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

  19. Dynamics and cellular localization of Bmp2, Bmp4, and Noggin transcription in the postnatal mouse skeleton.

    Science.gov (United States)

    Pregizer, Steven K; Mortlock, Douglas P

    2015-01-01

    Transcription of BMPs and their antagonists in precise spatiotemporal patterns is essential for proper skeletal development, maturation, maintenance, and repair. Nevertheless, transcriptional activity of these molecules in skeletal tissues beyond embryogenesis has not been well characterized. In this study, we used several transgenic reporter mouse lines to define the transcriptional activity of two potent BMP ligands, Bmp2 and Bmp4, and their antagonist, Noggin, in the postnatal skeleton. At 3 to 4 weeks of age, Bmp4 and Noggin reporter activity was readily apparent in most cells of the osteogenic or chondrogenic lineages, respectively, whereas Bmp2 reporter activity was strongest in terminally differentiated cells of both lineages. By 5 to 6 months, activity of the reporters had generally abated; however, the Noggin and Bmp2 reporters remained remarkably active in articular chondrocytes and persisted there indefinitely. We further found that endogenous Bmp2, Bmp4, and Noggin transcript levels in postnatal bone and cartilage mirrored the activity of their respective reporters in these tissues. Finally, we found that the activity of the Bmp2, Bmp4, and Noggin reporters in bone and cartilage at 3 to 4 weeks could be recapitulated in both osteogenic and chondrogenic culture models. These results reveal that Bmp2, Bmp4, and Noggin transcription persists to varying degrees in skeletal tissues postnatally, with each gene exhibiting its own cell type-specific pattern of activity. Illuminating these patterns and their dynamics will guide future studies aimed at elucidating both the causes and consequences of aberrant BMP signaling in the postnatal skeleton.

  20. BMP2 genetically engineered MSCs and EPCs promote vascularized bone regeneration in rat critical-sized calvarial bone defects.

    Directory of Open Access Journals (Sweden)

    Xiaoning He

    Full Text Available Current clinical therapies for critical-sized bone defects (CSBDs remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2 gene-modified MSCs and endothelial progenitor cells (EPCs could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

  1. Safety and Efficacy of Bioabsorbable Cervical Spacers and Low-Dose rhBMP-2 in Multi-Level ACDF

    OpenAIRE

    2014-01-01

    Introduction Many options for interbody spacer and graft biologic exist for multilevel anterior cervical discectomy and fusion (ACDF). The objective of this study is to evaluate the safety and efficacy of a bioabsorbable cervical spacer (BCS) (Cornerstone HSR, Medtronic Sofamor Danek) filled with low-dose rhBMP-2 (INFUSE, Medtronic Sofamor Danek) in multilevel ACDF. Methods 72 consecutive patients treated with a multi-level ACDF using BCS and rhBMP-2 (dosage between 0.5 to 0.7 mg per level) a...

  2. Dual Delivery of EPO and BMP2 from a Novel Modular Poly-ɛ-Caprolactone Construct to Increase the Bone Formation in Prefabricated Bone Flaps.

    Science.gov (United States)

    Patel, Janki Jayesh; Modes, Jane E; Flanagan, Colleen L; Krebsbach, Paul H; Edwards, Sean P; Hollister, Scott J

    2015-09-01

    Poly-ɛ-caprolactone (PCL) is a biocompatible polymer that has mechanical properties suitable for bone tissue engineering; however, it must be integrated with biologics to stimulate bone formation. Bone morphogenetic protein-2 (BMP2) delivered from PCL produces bone when implanted subcutaneously, and erythropoietin (EPO) works synergistically with BMP2. In this study, EPO and BMP2 are adsorbed separately on two 3D-printed PCL scaffold modules that are assembled for codelivery on a single scaffold structure. This assembled modular PCL scaffold with dual BMP2 and EPO delivery was shown to increase bone growth in an ectopic location when compared with BMP2 delivery along a replicate scaffold structure. EPO (200 IU/mL) and BMP2 (65 μg/mL) were adsorbed onto the outer and inner portions of a modular scaffold, respectively. Protein binding and release studies were first quantified. Subsequently, EPO+BMP2 and BMP2 scaffolds were implanted subcutaneously in mice for 4 and 8 weeks, and the regenerated bone was analyzed with microcomputed tomography and histology; 8.6±1.4 μg BMP2 (22%) and 140±29 IU EPO (69.8%) bound to the scaffold and EPO was released in 7 days. Increased endothelial cell proliferation on EPO-adsorbed PCL discs indicated protein bioactivity. At 4 and 8 weeks, dual BMP2 and EPO delivery regenerated more bone (5.1±1.1 and 5.5±1.6 mm(3)) than BMP2 alone (3.8±1.1 and 4.3±1.7 mm(3)). BMP2 and EPO scaffolds had more ingrowth (1.4%±0.6%) in the outer module when compared with BMP2 (0.8%±0.3%) at 4 weeks. Dual delivery produced more dense cellular marrow, while BMP2 had more fatty marrow. Dual EPO and BMP2 delivery is a potential method to regenerate bone faster for prefabricated flaps.

  3. Reduction of Adipose Tissue Formation by the Controlled Release of BMP-2 Using a Hydroxyapatite-Coated Collagen Carrier System for Sinus-Augmentation/Extraction-Socket Grafting

    Directory of Open Access Journals (Sweden)

    Jung-Seok Lee

    2015-11-01

    Full Text Available The effects of hydroxyapatite (HA-coating onto collagen carriers for application of recombinant human bone morphogenetic protein 2 (rhBMP-2 on cell differentiation in vitro, and on in vivo healing patterns after sinus-augmentation and alveolar socket-grafting were evaluated. In vitro induction of osteogenic/adipogenic differentiation was compared between the culture media with rhBMP-2 solution and with the released rhBMP-2 from the control collagen and from the HA-coated collagen. Demineralized bovine bone and collagen/HA-coated collagen were grafted with/without rhBMP-2 in sinus-augmentation and tooth-extraction-socket models. Adipogenic induction by rhBMP-2 released from HA-coated collagen was significantly reduced compared to collagen. In the sinus-augmentation model, sites that received rhBMP-2 exhibited large amounts of vascular tissue formation at two weeks and increased adipose tissue formation at eight weeks; this could be significantly reduced by using HA-coated collagen as a carrier for rhBMP-2. In extraction-socket grafting, dimensional reduction of alveolar ridge was significantly decreased at sites received rhBMP-2 compared to control sites, but adipose tissue was increased within the regenerated socket area. In conclusion, HA-coated collagen carrier for Escherichia coli-derived rhBMP-2 (ErhBMP-2 may reduce in vitro induction of adipogenic differentiation and in vivo adipose bone marrow tissue formation in bone tissue engineering by ErhBMP-2.

  4. 颌骨牙骨质化纤维瘤中BMP-2mRNA的表达%Expression of BMP-2mRNA in Cementifying Fibroma of the Jaw

    Institute of Scientific and Technical Information of China (English)

    黄宏杰; 平飞云; 彭加萍

    2006-01-01

    目的:检测颌骨牙骨质化纤维瘤(cementifying fibroma,CF)中BMP-2mRNA的表达,分析其与牙骨质小体及牙骨质样物质形成的关系.方法:采用原位杂交方法检测10例CF标本中BMP-2mRNA的表达.结果:10例CF标本中9例有BMP-2mRNA表达,阳性信号位于牙骨质小体及牙骨质样物质周围环绕的短梭形细胞和瘤样增生的纤维组织中的多数纤维母细胞和部分纤维细胞胞浆中.结论:BMP-2mRNA在CF中的表达可能与牙骨质小体及牙骨质样物质的形成有关.

  5. Expression of TGFβ1 and BMP-2 in the experimental ligamentum flavum of the unstable cervical spine%颈椎不稳黄韧带TGFβ1、BMP2表达的实验研究

    Institute of Scientific and Technical Information of China (English)

    张喜善; 蔡国栋; 李建民; 杨明峰

    2007-01-01

    目的 探讨颈椎不稳对于黄韧带中转化生长因子β1(TGFβ1)、骨形态发生蛋白2(BMP2)基因表达的影响及其意义.方法 采用逆转录聚合酶联反应技术(RT-PCR)对正常与颈椎不稳定动物模型的黄韧带中不同时间点TGFβ1、BMP2的表达水平进行测量比较.结果 在不同时间点,颈椎不稳组黄韧带中TGFβ1、BMP2的基因表达较正常黄韧带组均增高(P<0.01).结论 TGFβ1、BMP2在颈椎不稳大鼠黄韧带的表达在不同时间点有明显变化,这种变化可能在加速黄韧带的退变中起重要作用.

  6. BMP2 mRNA在大鼠再生肝组织的表达与意义%Expression of BMP2 mRNA in regenerated liver tissue of rats and its significance

    Institute of Scientific and Technical Information of China (English)

    冀文敏; 许翠萍

    2008-01-01

    目的:在基因水平上观察BMP2在肝组织的表达和肝再生过程中的动态变化特征,并结合NF-κB在肝再生过程中的表达变化特征,探讨BMP2对肝再生的影响.方法:健康成年♂Wistar大鼠54只,随机分为3组:正常对照(NC)组,即0 h组(n=6),假手术(SO)组(n=24),肝部分切除术(PH)组(n=24).采用原位杂交和免疫组化的方法分别测定大鼠再生肝脏组织中BMP2 mRNA和NF-κB的表达.结果:BMP2 mRNA表达在SO组与PH组6、12、24 h时间点组间存在统计学差异(灰度值:99.74+6.85 vs 114.41±5.12,130.59±6.74,113.74±7.32,均P<0.05).SO组与NC组无统计学意义.NC组肝组织内NF-κB阴性,SO组术后各时相点未见特异性染色.PH组NF-κB最初少量弱阳性表达,术后6、12、24 h,NF-κB表达逐步增强,灰度值降低(96.22±3.12,89.59±3.24,83.72±4.32),有统计学差异(P<0.05).结论:BMP2在正常大鼠肝组织中有表达且在肝再生过程中BMP2表达呈现先减弱后增强的趋势.NF-κB在正常大鼠肝脏组织中无表达,在肝再生过程中NFκB表达逐渐增强.BMP2可能抑制肝再生.

  7. Clinical significance of serum BMP2 and BMP4 in patients with chronic hepatitis virus infection.%检测慢性肝病患者血清BMP2和BMP4的临床价值

    Institute of Scientific and Technical Information of China (English)

    温彪; 许翠萍

    2012-01-01

    Objective To study the significance of serum bone morphogenetic protein 2 ( BMP2 ) and bone morphogenetic protein 4 ( BMP4 ) in diagnosis and differential diagnosis of primary hepatic carcinoma ( PHC ). Methods Blood samples were drawn from 60 inpatients. According to their pathological diagnosis or clinical diagnosis, they were divided into three groups: 25 in primary hepatic carcinoma group, 17 in hepatic cirrhosis group and 18 in chronic hepatitis group. The control blood samples were collected from 9 healthy persons in health examination department. The serum levels of AFP, BMP2 and BMP4 were detected in all these groups. Results (T) The serum levels of BMP2 in PHC patients were lower than those of normal controls and patients with chronic hepatitis and hepatic cirrhosis. ( P <0.05 ). (2) The serum levels of BMP4 in patients with PHC were lower than those in other groups. ( P <0.05 ). (3) The sensitivity, specificity and veracity of serum BMP2 in diagnosis of PHC were 92.00% , 100% and 97.10% respectively. The sensitivity, specificity and veracity of serum BMP4 in diagnosis of PHC were 80.00% , 100% and 92.75% respectively. Conclusion There is certain value of serum BMP2 and BMP4 in diagnosis and differential diagnosis of PHC.%目的 研究慢性肝病患者血清中的骨形态发生蛋白2(BMP2)和骨形态发生蛋白4(BMP4)的表达,了解两者在诊断和鉴别诊断原发性肝癌(PHC)中的临床价值.方法 按照病理学诊断或者临床资料诊断,对住院的60例患者进行分组,PHC组25例,肝硬化组17例,肝炎组18例.9例健康对照组采自同期健康体检者.分别检测各组患者及正常者血清甲胎蛋白(AFP)、BMP2和BMP4.结果 ①PHC组患者血清中的BMP2较健康对照组、肝炎组、肝硬化组低,差异都有统计学意义(P<0.05).②PHC组患者血清BMP4较其余3组组低,差异都有统计学意义(P<0.01).③BMP2诊断PHC的敏感性为92.00%,特异性为100%,准确率是97.10%;BMP4

  8. 消骨痛汤对骨关节炎大鼠 TGFβ1和 BMP2表达观察%The observation of Xiaogutong decoction in TGFβ1 and BMP2 of rats with osteoarthritis

    Institute of Scientific and Technical Information of China (English)

    郝亚明; 胡永红

    2014-01-01

    目的:观察中药方剂消骨痛汤对骨关节炎(OA)大鼠模型中软骨和滑膜中转化生长因子β1(TGFβ1)和骨形成蛋白2(BMP2)的表达。方法将40只Wistar大鼠随机分为4组,每组10只。正常对照组不予干预,其余3组采用膝关节腔内注射木瓜蛋白酶的方法建立骨关节炎大鼠模型,造模2周后开始给药,分别给予生理盐水(模型组)、双氯芬酸钠(西药组)和中药消骨痛汤(中药组),灌胃8周后取软骨及滑膜标本。用免疫组化方法检测TGFβ1和BMP2在软骨和滑膜中的蛋白表达。结果模型组和西药组软骨、滑膜中TGFβ1、BMP2水平低于中药组和正常组,差异均有统计学意义(P<0.05)。中药组和正常组TGFβ1和BMP2水平差异无统计学意义(P>0.05)。结论消骨痛汤能上调OA大鼠软骨和滑膜中TGFβ1和BMP2的蛋白表达。%Objective To observe the influence of Xiaogutong decoction in TGFβ1 and BMP2 of rats with osteoarthri-tis.Methods 40 cases rats were randomly divided into 4 groups,each of 10 cases.Normal control group with no intervention. The remaining 3 groups to establish osteoarthritis rat model.Dosing in 2 weeks after modeling.Respectively treated by normal saline,diclofenac and Xiaogutong decoction.After 8 weeks,taken the cartilage and synovial samples.TGFβ1 and BMP2 were de-tected by immunohistochemistry.Compared the level of TGFβ1 and BMP2 of 4 groups.Results The level of TGFβ1 and BMP2 in model group and western medicine group were lower than that of normal group and chinese medicine group,the difference was statistically significant(P0.05).Conclusion Xiaogutong decoction can improve the expression of the TGFβ1 and BMP2 pro-tein of cartilage and synovial of OA.

  9. Simulated Microgravity Regulates Osteoblast BMP2-Smad Signaling via Actin Microfilament%模拟微重力效应通过微丝骨架调控成骨细胞BMP2-Smad信号

    Institute of Scientific and Technical Information of China (English)

    徐洪杰; 戴钟铨; 吴峰; 张洪玉; 杨超; 商澎; 李莹辉

    2014-01-01

    细胞微丝骨架在力信号传导和基因表达调控中起重要作用.为了研究微丝骨架在模拟微重力效应调控成骨细胞BMP2-Smad信号中的作用,作者通过构建反映Smad活性的报告基因载体转染MC3T3-E1细胞,并通过报告基因活性分析、Western blot等方法检测了微丝骨架解聚剂和回转模拟微重力效应对BMP2诱导Samd磷酸化、核质分布和转录活性的作用.结果显示,构建的报告基因载体在成骨细胞中正确表达并响应BMP2;破坏微丝骨架会抑制BMP2诱导的Smad1/5/8蛋白磷酸化、入核及转录活性;回转抑制Smad1/5/8磷酸化、入核及其转录活性,而微丝骨架稳定剂可对抗回转的抑制作用.因此,认为回转模拟微重力效应可通过解聚微丝骨架抑制BMP2-Smad信号传导.

  10. Critical assessment of rhBMP-2 mediated bone induction: an in vitro and in vivo evaluation

    NARCIS (Netherlands)

    Kisiel, M.; Ventura, M.; Oommen, O.P.; George, A.; Walboomers, X.F.; Hilborn, J.; Varghese, O.P.

    2012-01-01

    Understanding the influence of formulation and storage conditions on rhBMP-2 bioactivity is extremely important for its clinical application. Reports in the literature show that different research groups employ different parameters such as formulation conditions, storage, doses for in vivo applicati

  11. Combinatorial therapeutic targeting of BMP2 and MEK-ERK pathways in NF1-associated malignant peripheral nerve sheath tumors

    Science.gov (United States)

    Ahsan, Sidra; Ge, Yubin; Tainsky, Michael A.

    2016-01-01

    The clinical management of malignant peripheral nerve sheath tumors (MPNSTs) is challenging not only due to its aggressive and invasive nature, but also limited therapeutic options. Using gene expression profiling, our lab identified BMP2-SMAD1/5/8 pathway as a potential therapeutic target for treating MPNSTs. In this study, we explored the therapeutic impact of targeting BMP2-SMAD1/5/8 pathway in conjunction with RAS-MEK-ERK signaling, which is constitutively activated in MPNSTs. Our results indicated that single agent treatment with LDN-193189, a BMP2 Type I receptor inhibitor, did not affect the growth and survival of MPNST cells at biochemically relevant inhibitory concentrations. However, addition of a MEK1/2 inhibitor, selumetinib, to LDN-193189-treated cells resulted in significant inhibition of cell growth and induction of cell death. LDN-193189 at biochemically effective concentrations significantly inhibited motility and invasiveness of MPNST cells, and these effects were enhanced by the addition of selumetinib. Overall, our results advocate for a combinatorial therapeutic approach for MPNSTs that not only targets the growth and survival via inhibition of MEK1/2, but also its malignant spread by suppressing the activation of BMP2-SMAD1/5/8 pathway. Importantly, these studies were conducted in low-passage patient-derived MPNST cells, allowing for an investigation of the effects of the proposed drug treatments in a biologically-relevant context. PMID:27494873

  12. RAS/MEK-independent gene expression reveals BMP2-related malignant phenotypes in the Nf1-deficient MPNST.

    Science.gov (United States)

    Sun, Daochun; Haddad, Ramsi; Kraniak, Janice M; Horne, Steven D; Tainsky, Michael A

    2013-06-01

    Malignant peripheral nerve sheath tumor (MPNST) is a type of soft tissue sarcoma that occurs in carriers of germline mutations in Nf1 gene as well as sporadically. Neurofibromin, encoded by the Nf1 gene, functions as a GTPase-activating protein (GAP) whose mutation leads to activation of wt-RAS and mitogen-activated protein kinase (MAPK) signaling in neurofibromatosis type I (NF1) patients' tumors. However, therapeutic targeting of RAS and MAPK have had limited success in this disease. In this study, we modulated NRAS, mitogen-activated protein/extracellular signal-regulated kinase (MEK)1/2, and neurofibromin levels in MPNST cells and determined gene expression changes to evaluate the regulation of signaling pathways in MPNST cells. Gene expression changes due to neurofibromin modulation but independent of NRAS and MEK1/2 regulation in MPNST cells indicated bone morphogenetic protein 2 (Bmp2) signaling as a key pathway. The BMP2-SMAD1/5/8 pathway was activated in NF1-associated MPNST cells and inhibition of BMP2 signaling by LDN-193189 or short hairpin RNA (shRNA) to BMP2 decreased the motility and invasion of NF1-associated MPNST cells. The pathway-specific gene changes provide a greater understanding of the complex role of neurofibromin in MPNST pathology and novel targets for drug discovery.

  13. Combinatorial therapeutic targeting of BMP2 and MEK-ERK pathways in NF1-associated malignant peripheral nerve sheath tumors.

    Science.gov (United States)

    Ahsan, Sidra; Ge, Yubin; Tainsky, Michael A

    2016-08-30

    The clinical management of malignant peripheral nerve sheath tumors (MPNSTs) is challenging not only due to its aggressive and invasive nature, but also limited therapeutic options. Using gene expression profiling, our lab identified BMP2-SMAD1/5/8 pathway as a potential therapeutic target for treating MPNSTs. In this study, we explored the therapeutic impact of targeting BMP2-SMAD1/5/8 pathway in conjunction with RAS-MEK-ERK signaling, which is constitutively activated in MPNSTs. Our results indicated that single agent treatment with LDN-193189, a BMP2 Type I receptor inhibitor, did not affect the growth and survival of MPNST cells at biochemically relevant inhibitory concentrations. However, addition of a MEK1/2 inhibitor, selumetinib, to LDN-193189-treated cells resulted in significant inhibition of cell growth and induction of cell death. LDN-193189 at biochemically effective concentrations significantly inhibited motility and invasiveness of MPNST cells, and these effects were enhanced by the addition of selumetinib. Overall, our results advocate for a combinatorial therapeutic approach for MPNSTs that not only targets the growth and survival via inhibition of MEK1/2, but also its malignant spread by suppressing the activation of BMP2-SMAD1/5/8 pathway. Importantly, these studies were conducted in low-passage patient-derived MPNST cells, allowing for an investigation of the effects of the proposed drug treatments in a biologically-relevant context.

  14. Immunohistological Localization of BMP-2, BMP-7, and Their Receptors in Knee Joints with Focal Cartilage Lesions

    Directory of Open Access Journals (Sweden)

    Hagen Schmal

    2012-01-01

    Full Text Available Introduction. Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. Methods. Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. Results. BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P=0.002. BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P<0.02, but there was no influence of these parameters on protein expression. Conclusions. BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

  15. A novel BMP2-coprecipitated, layer-by-layer assembled biomimetic calcium phosphate particle: a biodegradable and highly efficient osteoinducer

    NARCIS (Netherlands)

    Zheng, Y.; Wu, G.; Liu, T.; Liu, Y.; Wismeijer, D.; Liu, Y.

    2014-01-01

    Purpose To repair large-size bone defects, most bone-defect-filling materials in clinic need to obtain osteoinductivity either by mixing them with particulate autologous bone or adsorbing bone morphogenetic protein 2 (BMP2). However, both approaches encounter various limitations. In this study, we h

  16. Immunohistological localization of BMP-2, BMP-7, and their receptors in knee joints with focal cartilage lesions

    DEFF Research Database (Denmark)

    Schmal, Hagen; Mehlhorn, Alexander T; Pilz, Ingo H

    2012-01-01

    , but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression...

  17. 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

  18. 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.

  19. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  20. BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

    Science.gov (United States)

    Yoo, Su-Hyang; Kim, Jae Goo; Kim, Beom-Su; Lee, Jun; Pi, Sung-Hee; Lim, Hyun-Dae; Shin, Hong-In; Cho, Eui-Sic; You, Hyung-Keun

    2016-01-01

    The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

  1. Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole

    Directory of Open Access Journals (Sweden)

    Byung-Chul Jeong

    2015-01-01

    Full Text Available Recently a submicron particle of biphasic calcium phosphate ceramic (BCP with through-hole (donut-shaped BCP (d-BCP was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM, energy dispersive spectroscopy (EDS, and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

  2. 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

    Directory of Open Access Journals (Sweden)

    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.

  3. Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole.

    Science.gov (United States)

    Jeong, Byung-Chul; Choi, Hyuck; Hur, Sung-Woong; Kim, Jung-Woo; Oh, Sin-Hye; Kim, Hyun-Seung; Song, Soo-Chang; Lee, Keun-Bae; Park, Kwang-Bum; Koh, Jeong-Tae

    2015-01-01

    Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

  4. rhBMP-2/CPC强化骨质疏松椎体力学强度的实验研究%The experimental study of the effect of rhBMP-2/CPC on biomechanical intensity for osteoporotic vertebrae

    Institute of Scientific and Technical Information of China (English)

    罗志强

    2011-01-01

    Objective To observe the dynamic efficacy of recombinant human bone morphogenetic protein-2/calcium phosphate cement ( rhBMP-2/CPC ) on the augment of biomechanical intensity of the lumbar vertebrae in ovariectomized sheep. Methods Twelve adult female sheep were ovariectomized and raised for 1 year after the surgery. Bone mineral densities ( BMD) of the lumbar vertebrae were measured before and after the surgery. L1-L6 were the experimental objects. The sheep in control group were offered no treatment. The sheep in CPC group were injected with CPC (2.0ml) via the transpedicular of the vertebra. The sheep in rhBMP-2/CPC group were injected with rhBMP-2/CPC (2. 0ml) via the transpedicular of the vertebra. Every 3 sheep were randomly killed on day 1, week 6, week 12, and week 24 after the surgery. The vertebra compression test was performed. The ultimate compressive stress ( σalt ) and energy absorption value ( EAV ) of the vertebrae in every group were measured. The biomechanical indexes were compared and analyzed among different methods at the same time and among different time points using same method. Results BMO of the sheep vertebrae significantly decreased at 1 year after the surgery ( P 0.05). However, aalt in rhBMP-2/CPC group was significantly higher than that in CPC group on week 24 ( P 0. 05). However, σalt in rhBMP-2/ CPC group on week 24 was significantly higher than that on other three time points in the same group ( P < 0. 05). Conclusion rhBMP-2/CPC not only improved the immediate mechanical strength of osteoporotic vertebrae, but also maintained the dynamic mechanical strength and further enhanced the lone-term mechanical strength of osteoporotic vertebrae, which could provide an ideal mechanical condition for firm bone fusion of the spine.%目的 观察人重组骨形态发生蛋白-2复合磷酸钙骨水泥(recombinant human bonemorphogenetic protein-2/calcium phosphate cement,rhBMP-2/CPC)强化骨质疏松绵羊腰椎生物力学

  5. BMP2/BMP4 colorectal cancer susceptibility loci in northern and southern European populations.

    Science.gov (United States)

    Fernandez-Rozadilla, Ceres; Palles, Claire; Carvajal-Carmona, Luis; Peterlongo, Paolo; Nici, Carmela; Veneroni, Silvia; Pinheiro, Manuela; Teixeira, Manuel R; Moreno, Victor; Lamas, Maria-Jesus; Baiget, Montserrat; Lopez-Fernandez, L A; Gonzalez, Dolors; Brea-Fernandez, Alejandro; Clofent, Juan; Bujanda, Luis; Bessa, Xavier; Andreu, Montserrat; Xicola, Rosa; Llor, Xavier; Jover, Rodrigo; Castells, Antoni; Castellvi-Bel, Sergi; Carracedo, Angel; Tomlinson, Ian; Ruiz-Ponte, Clara

    2013-02-01

    Genome-wide association studies have successfully identified 20 colorectal cancer susceptibility loci. Amongst these, four of the signals are defined by tagging single nucleotide polymorphisms (SNPs) on regions 14q22.2 (rs4444235 and rs1957636) and 20p12.3 (rs961253 and rs4813802). These markers are located close to two of the genes involved in bone morphogenetic protein (BMP) signaling (BMP4 and BMP2, respectively). By investigating these four SNPs in an initial cohort of Spanish origin, we found substantial evidence that minor allele frequencies (MAFs) may be different in northern and southern European populations. Therefore, we genotyped three additional southern European cohorts comprising a total of 2028 cases and 4273 controls. The meta-analysis results show that only one of the association signals (rs961253) is effectively replicated in the southern European populations, despite adequate power to detect all four. The other three SNPs (rs4444235, rs1957636 and rs4813802) presented discordant results in MAFs and linkage disequilibrium patterns between northern and southern European cohorts. We hypothesize that this lack of replication could be the result of differential tagging of the functional variant in both sets of populations. Were this true, it would have complex consequences in both our ability to understand the nature of the real causative variants, as well as for further study designs.

  6. Fabrication of Core-Shell PEI/pBMP2-PLGA Electrospun Scaffold for Gene Delivery to Periodontal Ligament Stem Cells

    Directory of Open Access Journals (Sweden)

    Qiao Xie

    2016-01-01

    Full Text Available Bone tissue engineering is the most promising technology for enhancing bone regeneration. Scaffolds loaded with osteogenic factors improve the therapeutic effect. In this study, the bioactive PEI (polyethylenimine/pBMP2- (bone morphogenetic protein-2 plasmid- PLGA (poly(D, L-lactic-co-glycolic acid core-shell scaffolds were prepared using coaxial electrospinning for a controlled gene delivery to hPDLSCs (human periodontal ligament stem cells. The pBMP2 was encapsulated in the PEI phase as a core and PLGA was employed to control pBMP2 release as a shell. First, the scaffold characterization and mechanical properties were evaluated. Then the gene release behavior was analyzed. Our results showed that pBMP2 was released at high levels in the first few days, with a continuous release behavior in the next 28 days. At the same time, PEI/pBMP2 showed high transfection efficiency. Moreover, the core-shell electrospun scaffold showed BMP2 expression for a much longer time (more than 28 days compared with the single axial electrospun scaffold, as evaluated by qRT-PCR and western blot after culturing with hPDLSCs. These results suggested that the core-shell PEI/pBMP2-PLGA scaffold fabricated by coaxial electrospinning had a good gene release behavior and showed a prolonged expression time with a high transfection efficiency.

  7. Proliferation and odontogenic differentiation of BMP2 gene‑transfected stem cells from human tooth apical papilla: an in vitro study.

    Science.gov (United States)

    Zhang, Wen; Zhang, Xiaolei; Ling, Junqi; Liu, Wei; Zhang, Xinchun; Ma, Jinglei; Zheng, Jianmao

    2014-10-01

    Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue‑derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral‑mediated BMP2 gene‑transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector‑transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK‑8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: ⅰ) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ⅱ) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; ⅲ) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro.

  8. Evaluation of rhBMP-2/collagen/TCP-HA bone graft with and without bone marrow cells in the canine femoral multi defect model.

    Science.gov (United States)

    Luangphakdy, V; Shinohara, K; Pan, H; Boehm, C; Samaranska, A; Muschler, G F

    2015-01-12

    Recombinant human bone morphogenetic protein-2, when applied to an absorbable type 1 bovine collagen sponge (rhBMP-2/ACS) is an effective therapy in many bone grafting settings. Bone marrow aspirate (BMA) has also been used as a source of transplantable osteogenic connective tissue progenitors. This study was designed to characterize the performance of a scaffold comprising rhBMP-2/ACS in which the sponge wraps around tri-calcium phosphate hydroxyapatite granules (rhBMP-2/ACS/TCP-HA) and to test the hypothesis that addition of BMA will improve the performance of this construct in the Canine Femoral Multi Defect Model. In each subject, two sites were grafted with rhBMP-2/ACS/TCP-HA scaffold loaded with BMA clot and two other sites with rhBMP-2/ACS/TCP-HA scaffold loaded with wound blood (WB). After correction for unresorbed TCP-HA granules, sites grafted with rhBMP-2/ACS/TCP-HA+BMA and rhBMP-2/ACS/TCP-HA+WB were similar, with mean percent bone volumes of 10.9 %±1.2 and 11.2 %±1.2, respectively. No differences were seen in quantitative histomorphometry. While bone formation using both constructs was robust, this study did not support the hypothesis that the addition of unprocessed bone marrow aspirate clot improved bone regeneration in a site engrafted with rhBMP-2/ACS/TCP-HA+BMA. In contrast to prior studies using this model, new bone formation was greater at the center of the defect where TCP-HA was distributed. This finding suggests a potential synergy between rhBMP-2 and the centrally placed ceramic and cellular components of the graft construct. Further optimization may also require more uniform distribution of TCP-HA, alternative cell delivery strategies, and a more rigorous large animal segmental defect model.

  9. Changes with Age and the Effect of Recombinant Human BMP-2 on Proteoglycan and Collagen Gene Expression in Rabbit Anulus Fibrosus Cells

    Institute of Scientific and Technical Information of China (English)

    Qin-Ming FEI; Xiao-Xing JIANG; Tong-Yi CHEN; Jun LI; Hideki MURAKAMI; Kai-Jow TSAI; William C. HUTTON

    2006-01-01

    In order to compare the difference between young and old intervertebral disc cells and their responsiveness to recombinant human bone morphogenetic protein-2 (rhBMP-2), disc cells were isolated from the anulus fibrosus (AF) and transition zones of lumbar discs from eight old and eight young New Zealand white rabbits. Compared with the cells from the young rabbits, cells from old rabbits respond less to rhBMP-2 treatment with respect to sulfated-glycosaminoglycan (sGAG) synthesis and aggrecan gene expression. But in collagen Ⅰ and collagen Ⅱ gene expressions, there are no significant differences between the old and the young. When comparing sGAG content, aggrecan, and collagen Ⅱ gene expression of the old AF cells after rhBMP-2 treatment with that of the young AF cells without rhBMP-2 treatment, the old AF cells with rhBMP-2 treatment have a greater capacity to synthesize sGAG bound in the cells and to release sGAG in the media, as well as to express aggrecan and collagen Ⅱ gene. It can be concluded that old AF cells after rhBMP-2 treatment have a greater capacity to synthesize sGAG and express aggrecan and collagen Ⅱ as compared to young AF cells without rhBMP-2 treatment. Thus rhBMP-2 can reverse the decline in the anabolic capacity of the disc cells with ageing. So it seems that rhBMP-2 has potential for use as an agent to retard a key component of disc degeneration and loss of disc matrix.

  10. 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

    Directory of Open Access Journals (Sweden)

    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.

  11. Effect of implantation of biodegradable magnesium alloy on BMP-2 expression in bone of ovariectomized osteoporosis rats

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Yue, E-mail: 373073766@qq.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Ren, Ling, E-mail: lren@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Liu, Chang, E-mail: meixifan1971@163.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Yuan, Yajiang, E-mail: yuan925@163.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Lin, Xiao, E-mail: linx@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Tan, Lili, E-mail: lltan@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Chen, Shurui, E-mail: 272146792@qq.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China); Yang, Ke, E-mail: kyang@imr.ac.cn [Institute of Metal Research, Chinese Academy of Sciences, 72 Wenhua Road, Shenyang, 110016 (China); Mei, Xifan, E-mail: meixifan1971@163.com [Liaoning Medical University, 40 Songpo Road, Jinzhou, 121000 (China)

    2013-10-01

    The study was focused on the implantation of a biodegradable AZ31 magnesium alloy into the femoral periosteal of the osteoporosis modeled rats. The experimental results showed that after 4 weeks implantation of AZ31 alloy in the osteoporosis modeled rats, the expression of BMP-2 in bone tissues of the rats was much enhanced, even higher than the control group, which should promote the bone formation and be beneficial for reducing the harmful effect of osteoporosis. Results of HE stains showed that the implantation of AZ31 alloy did not have obvious pathological changes on both the liver and kidney of the animal. - Highlights: • Mg alloy greatly increased expression of BMP-2 in osteoporosis modeled rat bone. • Mg alloy showed good biological safety. • Mg alloy is beneficial for reducing the symptom of osteoporosis.

  12. Induction of osteoconductivity by BMP-2 gene modification of mesenchymal stem cells combined with plasma-sprayed hydroxyapatite coating

    Science.gov (United States)

    Wu, Jiang; Guo, Ying-qiang; Yin, Guang-fu; Chen, Huai-qing; Kang, Yunqing

    2008-11-01

    Success in bone implant depends greatly on the composition and surface features of the implant. The surface-modification measures not only favor the implant's osteoconductivity, but also promote both bone anchoring and biomechanical stability. This paper reports an approach to combine a hydroxyapatite (HA) coated substrate with a cellular vehicle for the delivery of bone morphogenetic protein-2 (BMP-2) synergistically enhancing the osteoconductivity of implant surfaces. We examined the attachment, growth and osteoinductive activity of transfected BMP-producing bone marrow mesenchymal stem cells (BMSCs) on a plasma-sprayed HA coated substrate. It was found that the HA coated substrate could allow the attachment and growth of BMP-2 gene modified BMSCs, and this combined application synergistically enhanced osteconductivity of the substrate surface. This synergistic method may be of osseointegration value in orthopedic and dental implant surgery.

  13. In Vitro Evaluation of an Injectable Chitosan Gel for Sustained Local Delivery of BMP-2 for Osteoblastic Differentiation

    Science.gov (United States)

    2011-11-01

    an attrac- tive biopolymer due to its biodegradability and biocompatibil- ity. In particular, chitosan based delivery systems have gained FIGURE 6...dimers and a mixture of BMP-2/BMP-7 homodimers on osteoblast adhesion and growth following culture on a collagen scaffold . Biomed Mater 2008;3:1–10. 9...In vitro cellular responses to scaffolds containing two microencapsulated growth factors. Biomaterials 2009;30:5215–5224. 16. Park KH, Kim H, Moon S

  14. Methods to Analyze Bone Regenerative Response to Different rhBMP-2 Doses in Rabbit Craniofacial Defects

    Science.gov (United States)

    2014-02-28

    response of increasing regenerated bone volume with rhBMP-2 loaded on the TyrPC scaffolds after 16 weeks of implantation . Significantly more bone...Rutgers, The State University of New Jersey. 1Department of Craniomaxillofacial Regenerative Medicine, Dental and Trauma Research Detachment, United States...augmentation prior to implant placement, fracture repair, healing large defects stemming from traumatic injuries, cancer resections, or congenital

  15. Irx3 and Bmp2 Regulate Mouse Mesenchymal Cell Chondrogenic Differentiation in Both a Sox9-Dependent and -Independent Manner.

    Science.gov (United States)

    Tamamura, Yoshihiro; Katsube, Kenichi; Mera, Hisashi; Itokazu, Maki; Wakitani, Shigeyuki

    2017-01-06

    Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation. This article is protected by copyright. All rights reserved.

  16. Research to Influence of Buyang Huanwu Tang on Axonal Remyellnation Mediated by BMP2/4%补阳还五汤对BMP2/4介导轴突再髓鞘化的影响

    Institute of Scientific and Technical Information of China (English)

    赵鹏; 饶耀剑; 崔泽升; 崔家伟

    2015-01-01

    目的 通过研究补阳还五汤对BMP2/4的干预作用,来探讨补阳还五汤对大鼠脊髓损伤后残存轴突再髓鞘化的影响.方法 将60只大鼠分为假手术组、模型组和治疗组(n=20);假手术组只打开胸10节段椎板,不损伤脊髓;模型组和治疗组采用改良Allen垂直打击法制作胸10节段脊髓损伤模型;术后治疗组给予补阳还五汤浓缩液灌胃,连续灌胃28天;分别于造模前、造模后当天、造模后7天、造模后14天、造模后28天评估每只大鼠的BBB评分,并于造模后28天取大鼠胸10节段脊髓组织,检测BMP2/4的表达变化.结果 造模后28天治疗组大鼠BBB评分明显高于模型组,P<0.05(差异有统计学意义).分析免疫印迹检测结果提示:造模后28天治疗组和模型组脊髓损伤节段BMP2/4表达量大量增加,假手术组BMP2/4少量表达,治疗组BMP2/4表达少于模型组,各组组间差异具有统计学意义(P.<0.05).结论 补阳还五汤可有效抑制大鼠脊髓损伤局部BMP2/4的表达,可能通过此种途径来促进脊髓损伤后残存轴突的再髓鞘化.

  17. Sustained delivery of BMP-2 enhanced osteoblastic differentiation of BMSCs based on surface hydroxyapatite nanostructure in chitosan-HAp scaffold.

    Science.gov (United States)

    Wang, Guancong; Qiu, Jichuan; Zheng, Lin; Ren, Na; Li, Jianhua; Liu, Hong; Miao, Junying

    2014-01-01

    The surface characteristics of biomaterials, especially regarding the sustained delivery of bone morphogenetic protein-2 (BMP-2), can possibly provide a novel and effective drug delivery system that can enhance osteogenesis. In this study, we evaluated the BMP-2 adsorption and release ability of the surface biomimetic hydroxyapatite (HAp) nanostructure on a new HAp-coated genipin-chitosan conjugation scaffold (HGCCS), and the resulting osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. HGCCS exhibited a loading efficiency of 65% (1.30 μg), which is significantly higher than 28% (0.56 μg, p nanostructure of HGCCS used as a delivery system for BMP-2 is capable of promoting osteogenic differentiation in vitro. These findings demonstrated that HAp nanostructure assembled on organic porous scaffold could work as both calcium source and absorption/release platform, which opened a new research avenue for cell growth factor release, and provided a promising strategy for design and preparation of bioactive scaffold for bone tissue engineering.

  18. Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels

    Directory of Open Access Journals (Sweden)

    J van der Stok

    2015-03-01

    Full Text Available Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2. This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting load-bearing defects in trauma and orthopaedic surgery.

  19. Repair of Rat Segmental Defects with Mineralized Collagen Grafts Combined with or without Mesenchymal Stem Cells and BMP-2

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The aim of the present study was to investigate and compare the bone formation capacity with three different grafts. Four millimeter segmental defects were created in adult rat tibias and were either left empty(control defects) or implanted with (1) nano-hydroxyapatite/collagen/PLA (nHAC/PLA) composite, (2)nHAC/ PLA composite added with bone marrow mesenchymal stem cells ( BMSCs ), (3) nHAC/PLA composite added with bone morphogenetic protein 2 ( BMP- 2). Radiographs of the defects were taken weekly post-surgery.After 1 or 2 months, the rats were euthanized. Histologic analyses were performed on the harvested tissue.nHAC/ PLA composite could enhance the repair of rat tibia segmental defects. Addition of BMSCs or BMP- 2 to nHAC/ PLA led to an increase in osteogenesis. nHAC/ PLA composite could be an ideal alternative bone-graft material and it could also be used as an ideal carrier of BMSCs or BMP- 2.

  20. BMP2 mRNA 在颌骨骨肉瘤及软骨肉瘤中的扩增及其对肿瘤增殖活性的影响%Expression of BMP2 mRNA in osteosarcoma and chondrosarcoma of jaw and it's effect on tumor proliferating activity

    Institute of Scientific and Technical Information of China (English)

    岳文; 杨连甲; 金岩; 董绍忠

    2000-01-01

    目的研究BMP2 mRNA 在颌骨骨肉瘤及软骨肉瘤中的扩增及其对肿瘤预后和增殖活性的影响.方法应用 BMP 2 的 cDNA探针,原位杂交检测 mRNA 的扩增.结果 32例标本中有2 1例表达BMP2 mRNA,有BMP2 mRNA表达的病例更容易发生淋巴结转移,且有较高增殖活性. 结论 BMP2 可能在骨肿瘤的发生和发展过程中起一定作用,可以作为估计骨肉瘤预后的参考指标.

  1. Experimental and computational investigation of the effect of hydrophobicity on aggregation and osteoinductive potential of BMP-2-derived peptide in a hydrogel matrix.

    Science.gov (United States)

    Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K; Karimi, Tahereh; Jabbari, Esmaiel

    2015-01-01

    An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73-92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005-0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the

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

    Energy Technology Data Exchange (ETDEWEB)

    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.)

  3. Medium-Term Function of a 3D Printed TCP/HA Structure as a New Osteoconductive Scaffold for Vertical Bone Augmentation: A Simulation by BMP-2 Activation

    Directory of Open Access Journals (Sweden)

    Mira Moussa

    2015-04-01

    Full Text Available Introduction: A 3D-printed construct made of orthogonally layered strands of tricalcium phosphate (TCP and hydroxyapatite has recently become available. The material provides excellent osteoconductivity. We simulated a medium-term experiment in a sheep calvarial model by priming the blocks with BMP-2. Vertical bone growth/maturation and material resorption were evaluated. Materials and methods: Titanium hemispherical caps were filled with either bare- or BMP-2 primed constructs and placed onto the calvaria of adult sheep (n = 8. Histomorphometry was performed after 8 and 16 weeks. Results: After 8 weeks, relative to bare constructs, BMP-2 stimulation led to a two-fold increase in bone volume (Bare: 22% ± 2.1%; BMP-2 primed: 50% ± 3% and a 3-fold decrease in substitute volume (Bare: 47% ± 5%; BMP-2 primed: 18% ± 2%. These rates were still observed at 16 weeks. The new bone grew and matured to a haversian-like structure while the substitute material resorbed via cell- and chemical-mediation. Conclusion: By priming the 3D construct with BMP-2, bone metabolism was physiologically accelerated, that is, enhancing vertical bone growth and maturation as well as material bioresorption. The scaffolding function of the block was maintained, leaving time for the bone to grow and mature to a haversian-like structure. In parallel, the material resorbed via cell-mediated and chemical processes. These promising results must be confirmed in clinical tests.

  4. BMP2基因转染犬牙髓成纤维细胞与异种烧结骨复合组织工程的实验研究%THE EXPERIMENTAL STUDY OF TISSUE ENGINEERING OF DOG DENTAL PULP CELLS TRANSFECTED BY BMP2 GENE AND CURLURED WITH XENOGENEIC BONE CERAMICE

    Institute of Scientific and Technical Information of China (English)

    冯艳红; 刁志虹; 高毅; 李威

    2011-01-01

    目的 构建骨形成蛋白2(bone morphogenetic proteins 2,BMP2)绿色荧光融合蛋白pEGFP-N1-BMP2真核表达质粒,用其在体外转染犬牙髓细胞(dog dental pulp cells,DDPCs),利用构建的种子细胞BMP2-DDPCs与异种烧结骨(xenogeneic bone ceramice,XBC)复合,检测BMP2-DDPCs增殖活性,扫描电镜观察BMP2-DDPCs在异种烧结骨中的生长情况,评价异种烧结骨作为支架材料的可行性,为将人BMP2基因转染的牙髓成纤维细胞与异种烧结骨复合进行牙体修复的研究奠定基础.方法 构建pEGFP-N1-BMP2真核表达质粒,采用阳离子脂质体转染法将BMP2基因转染体外培养的犬牙髓细胞,将构建的种子细胞BMP2-DDPCs与异种烧结骨复合,MTT法检测BMP2-DDPCs增殖活性以及应用扫描电镜观察BMP2-DDPCs在异种烧结骨中的生长情况.结果 成功构建pEGFP-N1-BMP2真核表达质粒,并成功转染犬牙髓细胞.BMP2-DDPCs复合异种烧结骨组的细胞增殖活性与单层贴壁培养BMP2-DDPCs组的细胞增殖情况差异无统计学意义(P>0.05).说明异种烧结骨上黏附的牙髓细胞增殖情况良好.扫描电镜观察异种烧结骨上BMP2-DDPCs黏附、生长的情况,可见支架材料的孔径为100~600μm,材料表面粗糙,利于细胞的黏附生长;孔隙中可见大量BMP2-DDPCs贴附于材料上,生长旺盛、伸展良好.结论 BMP2-DDPCs可在异种烧结骨表面及孔隙中生长、增殖,异种烧结骨可以作为牙组织工程的支架材料.%Objective To construct recombinant plasmid pEGFP - N1 - BMP2 and transfect dog dental pulp cells( DDPCs )with pEGFP - N1 - BMP2. BMP2 - DDPCs were seeded onto xenogeneic bone ceramice ( XBC ) scaffold as seed cells. To evaluate the feasibility of using XBC as scaffold for tissue engineered dentin, the biocompatibility between the seed cells( BMP2 - DDPCs ) and scaffold was detected by scanning electron microscopy and methyl thiazolyl tetrazolium( MTT ) analysis. Methods The full length dog BMP2 c

  5. Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    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

  6. Construction and identification of recombinant adenovirus vector co-expressing VEGF121 and BMP2 genes and its expression in HEK293 cells%VEGF121和BMP2双基因共表达重组腺病毒载体的构建及其在HEK293中的表达

    Institute of Scientific and Technical Information of China (English)

    栗刚; 吴秀成; 钟声; 王巍; 李媛; 刘丹平

    2011-01-01

    目的 构建人血管内皮生长因子121(VEGF121)与人骨形态发生蛋白2(BMP2)双基因共表达腺病毒载体Adv-BMP2-IRES-VEGF121,并观察其在人胚肾细胞株(HEK293)中的表达情况.方法 对腺病毒质粒pShuttle-CMV-BMP2的目的基因BMP2进行PCR扩增.腺病毒质粒pShuttle-CMV-VEGF121-IRES-hrGFP-1经Kpn I/Xba I酶切后,将BMP2片段定向导入pShuttle-CMV-VEGF121-IRES,构建pShuttle-CMV-V EGF121-IRES-BMP2,并注入大肠杆菌DH5a中扩增,提取质粒.通过酶切分析、PCR检测和序列分析进行鉴定.将构建所得的质粒转染HEK293,采用RT-PCR法检测HEK293中的BMP2、VEGF121 mRNA,Western blot法检测其蛋白.结果 成功构建了Adv-BMP2-IRES-VEGF121.酶切分析及DNA序列测定证实重组质粒构建正确.质粒转染后的HEK293 BMP2和VEGF121表达阳性.结论 成功构建了Adv-BMP2-IRES-VEGF121,其转染HEK293后,VEGF121、BMP2在HEK293中共表达阳性.%Objective To construct and identify the adenovirus shuttle plasmid pShuttle-CMV-VEGF121-IRES-BMP2 and its express in HEK293 cells.Methods The DNA fragments of human BMP2 gene were changed restriction sites and subcloned by PCR.The human BMP2 genes and pShuttle-CMV-VEGF121-IRES were ligated into the plasmid by directional cloning method.The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing.The correct recombinant express plasmid was transfected to HEK293 cells.The expression of VEGF121, BMP2 mRNA were detected by RT-PCR, the VEGF121, BMP2 protein were detected by Western blotting.Results The adenovirus shuttle plasmid was constructed correctly.The VEGF121, BMP2 mRNA and protein were expressed in HEK293 cells.Conclusion The adenovirus shuttle plasmid is constructed, VEGF121, BMP2 mRNA and protein are successfully expressed in HEK293 cells.

  7. Regulation of expression of osteoblast transcription factor SATB2 by BMP2 in osteoblastic differentiation of MSCs%MSCs成骨分化中BMP2对成骨转录因子SATB2表达的调控作用

    Institute of Scientific and Technical Information of China (English)

    左炽健; 剌婷; 张宁; 戴尅戎; 张晓玲

    2011-01-01

    目的 探索间充质干细胞(MSCs)成骨分化中骨形态发生蛋白2(BMP2)对成骨转录因子SATB2表达的调控作用.方法 体外培养小鼠间充质细胞系C2C12,腺病毒介导的BMP2 (Adv-BMP2)诱导其向成骨细胞分化,建立并验证C2C12细胞成骨分化细胞模型.Real-Time PCR和Western blotting分别检测C2C12细胞成骨分化过程中经不同浓度Adv-BMP2处理不同时间时SATB2 mRNA和SATB2蛋白表达;以经相应浓度Adv-β-Gal处理细胞作对照.结果 经150 pfu/cell Adv-BMP2处理C2C12细胞5d后,成骨细胞标志基因Ⅰ型胶原,骨唾液酸蛋白和骨钙素表达以及碱性磷酸酶活性均显著增加,MSCs成骨分化模型构建成功.150 pfu/cell Adv-BMP2诱导C2C12细胞成骨分化过程中,SATB2 mRNA和SATB蛋白表达随分化进程而增加;Adv-BMP2浓度为0~225 pfu/cell时,SATB2表达随Adv-BMP2浓度升高而增加.结论BMP2可调控SATB2的表达,从而影响MSCs成骨分化.%Objective To investigate the role of bone morphogenetic protein 2 (BMP2) in the regulation of expression of osteoblast transcription factor SATB2 in osteoblastic differentiation of mesenchymal stem cells ( MSCs). Methods Mesenchymal cell line C2C12 was stimulated by adenovirus-derived BMP2 (Adv-BMP2) for osteoblastic differentiation, and model of osteoblastic differentiation of C2C12 cells was established and verified. Real-Time PCR and Western blotting were employed to detect the expression of SATB2 mRNA and SATB2 protein respectively in osteoblastic differentiation treated by Adv-BMP2 for different time and by Adv-BMP2 of different concentrations. C2CI2 cells treated with Adv-β-Gal were served as controls. Results After treatment by ISO pfu/cell Adv-BMP2 for 5 d, the expression of collagen type I , bone sialoprotein and osteocalcin and the activity of alkaline phosphatase in C2C12 cells significantly increased, and the model of osteoblastic differentiation of C2C12 cells was established. The expression of SATB2 mRNA and

  8. 腺病毒穿梭质粒pShuttle CMV-BMP2+-IRES-hrGFP-1的构建和鉴定%Construction and Identification of Recombinant Adenovirus Shuttle Plasimid pShuttle CMV-BMP2 +-IRES-GFP-1

    Institute of Scientific and Technical Information of China (English)

    张正; 刘丹平; 蒲勤; 郭韬; 张男

    2005-01-01

    目的构建腺病毒穿梭质粒pShuttle CMV-BMP2+-IRES-hrGFP-1,为构建表达具有抗原表位标记的骨形态发生蛋白2(bone morpbogenetic protein 2,BMP-2),并同时表达绿色荧光蛋白(green fluorescent protein,GFP)报告分子的腺病毒真核细胞表达载体打下基础.方法对目的基因供体质粒pcDNA3-BMP2携带的BMP2基因测序和序列内部存在的限制性内切酶识别位点进行分析,利用PCR(polymeras chain reaction,PCR)技术对pcDNA3-BMP2携带的BMP2基因突变,以去除翻译终止密码子后的基因序列并添加新的酶切识别位点Xho Ⅰ.测序检测突变情况,将突变后的BMP2基因(BMP2+基因)定向连入腺病毒穿梭质粒pShuttleCMV-IRES-hrGFP-1,通过限制性内切酶酶切图谱分析鉴定获得的重组质粒.结果重组质粒经双酶切鉴定图谱正确.结论成功构建了pShuttle CMV-BMP2+-IRES-hrGFP-1.

  9. 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

  10. Genetic Variants of BMP2 and Their Association with the Risk of Non-Syndromic Tooth Agenesis

    Science.gov (United States)

    Wang, Yuting; Gu, Ning; Ma, Lan; Xu, Min; Ma, Junqing; Zhang, Weibing; Pan, Yongchu; Wang, Lin

    2016-01-01

    Non-syndromic tooth agenesis (or non-syndromic congenitally missing tooth) is one of the most common congenital defects in humans affecting the craniofacial function and appearance. Single nucleotide polymorphisms (SNPs) have been associated with an individual’s susceptibility to these anomalies. The aim of the present study was therefore to investigate the roles of the potentially functional SNPs of BMP2 in the occurrence of tooth agenesis. Overall, four potentially functional SNPs of BMP2 (rs15705, rs235768, rs235769 and rs3178250) were selected, and their associations with the susceptibility of tooth agenesis were evaluated in a case-control study of 335 non-syndromic tooth agenesis cases and 444 healthy controls. The SNPs rs15705 and rs3178250 were found to be associated with an individual’s risk of tooth agenesis (P = 0.046 and P = 0.039, respectively). Both SNPs showed an increased risk of mandibular incisor agenesis (rs15705, AA/AC vs. CC = 1.58, 95% CI = [1.06–2.34], P = 0.024; rs3178250, TT/TC vs. CC = 1.60, 95% CI = [1.08–2.37], P = 0.020). Bioinformatics analysis indicated that these two SNPs located at the 3’-untranslated region (3’-UTR) of BMP2 might alter the binding ability of miR-1273d and miR-4639-5p, respectively, which was confirmed by luciferase activity assays in the 293A and COS7 cell lines (P < 0.001 in 293A and P < 0.01 in COS7 for miR-1273d; and P < 0.001 in both cells for miR-4639-5p). Furthermore, BMP2 mRNA expression decreased after transfecting either miR-1273d or miR-4639-5p into these two cell lines (P < 0.01 in 293A and P < 0.001 in COS7 for miR-1273d, and P < 0.01 in both cell lines for miR-4639-5p). Taken together, our findings indicate that rs15705 and rs317250 are associated with the susceptibility of non-syndromic tooth agenesis by possibly affecting miRNAs and mRNA interaction. PMID:27362534

  11. Genetic Variants of BMP2 and Their Association with the Risk of Non-Syndromic Tooth Agenesis.

    Directory of Open Access Journals (Sweden)

    Yun Lu

    Full Text Available Non-syndromic tooth agenesis (or non-syndromic congenitally missing tooth is one of the most common congenital defects in humans affecting the craniofacial function and appearance. Single nucleotide polymorphisms (SNPs have been associated with an individual's susceptibility to these anomalies. The aim of the present study was therefore to investigate the roles of the potentially functional SNPs of BMP2 in the occurrence of tooth agenesis. Overall, four potentially functional SNPs of BMP2 (rs15705, rs235768, rs235769 and rs3178250 were selected, and their associations with the susceptibility of tooth agenesis were evaluated in a case-control study of 335 non-syndromic tooth agenesis cases and 444 healthy controls. The SNPs rs15705 and rs3178250 were found to be associated with an individual's risk of tooth agenesis (P = 0.046 and P = 0.039, respectively. Both SNPs showed an increased risk of mandibular incisor agenesis (rs15705, AA/AC vs. CC = 1.58, 95% CI = [1.06-2.34], P = 0.024; rs3178250, TT/TC vs. CC = 1.60, 95% CI = [1.08-2.37], P = 0.020. Bioinformatics analysis indicated that these two SNPs located at the 3'-untranslated region (3'-UTR of BMP2 might alter the binding ability of miR-1273d and miR-4639-5p, respectively, which was confirmed by luciferase activity assays in the 293A and COS7 cell lines (P < 0.001 in 293A and P < 0.01 in COS7 for miR-1273d; and P < 0.001 in both cells for miR-4639-5p. Furthermore, BMP2 mRNA expression decreased after transfecting either miR-1273d or miR-4639-5p into these two cell lines (P < 0.01 in 293A and P < 0.001 in COS7 for miR-1273d, and P < 0.01 in both cell lines for miR-4639-5p. Taken together, our findings indicate that rs15705 and rs317250 are associated with the susceptibility of non-syndromic tooth agenesis by possibly affecting miRNAs and mRNA interaction.

  12. Elucidation of a novel pathway through which HDAC1 controls cardiomyocyte differentiation through expression of SOX-17 and BMP2.

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    Eneda Hoxha

    Full Text Available Embryonic Stem Cells not only hold a lot of potential for use in regenerative medicine, but also provide an elegant and efficient way to study specific developmental processes and pathways in mammals when whole animal gene knock out experiments fail. We have investigated a pathway through which HDAC1 affects cardiovascular and more specifically cardiomyocyte differentiation in ES cells by controlling expression of SOX17 and BMP2 during early differentiation. This data explains current discrepancies in the role of HDAC1 in cardiovascular differentiation and sheds light into a new pathway through which ES cells determine cardiovascular cell fate.

  13. The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model.

    Science.gov (United States)

    Zwingenberger, Stefan; Langanke, Robert; Vater, Corina; Lee, Geoffrey; Niederlohmann, Eik; Sensenschmidt, Markus; Jacobi, Angela; Bernhardt, Ricardo; Muders, Michael; Rammelt, Stefan; Knaack, Sven; Gelinsky, Michael; Günther, Klaus-Peter; Goodman, Stuart B; Stiehler, Maik

    2016-09-01

    The treatment of critical size bone defects represents a challenge. The growth factor bone morphogenetic protein 2 (BMP-2) is clinically established but has potentially adverse effects when used at high doses. The aim of this study was to evaluate if stromal derived factor-1 alpha (SDF-1α) and BMP-2 released from heparinized mineralized collagen type I matrix (MCM) scaffolds have a cumulative effect on bone regeneration. MCM scaffolds were functionalized with heparin, loaded with BMP-2 and/or SDF-1α and implanted into a murine critical size femoral bone defect (control group, low dose BMP-2 group, low dose BMP-2 + SDF-1α group, and high dose BMP-2 group). After 6 weeks, both the low dose BMP-2 + SDF-1α group (5.8 ± 0.6 mm³, p = 0.0479) and the high dose BMP-2 group (6.5 ± 0.7 mm³, p = 0.008) had a significantly increased regenerated bone volume compared to the control group (4.2 ± 0.5 mm³). There was a higher healing score in the low dose BMP-2 + SDF-1α group (median grade 8; Q1-Q3 7-9; p = 0.0357) than in the low dose BMP-2 group (7; Q1-Q3 5-9) histologically. This study showed that release of BMP-2 and SDF-1α from heparinized MCM scaffolds allows for the reduction of the applied BMP-2 concentration since SDF-1α seems to enhance the osteoinductive potential of BMP-2. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2126-2134, 2016.

  14. BMP-2,3,4,5在颌面部神经鞘瘤中的表达分析%Analysis of the Expression of BMP-2,3,4,5 in Nerve Sheath Tumors of Maxillofacial Region

    Institute of Scientific and Technical Information of China (English)

    金岩; 吕红兵; Tipoe GL; 李媛

    2000-01-01

    目的:探讨BMPs家族成员与外周神经肿瘤的关系。方法:用原位杂交方法对BMP-2,3,4,5在人良、恶性神经鞘瘤中的表达进行观察。结果:显示BMP-2,3,4,5 mRNA在外周神经的良恶性神经鞘瘤中均有分布。良性肿瘤中,呈栅栏状排列,肿瘤细胞聚集的区域BMPs表达升高;恶性肿瘤中BMPs的表达高于良性肿瘤。结论:确定了BMPs在外周神经肿瘤中的表达和分布,揭示部分BMPs可能参与了外周神经肿瘤的发生发展过程。

  15. Research on the Positioning and Quantitative Expression of BMP2 Gene in Chest, Abdomen and Back Skin of Goose%BMP2基因在鹅胸、腹、背部皮肤中的定位定量达研究

    Institute of Scientific and Technical Information of China (English)

    牟玉婷; 吴伟; 孙永峰; 宋佳巍; 孙远; 吴文立; 李瑶

    2013-01-01

    The expression of BMP2 mRNA in chest, abdomen and back skin of jilin white goose in postnatal period was studied by immunohistochemistry and quantitative PCR. The results showed that BMP2 mRNA was expressed in all skin tissues during each stage. BMP2 was expressed in barb ridge or rachidial ridge in feather follicles, and the barb ridge developed into barbs based on developmental age, which indicates that BMP2 may play critical roles in barb growth. The relative expression quantity in back was 5.521 7 in D7, 3.42 times of chest, 7.56 times of abdomen. The feather follicles of chest and abdomen were in the stage of rapid development, and BMP2 inhibited feather follicle development. The chest and back in D28 expressed lower levels of BMP2 mRNA compared with feather follicles of abdomen. At this point, the hooklets appeared in abdomen, which indicates that BMP2 may be related to feather branching . BMP2 mRNA was lower in feather follicles of abdomen and back compared with chest at D63, and feather development had basically matured in chest. Abdomen and back would mature in 1—2 weeks.%运用免疫组化和荧光定量PCR方法,研究了吉林白鹅生后期胸、腹、背皮肤中BMP2基因的定位表达及mRNA变化规律.结果表明:BMP2基因在鹅胸、腹、背部皮肤初级毛囊和次级毛囊发育过程中均有表达,BMP2在毛囊中羽枝嵴和羽轴嵴部位表达,而羽枝嵴将来发育成羽枝,推断BMP2与羽枝发育有关.7日龄时背部BMP2的mRNA相对基因表达量为5.521 7,分别是胸部的3.42倍和腹部的7.56倍.此时胸腹部毛囊处于快速发育阶段,BMP2则抑制毛囊发育;28日龄腹部BMP2相对基因表达量明显高于背部和胸部,此时腹部已有羽小钩出现,BMP2可能参与羽毛分支;63日龄BMP2的mRNA相对表达量胸部高于腹部和背部,此时胸部羽毛发育已基本成熟,而腹部和背部晚1~2周成熟.

  16. Effect of rhBMP- 2 on bone regeneration and osseointegration in peri- implant defects- histological observation%rhBMP-2在种植体周围骨缺损修复中应用的组织学观察

    Institute of Scientific and Technical Information of China (English)

    黄元瑾; 章锦才; 刘曙光; 蔡德鸿

    2012-01-01

    目的:研究rhBMP- 2及不同载体在种植体周围骨缺损修复中的应用.方法:在beagle犬下颌骨植入种植体,颊侧形成裂开性骨缺损,置入复合了不同浓度rhBMP- 2的珊瑚羟基磷灰石人造骨(CHA)或可吸收胶原海绵(ACS).种植体植入后2、4、8、12 周,获取含种植体骨标本,进行组织学观察.结果:2 周时,rhBMP- 2组可见极少量的新生骨组织.4 周时,rhBMP- 2/ACS组新骨组织由牙槽骨顶端向缺损区中心方向生长;rhBMP- 2/CHA组人造骨颗粒内部和周围出现呈岛状生长的新生骨组织.8 周时,rhBMP- 2/ACS组的新骨形成大片状结构;rhBMP- 2/CHA组人造骨颗粒周围较多骨岛形成.12 周时,rhBMP- 2组的缺损区内骨量和骨高度进一步增加,与种植体形成骨性结合.浓度为0.05 mg/ml和0.2 mg/ml,载体为CHA或ACS促进骨再生作用差异无统计学意义.结论:以CHA或ACS为载体rhBMP- 2能促进种植体周围骨缺损区内的骨组织再生并与种植体表面较好地结合.%Objective: To investigate the effects of rhBMP-2 in bone formation and osseointegration in peri-implant defects. Methods: 8 implants were placed in each of 8 beagle dogs. Dehiscence defects were surgically created on the buccal side of each implant. rhBMP-2 at 0. 05 mg/ml or 0. 2 mg/ml was combined into absorbable collagen sponge( ACS ) or coraline hydroxyapatite( CHA ) respectively and applied into the defects. ACS or CHA alone were used as the control. Animals were sacrificed 2,4,8 and 12 weeks after implantation respectively, non- decalcified ground sections of the bone samples with the implants were made and observed under microscope. Results: 2 weeks after implantation, the newly generated bone was minimal. 4 weeks after implantation, in rhBMP-2/ACS group formations of trabeculae of woven bone could be seen in the defects, in rhBMP-2/CHA the CHA particles were surrounded by newly formed bone. 8 weeks after implantation, in rhBMP-2/ACS group the newly formed bone

  17. Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors.

    Science.gov (United States)

    Chen, Jiaxuan; Dosier, Christopher R; Park, Jung Hwa; De, Subhendu; Guldberg, Robert E; Boyan, Barbara D; Schwartz, Zvi

    2016-01-01

    1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), Pdia3-silenced (Sh-Pdia3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε-caprolactone)-collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (μCT) showed that the mineralized volume of untreated Sh-Pdia3 and Sh-VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3.

  18. Purification and regeneration of rhBMP-2%重组人骨形成蛋-2的纯化和复性

    Institute of Scientific and Technical Information of China (English)

    孙叶芳; 赵艳萍; 刘晗; 李强; 史俊南

    2001-01-01

    目的:探索原核表达重组人骨形成蛋白 -2(rhBMP-2)的纯化和复性方法。方法:对携带有pBV220-hBMP-2的 工程 菌进行发酵,收集菌体,超声破碎菌体后制备包涵体。包涵体经进一步超声裂解后过凝胶色 谱和离子交换色谱,对获得的高纯度蛋白进行不同条件下的复性。rhBMP-2产物作用于培养 的人牙乳头细胞,检测ALP活性。结果:产物经过凝胶色谱后纯度达 到90%,复性后再经离子交换和凝胶色谱纯度为85%以上,并且提高了生物学活性。结论:rhBMP-2产物经过离子交换色谱和凝胶色谱获得较高纯度的重组蛋白; 3mol/L 尿素和0.75~1.25mol/L的NaCl能显著提高产物生物学活性。%AIM: To study methods for purificat ion and regeneration of E.coli generated rhBMP-2. METHODS: TOP10 strain carrying pBV220-rhBMP-2 recombinant construct was fermented and lysised to prepare inclusion bodies which was then further lysised by ultrosonic followe d by Gel chromatography and ion-chromatographyprocessures to generate high puri ty protein product. different regenerative conditions were investigated to obtai n rhBMP-2 product of higher biological activity measured by ALP activity analysi s. RESULTS: The purity of primitive rhBMP-2 product increa sed by 90% after Gel chromatography and the regenerated protein increased its pu rity by 85% after ion-exchange chromatography. CONCLUSIONS: Gel chromatography and ion-exchange Chromatogaphy help obtain rhBMP product of high purity; 3mol/L urea and 0.75~1.25mol/L NaCl can regenerate rhBMP-2 to a higher level of biological activity.

  19. Cardiogenic induction of pluripotent stem cells streamlined through a conserved SDF-1/VEGF/BMP2 integrated network.

    Directory of Open Access Journals (Sweden)

    Anca Chiriac

    Full Text Available BACKGROUND: Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output. METHODS AND RESULTS: To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes. The streamlined pool of 288 genes organized into a core biological network that prioritized the "Cardiovascular Development" function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield. CONCLUSIONS: Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis.

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

    Science.gov (United States)

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

    2016-01-01

    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;Bmp2f/f;Bmp4f/fameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

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

    Science.gov (United States)

    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.

  2. 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 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.

  3. Interplay between self-assembled structure of bone morphogenetic protein-2 (BMP-2) and osteoblast functions in three-dimensional titanium alloy scaffolds: Stimulation of osteogenic activity.

    Science.gov (United States)

    Nune, K C; Kumar, A; Murr, L E; Misra, R D K

    2016-02-01

    Three-dimensional cellular scaffolds are receiving significant attention in bone tissue engineering to treat segmental bone defects. However, there are indications of lack of significant osteoinductive ability of three-dimensional cellular scaffolds. In this regard, the objective of the study is to elucidate the interplay between bone morphogenetic protein (BMP-2) and osteoblast functions on 3D mesh structures with different porosities and pore size that were fabricated by electron beam melting. Self-assembled dendritic microstructure with interconnected cellular-type morphology of BMP-2 on 3D scaffolds stimulated osteoblast functions including adhesion, proliferation, and mineralization, with prominent effect on 2-mm mesh. Furthermore, immunofluorescence studies demonstrated higher density and viability of osteoblasts on lower porosity mesh structure (2 mm) as compared to 3- and 4-mm mesh structures. Enhanced filopodia cellular extensions with extensive cell spreading was observed on BMP-2 treated mesh structures, a behavior that is attributed to the unique self-assembled structure of BMP-2 that effectively communicates with the cells. The study underscores the potential of BMP-2 in imparting osteoinductive capability to the 3D printed scaffolds.

  4. Sustained release of VH and rhBMP-2 from nanoporous magnesium-zinc-silicon xerogels for osteomyelitis treatment and bone repair.

    Science.gov (United States)

    Li, Fengqian; Wu, Wen; Xiang, Li; Weng, Gan; Hong, Hua; Jiang, Hong; Qian, Jun

    2015-01-01

    Nanoporous magnesium-zinc-silicon (n-MZS) xerogels with a pore size ∼4 nm, a surface area of 718 cm(2)/g, and a pore volume of 1.24 cm(3)/g were synthesized by a sol-gel method. The n-MZS xerogels had high capacity to load vancomycin hydrochloride (VH) and human bone morphogenetic protein-2 (rhBMP-2), after soaking in phosphate buffered saline (PBS) for 24 hours (1.5 and 0.8 mg/g, respectively). Moreover, the n-MZS xerogels exhibited the sustained release of VH and rhBMP-2 as compared with magnesium-zinc-silicon (MZS) xerogels without nanopores (showing a burst release). The VH/rhBMP-2/n-MZS system not only exhibited a good antibacterial property but also promoted the MG63 cell proliferation and differentiation demonstrating good bactericidal activity and cytocompatibility. The results suggested that n-MZS with larger surface area and high pore volume might be a promising carrier for loading and sustained release of VH and rhBMP-2. Hence, the VH/rhBMP-2/n-MZS system might be one of the promising biomaterials for osteomyelitis treatment and bone repair.

  5. 地塞米松预培养兔BMSCs促进腺病毒介导BMP2转基因的高效表达

    Institute of Scientific and Technical Information of China (English)

    王栋梁; 刘丹平; 张正; 胡汉祥

    2007-01-01

    目前,骨形态发生蛋白2(BMP2)的基因治疗在骨组织工程中正成为广泛研究的热点。国外最新研究表明,以腺病毒为载体转染骨髓间充质干细胞(BMSCs)表达BMP2的效率取决于细胞的分化状态,1μmol/L地塞米松诱导培养人BMSCs后再进行腺病毒转染,其BMP2的表达量是单纯转染组的20倍。在此基础上,笔者首次利用同时表达绿色荧光蛋白(GFP)和BMP2的AdCMV—hBMP2-IRES—GFP-1转染兔BMSCs,通过流式细胞仪等方法,

  6. Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) for the Treatment of Nonunion of the Femur in Children and Adolescents: A Retrospective Analysis

    Science.gov (United States)

    Stiel, Norbert; Babin, Kornelia; Rupprecht, Martin; Rueger, Johannes M.; Stuecker, Ralf

    2017-01-01

    Background. The aim of this study was to examine clinical and radiographic healing after rhBMP-2 application in children and adolescents presenting with nonunion of the femur and to investigate the safety of rhBMP-2 use in these cases. Materials and Methods. We reviewed the medical records of five patients with a mean age of 11 years (5.4 to 16.2) with nonunion of the femur who were treated with rhBMP-2 and internal fixation using a locking plate at a single institution. Particular attention was paid to identify all adverse events that may be due to rhBMP-2 use. Results. Union occurred in four of five patients at a mean of 12.1 months (7.9 to 18.9). The locking plates were removed after a mean of 16 months (11 to 23). One patient had nonunion due to deep infection. After a mean follow-up of 62.5 months (17 to 100), union was still evident in all four patients and they were fully weight-bearing without pain. Discussion. In this retrospective study, rhBMP-2 combined with a locking plate has been used successfully to treat children and adolescents with nonunion of the femur in four of five cases. One major complication was thought to be possibly related to its use.

  7. Construction and identification of recombinant adenovirus shuttle plasimid expressing FLAG labeled BMP2 and traced by GFP%GFP示踪FLAG抗原表位标记BMP2转基因腺病毒穿梭质粒的构建

    Institute of Scientific and Technical Information of China (English)

    张正; 李谌; 陈峻江; 刘丹平

    2011-01-01

    Objective To construct a novel recombinant adenovirus shuttle plasimid expressing the BMP2 fused to FLAG epitope and green fluorescent protein(GFP) as a tracer protein of the recombinant adenovirus on the same transcript.Methods The base pairs behind the translation stop codon TAG were removed and a Xho I restriction site was added following the 3'end of the mutant through PCR. After being tested through sequencing , the mutant of BMP2 gene ( BMP2+ gene)was ligated into the multiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP-1 by the directional cloning method. The analysis of restricion map was adopted to identify the correct recombinants to monitor the expression of BMP2 and GFP in the MSCs and the HEK293A, the recombinants were transfected, fluorescence microscope and immunol histochemistry were employed. Rsults The plasimid (pShutfle CMV-BMP2 + -IRES-hrGFP-1 ) was centrcted correctly by two kinds of rectriction endnoucleases and sequence of the recombinant. The GFP and BMP2 were expressed in HEK293A and MSCs. Conclusion The adenovirus shuttle plasmid pShuttle CMV-BMP2+-IRES-hrGFP-1 is constructed successfully.%目的 构建同时表达具有抗原表位FLAG标记的重组人骨形态发生蛋白2(rhBMP2)目的蛋白和示踪绿色荧光蛋白(GFP)报告分子的腺病毒穿梭质粒pShuttle CMV-BMP2+-IRES-hrGFP-1.方法 采用PCR技术对pcDNA3-BMP2携带的BMP2基因诱变,去除翻译终止密码子后的基因序列并添加新的酶切识别位点XhoⅠ.测序检测诱变情况,将诱变后的BMP2基因定向导入pShuttle CMV-IRES-hrGFP-1,将质粒分别转染人胚肾细胞HEK293A和兔骨髓间充质干细胞(MSCs).通过限制性内切酶酶切图谱分析该质粒;采用荧光显微镜检查和免疫组化SP法测定HEK293A中的GFP和MSCs中的BMP2,行重组腺病毒穿梭质粒鉴定.结果 质粒pShuttle CMV-BMP2+-IRES-hrGFP-1经双酶切鉴定图谱分析构建正确,HEK293A、MSCs中均有GFP和BMP2表达.结论 p

  8. Acceleration of bone formation and implant osseointegration by BMP-2/7 heterodimers in vivo%重组BMP2/7诱导成骨和种植体骨整合的动物实验研究

    Institute of Scientific and Technical Information of China (English)

    王靖虓; 谷志远; 赵娟; 郑园娜; 刘铁; 毛英杰; 张欣; 高丽霞

    2009-01-01

    目的:探讨重组BMP2/7异二聚体在诱导成骨和骨整合中的作用.方法:10只广西巴马小型猪颅骨上,用空心钻造成直径8 mm、深4 mm的7个骨缺损.在缺损中心植入直径2.8 mm、长10mm的经喷砂处理纯钛种植体,缺损内随机填塞BMP2/7胶原海绵、BMP2胶原海绵、胶原海绵,分为BMP2/7、BMP2、胶原海绵对照组.于术后14天和21天分别处死5只动物、取材,行硬组织切片和显微CT检查.结果:术后14天BMP2/7组BV(Bone Volume)均值为45.4215mm3、BV/TV(TissueVolume)为0.2872,BMP2组BV均值为29.0673mm3、BV/TV为0.1991,胶原海绵组BV均值为17.1824mm3、BV/TV为0.1167.术后21天BMP2/7组BV均值为67.3024mm3、BV/TV为0.4610,BMP2组BV均值为52.1323mm3、BV/TV为0.3373,胶原海绵组BV均值为34.0631mm3、BV/TV为0.2309.经统计学检查,三组之间存在明显差异(P<0.05).结论:BMP2/7比BMP2有更好的诱导成骨和种植体骨整合作用.

  9. Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP2 Gene%携带IRES的BMP2绿色荧光蛋白表达载体的构建和表达

    Institute of Scientific and Technical Information of China (English)

    邹海波; 安洪; 蒋电明; 刘建伟; 周亚莉

    2004-01-01

    目的:构建携带有BMP2基因的pIRES2绿色荧光蛋白表达载体,为BMP2在体内外表达及蛋白定位提供标记.方法:酶切pCDAN3.1-BMP2质粒,获得BMP2cDNA片段并亚克隆至pUC18载体上,随后用SalⅠ单酶切pUC18-BMP2,再次获得BMP2cDNA,同时用SalⅠ将载体pIRES2-EGFP线性化后去磷酸化修饰.连接二者构建重组质粒.酶切筛选阳性克隆并鉴定插入的方向.重组质粒转染细胞,用激光共聚焦显微镜、RT-PCR及流式细胞仪检测基因在细胞内的定位、表达及转染效率;结果:重组载体经酶切和测序证明构建正确,并在细胞中表达.结论:成功构建了pIRES2-BMP2-EGFP表达载体,为研究BMP2的细胞内定位提供了一个重要而方便的工具.

  10. 新型人骨形成蛋白2逆转录病毒载体的构建及活性检测%Construction and identification of recombinant retroviral vector expressing BMP2 gene

    Institute of Scientific and Technical Information of China (English)

    段有文; 赤人杰; 陈晓春; 关景玉; 白宏治; 常洪涛

    2008-01-01

    目的 构建表达重组人骨形成蛋白2(BMP2)基因的重组逆转录病毒,对其在成骨细胞中的生物学作用进行探讨.方法 克隆BMP2基因,与pDNR-CMV连接构成pDNR-CMV-BMP2,然后将重组质粒pDNR-CMV-BMP2和逆转录病毒质粒pLP-LNCX以loxP位点进行同源重组,构成逆转录病毒载体pLP-LNCX-BMP2,转染包装细胞PT67进行病毒包装,NIH3T3细胞测定病毒滴度;将逆转录病毒感染人成骨细胞,噻唑蓝(MTT)法检测细胞生长变化,Western blot检测BMP2蛋白表达.结果 重组逆转录病毒载体pLP-LNCX-BMP2经鉴定连接正确;病毒载体pLP-LNCX-BMP2转染PT67后,上清液中病毒滴度可达到5×108pfu;MTT检测见逆转录病毒组与对照组比,48和72h细胞抑制率(5.1%比5.3%,8.5%比8.3%)差异无统计学意义(P>0.05),转染48 h后Westernblot可见BMP2蛋白高表达.结论 成功构建了BMP2逆转录病毒,为BMP2基因治疗及其功能研究提供了有效的手段.%Objective To construct recombinant retrovirus expressing human bone morphogenefic protein-2 gene ( BMP2 ) and investigate its biological function in osteoblasts. Methods BMP2 gene was amplified and reconstructed with pDNR-CMV into pDNR-CMV-BMP2 plasmid. Recombinant plasmid pD-NR-CMV-BMP2 and retroviral plasmid pLP-LNCX were recombinated homologously in loxP sites into pLP-LNCX-BMP2 plasmid transferred into packing cell line PT67. The viral titer was tested by NIH3T3 cells.Human osteoblasts were transfected with retrovirus. By using MTT assay, the changes in cell growth were measured. The expression of BMP2 protein was detected by Western blot. Results Recombinant retrovirus vector pLP-LNCX-BMP2 was constructed successfully. After transfection of pLP-LNCX-BMP2 into PT67,viral titer in the supernatant was up to 5 × 108 pfu. The cell growth inhibition rate at 48 and 72 h had no significant difference between retrovirus group and control group (5.1% vs 5.3% ,8.5% vs 8.3% ,P >0.05). After transfection for 48 h, the expression of

  11. Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration

    Science.gov (United States)

    Kim, Tae-Hyun; Singh, Rajendra K.; Kang, Min Sil; Kim, Joong-Hyun; Kim, Hae-Won

    2016-04-01

    The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ~73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.The recent development of bioactive glasses with nanoscale morphologies has

  12. In situ osteogenesis: regeneration of 10-cm mandibular defect in porcine model using recombinant human bone morphogenetic protein-2 (rhBMP-2) and Helistat absorbable collagen sponge.

    Science.gov (United States)

    Carstens, Michael H; Chin, Martin; Li, X Jian

    2005-11-01

    Traditional bone grafting relies upon the incorporation of a bone-cell bearing structure into a recipient site. The graft serves as a scaffold that is eventually replaced and remodeled. This process is known as osteoconduction. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is commercially available as an acellular implant in which the protein is bound to an absorbable collagen sponge (ACS). The rhBMP-2/ACS implant converts undifferentiated mesenchymal stem cells into osteoblasts and promotes an intense local neovascular response. This process, known as osteoinduction, produces bone via membranous, chondroid, or endochondral ossification. The type of bone synthesis depends upon the mesenchymal substrate and the local cellular environment. Using this simple technique, bone defects can be resynthesized with good outcomes and a significant reduction in donor site morbidity. Repair of a critical-sized mandibular resection defect with ISO is described. Basic science concepts of rhBMP-2, relevant histopathologic findings, and clinical application are described.

  13. Expression of BMP2 and vegf165 genes simultaneity transfected mouse bone marrow mesenchymal stem cells%BMP2、VEGF165双基因共表达质粒在小鼠骨髓基质干细胞的表达

    Institute of Scientific and Technical Information of China (English)

    张恒; 田晓滨; 孙立; 杨述华; 胡如印; 汪雷; 陆延盛; 张宇坤; 傅德皓

    2009-01-01

    目的 观察转染BMP2和VEGF165双基因共表达质粒在小鼠骨髓基质干细胞的表达情况.方法 脂质体介导下将双基因真核表达质粒pIRES-BMP2-VEGF165导入小鼠骨髓基质干细胞,用RT-PCR和免疫组织化学方法观察BMP2和VEGF165双基因在小鼠骨髓基质干细胞内的表达.结果 转染BMP2、VEGF165的小鼠骨髓基质干细胞有明显的BMP2和VEGF165mRNA及其蛋白表达.结论 转染BMP2和VEGF165双基因的小鼠骨,髓基质干细胞能同时表达以上两种基因.

  14. Mesoporous calcium-silicon xerogels with mesopore size and pore volume influence hMSC behaviors by load and sustained release of rhBMP-2.

    Science.gov (United States)

    Song, Wenhua; Li, Xiangde; Qian, Jun; Lv, Guoyu; Yan, Yonggang; Su, Jiacan; Wei, Jie

    2015-01-01

    Mesoporous calcium-silicon xerogels with a pore size of 15 nm (MCS-15) and pore volume of 1.43 cm(3)/g were synthesized by using 1,3,5-mesitylene (TMB) as the pore-expanding agent. The MCS-15 exhibited good degradability with the weight loss of 50 wt% after soaking in Tris-HCl solution for 56 days, which was higher than the 30 wt% loss shown by mesoporous calcium-silicon xerogels with a pore size of 4 nm (MCS-4). The pore size and pore volume of MCS-15 had significant influences on load and release of recombinant human bone morphogenetic protein-2 (rhBMP-2). The MCS-15 had a higher capacity to encapsulate a large amount of rhBMP-2; it could adsorb 45 mg/g of rhBMP-2 in phosphate-buffered saline after 24 hours, which was more than twice that with MCS-4 (20 mg/g). Moreover, the MCS-15 system exhibited sustained release of rhBMP-2 as compared with MCS-4 system (showing a burst release). The MCS-15/rhBMP-2 system could promote the proliferation and differentiation of human mesenchymal stem cells, showing good cytocompatibility and bioactivity. The results indicated that MCS-15, with larger mesopore size and higher pore volume, might be a promising carrier for loading and sustained release of rhBMP-2, which could be used as bone repair material with built-in osteoinduction function in bone reconstruction.

  15. Effects of BMP-2 and dexamethasone on osteogenic differentiation of rat dental follicle progenitor cells seeded on three-dimensional beta-TCP

    Energy Technology Data Exchange (ETDEWEB)

    Xu Lulu; Jin Zuolin; Duan Yinzhong [Department of Orthodontics, Stomatological College, Fourth Military Medical University, Xi' an 710032 (China); Liu Hongchen; Wang Dongsheng; E Lingling [Department of Stomatology, China PLA General Hospital, Beijing 100853 (China); Xu Lin, E-mail: jinzuolin88@yahoo.com.c, E-mail: duanyinzhong@yahoo.com.c [Department of Stomatology, the First Hospital of PLA, Lanzhou 730000 (China)

    2009-12-15

    The aim of this study was to investigate the effects of BMP-2 and dexamethasone (Dex) on osteogenic differentiation of rat dental follicle progenitor cells (RDFCs) seeded on three-dimensional beta-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial beta-TCP after 1-3, 3-7, 7-14 days of culture were examined respectively. The growth of cells on the scaffolds was observed by scanning electron microscope (SEM) after 3, 7 days of culture and by implanting in the backs of severe combined immunodeficient (SCID) mice for bone regeneration. The third passage RDFCs could be seen adhered, extended and proliferated on the beta-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10{sup -8} M) or/and BMP-2 (100 ng ml{sup -1}) were significantly higher than their existence in the control group. They were the significantly highest among four groups after joint application of BMP-2 and dexamethasone. After 8 weeks of implantation, the percentage of the new bones formed area in the RDFCs+beta-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+beta-TCP+BMP-2 group. In contrast, beta-TCP, RDFCs+beta-TCP+Dex and control constructs lacked new bone formation by histological staining and histomorphometric analysis. The BMP-2+Dex could significantly promote osteogenic differentiation of RDFCs on beta-TCP. beta-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

  16. Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing

    Directory of Open Access Journals (Sweden)

    M.T. Xu

    2016-01-01

    Full Text Available Transforming growth factor beta 1 (TGF-β1 and bone morphogenetic protein-2 (BMP-2 are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05. TGF-β1 and BMP-2 expression were significantly lower (P<0.05 in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-β1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-β1 and BMP-2.

  17. Off-label innovation: characterization through a case study of rhBMP-2 for spinal fusion.

    Science.gov (United States)

    Schnurman, Zane; Smith, Michael L; Kondziolka, Douglas

    2016-09-01

    OBJECTIVE Off-label therapies are widely used in clinical practice by spinal surgeons. Some patients and practitioners have advocated for increased regulation of their use, and payers have increasingly questioned reimbursment for off-label therapies. In this study, the authors applied a model that quantifies publication data to analyze the developmental process from initial on-label use to off-label innovation, using as an example recombinant human bone morphogenetic protein 2 (rhBMP-2) because of its wide off-label use. METHODS As a case study of off-label innovation, the developmental patterns of rhBMP-2 from FDA-approved use for anterior lumbar interbody fusion to several of its off-label uses, including posterolateral lumbar fusion, anterior cervical discectomy and fusion, and posterior lumbar interbody fusion/transforaminal lumbar interbody fusion, were evaluated using the "progressive scholarly acceptance" (PSA) model. In this model, PSA is used as an end point indicating acceptance of a therapy or procedure by the relevant scientific community and is reached when the total number of peer-reviewed studies devoted to refinement or improvement of a therapy surpasses the total number assessing initial efficacy. Report characteristics, including the number of patients studied and study design, were assessed in addition to the time to and pattern of community acceptance, and results compared with previous developmental study findings. Disclosures and reported conflicts of interest for all articles were reviewed, and these data were also used in the analysis. RESULTS Publication data indicated that the acceptance of rhBMP-2 off-label therapies occurred more rapidly and with less evidence than previously studied on-label therapies. Additionally, the community appeared to respond more robustly (by rapidly changing publication patterns) to reports of adverse events than to new questions of efficacy. CONCLUSIONS The development of off-label therapies, including the

  18. Gene Delivery of TGF-β3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.

    Science.gov (United States)

    Gonzalez-Fernandez, Tomas; Tierney, Erica G; Cunniffe, Grainne M; O'Brien, Fergal J; Kelly, Daniel J

    2016-05-01

    Incorporating therapeutic genes into three-dimensional biomaterials is a promising strategy for enhancing tissue regeneration. Alginate hydrogels have been extensively investigated for cartilage and bone tissue engineering, including as carriers of transfected cells to sites of injury, making them an ideal gene delivery platform for cartilage and osteochondral tissue engineering. The objective of this study was to develop gene-activated alginate hydrogels capable of supporting nanohydroxyapatite (nHA)-mediated nonviral gene transfer to control the phenotype of mesenchymal stem cells (MSCs) for either cartilage or endochondral bone tissue engineering. To produce these gene-activated constructs, MSCs and nHA complexed with plasmid DNA (pDNA) encoding for transforming growth factor-beta 3 (pTGF-β3), bone morphogenetic protein 2 (pBMP2), or a combination of both (pTGF-β3-pBMP2) were encapsulated into alginate hydrogels. Initial analysis using reporter genes showed effective gene delivery and sustained overexpression of the transgenes were achieved. Confocal microscopy demonstrated that complexing the plasmid with nHA before hydrogel encapsulation led to transport of the plasmid into the nucleus of MSCs, which did not happen with naked pDNA. Gene delivery of TGF-β3 and BMP2 and subsequent cell-mediated expression of these therapeutic genes resulted in a significant increase in sulfated glycosaminoglycan and collagen production, particularly in the pTGF-β3-pBMP2 codelivery group in comparison to the delivery of either pTGF-β3 or pBMP2 in isolation. In addition, stronger staining for collagen type II deposition was observed in the pTGF-β3-pBMP2 codelivery group. In contrast, greater levels of calcium deposition were observed in the pTGF-β3- and pBMP2-only groups compared to codelivery, with a strong staining for collagen type X deposition, suggesting these constructs were supporting MSC hypertrophy and progression along an endochondral pathway. Together, these

  19. Genetic Dissection of Ventral Folding Morphogenesis in Mouse: Embryonic Visceral Endoderm-supplied BMP2 Positions Head and Heart

    Science.gov (United States)

    Gavrilov, Svetlana; Lacy, Elizabeth

    2013-01-01

    Ventral folding morphogenesis, a vital morphogenetic process in amniotes, mediates gut endoderm internalization, linear heart tube formation, ventral body wall closure and encasement of the fetus in extraembryonic membranes. Aberrant ventral folding morphogenesis underlies a number of birth defects, such as gastroschisis and ectopia cordis in human and misplacement of head and heart in mouse. Recent cell lineage-specific mouse mutant analyses identified the Bone Morphogenetic Protein (BMP) pathway and Anterior Visceral Endoderm (AVE) as key regulators of anterior ventral folding morphogenesis. Loss of BMP2 expression solely from embryonic visceral endoderm (EmVE) and the AVE blocks formation of foregut invagination, and simultaneously, aberrantly positions the heart anterior/dorsal to the head, suggesting a mechanistic link between foregut and head/heart morphogenesis. PMID:23706163

  20. Differential expression of Bmp2, Bmp4 and Bmp3 in embryonic development of mouse anterior and posterior palate

    Institute of Scientific and Technical Information of China (English)

    NIE Xu-guang

    2005-01-01

    Background The palate is differently regulated and developed along the anterior-posterior axis. The Bmp signal pathway plays a crucial role in palatogenesis. Conditioned-inactivation of Bmp type I receptor Alk2 or Alk3 in the neural crest or craniofacial region leads to palatal cleft in mice. However, how different Bmp members are involved in palatogenesis remains to be elucidated. In the present study, mRNA expression patterns of Bmp2, Bmp3 and Bmp4 in the developing anterior and posterior palates were examined and compared, focusing on the fusion stage. Methods To detect the expression of Bmp mRNA, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on sagital and coronal sections of mice head from E13 to E18. Results The expression of these Bmps were developmentally regulated in the anterior and posterior palates prior to, during and after palatal fusion. During palatal fusion, Bmp4 expression shifted from the anterior to the posterior palate, Bmp2 was highly expressed in both the anterior and posterior palates in this process, whereas Bmp3 was only localized in the posterior palate. They showed generally non-overlapping pattern in their expression domains. Thereafter, their expression was detected in both the anterior and posterior palates regulating osteogenesis and myogenesis respectively. Conclusions Bmp signalling is involved in palatogenesis in multiple stages and has multiple roles in regulating anterior and posterior palatal development. Disturbances of Bmp signalling during palatogenesis might be a possible mechanism of cleft palate.

  1. Heterodimeric BMP-2/7 antagonizes the inhibition of all-trans retinoic acid and promotes the osteoblastogenesis.

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    Wenjuan Bi

    Full Text Available OBJECTIVES: Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration. MATERIALS AND METHODS: We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation, alkaline phosphatase activity (a marker for early differentiation, osteocalcin (a marker for late differentiation, calcium deposition (a marker for final mineralization and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin at different time points. RESULTS: All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28(th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7. CONCLUSIONS: Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism.

  2. BMP2、VEGF165双基因修饰的小鼠BMSCs复合人工骨的体内诱导成骨%In vivo osteogenic potential of calcium phosphate cement seeded with BMP2-and VEGF165-transduced mice BMSCs

    Institute of Scientific and Technical Information of China (English)

    孙立; 姜小峰; 田晓滨; 田家亮; 杨先腾; 胡如印; 张一; 韩伟; 陈涛

    2014-01-01

    目的 观察BMP2、VEGF165双基因转染的小鼠BMSCs复合自固化磷酸钙人工骨后的异位诱导成骨能力. 方法 脂质体介导下将含BMP2、VEGF165双基因的真核表达质粒pIRES转染小鼠BMSCs,转染空白质粒作阴性对照.用免疫组织化学和Western blot方法观察BMP2和VEGF165双基因在小鼠BMSCs内的表达;以自固化磷酸钙人工骨为支架,建立真核表达质粒pIRES-BMP2-VEGF165双基因修饰的组织工程骨植入小鼠右侧股骨肌袋内;将复合转染空白质粒小鼠BMSCs的自固化磷酸钙人工骨植入小鼠左侧股骨肌袋内.于术后第3天、第2,4,6周进行X线摄影对比后处死小鼠,取肌袋内组织行病理切片染色,观察成骨情况. 结果 小鼠BMSCs在转染BMP2、VEGF165基因后相关蛋白显著表达,由真核表达质粒pIRES-BMP2-VEGF165双基因修饰的组织工程骨在X线和病理学检查中相对于复合转染空白质粒小鼠BMSCs的组织工程骨具有更明显的异位成骨能力. 结论 由BMP2、VEGF165双基因质粒构建的组织工程骨有明显的异位成骨能力.%Objective To evaluate the ectopic osteogenic potential of calcium phosphate cement seeded with BMP2-and VEGF165-transduced mice BMSCs.Methods BMSCs transfected with recombinant plasmid pIRES-BMP2-VEGF165 using a liposome method were implanted into the muscle pouches of right thigh of the mice.BMSCs transfected only with pIRES served as a negative control and implanted into the muscle pouches of right thigh of the mice.Expressions of BMP2 and VEGF165 genes were assayed by immunohistochemical analysis and Western blot.Ectopic bone formation was evaluated by radiography and histology at 3 days and 2,4,and 6 weeks after implantation.Results Expressions of BMP2 and VEGF165 proteins were highly improved in BMP2-and VEGF165-transduced BMSCs.Calcium phosphate cement with BMSCs transfected with pIRES-BMP2-VEGF165 provided better ectopic osteogenesis compared to control group.Conclusion Tissue

  3. Effects of rhBMP-2 on Sandblasted and Acid Etched Titanium Implant Surfaces on Bone Regeneration and Osseointegration: Spilt-Mouth Designed Pilot Study

    Directory of Open Access Journals (Sweden)

    Nam-Ho Kim

    2015-01-01

    Full Text Available This study was conducted to evaluate effects of rhBMP-2 applied at different concentrations to sandblasted and acid etched (SLA implants on osseointegration and bone regeneration in a bone defect of beagle dogs as pilot study using split-mouth design. Methods. For experimental groups, SLA implants were coated with different concentrations of rhBMP-2 (0.1, 0.5, and 1 mg/mL. After assessment of surface characteristics and rhBMP-2 releasing profile, the experimental groups and untreated control groups (n = 6 in each group, two animals in each group were placed in split-mouth designed animal models with buccal open defect. At 8 weeks after implant placement, implant stability quotients (ISQ values were recorded and vertical bone height (VBH, mm, bone-to-implant contact ratio (BIC, %, and bone volume (BV, % in the upper 3 mm defect areas were measured. Results. The ISQ values were highest in the 1.0 group. Mean values of VBH (mm, BIC (%, and BV (% were greater in the 0.5 mg/mL and 1.0 mg/mL groups than those in 0.1 and control groups in buccal defect areas. Conclusion. In the open defect area surrounding the SLA implant, coating with 0.5 and 1.0 mg/mL concentrations of rhBMP-2 was more effective, compared with untreated group, in promoting bone regeneration and osseointegration.

  4. Improved anchorage of Ti6Al4V orthopaedic bone implants through oligonucleotide mediated immobilization of BMP-2 in osteoporotic rats.

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    Julia V Wölfle

    Full Text Available The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo.

  5. Improved Anchorage of Ti6Al4V Orthopaedic Bone Implants through Oligonucleotide Mediated Immobilization of BMP-2 in Osteoporotic Rats

    Science.gov (United States)

    Wölfle, Julia V.; Fiedler, Jörg; Dürselen, Lutz; Reichert, Judith; Scharnweber, Dieter; Förster, Anne; Schwenzer, Bernd; Reichel, Heiko; Ignatius, Anita; Brenner, Rolf E.

    2014-01-01

    The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo. PMID:24465929

  6. Intestinal Mucosal Barrier Is Injured by BMP2/4 via Activation of NF-κB Signals after Ischemic Reperfusion

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    Kang Chen

    2014-01-01

    Full Text Available Intestinal ischemic reperfusion (I/R can cause dysfunction of the intestinal mucosal barrier; however, the mechanism of the intestinal mucosal barrier dysfunction caused by I/R remains unclear. In this study, using intestinal epithelial cells under anaerobic cultivation and an in vivo rat intestinal I/R model, we found that hypoxia and I/R increased the expression of BMP2/4 and upregulated BMP type Ia receptor and BMP type II receptor expression. We also found that exogenous BMP2/4 can activate the ERK and AKT signaling pathways in rat small intestine (IEC-6 cells, thereby activating NF-κB signaling, which leads to increased levels of inflammatory factors, such as TNF-α and IL-6. Furthermore, recombinant BMP2/4 decreased the expression of the tight junction protein occludin via the activation of the NF-κB pathway; these effects were abolished by treatment with the BMP-specific antagonist noggin or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC. All these factors can destroy the intestinal mucosal barrier, thereby leading to weaker barrier function. On the basis of these data, we conclude that BMP2/4 may act as the pathogenic basis for intestinal mucosal barrier dysfunction when the intestines suffer an I/R injury. Our results provide background for the development pharmacologic interventions in the management of I/R injury.

  7. TNF-α对大鼠牙乳头细胞中BMP2及相关蛋白的表达调节研究

    Institute of Scientific and Technical Information of China (English)

    万艳

    2013-01-01

    目的:在大鼠牙乳头间充质细胞中探讨肿瘤坏死因子α(TNF-α)对骨形态发生蛋白2(BMP2)及矿化相关蛋白的表达调控.方法:体外分离培养SD新生大鼠牙乳头间充质细胞,在添加 TNF-α梯度的血清培养基中诱导培养,RT-PCR 、WB及ELISA方法检测骨形态发生蛋白2(BMP2)表达情况;添加 TNF-α不同时间梯度,RT-PCR、WB检测成骨分化标志蛋白碱性磷酸酶(ALP)、骨涎蛋白(BSP)表达变化.结果:TNF-α处理后,大鼠牙乳头间充质细胞中BMP2表达上调,ALP、BSP表达有所提高.结论:在大鼠牙乳头细胞中,TNF-α能够上调BMP2表达,进而诱导成牙骨质细胞矿化相关蛋白ALP,BSP表达,对牙齿发育矿化可能起到促进作用.

  8. TNF-α对大鼠牙乳头细胞中BMP2及相关蛋白的表达调节研究

    Institute of Scientific and Technical Information of China (English)

    万艳

    2013-01-01

    目的:在大鼠牙乳头间充质细胞中探讨肿瘤坏死因子α(TNF-α)对骨形态发生蛋白2(BMP2)及矿化相关蛋白的表达调控。方法:体外分离培养SD新生大鼠牙乳头间充质细胞,在添加TNF-α梯度的血清培养基中诱导培养,RT-PCR、WB及ELISA方法检测骨形态发生蛋白2(BMP2)表达情况;添加TNF-α不同时间梯度,RT-PCR、WB检测成骨分化标志蛋白碱性磷酸酶(ALP)、骨涎蛋白(BSP)表达变化。结果:TNF-α处理后,大鼠牙乳头间充质细胞中BMP2表达上调,ALP、BSP表达有所提高。结论:在大鼠牙乳头细胞中,TNF-α能够上调BMP2表达,进而诱导成牙骨质细胞矿化相关蛋白ALP,BSP表达,对牙齿发育矿化可能起到促进作用。

  9. Improved Bone Formation in Osteoporotic Rabbits with the Bone Morphogenetic Protein-2 (rhBMP-2 Coated Titanium Screws Which Were Coated By Using Plasma Polymerization Technique

    Directory of Open Access Journals (Sweden)

    Salih Gulsen

    2014-06-01

    Full Text Available Delaying of bone fusion in osteoporotic patients underwent spinal stabilization surgery leads to screw loosening, and this causes pseudoarticulation, mobility and fibrosis at vertebral segments. To prevent these complications, the screws coated with recombinant bone morphogenetic protein-2 (rhBMP-2 could be used. To verify this hypothesis, we coated 5 Titanium screws with rhBMP-2 using plasma polymerization method, and also used 10 uncoated screws for making comparison between coated and uncoated screws in different groups. And 15 skeletally mature white New Zealand female rabbits were assigned into three different groups: Group 1(N = 5: No osteoporosis induction and insertion of uncoated Titanium screw into right sacrum of each rabbit in group 1; group 2 (N = 5: Osteoporosis induction and insertion of uncoated Titanium screw into right sacrum of each rabbit in group 2; group 3 (N = 5 rhBMP-2 coated Titanium screw inserted into right sacrum of each rabbit in group 3. In summary, using of these coated screws provides new bone formation, but causes less fibrosis and less inflammation than uncoated screws at the interface between the coated screw and bone. Then the plasma polymerization technique provides controlled releasing of rhBMP-2 from the screw to the bone tissue in osteoporotic rabbits.

  10. Immunohistochemical response in rats of beta-tricalcium phosphate (TCP) with or without BMP-2 in the production of collagen matrix critical defects.

    Science.gov (United States)

    Luvizuto, Eloá Rodrigues; de Oliveira, Júlio César Silva; Gomes-Ferreira, Pedro Henrique Silva; Pereira, Cassiano Costa Silva; Faverani, Leonardo Perez; Antoniali, Cristina; Okamoto, Roberta

    2017-03-02

    This study aimed to assess the biological response of BMP-2 (bone morphogenetic protein-2) in supplementation with β-tricalcium phosphate (TCP) as a carrier in the bone healing of surgical defects in rats' calvaria. A critical-size defect (5mm in diameter) was filled with β-TCP alone or added with that plus 5mg of BMP-2 at 5, 15, and 30 postoperative days. Histomorphometric and immunohistochemical (osteocalcin, collagen type I, and metalloproteinase-9) analysis was performed to assess the features of bone healing. Histological behavior and collagen type I labeling showed increased formation of the collagen matrix, leading to a higher percentage of newly formed bone and biomaterial for tissue and more total mineralization of pure TCP when compared to the other groups. The supplementation with BMP-2 promoted faster TCP remodeling; however, there was no statistically significant difference for the bone formed in both groups (P>0.05). Collagen-matrix formation and new bone formation reached maximum levels when the defects were filled with pure TCP, even exceeding the levels from BMP-2 supplementation.

  11. 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

  12. Mesoporous calcium–silicon xerogels with mesopore size and pore volume influence hMSC behaviors by load and sustained release of rhBMP-2

    Directory of Open Access Journals (Sweden)

    Song W

    2015-03-01

    Full Text Available Wenhua Song,1,* Xiangde Li,1,* Jun Qian,1 Guoyu Lv,2 Yonggang Yan,2 Jiacan Su,3 Jie Wei1 1Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai, People’s Republic of China; 2College of Physical Science and Technology, Sichuan University, Chengdu, People’s Republic of China; 3Changhai Hospital, Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this paper Abstract: Mesoporous calcium–silicon xerogels with a pore size of 15 nm (MCS-15 and pore volume of 1.43 cm3/g were synthesized by using 1,3,5-mesitylene (TMB as the pore-expanding agent. The MCS-15 exhibited good degradability with the weight loss of 50 wt% after soaking in Tris-HCl solution for 56 days, which was higher than the 30 wt% loss shown by mesoporous calcium–silicon xerogels with a pore size of 4 nm (MCS-4. The pore size and pore volume of MCS-15 had significant influences on load and release of recombinant human bone morphogenetic protein-2 (rhBMP-2. The MCS-15 had a higher capacity to encapsulate a large amount of rhBMP-2; it could adsorb 45 mg/g of rhBMP-2 in phosphate-buffered saline after 24 hours, which was more than twice that with MCS-4 (20 mg/g. Moreover, the MCS-15 system exhibited sustained release of rhBMP-2 as compared with MCS-4 system (showing a burst release. The MCS-15/rhBMP-2 system could promote the proliferation and differentiation of human mesenchymal stem cells, showing good cytocompatibility and bioactivity. The results indicated that MCS-15, with larger mesopore size and higher pore volume, might be a promising carrier for loading and sustained release of rhBMP-2, which could be used as bone repair material with built-in osteoinduction function in bone reconstruction. Keywords: mesoporous calcium–silicon xerogels, pore size, pore volume, load-release, rhBMP-2

  13. 生物力学因素与BMP2单核苷酸多态性对颈椎后纵韧带骨化成骨作用的影响%Mutual Effect of Biomechanical Factor and BMP2 Gene Nucleotide Polymorphisms on Osteogenic Differentiation in Ossification of the Posterior Longitudinal Ligament (OPLL)

    Institute of Scientific and Technical Information of China (English)

    张耀; 刘宝戈; 林欣; 宋磊; 李家谋; 赵虎成

    2014-01-01

    目的 探讨生物力学因素与骨形态发生蛋白-2(bone morphogenetic protein 2,BMP2)单核苷酸多态性对颈椎后纵韧带骨化(ossification of the posterior longitudinal ligament,OPLL)成骨作用的影响,为进一步发现OPLL多因素共同致病的具体机制奠定基础.方法 采集颈椎OPLL病变组手术减压过程中骨化部位的后纵韧带和颈椎外伤等手术减压过程中的正常对照组后纵韧带.利用PCR和直接测序法分析BMP2上2个单核苷酸多态性位点109T>G(rs2273073),570A>T(rs235768)基因型及等位基因型的分布.Masson三色染色观察组织学结构改变.免疫组织化学染色和Western blot法检测BMP2的分布和表达.用C3H10T1/2细胞做转染构建细胞模型:正常组、空载体组、BMP2野生型组、BMP2(rs2273073)单突变组、BMP2(rs235768)单突变组、BMP2(rs2273073,rs235768)双突变组.采用机械应力装置对接种于Flexercell板的细胞施加10%、0.5Hz的机械应力,持续加载24h.以同样接种于Flexereell板未施加机械应力的细胞作为对照组.采用Western blot法检测机械应力加载前后各组BMP2表达情况.结果 Masson三色染色组织学观察显示病变组有成骨结构改变,免疫组织化学染色和Western blot法显示病变组BMP2的表达增加.机械应力加载后,细胞转染模型BMP2(rs2273073)单突变组和BMP2(rs2273073,rs235768)双突变组的BMP2表达较未施加应力组明显增加,差异具有统计学意义(P<0.05).结论 BMP2单核苷酸多态性位点109T>G(rs2273073)的突变不仅能提高人们对OPLL的易感性,同时能提高OPLL患者对机械应力的敏感度,从而加速OPLL的进展.

  14. BMP2 expression impacts the growth and osteogenic differentiation of BMSCs in the co-culture system%BMP2的表达对联合培养体系中骨髓间充质干细胞增殖和成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    董苑; 杨瑞年; 王福科; 刘流

    2013-01-01

    目的 研究BMP2在hBMSCs和hUVECs体外联合培养系统中对细胞增殖和成骨分化的调控作用. 方法 选用第三代hUVECs、hBMSCs细胞以及经BMP2特异性静默干扰处理的hUVECs细胞建立联合培养体系.观察不同培养组单独hBMSCs组、hUVECs与hBMSCs细胞联合培养组以及经BMP2蛋白静默干扰处理hUVECs与hBMSCs细胞联合培养干扰组中BMSCs数量,运用酶活性法和放射免疫法分别对碱性磷酸酶及骨钙素的表达进行检测. 结果 在BMP2静默的hUVECs联合hBMSCs培养干扰组观察到随BMP2蛋白分泌减少,hBMSCs的细胞数目下降,但仍高于单独hBMSCs培养组,差异有统计学意义(P<0.05).联合培养体系中运用RNA干扰技术静默血管内皮细胞对BMP2的表达后,联合培养干扰组内hBMSCs对碱性磷酸酶、骨钙素在同一时间点表达低于联合培养组,但仍然高于单独间充质干细胞培养组,差异有统计学意义(P<0.05). 结论 联合培养体系中血管内皮细胞分泌的BMP2在BMSCs增殖和成骨分化诱导方面起着重要作用.%Objective To investigate the effects of BMP2 in the co-culture system of hBMSCs and hUVECs.Methods Based on the co-culture system of cells,a simple culture group (hBMSCs group),a combined culture group (BMSCs + hUVECs group) and a BMP2 silenced group (BMSCs + BMP2 silenced hUVECs) were established.On days 4,6,8,10,cells were counted for drawing cell growth curves.In addition,ALP and OC expression in BMSCs was detected in each group.Results At each time point,cell number in the BMSCs + hUVECs group were significantly greater than in the BMSCs group,while the BMP2 silenced group were in the median.ALP and OC expression in the BMSCs + hUVECs group were significantly greater than in the BMSCs group,while the BMP2 silenced group were in the median.Conclusion BMP2 plays an important role in the co-culture system,which can promote osteogenic differentiation and the growth of BMSCs.

  15. Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway

    Institute of Scientific and Technical Information of China (English)

    Jing Yang; Ling Ye; Tian-Qian Hui; Dong-Mei Yang; Ding-Ming Huang; Xue-Dong Zhou; Jeremy J Mao; Cheng-Lin Wang

    2015-01-01

    Both bone morphogenetic protein 2 (BMP2) and the wingless-type MMTV integration site (WNT)/b-catenin signalling pathway play important roles in odontoblast differentiation and dentinogenesis. Cross-talk between BMP2 and WNT/b-catenin in osteoblast differentiation and bone formation has been identified. However, the roles and mechanisms of the canonical WNT pathway in the regulation of BMP2 in dental pulp injury and repair remain largely unknown. Here, we demonstrate that BMP2 promotes the differentiation of human dental pulp cells (HDPCs) by activating WNT/b-catenin signalling, which is further mediated by p38 mitogen-activated protein kinase (MAPK) in vitro. BMP2 stimulation upregulated the expression of b-catenin in HDPCs, which was abolished by SB203580 but not by Noggin or LDN193189. Furthermore, BMP2 enhanced cell differentiation, which was not fully inhibited by Noggin or LDN193189. Instead, SB203580 partially blocked BMP2-induced b-catenin expression and cell differentiation. Taken together, these data suggest a possible mechanism by which the elevation of b-catenin resulting from BMP2 stimulation is mediated by the p38 MAPK pathway, which sheds light on the molecular mechanisms of BMP2-mediated pulp reparative dentin formation.

  16. rhBMP-2 protects against reoperation for pseudoarthrosis and/or instrumentation failure: A matched case-control study of 448 patients.

    Science.gov (United States)

    Macki, Mohamed; Syeda, Sbaa; Kerezoudis, Panagiotis; Bydon, Ali; Witham, Timothy F; Sciubba, Daniel M; Wolinsky, Jean-Paul; Bydon, Mohamad; Gokaslan, Ziya

    2016-10-01

    The objective of this independent study is to determine the impact of recombinant human bone morphogenetic protein 2 (rhBMP-2) on reoperation for pseudarthrosis and/or instrumentation failure. A nested case-control study of first-time posterolateral, instrumented fusion of the lumbar spine for degenerative spinal disease was undertaken. Cases of reoperation for pseudoarthrosis and/or instrumentation failure were assigned to controls, who did not experience the primary outcome measure at the time of reoperation. Cases and controls were matched on number of interspaces fused and inclusion of interbody. Predictors of reoperation for pseudoarthrosis and/or instrumentation failure were assessed with a conditional logistical regression controlling for rhBMP-2, age, obesity, and smoking. Of the 448 patients, 155 cases of reoperation for pseudoarthrosis and/or instrumentation were matched with 293 controls. Twenty-six percent of first-time surgeries included rhBMP-2, which was statistically more commonly used in the control cohort (33.11%) versus the case cohort (12.90%) (Unadjusted odds ratio [ORunadj]=0.28) (95% confidence interval [CI]: 0.16-0.49). Following a multivariate analysis controlling for age, obesity, and smoking, the rhBMP-2 recipients incurred a 73% lower odds of reoperation for pseudoarthrosis and/or instrumentation failure (95% CI, 0.15-0.48). Neither sarcomatous nor osseous neoplasm was detected in the study population. Mean follow up did not differ between the cases (81.57±standard deviation [SD] 4.98months) versus controls (74.75±2.49month) (ORunadj=1.01) (95% CI: 1.00-1.01). rhBMP-2 in lumbar fusion constructs protects against reoperation for pseudoarthrosis and/or instrumentation failure. However, the decision to include fusion supplements should be weighted between surgical determinants and clinical outcomes.

  17. Expression patterns indicate that BMP2/4 and Chordin, not BMP5-8 and Gremlin, mediate dorsal-ventral patterning in the mollusk Crassostrea gigas.

    Science.gov (United States)

    Tan, Sujian; Huan, Pin; Liu, Baozhong

    2016-12-16

    Though several bilaterian animals use a conserved BMP2/4-Chordin antagonism to pattern the dorsal-ventral (DV) axis, the only lophotrochozoan species in which early DV patterning has been studied to date, the leech Helobdella robusta, appears to employ BMP5-8 and Gremlin. These findings call into question the conservation of a common DV patterning mechanism among bilaterian animals. To explore whether the unusual DV patterning mechanism in H. robusta is also used in other lophotrochozoan species, we investigated the expression of orthologous genes in the early embryo of a bivalve mollusk, Crassostrea gigas. Searching of the genome and phylogenetic analysis revealed that C. gigas possesses single orthologs of BMP2/4, Chordin, and BMP5-8 and no Gremlin homolog. Whole mount in situ hybridization revealed mRNA localization of BMP2/4 and Chordin on the opposite sides of embryos, suggesting the potential involvement of a BMP2/4-Chordin antagonism in DV patterning in this species. Furthermore, universal BMP5-8 expression and the absence of a Gremlin homolog in the C. gigas genome called into question any major contribution by BMP5-8 and Gremlin to early DV patterning in this species. Additionally, we identified seven genes showing asymmetric expression along the DV axis, providing further insight into DV patterning in C. gigas. We present the first report of a Chordin gene in a lophotrochozoan species and of the opposite expression of BMP2/4 (dorsal) and Chordin (ventral) along the D/V axis of a lophotrochozoan embryo. The findings of this study further the knowledge of axis formation in lophotrochozoan species and provide insight into the evolution of the animal DV patterning mechanism.

  18. BMP-2基因修饰自体BMSCs移植促进兔下颌骨牵张成骨新骨形成的实验研究%EXPERIMENTAL STUDY ON TRANSPLANTATION OF BMP-2 GENE TRANSFECTED AUTOGENOUS BONE MESENCHYMAL STEM CELLS FOR PROMOTING BONE REGENERATION IN RABBIT MANDIBULAR DISTRACTION OSTEOGENESIS

    Institute of Scientific and Technical Information of China (English)

    黄旋平; 周诺; 江献芳; 杨媛媛; 李华; 谢庆条

    2012-01-01

    目的:检测骨形态发生蛋白-2(BMP-2)基因mRNA及其蛋白的表达,探讨BMP-2基因修饰自体骨髓间充质干细胞(BMSCs)移植对兔下颌骨牵张成骨新骨形成的促进作用.方法:取新西兰白兔36只随机分为3组,每组12只.建立牵张成骨动物模型,在固定期第2天,实验组于牵张间隙注射200 μL的BMP-2基因修饰的自体BMSCs液;对照组注射等量自体BMSCs液;空白组注射等量生理盐水.分别于固定2,6周通过逆转录多聚酶链式反应(RT-PCR)、免疫组化等手段检测BMP-2基因mRNA及其蛋白的表达情况.结果:实验组牵张间隙新生骨组织均可见BMP-2基因mRNA和其蛋白强阳性表达.结论:BMP-2基因修饰的自体BMSCs移植能有效促进兔下颌骨牵张成骨新骨形成.%Objective:To examine the expressions of both bone morphogenetic protein-2 (BMP-2) gene mR-NA and related proteins and investigate the promotive effect of transplantation of BMP-2 gene transfected autogenous bone mesenchymal stem cells on bone regeneration in rabbit mandibular distraction osteogene-sis. Methods: Thirty-six New Zealand's white rabbits were randomly divided into three groups with twelve in each. All objects -were prepared into distraction osteogenesis surgical model on right mandibles. On the 2nd day of consolidation, experimental, control, and blank groups -were injected -with the same amount of 200 juL of the solution with BMP-2 gene transfected autogenous bone mesenchymal stem cells, the solution with autogenous bone mesenchymal stem cells, and physiological saline at distraction gap, respectively. The expressions of BMP-2 mRNA and related proteins -were examined by RT-PCR and immunohistochem-istry at the ends of the 2nd and 6th -week consolidations, respectively. Results: Strongly positive expression of both BMP-2 gene mRNA and related proteins were confirmed on regenerated bone in distraction gap. Conclusion: The transplantation of BMP-2 gene transfected autogenous bone

  19. Radiographic, biomechanical, and histological evaluation of rhBMP-2 in a 3-level intertransverse process spine fusion: an ovine study.

    Science.gov (United States)

    Toth, Jeffrey M; Wang, Mei; Lawson, Joshua; Badura, Jeffrey M; DuBose, Kimberly Bailey

    2016-12-01

    OBJECTIVE The objective of this study was to evaluate bone grafts consisting of rhBMP-2 on an absorbable collagen sponge with a ceramic composite bulking agent, rhBMP-2, directly on a ceramic-collagen sponge carrier or iliac crest bone graft (ICBG) in combination with local bone graft to effect fusion in a multisegmental instrumented ovine lumbar intertransverse process fusion model. METHODS Thirty-six sheep had a single treatment at 3 spinal levels in both the right and left intertransverse process spaces. Group 1 sheep were treated with 7.5 cm(3) of autograft consisting of ICBG plus local bone for each intertransverse process space. For Groups 2-4, 4 cm(3) of local bone was placed within the intertransverse process space followed by 4.5-5 cm(3) of the rhBMP-2 graft material. Group 2 animals received 1.5 mg/cm(3) rhBMP-2 on an absorbable collagen sponge with a commercial bone void filler consisting of Type I lyophilized collagen with a biphasic hydroxyapatite/β-tricalcium phosphate ceramic with local bone. Group 3 animals received 0.75 mg/m cm(3) of rhBMP-2 on a collagen ceramic sponge carrier with local bone. Group 4 animals received 1.35 mg/cm(3) of rhBMP-2 on the same collagen ceramic sponge carrier with local bone. Sheep were euthanized 6 months postoperatively. Manual palpation, biomechanical testing, CT, radiography, and undecalcified histology were performed to assess the presence of fusion associated with the treatments. RESULTS All animals in Groups 2-4 that received grafts containing rhBMP-2 achieved radiographic and CT fusion at all 3 levels. In Group 1 (bone autograft alone), only 19% of the levels demonstrated radiographic fusion, 14% resulted in possible radiographic fusion, and 67% of the levels demonstrated radiographic nonfusion. Biomechanical testing showed that Groups 2-4 demonstrated similar stiffness of the L2-5 segment in all 6 loading directions, with each of the 3 groups having significantly greater stiffness than the autograft-only group

  20. Influence of age on the amount of MSCs and values of BMP2 in serum and cortical bone in rats%年龄因素对大鼠骨髓间充质干细胞数量及组织中BMP2含量的影响

    Institute of Scientific and Technical Information of China (English)

    汤亭亭; 岳冰; 陆斌; 郁朝锋; 楼觉人; 戴尅戎

    2005-01-01

    目的观察增龄对大鼠骨髓间充质干细胞(mesenchymal stromal cells,MSCs)数量及组织中骨形态发生蛋白2(bone morphogenetic proteins 2,BMP2)含量的影响,分析MSCs数量和BMP2含量变化与老年人骨量丢失及老年性骨质疏松症间的关系.方法取3种年龄层次的大鼠(1月龄、9月龄、24月龄),抽取骨髓进行体外培养,计算纤维细胞集落形成单位(colony forming units dfibroblastic,CFU-F)的数量;应用ELISA技术定量检测大鼠外周血以及股骨皮质骨中BMP2的含量.结果 MSCs数量随增龄显著减少.1月龄组皮质骨中以及血清中BMP2含量均明显高于其它两组,9月龄与24月龄大鼠间没有显著差异.结论骨髓中MSCs的数量随增龄而减少.外周血和皮质骨中BMP2的含量随增龄而减少.骨髓MSCs数量的减少及组织BMP2含量的降低可能是老年骨量丢失与老年性骨质疏松发病的重要原因.

  1. Cem-Ostetic/rhBMP-2复合物促进即刻种植体周围骨组织再生的研究%Study on Cem-Ostetic/rhBMP-2 Complex to Promote Bone Regeneration around Immediate Implants

    Institute of Scientific and Technical Information of China (English)

    郭盛; 史久慧; 王屹博; 丁超

    2011-01-01

    目的:研究Cem- Ostetic/rhBMP-2对即刻种植骨缺损修复的生物学特性.方法:将6只狗随即分成2组,拔除犬双侧下颌第二、三、四前磨牙,即刻植入种植体,每个种植体颊侧制备标准缺损,分别植入Cem- Ostetic/rhBMP-2、Cem- Ostetic、自体骨.术后6、12周分期处死,进行大体、影像学、组织学观测.结果:术后6、12周各组均有不同程度的骨组织再生.各个时期观察显示Cem- Ostetic/rhBMP-2组新生骨与种植体间的骨结合较其他组多.结论:Cem-Ostetic/rhBMP-2能有效促进种植体周围骨缺损修复,并促进种植体-骨界面形成较完善的骨整合.%Objective: To study the biologic effects of Cem-Ostetic/BMP-2 in rehabilitating bone defects during immediate implantation. Methods: Six dogs were divided into two groups randomly. We extracted each dog mandib-ular second third and fourth premolars to create edentulous regions. Immediate implantation was placed in the medial root site. Standard bone defects were prepared on the buccal side of the implants. The defects were filled with a composite consisting of Cem-Ostetic/rhBMP-2 , autologous bone graft and pure Cem-Ostetic. At 6 weeks and 12 weeks postoperative, the dogs were sacrificed for clinical check, radiological, and histological analysis. Results: At 6,12 weeks, various amount of new bone appeared in each group. Each time observation demonstrated that Cem - Ostetic/BMP-2 group had more new bone and osseointegration than the other groups. Conclusion: The Cem - Ostetic/ rhBMP - 2 composite can effectively accelerate the reconstruction of peri-implant bone defects and improve the osseointegration at the interface between the implant and bone.

  2. The relationship between perioperative serum PCT, BMP2 and postoperative hip functional recovery for patients with hip replacement%髋关节置换围术期血清PCT、BMP2水平与术后髋关节功能恢复的关系

    Institute of Scientific and Technical Information of China (English)

    叶握球; 冯奕鑫

    2015-01-01

    Objective To investigate the relationship between perioperative serum procalcitonin (PCT),bone morphogenetic protein 2 (BMP2) and postoperative recovery of hip function for patients with hip replacement.Methods A total of 148 patients with femoral neck fractures who underwent hip replacement from January 2013 to October 2014 in Puning People's Hospital,were randomly selected as the observation group,and 30 healthy who underwent physical examination during the same period were selected as the control group.The serum levels of PCT and BMP2 were detected by enzyme-linked immunosorbent assay (ELISA) before the operation and 1,3,5,7,14 days after the operation.Recovery of hip function was evaluated by Harris hip score at 1,3,6 months postoperatively.Pearson correlation analysis was applied to analyze the relationship between perioperative serum PCT,BMP2 levels and postoperative recovery of hip joint function.Results The levels of serum PCT and BMP2 in the observation group were higher than those in the control group at the different time-points (P <0.05).In observation group,preoperative serum levels of PCT and BMP2 were higher than those at 3,5,7 and 14 d postoperatively (P <0.05).Harris hip score after 6 months was as follows: 69 excellent,51 good,22 fair and 6 poor.The levels of serum PCT in the patients who evaluated as good and excellent hip function were lower,while levels of BMP2 were higher when compared with the patients with fair or poor hip function (P <0.05); Postoperative function of hip joint was negatively correlated with serum PCT levels,while was positively correlated with serum BMP2 levels (P <0.05).Conclusion For patients with hip replacement,serum PCT,BMP2 levels can be used as reference indexes to evaluate the recovery of hip function,which patients with high serum PCT and low serum BMP2 may show poor hip function recovery.%目的 探讨髋关节置换围术期血清降钙素原(PCT)和骨形态发生蛋白2(BMP2)水平与术后髋关节功能恢

  3. Effects of lentivirus-mediated co-transfection of BMP2 gene and VEGF165 gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells%慢病毒介导BMP2和VEGF165基因共转染对骨髓基质干细胞成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    蒋佳; 范存义; 曾炳芳

    2011-01-01

    Objective To investigate the effects of lentivirus-mediated co-transfection of bone morphogenetic protein 2 (BMP2) gene and vascular endothelial growth factor 165 ( VEGF165) gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells ( MSCs). Methods The expression lentivirus vectors carrying VEGF165, BMP2 or green fluorescent protein (GFP) gene were constructed respectively, and recombinant lentivirus carrying VEGF165 ( Lv-VEGF), BMP2 ( Lv-BMP) or GFP ( Lv-GFP) were packaged and produced respectively. Rat bone marrow-derived MSCs were isolated and cultured in vitro, and were transfected with Lv-VEGF (VEGF group), Lv-BMP ( BMP group) or Lv-GFP (GFP group), co-transfected with Lv-VEGF and Lv-BMP (BMP + VEGF group), or transfected with no virus ( control group). The expression of VEGF165 and BMP2 mRNA 7 d after transfection and that of osteocalcin (OCN) mRNA 1, 2 and 4 weeks after transfection in MSCs was detected by RT-PCR in each group. The expression of VEGF165 and BMP2 protein 1, 4 and 8 weeks after transfection and that of OCN protein 1, 2 and 4 weeks after transfection in supernatant fluid of culture was detected by ELISA in each group. Alkaline phosphatase (ALP) staining was conducted, and ALP activity of MSCs was measured 14 d after transfection in each group. Results VEGF165 and BMP2 mRNA and protein effectively co-expressed in BMP + VEGF group. There was no significant difference in the expression of BMP2 mRNA and protein between BMP + VEGF group and BMP group (P > 0. 05), and there was also no significant difference in the expression of VEGF165 mRNA and protein between BMP + VEGF group and VEGF group (P >0.05). There was no expression of BMP2 mRNA and protein in control group, GFP group and VEGF group, and there was also no expression of VEGF165 mRNA and protein in control group, GFP group and BMP group. The expression of OCN mRNA and protein in BMP + VEGF group was significantly higher than that in the other groups (P <0.01). The

  4. Efficacy of rhBMP-2 loaded PCL/PLGA/β-TCP guided bone regeneration membrane fabricated by 3D printing technology for reconstruction of calvaria defects in rabbit.

    Science.gov (United States)

    Shim, Jin-Hyung; Yoon, Min-Chul; Jeong, Chang-Mo; Jang, Jinah; Jeong, Sung-In; Cho, Dong-Woo; Huh, Jung-Bo

    2014-11-10

    We successfully fabricated a three-dimensional (3D) printing-based PCL/PLGA/β-TCP guided bone regeneration (GBR) membrane that slowly released rhBMP-2. To impregnate the GBR membrane with intact rhBMP-2, collagen solution encapsulating rhBMP-2 (5 µg ml(-1)) was infused into pores of a PCL/PLGA/β-TCP membrane constructed using a 3D printing system with four dispensing heads. In a release profile test, sustained release of rhBMP-2 was observed for up to 28 d. To investigate the efficacy of the GBR membrane on bone regeneration, PCL/PLGA/β-TCP membranes with or without rhBMP-2 were implanted in an 8 mm calvaria defect of rabbits. Bone formation was evaluated at weeks 4 and 8 histologically and histomorphometrically. A space making ability of the GBR membrane was successfully maintained in both groups, and significantly more new bone was formed at post-implantation weeks 4 and 8 by rhBMP-2 loaded GBR membranes. Interestingly, implantation with rhBMP-2 loaded GBR membranes led to almost entire healing of calvaria defects within 8 weeks.

  5. Effect of soybean isoflavonet on bone morphogenetic protein 2 expression of cultured osteoblasts from rat%大豆异黄酮与大鼠成骨细胞BMP2PCR产物表达

    Institute of Scientific and Technical Information of China (English)

    李万里; 余冰; 田玉慧; 杨献军; 沈关心

    2006-01-01

    目的探讨大豆异黄酮对体外培养大鼠成骨细胞骨形态发生蛋白2(BMP2)表达的影响.方法制作新生大鼠颅骨成骨细胞体外培养模型,用不同浓度的大豆异黄酮刺激分离培养的大鼠成骨细胞,提取细胞总RNA,RT-PCR扩增BMP2基因cDNA,同时扩增β-actin cDNA作为内对照,扫描RCR产物电泳照片,计算BMP-2/β-actin cDNA的积分吸光度比值,推算BMP2基因相对表达水平;并通过免疫组化方法观察成骨细胞BMP2的表达,用HPIAS-2000图象分析仪对各组表达进行定量分析.结果大豆异黄酮增加体外培养成骨细胞BMP2的水平,并且在基因转录水平促进大鼠成骨细胞BMP2表达,呈剂量-效应关系(P《0.01).结论大豆异黄酮对成骨细胞形成BMP2表达有显著促进作用.提示大豆异黄酮促进成骨细胞增殖、分化的作用可能与通过增加BMP2基因的表达有关.

  6. The effects of substrate-streching strain on the BMP-2 mRNA expression in three kinds of mouse cell lines%基底拉伸应变对小鼠三种骨组织细胞BMP-2 mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    闫玉仙; 宋梅; 郭春; 郭勇; 宫元伟; 李瑞欣; 张西正

    2010-01-01

    目的 研究基底拉伸应变对小鼠成骨细胞系MC3T3-E1、破骨细胞系RAW264.7及骨细胞MLO-Y4三种细胞BMP-2 mRNA表达的影响.方法 三种细胞随机分为0 με、1 000 με、1 500 με、2 000 με、2 500 με和5 000 με组,最佳拉伸时间和周期为1次/d,每次1 h,连续3 d,频率为0.5 Hz.采用卫生装备研究所自行设计研制的四点弯曲装置对小鼠三种细胞进行拉伸加载.采用RT-PCR技术分别研究不同应变对小鼠三种细胞BMP-2 mRNA表达.结果 MC3T3-E1细胞RT-PCR结果显示:1 500 με、2 000 με组和2 500 με组与0 με组相比BMP-2 mRNA表达显著增强(P<0.01);5 000 με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);RAW264.7细胞RT-PCR结果显示:1 500 με、2 000 με组和2 500 με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);5 000με组与0 με组相比BMP-2 mRNA表达显著降低(P<0.01);MLO-Y4细胞BMP2基因表达结果与MC3T3-E1一致.结论 ①BMP-2在成骨细胞系MC3T3-E1、破骨细胞系RAW-264.7及骨细胞系MLO-Y4三种细胞中均有表达;②1 500 με、2 000 με、2 500 με三种生理剂量的拉伸应变可以显著增加MC3T3-E1、MLO-Y4 细胞BMP-2的表达,并呈剂量依赖性,超生理剂量5 000 με可以显著降低MC3T3-E1、MLO-Y4细胞BMP-2的表达;③相同的力学拉伸作用条件下,BMP-2在RAW-264.7细胞中表达与MC3T3-E1、MLO-Y4细胞的表达趋势相反.

  7. Effects of different compatibility of TCM on BMP-2 expression at fracture site and healing of early radius fracture of rats%桃仁、木香、黄芪分别与红花配伍对大鼠早期桡骨骨折愈合过程中BMP-2表达的影响

    Institute of Scientific and Technical Information of China (English)

    王轩; 潘琪; 张慧萍; 李引刚

    2011-01-01

    目的:研究桃仁、木香、黄芪分别与红花配伍对实验性SD大鼠早期桡骨骨折愈合过程中骨痂厚度及BMP-2表达的影响,探讨骨折三期辨证的治疗方法.方法:756只SD大鼠造模后随机分为模型组、桃仁红花组、木香红花组、黄芪红花组4组,每组各64只,每组再随机平均分为4个小组,分别作为骨折后第3、7、10、14d组,每小组16只.于造模后第3、7、10、14d,处死1小组大鼠.取材进行常规HE染色观察骨痂厚度、BMP-2-DAB显色计数观察BMP-2阳性细胞数.结果:造模后第3d,各给药组与模型组比较,标本BMP-2阳性细胞数及骨痂厚度差异无统计学意义;造模后第7、10、14d,各给药组与模型组比较,标本BMP-2阳性细胞数及骨痂厚度差异有统计学意义(P<0.05,P<0.01),桃仁红花组、黄芪红花组与木香红花组比较,标本BMP-2阳性细胞数及骨痂厚度差异有统计学意义(P<0.05).结论:木香与红花配伍能显著提高SD大鼠早期桡骨骨折断端局部BMP-2的表达水平,增加骨痂厚度,就促进SD大鼠骨折愈合过程中BMP-2表达作用而言,理气活血法可能是四肢闭合骨折早期的较佳治法.%Objective: To observe the effect of different compatibility of herbs on BMP-2 expression at fracture site and thickness of callus of early radius fracture rat models. Methods: 256 SD rats were divided into four groups randomly: model group,Semen Persicae and Flos Carthami group, Radix Aucklandiae and Flos Carthami group, Radix Astragali and Flos Carthami group.Left radius fracture rat models were established. Conventional haematoxylin and eosin staining and immunostaining for bone section were carried out on days 3,7,10 and 14 after model establishment. Results: On days 7, 10 and 14 after model establishment,compared with model group, BMP-2 positive cells at fracture site and thickness of callus were significantly increased in all the treatment groups; compared with Radix Aucklandiae and Flos

  8. BMP-2和地塞米松联合诱导大鼠牙囊细胞与β-磷酸三钙生物陶瓷相容性的实验研究%To investigate the adhesion and growth of RDFCs combined onto β-tricalcium phosphate(β-TCP)in vitro

    Institute of Scientific and Technical Information of China (English)

    徐璐璐; 刘洪臣; 王东胜; 鄂玲玲

    2012-01-01

    Objective: To construct a three-dimensional culture model of RDFCs by using porous β-tricalcium phosphate (β-TCP) as scaffold and RDFCs as seed cells. To investigate the possibility and the biological capability of the reconstruction of the combined tissues by using RDFCs with β-TCP in vitro. Method: RDFCs of the third passage were induced by BMP-2 (l00ng/ml), Dex (l0-8moll/ml) and a mixture of BMP-2 (l00ng/ml) and Dex (l0-8mol/ml) in DMEM. After 3 days, the control cells were collected for combination with β-TCP scaffold and also cultured in DMEM solution. On the 3rd day and 7th day, RDFCs adhesion and growth were observed by scanning electronic microscope (SEM), the proliferation of the cells was evaluated by cell counting. Results: SEM showed that the multi-porus scaffold had 3 morphologically distinct pore types. The third passage rat dental follicle cells could be adhered and extended and proliferation on the β-TCP by SEM. Conclusion: The pours scaffold β-TCP had good 3-D structure and biocompatibility, which provided safe surface for RDFCs to adhere to and proliferate. It can be used as biomaterials in bone tissue engineering.%目的:以BMP-2和地塞米松诱导的大鼠牙囊细胞作为种子细胞,β-磷酸三钙作为细胞支架材料,体外构建细胞生物材料复合体,观察大鼠牙囊细胞在β-磷酸三钙生物陶瓷材料的贴附、增殖情况.方法:收集培养的第3代RDFCs,血清饥饿同步化后,细胞分为四组,分别为BMP-2、Dex、BMP-2+Dex诱导组及空白对照组.诱导3d后,将密度为4×106/ml的细胞悬液0.1ml均匀接种到预制好的β-TCP支架材料上,分别于体外培养3d、7d取细胞材料复合体,通过细胞计数、扫描电子显微镜观察细胞在材料表面的贴附、增殖情况.结果:细胞计数发现,7d时BMP-2+Dex诱导组细胞数量显著高于BMP-2诱导组、Dex诱导组(P<0.01).扫描电镜观察,β-TCP表面呈多孔三维立体网状结构.细胞接种3 d和7d时,单位面积内BMP

  9. Posterior tooth replacement with dental implants in sites augmented with rhBMP-2 at time of extraction--a case series.

    Science.gov (United States)

    Levin, Barry P; Tawil, Peter

    2012-02-01

    This case series demonstrates seven molar-site implants placed in six consecutively treated patients. All sites were augmented with rhBMP-2 (1.50 mg/cc)/ACS (recombinant human Bone Morphogenetic Protein-2/Absorbable Collagen Sponge) at extraction to regenerate bone-facilitating implant placement. In four patients, osteotomies were initiated with trephines to evaluate qualitatively for native bone and for the absence of residual ACS. All sites facilitated implant placement after augmentation. All seven implants achieved primary stabilization and were functionally loaded. No implants were lost or developed complications. It can be concluded that augmenting molar extraction sockets with rhBMP-2/ACS can allow standard implant placement in the posterior dentition that is capable of withstanding a functional load.

  10. BMP2在热应激导致小鼠神经管畸形发生中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    龙治峰; 谢远杰; 莫中成; 赵国军

    2009-01-01

    观察骨形态蛋白2(BMP2)在正常小鼠神经管和热应激后神经管的表达情况,探讨BMP2与神经管畸形(NTDs)发生的关系。本实验用昆明小鼠40只,实验组20只小鼠于妊娠后8.5天(E8.5)在42℃温箱中喂养30分钟,进行热应激处理,建立小鼠胚胎NTDs模型。对照组不处理。通过体视显微镜和组织学切片观察实验组和对照组神经管发育情况,

  11. The Effects of rhBMP-2 Released from Biodegradable Polyurethane/Microsphere Composite Scaffolds on New Bone Formation in Rat Femora

    Science.gov (United States)

    2009-09-17

    of 33 wt% triethylene diamine (TEDA) in dirpropylene glycol , was received from Goldschmidt (Hopewell, VA) as a gift. Polyethylene glycol (PEG, 600 Da...angiogenesis, and cell signaling at the initiation of fracture healing . A number of studies have suggested that sustained release of rhBMP-2 [17–19] is more...after surgery resulted in enhanced fracture healing relative to injectionwithin one day in a primatemodel [20]. The improvement in healing associated

  12. Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2 carriers: in vitro and in vivo evaluation.

    Science.gov (United States)

    Fu, Yin-Chih; Chen, Chung-Hwan; Wang, Chau-Zen; Wang, Yan-Hsiung; Chang, Je-Ken; Wang, Gwo-Jaw; Ho, Mei-Ling; Wang, Chih-Kuang

    2013-11-01

    Porous biphasic calcium phosphates (BCP) were fabricated using reverse thermo-responsive hydrogels with hydroxyapatite (HAp) and β-tricalcium (β-TCP) powder and planetary centrifugal mixer. This hydrogel mixture slurry will shrink and compress the HAp powder during the sintering process. The porous bioceramics are expected to have good mechanical properties after sintering at 1200°C. Reverse thermo-responsive hydrogels of poly[(N-isopropylacrylamide)-co-(methacrylic acid)] p(NiPAAm-MAA) were synthesized by free-radical cross-linking copolymerization, and their chemical properties were evaluated by nuclear magnetic resonance spectroscopy, infrared spectroscopy, and electrospray-ionization mass spectrometry. The lower critical solution temperature (LCST) of the hydrogel was determined using turbidity measurements. A thermogravimetric analysis was used to examine the thermal properties. The porous bioceramic properties were analyzed by X-ray diffraction, scanning electron microscopy, bulk density, compressive strength testing and cytotoxicity. The compressive strength and average porosity of the porous bioceramics were examined at approximately 6.8MPa and 66% under 10wt% p(NiPAAm-MAA)=99:1 condition. The ratio of HAp/β-TCP can adjust two different compositional behaviors during the 1200°C sintering process without resulting in cell toxicity. The (rhBMP-2)-HAp-PLGA carriers were fabricated as in our previous study of the double emulsion and drop-coating technique. Results of animal study included histological micrographs of the 1-mm defect in the femurs, with the rhBMP-2 carrier group, the bioceramic spacer group and the bioceramic spacer with rhBMP-2 carriers group showing better callus formation around the femur defect site than the control group. The optimal dual effects of the bone growth factors from osteoconductive bioceramics and osteoinductive rhBMP-2 carriers produced better bone formation.

  13. The Value of SPECT/CT in Monitoring Prefabricated Tissue-Engineered Bone and Orthotopic rhBMP-2 Implants for Mandibular Reconstruction.

    Directory of Open Access Journals (Sweden)

    Miao Zhou

    Full Text Available Bone tissue engineering shows good prospects for mandibular reconstruction. In recent studies, prefabricated tissue-engineered bone (PTEB by recombinant human bone morphogenetic proteins (rhBMPs applied in vivo has found to be an effective alternative for autologous bone grafts. However, the optimal time to transfer PTEB for mandibular reconstruction is still not elucidated. Thus, here in an animal experiment of rhesus monkey, the suitable transferring time for PTEB to reconstruct mandibular defects was evaluated by 99mTc-MDP SPECT/CT, and its value in monitoring orthotopic rhBMP-2 implants for mandibular reconstruction was also evaluated. The result of SPECT/CT showed higher 99mTc-MDP uptake, indicating osteoinductivity, in rhBMP-2 incorporated demineralized freeze-dried bone allograft (DFDBA and coralline hydroxyapatite (CHA implants than those without BMP stimulation. 99mTc-MDP uptake of rhBMP-2 implant peaked at 8 weeks following implantation while CT showed the density of these implants increased after 13 weeks' prefabrication. Histology confirmed that mandibular defects were repaired successfully with PTEB or orthotopically rhBMP-2 incorporated CHA implants, in accordance with SPECT/CT findings. Collectively, data shows 99mTc-MDP SPECT/CT is a sensitive and noninvasive tool to monitor osteoinductivity and bone regeneration of PTEB and orthotopic implants. The PTEB achieved peak osteoinductivity and bone density at 8 to 13 weeks following ectopic implantation, which would serve as a recommendable time frame for its transfer to mandibular reconstruction.

  14. Serum levels of BMP-2, 4, 7 and AHSG in patients with degenerative joint disease requiring total arthroplasty of the hip and temporomandibular joints.

    Science.gov (United States)

    Albilia, Jonathan B; Tenenbaum, Howard C; Clokie, Cameron M L; Walt, David R; Baker, Gerald I; Psutka, David J; Backstein, David; Peel, Sean A F

    2013-01-01

    To date, there is no objective or reliable means of assessing the severity of degenerative joint disease (DJD) and need for joint replacement surgery. Hence, it is difficult to know when an individual with DJD has reached a point where total arthroplasty is indicated. The purpose of the present study is to determine whether serum levels of Alpha-2 HS-glycoprotein (AHSG) as well as bone morphogenetic proteins (BMP-2, 4, 7) can be used to predict the presence of severe DJD of the hip and/or temporomandibular joint (TMJ) (specifically: joints that require replacement). A total of 30 patients scheduled for arthroplasty (diseased) (15 HIP, 15 TMJ) and 120 age-matched controls (healthy/non-diseased) were included. Blood samples were collected from all patients ≥8 weeks after the last arthroplasty. Concentrations of serum analytes were measured using enzyme-linked immunosorbent assays, and these were compared between the Diseased and Healthy groups, utilizing the Mann-Whitney U-test. Patients with disease had significantly higher levels of BMP-2 and BMP-4 and lower levels of AHSG in serum compared to non-diseased humans (p < 0.01). Higher levels of BMP-2, 4 and reduced levels of AHSG appear to characterize patients who have DJD that is severe enough to require total joint replacement. Perhaps measurements of these proteins can be used to make objective decisions regarding the need for total arthroplasty as opposed to the current subjective approaches.

  15. RhBMP-2 loaded 3D-printed mesoporous silica/calcium phosphate cement porous scaffolds with enhanced vascularization and osteogenesis properties

    Science.gov (United States)

    Li, Cuidi; Jiang, Chuan; Deng, Yuan; Li, Tao; Li, Ning; Peng, Mingzheng; Wang, Jinwu

    2017-01-01

    A major limitation in the development of effective scaffolds for bone regeneration has been the limited vascularization of the regenerating tissue. Here, we propose the development of a novel calcium phosphate cement (CPC)-based scaffold combining the properties of mesoporous silica (MS) with recombinant human bone morphogenic protein-2 (rhBMP-2) to facilitate vascularization and osteogenesis. Specifically, the development of a custom MS/CPC paste allowed the three-dimensional (3D) printing of scaffolds with a defined macroporous structure and optimized silicon (Si) ions release profile to promote the ingrowth of vascular tissue at an early stage after implantation in support of tissue viability and osteogenesis. In addition, the scaffold microstructure allowed the prolonged release of rhBMP-2, which in turn significantly stimulated the osteogenesis of human bone marrow stromal cells in vitro and of bone regeneration in vivo as shown in a rabbit femur defect repair model. Thus, the combination MS/CPC/rhBMP-2 scaffolds might provide a solution to issues of tissue necrosis during the regeneration process and therefore might be able to be readily developed into a useful tool for bone repair in the clinic. PMID:28128363

  16. Maximizing bone formation in posterior spine fusion using rhBMP-2 and zoledronic acid in wild type and NF1 deficient mice.

    Science.gov (United States)

    Bobyn, Justin; Rasch, Anton; Kathy, Mikulec; Little, David G; Schindeler, Aaron

    2014-08-01

    Spinal pseudarthrosis is a well described complication of spine fusion surgery in NF1 patients. Reduced bone formation and excessive resorption have been described in NF1 and anti-resorptive agents may be advantageous in these individuals. In this study, 16 wild type and 16 Nf1(+/-) mice were subjected to posterolateral fusion using collagen sponges containing 5 µg rhBMP-2 introduced bilaterally. Mice were dosed twice weekly with 0.02 mg/kg zoledronic acid (ZA) or sterile saline. The fusion mass was assessed for bone volume (BV) and bone mineral density (BMD) by microCT. Co-treatment using rhBMP-2 and ZA produced a significant increase (p Nf1(+/-) mice (+174%). Co-treatment also produced a significantly higher total BMD of the fusion mass compared to rhBMP-2 alone in both groups (p Nf1(+/-) deficient mice still generated less bone than wild type controls. TRAP staining on histological sections indicated an increased osteoclast surface/bone surface (Oc.S/BS) in Nf1(+/-) mice relative to wild type mice, and this was reduced with ZA treatment.

  17. The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds.

    Science.gov (United States)

    Wu, Gang; Liu, Yuelian; Iizuka, Tateyuki; Hunziker, Ernst Bruno

    2010-10-01

    We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb, PLGA and Polyactive) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r(2) = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r(2) = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.

  18. Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton

    Directory of Open Access Journals (Sweden)

    Włodarski Krzysztof

    2007-12-01

    Full Text Available Abstract Background Differences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear. Methods In this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs, whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I. Results It was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2. Conclusion It was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton.

  19. RhBMP-2 loaded 3D-printed mesoporous silica/calcium phosphate cement porous scaffolds with enhanced vascularization and osteogenesis properties

    Science.gov (United States)

    Li, Cuidi; Jiang, Chuan; Deng, Yuan; Li, Tao; Li, Ning; Peng, Mingzheng; Wang, Jinwu

    2017-01-01

    A major limitation in the development of effective scaffolds for bone regeneration has been the limited vascularization of the regenerating tissue. Here, we propose the development of a novel calcium phosphate cement (CPC)-based scaffold combining the properties of mesoporous silica (MS) with recombinant human bone morphogenic protein-2 (rhBMP-2) to facilitate vascularization and osteogenesis. Specifically, the development of a custom MS/CPC paste allowed the three-dimensional (3D) printing of scaffolds with a defined macroporous structure and optimized silicon (Si) ions release profile to promote the ingrowth of vascular tissue at an early stage after implantation in support of tissue viability and osteogenesis. In addition, the scaffold microstructure allowed the prolonged release of rhBMP-2, which in turn significantly stimulated the osteogenesis of human bone marrow stromal cells in vitro and of bone regeneration in vivo as shown in a rabbit femur defect repair model. Thus, the combination MS/CPC/rhBMP-2 scaffolds might provide a solution to issues of tissue necrosis during the regeneration process and therefore might be able to be readily developed into a useful tool for bone repair in the clinic.

  20. Sequestration of rhBMP-2 into self-assembled polyelectrolyte complexes promotes anatomic localization of new bone in a porcine model of spinal reconstructive surgery.

    Science.gov (United States)

    Abbah, Sunny-Akogwu; Lam, Wing Moon Raymond; Hu, Tao; Goh, James; Wong, Hee-Kit

    2014-06-01

    Efficient and therapeutically safe delivery of recombinant human bone morphogenetic protein 2 (rhBMP-2) continues to be a central issue in bone tissue engineering. Recent evidence indicates that layer-by-layer self-assembly of polyelectrolyte complexes (PECs) can be used to recreate synthetic matrix environments that would act as tuneable reservoirs for delicate biomolecules and cells. Although preliminary in vitro as well as small-animal in vivo studies support this premise, translation into clinically relevant bone defect volumes in larger animal models remains unreported. Here we explored the use of native heparin-based PEC, deposited on a hydrated alginate gel template, to load bioactive rhBMP-2 and to facilitate lumbar interbody spinal fusion in pigs. We observed that triple PEC deposits with the highest protein sequestration efficiency and immobilization capacity promoted higher volume of new bone formation when compared with single PEC with low sequestration efficiency and immobilization capacity. This also resulted in a significantly enhanced biomechanical stability of the fused spinal segment when compared with PEC carriers with relatively low protein sequestration and immobilization capacities (pcollagen sponge carriers. We conclude that this growth factor sequestration platform is effective in the healing of clinically relevant bone defect volume and could overcome some of the safety concerns and limitations currently associated with rhBMP-2 therapy such as excessive heterotopic ossification.

  1. Effect of exogenous transforming growth factor-beta 2 (TGF-β2) and bone morphogenetic protein 2 (BMP2 ) and allografts bone on radius defects healing of experimental rabbits%大段同种异体骨移植复合BMP2和TGF-β2对兔桡骨中段骨缺损愈合的影响

    Institute of Scientific and Technical Information of China (English)

    刘亮; 王东

    2007-01-01

    [目的]探讨外源性TGF-β2和BMP2复合同种异体骨对骨缺损愈合的作用.[方法]用兔桡骨骨缺损模型,在骨缺损局部单独或联合应用TGF-β2和BMP2与同种异体骨复合,A组:同种异体骨与BMP2复合;B组(对照组):自体骨;C组:同种异体骨与TGF-β2复合;D组:同种异体骨与TGF-β2、BMP2复合;通过不同时间X线片、生物力学、骨密度和骨痂钙含量的检测对骨缺损愈合情况进行评估.[结果]B组的骨痂钙含量、骨缺损愈合情况和愈合后的力学强度明显优于A、C、D组(P<0.01),D组优于A、C组(P<0.05),C组优于A组(P<0.01).[结论]TGF-β2和BMP2在骨缺损愈合过程中均发挥了重要的作用.在兔骨缺损周围局部应用外源性TGF-β2和BMP2与同种异体骨复合,可明显促进骨缺损愈合,使骨痂量增加,增强骨折愈合后的力学强度.并且在骨折愈合时间上接近自体骨移植骨折愈合的时间,从而在临床上缩短了需要大段植骨患者治疗时间,减轻了患者痛苦.单用TGF-β2的作用强于BMP2.它们联合应用时,这种作用进一步增强,在促进骨缺损愈合方面具有协同作用.同时也为异体骨复合何种因子及因子的剂量提供可靠的参考指标.

  2. Impact of stress to BMP-2 expression in repairing tendon-bone after operation to rabbit supraspinatus tendon of acute rupture%被动训练对兔冈上肌腱急性断裂术后腱-骨修复中BMP-2表达的影响

    Institute of Scientific and Technical Information of China (English)

    李森; 靳安民; 付国建; 王鹏程; 张辉; 闵少雄

    2010-01-01

    目的 探讨应力刺激在肩袖损伤术后腱-骨修复中的作用.方法 选取18只成年雄性新西兰白兔,术前随机选取2只白兔处死,以熟悉解剖结构及对照用.将其余16只白兔随机分为两组,即应力刺激组(A组)与非应力刺激组(B组),每组各8只.A组术后第2周开始被动训练(CPM);B组正常笼养.分别于术后2、4、6、8周训练结束后处死,取材,每次每组各处死2只,检测BMP-2表达.结果 术后2周,两组BMP-2染色均呈阳性,组间比较无明显差别.术后4周,两组BMP-2染色阳性,A组稍显均匀.术后6周,两组BMP-2染色阳性分布均较前广泛,A组较B组明显,且相对均匀有序.术后8周,A组比B组BMP-2染色更为有序,靠近骨面BMP-2表达程度与靠近腱性部分比较,差别较前减小.结论 CPM训练能提高兔冈上肌腱急性断裂术后腱-骨界面BMP-2表达,从而增进胶原的合成,促进肩袖损伤术后腱-骨修复.

  3. 前列腺癌组织BMP2表达及其与DIF-1的相关性%EXPRESSION OF BONE MORPHOGENETIC PROTEIN 2 AND ITS ASSOCIATION WITH DIFFERENTIATION INHIBITING FACTOR 1 IN PROSTATE CANCER

    Institute of Scientific and Technical Information of China (English)

    刘玉波; 于小玲; 赵辉

    2011-01-01

    Objective To investigate the expression of bone morphogenetic protein 2 (BMP2) in prostate cancer (PCa)and benign prostatic hyperplasia (BPH), and its association with cell differentiation, and differentiation inhibiting factor 1 (DIF-1). Methods Expressions of BMP2 and DIF-1 PCa and BPH were detected using immunohistochemical streptavidin peroxidase (SP) method. The positive expressions were analyzed using image analysis software. Linear correlation analysis was applied to analyze the correlation of BMP2 with DIF-1. Results In BPH, weak or negative expression of both BMP2 and DIF-1 was observed;In PCa, both BMP2 and DIF-1 showed positive expression. In PCa, the expressions of BMP2 and DIF-1 were positively correlated with Gleason score (rs=O.61, 0.63;P<0.01), and BMP2 was positively correlated with DIF-1 (r=0.92,P<0.01). Conclusion In prostate cancer, there is a correlation between the expressions of BMP2 and DIF-1, their expressions are closely related with the extent of malignancy, which indicates that both parameters are involved in the occurrence and development of this malignant tumor.%目的 探讨骨形态发生蛋白2(BMP2)在前列腺癌(PCa)及前列腺增生(BPH)组织中的表达及其与细胞分化的关系,并进一步分析其与分化抑制因子1(DIF-1)的相关性.方法 采用免疫组化SP法,分别检测PCa和BPH组织中BMP2和DIF-1的表达.采用图像分析软件分析阳性表达的灰度和面积,并采用直线相关方法分析BMP2和DIF-1的相关性.结果 BPH组织中BMP2和DIF-1呈阴性或弱阳性表达;PCa组织中BMP2和DIF-1均呈阳性表达.在PCa组织中.BMP2和DIF-1的表达水平与Gleason评分呈正相关(r=0.61、0.63,P<0.01);BMP2与DIF-1的表达也呈正相关(r=0.92,P<0.01).结论 在PCa组织中BMP2与DIF-1的表达存在相关性,且二者的表达均与PCa的恶性程度密切相关,提示二者可能与PCa的发生发展有关.

  4. 前列腺癌骨转移灶中BMP-2、BMP-4、BMP-7的表达及临床意义%Expression of BMP-2, BMP-4 and BMP-7 in human metastatic prostate cancer and their clinical significance

    Institute of Scientific and Technical Information of China (English)

    宫丽华; 孙晓淇; 刘宝岳; 黄啸原

    2012-01-01

    目的 检测BMP-2、BMP-4、BMP-7在前列腺癌骨转移灶中的表达,探讨其在前列腺癌成骨性转移中的作用.方法 采用免疫组化EnVision法检测28例前列腺癌骨转移病例及17例良性前列腺增生(benign prostate hyperplasia,BPH)病例中BMP-2、BMP-4、BMP-7的表达并对其进行对比分析.结果 BMP-2在所有前列腺癌骨转移灶及BPH病例中均表达,二者中其阳性率及表达强度无明显差异(P>0.05).BMP-4在前列腺癌骨转移灶及BPH中的阳性率无明显差异(P>0.05),但在前者中BMP-4的表达强度明显高于后者(P<0.05).BMP-7在前列腺癌骨转移灶中的阳性率及表达强度均明显高于BPH (P<0.05).在BPH的阳性表达病例中,BMP-2、BMP-4、BMP-7细胞质与细胞核同时阳性的表达率分别为13.3%、7.1%和11.1%,在前列腺癌骨转移灶的阳性表达病例中,BMP-2、BMP-4、BMP-7细胞质与细胞核的同时阳性的表达率均为100%,且细胞核的表达强度明显高于细胞质.结论 BMP-4、BMP-7在前列腺癌骨转移灶中高表达,提示其在前列腺癌的成骨性转移中可能起重要作用.%Purpose To analyze the expression of BMP-2, BMP-4, BMP-7 in prostate cancer with bone metastasis. Methods Iininu-nohistochemical technique was used to detect the expression of BMP-2, BMP-4 and BMP-7 in the bone osteobastic metastastic lesions of prostate cancer and the benign prostate hyperplasia ( BPH ). Results The expression of BMP-2 was detected in all samples and there was no difference between prostate cancer and BPH. The intensity of positivity of BMP-4 was higher in prostate cancer than that in BPH ( P < 0. 05 ), but the difference of positive rate between these two groups showed no significance. The intensity of positivity and the positive rate of BMP-7 were higher in prostate cancer than that in BPH ( P < 0. 05 ). The positive rate of staining involving both cyto-plasm and nuclear in BMP-2, BMP-4 and BMP-7-positive samples in BPH was respectively 13. 3

  5. BMP-2在减阻牵张快速牙移动不同加力方式下的表达及对牙移动的影响%The effects of BMP-2 expression in the related periodontal tissue on bone remolding of rapid tooth movement by resistance reduction and distration method

    Institute of Scientific and Technical Information of China (English)

    彭早霞; 李宁; 李佩; 李美静; 杨乐乐; 陈曦; 米丛波

    2016-01-01

    目的:观察不同牵张力下减阻牵张快速牙移动中 BMP-2的表达。方法:Beagle 犬12只,随机均分为加力5、15 d、加力15 d 保持固定10、90 d 4组。以44为移动牙,每个组3只犬的6颗移动牙随机采用减阻-牵张方法、减阻-常规方法和常规方法各2颗。各组犬按预定时间处死并获取移动牙牙周组织块,免疫组化法染色并观察 BMP-2表达。结果:各加力方式下 BMP-2阳性表达分布区域相似,均在加力结束时达峰值,其中减阻牵张组峰值最大(P <0.05);牙移动距离最大(P <0.01)。加力各时间点,减阻常规组 BMP-2阳性表达均强于常规方法组但不及减阻牵张组显著;保持固定90 d,3组无差异(P >0.05)。结论:减阻措施配合持续强牵张力可显著提高移动牙牵张新骨区 BMP-2阳性表达,加速牙周组织新骨形成。%Objective:To investigate the effects of bone morphogenetic protein 2(BMP-2)expression in the related periodontal tissue on bone remodeling under different distracting force during rapid tooth movement by resistance reduction and distraction. Methods:1 2 Beagle dogs were randomly divided into 4 groups as follows:5 d distraction,1 5 d distraction,1 5 d distraction and 1 0 d retaining and 1 5 d distraction and 90 d retaining.4 4 were distalized.6 teeth in each group were randomly assigned to re-sistance and distracting method,resistance and conventional method and conventional method,and there were 2 teeth in each group.Moving teeth models were prepared regularly.BMP-2 expression was examined by immunohistochemistry.Results:The BMP-2 positive expression of the 3 groups of different distraction schedule showed similar distribution area,and it reached peak at the end of 1 5-day distration,but the group of resistance and distracting method showed the maximum peak(P 0.05).Conclusion:Resistance reduction with sustained strong distracting force can significantly increased the

  6. Expression of BMP2/Smad1/Rnnx2 Signal Pathway in Renal Artery of Rat with Vascular Calcification%血管钙化大鼠肾动脉BMP2/Smad1/Runx2信号通路的表达

    Institute of Scientific and Technical Information of China (English)

    覃春美; 魏晓; 龚财判; 吴蔚桦; 刘建; 樊均明; 欧三桃

    2016-01-01

    目的 观察血管钙化大鼠模型肾动脉上骨形态蛋白2 (BMP2)/Smad1/Runt相关转录因子2(Runx2)信号通路的表达及其变化规律,探讨BMP2/Smad1/Runx2信号通路激活在肾动脉钙化中的作用.方法 将24只雄性SD大鼠随机分为对照组和钙化组,钙化组建立维生素D3和尼古丁诱导的大鼠血管钙化模型,对照组给予生理盐水和花生油.6周后采用Von Kossa染色检测肾动脉钙化程度,钙离子试剂盒测定大鼠肾动脉钙含量,实时荧光定量PCR检测肾动脉组织中BMP2、Smad1、Runx2 mRNA水平,免疫组化法观察BMP2、Smad1、Runx2及α-平滑肌肌动蛋白(α-SMA)的表达.结果 Von Kossa染色见钙化组大鼠肾动脉有大量黑色颗粒沉淀.钙化组血管钙含量高于对照组(P<0.05).与对照组相比,钙化组大鼠肾动脉组织BMP2、Smad1、Runx2 mRNA的水平升高(P<0.05),BMP2/Smad1/Runx2信号通路蛋白在肾动脉的表达亦同步上调,而α-SMA的表达较对照组下降(P<0.05).相关分析表明,大鼠肾血管钙含量与肾动脉组织BMP2 mRNA(r=0.655,P<0.05)、Smad1 mRNA (r=0.735,P<0.05)、Runx2 mRNA (r=0.734,P<0.05)均呈正相关.结论 BMP2/Smad1/Runx2通路的表达变化与肾动脉钙化的严重程度相关,提示BMP2/Smad1/Runx2信号通路参与了肾动脉钙化的发生发展.

  7. The effect of vancomycin powder on bone healing in a rat spinal rhBMP-2 model.

    Science.gov (United States)

    Mendoza, Marco C; Sonn, Kevin A; Kannan, Abhishek S; Bellary, Sharath S; Mitchell, Sean M; Singh, Gurmit; Park, Christian; Yun, Chawon; Stock, Stuart R; Hsu, Erin L; Hsu, Wellington K

    2016-08-01

    OBJECTIVE This study aims to quantify the impact of vancomycin powder application on new bone formation and spine fusion rates in a rat posterolateral arthrodesis model. METHODS Thirty-six female Sprague-Dawley rats underwent a posterolateral lumbar spinal fusion (PLF) at the L-4 and L-5 vertebrae. Fusion was elicited via implantation of an absorbable collagen sponge containing 3 µg rhBMP-2. Rats were divided into 3 groups: no vancomycin (control), standard-dose vancomycin, and high-dose vancomycin, based on what was applied to the fusion bed. Clinical studies typically describe the application of 1 g vancomycin into the surgical wound. Presuming an average individual patient weight of 70 kg, a weight-based equivalent dose of vancomycin powder was applied subfascially in the PLF model constituting a "standard-dose" treatment group (14.3 mg/kg, n = 12). To determine whether there is a critical threshold beyond which vancomycin increases the risk of pseudarthrosis, a 10-fold higher dose was administered to a "high-dose" treatment group (143 mg/kg, n = 12). No vancomycin powder was applied to the surgical site in the control group (n = 12). Fusion was evaluated with plain radiographs at 4 and 8 weeks after surgery. The spines were harvested after the 8-week radiographs were obtained and evaluated using manual palpation, microCT analysis, and histological analysis. RESULTS Radiographs demonstrated equivalent bridging bone formation in all groups. No significant differences in fusion scores were seen in the standard-dose (mean 2.25) or high-dose (2.13) treatment groups relative to untreated control animals (1.78). Similarly, fusion rates did not differ significantly different between vancomycin-treated animals (100% for both groups) and control animals (92%). Quantification of new bone formation via microCT imaging revealed no significant between-groups differences in the volume of newly regenerated bone (control vs standard-dose vancomycin, p = 0.57; control vs high

  8. Comparison of large-scale preparation of recombinant human BMP-4 and BMP-2 mature peptide expressed in E.coli%大肠杆菌表达的重组人BMP-2和hBMP-4成熟肽大规模制备方法的比较

    Institute of Scientific and Technical Information of China (English)

    王涛; 陈苏民; 陈南春; 赵伟钦; 张晓楠

    2006-01-01

    目的:大规模制备人骨形成蛋白成熟肽(hBMP-m):hBMP-4m和hBMP-2m.方法:两种工程菌株分别含有能够高表达hBMP-4m和hBMP-2m的质粒,分别导入15 L NBS发酵罐中进行恒溶氧高密度发酵和诱导表达,离心收集菌体,悬浮所收集的菌体,裂菌,洗涤4次.预先取少量样品进行探索实验后,全部包涵体分别用8 mol/L尿素缓冲液溶解,上Sepharose SP-FF阳离子柱.结果:发酵菌液A600 nm值分别为28.8和26.3.SOS-PAGE电泳后吸光度扫描表明hBMP-4m占细菌总蛋白量的40.0%,hBMP-2m占细菌总蛋白量的47.2%.洗涤4次后的包涵体中hBMP-4m占蛋白量的82.9%,hBMP-2m占蛋白量的84.5%.上Sepharose SP-FF阳离子柱后,分别收集0.35 mol/L NaCl和0.20 mol/L NaCl洗脱部分,获得纯度为96%的hBMP-4m及纯度为95%的hBMP-2m.其收获量分别为1.30 g/L发酵液和1.25 g/L发酵液;收得率分别为34.33%和34.81%.结论:大规模制备hBMP-4m和hBMP-2m时,使用相同的发酵程序及相近的纯化方法,都能够得到较好的收得率、较高的收获量和纯度.

  9. Prolonged osteogenesis from human mesenchymal stem cells implanted in immunodeficient mice by using coralline hydroxyapatite incorporating rhBMP2 microspheres.

    Science.gov (United States)

    Fu, Kun; Xu, Qingguo; Czernuszka, Jan; McKenna, Charles E; Ebetino, Frank H; Russell, R Graham G; Triffitt, James T; Xia, Zhidao

    2010-03-15

    The local environment plays an important role in osteogenic tissue regeneration. Our previous studies have shown that xenogenic transplantation of human mesenchymal stem cells (hMSCs) alone into immunodeficient mice did not result in long-term bone formation. This study investigates whether bone formation can be prolonged by incorporating human mesenchymal stem cells in mineralized scaffolds together with controlled delivery of a growth factor, BMP2. A composite of coralline hydroxyapatite (CHA) with poly(lactic-co-glycolic acid) (PLGA)-encapsulated rhBMP2 was incorporated with hMSCs in vitro. After 2 weeks in vitro culture the constructs were implanted subcutaneously in CB17 scid beige mice and harvested 10 weeks after implantation. The mineralized tissues were stained by using a fluorescent marker, 5FAM-risedronate, followed by observation with fluorescence microscopy, histology, histomorphometry, mouse-anti-human vimentin immunohistochemistry, and scanning microscopy. The results showed that compared with control materials in which only fibrous tissue formed following implantation of coralline scaffolds, bone-like tissue formed within the CHA composite containing PLGA encapsulated rhBMP2 and hMSCs for up to 10 weeks after implantation. Human cells, identified by the human vimentin-specific monoclonal antibody were seen within the bone-like tissue. In conclusion, incorporation of hMSCs into CHA with controlled delivery of BMP showed prolonged bone formation in immunodeficient mice. Further research is required to optimize the growth factor delivery system and to understand the underlying cellular and molecular mechanisms involved.

  10. A Suppressive Antagonism Evidences Progesterone and Estrogen Receptor Pathway Interaction with Concomitant Regulation of Hand2, Bmp2 and ERK during Early Decidualization.

    Directory of Open Access Journals (Sweden)

    Ana C Mestre-Citrinovitz

    Full Text Available Progesterone receptor and estrogen receptor participate in growth and differentiation of the different rat decidual regions. Steroid hormone receptor antagonists were used to study steroid regulation of decidualization. Here we describe a suppressive interaction between progesterone receptor (onapristone and estrogen receptor (ICI182780 antagonists and their relation to a rescue phenomenon with concomitant regulation of Hand2, Bmp2 and p-ERK1/2 during the early decidualization steps. Phenotypes of decidua development produced by antagonist treatments were characterized by morphology, proliferation, differentiation, angiogenesis and expression of signaling molecules. We found that suppression of progesterone receptor activity by onapristone treatment resulted in resorption of the implantation sites with concomitant decrease in progesterone and estrogen receptors, PCNA, KI67 antigen, DESMIN, CCND3, CX43, Prl8a2, and signaling players such as transcription factor Hand2, Bmp2 mRNAs and p-ERK1/2. Moreover, FGF-2 and Vegfa increased as a consequence of onapristone treatment. Implantation sites from antagonist of estrogen receptor treated rats developed all decidual regions, but showed an anomalous blood vessel formation at the mesometrial part of the decidua. The deleterious effect of onapristone was partially counteracted by the impairment of estrogen receptor activity with rescue of expression levels of hormone steroid receptors, proliferation and differentiation markers, and the induction of a probably compensatory increase in signaling molecules Hand2, Bmp2 and ERK1/2 activation compared to oil treated controls. This novel drug interaction during decidualization could be applied to pathological endometrial cell proliferation processes to improve therapies using steroid hormone receptor targets.

  11. The toxic effects of Tris-(2,3-dibromopropyl)isocyanurate(TBC) on genes expression of bmp2b and bmp4 of zebrafish embryos

    Institute of Scientific and Technical Information of China (English)

    JIA Wan-jun

    2016-01-01

    We exposed zebrafish embryos to Tris-(2,3-dibromopropyl)isocyanurate(TBC)at the concentration of 20ppb, 100ppb, 400ppb, 1000ppb for 120h and 0.1%DMSO was set as the control group. Bmp2b and bmp4 were chosen perform RT-PCR to determine their genes expression level. The results showed that, TBC influenced their genes expression level in some extent and it significantly raised the genes expression level at the concentration of 20ppb.

  12. 骨形成蛋白(BMP)-2/4,-5与IA型BMP受体在口腔正常上皮、良性病变及癌变中的表达分析%EXPRESSIONS OF BMP-2/4,-5 and BMPR-IA RECEPTOR IN NORMAL, BENIGN AND MALIGNANT LESIONS OF ORAL EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The aim of the present study was investigate the relationshipsbetween bone morphogenetic proteins (BMPs), BMP receptor type IA and carcinogenesis of oral epithelia. Expressions of BMP-2/4,-5 and BMPR-IA were analyzed in the specimens obtained from buccal mucosa, including 9 cases of normal mucosa (NB), 8 cases of chronic inflammation (NCI), 7 cases of hyperkeratosis (HK), 5 cases of squamous cell papilloma (SCP), 29 cases of squamous cell carcinoma (SCC), 10 cases of epithelium adjacent to carcinoma (EAC) and 6 cases of normal mucosa from hard palate (NHP) by using immunohistochemistry. Results demonstrated that the immunostaining of BMP-2/4,BMP-5 and BMPR-IA was weak and not consistent in normal and benign groups. Except for 3 cases of SCC, all cases of SCC showed positive staining for BMP-2/4, BMP-5 and BMPR-IA. The staining intensity and proportion of the positively stained cells in SCC were markedly increased than in normal and benign groups except for EAC. The metastatic carcinoma cells in lymph nodes were strongly and positively stained for BMP-2/4,BMP-5 and BMPR-IA might be involved in the carcinogenesis of oral epithelia.%认识BMP及其受体与口腔正常上皮及其癌变的关系,有助于深入了解口腔上皮癌变的机理。本文用免疫组织化学方法对BMP-2/4,-5与BMP受体BMPR-IA在口腔颊部粘膜正常上皮、良性病变和癌变中的表达进行观察和半定量分析。标本包括:9例正常上皮(normalbuccal mucosa, NB)、8例慢性炎症(nonspecific chronic inflammation,NCI)、7例过度角化(hyperkeratosis, HK)、5例乳头状瘤(squamous cell papilloma,SCP)、29例鳞癌(squamous cell carcinoma,SCC)、10例癌旁上皮(epithelium immediately adjacent to carcinoma,EAC)以及6例硬腭粘膜上皮(normal mucosa of hard palate,NHP)。结果显示:BMP-2/4,-5与BMPR-IA在口腔粘膜的正常与良性病变上皮中有弱的和不均一的阳性表达,NB与NHP无明显差别。而除3例SCC外,其它SCC

  13. Research of quaternized chitosan based hybrid scaffold as a delivery system for rhBMP-2%壳聚糖季铵盐基杂化支架的制备及其作为rhBMP-2载体的研究*

    Institute of Scientific and Technical Information of China (English)

    胡园园; 王靖; 刘昌胜

    2014-01-01

    Quaternized chitosan was synthesized by the reaction of chitosan and glycidyltrimethylammonium chloride (GTMAC)and named as N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan chloride (HTCC). A hydrogel system composed of HTCC andβ-glycerophosphate (β-GP)was obtained by intermolecular forces. Introduction ofβ-TCP granules improved the mechanical strength and controlled the swelling behavior and pro-longed the releasing period for rhBMP-2 of the hydrogel scaffold.Moreover,rhBMP-2 loaded scaffolds were as-sessed in ectopic bone formation.The results indicate that porous HTCC/β-GP/β-TCP/rhBMP-2 scaffolds are promising candidates for application in tissue engineering of bone.%采用环氧衍生物开环法制备壳聚糖季铵盐,通过其与甘油磷酸钠的分子间作用力交联成凝胶,冻干后得多孔支架,用于装载骨形态发生蛋白-2应用于骨修复领域;同时引入β-TCP 作为物理交联点,在提高支架力学强度的同时,更好地调控支架的溶胀行为,延长rhBMP-2的释放时间,并且在异位诱导成骨的动物实验中取得良好效果。

  14. Effects of Acteoside and Echinacoside on the Expression of the BMP2 in Rat Osteoblast%麦角甾苷及松果菊苷对体外培养大鼠成骨细胞BMP2基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    邢晓旭; 刘钟杰; 韩博

    2011-01-01

    为了观察麦角甾苷和松果菊苷对体外培养大鼠成骨细胞BMP2基因表达的影响.本试验采用酶消化法分离得到大鼠颅骨成骨细胞,在第3代成骨细胞中分别加入含有不同浓度麦角甾苷和松果菊苷的培养液进行培养,在培养的不同时间收集细胞、提取细胞总RNA,采用实时荧光定量PCR方法观察对成骨细胞BMP2基因表达的影响.结果表明,麦角甾苷和松果菊苷均能促进大鼠成骨细胞BMP2基因的表达.

  15. Expression and localization of macrophages and BMP2 in mucosa of tympanosclerosis%巨噬细胞和骨形成蛋白2在鼓室硬化中的定位和表达

    Institute of Scientific and Technical Information of China (English)

    刘翔; 李源; 郑亿庆; 张志钢

    2009-01-01

    目的:检测巨噬细胞、骨形成蛋白2(BMP2)在鼓室硬化的中耳黏膜中的定位和表达,探讨巨噬细胞、BMP2在鼓室硬化发生机制中的作用.方法:选取17例鼓室硬化患者(鼓室硬化组),以及17例年龄、性别、病程相匹配的单纯慢性化脓性中耳炎患者(中耳炎组),应用免疫组织化学的方法检测病变黏膜的巨噬细胞标志CD68以及BMP2蛋白在两组标本的定位和表达情况.结果: 两组中CD68阳性染色细胞均主要分布于黏膜下层中,黏膜层中仅有少量分布.两组阳性患者均为12例(70.59%),两组患者的CD68阳性染色细胞数量无差别(P=0.79),鼓室硬化组为6.94±6.08,中耳炎组为7.59±7.84.两组均有BMP2阳性染色细胞表达,BMP2蛋白广泛表达于中耳黏膜层、黏膜下层.两组的BMP2阳性染色细胞数及平均吸收度值均有显著差异(P<0.01).在鼓室硬化组中,钙化斑周围有巨噬细胞浸润和大量BMP2阳性着色,钙化斑周围的部分细胞同时有CD68和BMP2阳性着色.结论:巨噬细胞参与了鼓室硬化, 可能通过分泌BMP2在鼓室硬化形成中起重要作用.

  16. Hereditary hemochromatosis type 1 phenotype modifiers in Italian patients. The controversial role of variants in HAMP, BMP2, FTL and SLC40A1 genes.

    Science.gov (United States)

    Radio, Francesca Clementina; Majore, Silvia; Aurizi, Caterina; Sorge, Fiammetta; Biolcati, Gianfranco; Bernabini, Sara; Giotti, Irene; Torricelli, Francesca; Giannarelli, Diana; De Bernardo, Carmelilia; Grammatico, Paola

    2015-06-01

    Hereditary hemochromatosis (HH) is a heterogeneous disorder of iron metabolism. The most common form of the disease is Classic or type 1 HH, mainly caused by a biallelic missense p.Cys282Tyr (c.845G>A) mutation in the HFE gene. However, the penetrance of p.Cys282Tyr/p.Cys282Tyr genotype is incomplete in terms of both biochemical and clinical expressivity. Lack of penetrance is thought to be caused by several genetic and environmental factors. Recently, a lot of evidences on HH genetic modifiers were produced, often without conclusive results. We investigated 6 polymorphisms (rs10421768 in HAMP gene, rs235756 in BMP2 gene, rs2230267 in FTL gene, rs1439816 in SLC40A1 gene, rs41295942 in TFR2 gene and rs2111833 in TMPRSS6 gene) with uncertain function in order to further evaluate their role in an independent cohort of 109 HH type 1 patients. Our results make it likely the role of rs10421768, rs235756, rs2230267 and rs1439816 polymorphisms, respectively in HAMP, BMP2, FTL and SLC40A1 genes in HH expressivity. In addition, previous and our findings support a hypothetical multifactorial model of HH, characterized by a principal gene (HFE in HH type 1) and minor genetic and environmental factors that still have to be fully elucidated.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. The regulation of osteogenic differentiation by BMP2 and the canonical Wnt signaling pathway and the crosstalk between them%BMP2信号通路与经典 Wnt 信号通路及相互交联对成骨分化的调控

    Institute of Scientific and Technical Information of China (English)

    刘光源; 田发明; 张柳; 刘家寅

    2014-01-01

    The osteogenic differentiation process in mesenchymal stem cells and other lineages that are capable of differentiating into osteoblasts, is regulated by several signal transduction pathways and the dynamic regulatory networks consisted of them.In this process, more and more attention has been paid to the role of BMP2 signaling pathway and canonical Wnt signaling pathway. Previous studies have shown that BMP2 and canonical Wnt signaling pathway can regulate the osteogenic differentiation process in target cells, during which the expression level and functional activity of key molecules in one signaling pathway are regulated by the other.Moreover, they also exhibit cooperative or antagonize interaction on the regulation of osteogenic differentiation biomarkers. This paper briefly summarizes the role of BMP2 and canonical Wnt signaling pathway in regulating osteogenic differentiation and the crosstalk between the two signaling pathways in this process.%在以间充质干细胞为代表的具有成骨分化潜能的细胞中,多条信号传导通路及其构成的动态调控网络参与调控其成骨分化过程。其中BMP2和经典Wnt信号通路在此过程中的作用日益受到关注。国内外研究表明,BMP2和经典Wnt信号通路在调控靶细胞成骨分化过程中伴有彼此通路中的关键分子的表达水平和功能活性的改变,并在一定条件下表现出对成骨分化特异性标志物的协同和拮抗调控。成骨增殖分化过程是一个动态过程,在成骨分化的不同阶段,分子通路的作用不同,相互之间的调节也动态变化的,研究BMP2和经典Wnt信号通路在成骨分化过程中的相互调节作用,对于指导治疗骨质疏松和骨折有重要的作用,本文就BMP2和经典Wnt信号通路对靶细胞成骨分化的调控作用以及在此过程中两条信号通路之间的交联作一简要综述。

  19. rhBMP2作用下人牙周膜成纤维细胞中OCIF、ODF的表达研究%Study of the expression variety of OCIF and ODF in human periodontal ligament fibroblasts with rhBMP2 reaction

    Institute of Scientific and Technical Information of China (English)

    吉玲玲; 鲍庆江; 李昂; 饶国州; 周洪

    2011-01-01

    Objective By RT-PCR to investigating the effects of rhBMPs in different concentration on the expression of OCIF,ODF in human periodontal ligament fibroblasts. To clarify the molecular mechanism of BMPs' regulation to the HPDLfs, which contribute to the bone rebuilding. Methods The 6th generation well-grown primary culture fibrocyte from HPDLfs were used in the experiment. The cells were subjected to different doses of rhBMP2 (25ng/ml,50ng/ml and 100ng/ml) for 1,3,5,7 days in this experiment. mRNA expression of OCIFsODF were determined by semiquantitative RT-PCR approach, electrophoresis tape brightness was analyzed by graphical analysis software Image Pro Plus5.0. Results The expression varieties between cbfart and OCIF in different concentration were compared. At primary stage, their expression varieties were concordance and presented positive correlation. But the variety correlation between cbfort and OCIF was no more definitude after OCIF reaching peak value with the action time prolonging. Conclusion Based on the varieties of expression between OCIF and ODFjt showed that the formation of osteclasts was restrained intensively by HPDLfs at primary stage with the action of rhBMP2. With the action time prolonging.the restraining effect was changed into the activation effect.%目的:本研究通过反转录聚合酶链反应来测定不同浓度的重组人骨形成蛋白2(rhBMP2)对人牙周膜成纤维细胞中OCIF、0DFmRNA在不同作用时间点表达的影响,了解rhBMP2对骨改建相关基因的调控方式。方法:原代培养人牙周膜成纤维细胞,取生长良好的第六代细胞,分别用25ng/ml、50 ng/ml及100 ng/ml的rhBMP2作用于细胞,于作用1、3、5、7天后收集细胞。反转录聚合酶链反应检测各组细胞四个时间作用点OCIF、ODF mRNA含量,PCR产物1%琼脂糖凝胶电泳后,采用Image Pro Plus5.0图像分析软件对电泳胶带亮度进行分析。结果:在rhBMP2作用初期,由于OCIF m

  20. 辽宁绒山羊BMP-2基因的克隆及序列比较分析%Cloning and Sequence Analyzing of B MP-2 Gene from Liaoning Cashmere Goats

    Institute of Scientific and Technical Information of China (English)

    薛冰; 郭丹; 王春艳; 郑旭; 高月; 张世伟

    2012-01-01

    根据GenBank上绵羊的BMP-2基因序列设计特异性引物,以辽宁绒山羊基因组DNA为模板,利用聚合酶链式反应,成功克隆了常年长绒型和季节长绒型辽宁绒山羊BMP-2部分基因片段,丰富了绒山羊BMP-2基因序列。经与绵羊、牛、鼠、猪和人的BMP-2基因进行的比对结果表明,季节与常年长绒型辽宁绒山羊的BMP-2基因同源片段的同源性达到99.7%,二者与绵羊同源性为98.2%和98.4%;与牛同源性为98.2%和97.9%;与鼠同源性为86.3%和86%;与人同源性为88.1%和88.1%。结果表明,辽宁绒山羊BMP-2基因部分核苷酸序列与其他哺乳动物同源性很高,与绵羊、牛的同源性高达97%以上,这与它们的种属关系相近一致。与人、鼠的同源性也在86%以上,说明BMP-2基因在不同物种之间具有较高的保守性。%According to the BMP-2 gene sequence of sheep on the GenBank, specific primers were designed and perennial and seasonal long-staple Cashmere goat BMP type-2 partial gene fragments were successfully cloned from the Liaoning cashmere goat genomic DNA, using the polymerase chain reaction. With the sheep, cat- tie, rats, pigs and human BMP-2 gene than on the results show that seasonal and perennial long-staple type of Liaoning cashmere goats homologous fragment of BMP-2 homology 99.7%, respeciively homology with the sheep was 98.2% and 98.4%; and bovine homology was 98.2% and 97.9%; and rat homolgy 86.3% and 86%; and human homology was 88.1% and 88.1%. The results showed that BMP-2 in Liaoning Cashmere goat gene partial nucleotide sequence homology with other mammals, which is similar to their relationship of species, and BMP-2 gene were conserved in different species.

  1. 骨形成蛋白BMP2基因在人骨髓基质细胞中的表达及对其成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    李军; 范清宇; 范德刚; 张鹏; 文艳华

    2005-01-01

    目的:利用构建的人骨形成蛋白BMP2真核表达载体pcDNA3/BMP2,检测其转染人骨髓基质细胞后的表达及对其成骨分化的影响.方法:酶切鉴定构建的真核表达载体pcDNA3/BMV2,利用脂质体介导的转染技术,将所构建的载体导入骨髓基质细胞中,体外单层培养.分别于转染后48h和4wk,采用原位杂交、免疫组化和碱性磷酸酶、钙化学染色方法检测BMV2的基因蛋白表达以及对骨髓基质细胞成骨分化的影响.结果:pcDNA3/BMP2酶切片段的大小与理论相符,转染后细胞能检测到BMP2基因和BMP2蛋白表达,并促进成骨转化.结论:pcDNA3/BMP2转染骨髓基质干细胞中可获得短暂和长期表达,并加强骨髓基质细胞的成骨分化能力。

  2. 丝素蛋白增强型磷酸钙复合rhBMP-2用于绵羊腰椎椎体间融合的实验研究%Experimental study on lumbar interbody fusion with silk fibroin enhanced calcium phosphate cement composite loaded with recombinant human bone morphogenetic protein-2 in sheep

    Institute of Scientific and Technical Information of China (English)

    陈亮; 顾勇; 陈晓庆; 干旻峰; 朱雪松; 杨惠林; 唐天驷

    2010-01-01

    Objective To evaluate the osteogenic characteristics of an injectable silk fibroin (SF) enhanced calcium phosphate cement (CPC) composite loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) on lumbar interbody fusion in sheep. Methods Twenty-four mature sheep were randomly divided into two groups. Each sheep underwent L1.2, L3.4 and L5.6 lumber interbody fusion, and the three disc spaces were randomly implanted with three of the following materials: SF/CPC, CPC/rhBMP-2, SF/CPC/rhBMP2 and autogenous iliac crest bone. One group was killed at 6 months and the other at 12 months. The fusion segments were observed and analyzed by manual palpation, CT scan, undestructive biomechanical testing, undecalcified histology, and histomorphology. Results The fusion rates of SF/CPC, CPC/rhBMP-2, SF/CPC/rhBMP-2 and autogenous bone assessed by manual palpation were 0, 33.33%, 55.56% and 77.78% respectively at 6 months. At 12 months, the fusion rates improved to 11.11%, 44.44%, 77.78% and 77.78%, respectively.The biomechanical results showed that fusion stiffness was significantly greater in autograft compared with SF/CPC/rhBMP-2, CPC/rhBMP-2, and SF/CPC in 4 degrees of freedom (flexion, extension, right bending, and left bending) at 6 months. The SF/CPC/rhBMP-2 composite showed similar stiffness as autograft, which was significantly greater than CPC/rhBMP-2 and SF/CPC at 12 nonths. Both CPC/rhBMP-2 and SF/CPC/rhBMP-2 showed significantly greater stiffness at 12 months compared with that of at 6 months. The results showed that bone volume was significantly greater in autograft compared with SF/CPC/rhBMP-2, CPC/rhBMP-2, and SF/CPC at 6 months. There was significant difference among ceramic residue among SF/CPC, CPC/rhBMP-2 and SF/CPC/rhBMP-2, with SF/CPC the greatest and SF/CPC/thBMP-2 the least. At 12 months, the bone volume of SF/CPC/rhBMP-2 composite was comparable with autograft, and greater than that of CPC/rhBMP-2 and SF/CPC. The bone volume of SF/CPC, CPC/rhBMP

  3. A novel therapeutic approach with Caviunin-based isoflavonoid that en routes bone marrow cells to bone formation via BMP2/Wnt-β-catenin signaling.

    Science.gov (United States)

    Kushwaha, P; Khedgikar, V; Gautam, J; Dixit, P; Chillara, R; Verma, A; Thakur, R; Mishra, D P; Singh, D; Maurya, R; Chattopadhyay, N; Mishra, P R; Trivedi, R

    2014-09-18

    Recently, we reported that extract of Dalbergia sissoo made from leaves and pods have antiresorptive and bone-forming effects. The positive skeletal effect attributed because of active molecules present in the extract of Dalbergia sissoo. Caviunin 7-O-[β-D-apiofuranosyl-(1-6)-β-D-glucopyranoside] (CAFG), a novel isoflavonoid show higher percentage present in the extract. Here, we show the osteogenic potential of CAFG as an alternative for anabolic therapy for the treatment of osteoporosis by stimulating bone morphogenetic protein 2 (BMP2) and Wnt/β-catenin mechanism. CAFG supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur and decreased bone turnover markers better than genistein. Oral administration of CAFG to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased the expression of osteogenic genes in femur and show new bone formation without uterine hyperplasia. CAFG increased mRNA expression of osteoprotegerin in bone and inhibited osteoclast activation by inhibiting the expression of skeletal osteoclastogenic genes. CAFG is also an effective accelerant for chondrogenesis and has stimulatory effect on the repair of cortical bone after drill-hole injury at the tissue, cell and gene level in mouse femur. At cellular levels, CAFG stimulated osteoblast proliferation, survival and differentiation. Signal transduction inhibitors in osteoblast demonstrated involvement of p-38 mitogen-activated protein kinase pathway stimulated by BMP2 to initiate Wnt/β-catenin signaling to reduce phosphorylation of GSK3-β and subsequent nuclear accumulation of β-catenin. Osteogenic effects were abrogated by Dkk1, Wnt-receptor blocker and FH535, inhibitor of TCF-complex by reduction in β-catenin levels. CAFG modulated MSC responsiveness to BMP2, which promoted osteoblast differentiation via Wnt/β-catenin mechanism. CAFG at 1 mg/kg(/)day dose in

  4. Expression of PCNA,BCL-2 and BMP2/4 in diagnosis and prognosis of adenocarcinoma of the duodenum value%PCNA、BCL-2及 BMP2/4的表达对十二指肠腺癌的诊断意义及预后价值

    Institute of Scientific and Technical Information of China (English)

    李忠辉; 张晋冀; 石书玮; 邰智慧

    2016-01-01

    Objective To observe the expression of PCNA,BCL-2 and BMP2/4,in patients with duodenal adenocar-cinoma,to further explore the clinical significance of PCNA,BCL-2 and BMP2/4 in the diagnosis and prognosis.Meth-ods From 2005 January to 2014 November in our hospital check biopsy diagnosed 45 cases of duodenal adenocarcinoma patients,according to the Joint Committee American cancer AJCC staging criteria above,the patients were divided into group Ⅰ(7 cases),group Ⅱ(16 cases),group Ⅲ(13 cases)and IV (9 cases),using immunohistochemical staining SP method,the expression of PCNA,BCL-2 and BMP2/4detection in patients with biopsy is different,and the control group (45 cases)were analyzed by statistical software,calculation,the relationship between the expression of further a-nalysis of above factors and duodenal adenocarcinoma.Results The expression level of PCNA,BCL-2 and BMP2/4 in patients with duodenal adenocarcinoma,increased significantly compared with the control group (P <0.05),Ⅰ,Ⅱ,Ⅲ,Ⅳ,the difference between the four groups was significantly(P <0.05).Conclusion Expression of PCNA,BCL-2 and BMP2/4 of duodenal adenocarcinoma tissues is closely related to the disease,at the same time with the biological behav-ior and pathology of the disease the academic stages of relevance,it has certain guiding significance for the diagnosis of duodenaladenocarcinoma and prognosis.%目的:观察十二指肠腺癌患者体内 PCNA、BCL-2及 BMP2/4的表达情况,进一步探讨 PCNA、BCL-2及BMP2/4在该疾病诊断及预后中的临床意义。方法选取2005年1月至2014年11月间在我院检查取组织活检确诊为十二指肠腺癌患者45例,根据美国癌症联合委员会 AJCC 的分期标准,将以上患者分为Ⅰ组(7例)、Ⅱ组(16例)、Ⅲ组(13例)以及Ⅳ组(9例),采用免疫组织化学染色 SP 法,检测不同患者活检组织中的 PCNA、BCL-2及BMP2/4的表达情况,并与对照组(45例)进行对

  5. Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation.

    Science.gov (United States)

    Higashino, Kosaku; Viggeswarapu, Manjula; Bargouti, Maggie; Liu, Hui; Titus, Louisa; Boden, Scott D

    2011-02-01

    The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.

  6. Construction and identification of human BMP2-IRES-HIF1αmu adenovirus expressing carrier and its expression in HEK293 cells%人BMP2-IRES-HIF1αmu腺病毒表达载体的构建及其在HEK293细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    李全营; 李谌; 郭威; 吴秀成; 王巍; 刘丹平

    2011-01-01

    目的 构建人BMP2-IRES-HIF1 αmu腺病毒表达载体,并转染HEK293细胞,为下一步转染骨髓基质细胞和体内实验打下基础.方法 PCR扩增HIF1αmu片段,用BstXⅠ和XbaⅠ双酶切回收目的片段.pIRES2-EGFP用BstXⅠ和Xba Ⅰ进行双酶切后回收大片段.将上述回收的目的基因与载体片段连接,然后转化感受态大肠杆菌DH5α扩增;PCR扩增BMP2片段,用Nhe Ⅰ和BamH Ⅰ双酶切后回收目的片段.把目的基因与载体片段连接,转化感受态大肠杆菌DH5α扩增重组腺病毒表达载体,通过酶切分析、PCR和测序进行鉴定.将构建好的质粒转染HEK293细胞,检测病毒液滴度.结果 构建了人BMP2-1RES-HIF1 αmu腺病毒表达载体,转染HEK293细胞见绿色荧光表达.结论 成功构建了人BMP2 -IRES-HIF1 αmu腺病毒表达载体,酶切分析及DNA测序证实质粒构建正确,质粒成功转染HEK293细胞,并见绿色荧光蛋白表达.%Objective To construct and identify human BMP2-IRES-HIFlotmu Adenovirus expressing carrier, trans-feet it in HEK293 cells, and determinate the virus droplet degrees. Methods PCR was used to amplify HIFlamu segments, BstX I and Xba I double enzyme cut pIRES2-EGFP and recycling purpose extract. Connecting the recovery target gene with carrier segment. Then introduced it into E. Coli for amplification. PCR was used to amplify BMP2 segments, Nhe I and BamH I double enzyme cut and recycling purpose extract. Connecting the recovery target gene with carrier segment. Then introduced it into E. Coli for amplification and restructuring adenovirus expressing carrier. Using the enzyme cut analysis, PCR for identification. The correct recombinant express plasmid was transfected into HEK293 cells, and detecting the virus droplet degrees. Results The adenovirus shuttle plasmid was constructed. Green fluorescent expression was seen in transfected HEK293 cells. Conclusion The adenovirus shuttle plasmid is constructed, it is successfully expressed in HEK

  7. Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm.