WorldWideScience

Sample records for betacellulin inhibits osteogenic

  1. Inhibition of Histone Deacetylases Potentiates BMP9-Induced Osteogenic Signaling in Mouse Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Ning Hu

    2013-08-01

    Full Text Available Background/Aims: We have demonstrated that bone morphogenetic protein 9 (BMP9 is one of the most potent BMPs in regulating osteoblast differentiation of mesenchymal stem cells (MSCs although the molecular mechanism underlying BMP9-induced osteogenesis remains to be fully elucidated. It is known that epigenetic regulations play an important role in regulating the stem cell potency and lineage commitment. Here, we investigate if the inhibition of histone deacetylases (Hdacs affects BMP9-induced osteogenic differentiation of MSCs. Methods: Using the Hdac inhibitor trichostatin A (TSA, we assess that TSA enhances BMP9-mediated osteogenic markers and matrix mineralization in MSCs, and bone formation in mouse embryonic limb explants. Results: We find that the endogenous expression of most of the 11 Hdacs is readily detectable in MSCs. BMP9 is shown to induce most Hdacs in MSCs. We demonstrate that TSA potentiates BMP9-induced early osteogenic marker alkaline phosphatase (ALP activity in MSCs, as well as late osteogenic markers osteopontin (OPN and osteocalcin (OCN and matrix mineralization. Fetal limb explant culture studies reveal that TSA potentiates BMP9-induced endochondral bone formation, possibly by expanding hypertrophic chondrocyte zone of growth plate. Conclusion: Our findings strongly suggest histone deacetylases may play an important role in fine-tuning BMP9-mediated osteogenic signaling through a negative feedback network in MSCs. Thus, Hdac inhibitors may be used as novel therapeutics for bone fracture healing.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models.

    Science.gov (United States)

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; van de Klundert, Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; van Blitterswijk, Clemens; de Boer, Jan

    2009-08-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications. PMID:19231969

  4. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells

    DEFF Research Database (Denmark)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias;

    2016-01-01

    in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients....... As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation...... by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor...

  5. Azelnidipine inhibits Msx2-dependent osteogenic differentiation and matrix mineralization of vascular smooth muscle cells.

    Science.gov (United States)

    Shimizu, Takehisa; Tanaka, Toru; Iso, Tatsuya; Kawai-Kowase, Keiko; Kurabayashi, Masahiko

    2012-01-01

    Vascular calcification is an active and regulated process that is similar to bone formation. While calcium channel blockers (CCBs) have been shown to improve outcomes in atherosclerotic vascular disease, it remains unknown whether CCBs have an effect on the process of vascular calcification. Here we investigated whether CCBs inhibit osteogenic differentiation and matrix mineralization of vascular smooth muscle cells induced by Msx2, a key factor of vascular calcification. Human aortic smooth muscle cells (HASMCs) were transduced with adenovirus expressing MSX2 and were treated with 3 distinct CCBs. Azelnidipine, a dihydropyridine subclass of CCBs, significantly decreased alkaline phosphatase (ALP) activity of Msx2-overexpressed HASMCs, whereas verapamil and diltiazem had no effect. Furthermore, azelnidipine, but not verapamil and diltiazem, significantly decreased matrix mineralization of Msx2-overexpressing HASMCs. Azelnidipine significantly attenuated the induction of ALP gene expression by Msx2, a key transcription factor in osteogenesis, while it did not reduce enzymatic activity of ALP. Furthermore, azelnidipine inhibited the ability of Msx2 to activate the ALP gene, but had no effect on Notch-induced Msx2 expression. Given that L-type calcium channels are equally blocked by these CCBs, our results suggest that azelnidipine inhibits the Msx2-dependent process of vascular calcification by mechanisms other than inhibition of calcium channel activity.

  6. Secreted microvesicular miR-31 inhibits osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Weilner, Sylvia; Schraml, Elisabeth; Wieser, Matthias; Messner, Paul; Schneider, Karl; Wassermann, Klemens; Micutkova, Lucia; Fortschegger, Klaus; Maier, Andrea B; Westendorp, Rudi; Resch, Heinrich; Wolbank, Susanne; Redl, Heinz; Jansen-Dürr, Pidder; Pietschmann, Peter; Grillari-Voglauer, Regina; Grillari, Johannes

    2016-08-01

    Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor. PMID:27146333

  7. Inhibiting PPARγ by erythropoietin while upregulating TAZ by IGF1 synergistically promote osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Zhou, Jianwei; Wei, Fangyuan; Ma, Yuquan

    2016-09-01

    Erythropoietin (EPO) is reported to promote osteogenesis and inhibit adipogenesis of mesenchymal stem cells (MSC) through inhibiting PPARγ, while insulin-like growth factor 1 (IGF1) is able to enhance osteogenesis via upregulating transcriptional coactivator with PDZ-binding motif (TAZ). The different targets of EPO and IGF1 suggested their potential synergism to enhance osteogenesis. In this study, we aimed to determine the potential synergism of EPO and IGF1 and its efficacy on MSC differentiation. Rat adipose-derived mesenchymal stem cells (ADSCs) were separately treated with EPO, IGF1 and EPO/IGF1. It was observed that the co-treatment using EPO and IGF1 was able to potently promote the osteogenic differentiation of rat ADSCs compared with EPO or IGF1 alone, which offered a promising effective option to strengthen bone tissue regeneration for bone defects. Further, we demonstrated that the enhanced osteogenic differentiation by EPO and IGF1 co-treatment was almost counteracted by activating PPARγ through PPARγ agonist, RSG, and blocking TAZ through TAZ silencing RNA, siTAZ. Thus, it could be concluded that EPO and IGF1 possessed a potent synergism in promoting osteogenic differentiation, and the synergism was mainly attributed to co-regulation of different osteogenic regulators PPARγ and TAZ, which were targeted genes of EPO and IGF1 respectively. PMID:27422606

  8. Combined effects of proinflammatory cytokines and intermittent cyclic mechanical strain in inhibiting osteogenicity in human periodontal ligament cells.

    Science.gov (United States)

    Sun, Chaofan; Chen, Lijiao; Shi, Xinlian; Cao, Zhensheng; Hu, Bibo; Yu, Wenbin; Ren, Manman; Hu, Rongdang; Deng, Hui

    2016-09-01

    Mechanical strain plays an important role in bone formation and resorption during orthodontic tooth movement. The mechanism has not been fully studied, and the process becomes complex with increased amounts of periodontal patients seeking orthodontic care. Our aims were to elucidate the combined effects of proinflammatory cytokines and intermittent cyclic strain (ICS) on the osteogenic capacity of human periodontal ligament cells. Cultured human periodontal ligament cells were exposed to proinflammatory cytokines (interleukin-1β 5 ng/mL and tumor necrosis factor-α 10 ng/mL) for 1 and 5 days, and ICS (0.5 Hz, 12% elongation) was applied for 4 h per day. The autocrine of inflammatory cytokines was measured by enzyme-linked immunosorbent assay. The expression of osteoblast markers runt-related transcription factor 2 and rabbit collagen type I was determined using real-time polymerase chain reaction and Western blot. The osteogenic capacity was also detected by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. We demonstrated that ICS impaired the osteogenic capacity of human periodontal ligament cells when incubated with proinflammatory cytokines, as evidenced by the low expression of ALP staining, low ALP activity, reduced alizarin red staining, and reduced osteoblast markers. These data, for the first time, suggest that ICS has a negative effect on the inductive inhibition of osteogenicity in human PDL cells mediated by proinflammatory cytokines. PMID:27357508

  9. Naringin protects human adipose-derived mesenchymal stem cells against hydrogen peroxide-induced inhibition of osteogenic differentiation.

    Science.gov (United States)

    Wang, Lei; Zhang, Yu-Ge; Wang, Xiu-Mei; Ma, Long-Fei; Zhang, Yuan-Min

    2015-12-01

    Extensive evidence indicates that oxidative stress plays a pivotal role in the development of osteoporosis. We show that naringin, a natural antioxidant and anti-inflammatory compound, effectively protects human adipose-derived mesenchymal stem cells (hADMSCs) against hydrogen peroxide (H2O2)-induced inhibition of osteogenic differentiation. Naringin increased viability of hAMDSCs and attenuated H2O2-induced cytotoxicity. Naringin also reversed H2O2-induced oxidative stress. Oxidative stress induced by H2O2 inhibits osteogenic differentiation by decreasing alkaline phosphatase (ALP) activity, calcium content and mRNA expression levels of osteogenesis marker genes RUNX2 and OSX in hADMSCs. However, addition of naringin leads to a significant recovery, suggesting the protective effects of naringin against H2O2-induced inhibition of osteogenic differentiation. Furthermore, the H2O2-induced decrease of protein expressions of β-catenin and clyclin D1, two important transcriptional regulators of Wnt-signaling, was successfully rescued by naringin treatment. Also, in the presence of Wnt inhibitor DKK-1, naringin is no longer effective in stimulating ALP activity, increasing calcium content and mRNA expression levels of RUNX2 and OSX in H2O2-exposed hADMSCs. These data clearly demonstrates that naringin protects hADMSCs against oxidative stress-induced inhibition of osteogenic differentiation, which may involve Wnt signaling pathway. Our work suggests that naringin may be a useful addition to the treatment armamentarium for osteoporosis and activation of Wnt signaling may represent attractive therapeutic strategy for the treatment of degenerative disease of bone tissue. PMID:26482937

  10. Simulated microgravity inhibits osteogenic differentiation of mesenchymal stem cells via depolymerizing F-actin to impede TAZ nuclear translocation

    Science.gov (United States)

    Chen, Zhe; Luo, Qing; Lin, Chuanchuan; Kuang, Dongdong; Song, Guanbin

    2016-01-01

    Microgravity induces observed bone loss in space flight, and reduced osteogenesis of bone mesenchymal stem cells (BMSCs) partly contributes to this phenomenon. Abnormal regulation or functioning of the actin cytoskeleton induced by microgravity may cause the inhibited osteogenesis of BMSCs, but the underlying mechanism remains obscure. In this study, we demonstrated that actin cytoskeletal changes regulate nuclear aggregation of the transcriptional coactivator with PDZ-binding motif (TAZ), which is indispensable for osteogenesis of bone mesenchymal stem cells (BMSCs). Moreover, we utilized a clinostat to model simulated microgravity (SMG) and demonstrated that SMG obviously depolymerized F-actin and hindered TAZ nuclear translocation. Interestingly, stabilizing the actin cytoskeleton induced by Jasplakinolide (Jasp) significantly rescued TAZ nuclear translocation and recovered the osteogenic differentiation of BMSCs in SMG, independently of large tumor suppressor 1(LATS1, an upstream kinase of TAZ). Furthermore, lysophosphatidic acid (LPA) also significantly recovered the osteogenic differentiation of BMSCs in SMG through the F-actin-TAZ pathway. Taken together, we propose that the depolymerized actin cytoskeleton inhibits osteogenic differentiation of BMSCs through impeding nuclear aggregation of TAZ, which provides a novel connection between F-actin cytoskeleton and osteogenesis of BMSCs and has important implications in bone loss caused by microgravity. PMID:27444891

  11. Magnesium inhibits Wnt/β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

    Science.gov (United States)

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M; Madueño, Juan A; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  12. Lithium inhibits palatal fusion and osteogenic differentiation in palatal shelves in vitro

    NARCIS (Netherlands)

    Meng, L.; Wang, X.; Torensma, R.; Hoff, J.W. Von den; Bian, Z.

    2014-01-01

    OBJECTIVE: Glycogen synthase kinase-3beta (Gsk-3beta)/beta-catenin signaling regulates development of the secondary palate. It has been unclear about the effects of Gsk-3beta/beta-catenin signaling on palatal fusion and osteogenic differentiation in palatal shelves. DESIGN: In this study, palatal sh

  13. Lithium inhibits palatal fusion and osteogenic differentiation in palatal shelves in vitro

    NARCIS (Netherlands)

    Meng, L.; Wang, X.; Torensma, R.; Hoff, J.W. Von den; Bian, Z.

    2015-01-01

    OBJECTIVE: Glycogen synthase kinase-3beta (Gsk-3beta)/beta-catenin signaling regulates development of the secondary palate. It has been unclear about the effects of Gsk-3beta/beta-catenin signaling on palatal fusion and osteogenic differentiation in palatal shelves. DESIGN: In this study, palatal sh

  14. Androgens inhibit the osteogenic response to mechanical loading in adult male mice.

    Science.gov (United States)

    Sinnesael, Mieke; Laurent, Michaël R; Jardi, Ferran; Dubois, Vanessa; Deboel, Ludo; Delisser, Peter; Behets, Geert J; D'Haese, Patrick C; Carmeliet, Geert; Claessens, Frank; Vanderschueren, Dirk

    2015-04-01

    Androgens are well known to enhance exercise-induced muscle hypertrophy; however, whether androgens also influence bone's adaptive response to mechanical loading remains unclear. We studied the adaptive osteogenic response to unilateral in vivo mechanical loading of tibia in adult male mice in both a long- and a short-term experimental set-up. Mice were divided into four groups: sham operated, orchidectomized (ORX), T (ORX+T), or nonaromatizable dihydrotestosterone (ORX+DHT) replacement. Significant interactions between androgen status and osteogenic response to mechanical loading were observed. Cortical thickness increased by T (0.14 vs 0.11 mm sham, P<.05) and DHT (0.17 vs 0.11 mm sham, P<.05). However, T partially (+36%) and DHT completely (+10%) failed to exhibit the loading-related increase observed in sham (+107%) and ORX (+131%, all P<.05) mice. ORX decreased periosteal bone formation, which was restored to sham levels by T and DHT. However, both androgens completely suppressed the loading-related increase in periosteal bone formation. Short-term loading decreased the number of sclerostin-positive osteocytes in sham, whereas in control fibulas, ORX decreased and T increased the number of sclerostin-positive osteocytes. Loading no longer down-regulated sclerostin in the ORX or T groups. In conclusion, both T and DHT suppress the osteogenic response to mechanical loading.

  15. Magnesium inhibits Wnt/β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Addy Montes de Oca

    Full Text Available Magnesium reduces vascular smooth muscle cell (VSMC calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a whether inhibition of magnesium transport into the cell influences VSMC calcification, b whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM and moderately elevated magnesium (1.4 mM significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP and osteoprotegerin (OPG. The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]. High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in

  16. MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells

    OpenAIRE

    Huszar, Jessica M.; Payne, Christopher J.

    2014-01-01

    Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A...

  17. MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes

    Science.gov (United States)

    Qu, Xiaochen; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zeng, Yan

    2016-01-01

    Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6. PMID:27556448

  18. The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3.

    Science.gov (United States)

    Klingelhöffer, C; Reck, A; Ettl, T; Morsczeck, C

    2016-08-01

    The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: pHedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs. PMID:27368119

  19. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Jian Wang

    2016-01-01

    Full Text Available After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF, Adipose-derived Stem Cells (ASCs and those labeled by superparamagnetic iron oxide (SPIO nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT assay, proliferation by cell counting and bromodeoxyuridine (BrdU incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF, Insulin-like Growth Factor-1 (IGF-1, Transforming Growth Factor Beta 1 (TGF-β1, genetic markers comprising Stem Cell Antigen-1 (Sca1, Octamer-4 (Oct-4, ATP-binding Cassette Subfamily B Member 1 (ABCB1, adipogenic marker genes containing Lipoprotein Lipase (LPL, Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ, and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1 and Osterix (OSX. Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling.

  20. Analysis of coding variants in the betacellulin gene in type 2 diabetes and insulin secretion in African American subjects

    Directory of Open Access Journals (Sweden)

    Karim Mohammad A

    2006-07-01

    Full Text Available Abstract Background Betacellulin is a member of the epidermal growth factor family, expressed at the highest levels predominantly in the pancreas and thought to be involved in islet neogenesis and regeneration. Nonsynonymous coding variants were reported to be associated with type 2 diabetes in African American subjects. We tested the hypotheses that these previously identified variants were associated with type 2 diabetes in African Americans ascertained in Arkansas and that they altered insulin secretion in glucose tolerant African American subjects. Methods We typed three variants, exon1 Cys7Gly (C7G, exon 2 Leu44Phe (L44F, and exon 4 Leu124Met (L124M, in 188 control subjects and 364 subjects with type 2 diabetes. We tested for altered insulin secretion in 107 subjects who had undergone intravenous glucose tolerance tests to assess insulin sensitivity and insulin secretion. Results No variant was associated with type 2 diabetes, and no variant altered insulin secretion or insulin sensitivity. However, an effect on lipids was observed for all 3 variants, and variant L124M was associated with obesity measures. Conclusion We were unable to confirm a role for nonsynonymous variants of betacellulin in the propensity to type 2 diabetes or to impaired insulin secretion.

  1. Super pharmacological levels of calcitriol (1,25-(OH)2D3) inhibits mineral deposition and decreases cell proliferation in a strain dependent manner in chicken mesenchymal stem cells undergoing osteogenic differentiation in vitro

    Science.gov (United States)

    Pande, Vivek V.; Chousalkar, Kapil C.; Bhanugopan, Marie S.; Quinn, Jane C.

    2015-01-01

    The biologically active form of vitamin D3, calcitriol (1,25-(OH)2D3), plays a key role in mineral homeostasis and bone formation and dietary vitamin D3 deficiency is a major cause of bone disorders in poultry. Supplementary dietary cholecalciferol (25-hydroxyvitamin D, 25-OH), the precursor of calcitriol, is commonly employed to combat this problem; however, dosage must be carefully determined as excess dietary vitamin D can cause toxicity resulting in a decrease in bone calcification, hypercalcinemia and renal failure. Despite much research on the therapeutic administration of dietary vitamin D in humans, the relative sensitivity of avian species to exogenous vitamin D has not been well defined. In order to determine the effects of exogenous 1,25-(OH)2D3 during avian osteogenesis, chicken bone marrow-derived mesenchymal stem cells (BM-MSCs) were exposed to varying doses of 1,25-(OH)2D3 during in vitro osteogenic differentiation and examined for markers of early proliferation and osteogenic induction. Similar to humans and other mammals, poultry BM-MSCs were found to be highly sensitive to exogenous 1,25-(OH)2D3 with super pharmacological levels exerting significant inhibition of mineralization and loss of cell proliferation in vitro. Strain related differences were apparent, with BM-MCSs derived from layers strains showing a higher level of sensitivity to 1,25-(OH)2D3 than those from broilers. These data suggest that understanding species and strain specific sensitivities to 1,25-(OH)2D3 is important for optimizing bone health in the poultry industry and that use of avian BM-MSCs are a useful tool for examining underlying effects of genetic variation in poultry. PMID:26500277

  2. Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

    Science.gov (United States)

    Zheng, Dong; Neoh, Koon Gee; Kang, En-Tang

    2016-01-01

    In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50 μg/cm2 resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1 h immersion in 70% ethanol and autoclaving at 121 °C for 20 min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.

  3. Matrix stiffness regulation of integrin-mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Shih, Yu-Ru V; Tseng, Kuo-Fung; Lai, Hsiu-Yu; Lin, Chi-Hung; Lee, Oscar K

    2011-04-01

    Mesenchymal stem cells (MSCs) cultured on extracellular matrices with different stiffness have been shown to possess diverse lineage commitment owing to the extracellular mechanical stimuli sensed by the cells. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through the mechanotransducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MSCs. MSCs were cultured in osteogenic medium on tunable polyacrylamide hydrogels coated with type I collagen with elasticities corresponding to Young's modulus of 7.0 ± 1.2 and 42.1 ± 3.2 kPa. Osteogenic differentiation was increased on stiffer matrices, as evident by type I collagen, osteocalcin, and Runx2 gene expressions and alizarin red S staining for mineralization. Western blot analysis demonstrated an increase in kinase activities of ROCK, FAK, and ERK1/2 on stiffer matrices. Inhibition of FAK, an important mediator of osteogenic differentiation, and inhibition of ROCK, a known mechanotransducer of matrix stiffness during osteogenesis, resulted in decreased expression of osteogenic markers during osteogenic induction. In addition, FAK affects osteogenic differentiation through ERK1/2, whereas ROCK regulates both FAK and ERK1/2. Furthermore, α(2)-integrin was upregulated on stiffer matrices during osteogenic induction, and its knockdown by siRNA downregulated the osteogenic phenotype through ROCK, FAK, and ERK1/2. Taken together, our results provide evidence that the matrix rigidity affects the osteogenic outcome of MSCs through mechanotransduction events that are mediated by α(2)-integrin.

  4. Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

    Directory of Open Access Journals (Sweden)

    Teruyo Oishi

    Full Text Available Heterotopic ossification (HO is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+ and PDGFRα(+ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+ cells and PDGFRα(+ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+ cells formed bone-like tissue and showed successful engraftment, while CD56(+ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+ cells. Our results suggest that PDGFRα(+ cells may be the major source of HO and that the newly identified mi

  5. MicroRNA-24 Regulates Osteogenic Differentiation via Targeting T-Cell Factor-1

    Directory of Open Access Journals (Sweden)

    Weigong Zhao

    2015-05-01

    Full Text Available MicroRNAs (miRNAs have been reported to have diverse biological roles in regulating many biological processes, including osteogenic differentiation. In the present study, we identified that miR-24 was a critical regulator during osteogenic differentiation. We found that overexpression of miR-24 significantly inhibited osteogenic differentiation, which decreased alkaline phosphatase activity, matrix mineralization and the expression of osteogenic differentiation markers. In contrast, inhibition of miR-24 exhibited an opposite effect. Furthermore, we delineated that miR-24 regulates post-transcriptionals of T-cell factor-1 (Tcf-1 via targeting the 3'-untranslated region (UTR of Tcf-1 mRNA. MiR-24 was further found to regulate the protein expression of Tcf-1 in the murine osteoprogenitors cells and bone mesenchymal stem cells. Additionally, the positive effect of miR-24 suppression on osteoblast differentiation was apparently abrogated by Tcf-1 silencing. Taken together, our data suggest that miR-24 participates in osteogenic differentiation by targeting and regulating Tcf-1 expression in osteoblastic cells.

  6. Primary osteogenic sarcoma of the breast

    Directory of Open Access Journals (Sweden)

    Akang Effiong E

    2006-12-01

    Full Text Available Abstract Background Primary extra-osseous osteogenic sarcomas have been reported in many tissues of the body but their occurrence in the breast is extremely rare. It can arise as a result of osseous metaplasia in a pre-existing benign or malignant neoplasm of the breast or as non-phylloides sarcoma from the soft tissue of a previously normal breast. Case presentation A 40 year-old Nigerian woman was clinically diagnosed to have carcinoma of the left breast. The histology report of core-needle biopsy of the mass showed a malignant neoplasm comprising islands of chondroblastic and osteoblastic stromal cells. This report changed the diagnosis from carcinoma to osteogenic sarcoma of the breast. She had a left modified radical mastectomy, however there was significant post surgery skin deficit. A latissimus dorsi musculocutaneous flap was used to cover the anterior chest wall defect. Sections from the mastectomy specimen confirmed the diagnosis of osteogenic sarcoma. She died six months after mastectomy. Conclusion A diagnosis of osteogenic sarcoma of the breast was made based on histology report and after excluding an osteogenic sarcoma arising from underlying ribs and sternum. This is the second documented case of primary osteogenic sarcoma of the breast coming from Nigeria

  7. Bone Marrow Stress Decreases Osteogenic Progenitors.

    Science.gov (United States)

    Ng, Adeline H; Baht, Gurpreet S; Alman, Benjamin A; Grynpas, Marc D

    2015-11-01

    Age-related bone loss may be a result of declining levels of stem cells in the bone marrow. Using the Col2.3Δtk (DTK) transgenic mouse, osteoblast depletion was used as a source of marrow stress in order to investigate the effects of aging on osteogenic progenitors which reside in the marrow space. Five-month-old DTK mice were treated with one or two cycles of ganciclovir to conditionally ablate differentiated osteoblasts, whereas controls were saline-treated. Treatment cycles were two weeks in length followed by four weeks of recovery. All animals were sacrificed at 8 months of age; bone marrow stromal cells (BMSCs) were harvested for cell culture and whole bones were excised for bone quality assessment. Colony-forming unit (CFU) assays were conducted to investigate the osteogenic potential of BMSC in vitro, and RNA was extracted to assess the expression of osteoblastic genes. Bone quality assessments included bone histomorphometry, TRAP staining, microcomputed tomography, and biomechanical testing. Osteoblast depletion decreased CFU-F (fibroblast), CFU-ALP (alkaline phosphatase), and CFU-VK (von Kossa) counts and BMSC osteogenic capacity in cell culture. Ex vivo, there were no differences in bone mineral density of vertebrae or femurs between treatment groups. Histology showed a decrease in bone volume and bone connectivity with repeated osteoblast depletion; however, this was accompanied by an increase in bone formation rate. There were no notable differences in osteoclast parameters or observed bone marrow adiposity. We have developed a model that uses bone marrow stress to mimic age-related decrease in osteogenic progenitors. Our data suggest that the number of healthy BMSCs and their osteogenic potential decline with repeated osteoblast depletion. However, activity of the remaining osteoblasts increases to compensate for this loss in progenitor osteogenic potential. PMID:26220824

  8. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  9. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Science.gov (United States)

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  10. Osteogenic sarcoma with skeletal muscle metastases

    Energy Technology Data Exchange (ETDEWEB)

    Peh, W.C.G. [Department of Diagnostic Radiology, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Shek, T.W.H. [Department of Pathology, The University of Hong Kong, Queen Mary Hospital (Hong Kong); Wang Shihchang [Department of Diagnostic Imaging, National University of Singapore, National University Hospital (Singapore); Wong, J.W.K.; Chien, E.P. [Department of Orthopaedic Surgery, The University of Hong Kong, Queen Mary Hospital (Hong Kong)

    1999-05-01

    Two cases of osteogenic sarcoma with skeletal muscle metastases are described. A 40-year-old woman presented with progressive swelling of both calves and a soft tissue back lump. She had been diagnosed with mandibular chondroblastic osteogenic sarcoma 6 years earlier. Radiographs showed calcified masses. MRI scans and bone scintigraphy revealed multiple soft tissue masses in both calves. Bone scintigraphy also showed uptake in the back lump, right thigh and left lung base. Biopsy confirmed metastatic chondroblastic osteogenic sarcoma, which initially responded well to chemotherapy. However, the metastatic disease subsequently progressed rapidly and she died 21 months after presentation. The second case concerns a 20-year-old man who presented with a pathologic fracture of the humerus, which was found to be due to osteoblastic osteogenic sarcoma. He developed cerebral metastases 17 months later, followed by metastases at other sites. Calcified masses were subsequently seen on radiographs of the abdomen and chest. CT scans confirmed the presence of densely calcified muscle metastases in the abdominal wall, erector spinae and gluteal muscles. The patient`s disease progressed rapidly and he died 30 months after presentation. (orig.) With 6 figs., 29 refs.

  11. Mechanisms of stem cell osteogenic differentiation on TiO2 nanotubes.

    Science.gov (United States)

    Yu, Weiqiang; Qian, Chao; Jiang, Xinquan; Zhang, Fuqiang; Weng, Weimin

    2015-12-01

    TiO2 nanotubes could stimulate osteogenic differentiation of stem cells, but the molecular mechanisms underlying the interactions between nanotubes and stem cells remain unclear. In this study, we investigated the response of bone marrow stromal cells to nanotubes of different diameters using microarray-based bioinformatics approach. Gene ontology (GO) and GO enrichment network analysis indicated that larger TiO2 nanotubes were more potent than smaller nanotubes in inducing the expression of genes involved in cell proliferation, differentiation, and immune responses, and inhibiting that of genes responsible for cell adhesion. The analysis of the signaling network containing significantly affected genes suggested that Na(+)/K(+) transporting ATPases ATP1A2 (alpha 2 polypeptide) and ATP1A3 (alpha 3 polypeptide), and MAP3K11 (mitogen-activated protein kinase kinase kinase 11) were important for inducing osteogenic differentiation of bone marrow stromal cells without additional osteogenic stimuli. The upregulation of the ATP1A2 and MAP3K11 genes confirmed by real-time PCR indicates that the response of bone marrow stromal cells to nanotube cues may be mediated by the pathways previously implicated in transducing mechanical stress signals. Our results revealed some molecular mechanisms by which TiO2 nanotubes may direct osteogenic differentiation of stem cells.

  12. Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Maroni, Paola [Istituto Ortopedico Galeazzi, Milano (Italy); Brini, Anna Teresa [Istituto Ortopedico Galeazzi, Milano (Italy); Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano (Italy); Arrigoni, Elena [Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano (Italy); Girolamo, Laura de [Istituto Ortopedico Galeazzi, Milano (Italy); Niada, Stefania [Istituto Ortopedico Galeazzi, Milano (Italy); Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano (Italy); Matteucci, Emanuela; Bendinelli, Paola [Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universita degli Studi di Milano, Milano (Italy); Desiderio, Maria Alfonsina, E-mail: a.desiderio@unimi.it [Dipartimento di Scienze Biomediche per la Salute, Molecular Pathology Laboratory, Universita degli Studi di Milano, Milano (Italy)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Acetylation affected hASCs osteodifferentiation through Runx2-PPAR{gamma}. Black-Right-Pointing-Pointer HDACs knocking-down favoured the commitment effect of osteogenic medium. Black-Right-Pointing-Pointer HDACs silencing early activated Runx2 and ALP. Black-Right-Pointing-Pointer PPAR{gamma} reduction and calcium/collagen deposition occurred later. Black-Right-Pointing-Pointer Runx2/PPAR{gamma} target genes were modulated in line with HDACs role in osteo-commitment. -- Abstract: The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) {gamma}. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPAR{gamma} and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPAR{gamma}/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPAR{gamma} target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal

  13. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1.

    Science.gov (United States)

    Wang, J; Wang, C D; Zhang, N; Tong, W X; Zhang, Y F; Shan, S Z; Zhang, X L; Li, Q F

    2016-01-01

    Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation.

  14. Periodontally accelerated osteogenic orthodontics (PAOO) - a review

    OpenAIRE

    Amit, Goyal; JPS, Kalra; Pankaj, Bhatiya; Suchinder, Singla; Parul, Bansal

    2012-01-01

    With an increasing number of adult patients coming to the orthodontic clinic, the orthodontic professional is constantly looking for ways to accelerate tooth movement. Surgical intervention to affect the alveolar housing and tooth movement has been described in various forms for over a hundred years. However, it is the spirit of interdisciplinary collaboration in orthodontics has expanded the realm of traditional orthodontic tooth movement protocols. Periodontal accelerated osteogenic orthodo...

  15. Biocompatibility and osteogenic properties of porous tantalum

    OpenAIRE

    Wang, Qian; Zhang, Hui; LI, QIJIA; Ye,Lei; GAN, HONGQUAN; Liu, Yingjie; Wang, Hui; Wang, Zhiqiang

    2015-01-01

    Porous tantalum has been reported to be a promising material for use in bone tissue engineering. In the present study, the biocompatibility and osteogenic properties of porous tantalum were studied in vitro and in vivo. The morphology of porous tantalum was observed using scanning electron microscopy (SEM). Osteoblasts were cultured with porous tantalum, and cell morphology, adhesion and proliferation were investigated using optical microscopy and SEM. In addition, porous tantalum rods were i...

  16. Methods for analyzing microRNA expression and function during osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Kim, Yeon Jeong; Jung, Jin Sup

    2011-01-01

    MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length that regulate gene expression at the posttranscriptional level. They may play important roles during osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (hASC). In this chapter, we focus on the methods and strategies for elucidating miRNA function during osteogenic differentiation. We describe a miRNA expression analysis protocol, and a lentiviral vector strategy for the ectopic expression of miRNA in hASC to determine the role of miRNA during osteogenic differentiation. We also describe miRNA inhibition to further determine the role of miRNA during osteogenic differentiation, and a luciferase assay to demonstrate direct binding between a specific miRNA and its putative target.

  17. Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway.

    Science.gov (United States)

    Brum, Andrea M; van de Peppel, Jeroen; van der Leije, Cindy S; Schreuders-Koedam, Marijke; Eijken, Marco; van der Eerden, Bram C J; van Leeuwen, Johannes P T M

    2015-10-13

    Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs). Gene expression profiling was performed in hMSCs differentiated toward osteoblasts (at 6 h). Significantly regulated genes were analyzed in silico, and the Connectivity Map (CMap) was used to identify candidate bone stimulatory compounds. The signature of parbendazole matches the expression changes observed for osteogenic hMSCs. Parbendazole stimulates osteoblast differentiation as indicated by increased alkaline phosphatase activity, mineralization, and up-regulation of bone marker genes (alkaline phosphatase/ALPL, osteopontin/SPP1, and bone sialoprotein II/IBSP) in a subset of the hMSC population resistant to the apoptotic effects of parbendazole. These osteogenic effects are independent of glucocorticoids because parbendazole does not up-regulate glucocorticoid receptor (GR) target genes and is not inhibited by the GR antagonist mifepristone. Parbendazole causes profound cytoskeletal changes including degradation of microtubules and increased focal adhesions. Stabilization of microtubules by pretreatment with Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Cotreatment with the BMP-2 antagonist DMH1 limits, but does not block, parbendazole-induced mineralization. Using the CMap we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteogenic differentiation through a combination of cytoskeletal changes and increased BMP-2 activity. PMID:26420877

  18. The effects of acellular amniotic membrane matrix on osteogenic differentiation and ERK1/2 signaling in human dental apical papilla cells.

    Science.gov (United States)

    Chen, Yi-Jane; Chung, Min-Chun; Jane Yao, Chung-Chen; Huang, Chien-Hsun; Chang, Hao-Hueng; Jeng, Jiiang-Huei; Young, Tai-Horng

    2012-01-01

    The amniotic membrane (AM) has been widely used in the field of tissue engineering because of the favorable biological properties for scaffolding material. However, little is known about the effects of an acellular AM matrix on the osteogenic differentiation of mesenchymal stem cells. In this study, it was found that both basement membrane side and collagenous stroma side of the acellular AM matrix were capable of providing a preferential environment for driving the osteogenic differentiation of human dental apical papilla cells (APCs) with proven stem cell characteristics. Acellular AM matrix potentiated the induction effect of osteogenic supplements (OS) such as ascorbic acid, β-glycerophosphate, and dexamethasone and enhanced the osteogenic differentiation of APCs, as seen by increased core-binding factor alpha 1 (Cbfa-1) phosphorylation, alkaline phosphatase activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Even in the absence of soluble OS, acellular AM matrix also could exert the substrate-induced effect on initiating APCs' differentiation. Especially, the collagenous stroma side was more effective than the basement membrane side. Moreover, the AM-induced effect was significantly inhibited by U0126, an inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. Taken together, the osteogenic differentiation promoting effect on APCs is AM-specific, which provides potential applications of acellular AM matrix in bone/tooth tissue engineering.

  19. 不同品种醋炮制元胡对延胡索乙素提取率和固形物的影响%Influence of Different Vinegar-prepared Corydalis Tuber on Betacellulin and Solid Matter

    Institute of Scientific and Technical Information of China (English)

    刘英杰; 毛淑杰; 陈金春; 陈春生

    2011-01-01

    [Objective] To discuss the effect of different vinegar-prepared corydalis tuber on betaeellulin and solid Matter. [Method] Select real dry crude drugs corydalis tuber for preparation separately with 18℃ Qinghua white vinegar, 4℃ Longmen rice vinegar, Zhenjiang rice vinegar, Tianjin Cuibo rice vinegar, 4℃ Zhenji and Shanghai Tianyu rice vinegar under “China Pharmacopoeia"; take LC to measure the contents of betacellulin and .solid matter. [Result] The different influences on betacellulin are as follows: 18℃ Qinghua white vinegar>4℃ Longrnen rice vinegar> Zhenjiang rice vinegar> 4℃ Zhenji> Shanghai Tianyu rice vinegar; on solid matter: Shanghai Tianyu rice vinegar > Tianjin Cuibo rice vinegar> Zhenjiang rice vinegar>4℃ Zhenji>4℃ Longmen rice vinegar> 18℃ Qinghua white vinegar; the differences are very marked. [Conclusion] The different influence will affect the medicinal values.%[目的]探讨不同品种醋炮制元胡对延胡索乙素提取率和固形物的影响.[方法]选用延胡索正品药材的干燥块茎分别用18℃青花白醋、4℃龙门米醋、镇江米醋、天津翠波米醋、4℃珍极、上海天鱼米醋,按2005版延胡索炮制方法制备:每种醋平行炮制6份,每份500g元胡;采用液相色谱法测定元胡炮制品中延胡索乙素及固形物的含量.[结果]不同品种醋对元胡药材中延胡索乙素提取率的影响:18℃青花白醋>4℃龙门米醋>镇江米醋>天津翠波米醋>4℃珍极>上海天鱼米醋,对固形物得率的影响:上海天鱼米醋>天津翠波米醋>镇江米醋>4℃珍极>4℃龙门米醋>8℃青花白醋,且不同品种醋对元胡药材中延胡索乙素提取率和固形物得率的影响,存在显著性差异(P<0.05).[结论]不同品种醋炮制元胡可影响其有效成分延胡索乙素的提取率和固形物的得率,从而影响其药用价值.

  20. Sirtuin1 promotes osteogenic differentiation through downregulation of peroxisome proliferator-activated receptor γ in MC3T3-E1 cells.

    Science.gov (United States)

    Qu, Bo; Ma, Yuan; Yan, Ming; Gong, Kai; Liang, Feng; Deng, Shaolin; Jiang, Kai; Ma, Zehui; Pan, Xianming

    2016-09-01

    Osteoporosis is a skeletal disorder characterized by bone loss, resulting in architectural deterioration of the skeleton, decreased bone strength and an increased risk of fragility fractures. Strengthening osteogenesis is an effective way to relieve osteoporosis. Sirtuin1 (Sirt1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, which is reported to be involved in improving osteogenesis. Sirt1 targets peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of adipose tissues; however, the molecular mechanism of Sirt1 in osteogenic differentiation is still unknown. PPARγ tends to induce more adipogenic differentiation rather than osteogenic differentiation. Hence, we hypothesized that Sirt1 facilitates osteogenic differentiation through downregulation of PPARγ signaling. Mouse pre-osteoblastic MC3T3-E1 cells were cultured under osteogenic medium. Sirt1 was overexpressed through plasmid transfection. The results showed that high expression of Sirt1 was associated with increased osteogenic differentiation, as indicated by quantitative PCR and Western blot analysis of osteogenic markers, and Von Kossa staining. Sirt1 overexpression also directly and negatively regulated the expression of PPARγ and its downstream molecules. Use of the PPARγ agonist Rosiglitazone, reversed the effects of Sirt1 on osteogenic differentiation. Using constructed luciferase plasmids, we demonstrated a role of Sirt1 in inhibiting PPARγ-induced activity and expression of adipocyte-specific genes, including acetyl-coenzyme A carboxylase (Acc) and fatty acid binding protein 4 (Fabp4). The interaction between Sirt1 and PPARγ was further confirmed using co-immunoprecipitation analysis. Together, these results reveal a novel mechanism for Sirt1 in osteogenic differentiation through downregulation of PPARγ activity. These findings suggest that the Sirt1-PPARγ pathway may represent a potential target for enhancement of osteogenesis and treatment of

  1. Bovine endometrial stromal cells display osteogenic properties

    Directory of Open Access Journals (Sweden)

    Cavirani Sandro

    2008-12-01

    Full Text Available Abstract The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.

  2. Osteogenic signaling on silk-based matrices.

    Science.gov (United States)

    Midha, Swati; Murab, Sumit; Ghosh, Sourabh

    2016-08-01

    Bone tissue engineering has mainly focused on generating 3D grafts to repair bone defects. However, the underlying signaling mechanisms responsible for development of such 3D bone equivalents have largely been ignored. Here we describe the crucial aspects of embryonic osteogenesis and bone development including cell sources and general signaling cascades that guide mesenchymal progenitors towards osteogenic lineage. Drawing from the knowledge of developmental biology, we then review how silk biomaterial can regulate osteogenic signaling by focusing on the expression of cell surface markers, functional genomic information (mRNA) of stem cells cultured on silk matrices. In an attempt to recapitulate exact in vivo microenvironment of osteogenesis, role of scaffold architecture and material chemistry in regulating cellular differentiation is elaborated. The generated knowledge will not only improve our understanding of cell-material interactions but reveal newer strategies beyond a conventional tissue engineering paradigm and open new prospects for developing silk-based therapies against clinically relevant bone disorders. PMID:27163625

  3. Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold

    International Nuclear Information System (INIS)

    Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium has an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. In this study, the effects of strontium phosphate on the osteogenic differentiation of SHEDs were investigated. Strontium phosphate was found to enhance the osteogenic differentiation of SHEDs with up-regulated osteoblast-related gene expression. The proliferation of SHEDs was slightly inhibited by chitosan scaffolds; however, type-I collagen expression, alkaline phosphatase activity, and calcium deposition on chitosan scaffolds containing strontium were significantly enhanced. Furthermore, cells seeded in a 3D scaffold under dynamic culture at an optimal fluid rate might enhance cellular differentiation than static culture in osteoblastic gene expression. This experiment might provide a useful cell resource and dynamic 3D culture for tissue engineering and bone repair. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering • Strontium phosphate can enhance the osteogenic differentiation of SHEDs • 3D scaffold under dynamic culture with optimal fluid rate enhance cellular differentiation

  4. Osteogenic differentiation and mineralization of human exfoliated deciduous teeth stem cells on modified chitosan scaffold

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Wu, Pai-Shuen [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Ko, Chih-Sheng [PhytoHealth Corporation, Maywufa Biopharma Group, Taipei, Taiwan (China); Huang, Te-Yang [Mackay Memorial Hospital, Taipei, Taiwan (China)

    2014-08-01

    Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages. Strontium has an important function in bone remodeling because it can simulate bone formation and decrease bone resorption. In this study, the effects of strontium phosphate on the osteogenic differentiation of SHEDs were investigated. Strontium phosphate was found to enhance the osteogenic differentiation of SHEDs with up-regulated osteoblast-related gene expression. The proliferation of SHEDs was slightly inhibited by chitosan scaffolds; however, type-I collagen expression, alkaline phosphatase activity, and calcium deposition on chitosan scaffolds containing strontium were significantly enhanced. Furthermore, cells seeded in a 3D scaffold under dynamic culture at an optimal fluid rate might enhance cellular differentiation than static culture in osteoblastic gene expression. This experiment might provide a useful cell resource and dynamic 3D culture for tissue engineering and bone repair. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering • Strontium phosphate can enhance the osteogenic differentiation of SHEDs • 3D scaffold under dynamic culture with optimal fluid rate enhance cellular differentiation.

  5. Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

    Science.gov (United States)

    Chao, Yuan-Hung; Huang, Shih-Yung; Yang, Ruei-Cheng; Sun, Jui-Sheng

    2016-07-01

    Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum.

  6. The inhibitory effect of superparamagnetic iron oxide nanoparticle (Ferucarbotran) on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Superparamagnetic iron oxide (SPIO) nanoparticles are very useful for monitoring cell trafficking in vivo and distinguish whether cellular regeneration originated from an exogenous cell source, which is a key issue for developing successful stem cell therapies. However, the impact of SPIO labeling on stem cell behavior remains uncertain. Here, we show the inhibitory effect of Ferucarbotran, an ionic SPIO, on osteogenic differentiation and its signaling mechanism in human mesenchymal stem cells. Ferucarbotran caused a dose-dependent inhibition of osteogenic differentiation, abolished the differentiation at high concentration, promoted cell migration, and activated the signaling molecules, β-catenin, a cancer/testis antigen, SSX, and matrix metalloproteinase 2 (MMP2). An iron chelator, desferrioxamine, suppressed all the above Ferucarbotran-induced actions, demonstrating an important role of free iron in the inhibition of osteogenic differentiation that is mediated by the promotion of cell mobilization, involving the activation of a specific signaling pathway.

  7. Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis.

    Directory of Open Access Journals (Sweden)

    Federica Viti

    Full Text Available The culture of progenitor mesenchymal stem cells (MSC onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds, together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone

  8. Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis.

    Science.gov (United States)

    Viti, Federica; Landini, Martina; Mezzelani, Alessandra; Petecchia, Loredana; Milanesi, Luciano; Scaglione, Silvia

    2016-01-01

    The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo

  9. Soft matrix supports osteogenic differentiation of human dental follicle cells

    Energy Technology Data Exchange (ETDEWEB)

    Viale-Bouroncle, Sandra; Voellner, Florian [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany); Moehl, Christoph; Kuepper, Kevin [Institute of Complex Systems, ICS7: Biomechanics, Forschungszentrum Juelich, Juelich (Germany); Brockhoff, Gero [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reichert, Torsten E. [Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg (Germany); Schmalz, Gottfried [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany); Morsczeck, Christian, E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University Hospital Regensburg, Regensburg (Germany)

    2011-07-08

    Highlights: {yields} Rigid stiffness supports osteogenic differentiation in mesenchymal stem cells (MSCs). {yields} Our study examined stiffness and differentiation of dental follicle cells (DFCs). {yields} Soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs. {yields} DFCs and MSCs react contrarily to soft and rigid surface stiffness. -- Abstract: The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

  10. Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Huanchen Cheng; Lin Qiu; Hao Zhang; Mei Cheng; Wei Li; Xuefei Zhao; Keyu Liu; Lei Lei; Jun Ma

    2011-01-01

    Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells.Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However,reports about the effects of ATO on MSCs are limited. In this study, we found that 1μM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.

  11. Endoplasmic reticulum (ER stress inducible factor cysteine-rich with EGF-like domains 2 (Creld2 is an important mediator of BMP9-regulated osteogenic differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Jiye Zhang

    Full Text Available Mesenchymal stem cells (MSCs are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2 in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.

  12. Different roles of GNAS and cAMP signaling during early and late stages of osteogenic differentiation.

    Science.gov (United States)

    Zhang, S; Kaplan, F S; Shore, E M

    2012-09-01

    Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr. PMID:22903279

  13. Differentiation of skeletal osteogenic progenitor cells to osteoblasts with 3,4-diarylbenzopyran based amide derivatives: Novel osteogenic agents.

    Science.gov (United States)

    Gupta, Atul; Ahmad, Imran; Kureel, Jyoti; John, Aijaz A; Sultan, Eram; Chanda, Debabrata; Agarwal, Naresh Kumar; Alauddin; Wahajuddin; Prabhaker, S; Verma, Amita; Singh, Divya

    2016-10-01

    A series of 3,4-diarylbenzopyran based amide derivatives was synthesized and evaluated for osteogenic activity in in vitro and in vivo models of osteoporosis. Compounds 17a, 21b-c and 22a-b showed significant osteogenic activity in osteoblast differentiation assay. Among the synthesized compounds, 22b was identified as lead molecule which showed significant osteogenic activity at 1 pM concentration in osteoblast differentiation assay and at 1 mg kg(-1) body weight dose in estrogen deficient balb/c mice model. In vitro bone mineralization and expression of osteogenic marker genes viz BMP-2, RUNX-2, OCN, and collagen type 1 further confirmed the osteogenic potential of 22b. Gene expression study for estrogen receptor α and β (ER-α and ER-β) in mouse calvarial osteoblasts (MCOs) unveiled that possibly 22b exerted osteogenic efficacy via activation of Estrogen receptor-β preferentially. In vivo pharmacokinetic, estrogenicity and acute toxicity studies of 22b showed that it had good bioavailability and was devoid of uterine estrogenicity at 1 mg kg(-1) and inherent toxicity up to 1000 mg kg(-1) body weight dose respectively. PMID:27236065

  14. Calcific bursitis mimicking a parosteal osteogenic sarcoma

    International Nuclear Information System (INIS)

    A 43-year-old woman with no history of trauma or major medical illness, presented with a ten day history of right hip and thigh pain. The pain was described as constant, dull, and aching. It was nonradiating and was not relieved by analgesics. Physical examination revealed diffuse tenderness over the right hip and right lateral thigh region; no mass was palpable. The CBC, serum electrolytes, calcium, phosphorus, and alkaline phosphatase determinations were all normal. Radiographs of the right hip demonstrated amorphous soft tissue calcification adjacent to the lateral aspect of the right femur as well as periosteal reaction and apparent destruction in the adjacent bone. Because of these suspicious X-rays findings, the initial working diagnosis was parosteal osteogenic sarcoma. A bone scan was performed two hours after the intravenous administration of 15 millicuries of Tc-99m-MDP. It showed focal uptake overlying the upper femur, approximately where the X-ray had shown periosteal reaction and apparent bony destruction. In addition, the bone images also demonstrated a linear band of activity extending through the soft tissues from the greater trochanter to the lower lateral thigh. Because of the unexpected and quite extensive soft tissue uptake seen on the scan, the possibility that a benign process was involved was then considered seriously for the first time. An open biopsy was then performed. It revealed acute calcific trochanteric bursitis; there was no evidence of bone involvement. The patient was treated conservatively and symptoms gradually resolved. (orig.)

  15. Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage.

    Science.gov (United States)

    Quiroz, Felipe Garcia; Posada, Olga M; Gallego-Perez, Daniel; Higuita-Castro, Natalia; Sarassa, Carlos; Hansford, Derek J; Agudelo-Florez, Piedad; López, Luis E

    2010-04-01

    Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of beta-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC osteogenic markers more consistent with the observed differentiation process. The results suggest that beta-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.

  16. Microbioreactor array screening of Wnt modulators and microenvironmental factors in osteogenic differentiation of mesenchymal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jessica E Frith

    Full Text Available Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs, which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1 and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1 were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to

  17. Osteogenic Responses to Zirconia with Hydroxyapatite Coating by Aerosol Deposition

    OpenAIRE

    Cho, Y.; Hong, J.; Ryoo, H.; Kim, D.; Park, J.; Han, J.

    2015-01-01

    Previously, we found that osteogenic responses to zirconia co-doped with niobium oxide (Nb2O5) or tantalum oxide (Ta2O5) are comparable with responses to titanium, which is widely used as a dental implant material. The present study aimed to evaluate the in vitro osteogenic potential of hydroxyapatite (HA)-coated zirconia by an aerosol deposition method for improved osseointegration. Surface analysis by scanning electron microscopy and x-ray diffraction proved that a thin as-deposited HA film...

  18. Role of MSX1 in Osteogenic Differentiation of Human Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Noriko Goto

    2016-01-01

    Full Text Available Msh homeobox 1 (MSX1 encodes a transcription factor implicated in embryonic development of limbs and craniofacial tissues including bone and teeth. Although MSX1 regulates osteoblast differentiation in the cranial bone of young animal, little is known about the contribution of MSX1 to the osteogenic potential of human cells. In the present study, we investigate the role of MSX1 in osteogenic differentiation of human dental pulp stem cells isolated from deciduous teeth. When these cells were exposed to osteogenesis-induction medium, runt-related transcription factor-2 (RUNX2, bone morphogenetic protein-2 (BMP2, alkaline phosphatase (ALPL, and osteocalcin (OCN mRNA levels, as well as alkaline phosphatase activity, increased on days 4–12, and thereafter the matrix was calcified on day 14. However, knockdown of MSX1 with small interfering RNA abolished the induction of the osteoblast-related gene expression, alkaline phosphatase activity, and calcification. Interestingly, DNA microarray and PCR analyses revealed that MSX1 knockdown induced the sterol regulatory element-binding protein 2 (SREBP2 transcriptional factor and its downstream target genes in the cholesterol synthesis pathway. Inhibition of cholesterol synthesis enhances osteoblast differentiation of various mesenchymal cells. Thus, MSX1 may downregulate the cholesterol synthesis-related genes to ensure osteoblast differentiation of human dental pulp stem cells.

  19. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Directory of Open Access Journals (Sweden)

    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  20. Overexpression of Bmi1 in Lymphocytes Stimulates Skeletogenesis by Improving the Osteogenic Microenvironment

    Science.gov (United States)

    Zhou, Xichao; Dai, Xiuliang; Wu, Xuan; Ji, Ji; Karaplis, Andrew; Goltzman, David; Yang, Xiangjiao; Miao, Dengshun

    2016-01-01

    To investigate whether overexpression of Bmi1 in lymphocytes can stimulate skeletogenesis by improving the osteogenic microenvironment, we examined the skeletal phenotype of EμBmi1 transgenic mice with overexpression of Bmi1 in lymphocytes. The size of the skeleton, trabecular bone volume and osteoblast number, indices of proliferation and differentiation of bone marrow mesenchymal stem cells (BM-MSCs) were increased significantly, ROS levels were reduced and antioxidative capacity was enhanced in EμBmi1 mice compared to WT mice. In PTHrP1–84 knockin (PthrpKI/KI) mice, the expression levels of Bmi1 are reduced and potentially can mediate the premature osteoporosis observed. We therefore generated a PthrpKI/KI mice overexpressing Bmi1 in lymphocytes and compared them with PthrpKI/KI and WT littermates. Overexpression of Bmi1 in PthrpKI/KI mice resulted in a longer lifespan, increased body weight and improvement in skeletal growth and parameters of osteoblastic bone formation with reduced ROS levels and DNA damage response parameters. Our results demonstrate that overexpression of Bmi1 in lymphocytes can stimulate osteogenesis in vivo and partially rescue defects in skeletal growth and osteogenesis in PthrpKI/KI mice. These studies therefore indicate that overexpression of Bmi1 in lymphocytes can stimulate skeletogenesis by inhibiting oxidative stress and improving the osteogenic microenvironment. PMID:27373231

  1. Cytocompatibility with osteogenic cells and enhanced in vivo anti-infection potential of quaternized chitosan-loaded titania nanotubes.

    Science.gov (United States)

    Yang, Ying; Ao, Haiyong; Wang, Yugang; Lin, Wentao; Yang, Shengbing; Zhang, Shuhong; Yu, Zhifeng; Tang, Tingting

    2016-01-01

    Infection is one of the major causes of failure of orthopedic implants. Our previous study demonstrated that nanotube modification of the implant surface, together with nanotubes loaded with quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan, HACC), could effectively inhibit bacterial adherence and biofilm formation in vitro. Therefore, the aim of this study was to further investigate the in vitro cytocompatibility with osteogenic cells and the in vivo anti-infection activity of titanium implants with HACC-loaded nanotubes (NT-H). The titanium implant (Ti), nanotubes without polymer loading (NT), and nanotubes loaded with chitosan (NT-C) were fabricated and served as controls. Firstly, we evaluated the cytocompatibility of these specimens with human bone marrow-derived mesenchymal stem cells in vitro. The observation of cell attachment, proliferation, spreading, and viability in vitro showed that NT-H has improved osteogenic activity compared with Ti and NT-C. A prophylaxis rat model with implantation in the femoral medullary cavity and inoculation with methicillin-resistant Staphylococcus aureus was established and evaluated by radiographical, microbiological, and histopathological assessments. Our in vivo study demonstrated that NT-H coatings exhibited significant anti-infection capability compared with the Ti and NT-C groups. In conclusion, HACC-loaded nanotubes fabricated on a titanium substrate show good compatibility with osteogenic cells and enhanced anti-infection ability in vivo, providing a good foundation for clinical application to combat orthopedic implant-associated infections. PMID:27672479

  2. Graphene/single-walled carbon nanotube hybrids promoting osteogenic differentiation of mesenchymal stem cells by activating p38 signaling pathway

    Science.gov (United States)

    Yan, Xinxin; Yang, Wen; Shao, Zengwu; Yang, Shuhua; Liu, Xianzhe

    2016-01-01

    Carbon nanomaterials are becoming increasingly significant in biomedical fields since they exhibit exceptional physicochemical and biocompatible properties. Today, the stem cells offer potentially new therapeutic approaches in tissue engineering and regenerative medicine. However, the induction of differentiation into specific lineages remains challenging, which provoked us to explore the biomedical applications of carbon nanomaterials in stem cells. In this study, we investigated the interactions between graphene/single-walled carbon nanotube (G/SWCNT) hybrids and rat mesenchymal stem cells (rMSCs) and focused on the proliferation and differentiation of rMSCs treated with G/SWCNT hybrids. Cell viability and morphology were evaluated using cell counting kit-8 assay and immunofluorescence staining, respectively. Osteogenic differentiation evaluated by alkaline phosphatase activity of MSCs proved to be higher after treatment with G/SWCNT hybrids, and the mineralized matrix nodule formation was also enhanced. In addition, the expression levels of osteogenic-associated genes were upregulated, while the adipocyte-specific markers were downregulated. Consistent with these results, we illustrated that the effect of G/SWCNT hybrids on the process of osteogenic differentiation of rMSCs can be modulated by activating the p38 signaling pathway and inhibiting the extracellular signal-regulated kinase 1/2 pathway. Nevertheless, our study suggests that carbon nanomaterials offer a promising platform for regenerative medicine in the near future.

  3. Multifunction Sr, Co and F co-doped microporous coating on titanium of antibacterial, angiogenic and osteogenic activities

    Science.gov (United States)

    Zhou, Jianhong; Zhao, Lingzhou

    2016-01-01

    Advanced multifunction titanium (Ti) based bone implant with antibacterial, angiogenic and osteogenic activities is stringently needed in clinic, which may be accomplished via incorporation of proper inorganic bioactive elements. In this work, microporous TiO2/calcium-phosphate coating on Ti doped with strontium, cobalt and fluorine (SCF-TiCP) was developed, which had a hierarchical micro/nano-structure with a microporous structure evenly covered with nano-grains. SCF-TiCP greatly inhibited the colonization and growth of both gram-positive and gram-negative bacteria. No cytotoxicity appeared for SCF-TiCP. Furthermore, SCF-TiCP stimulated the expression of key angiogenic factors in rat bone marrow stem cells (MSCs) and dramatically enhanced MSC osteogenic differentiation. The in vivo animal test displayed that SCF-TiCP induced more new bone and tighter implant/bone bonding. In conclusion, multifunction SCF-TiCP of antibacterial, angiogenic and osteogenic activities is a promising orthopedic and dental Ti implant coating for improved clinical performance. PMID:27353337

  4. MicroRNA-200c Represses IL-6, IL-8, and CCL-5 Expression and Enhances Osteogenic Differentiation

    Science.gov (United States)

    Sharp, Thad; Khorsand, Behnoush; Fischer, Carol; Eliason, Steven; Salem, Ali; Akkouch, Adil; Brogden, Kim; Amendt, Brad A.

    2016-01-01

    MicroRNAs (miRs) regulate inflammation and BMP antagonists, thus they have potential uses as therapeutic reagents. However, the molecular function of miR-200c in modulating proinflammatory and bone metabolic mediators and osteogenic differentiation is not known. After miR-200c was transduced into a human embryonic palatal mesenchyme (HEPM) (a cell line of preosteoblasts), using lentiviral vectors, the resulting miR-200c overexpression increased osteogenic differentiation biomarkers, including osteocalcin (OCN) transcripts and calcium content. miR-200c expression also down-regulated interleukin (IL)-6, IL-8, and chemokine (C-C motif) ligand (CCL)-5 under lipopolysaccharide (LPS) stimulation and increased osteoprotegerin (OPG) in these cells. miR-200c directly regulates the expression of IL-6, IL-8 and CCL-5 transcripts by binding to their 3’UTRs. A plasmid-based miR-200c inhibitor effectively reduces their binding activities. Additionally, miR-200c delivered using polyethylenimine (PEI) nanoparticles effectively inhibits IL-6, IL-8 and CCL-5 in primary human periodontal ligament fibroblasts and increases the biomarkers of osteogenic differentiation in human bone marrow mesenchymal stem cells (MSCs), including calcium content, ALP, and Runx2. These data demonstrate that miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. miR-200c may potentially be used as an effective means to prevent periodontitis-associated bone loss by arresting inflammation and osteoclastogenesis and enhancing bone regeneration. PMID:27529418

  5. Crosstalk between Wnt/β-catenin and estrogen receptor signaling synergistically promotes osteogenic differentiation of mesenchymal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Yanhong Gao

    Full Text Available Osteogenic differentiation from mesenchymal progenitor cells (MPCs are initiated and regulated by a cascade of signaling events. Either Wnt/β-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER signaling by estradiol (E2 or exogenously expressed ERα in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ERα-mediated synergy can be effectively blocked by ERα antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ERα significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on β-catenin/Tcf reporter activity. However, ERα expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ERβ expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ERα expression and down-regulating ERβ expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.

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

    Directory of Open Access Journals (Sweden)

    Shaohui Yuan

    2013-01-01

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

  7. Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone

    Directory of Open Access Journals (Sweden)

    Yoshinobu Uchihara

    2015-01-01

    Full Text Available Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.

  8. Periostin: A Downstream Mediator of EphB4-Induced Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Fei Zhang

    2016-01-01

    Full Text Available Erythropoietin-producing hepatocyte B4 (EphB4 has been reported to be a key molecular switch in the regulation of bone homeostasis, but the underlying mechanism remains poorly understood. In this study, we investigated the role of EphB4 in regulating the expression of periostin (POSTN within bone marrow-derived mesenchymal stem cells (MSCs and assessed its effect and molecular mechanism of osteogenic induction in vitro. Treatment with ephrinB2-FC significantly increased the expression of POSTN in MSCs, and the inhibition of EphB4 could abrogate this effect. In addition, osteogenic markers were upregulated especially in MSCs overexpressing EphB4. To elucidate the underlying mechanism of cross talk between EphB4 and the Wnt pathway, we detected the change in protein expression of phosphorylated-glycogen synthase kinase 3β-serine 9 (p-GSK-3β-Ser9 and β-catenin, as well as the osteogenic markers Runx2 and COL1. The results showed that GSK-3β activation and osteogenic marker expression levels were downregulated by ephrinB2-FC treatment, but these effects were inhibited by blocking integrin αvβ3 in MSCs. Our findings demonstrate that EphB4 can promote osteogenic differentiation of MSCs via upregulation of POSTN expression. It not only helps to reveal the interaction mechanism between EphB4 and Wnt pathway but also brings a better understanding of EphB4/ephrinB2 signaling in bone homeostasis.

  9. Effect of advanced oxidation protein products on the proliferation and osteogenic differentiation of rat mesenchymal stem cells.

    Science.gov (United States)

    Sun, Nan; Yang, Li; Li, Yingbin; Zhang, Hua; Chen, Hong; Liu, Duan; Li, Qingnan; Cai, Dehong

    2013-08-01

    Advanced oxidation protein products (AOPPs) as a novel marker of oxidative stress, are involved in a variety of diseases, including osteoporosis. Although a number of studies have shown the possible functions of AOPPs in biological processes, little is known about the role of AOPPs in the pathogenesis of osteoporosis. In this study, we aimed to investigate the effect of AOPPs on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (MSCs). MSCs, isolated from bone marrow, were cultured in the absence or presence of AOPPs (50, 100, 200 and 400 mg/ml). MTT assay was used to determine the proliferative ability of the cells. Alkaline phosphatase (ALP) activity, the mRNA expression of ALP and collagen I and bone nodule formation were detected to assess osteogenic differentiation. Reactive oxygen species (ROS) generation was analyzed with the probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The expression of receptor of advanced glycation end-products (RAGE) at the mRNA and protein level was detected by real-time PCR and western blot analysis, respectively. Compared with the control group, AOPPs inhibited MSC proliferation in a dose- and time-dependent manner. Moreover, AOPPs induced a significant reduction in ALP activity, as well as a decrease in ALP and collagen I mRNA levels in the MSCs; bone nodule formation was also inhibited. Furthermore, AOPPs increased ROS generation in the MSCs, and upregulated the expression of RAGE at the mRNA and protein level. These results suggest that AOPPs inhibit the proliferation and osteogenic differentiation of MSCs, possibly through the AOPPs-RAGE-ROS pathway; this may be an important mechanism in the development of osteoporosis.

  10. An integrated study of natural hydroxyapatite-induced osteogenic differentiation of mesenchymal stem cells using transcriptomics, proteomics and microRNA analyses

    International Nuclear Information System (INIS)

    This work combined transcriptomics, proteomics, and microRNA (miRNA) analyses to elucidate the mechanism of natural hydroxyapatite (NHA)-induced osteogenic differentiation of mesenchymal stem cells (MSCs). First, NHA powder was obtained from pig bones and fabricated into disc-shaped samples. Subsequently, the proliferation and osteogenic differentiation of MSCs cultured on NHA were investigated. Then, proteomics was employed to detect the protein expression profiles of MSCs cultured on NHA, and the effect of NHA on MSCs was analyzed through an integrated pathway analysis (including proteomics and previous transcriptomics data) in which specific NHA-induced differentiation pathways were analyzed. The pathway nodes with expression data at both the mRNA and protein levels (mRNA–protein pairs) were filtered in differentiation-related pathways. miRNAs corresponding to these target mRNA–protein pairs were predicted, screened and tested, and the regulatory effects of miRNAs on mRNA–protein pairs were analyzed. Finally, the NHA-induced osteogenic pathways were verified. The results of an MTT assay and alkaline phosphatase (ALP) staining showed that the cell proliferation rate decreased and the osteogenic performance improved in the presence of NHA. By integrating transcriptomics and proteomics, the genes and proteins involved in 89 pathways were shown to be differentially expressed. Among them, 5 differentiation-associated pathways, in which 9 miRNAs and 8 regulated-target mRNA–protein zby inhibiting the target mRNA–protein pair HSPA8 in the MAPK signaling pathway, and miR-26a and miR-26b might inhibit adipogenic differentiation by repressing the target mRNA–protein pair HMGA1 in the adipogenesis pathway. A verification experiment for the osteogenic pathway indicated that the ERK1/2 or JNK MAPK pathways might play an important role in NHA-induced osteogenic differentiation. In conclusion, NHA affected MSCs at both the transcriptional and translational levels

  11. Glimepiride promotes osteogenic differentiation in rat osteoblasts via the PI3K/Akt/eNOS pathway in a high glucose microenvironment.

    Science.gov (United States)

    Ma, Pan; Gu, Bin; Xiong, Wei; Tan, Baosheng; Geng, Wei; Li, Jun; Liu, Hongchen

    2014-01-01

    Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment. PMID:25391146

  12. Glimepiride promotes osteogenic differentiation in rat osteoblasts via the PI3K/Akt/eNOS pathway in a high glucose microenvironment.

    Directory of Open Access Journals (Sweden)

    Pan Ma

    Full Text Available Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment.

  13. Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells by activating the APPL1-AMPK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Tong; Wu, Yu-wei; Lu, Hui; Guo, Yuan [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China); Tang, Zhi-hui, E-mail: tang_zhihui@live.cn [Second Dental Center, Peking University School and Hospital of Stomatology, Beijing (China); National Engineering Laboratory for Digital and Material Technology of Stomatology, Peking University School and Hospital of Stomatology, Beijing (China)

    2015-05-29

    Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with multi-lineage differentiation potential including osteogenesis and adipogenesis. While significant progress has been made in understanding the transcriptional control of hASC fate, little is known about how hASC differentiation is regulated by the autocrine loop. The most abundant adipocytokine secreted by adipocytes, adiponectin (APN) plays a pivotal role in glucose metabolism and energy homeostasis. Growing evidence suggests a positive association between APN and bone formation yet little is known regarding the direct effects of APN on hASC osteogenesis. Therefore, this study was designed to investigate the varied osteogenic effects and regulatory mechanisms of APN in the osteogenic commitment of hASCs. We found that APN enhanced the expression of osteoblast-related genes in hASCs, such as osteocalcin, alkaline phosphatase, and runt-related transcription factor-2 (Runx2, also known as CBFa1), in a dose- and time-dependent manner. This was further confirmed by the higher expression levels of alkaline phosphatase and increased formation of mineralization nodules, along with the absence of inhibition of cell proliferation. Importantly, APN at 1 μg/ml was the optimal concentration, resulting in maximum deposition of calcium nodules, and was significant superior to bone morphogenetic protein 2. Mechanistically, we found for the first time that APN increased nuclear translocation of the leucine zipper motif (APPL)-1 as well as AMP-activated protein kinase (AMPK) phosphorylation, which were reversed by pretreatment with APPL1 siRNA. Our results indicate that APN promotes the osteogenic differentiation of hASCs by activating APPL1-AMPK signaling, suggesting that manipulation of APN is a novel therapeutic target for controlling hASC fate. - Highlights: • Adiponectin enhances osteogenic differentiation in human adipose-derived stem cells. • The knock-down of APPL1 block the enhancement of

  14. Effect of Dy3+ on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and adipocytic trans-differentiation of mouse primary osteoblasts

    Institute of Scientific and Technical Information of China (English)

    ZHANG dinChao; LIU DanDan; SUN ding; ZHANG DaWei; SHEN ShiGang; YANG MengSu

    2009-01-01

    A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT) test,alkaline phosphatase (ALP) activity measurement,mineralized function,Oil Red O stain and measurement were employed to assess the effect of Dy3+ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differ-entiation of mouse primary osteoblasts (Obs).The results showed that Dy3+ had no effect on BMSC proliferation at concentrations of 1×10-8 and 1×10-5 mol/L,but inhibited BMSC proliferation at other concentrations.Dy3+ had no effect on OB proliferation at concentrations of 1×10-10 and 1×10-9 mol/L,but inhibited OB proliferation at other concentrations.Dy3+ had no effect on the osteogenic differentia-tion of BMSCs at concentrations of 1×10-9 and 1×10-7 mol/L,and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day.The osteogenic differentiation of BMSCs was inhibited by Dy3+ at concentration of 1×10-5 mol/L at the 14th day,but promoted osteogenic differentiation of BMSCs at concentrations of 1×10-9,1×10-8,1×10-7 and 1×10-6 mol/L with the maximal effect at concen-tration of 10-6 mol/L.Dy3+ promoted mineralized function of BMSCs at any concentration.Dy3+ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10-7 mol/L,but inhibited adipogenic differentiation of BMSCs at other concentrations.Dy3+ inhibited adipocytic trans-differentiation of Obs at any concentration,suggesting that Dy3+ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of Obs which may promote differentiation and mineralization of Obs.These results may be valuable for better understanding the mechanism of the effect of Dy3+ on pathogenesis of osteoporosis.

  15. Activation of the ERK1/2 Signaling Pathway during the Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Substrates Modified with Various Chemical Groups

    Directory of Open Access Journals (Sweden)

    Bing Bai

    2013-01-01

    Full Text Available The current study examined the influence of culture substrates modified with the functional groups –OH, –COOH, –NH2, and –CH3 using SAMs technology, in conjunction with TAAB control, on the osteogenic differentiation of rabbit BMSCs. The CCK-8 assay revealed that BMSCs exhibited substrate-dependent cell viability. The cells plated on –NH2- and –OH-modified substrates were well spread and homogeneous, but those on the –COOH- and –CH3-modified substrates showed more rounded phenotype. The mRNA expression of BMSCs revealed that –NH2-modified substrate promoted the mRNA expression and osteogenic differentiation of the BMSCs. The contribution of ERK1/2 signaling pathway to the osteogenic differentiation of BMSCs cultured on the –NH2-modified substrate was investigated in vitro. The –NH2-modified substrate promoted the expression of integrins; the activation of FAK and ERK1/2. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked ERK1/2 activation in a dose-dependent manner, as revealed for expression of Cbfα-1 and ALP. Blockade of ERK1/2 phosphorylation in BMSCs by PD98059 suppressed osteogenic differentiation on chemical surfaces. These findings indicate a potential role for ERK in the osteogenic differentiation of BMSCs on surfaces modified by specific chemical functional groups, indicating that the microenvironment affects the differentiation of BMSCs. This observation has important implications for bone tissue engineering.

  16. Circulating osteogenic cells: implications for injury, repair, and regeneration

    DEFF Research Database (Denmark)

    Pignolo, Robert J; Kassem, Moustapha

    2011-01-01

    The aim of this review is to provide a critical reading of recent literature pertaining to the presence of circulating, fluid-phase osteoblastic cells and their possible contribution to bone formation. We have termed this group of cells collectively as circulating osteogenic precursor (COP) cells....... We present evidence for their existence, methods used for their isolation and identification, possible physiological and pathophysiological roles, cellular origins, and possible mechanisms for their migration to target tissues....

  17. Osteogenic potential of sorted equine mesenchymal stem cell subpopulations

    OpenAIRE

    Radtke, Catherine L.; Nino-Fong, Rodolfo; Rodriguez-Lecompte, Juan Carlos; Esparza Gonzalez, Blanca P.; Stryhn, Henrik; McDuffee, Laurie A.

    2015-01-01

    The objectives of this study were to use non-equilibrium gravitational field-flow fractionation (GrFFF), an immunotag-less method of sorting mesenchymal stem cells (MSCs), to sort equine muscle tissue-derived mesenchymal stem cells (MMSCs) and bone marrow-derived mesenchymal stem cells (BMSC) into subpopulations and to carry out assays in order to compare their osteogenic capabilities. Cells from 1 young adult horse were isolated from left semitendinosus muscle tissue and from bone marrow asp...

  18. Osteogenic responses to zirconia with hydroxyapatite coating by aerosol deposition.

    Science.gov (United States)

    Cho, Y; Hong, J; Ryoo, H; Kim, D; Park, J; Han, J

    2015-03-01

    Previously, we found that osteogenic responses to zirconia co-doped with niobium oxide (Nb2O5) or tantalum oxide (Ta2O5) are comparable with responses to titanium, which is widely used as a dental implant material. The present study aimed to evaluate the in vitro osteogenic potential of hydroxyapatite (HA)-coated zirconia by an aerosol deposition method for improved osseointegration. Surface analysis by scanning electron microscopy and x-ray diffraction proved that a thin as-deposited HA film on zirconia showed a shallow, regular, crater-like surface. Deposition of dense and uniform HA films was measured by SEM, and the contact angle test demonstrated improved wettability of the HA-coated surface. Confocal laser scanning microscopy indicated that MC3T3-E1 pre-osteoblast attachment did not differ notably between the titanium and zirconia surfaces; however, cells on the HA-coated zirconia exhibited a lower proliferation than those on the uncoated zirconia late in the culture. Nevertheless, ALP, alizarin red S staining, and bone marker gene expression analysis indicated good osteogenic responses on HA-coated zirconia. Our results suggest that HA-coating by aerosol deposition improves the quality of surface modification and is favorable to osteogenesis. PMID:25586588

  19. Osteogenic Responses to Zirconia with Hydroxyapatite Coating by Aerosol Deposition

    Science.gov (United States)

    Cho, Y.; Hong, J.; Ryoo, H.; Kim, D.; Park, J.

    2015-01-01

    Previously, we found that osteogenic responses to zirconia co-doped with niobium oxide (Nb2O5) or tantalum oxide (Ta2O5) are comparable with responses to titanium, which is widely used as a dental implant material. The present study aimed to evaluate the in vitro osteogenic potential of hydroxyapatite (HA)-coated zirconia by an aerosol deposition method for improved osseointegration. Surface analysis by scanning electron microscopy and x-ray diffraction proved that a thin as-deposited HA film on zirconia showed a shallow, regular, crater-like surface. Deposition of dense and uniform HA films was measured by SEM, and the contact angle test demonstrated improved wettability of the HA-coated surface. Confocal laser scanning microscopy indicated that MC3T3-E1 pre-osteoblast attachment did not differ notably between the titanium and zirconia surfaces; however, cells on the HA-coated zirconia exhibited a lower proliferation than those on the uncoated zirconia late in the culture. Nevertheless, ALP, alizarin red S staining, and bone marker gene expression analysis indicated good osteogenic responses on HA-coated zirconia. Our results suggest that HA-coating by aerosol deposition improves the quality of surface modification and is favorable to osteogenesis. PMID:25586588

  20. Measurement of osteogenic exercise – How to interpret accelerometric data?

    Directory of Open Access Journals (Sweden)

    Timo eJämsä

    2011-10-01

    Full Text Available Bone tissue adapts to its mechanical loading environment. We review here the accelerometric measurements with special emphasis on osteogenic exercise. The accelerometric method offers a unique opportunity to assess the intensity of mechanical loadings. We present methods to interpret accelerometric data, reducing it to the daily distributions of magnitude, slope, area and energy of signal. These features represent the intensity level of physical activities, and were associated with the changes in bone density, bone geometry, physical performance and metabolism in healthy premenopausal women. Bone adaptations presented a dose- and intensity dependent relationship with impact loading. Changes in hip were threshold dependent, indicating the importance of high impacts exceeding acceleration of 4 g or slope of 100 g/s as an osteogenic stimulus. The number of impacts needed was 60 per day. We also present the Daily Impact Score to describe the osteogenic potential of daily mechanical loading with a single score. The methodology presented here can be used to study musculoskeletal adaptation to exercise in other target groups as well.

  1. Juxtacortical osteogenic sarcoma of chondroblastic type on the maxilla

    International Nuclear Information System (INIS)

    39-year-old female had been treated for the exophytic mass on buccal aspect of the left maxillary posterior area 2 years and 8 months ago. Tentative diagnosis was obtained as fibrous dysplasia on clinical and radiographic examinations and histopathologic findings revealed as osteochondroma after bone trimming at that time. She revisited for the treatment of recurred lesions. We reviewed this case with clinical, radiologic and histopathologic standpoints retrospectively, and came to a conclusion that the tumor primarily occurred was juxtacortical osteogenic sarcoma and recurred due to inadequate treatment and then expanded over intramedullary. This case shows that the diagnosis of osteosarcoma should take account of the patient history, clinical, radiographic and histopathologic findings and it requires attentive follow up check. Retrospectively reviewed results were as follows ; At first visit, oral examination revealed a bony hard swelling on the buccal aspect of the left maxillary posterior area. Radiographically, a dense radiopaque mass was noted on the site. The lesion showed hot uptake of 99mTc-MDP. Histopathologic diagnosis was done as osteochondroma, but it was considered as osteogenic sarcoma when compared with the recurrent lesion. When she revisited for the treatment of multiple bony swelling on the left maxilla, radiograms showed typical features of malignancy such as widening of periodontal ligament space and sun-ray appearance, and coincided with benign characters as follows ; relatively well circumscribed lesion and expansion and displacement of the adjacent structures. Finally, histopathologic findings of the lesion was well differentiated chondroblastic osteogenic sarcoma.

  2. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    Science.gov (United States)

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

  3. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    OpenAIRE

    Li Zou; Fahad K. Kidwai; Ross A. Kopher; Jason Motl; Cory A. Kellum; Jennifer J. Westendorf; Dan S. Kaufman

    2015-01-01

    Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow me...

  4. [Regulation of osteogenic differentiation of mesenchimal stem sells of bone marrow].

    Science.gov (United States)

    Zakharov, Iu M; Makarova, E B

    2013-04-01

    The review describes the mechanisms of hormonal, autocrine, paracrine and intracellular regulation in osteogenic differentiation of mesenchymal stem cells, the role of negative feedbacks in this process.

  5. Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study.

    Science.gov (United States)

    Jin, Pan; Wu, Huayu; Xu, Guojie; Zheng, Li; Zhao, Jinmin

    2014-05-01

    The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 μM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.

  6. Famotidine suppresses osteogenic differentiation of tendon cells in vitro and pathological calcification of tendon in vivo.

    Science.gov (United States)

    Yamamoto, Kenichi; Hojo, Hironori; Koshima, Isao; Chung, Ung-il; Ohba, Shinsuke

    2012-12-01

    Heterotopic ossification or calcification follows any type of musculoskeletal trauma and is known to occur after arthroplasties of hip, knee, shoulder, or elbow; fractures; joint dislocations; or tendon ruptures. Histamine receptor H2 (Hrh2) has been shown to be effective for reducing pain and decreasing calcification in patients with calcifying tendinitis, which suggested that H2 blockers were effective for the treatment of tendon ossification or calcification. However, the detailed mechanisms of its action on tendon remain to be clarified. We investigated the mechanisms underlying H2 blocker-mediated suppression of tendon calcification, with a focus on the direct action of the drug on tendon cells. Famotidine treatment suppressed the mRNA expressions of Col10a1 and osteocalcin, ossification markers, in a tendon-derived cell line TT-D6, as well as a preosteoblastic one MC3T3-E1. Both of the cell lines expressed Hrh2; histamine treatment induced osteocalcin expression in these cells. Famotidine administration suppressed calcification in the Achilles tendon of ttw mice, a mouse model of ectopic ossification. These data suggest that famotidine inhibits osteogenic differentiation of tendon cells in vitro, and this inhibition may underlie the anti-calcification effects of the drug in vivo. This study points to the use of H2 blockers as a promising strategy for treating heterotopic ossification or calcification in tendon, and provides evidence in support of the clinical use of famotidine.

  7. Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway

    OpenAIRE

    Guo-yong Yu; Gui-zhou Zheng; Bo Chang; Qin-xiao Hu; Fei-xiang Lin; De-zhong Liu; Chu-cheng Wu; Shi-xin Du; Xue-dong Li

    2016-01-01

    Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs) were cultured in osteogenic ...

  8. Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Li Zou

    2015-02-01

    Full Text Available We generated a RUNX2-yellow fluorescent protein (YFP reporter system to study osteogenic development from human embryonic stem cells (hESCs. Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

  9. Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo

    Institute of Scientific and Technical Information of China (English)

    Song XU; Kim DE VEIRMAN; Holly EVANS; Gaia Cecilia SANTINI; Isabelle VANDE BROEK; Xavier LELEU; Ann DE BECKER

    2013-01-01

    Vorinostat,a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients,has been reported to cause bone loss.The purpose of this study was to test whether,and to what extent,vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo.Methods:Bone marrow-derived MSCs were prepared from both normal donors and MM patients.The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation,which was evaluated by alkaline phosphatase (ALP) staining,Alizarin Red S staining and the mRNA expression of osteogenic markers.Naive mice were administered vorinostat (100 mg/kg,ip) every other day for 3 weeks.After the mice were sacrificed,bone formation was assessed based on serum osteocalcin level and histomorphometric analysis.Results:Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L).The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs,whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L).In bone marrow-derived hMSCs from both normal donors and MM patients,vorinostat (1 μmol/L) significantly increased ALP activity,mRNA expression of osteogenic markers,and matrix mineralization.These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation.Importantly,the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen.Conclusion:Vorinostat,known as a potent anti-myeloma drug,stimulates MSC osteogenesis in vitro.With the optimized treatment regimen,any decrease in bone formation was not observed in vivo.

  10. Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Yamada

    2016-01-01

    Full Text Available We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5, which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine.

  11. Osteogenic differentiation of preosteoblasts on a hemostatic gelatin sponge

    Science.gov (United States)

    Kuo, Zong-Keng; Lai, Po-Liang; Toh, Elsie Khai-Woon; Weng, Cheng-Hsi; Tseng, Hsiang-Wen; Chang, Pei-Zen; Chen, Chih-Chen; Cheng, Chao-Min

    2016-01-01

    Bone tissue engineering provides many advantages for repairing skeletal defects. Although many different kinds of biomaterials have been used for bone tissue engineering, safety issues must be considered when using them in a clinical setting. In this study, we examined the effects of using a common clinical item, a hemostatic gelatin sponge, as a scaffold for bone tissue engineering. The use of such a clinically acceptable item may hasten the translational lag from laboratory to clinical studies. We performed both degradation and biocompatibility studies on the hemostatic gelatin sponge, and cultured preosteoblasts within the sponge scaffold to demonstrate its osteogenic differentiation potential. In degradation assays, the gelatin sponge demonstrated good stability after being immersed in PBS for 8 weeks (losing only about 10% of its net weight and about 54% decrease of mechanical strength), but pepsin and collagenases readily biodegraded it. The gelatin sponge demonstrated good biocompatibility to preosteoblasts as demonstrated by MTT assay, confocal microscopy, and scanning electron microscopy. Furthermore, osteogenic differentiation and the migration of preosteoblasts, elevated alkaline phosphatase activity, and in vitro mineralization were observed within the scaffold structure. Each of these results indicates that the hemostatic gelatin sponge is a suitable scaffold for bone tissue engineering. PMID:27616161

  12. Optical diagnostics of osteoblast cells and osteogenic drug screening

    Science.gov (United States)

    Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

    2016-02-01

    Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

  13. Exosomes derived from mineralizing osteoblasts promote ST2 cell osteogenic differentiation by alteration of microRNA expression.

    Science.gov (United States)

    Cui, Yazhou; Luan, Jing; Li, Haiying; Zhou, Xiaoyan; Han, Jinxiang

    2016-01-01

    Mineralizing osteoblasts (MOBs) can release exosomes, although the functional significance remains unclear. In the present study, we demonstrate that exosomes derived from mineralizing pre-osteoblast MC3T3-E1 cells can promote bone marrow stromal cell (ST2) differentiation to osteoblasts. We reveal that MOB-derived exosomes significantly influence miRNA profiles in recipient ST2 cells, and these changes tend to activate the Wnt signaling pathway by inhibiting Axin1 expression and increasing β-catenin expression. We also suggest that MOB derived-exosomes partly induce the variation in miRNA expression in recipient ST2 cells by exosomal miRNA transfer. These findings suggest an exosome-mediated mode of cell-to-cell communication in the osteogenic microenvironment, and also indicate the potential of MOB exosomes in bone tissue engineering.

  14. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sheehy, Eamon J.; Buckley, Conor T. [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland); Kelly, Daniel J., E-mail: kellyd9@tcd.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Expansion in low oxygen enhances MSC proliferation and osteogenesis. Black-Right-Pointing-Pointer Differentiation in low oxygen enhances chondrogenesis and suppresses hypertrophy. Black-Right-Pointing-Pointer Oxygen can regulate the MSC phenotype for use in tissue engineering applications. -- Abstract: The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO{sub 2}) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO{sub 2}). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO{sub 2} proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO{sub 2} also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO{sub 2} was found to be a more potent promoter of chondrogenesis than expansion at 5% pO{sub 2}. Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO{sub 2} compared to 5% pO{sub 2}. Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO{sub 2} also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a

  15. Insulin-like growth factor binding protein-3 affects osteogenic efficacy on dental implants in rat mandible

    International Nuclear Information System (INIS)

    Insulin like growth factor binding protein-3 (IGFBP-3) in bone cells and its utilization in dental implants have not been well studied. The aim of this study was to determine the osteogenic efficacy of chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 coated titanium (Ti) implants. Ch-GNPs were conjugated with IGFBP-3 plasmid DNA through a coacervation process. Conjugation was cast over Ti surfaces, and cells were seeded on coated surfaces. For in vitro analysis the expression of different proteins was analyzed by immunoblotting. For in vivo analysis, Ch-GNP/IGFBP-3 coated implants were installed in rat mandibles. Four weeks post-implantation, mandibles were examined by microcomputed tomography (μCT), immunohistochemistry, hematoxylin & eosin and tartrate resistance acid phosphatase staining. In vitro overexpressed Ch-GNP/IGFBP-3 coated Ti surfaces was associated with activation of extracellular signal related kinase (ERK), inhibition of the stress activated protein c-Jun N-terminal kinase (JNK) and enhanced bone morphogenetic protein (BMP)-2 and 7 compared to control. Further, in vivo, Ch-GNP/IGFBP-3 coated implants were associated with inhibition of implant induced osteoclastogenesis molecules, receptor activator of nuclear factor kappa-B ligand (RANKL) and enhanced expression of osteogenic molecules including BMP2/7 and osteopontin (OPN). The μCT analysis demonstrated that IGFBP-3 increased the volume of newly formed bone surrounding the implants compared to control (n = 5; p < 0.05). These results support the view that IGFBP-3 overexpression diminishes osteoclastogenesis and enhances osteogenesis of Ti implants, and can serve as a potent molecule for the development of good implantation. - Highlights: • Chitosan gold nanoparticles were conjugated with IGFBP-3 and coated onto surface of the titanium implants for gene delivery to bone. • Implants were inserted in rat mandible for 4 weeks. • Parameters studied: histopathology and radiology.

  16. Insulin-like growth factor binding protein-3 affects osteogenic efficacy on dental implants in rat mandible

    Energy Technology Data Exchange (ETDEWEB)

    Bhattarai, Govinda; Lee, Young-Hee [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Lee, Min-Ho [Department of Dental Materials, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of); Park, Il-Song [Division of Advanced Materials Engineering, Research Center for Advanced Materials, Development and Institute of Biodegradable Materials, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Yi, Ho-Keun, E-mail: yihokn@chonbuk.ac.kr [Department of Oral Biochemistry, Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju (Korea, Republic of)

    2015-10-01

    Insulin like growth factor binding protein-3 (IGFBP-3) in bone cells and its utilization in dental implants have not been well studied. The aim of this study was to determine the osteogenic efficacy of chitosan gold nanoparticles (Ch-GNPs) conjugated with IGFBP-3 coated titanium (Ti) implants. Ch-GNPs were conjugated with IGFBP-3 plasmid DNA through a coacervation process. Conjugation was cast over Ti surfaces, and cells were seeded on coated surfaces. For in vitro analysis the expression of different proteins was analyzed by immunoblotting. For in vivo analysis, Ch-GNP/IGFBP-3 coated implants were installed in rat mandibles. Four weeks post-implantation, mandibles were examined by microcomputed tomography (μCT), immunohistochemistry, hematoxylin & eosin and tartrate resistance acid phosphatase staining. In vitro overexpressed Ch-GNP/IGFBP-3 coated Ti surfaces was associated with activation of extracellular signal related kinase (ERK), inhibition of the stress activated protein c-Jun N-terminal kinase (JNK) and enhanced bone morphogenetic protein (BMP)-2 and 7 compared to control. Further, in vivo, Ch-GNP/IGFBP-3 coated implants were associated with inhibition of implant induced osteoclastogenesis molecules, receptor activator of nuclear factor kappa-B ligand (RANKL) and enhanced expression of osteogenic molecules including BMP2/7 and osteopontin (OPN). The μCT analysis demonstrated that IGFBP-3 increased the volume of newly formed bone surrounding the implants compared to control (n = 5; p < 0.05). These results support the view that IGFBP-3 overexpression diminishes osteoclastogenesis and enhances osteogenesis of Ti implants, and can serve as a potent molecule for the development of good implantation. - Highlights: • Chitosan gold nanoparticles were conjugated with IGFBP-3 and coated onto surface of the titanium implants for gene delivery to bone. • Implants were inserted in rat mandible for 4 weeks. • Parameters studied: histopathology and radiology.

  17. Endothelial cells influence the osteogenic potential of bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Arvidson Kristina

    2009-11-01

    Full Text Available Abstract Background Improved understanding of the interactions between bone cells and endothelial cells involved in osteogenesis should aid the development of new strategies for bone tissue engineering. The aim of the present study was to determine whether direct communication between bone marrow stromal cells (MSC and human umbilical vein endothelial cells (EC could influence the osteogenic potential of MSC in osteogenic factor-free medium. Methods After adding EC to MSC in a direct-contact system, cell viability and morphology were investigated with the WST assay and immnostaining. The effects on osteogenic differentiation of adding EC to MSC was systematically tested by the using Superarray assay and results were confirmed with real-time PCR. Results Five days after the addition of EC to MSC in a ratio of 1:5 (EC/MSC significant increases in cell proliferation and cellular bridges between the two cell types were detected, as well as increased mRNA expression of alkaline phosphatase (ALP. This effect was greater than that seen with addition of osteogenic factors such as dexamethasone, ascorbic acid and β-glycerophosphate to the culture medium. The expression of transcription factor Runx2 was enhanced in MSC incubated with osteogenic stimulatory medium, but was not influenced by induction with EC. The expression of Collagen type I was not influenced by EC but the cells grown in the osteogenic factor-free medium exhibited higher expression than those cultured with osteogenic stimulatory medium. Conclusion These results show that co-culturing of EC and MSC for 5 days influences osteogenic differentiation of MSC, an effect that might be independent of Runx2, and enhances the production of ALP by MSC.

  18. Vergleich von BMP-4 versus BMP-2 für die osteogene Differenzierung von Periostzellen

    OpenAIRE

    Klumpp, Florian (Alexander Stephan)

    2010-01-01

    Es ist heute bekannt, dass humane periostale mesenchymale Stammzellen (PMSCs) eine aussichtsreiche Grundlage für ein erfolgreiches Knochen Tissue Engineering darstellen. Dennoch ist die osteogene Differenzierung noch nicht vollständig be-schrieben. Da BMP-2 und BMP-4 nachweislich Regulatoren der Osteogenese sind, bestand die Aufgabe der vorliegenden Arbeit darin, die Wirkung derer auf die osteo-gene Differenzierung humaner PMSCs zu untersuchen. Isolierte humane PMSCs wurden mit Hilfe von o...

  19. Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices

    OpenAIRE

    Marita Westhrin; Minli Xie; Olderøy, Magnus Ø.; Pawel Sikorski; Strand, Berit L; Therese Standal

    2015-01-01

    Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and ...

  20. Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

    OpenAIRE

    Bab, I; Gazit, D.; Chorev, M; Muhlrad, A; Shteyer, A; Greenberg, Z; Namdar, M; Kahn, A.

    1992-01-01

    It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and incr...

  1. Electrospun biomimetic scaffold of hydroxyapatite/chitosan supports enhanced osteogenic differentiation of mMSCs

    Science.gov (United States)

    Peng, Hongju; Yin, Zi; Liu, Huanhuan; Chen, Xiao; Feng, Bei; Yuan, Huihua; Su, Bo; Ouyang, Hongwei; Zhang, Yanzhong

    2012-12-01

    Engaging functional biomaterial scaffolds to regulate stem cell differentiation has drawn a great deal of attention in the tissue engineering and regenerative medicine community. In this study, biomimetic composite nanofibrous scaffolds of hydroxyapatite/chitosan (HAp/CTS) were prepared to investigate their capacity for inducing murine mesenchymal stem cells (mMSCs) to differentiate into the osteogenic lineage, in the absence and presence of an osteogenic supplementation (i.e., ascorbic acid, β-glycerol phosphate, and dexamethasone), respectively. Using electrospun chitosan (CTS) nanofibrous scaffolds as the control, cell morphology, growth, specific osteogenic genes expression, and quantified proteins secretion on the HAp/CTS scaffolds were sequentially examined and assessed. It appeared that the HAp/CTS scaffolds supported better attachment and proliferation of the mMSCs. Most noteworthy was that in the absence of the osteogenic supplementation, expression of osteogenic genes including collagen I (Col I), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were significantly upregulated in mMSCs cultured on the HAp/CTS nanofibrous scaffolds. Also increased secretion of the osteogenesis protein markers of alkaline phosphatase and collagen confirmed that the HAp/CTS nanofibrous scaffold markedly promoted the osteogenic commitment in the mMSCs. Moreover, the presence of osteogenic supplementation proved an enhanced efficacy of mMSC osteogenesis on the HAp/CTS nanofibrous scaffolds. Collectively, this study demonstrated that the biomimetic nanofibrous HAp/CTS scaffolds could support and enhance the adhesion, proliferation, and particularly osteogenic differentiation of the mMSCs. It also substantiated the potential of using biomimetic nanofibrous scaffolds of HAp/CTS for functional bone repair and regeneration applications.

  2. Osteogenic Differentiation of Miniature Pig Mesenchymal Stem Cells in 2D and 3D Environment

    OpenAIRE

    Juhásová, J. (Jana); Juhás, Š. (Štefan); Klíma, J.; Strnádel, J. (Ján); Holubová, M. (Monika); Motlík, J. (Jan)

    2011-01-01

    Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the form...

  3. Extracellular Vesicles Derived from Osteogenically Induced Human Bone Marrow Mesenchymal Stem Cells Can Modulate Lineage Commitment

    Directory of Open Access Journals (Sweden)

    Margarida Martins

    2016-03-01

    Full Text Available The effective osteogenic commitment of human bone marrow mesenchymal stem cells (hBMSCs is critical for bone regenerative therapies. Extracellular vesicles (EVs derived from hBMSCs have a regenerative potential that has been increasingly recognized. Herein, the osteoinductive potential of osteogenically induced hBMSC-EVs was examined. hBMSCs secreted negatively charged nanosized vesicles (∼35 nm with EV-related surface markers. The yield of EVs over 7 days was dependent on an osteogenic stimulus (standard chemical cocktail or RUNX2 cationic-lipid transfection. These EVs were used to sequentially stimulate homotypic uncommitted cells during 7 days, matching the seeding density of EV parent cells, culture time, and stimuli. Osteogenically committed hBMSC-EVs induced an osteogenic phenotype characterized by marked early induction of BMP2, SP7, SPP1, BGLAP/IBSP, and alkaline phosphatase. Both EV groups outperformed the currently used osteoinductive strategies. These data show that naturally secreted EVs can guide the osteogenic commitment of hBMSCs in the absence of other chemical or genetic osteoinductors.

  4. A proteomic analysis during serial subculture and osteogenic differentiation of human mesenchymal stem cell.

    Science.gov (United States)

    Sun, Hyun Jin; Bahk, Young Yil; Choi, Yon Rak; Shim, Jung Hye; Han, Seung Hwan; Lee, Jin Woo

    2006-11-01

    Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 alpha subunit (TCP-1alpha), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing.

  5. Osteogenic sarcoma of the skull. A clinicopathologic study of 19 patients

    International Nuclear Information System (INIS)

    The authors studied 19 patients with well documented osteogenic sarcomas arising in the skull, which represent 1.6% of all osteogenic sarcomas registered during a 60-year period (1921-1981). Ten sarcomas were primary, de novo tumors. Nine others developed secondary osteogenic sarcomas; among these, six arose as a complication of Paget's disease, two followed irradiation, and one was associated with pre-existent fibrous dysplasia. The sarcomas arose in equal proportion in both sexes with the men being much older (mean age, 44 years) as compared to the women (mean age, 31 years). Patients with de novo osteogenic sarcomas were considerably younger than those with secondary lesions. Osteoblastic osteogenic sarcoma was by far the most common histologic variant in both the primary and the Paget's sarcomas. None of the patients with Paget's sarcoma lived longer than 1 year; the median survival here was 4 months. Patients with de novo osteogenic sarcomas fared much better and there are four long-term survivors (longer than 3 years) who are currently disease-free

  6. Effects of interleukin-17A on osteogenic differentiation of isolated human mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Bilal eOsta

    2014-09-01

    Full Text Available OBJECTIVES: Rheumatoid arthritis (RA is characterized by defective bone repair and excessive destruction, and ankylosing spondylitis (AS by increased ectopic bone formation with syndesmophytes. Since TNF-α and IL-17A are involved in both diseases, this study investigated their effects on the osteogenic differentiation of isolated human bone marrow derived mesenchymal stem cells (hMSCs. METHODS: Differentiation of hMSCs into osteoblasts was induced in the presence or absence of IL-17A and/or TNF-α. Matrix mineralization (MM was evaluated by alizarin red staining and alkaline phosphatase activity (ALP. mRNA expression was measured by qRT-PCR for BMP2 and Runx2, genes associated with osteogenesis, of DKK1, a negative regulator of osteogenesis, of Schnurri-3 and RANKL, associated with the cross talk with osteoclasts, and of TNFRI and TNFRII.RESULTS: TNF-α alone increased both MM and ALP activity. IL-17A alone increased ALP but not MM. Their combination was more potent. TNF-α alone increased BMP2 mRNA expression at 6 and 12 hr. These levels decreased in combination with IL-17A at 6 hr only. DKK1mRNA expression was inhibited by TNF-α and IL-17A either alone or combined. Supporting an imbalance towards osteoblastogenesis, RANKL expression was inhibited by TNF-α and IL-17A. However, TNF-α but not IL-17 alone decreased Runx2 mRNA expression at 6 hr. In parallel, TNF-α but not IL-17 alone increased Schnurri-3 expression with a synergistic effect with their combination. This may be related to an increase of TNFRII overexpression. CONCLUSION: IL-17 increased the effects of TNF-α on bone matrix formation by hMSCs. However, IL-17 decreased the TNF-α -induced BMP2 inhibition. Synergistic interactions between TNF-α and IL-17 were seen for RANKL inhibition and Schnurri-3 induction. Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA. Conversely, in the absence of osteoclasts, this could promote ectopic

  7. In Vitro Osteogenic Properties of Two Dental Implant Surfaces

    Directory of Open Access Journals (Sweden)

    Marta Monjo

    2012-01-01

    Full Text Available Current dental implant research aims at understanding the biological basis for successful implant therapy. The aim of the study was to perform a full characterization of the effect of two commercial titanium (Ti surfaces, OsseoSpeed and TiOblast, on the behaviour of mouse preosteoblast MC3T3-E1 cells. The effect of these Ti surfaces was compared with tissue culture plastic (TCP. In vitro experiments were performed to evaluate cytotoxicity, cell morphology and proliferation, alkaline phosphatase activity, gene expression, and release of a wide array of osteoblast markers. No differences were observed on cell viability and cell proliferation. However, changes were observed in cell shape after 2 days, with a more branched morphology on OsseoSpeed compared to TiOblast. Moreover, OsseoSpeed surface increased BMP-2 secretion after 2 days, and this was followed by increased IGF-I, BSP, and osterix gene expression and mineralization compared to TiOblast after 14 days. As compared to the gold standard TCP, both Ti surfaces induced higher osteocalcin and OPG release than TCP and differential temporal gene expression of osteogenic markers. The results demonstrate that the gain of using OsseoSpeed surface is an improved osteoblast differentiation and mineralization, without additional effects on cell viability or proliferation.

  8. Longitudinal analysis of osteogenic and angiogenic signaling factors in healing models mimicking atrophic and hypertrophic non-unions in rats.

    Directory of Open Access Journals (Sweden)

    Susann Minkwitz

    Full Text Available Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group to an open osteotomy (hypertrophy group led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.

  9. Effect of Nano-HA/Collagen Composite Hydrogels on Osteogenic Behavior of Mesenchymal Stromal Cells.

    Science.gov (United States)

    Hayrapetyan, Astghik; Bongio, Matilde; Leeuwenburgh, Sander C G; Jansen, John A; van den Beucken, Jeroen J J P

    2016-06-01

    This study aimed to comparatively evaluate the in vitro effect of nanosized hydroxyapatite and collagen (nHA/COL) based composite hydrogels (with different ratios of nHA and COL) on the behavior of human mesenchymal stromal cells (MSCs), isolated from either adipose tissue (AT-MSCs) or bone marrow (BM-MSCs). We hypothesized that (i) nHA/COL composite hydrogels would promote the osteogenic differentiation of MSCs in an nHA concentration dependent manner, and that (ii) AT-MSCs would show higher osteogenic potential compared to BM-MSCs, due to their earlier observed higher proliferation and osteogenic differentiation potential in 2D in vitro cultures [1]. The obtained results indicated that AT-MSCs show indeed high proliferation, differentiation and mineralization capacities in nHA/COL constructs compared to BM-MSCs, but this effect was irrespective of nHA concentration. Based on the results of alkaline phosphatase (ALP) activity and osteocalcin (OCN) protein level, the osteogenic differentiation of BM-MSCs started in the beginning of the culture period and for AT-MSCs at the end of the culture period. At a molecular level, both cell types showed high expression of osteogenic markers (bone morphogenic protein 2 [BMP2], runt-related transcription factor 2 [RUNX2], OCN or COL1) in both an nHA concentration and time dependent manner. In conclusion, AT-MSCs demonstrated higher osteogenic potential in nHA/COL based 3D micro-environments compared to BM-MSCs, in which proliferation and osteogenic differentiation were highly promoted in a time dependent manner, irrespective of nHA amount in the constructs. The fact that AT-MSCs showed high proliferation and mineralization potential is appealing for their application in future pre-clinical research as an alternative cell source for BM-MSCs. PMID:26803618

  10. Does tranexamic acid stabilised fibrin support the osteogenic differentiation of human periosteum derived cells?

    Directory of Open Access Journals (Sweden)

    J Demol

    2011-03-01

    Full Text Available Fibrin sealants have long been used as carrier for osteogenic cells in bone regeneration. However, it has not been demonstrated whether fibrin’s role is limited to delivering cells to the bone defect or whether fibrin enhances osteogenesis. This study investigated fibrin’s influence on the behaviour of human periosteum-derived cells (hPDCs when cultured in vitro under osteogenic conditions in two-dimensional (fibrin substrate and three-dimensional (fibrin carrier environments. Tranexamic acid (TEA was used to reduce fibrin degradation after investigating its effect on hPDCs in monolayer culture on plastic.TEA did not affect proliferation nor calcium deposition of hPDCs under these conditions. Expression profiles of specific osteogenic markers were also maintained within the presence of TEA, apart from reduced alkaline phosphatase (ALP expression (day 14. Compared to plastic, proliferation was upregulated on 2D fibrin substrates with a 220% higher DNA content by day 21. Gene expression was also altered, with significantly (p<0.05 decreased Runx2 (day 7 and ALP (day 14 expression and increased collagen I expression (day 14 and 21. In contrast to plastic, mineralisation was absent on fibrin substrates. Inside fibrin carriers, hPDCs were uniformly distributed. Moderate cell growth and reduced osteogenic marker expression was observed inside fibrin carriers. After 2 weeks, increased cell death was present in the carrier’s centre. In conclusion, fibrin negatively influences osteogenic differentiation, compared to culture plastic, but enhanced proliferation (at least in 2D cultures for hPDCs cultured in osteogenic conditions. TEA maintained the integrity of fibrin-based constructs, with minor effects on the osteogenic differentiation of hPDCs.

  11. Osteogenic cell cultures cannot utilize exogenous sources of synthetic polyphosphate for mineralization.

    Science.gov (United States)

    Ariganello, Marianne B; Omelon, Sidney; Variola, Fabio; Wazen, Rima M; Moffatt, Pierre; Nanci, Antonio

    2014-12-01

    Phosphate is critical for mineralization and deficiencies in the regulation of free phosphate lead to disease. Inorganic polyphosphates (polyPs) may represent a physiological source of phosphate because they can be hydrolyzed by biological phosphatases. To investigate whether exogenous polyP could be utilized for mineral formation, mineralization was evaluated in two osteogenic cell lines, Saos-2 and MC3T3, expressing different levels of tissue non-specific alkaline phosphatase (tnALP). The role of tnALP was further explored by lentiviral-mediated overexpression in MC3T3 cells. When cells were cultured in the presence of three different phosphate sources, there was a strong mineralization response with β-glycerophosphate (βGP) and orthophosphate (Pi) but none of the cultures sustained mineralization in the presence of polyP (neither chain length 17-Pi nor 42-Pi). Even in the presence of mineralizing levels of phosphate, low concentrations of polyP (50 μM) were sufficient to inhibit mineral formation. Energy-dispersive X-ray spectroscopy confirmed the presence of apatite-like mineral deposits in MC3T3 cultures supplemented with βGP, but not in those with polyP. While von Kossa staining was consistent with the presence or absence of mineral, an unusual Alizarin staining was obtained in polyP-treated MC3T3 cultures. This staining pattern combined with low Ca:P ratios suggests the persistence of Ca-polyP complexes, even with high residual ALP activity. In conclusion, under standard culture conditions, exogenous polyP does not promote mineral deposition. This is not due to a lack of active ALP, and unless conditions that favor significant processing of polyP are achieved, its mineral inhibitory capacity predominates.

  12. Boron Nitride Nanotubes Reinforce Tricalcium Phosphate Scaffolds and Promote the Osteogenic Differentiation of Mesenchymal Stem Cells.

    Science.gov (United States)

    Shuai, Cijun; Gao, Chengde; Feng, Pei; Xiao, Tao; Yu, Kun; Deng, Youwen; Peng, Shuping

    2016-05-01

    Incorporating boron nitride nanotubes (BNNTs) into ceramic matrices is a promising strategy for obtaining multifunctional composites. In this study, the application of BNNTs in reinforcing β-tricalcium phosphate (β-TCP) scaffolds manufactured using laser sintering is demonstrated. BNNTs contribute to the effective inhibition of both grain growth and phase transformation in β-TCP. Moreover, they can strengthen the grain boundaries and boost the fracture mode transition from intergranular to transgranular. BNNTs play an active role in reinforcing β-TCP in terms of load transfer and energy absorption by the synergistic mechanisms of pull-out, peel-off, crack bridging and deflection. With a BNNT content of 4 wt%, the elastic modulus, hardness, compressive strength and fracture toughness of β-TCP increase by 46%, 39%, 109% and 35%, respectively. Umbilical cord mesenchymal stem cells (UC-MSCs) were isolated with high purity, and surface molecule characterization revealed that they were CD90+, CD29+, CD73+, CD31-, CD34- and CD45-. UC-MSCs on BNNTs/β-TCP scaffolds were characterized by more positive Alizarin Red staining as well as up-regulated expression of osteoblast markers, as revealed by quantitative real-time reverse transcriptase polymerase chain reaction analysis and immunofluorescence staining. These results are the first to demonstrate that BNNTs promote the osteogenic differentiation of UC-MSCs, indicating good osteoinductive properties for use in bone scaffolds. This study paves the way for the potential use of a BNNT/β-TCP scaffold in bone repair. PMID:27305816

  13. cAMP/PKA Signaling Inhibits Osteogenic Differentiation and Bone Formation in Rodent Models

    NARCIS (Netherlands)

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; Klundert, van de Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; Blitterswijk, van Clemens; Boer, de Jan

    2009-01-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable

  14. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43‑ symmetric stretch vibrations at 959 cm‑1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue–implant-interfaces or disease diagnosis.

  15. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

  16. 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.

  17. Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

    Science.gov (United States)

    Westhrin, Marita; Xie, Minli; Olderøy, Magnus Ø.; Sikorski, Pawel

    2015-01-01

    Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering. PMID:25769043

  18. Osteogenic differentiation of human mesenchymal stem cells in mineralized alginate matrices.

    Directory of Open Access Journals (Sweden)

    Marita Westhrin

    Full Text Available Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST and dental matrix protein-1 (DMP1, markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

  19. Osteogenic potential of murine periosteum for critical-size cranial defects.

    Science.gov (United States)

    Ruvalcaba-Paredes, E K; Hidalgo-Bastida, L A; Sesman-Bernal, A L; Garciadiego-Cazares, D; Pérez-Dosal, M R; Martínez-López, V; Vargas-Sandoval, B; Pichardo-Bahena, R; Ibarra, C; Velasquillo, C

    2016-09-01

    Tissue engineering of bone has combined bespoke scaffolds and osteoinductive factors to maintain functional osteoprogenitor cells, and the periosteum has been confirmed as a satisfactory source of osteoblasts. Suitable matrices have been identified that support cell proliferation and differentiation, including demineralised bone matrix (both compatible and osteoinductive) and acellular human dermis. We have evaluated the osteogenic potential of an osteogenic unit, developed by combining periosteum, demineralised bone matrix, and acellular human dermis, in rodents with critical-size cranial defects. Briefly, remnants from the superior maxillary periosteum were used to harvest cells, which were characterised by flow cytometry and reverse retrotranscriptase-polymerase chain reaction (RT-PCR). Cells were cultured into the osteogenic unit and assessed for viability before being implanted into 3 rodents, These were compared with the control group (n=3) after three months. Histological analyses were made after staining with haematoxylin and eosin and Von Kossa, and immunostaining, and confirmed viable cells that stained for CD90, CD73, CD166, runt-related transcription factor, osteopontin, and collagen type I in the experimental group, while in the control group there was only connective tissue on the edges of the bone in the injury zone. We conclude that osteogenic unit constructs have the osteogenic and regenerative potential for use in engineering bone tissue. PMID:27282080

  20. In Vivo Osteogenic Differentiation of Human Embryoid Bodies in an Injectable in Situ-Forming Hydrogel

    Directory of Open Access Journals (Sweden)

    Moon Suk Kim

    2013-07-01

    Full Text Available In this study, we examined the in vivo osteogenic differentiation of human embryoid bodies (hEBs by using an injectable in situ-forming hydrogel. A solution containing MPEG-b-(polycaprolactone-ran-polylactide (MCL and hEBs was easily prepared at room temperature. The MCL solution with hEBs and osteogenic factors was injected into nude mice and developed into in situ-forming hydrogels at the injection sites; these hydrogels maintained their shape even after 12 weeks in vivo, thereby indicating that the in situ-forming MCL hydrogel was a suitable scaffold for hEBs. The in vivo osteogenic differentiation was observed only in the in situ gel-forming MCL hydrogel in the presence of hEBs and osteogenic factors. In conclusion, this preliminary study suggests that hEBs and osteogenic factors embedded in an in situ-forming MCL hydrogel may provide numerous benefits as a noninvasive alternative for allogeneic tissue engineering applications.

  1. In Vitro Study of the Effect of Vitamin E on Viability, Morphological Changes and Induction of Osteogenic Differentiation in Adult Rat Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    M Soleimani Mehranjani

    2014-10-01

    Full Text Available Introduction: Vitamin E as a strong antioxidant plays an important role in inhibiting free radicals. Therefore, this study aimed to investigate the effect of vitamin E on the viability, morphology and osteogenic differentiation in bone marrow mesenchymal stem cells of an adult rat. Methods: The bone marrow mesenchymal stem cells were extracted using the flashing-out method. At the end of the third passage, cells were divided into groups of control and experimental. Experimental cells were treated withVitamin E (5,10,15,25,50,100,150μM for a period of 21 days in the osteogenic media containing 10% of fetal bovine serum. The cell viability, bone matrix mineralization, intercellular and extracellular calcium deposition, alkaline phosphatase activity, expression of genes and synthesis of proteins of osteopontin and osteocalcin as well as morphological changes of the cells were investigated. The study data was analyzed using one-way ANOVA and T-Test setting the significant P value at P<0.05. Results: Within vitamin- E treated cells, the mean viability, mean bone matrix mineralization, calcium deposition, alkaline phosphatase activity, expression and synthesis of osteopontin and osteocalcin of the mesenchymal stem cells treated with vitamin E significantly increased in a dose dependent manner. Also cytoplasm extensions were observed in the cells treated with vitamin E. Conclusion: Since vitamin E caused a significant increase in cell viability and osteogenic differentiation in the mesenchymal stem cells, therefore it can be utilized in order to increase cell differentiation and cell survival.

  2. Small Buccal Fat Pad Cells Have High Osteogenic Differentiation Potential.

    Science.gov (United States)

    Tsurumachi, Niina; Akita, Daisuke; Kano, Koichiro; Matsumoto, Taro; Toriumi, Taku; Kazama, Tomohiko; Oki, Yoshinao; Tamura, Yoko; Tonogi, Morio; Isokawa, Keitaro; Shimizu, Noriyoshi; Honda, Masaki

    2016-03-01

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 μm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-μm nylon mesh filters: cell diameters less than 40 μm (small adipocytes: S-adipocytes) and cell diameters of 40-100 μm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells. PMID:26651216

  3. The Control of Mesenchymal Stromal Cell Osteogenic Differentiation through Modified Surfaces

    Directory of Open Access Journals (Sweden)

    Niall Logan

    2013-01-01

    Full Text Available Stem cells continue to receive widespread attention due to their potential to revolutionise treatments in the fields of both tissue engineering and regenerative medicine. Adult stem cells, specifically mesenchymal stromal cells (MSCs, play a vital role in the natural events surrounding bone healing and osseointegration through being stimulated to differentiate along their osteogenic lineage and in doing so, they form new cortical and trabecular bone tissue. Understanding how to control, manipulate, and enhance the intrinsic healing events modulated through osteogenic differentiation of MSCs by the use of modified surfaces and biomaterials could potentially advance the fields of both orthopaedics and dentistry. This could be by either using surface modification to generate greater implant stability and more rapid healing following implantation or the stimulation of MSCs ex vivo for reimplantation. This review aims to gather publications targeted at promoting, enhancing, and controlling the osteogenic differentiation of MSCs through biomaterials, nanotopographies, and modified surfaces for use in implant procedures.

  4. The effect of low static magnetic field on osteogenic and adipogenic differentiation potential of human adipose stromal/stem cells

    Science.gov (United States)

    Marędziak, Monika; Śmieszek, Agnieszka; Tomaszewski, Krzysztof A.; Lewandowski, Daniel; Marycz, Krzysztof

    2016-01-01

    The aim of this work was to investigate the effects of static magnetic field (SMF) on the osteogenic properties of human adipose derived mesenchymal stem cells (hASCs). In this study in seven days viability assay we examined the impact of SMF on cells proliferation rate, population doubling time, and ability to form single-cell derived colonies. We have also examined cells' morphology, ultrastructure and osteogenic properties on the protein as well as mRNA level. We established a complex approach, which enabled us to obtain information about SMF and hASCs potential in the context of differentiation into osteogenic and adipogenic lineages. We demonstrated that SMF enhances both viability and osteogenic properties of hASCs through higher proliferation factor and shorter population doubling time. We have also observed asymmetrically positioned nuclei and organelles after SMF exposition. With regards to osteogenic properties we observed increased levels of osteogenic markers i.e. osteopontin, osteocalcin and increased ability to form osteonodules with positive reaction to Alizarin Red dye. We have also shown that SMF besides enhancing osteogenic properties of hASCs, simultaneously decreases their ability to differentiate into adipogenic lineage. Our results clearly show a direct influence of SMF on the osteogenic potential of hASCs. These results provide key insights into the role of SMF on their cellular fate and properties.

  5. Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Guo-yong Yu

    2016-01-01

    Full Text Available Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling, the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1, adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 μg/mL increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway.

  6. Naringin Stimulates Osteogenic Differentiation of Rat Bone Marrow Stromal Cells via Activation of the Notch Signaling Pathway.

    Science.gov (United States)

    Yu, Guo-Yong; Zheng, Gui-Zhou; Chang, Bo; Hu, Qin-Xiao; Lin, Fei-Xiang; Liu, De-Zhong; Wu, Chu-Cheng; Du, Shi-Xin; Li, Xue-Dong

    2016-01-01

    Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs) were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling), the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1), adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 μg/mL) increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway. PMID:27069482

  7. Reduced graphene oxide-coated hydroxyapatite composites stimulate spontaneous osteogenic differentiation of human mesenchymal stem cells

    Science.gov (United States)

    Lee, Jong Ho; Shin, Yong Cheol; Jin, Oh Seong; Kang, Seok Hee; Hwang, Yu-Shik; Park, Jong-Chul; Hong, Suck Won; Han, Dong-Wook

    2015-07-01

    Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite the potential biomedical applications of graphene and its derivatives, only limited information is available regarding their osteogenic activity. This study concentrates upon the effects of reduced graphene oxide (rGO)-coated hydroxyapatite (HAp) composites on osteogenic differentiation of hMSCs. The average particle sizes of HAp and rGO were 1270 +/- 476 nm and 438 +/- 180 nm, respectively. When coated on HAp particulates, rGO synergistically enhanced spontaneous osteogenic differentiation of hMSCs, without hampering their proliferation. This result was confirmed by determining alkaline phosphatase activity and mineralization of calcium and phosphate as early and late stage markers of osteogenic differentiation. It is suggested that rGO-coated HAp composites can be effectively utilized as dental and orthopedic bone fillers since these graphene-based particulate materials have potent effects on stimulating the spontaneous differentiation of MSCs and show superior bioactivity and osteoinductive potential.Human mesenchymal stem cells (hMSCs) have great potential as cell sources for bone tissue engineering and regeneration, but the control and induction of their specific differentiation into bone cells remain challenging. Graphene-based nanomaterials are considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for SC differentiation and components of implantable devices, due to their biocompatible and bioactive properties. Despite

  8. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Highlights: ► We detect the functional Ca2+ currents and mRNA expression of VDCCL in rMSCs. ► Blockage of VDCCL exert antiproliferative and apoptosis-inducing effects on rMSCs. ► Inhibiting VDCCL can suppress the ability of rMSCs to differentiate into osteoblasts. ► α1C of VDCCL may be a primary functional subunit in VDCCL-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca2+ channels (VDCCL) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCCL expression was reported in mesenchymal stem cells (MSCs), but the role of VDCCL in MSCs is still undetermined. The aim of this study was to determine whether VDCCL may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca2+ currents (ICa) and mRNA expression of VDCCL in rMSCs, and then suppressed VDCCL using nifedipine (Nif), a VDCCL blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected α1C-siRNA into the cells to further confirm the role of VDCCL in rMSCs, and a similar effect on osteogenesis was found. These results suggest that VDCCL plays a crucial role in the proliferation and osteogenic differentiation of rMSCs.

  9. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Li [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Wang, Yu [Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Wang, Huan [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Kong, Lingmin [Department of Fundamental Medicine, Cell Engineering Research Centre, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Liang [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Chen, Xin [Department of General Dentistry, The 174th Hospital of Chinese PLA, Xiamen 361003 (China); Ding, Yin, E-mail: dingyin@fmmu.edu.cn [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We detect the functional Ca{sup 2+} currents and mRNA expression of VDCC{sub L} in rMSCs. Black-Right-Pointing-Pointer Blockage of VDCC{sub L} exert antiproliferative and apoptosis-inducing effects on rMSCs. Black-Right-Pointing-Pointer Inhibiting VDCC{sub L} can suppress the ability of rMSCs to differentiate into osteoblasts. Black-Right-Pointing-Pointer {alpha}1C of VDCC{sub L} may be a primary functional subunit in VDCC{sub L}-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca{sup 2+} channels (VDCC{sub L}) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC{sub L} expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC{sub L} in MSCs is still undetermined. The aim of this study was to determine whether VDCC{sub L} may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca{sup 2+} currents (I{sub Ca}) and mRNA expression of VDCC{sub L} in rMSCs, and then suppressed VDCC{sub L} using nifedipine (Nif), a VDCC{sub L} blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected {alpha}1C-siRNA into the cells to further confirm the role of VDCC{sub L} in rMSCs, and a similar effect on osteogenesis was found. These

  10. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1

    Directory of Open Access Journals (Sweden)

    Yin-Hua Zhao

    2015-05-01

    Full Text Available Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs. The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA and Ras-related C3 botulinum toxin substrate 1 (Rac1 in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs.

  11. Liver-derived IGF-I regulates cortical bone mass but is dispensable for the osteogenic response to mechanical loading in female mice.

    Science.gov (United States)

    Svensson, Johan; Windahl, Sara H; Saxon, Leanne; Sjögren, Klara; Koskela, Antti; Tuukkanen, Juha; Ohlsson, Claes

    2016-07-01

    Low circulating IGF-I is associated with increased fracture risk. Conditional depletion of IGF-I produced in osteoblasts or osteocytes inhibits the bone anabolic effect of mechanical loading. Here, we determined the role of endocrine IGF-I for the osteogenic response to mechanical loading in young adult and old female mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice, serum IGF-I reduced by ≈70%) and control mice. The right tibia was subjected to short periods of axial cyclic compressive loading three times/wk for 2 wk, and measurements were performed using microcomputed tomography and mechanical testing by three-point bending. In the nonloaded left tibia, the LI-IGF-I(-/-) mice had lower cortical bone area and increased cortical porosity, resulting in reduced bone mechanical strength compared with the controls. Mechanical loading induced a similar response in LI-IGF-I(-/-) and control mice in terms of cortical bone area and trabecular bone volume fraction. In fact, mechanical loading produced a more marked increase in cortical bone mechanical strength, which was associated with a less marked increase in cortical porosity, in the LI-IGF-I(-/-) mice compared with the control mice. In conclusion, liver-derived IGF-I regulates cortical bone mass, cortical porosity, and mechanical strength under normal (nonloaded) conditions. However, despite an ∼70% reduction in circulating IGF-I, the osteogenic response to mechanical loading was not attenuated in the LI-IGF-I(-/-) mice.

  12. Computed tomography in low-back-pain after femur-amputation for osteogenic sarcoma

    International Nuclear Information System (INIS)

    Following amputation of a lower extremity for osteogenic sarcoma, the lumbar muscles receive an asymmetric strain. This predisposes to low-back-pain. When this occurs, tumour-recurrence must be excluded. This report demonstrates the usefulness of high-resolution computer tomography (CT) in this clinical situation. (orig.)

  13. Graphene oxide nanoflakes incorporated gelatin-hydroxyapatite scaffolds enhance osteogenic differentiation of human mesenchymal stem cells

    Science.gov (United States)

    Nair, Manitha; Nancy, D.; Krishnan, Amit G.; Anjusree, G. S.; Vadukumpully, Sajini; Nair, Shantikumar V.

    2015-04-01

    In this study, graphene oxide (GO) nanoflakes (0.5 and 1 wt%) were incorporated into a gelatin-hydroxyapatite (GHA) matrix through a freeze drying technique and its effect to enhance mechanical strength and osteogenic differentiation was studied. The GHA matrix with GO demonstrated less brittleness in comparison to GHA scaffolds. There was no significant difference in mechanical strength between GOGHA0.5 and GOGHA1.0 scaffolds. When the scaffolds were immersed in phosphate buffered saline (to mimic physiologic condition) for 60 days, around 50-60% of GO was released in sustained and linear manner and the concentration was within the toxicity limit as reported earlier. Further, GOGHA0.5 scaffolds were continued for cell culture experiments, wherein the scaffold induced osteogenic differentiation of human adipose derived mesenchymal stem cells without providing supplements like dexamethasone, L-ascorbic acid and β glycerophosphate in the medium. The level of osteogenic differentiation of stem cells was comparable to those cultured on GHA scaffolds with osteogenic supplements. Thus biocompatible, biodegradable and porous GO reinforced gelatin-HA 3D scaffolds may serve as a suitable candidate in promoting bone regeneration in orthopaedics.

  14. Relation between in vitro and in vivo osteogenic potential of cultured human bone marrow stromal cells

    NARCIS (Netherlands)

    Mendes, SC; Tibbe, JM; Veenhof, M; Both, S; Oner, FC; van Blitterswijk, CA; de Bruijn, Joost D.

    2004-01-01

    The use of cell therapies in bone reconstruction has been the subject of extensive research. It is known that human bone marrow stromal cell (HBMSC) cultures contain a population of progenitor cells capable of differentiation towards the osteogenic lineage. In the present study, the correlation betw

  15. Osteogenic potential of bone-lining cells in the adult skeleton

    International Nuclear Information System (INIS)

    Radiation-induced osteogenic sarcomas are believed to arise from proliferating osteogenic precursor cells. The identity and location of these cells in the adult skeleton is not well understood. In order to determine reliable cell dose estimates, it is important to determine the osteogenic pathway in the adult skeleton. Bone-lining cells (BLCs) cover inactive endosteal surfaces in the adult skeleton of long-lived animals. BLCs are flat elongated cells which are directly apposed to the bone surface. They have cell processes extending into canaliculi and have gap junctions at some contacts with other bone-lining cells. The morphology of the bone-lining cell and its proximity to the bone surface can only be resolved at the ultrastructural level. These cells are a distinct morphologic phenotype but have been referred to by a variety of names including resting osteoblasts, surface osteocytes, and flattened mesenchymal cells. The BLC, as a distinct phenotype, should not be confused with the more descriptive term cells lining the bone surface of bone lining cells, sometimes used to include any cell near the bone. The purpose of the study was to determine what role, if any, the bone-lining cells have in the osteogenic process. Do these cells proliferate and contribute to the population of osteoblasts?

  16. Osteogenic Potential of Multipotent Adult Progenitor Cells for Calvaria Bone Regeneration

    Science.gov (United States)

    Lee, Dong Joon; Park, Yonsil; Hu, Wei-Shou; Ko, Ching-Chang

    2016-01-01

    Osteogenic cells derived from rat multipotent adult progenitor cells (rMAPCs) were investigated for their potential use in bone regeneration. rMAPCs are adult stem cells derived from bone marrow that have a high proliferation capacity and the differentiation potential to multiple lineages. They may also offer immunomodulatory properties favorable for applications for regenerative medicine. rMAPCs were cultivated as single cells or as 3D aggregates in osteogenic media for up to 38 days, and their differentiation to bone lineage was then assessed by immunostaining of osteocalcin and collagen type I and by mineralization assays. The capability of rMAPCs in facilitating bone regeneration was evaluated in vivo by the direct implantation of multipotent adult progenitor cell (MAPC) aggregates in rat calvarial defects. Bone regeneration was examined radiographically, histologically, and histomorphometrically. Results showed that rMAPCs successfully differentiated into osteogenic lineage by demonstrating mineralized extracellular matrix formation in vitro and induced new bone formation by the effect of rMAPC aggregates in vivo. These outcomes confirm that rMAPCs have a good osteogenic potential and provide insights into rMAPCs as a novel adult stem cell source for bone regeneration. PMID:27239552

  17. Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Pei-xun Zhang; Xiao-rui Jiang; Lei Wang; Fang-min Chen; Lin Xu; Fei Huang

    2015-01-01

    Preliminary animal experiments have conifrmed that sensory nerve ifbers promote osteoblast differentiation, but motor nerve ifbers have no promotion effect. Whether sensory neurons pro-mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons (sensory neurons) from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green lfuorescent protein 3 weeks after osteo-genic differentiationin vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera-tion of bone marrow mesenchymal stem cell-derived osteoblasts at 3 and 5 days of co-culture, as observed by lfuorescence microscopy. The levels of mRNAs for osteogenic differentiation-re-lated factors (including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2) in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our ifndings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro-vides a theoretical basis forin vitro experiments aimed at constructing tissue-engineered bone.

  18. Bone marrow concentrate for autologous transplantation in minipigs. Characterization and osteogenic potential of mesenchymal stem cells.

    Science.gov (United States)

    Herten, M; Grassmann, J P; Sager, M; Benga, L; Fischer, J C; Jäger, M; Betsch, M; Wild, M; Hakimi, M; Jungbluth, P

    2013-01-01

    Autologous bone marrow plays an increasing role in the treatment of bone, cartilage and tendon healing disorders. Cell-based therapies display promising results in the support of local regeneration, especially therapies using intra-operative one-step treatments with autologous progenitor cells. In the present study, bone marrow-derived cells were concentrated in a point-of-care device and investigated for their mesenchymal stem cell (MSC) characteristics and their osteogenic potential. Bone marrow was harvested from the iliac crest of 16 minipigs. The mononucleated cells (MNC) were concentrated by gradient density centrifugation, cultivated, characterized by flow cytometry and stimulated into osteoblasts, adipocytes, and chondrocytes. Cell differentiation was investigated by histological and immunohistological staining of relevant lineage markers. The proliferation capacity was determined via colony forming units of fibroblast and of osteogenic alkaline-phosphatase-positive-cells. The MNC could be enriched 3.5-fold in nucleated cell concentrate in comparison to bone marrow. Flow cytometry analysis revealed a positive signal for the MSC markers. Cells could be differentiated into the three lines confirming the MSC character. The cellular osteogenic potential correlated significantly with the percentage of newly formed bone in vivo in a porcine metaphyseal long-bone defect model. This study demonstrates that bone marrow concentrate from minipigs display cells with MSC character and their osteogenic differentiation potential can be used for osseous defect repair in autologous transplantations.

  19. Effects of substrate stiffness on adipogenic and osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Zhao, Wen; Li, Xiaowei; Liu, Xiaoyan; Zhang, Ning; Wen, Xuejun

    2014-07-01

    Substrate mechanical properties, in addition to biochemical signals, have been shown to modulate cell phenotype. In this study, we inspected the effects of substrate stiffness on human mesenchymal stem cells (hMSCs) derived from adult human bone marrow differentiation into adipogenic and osteogenic cells. A chemically modified extracellular matrix derived and highly biocompatible hydrogel, based on thiol functionalized hyaluronic acid (HA-SH) and thiol functionalized recombinant human gelatin (Gtn-SH), which can be crosslinked by poly (ethylene glycol) tetra-acrylate (PEGTA), was used as a model system. The stiffness of the hydrogel was controlled by adjusting the crosslinking density. Human bone marrow MSCs were cultured on the hydrogels with different stiffness under adipogenic and osteogenic conditions. Oil Red O staining and F-actin staining were applied to assess the change of cell morphologies under adipogenic and osteogenic differentiation, respectively. Gene expression of cells was determined with reverse transcription polymerase chain reaction (RT-PCR) as a function of hydrogel stiffness. Results support the hypothesis that adipogenic and osteogenic differentiation of hMSCs are inclined to occur on substrate with stiffness similar to their in vivo microenvironments. PMID:24857499

  20. Identification of novel osteogenic compounds by an ex-vivo sp7 : Luciferase zebrafish scale assay

    NARCIS (Netherlands)

    de Vrieze, Erik; Zethof, Jan; Schulte-Merker, Stefan; Flik, Gert; Metz, Juriaan R.

    2015-01-01

    Tight interactions among different cell types contributing to bone formation are of key importance in the maintenance of bone homeostasis. Based on the high similarity in responses to (anti)osteogenic signals between zebrafish scales and mammalian bone, we developed and validated a model to screen l

  1. Comparison of Effects of Mechanical Stretching on Osteogenic Potential of ASCs and BMSCs

    Institute of Scientific and Technical Information of China (English)

    Brian E. Grottkau; Xingmei Yang; Liang Zhang; Ling Ye; Yunfeng Lin

    2013-01-01

    Mechanical forces play critical roles in the development and remodeling processes of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress. Similarly, the osteogenic potential, stimulated by mechanical stress, should be compared with bone marrow stromal cells (BMSCs), which have been clinically used for bone tissue engineering. In this study, ASCs and BMSCs were osteogenic-induced for 48 hours, and then subjected to uniaxial mechanical stretching for 2 or 6 hours. Cell orientation, osteogenic regulatory genes, osteogenic genes and ALP activities were measured and compared between ASCs and BMSCs. ASCs could align in a perpendicular way to the direction of stretching stress, while BMSCs did not present a specific alignment. Both 2 and 6 hours mechanical stretching could enhance the mRNA expression of Osx and Runx2 in BMSCs and ASCs, while OCN mRNA only increased in ASCs after 6 hours mechanical loading. Mechanical stretching enhanced the BMP-2 mRNA expression in ASCs, while only after 6 hours of mechanical loading significantly increased the BMP-2 gene expression in BMSCs. Significant differences only exist between ASCs and BMSCs loaded at 2 hours of mechanical stretching. It is concluded that ASCs are more rapid responders to mechanical stress, and have greater potential than BMSCs in osteogenesis when stimulated by mechanical stretching, indicating their usefulness for bone study in a rat model.

  2. Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices

    Directory of Open Access Journals (Sweden)

    A. Pozzuoli

    2015-03-01

    Full Text Available Aim Tissue engineering is a rapidly expanding field with regard to the use of biomaterials and stem cells in the orthopedic surgery. Many experimental studies have been done to understand the best characteristics of cells, materials and laboratory methods for safe clinical applications. The aim of this study was to compare the ability of 2 different human demineralized bone matrices (DBMs, the one enriched and the other not enriched with hyaluronic acid, to stimulate in vitro the proliferation and the osteogenic differentiation of human adipose-derived stem cells (ADSCs seeded onto an osteoconductive scaffold. Materials and Methods ADSCs were isolated, by enzymatic digestion, from abdominal adipose tissue of 5 patients undergoing cosmetic lipoaspiration surgery. ADSCs were then seeded onto a 3D scaffold in the presence of the two different osteoinductive matrices of human demineralized bone and evaluated for proliferation and osteogenic differentiation. The safety of the methods was verified using array-Comparative Genomic Hybridization (array-CGH. Results ADSCs were able to differentiate in osteogenic sense. Both DBMs showed the ability to induce osteogenic differentiation of the cells. Conclusion array-CGH showed no changes at genome level, thus confirming the safety of materials and methods.

  3. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells' osteogenic differentiation.

    Science.gov (United States)

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration. PMID:26451103

  4. Transcriptome changes during TNF-α promoted osteogenic differentiation of dental pulp stem cells (DPSCs).

    Science.gov (United States)

    Liu, Ya-Ke; Zhou, Zhen-Yu; Liu, Fan

    2016-08-01

    Dental pulp stem cells (DPSCs), due to the ease of isolation and their capacities of multi-lineage differentiation, are considered as attractive resources for regenerative medicine. In a previous study, we showed that TNF-α promoted the osteogenic differentiation of DPSCs via the NF-κB signaling pathway. However, the mechanisms of such differentiation were largely unknown. Here, we examined the gene expression profiles between undifferentiated, partially differentiated and fully differentiated DPSCs induced by TNF-α by performing the next-generation sequencing technique (RNA-Seq). Our results revealed a continuous transition of the transcriptome changes during TNF-α promoted osteogenic differentiation of DPSC. Bioinformatics analysis revealed a relatively general to specific transformation of the involved signaling pathways from the early to late stages of differentiation. Gene regulatory network analysis highlighted novel, key genes that are essential for osteogenic differentiation at different time points. These results were further validated by quantitative RT-PCR, confirming the high reliability of the RNA-Seq. Our data therefore will not only provide novel insights into the molecular mechanisms that drive the osteogenic differentiation of DPSCs, but also promote the studies of bone tissue engineering that utilizes DPSCs as a crucial resource. PMID:27237976

  5. The size of surface microstructures as an osteogenic factor in calcium phosphate ceramics.

    Science.gov (United States)

    Zhang, Jingwei; Luo, Xiaoman; Barbieri, Davide; Barradas, Ana M C; de Bruijn, Joost D; van Blitterswijk, Clemens A; Yuan, Huipin

    2014-07-01

    The microporosity of calcium phosphate (CaP) ceramics has been shown to have an essential role in osteoinduction by CaP ceramics after ectopic implantation. Here we show that it is not the microporosity but the size of surface microstructural features that is the most likely osteogenic factor. Two tricalcium phosphate (TCP) ceramics, namely TCP-S and TCP-B, were fabricated with equivalent chemistry and similar microporosity but different sizes of surface microstructural features. TCP-S has a grain size of 0.99 ± 0.20 μm and a micropore size of 0.65 ± 0.25 μm, while TCP-B displays a grain size of 3.08 ± 0.52 μm and a micropore size of 1.58 ± 0.65 μm. In vitro, both cell proliferation and osteogenic differentiation were significantly enhanced when human bone marrow stromal cells were cultured on TCP-S without any osteogenic growth factors, compared to TCP-B ceramic granules. The possible involvement of direct contact between cells and the TCP ceramic surface in osteogenic differentiation is also shown with a trans-well culture model. When the ceramic granules were implanted in paraspinal muscle of dogs for 12 weeks, abundant bone was formed in TCP-S (21 ± 10% bone in the available space), whereas no bone was formed in any of the TCP-B implants. The current in vitro and in vivo data reveal that the readily controllable cue, i.e. the size of the surface microstructure, could be sufficient to induce osteogenic differentiation of mesenchymal stem cells, ultimately leading to ectopic bone formation in calcium phosphate ceramics.

  6. Fusion peptide P15-CSP shows antibiofilm activity and pro-osteogenic activity when deposited as a coating on hydrophilic but not hydrophobic surfaces.

    Science.gov (United States)

    Li, Xian; Contreras-Garcia, Angel; LoVetri, Karen; Yakandawala, Nandadeva; Wertheimer, Michael R; De Crescenzo, Gregory; Hoemann, Caroline D

    2015-12-01

    In the context of porous bone void filler for oral bone reconstruction, peptides that suppress microbial growth and promote osteoblast function could be used to enhance the performance of a porous bone void filler. We tested the hypothesis that P15-CSP, a novel fusion peptide containing collagen-mimetic osteogenic peptide P15, and competence-stimulating peptide (CSP), a cationic antimicrobial peptide, has emerging properties not shared by P15 or CSP alone. Peptide-coated surfaces were tested for antimicrobial activity toward Streptoccocus mutans, and their ability to promote human mesenchymal stem cell (MSC) attachment, spreading, metabolism, and osteogenesis. In the osteogenesis assay, peptides were coated on tissue culture plastic and on thin films generated by plasma-enhanced chemical vapor deposition to have hydrophilic or hydrophobic character (water contact angles 63°, 42°, and 92°, respectively). S. mutans planktonic growth was specifically inhibited by CSP, whereas biofilm formation was inhibited by P15-CSP. MSC adhesion and actin stress fiber formation was strongly enhanced by CSP, P15-CSP, and fibronectin coatings and modestly enhanced by P15 versus uncoated surfaces. Metabolic assays revealed that CSP was slightly cytotoxic to MSCs. MSCs developed alkaline phosphatase activity on all surfaces, with or without peptide coatings, and consistently deposited the most biomineralized matrix on hydrophilic surfaces coated with P15-CSP. Hydrophobic thin films completely suppressed MSC biomineralization, consistent with previous findings of suppressed osteogenesis on hydrophobic bioplastics. Collective data in this study provide new evidence that P15-CSP has unique dual capacity to suppress biofilm formation, and to enhance osteogenic activity as a coating on hydrophilic surfaces.

  7. Fusion peptide P15-CSP shows antibiofilm activity and pro-osteogenic activity when deposited as a coating on hydrophilic but not hydrophobic surfaces.

    Science.gov (United States)

    Li, Xian; Contreras-Garcia, Angel; LoVetri, Karen; Yakandawala, Nandadeva; Wertheimer, Michael R; De Crescenzo, Gregory; Hoemann, Caroline D

    2015-12-01

    In the context of porous bone void filler for oral bone reconstruction, peptides that suppress microbial growth and promote osteoblast function could be used to enhance the performance of a porous bone void filler. We tested the hypothesis that P15-CSP, a novel fusion peptide containing collagen-mimetic osteogenic peptide P15, and competence-stimulating peptide (CSP), a cationic antimicrobial peptide, has emerging properties not shared by P15 or CSP alone. Peptide-coated surfaces were tested for antimicrobial activity toward Streptoccocus mutans, and their ability to promote human mesenchymal stem cell (MSC) attachment, spreading, metabolism, and osteogenesis. In the osteogenesis assay, peptides were coated on tissue culture plastic and on thin films generated by plasma-enhanced chemical vapor deposition to have hydrophilic or hydrophobic character (water contact angles 63°, 42°, and 92°, respectively). S. mutans planktonic growth was specifically inhibited by CSP, whereas biofilm formation was inhibited by P15-CSP. MSC adhesion and actin stress fiber formation was strongly enhanced by CSP, P15-CSP, and fibronectin coatings and modestly enhanced by P15 versus uncoated surfaces. Metabolic assays revealed that CSP was slightly cytotoxic to MSCs. MSCs developed alkaline phosphatase activity on all surfaces, with or without peptide coatings, and consistently deposited the most biomineralized matrix on hydrophilic surfaces coated with P15-CSP. Hydrophobic thin films completely suppressed MSC biomineralization, consistent with previous findings of suppressed osteogenesis on hydrophobic bioplastics. Collective data in this study provide new evidence that P15-CSP has unique dual capacity to suppress biofilm formation, and to enhance osteogenic activity as a coating on hydrophilic surfaces. PMID:26097095

  8. Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: ccpperio@snu.ac.kr [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a

  9. Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

    Directory of Open Access Journals (Sweden)

    Jansen Justus HW

    2010-08-01

    Full Text Available Abstract Background Although pulsed electromagnetic field (PEMF stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation. Methods BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition, expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p Results Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control. Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation

  10. Osteogenic potential of bone marrow stromal cells on smooth, roughened, and tricalcium phosphate-modified titanium alloy surfaces.

    LENUS (Irish Health Repository)

    Colombo, John S

    2012-09-01

    This study investigated the influence of smooth, roughened, and tricalcium phosphate (TCP)-coated roughened titanium-aluminum-vanadium (Ti-6Al-4V) surfaces on the osteogenic potential of rat bone marrow stromal cells (BMSCs).

  11. Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

    OpenAIRE

    Endres, M; Hutmacher, D.W.; Salgado, A. J.; Kaps, C; RINGE, J; Reis, R. L.; Sittinger, M; Brandwood, A.; Schantz, J. T.

    2003-01-01

    The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated...

  12. Precursor cells from Atlantic salmon (Salmo salar visceral fat holds the plasticity to differentiate into the osteogenic lineage

    Directory of Open Access Journals (Sweden)

    Elisabeth Ytteborg

    2015-07-01

    Full Text Available In order to study the potential plasticity of Atlantic salmon (Salmo salar precursor cells (aSPCs from the adipogenic mesenchyme cell lineage to differentiate to the osteogenic lineage, aSPCs were isolated and cultivated under either osteogenic or adipogenic promoting conditions. The results strengthen the hypothesis that aSPCs most likely are predestined to the adipogenic lineage, but they also hold the flexibility to turn into other lineages given the right stimuli. This assumption is supported by the fact that the transcription factor pparγ , important for regulation of adiopogenesis, was silent in aSPCs grown in osteogenic media, while runx2, important for osteogenic differentiation, was not expressed in aSPCs cultivated in adipogenic media. After 2 weeks in osteogenic promoting conditions the cells started to deposit extracellular matrix and after 4 weeks, the cells started mineralizing secreted matrix. Microarray analyses revealed large-scale transcriptome responses to osteogenic medium after 2 days, changes remained stable at day 15 and decreased by magnitude at day 30. Induction was observed in many genes involved in osteogenic differentiation, growth factors, regulators of development, transporters and production of extracellular matrix. Transcriptome profile in differentiating adipocytes was markedly different from differentiating osteoblasts with far fewer genes changing activity. The number of regulated genes slowly increased at the mature stage, when adipocytes increased in size and accumulated lipids. This is the first report on in vitro differentiation of aSPCs from Atlantic salmon to mineralizing osteogenic cells. This cell model system provides a new valuable tool for studying osteoblastogenesis in fish.

  13. Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate

    Institute of Scientific and Technical Information of China (English)

    Jyun-Yi Wu; Chia-Hsin Chen; Li-Yin Yeh; Ming-Long Yeh; Chun-Chan Ting; Yan-Hsiung Wang

    2013-01-01

    Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J?cm22. Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J?cm22 significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J?cm22 showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.

  14. The NAD-Dependent Deacetylase Sirtuin-1 Regulates the Expression of Osteogenic Transcriptional Activator Runt-Related Transcription Factor 2 (Runx2 and Production of Matrix Metalloproteinase (MMP-13 in Chondrocytes in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Koh Terauchi

    2016-06-01

    Full Text Available Aging is one of the major pathologic factors associated with osteoarthritis (OA. Recently, numerous reports have demonstrated the impact of sirtuin-1 (Sirt1, which is the NAD-dependent deacetylase, on human aging. It has been demonstrated that Sirt1 induces osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the role of Sirt1 in the OA chondrocytes still remains unknown. We postulated that Sirt1 regulates a hypertrophic chondrocyte lineage and degeneration of articular cartilage through the activation of osteogenic transcriptional activator Runx2 and matrix metalloproteinase (MMP-13 in OA chondrocytes. To verify whether sirtuin-1 (Sirt1 regulates chondrocyte activity in OA, we studied expressions of Sirt1, Runx2 and production of MMP-13, and their associations in human OA chondrocytes. The expression of Sirt1 was ubiquitously observed in osteoarthritic chondrocytes; in contrast, Runx2 expressed in the osteophyte region in patients with OA and OA model mice. OA relating catabolic factor IL-1βincreased the expression of Runx2 in OA chondrocytes. OA chondrocytes, which were pretreated with Sirt1 inhibitor, inhibited the IL-1β-induced expression of Runx2 compared to the control. Since the Runx2 is a promotor of MMP-13 expression, Sirt1 inactivation may inhibit the Runx2 expression and the resultant down-regulation of MMP-13 production in chondrocytes. Our findings suggest thatSirt1 may regulate the expression of Runx2, which is the osteogenic transcription factor, and the production of MMP-13 from chondrocytes in OA. Since Sirt1 activity is known to be affected by several stresses, including inflammation and oxidative stress, as well as aging, SIRT may be involved in the development of OA.

  15. The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells.

    Science.gov (United States)

    Kwist, K; Bridges, W C; Burg, K J L

    2016-08-01

    Cell line passage number is an important consideration when designing an experiment. At higher passages, it is generally understood that cell health begins to decline and, when this occurs, the result can be variable data. However, there are no specific guidelines regarding optimal passage range, and this information is dependent on cell type. To explore these variabilities, low passage D1 cells were thawed (passage 3) and passaged serially until a much higher number (passage 34). Samples were taken every five passages and analyzed for alkaline phosphatase and triglyceride; also, the gene expression of both adipogenic and osteogenic markers was tested. The results indicate that the growth rate of these cells did slow down after passage 30. However, expression of the osteogenic characteristics seemed to cycle, with the highest levels seen at passage 4 and 24. The adipocyte expression levels remained the same throughout the study. PMID:26208915

  16. Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity

    DEFF Research Database (Denmark)

    Kraft, David C E; Bindslev, Dorthe A; Melsen, Birte;

    2010-01-01

    For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem...... cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC...... expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs...

  17. The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells.

    Science.gov (United States)

    Kwist, K; Bridges, W C; Burg, K J L

    2016-08-01

    Cell line passage number is an important consideration when designing an experiment. At higher passages, it is generally understood that cell health begins to decline and, when this occurs, the result can be variable data. However, there are no specific guidelines regarding optimal passage range, and this information is dependent on cell type. To explore these variabilities, low passage D1 cells were thawed (passage 3) and passaged serially until a much higher number (passage 34). Samples were taken every five passages and analyzed for alkaline phosphatase and triglyceride; also, the gene expression of both adipogenic and osteogenic markers was tested. The results indicate that the growth rate of these cells did slow down after passage 30. However, expression of the osteogenic characteristics seemed to cycle, with the highest levels seen at passage 4 and 24. The adipocyte expression levels remained the same throughout the study.

  18. Notch signaling stimulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LU Zhuozhuang; WU Zuze(WU Chutse); ZHANG Qunwei; WANG Hua; JIA Xiangxu; DUAN Haifeng; WANG Lisheng

    2004-01-01

    Notch signaling is one of the most important pathways mediating cell determination and differentiation. In this study, the roles of Notch signal in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were investigated. The expression of Notch1, Jagged1 and DTX1 detected by reverse transcription polymerase chain reaction (RT-PCR) suggested that Notch signal might exhibit a physiological regulatory role in the differentiation of MSCs. Constitutive expression of the intracellular domain of Notch1 (ICN), the active form of Notch1 protein, can activate Notch signal in cells without ligands' binding. hMSCs were isolated, expanded, and infected with retrovirus carrying green fluorescent protein (GFP) gene or ICN. Overexpression of ICN in hMSCs resulted in enhanced osteogenic differentiation induced by dexamethasone (Dex), which was characterized by an increase of cellular alkaline phosphatase (ALP) activity and calcium deposition. These results indicate that Notch stimulates differentiation of MSCs into osteoblasts.

  19. Osteogenic commitment of mesenchymal stem cells in apatite nanorod-aligned ceramics.

    Science.gov (United States)

    Chen, Ying; Sun, Zhihui; Li, Yanyan; Hong, Youliang

    2014-12-24

    It is significant to process the clinically used biomaterials into a scaffold with specific nanotopographies, which can act as physical cues to regulate the osteogenic commitment of mesenchymal stem cells. In this study, hydroxyapatite (HAP) was considered as the processed objective and a facile, hydrothermal method was developed to grow the vertically oriented HAP nanorods in porous HAP ceramics. Experiments demonstrated that the formation of the HAP nanorods in porous ceramics was decided by a novel epitaxial growth mechanism and length of nanorods could be well-controlled by the growth time. Cell experiments demonstrated that such novel stereotopographical cues could regulate bone marrow mesenchymal stem cells to differentiate into the osteogenic lineage, thereby displaying that the porous ceramics with the HAP nanorods-aligned stereotopographies have a good prospect for applications in regenerative medicine of hard tissues.

  20. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Ikbale El-Ayachi

    2016-03-01

    Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

  1. Morinda citrifolia leaves enhance osteogenic differentiation and mineralization of human periodontal ligament cells.

    Science.gov (United States)

    Boonanantanasarn, Kanitsak; Janebodin, Kajohnkiart; Suppakpatana, Prapan; Arayapisit, Tawepong; Rodsutthi, Jit-aree; Chunhabundit, Panjit; Boonanuntanasarn, Surintorn; Sripairojthikoon, Wanida

    2014-01-01

    This present study investigated the potential of Morinda citrifolia leaf aqueous extract to induce osteogenic differentiation and matrix mineralization of human periodontal ligament (hPDL) cells. Human periodontal ligament cells were cultured in complete medium, ascorbic acid with β-glycerophosphate, or Morinda citrifolia leaf aqueous extract. Morinda citrifolia leaf aqueous extract significantly increased alkaline phosphatase activity compared to culturing in complete medium or ascorbic acid with β-glycerophosphate. Matrixcontaining mineralized nodules were formed only when the cells were cultured in the presence of Morinda citrifolia leaf aqueous extract. These nodules showed positive alizarin red S staining and were rich in calcium and phosphorus according to energy dispersive X-ray analysis. In conclusion, Morinda citrifolia leaf extract promoted osteogenic differentiation and matrix mineralization in human periodontal ligament cells, a clear indication of the therapeutic potential of Morinda citrifolia leaves in bone and periodontal tissue regeneration.

  2. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.

    Science.gov (United States)

    Clough, Bret H; McCarley, Matthew R; Krause, Ulf; Zeitouni, Suzanne; Froese, Jeremiah J; McNeill, Eoin P; Chaput, Christopher D; Sampson, H Wayne; Gregory, Carl A

    2015-01-01

    Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.

  3. Regulating the osteogenic function of rhBMP 2 by different titanium surface properties.

    Science.gov (United States)

    Xiao, Ming; Biao, Meina; Chen, Yangmei; Xie, Meiju; Yang, Bangcheng

    2016-08-01

    Bone morphogenetic protein 2 (BMP-2) is important for regulating the osteogenic differentiation of mesenchymal stem cells and the response of bone tissue. It adsorbs on the surface of biomedical implants immediately and plays a role of mediator between the materials surfaces and the host cells. Studies usually connect the material surface properties and the new bone formation directly. However, interaction between the adsorbed BMP-2 on the implant surface and the cells in the tissue is the key to explaining the osteogenic properties of the material. So, in this article, we investigated the conformational and functional changes induced by the surface modified titanium metals. We found that the α-helix and β-sheet structure of rhBMP-2 can be well maintained on the anodic oxidation treated titanium surface. The osteogenic function of rhBMP-2 can sustain for a relatively long time even though there is less amount adhere to the surface compared with that on the acid alkali treated titanium. Surface properties, especially the morphology enable a larger amount of rhBMP-2 to adsorb to the surface of the acid alkali treated titanium, but the conformation of the protein is severely influenced. The percentage of α-helix structure is also significantly decreased so that the efficacy of rhBMP-2 is only maintained in the early time. This study indicated that different surface modification of the surface could regulate the structure of rhBMP-2 and then further influence its osteogenic function. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1882-1893, 2016. PMID:26991341

  4. Periodontally accelerated osteogenic orthodontics: An interdisciplinary approach for faster orthodontic therapy

    OpenAIRE

    Srikanth Adusumilli; Lohith Yalamanchi; Pallavi Samatha Yalamanchili

    2014-01-01

    Periodontally accelerated osteogenic orthodontics is a relatively new procedure designed to minimize the time taken for orthodontic treatment. The orthodontist avails of the aid of a periodontist to perform decortication of the bone and places bone graft for rapid orthodontic correction of malocclusion. A PubMed search was carried out to identify papers describing the procedure, and the data were organized in the current format.

  5. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  6. Differences in Valvular and Vascular Cell Responses to Strain in Osteogenic Media

    OpenAIRE

    Zannatul, Ferdous; Hanjoong, Jo; Robert M., Nerem

    2011-01-01

    Calcification is the primary cause of failure of bioprosthetic and tissue-engineered vascular and valvular grafts. We used tissue-engineered collagen gels containing human aortic smooth muscle cells (HASMC) and human aortic valvular interstitial cells (HAVIC) as a model to investigate cell-mediated differences in early markers of calcification. The HASMCs and HAVICs were isolated from non-sclerotic human tissues. After 21 days of culture in either regular or osteogenic media with or without 1...

  7. Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Zuo-lin JIN; Yong-kuan ZHANG; Hai-yan SUN; Zhu LIN; Ying-chun BI; Yin-zhong DUAN; Yin DING

    2008-01-01

    Aim:Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or os-teogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. Methods: Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. Results: Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregn-lated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-β superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. Conclusion: Our results demonstrated that hDFC dis-played osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.

  8. Asiaticoside induces type I collagen synthesis and osteogenic differentiation in human periodontal ligament cells.

    Science.gov (United States)

    Nowwarote, Nunthawan; Osathanon, Thanaphum; Jitjaturunt, Peachaya; Manopattanasoontorn, Sukuman; Pavasant, Prasit

    2013-03-01

    Asiaticoside, an active ingredient extracted from Centella asiatica, has been widely used to promote wound healing. In this study, the effects of asiaticoside on proliferation, protein synthesis, and osteogenic differentiation in human periodontal ligament cells (HPDLs) were investigated. HPDLs were treated with asiaticoside at concentrations of 25, 50, and 100 µg/mL. Cell number was determined by MTT assay. The mRNA expression was analyzed by reverse transcription-polymerase chain reaction. Western blot analysis and immunocytochemistry were used to confirm protein synthesis. Osteogenic differentiation was determined by alkaline phosphatase activity, osteoblast marker gene expression, and in vitro mineralization. The results showed that asiaticoside treatment, ranging from 25 to 100 mg/mL, had no effect on cytotoxicity or cell proliferation. When HPDLs were treated with asiaticoside in serum-free medium, dose-dependent increases in the levels of fibronectin and collagen type I mRNA and protein were observed at 72 h. Moreover, asiaticoside attenuated matrix metalloproteinase-1 but enhanced tissue inhibitor of metalloproteinase-1 mRNA expression. The addition of asiaticoside to osteogenic medium resulted in an increase in alkaline phosphatase enzymatic activity, up-regulation of osteoblast marker gene mRNA expression, and enhancement of mineralization by HPDLs. These results suggest the potential application of asiaticoside for enhancing periodontal tissue healing.

  9. 3D chitosan-gelatin-chondroitin porous scaffold improves osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Machado, C B [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Ventura, J M G [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Lemos, A F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Ferreira, J M F [Department of Ceramics and Glass Engineering, University of Aveiro (Portugal); Leite, M F [Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil); Goes, A M [Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (Brazil)

    2007-06-01

    A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

  10. Surface thermal oxidation on titanium implants to enhance osteogenic activity and in vivo osseointegration

    Science.gov (United States)

    Wang, Guifang; Li, Jinhua; Lv, Kaige; Zhang, Wenjie; Ding, Xun; Yang, Guangzheng; Liu, Xuanyong; Jiang, Xinquan

    2016-08-01

    Thermal oxidation, which serves as a low-cost, effective and relatively simple/facile method, was used to modify a micro-structured titanium surface in ambient atmosphere at 450 °C for different time periods to improve in vitro and in vivo bioactivity. The surface morphology, crystallinity of the surface layers, chemical composition and chemical states were evaluated by field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Cell behaviours including cell adhesion, attachment, proliferation, and osteogenic differentiation were observed in vitro study. The ability of the titanium surface to promote osseointegration was evaluated in an in vivo animal model. Surface thermal oxidation on titanium implants maintained the microstructure and, thus, both slightly changed the nanoscale structure of titanium and enhanced the crystallinity of the titanium surface layer. Cells cultured on the three oxidized titanium surfaces grew well and exhibited better osteogenic activity than did the control samples. The in vivo bone-implant contact also showed enhanced osseointegration after several hours of oxidization. This heat-treated titanium enhanced the osteogenic differentiation activity of rBMMSCs and improved osseointegration in vivo, suggesting that surface thermal oxidation could potentially be used in clinical applications to improve bone-implant integration.

  11. Autocrine fibroblast growth factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells.

    Science.gov (United States)

    Hamidouche, Zahia; Fromigué, Olivia; Nuber, Ulrike; Vaudin, Pascal; Pages, Jean-Christophe; Ebert, Regina; Jakob, Franz; Miraoui, Hichem; Marie, Pierre J

    2010-08-01

    The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV-FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV-FGF18 is mediated by activation of ERK1/2-MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2-mediated ERK1/2-MAPKs and PI3K signaling. PMID:20432451

  12. Gadolinium contrast agent-induced CD163+ ferroportin+ osteogenic cells in nephrogenic systemic fibrosis.

    Science.gov (United States)

    Swaminathan, Sundararaman; Bose, Chhanda; Shah, Sudhir V; Hall, Kimberly A; Hiatt, Kim M

    2013-09-01

    Gadolinium-based contrast agents are linked to nephrogenic systemic fibrosis in patients with renal insufficiency. The pathology of nephrogenic systemic fibrosis is characterized by abnormal tissue repair: fibrosis and ectopic ossification. The mechanisms by which gadolinium could induce fibrosis and ossification are not known. We examined in vitro the effect of a gadolinium-based contrast agent on human peripheral blood mononuclear cells for phenotype and function relevant to the pathology of nephrogenic systemic fibrosis using immunofluorescence, flow cytometry, real-time PCR, and osteogenic assays. We also examined tissues from patients with nephrogenic systemic fibrosis, using IHC to identify the presence of cells with phenotype induced by gadolinium. Gadolinium contrast induced differentiation of human peripheral blood mononuclear cells into a unique cellular phenotype--CD163(+) cells expressing proteins involved in fibrosis and bone formation. These cells express fibroblast growth factor (FGF)23, osteoblast transcription factors Runt-related transcription factor 2, and osterix, and show an osteogenic phenotype in in vitro assays. We show in vivo the presence of CD163(+)/procollagen-1(+)/osteocalcin(+) cells in the fibrotic and calcified tissues of nephrogenic systemic fibrosis patients. Gadolinium contrast-induced CD163(+)/ferroportin(+)/FGF23(+) cells with osteogenic potential may play a role in systemic fibrosis and ectopic ossification in nephrogenic systemic fibrosis.

  13. The Effects of High Glucose on Adipogenic and Osteogenic Differentiation of Gestational Tissue-Derived MSCs

    Directory of Open Access Journals (Sweden)

    Weerawan Hankamolsiri

    2016-01-01

    Full Text Available Most type 2 diabetic patients are obese who have increased number of visceral adipocytes. Those visceral adipocytes release several factors that enhance insulin resistance making diabetic treatment ineffective. It is known that significant percentages of visceral adipocytes are derived from mesenchymal stem cells and high glucose enhances adipogenic differentiation of mouse bone marrow-derived MSCs (BM-MSCs. However, the effect of high glucose on adipogenic differentiation of human bone marrow and gestational tissue-derived MSCs is still poorly characterized. This study aims to investigate the effects of high glucose on proliferation as well as adipogenic and osteogenic differentiation of human MSCs derived from bone marrow and several gestational tissues including chorion, placenta, and umbilical cord. We found that high glucose reduced proliferation but enhanced adipogenic differentiation of all MSCs examined. The expression levels of some adipogenic genes were also upregulated when MSCs were cultured in high glucose. Although high glucose transiently downregulated the expression levels of some osteogenic genes examined, its effect on the osteogenic differentiation levels of the MSCs is not clearly demonstrated. The knowledge gained from this study will increase our understanding about the effect of high glucose on adipogenic differentiation of MSCs and might lead to an improvement in the diabetic treatment in the future.

  14. [Bone and Stem Cells. The mechanism of osteogenic differentiation from mesenchymal stem cell].

    Science.gov (United States)

    Ohata, Yasuhisa; Ozono, Keiichi

    2014-04-01

    Osteoblasts and osteocytes originate from pluripotent mesenchymal stem cells. Mesenchymal stem cells commit to osteogenic lineage and differentiate into mature osteoblasts and osteocytes through osteoprogenitor cells and preosteoblasts in response to multiple stimuli. The osteoblast commitment, differentiation, and functions are governed by several transcription factors. Among these transcription factors, runt-related transcription factor 2 (Runx2) is a crucial factor in osteoblast differentiation and controls bone formation. Differentiation toward these osteogenic lineage is controlled by a multitude of cytokines including WNTs, bone morphogenetic protein (BMP) , transforming growth factor-β (TGF-β) , hedgehog, parathyroid hormone (PTH) /parathyroid hormone related protein (PTHrP) , insulin-like growth factor-1 (IGF-1) , fibroblast growth factor (FGF) , and Notch. Although regulation of Runx2 activity is a point of convergence of many of the signal transduction routes, there is also a high degree of cross-talk between these pathways. Thus, the combined action of the signal transduction pathways induced by some cytokines determines the commitment and differentiation of mesenchymal stem cells toward the osteogenic lineage. PMID:24681495

  15. Crucial role of Notch signaling in osteogenic differentiation of periodontal ligament stem cells in osteoporotic rats.

    Science.gov (United States)

    Li, Ying; Li, S Q; Gao, Y M; Li, Jin; Zhang, Bin

    2014-06-01

    Estrogen deficiency-induced osteoporosis typically occurs in postmenopausal women and has been strongly associated with periodontal diseases. Periodontal ligament stem cells (PDLSCs) isolated from the periodontal ligament can differentiate into many types of specialized cells, including osteoblast-like cells that contribute to periodontal tissue repair. The Notch signaling pathway is highly conserved and associated with self-renewal potential and cell-fate determination. Recently, several studies have focused on the relationship between Notch signaling and osteogenic differentiation. However, the precise mechanisms underlying this relationship are largely unknown. We have successfully isolated PDLSCs from both ovariectomized (OVX) and sham-operated rats. Both the mRNA and protein levels of Notch1 and Jagged1 were upregulated when PDLSCs were cultured in osteogenic induction media. Mineralization assays showed decreased calcium deposits in OVX-PDLSCs treated with a γ-secretase inhibitor compared with control cells. Thus Notch signaling is important in maintaining the osteogenic differentiation of PDLSCs in osteoporotic rats, which help in the development of a potential therapeutic strategy for periodontal disease in postmenopausal women.

  16. Human mesenchymal stromal cell-enhanced osteogenic differentiation by contact interaction with polyethylene terephthalate nanogratings.

    Science.gov (United States)

    Antonini, Sara; Meucci, Sandro; Parchi, Paolo; Pacini, Simone; Montali, Marina; Poggetti, Andrea; Lisanti, Michele; Cecchini, Marco

    2016-01-01

    Among the very large number of polymeric materials that have been proposed in the field of orthopedics, polyethylene terephthalate (PET) is one of the most attractive thanks to its flexibility, thermal resistance, mechanical strength and durability. Several studies have been proposed that interface nano- or micro-structured surfaces with mesenchymal stromal cells (MSCs), demonstrating the potential of this technology for promoting osteogenesis. All these studies were carried out on biomaterials other than PET, which remains almost uninvestigated in terms of cell shaping, alignment and differentiation. Here, we study the effect of PET 350-depth nanogratings (NGs) with a ridge and lateral groove size of 500 nm (T1) or 1 μm (T2), on bone marrow-derived human MSC (hMSC) differentiation in relation to the osteogenic fate. We demonstrate that these substrates, especially T2, can promote the osteogenic phenotype more efficiently than standard flat surfaces and that this effect is more marked if cells are cultured in osteogenic medium than in basal medium. Finally, we show that the shape and disposition of calcium hydroxyapatite granules on the different substrates was influenced by the substrate symmetry, being more elongated and spatially organized on NGs than on flat surfaces. PMID:27388559

  17. Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Han-Tsung; Liao; Chien-Tzung; Chen

    2014-01-01

    Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its’ clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in

  18. Enhancement of osteogenic differentiation and proliferation in human mesenchymal stem cells by a modified low intensity ultrasound stimulation under simulated microgravity.

    Directory of Open Access Journals (Sweden)

    Sardar M Z Uddin

    Full Text Available Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs. Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG and treated with LIPUS at 30mW/cm(2 for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01 and ALP activity by 22% (p<0.01, while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001 and mineralization by 45% (p<0.001 in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation.

  19. Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

    International Nuclear Information System (INIS)

    Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but

  20. Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Salamon, Achim, E-mail: achim.salamon@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Jonitz-Heincke, Anika, E-mail: anika.jonitz@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Adam, Stefanie, E-mail: stefanie.adam@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Rychly, Joachim, E-mail: joachim.rychly@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Müller-Hilke, Brigitte, E-mail: brigitte.mueller-hilke@med.uni-rostock.de [Institute of Immunology, Rostock University Medical Center, Schillingallee 68, D-18057 Rostock (Germany); Bader, Rainer, E-mail: rainer.bader@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Lochner, Katrin, E-mail: katrin.lochner@med.uni-rostock.de [Biomechanics and Implant Technology Research Laboratory, Department of Orthopedics, Rostock University Medical Center, Doberaner Straße 142, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients. - Highlights: • We analyze the mesenchymal differentiation capacity of cartilage-derived cells (CDC). • CDC express mesenchymal stem cell (MSC) markers CD29, CD44, CD105, and CD166. • CDC and MSC proliferation is reduced in adipogenesis and increased in osteogenesis. • Adipogenic differentiation is virtually absent in CDC, but

  1. Biomimetic hybrid nanofibrous substrates for mesenchymal stem cells differentiation into osteogenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Gandhimathi, Chinnasamy [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Venugopal, Jayarama Reddy [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Tham, Allister Yingwei [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore); Ramakrishna, Seeram [Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, National University of Singapore (Singapore); Kumar, Srinivasan Dinesh, E-mail: dineshkumar@ntu.edu.sg [Cellular and Molecular Epigenetics Lab, Lee Kong Chian School of Medicine, Nanyang Technological University (Singapore)

    2015-04-01

    Mimicking native extracellular matrix with electrospun porous bio-composite nanofibrous scaffolds has huge potential in bone tissue regeneration. The aim of this study is to fabricate porous poly(L-lactic acid)-co-poly-(ε-caprolactone)/silk fibroin/ascorbic acid/tetracycline hydrochloride (PLACL/SF/AA/TC) and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). Fabricated nanofibrous scaffolds were characterized for fiber morphology, hydrophilicity, porosity, mechanical test and chemical properties by FT-IR and EDX analysis. The results showed that the fiber diameter and pore size of scaffolds observed around 228 ± 62–320 ± 22 nm and 1.5–6.9 μm respectively. Resulting nanofibrous scaffolds are highly porous (87–94%) with ultimate tensile strength observed in the range of 1.51–4.86 MPa and also showed better hydrophilic properties after addition of AA, TC and n-HA. Human mesenchymal stem cells (MSCs) cultured on these bio-composite nanofibrous scaffolds and stimulated to osteogenic differentiation in the presence of AA/TC/n-HA for BTE. The cell proliferation and biomaterial interactions were studied using MTS assay, SEM and CMFDA dye exclusion methods. Osteogenic differentiation of MSCs was proven by using alkaline phosphatase activity, mineralization and double immunofluorescence staining of both CD90 and osteocalcin. The observed results suggested that the fabricated PLACL/SF/AA/TC/n-HA biocomposite hybrid nanofibrous scaffolds have good potential for the differentiation of MSCs into osteogenesis for bone tissue engineering. - Highlights: • We fabricated and characterized hybrid porous nanofibrous scaffolds. • PLACL/SF/AA/TC/n-HA scaffolds promote cell differentiation and mineralization. • Porous nanofibrous scaffolds initiate MSC differentiation into osteogenic cells. • Biomimetic nanofibrous scaffolds have good potential for bone tissue engineering.

  2. Antimicrobial and osteogenic effect of Ag-implanted titanium with a nanostructured surface

    Directory of Open Access Journals (Sweden)

    Zheng Y

    2012-02-01

    Full Text Available Yanhua Zheng1, Jinbo Li2, Xuanyong Liu2, Jiao Sun11Shanghai Biomaterials Research and Testing Center, Shanghai Key Laboratory of Stomatology, Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, People’s Republic of China; 2State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of ChinaAbstract: Ag-implanted titanium with a nanostructured surface was prepared by hydrothermal treatment with H2O2 followed by Ag plasma immersion ion implantation. Streptococcus mutans, Porphyromonas gingivalis and Candida albicans were chosen for antimicrobial tests. Genes related to microbial structure or adhesion, namely glucan-binding proteins B (GbpB, fimbria protein A (FimA, and agglutinin-like sequence4 (Als4, were examined. The osteoblast’s attachment, viability, and quantitative analysis of osteogenic gene expression (Alp, Ocn, RunX2 on titanium surfaces were evaluated. Scanning electron microscopy (SEM revealed that Ag nanoparticles of approximately 10 nm were incorporated on the nanostructured surface of titanium after Ag plasma immersion ion implantation. Trials showed that 93.99% of S. mutans, 93.57% of P. g, and 89.78% of C. albicans were killed on the Ag-implanted titanium with a nanostructured surface. Gene expressions from the three microorganisms confirmed the antimicrobial activities of the Ag-implanted titanium with a nanostructured surface. Furthermore, the adhesive images and viability assays indicated that the Ag-implanted titanium with a nanostructured surface did not impair osteoblasts. The expressions of osteoblast phenotype genes in cells grown on the Ag-implanted titanium surface were significantly increased. The results of this study suggest that the Ag-implanted titanium with a nanostructured surface displays good antimicrobial properties, reducing gene expressions of

  3. Controlling Growth and Osteogenic Differentiation of Osteoblasts on Microgrooved Polystyrene Surfaces.

    Science.gov (United States)

    Sun, Lanying; Pereira, Daniel; Wang, Qibao; Barata, David Baião; Truckenmüller, Roman; Li, Zhaoyuan; Xu, Xin; Habibovic, Pamela

    2016-01-01

    Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates.

  4. Controlling Growth and Osteogenic Differentiation of Osteoblasts on Microgrooved Polystyrene Surfaces

    Science.gov (United States)

    Sun, Lanying; Pereira, Daniel; Wang, Qibao; Barata, David Baião; Truckenmüller, Roman; Li, Zhaoyuan; Xu, Xin; Habibovic, Pamela

    2016-01-01

    Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates. PMID:27571520

  5. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    Science.gov (United States)

    Wang, Fangfang; Okawa, Hiroko; Kamano, Yuya; Niibe, Kunimichi; Kayashima, Hiroki; Osathanon, Thanaphum; Pavasant, Prasit; Saeki, Makio; Yatani, Hirofumi; Egusa, Hiroshi

    2015-01-01

    Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation

  6. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    Directory of Open Access Journals (Sweden)

    Fangfang Wang

    Full Text Available Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR. Expression vectors that contained the Tet operator and amelogenin-coding (Amelx cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx. MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP, osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional

  7. Utility of tricalcium phosphate and osteogenic matrix cellsheet constructs for bone defect reconstruction

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    AIM To determine the effects of transplanting osteogenicmatrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were preparedfrom bone marrow stromal cells (BMSCs), anda porous TCP ceramic was used as a scaffold. Threeexperimental groups were prepared, comprised of TCPscaffolds (1) seeded with BMSCs; (2) wrapped withosteogenic matrix cell sheets; or (3) both. Constructswere implanted into a femoral defect model in rats andbone growth was evaluated by radiography, histology,biochemistry, and mechanical testing after 8 wk.RESULTS: In bone defects, constructs implanted withcell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast toTCP seeded with BMSCs, which resulted in bone nonunion.Wrapping TCP constructs with osteogenic matrixcell sheets increased their osteogenic potential andresulting bone formation, compared with conventionalbone tissue engineering TCP scaffolds seeded withBMSCs. The compressive stiffness (mean ± SD) valueswere 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6MPa for BMSC/TCP/Sheet constructs with continuousbone formation, BMSC/TCP/Sheet constructs withsegmental bone formation, and BMSC/TCP constructs,respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation wassignificantly higher than those with segmental boneformation and BMSC/TCP constructs.CONCLUSION: This technique is an improvementover current methods, such as TCP substitution, andis useful for hard tissue reconstruction and inducingearlier bone union in defects.

  8. Biomimetic hybrid nanofibrous substrates for mesenchymal stem cells differentiation into osteogenic cells

    International Nuclear Information System (INIS)

    Mimicking native extracellular matrix with electrospun porous bio-composite nanofibrous scaffolds has huge potential in bone tissue regeneration. The aim of this study is to fabricate porous poly(L-lactic acid)-co-poly-(ε-caprolactone)/silk fibroin/ascorbic acid/tetracycline hydrochloride (PLACL/SF/AA/TC) and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). Fabricated nanofibrous scaffolds were characterized for fiber morphology, hydrophilicity, porosity, mechanical test and chemical properties by FT-IR and EDX analysis. The results showed that the fiber diameter and pore size of scaffolds observed around 228 ± 62–320 ± 22 nm and 1.5–6.9 μm respectively. Resulting nanofibrous scaffolds are highly porous (87–94%) with ultimate tensile strength observed in the range of 1.51–4.86 MPa and also showed better hydrophilic properties after addition of AA, TC and n-HA. Human mesenchymal stem cells (MSCs) cultured on these bio-composite nanofibrous scaffolds and stimulated to osteogenic differentiation in the presence of AA/TC/n-HA for BTE. The cell proliferation and biomaterial interactions were studied using MTS assay, SEM and CMFDA dye exclusion methods. Osteogenic differentiation of MSCs was proven by using alkaline phosphatase activity, mineralization and double immunofluorescence staining of both CD90 and osteocalcin. The observed results suggested that the fabricated PLACL/SF/AA/TC/n-HA biocomposite hybrid nanofibrous scaffolds have good potential for the differentiation of MSCs into osteogenesis for bone tissue engineering. - Highlights: • We fabricated and characterized hybrid porous nanofibrous scaffolds. • PLACL/SF/AA/TC/n-HA scaffolds promote cell differentiation and mineralization. • Porous nanofibrous scaffolds initiate MSC differentiation into osteogenic cells. • Biomimetic nanofibrous scaffolds have good potential for bone tissue engineering

  9. Sox9 Modulates Proliferation and Expression of Osteogenic Markers of Adipose-Derived Stem Cells (ASC

    Directory of Open Access Journals (Sweden)

    Sabine Stöckl

    2013-05-01

    Full Text Available Background: Mesenchymal stem cells (MSC are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC physiology in order to make them a safe tool for in vivo application. Methods/Results: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC. In comparison to rat bone marrow-derived stem cells (rBMSC, mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. Conclusions: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.

  10. Hydroxyapatite nanoparticle injectable hydrogel scaffold to support osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Thorpe, A A; Creasey, S; Sammon, C; Le Maitre, C L

    2016-01-01

    Bone loss associated with degenerative disease and trauma is a clinical problem increasing with the aging population. Thus, effective bone augmentation strategies are required; however, many have the disadvantages that they require invasive surgery and often the addition of expensive growth factors to induce osteoblast differentiation. Here, we investigated a LaponiteÒ crosslinked, pNIPAM-DMAc copolymer (L-pNIPAM-co-DMAc) hydrogel with hydroxyapatite nanoparticles (HAPna), which can be maintained as a liquid ex vivo, injected via narrow-gauge needle into affected bone, followed by in situ gelation to deliver and induce osteogenic differentiation of human mesenchymal stem cells (hMSC). L-pNIPAM-co-DMAc hydrogels were synthesised and HAPna added post polymerisation. Commercial hMSCs from one donor (Lonza) were incorporated in liquid hydrogel, the mixture solidified and cultured for up to 6 weeks. Viability of hMSCs was maintained within hydrogel constructs containing 0.5 mg/mL HAPna. SEM analysis demonstrated matrix deposition in cellular hydrogels which were absent in acellular controls. A significant increase in storage modulus (G') was observed in cellular hydrogels with 0.5 mg/mL HAPna. Semi-quantitative immunohistochemistry and histological analysis demonstrated that bone differentiation markers and collagen deposition was induced within 48 h, with increased calcium deposition with time. The thermally triggered hydrogel system, described here, was sufficient without the need of additional growth factors or osteogenic media to induce osteogenic differentiation of commercial hMSCs. Preliminary data presented here will be expanded on multiple patient samples to ensure differentiation is seen in these samples. This system could potentially reduce treatment costs and simplify the treatment strategy for orthopaedic repair and regeneration. PMID:27377664

  11. Controlling Growth and Osteogenic Differentiation of Osteoblasts on Microgrooved Polystyrene Surfaces.

    Science.gov (United States)

    Sun, Lanying; Pereira, Daniel; Wang, Qibao; Barata, David Baião; Truckenmüller, Roman; Li, Zhaoyuan; Xu, Xin; Habibovic, Pamela

    2016-01-01

    Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 μm and 4 μm) and distance between individual grooves (about 6 μm and 11 μm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 μm-grooves / 11 μm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 μm-grooves / 6 μm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates. PMID:27571520

  12. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    Science.gov (United States)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  13. Osteogenic ability of bone marrow stem cells intraoperatively enriched by a novel matrix

    OpenAIRE

    Ye, Qing; Chen, Kaining; HUANG, WU; HE, YUNSONG; NONG, MINGSHAN; LI, CHUNXIANG; LIANG, TIANSEN

    2014-01-01

    Poly-L-lysine (PLL) is commonly used as an adhibiting agent due to its good viscosity, and demineralized bone matrix (DBM) is a common enriched matrix for selective cell retention technology. Therefore, the aim of this study was to use PLL to coat the surface and interspaces of DBM to form a novel type of enriched matrix [DBM coated with PLL (PLL-DBM)], in order to effectively improve the enrichment effects of bone marrow stem cells and enhance their osteogenic ability. Electron microscope sc...

  14. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, H.; Stenvang, Jan;

    2011-01-01

    of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down......, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3' UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central...

  15. Synergistic effect of exogeneous and endogeneous electrostimulation on osteogenic differentiation of human mesenchymal stem cells seeded on silk scaffolds.

    Science.gov (United States)

    Çakmak, Anıl S; Çakmak, Soner; White, James D; Raja, Waseem K; Kim, Kyungsook; Yiğit, Sezin; Kaplan, David L; Gümüşderelioğlu, Menemşe

    2016-04-01

    Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10 nM) and glibenclamide (10 μM), along with external electrostimulation (60 kHz; 100-500 mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX-2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue-specific goals.

  16. Nicotine deteriorates the osteogenic differentiation of periodontal ligament stem cells through α7 nicotinic acetylcholine receptor regulating Wnt pathway.

    Directory of Open Access Journals (Sweden)

    Zhifei Zhou

    Full Text Available AIMS: Cigarette smoking is one of the high risk factors of adult chronic periodontitis and nicotine is the well established toxic substance in cigarette. However, the mechanism of nicotine induced periodontitis is still unknown. Here we studied whether nicotine impaired the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs through activating α7 nicotinic acetylcholine receptor (α7 nAChR. METHODS: hPDLSCs with multi differentiation potential and surface makers for mesenchymal stem cells were harvested by limiting dilution technique. The level of mineralized nodule formation was assessed by alizarin red S staining. Expression level of ostegenic related genes and proteins were detected by real-time PCR and western blot analysis. The expression of α7 nAChR and its downstream signaling pathway were examined by western blot. The role of the receptor and related signaling pathway in nicotine impairing the osteogenic potential of hPDLSCs were also studied in different levels. RESULTS: Nicotine deteriorated the ostegenic differentiation of hPDLSCs in a dose dependent manner. Activation of α7 nAChR by nicotine treatment activated wnt/β-catenin signaling pathway, leading to osteogenic deficiency of hPDLSCs. Blockage of α7 nAChR and wnt pathway inhibitor treatment rescued nicotine induced osteogenic differentiation deficiency. CONCLUSIONS: These data suggested that nicotine activated α7 nAChR expressed on PDLSCs and further activated wnt signaling downstream, thus deteriorating the osteogenic potential of PDLSCs. The impairment of osteogenic differentiation of PDLSCs by nicotine might lead to cigarette smoking related periodontitis.

  17. Abnormal osteogenic and chondrogenic differentiation of human mesenchymal stem cells from patients with adolescent idiopathic scoliosis in response to melatonin

    Science.gov (United States)

    Chen, Chong; Xu, Caixia; Zhou, Taifeng; Gao, Bo; Zhou, Hang; Chen, Changhua; Zhang, Changli; Huang, Dongsheng; Su, Peiqiang

    2016-01-01

    Abnormalities of membranous and endochondral ossification in patients with adolescent idiopathic scoliosis (AIS) remain incompletely understood. To investigate abnormalities in the melatonin signaling pathway and cellular response to melatonin in AIS, a case-control study of osteogenic and chondrogenic differentiation was performed using human mesenchymal stem cells (hMSCs). AIS was diagnosed by physical and radiographic examination. hMSCs were isolated from the bone marrow of patients with AIS and control subjects (n=12 each), and purified by density gradient centrifugation. The expression levels of melatonin receptors (MTs) 1 and 2 were detected by western blotting. Osteogenic and chondrogenic differentiation was induced by culturing hMSCs in osteogenic and chondrogenic media containing vehicle or 50 nM melatonin. Alkaline phosphatase (ALP) activity assays, quantitative glycosaminoglycan (GAG) analysis, and reverse transcription-quantitative polymerase chain reaction analysis were performed. Compared with controls, MT2 demonstrated low expression in the AIS group. Melatonin increased ALP activity, GAG synthesis and upregulated the expression of genes involved in osteogenic and chondrogenic differentiation including, ALP, osteopontin, osteocalcin, runt-related transcription factor 2, collagen type II, collagen type X, aggrecan and sex-determining region Y-box 9 in the normal control hMSCs, but did not affect the AIS groups. Thus, AIS hMSCs exhibit abnormal cellular responses to melatonin during osteogenic and chondrogenic differentiation, which may be associated with abnormal membranous and endochondral ossification, and skeletal growth. These results indicate a potential modulating role of melatonin via the MT2 receptor on abnormal osteogenic and chondrogenic differentiaation in patients with AIS. PMID:27314307

  18. Osteogenic differentiation of mesenchymal stem cells is regulated by osteocyte and osteoblast cells in a simplified bone niche

    Directory of Open Access Journals (Sweden)

    LM McNamara

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs within their native environment of the stem cell niche in bone receive biochemical stimuli from surrounding cells. These stimuli likely influence how MSCs differentiate to become bone precursors. The ability of MSCs to undergo osteogenic differentiation is well established in vitro;however, the role of the natural cues from bone’s regulatory cells, osteocytes and osteoblasts in regulating the osteogenic differentiation of MSCs in vivo are unclear. In this study we delineate the role of biochemical signalling from osteocytes and osteoblasts, using conditioned media and co-culture experiments, to understand how they direct osteogenic differentiation of MSCs. Furthermore, the synergistic relationship between osteocytes and osteoblasts is examined by transwell co-culturing of MSCs with both simultaneously. Osteogenic differentiation of MSCs was quantified by monitoring alkaline phosphatase (ALP activity, calcium deposition and cell number. Intracellular ALP was found to peak earlier and there was greater calcium deposition when MSCs were co-cultured with osteocytes rather than osteoblasts, suggesting that osteocytes are more influential than osteoblasts in stimulating osteogenesis in MSCs. Osteoblasts initially stimulated an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. Our novel co-culture system confirmed a synergistic relationship between osteocytes and osteoblasts in producing biochemical signals to stimulate the osteogenic differentiation of MSCs. This study provides important insights into the mechanisms at work within the native stem cell niche to stimulate osteogenic differentiation and outlines a possible role for the use of co-culture or conditioned media methodologies for tissue engineering applications.

  19. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

    Science.gov (United States)

    Ramasamy, Thamil Selvee; Karunanithi, Puvanan; Naveen, Sangeetha Vasudevaraj; Murali, Malliga Raman; Abbas, Azlina A.; Kamarul, Tunku

    2016-01-01

    Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red

  20. Osteogenic activity of cyclodextrin-encapsulated doxycycline in a calcium phosphate PCL and PLGA composite.

    Science.gov (United States)

    Trajano, V C C; Costa, K J R; Lanza, C R M; Sinisterra, R D; Cortés, M E

    2016-07-01

    Composites of biodegradable polymers and calcium phosphate are bioactive and flexible, and have been proposed for use in tissue engineering and bone regeneration. When associated with the broad-spectrum antibiotic doxycycline (DOX), they could favor antimicrobial action and enhance the action of osteogenic composites. Composites of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and a bioceramic of biphasic calcium phosphate Osteosynt® (BCP) were loaded with DOX encapsulated in β-cyclodextrin (βCD) and were evaluated for effects on osteoblastic cell cultures. The DOX/βCD composite was prepared with a double mixing method. Osteoblast viability was assessed with methyl tetrazolium (MTT) assays after 1day, 7day, and 14days of composite exposure; alkaline phosphatase (AP) activity and collagen production were evaluated after 7days and 14days, and mineral nodule formation after 14days. Composite structures were evaluated by scanning electron microscopy (SEM). Osteoblasts exposed to the composite containing 25μg/mL DOX/βCD had increased cell proliferation (pcomposite (pcomposite material revealed a surface topography with pore sizes suitable for growing osteoblasts. Together, these results suggest that osteoblasts are viable, proliferative, and osteogenic in the presence of a DOX/βCD-containing BCP ceramic composite.

  1. Hydroxyapatite coatings with oriented nanoplate and nanorod arrays: Fabrication, morphology, cytocompatibility and osteogenic differentiation.

    Science.gov (United States)

    Chen, Wei; Tian, Bo; Lei, Yong; Ke, Qin-Fei; Zhu, Zhen-An; Guo, Ya-Ping

    2016-10-01

    Hydroxyapatite (HA) crystals exhibit rod-like shape with c-axis orientation and plate-like shape with a(b)-axis orientation in vertebrate bones and tooth enamel surfaces, respectively. Herein, we report the synthesis of HA coatings with the oriented nanorod arrays (RHACs) and HA coatings with oriented nanoplate arrays (PHACs) by using bioglass coatings as sacrificial templates. After soaking in simulated body fluid (SBF) at 120°C, the bioglass coatings are hydrothermally converted into the HA coatings via a dissolution-precipitation reaction. If the Ca/P ratios in SBF are 2.50 and 1.25, the HA crystals on the coatings are oriented nanorod arrays and oriented nanoplate arrays, respectively. Moreover, the bioglass coatings are treated with SBF at 37°C, plate-like HA coatings with a low crystallinity (SHACs) are prepared. As compared with the Ti6Al4V and SHACs, the human bone marrow stromal cells (hBMSCs) on the RHACs and PHACs have better cell adhesion, spreading, proliferation and osteogenic differentiation because of their moderately hydrophilic surfaces and similar chemical composition, morphology and crystal orientation to human hard tissues. Notably, the morphologies of HA crystals have no obvious effects on cytocompatibility and osteogenic differentiation. Hence, the HA coatings with oriented nanoplate arrays or oriented nanorod arrays have a great potential for orthopedic applications. PMID:27287136

  2. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential.

    Science.gov (United States)

    Pipino, Caterina; Pandolfi, Assunta

    2015-05-26

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.

  3. Osteogenic differentiation of amniotic fluid mesenchymalstromal cells and their bone regeneration potential

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    In orthopedics, tissue engineering approach usingstem cells is a valid line of treatment for patients withbone defects. In this context, mesenchymal stromalcells of various origins have been extensively studiedand continue to be a matter of debate. Although mesenchymalstromal cells from bone marrow are alreadyclinically applied, recent evidence suggests that one mayuse mesenchymal stromal cells from extra-embryonictissues, such as amniotic fluid, as an innovative andadvantageous resource for bone regeneration. Theuse of cells from amniotic fluid does not raise ethicalproblems and provides a sufficientnumber of cellswithout invasive procedures. Furthermore, they donot develop into teratomas when transplanted, aconsequence observed with pluripotent stem cells.In addition, their multipotent differentiation ability,low immunogenicity, and anti-inflammatory propertiesmake them ideal candidates for bone regenerativemedicine. We here present an overview of the featuresof amniotic fluid mesenchymal stromal cells and theirpotential in the osteogenic differentiation process.We have examined the papers actually availableonthis regard, with particular interest in the strategiesapplied to improve in vitro osteogenesis. Importantly, adetailed understanding of the behavior of amniotic fluidmesenchymal stromal cells and their osteogenic abilityis desirable considering a feasible application in boneregenerative medicine.

  4. Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Chad M. Teven

    2011-01-01

    Full Text Available Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES cells. Mesenchymal stem cells (MSCs are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

  5. Effects of Plants on Osteogenic Differentiation and Mineralization of Periodontal Ligament Cells: A Systematic Review.

    Science.gov (United States)

    Costa, Cláudio Rodrigues Rezende; Amorim, Bruna Rabelo; de Magalhães, Pérola; De Luca Canto, Graziela; Acevedo, Ana Carolina; Guerra, Eliete Neves Silva

    2016-04-01

    This systematic review aimed to evaluate the effects of plants on osteogenic differentiation and mineralization of human periodontal ligament cells. The included studies were selected using five different electronic databases. The reference list of the included studies was crosschecked, and a partial gray literature search was undertaken using Google Scholar and ProQuest. The methodology of the selected studies was evaluated using GRADE. After a two-step selection process, eight studies were identified. Six different types of plants were reported in the selected studies, which were Morinda citrifolia, Aloe vera, Fructus cnidii, Zanthoxylum schinifolium, Centella asiatica, and Epimedium species. They included five types of isolated plant components: acemannan, osthole, hesperetin, asiaticoside, and icariin. In addition, some active substances of these components were identified as polysaccharides, coumarins, flavonoids, and triterpenes. The studies demonstrated the potential effects of plants on osteogenic differentiation, cell proliferation, mineral deposition, and gene and protein expression. Four studies showed that periodontal ligament cells induce mineral deposition after plant treatment. Although there are few studies on the subject, current evidence suggests that plants are potentially useful for the treatment of periodontal diseases. However, further investigations are required to confirm the promising effect of these plants in regenerative treatments. PMID:26822584

  6. Osteogenic differentiation of human mesenchymal stem cells promotes mineralization within a biodegradable peptide hydrogel

    Science.gov (United States)

    Castillo Diaz, Luis A; Elsawy, Mohamed; Saiani, Alberto; Gough, Julie E; Miller, Aline F

    2016-01-01

    An attractive strategy for the regeneration of tissues has been the use of extracellular matrix analogous biomaterials. Peptide-based fibrillar hydrogels have been shown to mimic the structure of extracellular matrix offering cells a niche to undertake their physiological functions. In this study, the capability of an ionic-complementary peptide FEFEFKFK (F, E, and K are phenylalanine, glutamic acid, and lysine, respectively) hydrogel to host human mesenchymal stem cells in three dimensions and induce their osteogenic differentiation is demonstrated. Assays showed sustained cell viability and proliferation throughout the hydrogel over 12 days of culture and these human mesenchymal stem cells differentiated into osteoblasts simply upon addition of osteogenic stimulation. Differentiated osteoblasts synthesized key bone proteins, including collagen-1 (Col-1), osteocalcin, and alkaline phosphatase. Moreover, mineralization occurred within the hydrogel. The peptide hydrogel is a naturally biodegradable material as shown by oscillatory rheology and reversed-phase high-performance liquid chromatography, where both viscoelastic properties and the degradation of the hydrogel were monitored over time, respectively. These findings demonstrate that a biodegradable octapeptide hydrogel can host and induce the differentiation of stem cells and has the potential for the regeneration of hard tissues such as alveolar bone. PMID:27493714

  7. EXPRESSION OF ALKALINE PHOSPHATASE DURING OSTEOGENIC DIFFERENTIATION OF RAT BONE MARROW STROMAL CELLS

    Directory of Open Access Journals (Sweden)

    AKBARI M

    2001-01-01

    Full Text Available Introduction: Bone marrow contains a population of stem cells capable of differentiating to osteoblast and forming the bone nodule by dexamethasone. Material and Methods: The stromal cells of bone marrow obtained from 4 to 6 weeks old Spruge-Dawely male rats were grown in primary culture for 7 days and subcultured for 18 days. The cells were cultured in either DMEM medium containing 15% fetal calf serum and antibiotics as the controls or the above medium supplemented with osteogenic supplements (OS: include 10 mM Na-beta glycerophosphate (Na-betaGp, 10 nM dexamethasone (Dex and 50 g/ml ascordic acid (AsA as the examined cultures. After 6, 12 and 18 days of grow up in subculture, the cultures were examined for mineralization and alkaline phosphatase (Apase expression. Results: Mesenchymal stem cells (MSCs in examined cultures underwent a dramatic change in cellular morphology and a significat increase in Apase activity by day 12. The deposition of a calcified matrix on the surface of the culture flasks became evident between days 12 and 18. Conclusion: The addition of osteogenic supplements (OS to MSCs cultures induced Apase expression that contributes to cellular differentiation and mineralization of extracellular matrix.

  8. 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. PMID:25609957

  9. Effects of Plants on Osteogenic Differentiation and Mineralization of Periodontal Ligament Cells: A Systematic Review.

    Science.gov (United States)

    Costa, Cláudio Rodrigues Rezende; Amorim, Bruna Rabelo; de Magalhães, Pérola; De Luca Canto, Graziela; Acevedo, Ana Carolina; Guerra, Eliete Neves Silva

    2016-04-01

    This systematic review aimed to evaluate the effects of plants on osteogenic differentiation and mineralization of human periodontal ligament cells. The included studies were selected using five different electronic databases. The reference list of the included studies was crosschecked, and a partial gray literature search was undertaken using Google Scholar and ProQuest. The methodology of the selected studies was evaluated using GRADE. After a two-step selection process, eight studies were identified. Six different types of plants were reported in the selected studies, which were Morinda citrifolia, Aloe vera, Fructus cnidii, Zanthoxylum schinifolium, Centella asiatica, and Epimedium species. They included five types of isolated plant components: acemannan, osthole, hesperetin, asiaticoside, and icariin. In addition, some active substances of these components were identified as polysaccharides, coumarins, flavonoids, and triterpenes. The studies demonstrated the potential effects of plants on osteogenic differentiation, cell proliferation, mineral deposition, and gene and protein expression. Four studies showed that periodontal ligament cells induce mineral deposition after plant treatment. Although there are few studies on the subject, current evidence suggests that plants are potentially useful for the treatment of periodontal diseases. However, further investigations are required to confirm the promising effect of these plants in regenerative treatments.

  10. Enhanced Stem Cell Osteogenic Differentiation by Bioactive Glass Functionalized Graphene Oxide Substrates

    Directory of Open Access Journals (Sweden)

    Xiaoju Mo

    2016-01-01

    Full Text Available An unmet need in engineered bone regeneration is to develop scaffolds capable of manipulating stem cells osteogenesis. Graphene oxide (GO has been widely used as a biomaterial for various biomedical applications. However, it remains challenging to functionalize GO as ideal platform for specifically directing stem cell osteogenesis. Herein, we report facile functionalization of GO with dopamine and subsequent bioactive glass (BG to enhance stem cell adhesion, spreading, and osteogenic differentiation. On the basis of graphene, we obtained dopamine functionalized graphene oxide/bioactive glass (DGO/BG hybrid scaffolds containing different content of DGO by loading BG nanoparticles on graphene oxide surface using sol-gel method. To enhance the dispersion stability and facilitate subsequent nucleation of BG in GO, firstly, dopamine (DA was used to modify GO. Then, the modified GO was functionalized with bioactive glass (BG using sol-gel method. The adhesion, spreading, and osteoinductive effects of DGO/BG scaffold on rat bone marrow mesenchymal stem cells (rBMSCs were evaluated. DGO/BG hybrid scaffolds with different content of DGO could influence rBMSCs’ behavior. The highest expression level of osteogenic markers suggests that the DGO/BG hybrid scaffolds have great potential or elicit desired bone reparative outcome.

  11. Induction of osteogenic differentiation of adipose derived stem cells by microstructured nitinol actuator-mediated mechanical stress.

    Directory of Open Access Journals (Sweden)

    Sarah Strauß

    Full Text Available The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi with adipose derived stem cells (ASCs opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.

  12. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

    NARCIS (Netherlands)

    O. Ghali (Olfa); O. Broux (Odile); G. Falgayrac (Guillaume); N. Haren (Nathalie); J.P.T.M. van Leeuwen (Hans); G. Penel (Guillaume); P. Hardouin (Pierre); C. Chauveau (Christophe)

    2015-01-01

    textabstractBackground: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipog

  13. Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation

    NARCIS (Netherlands)

    O. Ghali (Olfa); O. Broux (Odile); G. Falgayrac (Guillaume); N. Haren (Nathalie); J.P.T.M. van Leeuwen (Hans); G. Penel (Guillaume); P. Hardouin (Pierre); C. Chauveau (Christophe)

    2015-01-01

    textabstractBACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipog

  14. Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells.

    Science.gov (United States)

    Keller, Vivian; Deiwick, Andrea; Pflaum, Michael; Schlie-Wolter, Sabrina

    2016-10-01

    The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization.

  15. Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

    Directory of Open Access Journals (Sweden)

    Ketul C. Popat

    2012-11-01

    Full Text Available Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medium. Quantitative PCR, colorimetric assays, immunofluorescent labeling, and electron microscopy measured osteogenic cell responses to the HAp/PCL scaffolds. In maintenance conditions, both Hap/PCL scaffolds and control scaffolds supported cell colonization through seven days with minor differences. In osteogenic conditions, the 10% HAp scaffolds exhibited significantly increased ALP assay levels at week 3, consistent with previous reports. However, qPCR analysis demonstrated an overall decrease in bone matrix-associated genes on Hap/PCL scaffolds. Osteopontin and osteocalcin immunofluorescent microscopy revealed a trend that both mineralized scaffolds had greater amounts of both proteins, though qPCR results indicated the opposite trend for osteopontin. Additionally, type I collagen expression decreased on HAp scaffolds. These results indicate that cells are sensitive to minor changes in mineral content within nanofibers, even at just 1% w/w, and elucidating the sensing mechanism may lead to optimized osteogenic scaffold designs.

  16. Non-invasive monitoring of osteogenic differentiation on microtissue arrays under physiological conditions using scanning electrochemical microscopy

    NARCIS (Netherlands)

    Sridhar, Adithya; Berg, van den Albert; Le Gac, Séverine

    2014-01-01

    In this paper, we present a non-invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP-2), C2C12 microtissues differentiate and exp

  17. Semaphorin 3A Shifts Adipose Mesenchymal Stem Cells towards Osteogenic Phenotype and Promotes Bone Regeneration In Vivo

    Directory of Open Access Journals (Sweden)

    Xiangwei Liu

    2016-01-01

    Full Text Available Adipose mesenchymal stem cells (ASCs are considered as the promising seed cells for bone regeneration. However, the lower osteogenic differentiation capacity limits its therapeutic efficacy. Identification of the key molecules governing the differences between ASCs and BMSCs would shed light on manipulation of ASCs towards osteogenic phenotype. In this study, we screened semaphorin family members in ASCs and BMSCs and identified Sema3A as an osteogenic semaphorin that was significantly and predominantly expressed in BMSCs. The analyses in vitro showed that the overexpression of Sema3A in ASCs significantly enhanced the expression of bone-related genes and extracellular matrix calcium deposition, while decreasing the expression of adipose-related genes and thus lipid droplet formation, resembling a BMSCs phenotype. Furthermore, Sema3A modified ASCs were then engrafted into poly(lactic-co-glycolic acid (PLGA scaffolds to repair the critical-sized calvarial defects in rat model. As expected, Sema3A modified ASCs encapsulation significantly promoted new bone formation with higher bone volume fraction and bone mineral density. Additionally, Sema3A was found to simultaneously increase multiple Wnt related genes and thus activating Wnt pathway. Taken together, our study here identifies Sema3A as a critical gene for osteogenic phenotype and reveals that Sema3A-modified ASCs would serve as a promising candidate for bettering bone defect repair.

  18. Semaphorin 3A Shifts Adipose Mesenchymal Stem Cells towards Osteogenic Phenotype and Promotes Bone Regeneration In Vivo

    Science.gov (United States)

    Liu, Xiangwei; Tan, Naiwen; Zhou, Yuchao; Zhou, Xueying; Chen, Hui; Wei, Hongbo; Chen, Ji; Xu, Xiaoru; Zhang, Sijia

    2016-01-01

    Adipose mesenchymal stem cells (ASCs) are considered as the promising seed cells for bone regeneration. However, the lower osteogenic differentiation capacity limits its therapeutic efficacy. Identification of the key molecules governing the differences between ASCs and BMSCs would shed light on manipulation of ASCs towards osteogenic phenotype. In this study, we screened semaphorin family members in ASCs and BMSCs and identified Sema3A as an osteogenic semaphorin that was significantly and predominantly expressed in BMSCs. The analyses in vitro showed that the overexpression of Sema3A in ASCs significantly enhanced the expression of bone-related genes and extracellular matrix calcium deposition, while decreasing the expression of adipose-related genes and thus lipid droplet formation, resembling a BMSCs phenotype. Furthermore, Sema3A modified ASCs were then engrafted into poly(lactic-co-glycolic acid) (PLGA) scaffolds to repair the critical-sized calvarial defects in rat model. As expected, Sema3A modified ASCs encapsulation significantly promoted new bone formation with higher bone volume fraction and bone mineral density. Additionally, Sema3A was found to simultaneously increase multiple Wnt related genes and thus activating Wnt pathway. Taken together, our study here identifies Sema3A as a critical gene for osteogenic phenotype and reveals that Sema3A-modified ASCs would serve as a promising candidate for bettering bone defect repair. PMID:27721834

  19. NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

    Science.gov (United States)

    Spina, Anna; Montella, Roberta; Liccardo, Davide; De Rosa, Alfredo; Laino, Luigi; Mitsiadis, Thimios A.; La Noce, Marcella

    2016-01-01

    Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact. PMID:27594842

  20. Influence of select extracellular matrix proteins on mesenchymal stem cell osteogenic commitment in three-dimensional contexts.

    Science.gov (United States)

    Becerra-Bayona, Silvia; Guiza-Arguello, Viviana; Qu, Xin; Munoz-Pinto, Dany J; Hahn, Mariah S

    2012-12-01

    Growth factors have been shown to be powerful mediators of mesenchymal stem cell (MSC) osteogenic differentiation. However, their use in tissue engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically promote MSC osteogenic differentiation in the absence of exogenous growth factors via the manipulation of scaffold material properties would be beneficial. The current work examines the influence of select extracellular matrix (ECM) proteins on MSC osteogenesis toward the goal of developing scaffolds with intrinsically osteoinductive properties. Fibrinogen (FG), fibronectin (FN) and laminin-1 (LN) were chosen for evaluation due to their known roles in bone morphogenesis or bone fracture healing. These proteins were conjugated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels and their effects on encapsulated 10T½ MSCs were evaluated. Specifically, following 1week of culture, mid-term markers of various MSC lineages were examined in order to assess the strength and specificity of the observed osteogenic responses. PEG-LN gels demonstrated increased levels of the osteogenic transcription factor osterix relative to day 0 levels. In addition, PEG-FG and PEG-LN gels were associated with increased deposition of bone ECM protein osteocalcin relative to PEG-FN gels and day 0. Importantly, the osteogenic response associated with FG and LN appeared to be specific in that markers for chondrocytic, smooth muscle cell and adipocytic lineages were not similarly elevated relative to day 0 in these gels. To gain insight into the integrin dynamics underlying the observed differentiation results, initial integrin adhesion and temporal alterations in cell integrin profiles were evaluated. The associated results suggest that α(2), α(v) and α(6) integrin subunits may play key roles in integrin-mediated osteogenesis.

  1. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

  2. Evaluation of the Morphology and Osteogenic Potential of Titania-Based Electrospun Nanofibers

    Directory of Open Access Journals (Sweden)

    Xiaokun Wang

    2012-01-01

    Full Text Available Submicron-scale titania-based ceramic fibers with various compositions have been prepared by electrospinning. The as-prepared nanofibers were heat-treated at 700°C for 3 h to obtain pure inorganic fiber meshes. The results show that the diameter and morphology of the nanofibers are affected by starting polymer concentration and sol-gel composition. The titania and titania-silica nanofibers had the average diameter about 100–300 nm. The crystal phase varied from high-crystallized rutile-anatase mixed crystal to low-crystallized anatase with adding the silica addition. The morphology and crystal phase were evaluated by SEM and XRD. Bone-marrow-derived mesenchymal stem cells were seeded on titania-silica 50/50 fiber meshes. Cell number and early differentiation marker expressions were analyzed, and the results indicated osteogenic potential of the titania-silica 50/50 fiber meshes.

  3. Boron nitride nanotube-enhanced osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Li, Xia; Wang, Xiupeng; Jiang, Xiangfen; Yamaguchi, Maho; Ito, Atsuo; Bando, Yoshio; Golberg, Dmitri

    2016-02-01

    The interaction between boron nitride nanotubes (BNNTs) layer and mesenchymal stem cells (MSCs) is evaluated for the first time in this study. BNNTs layer supports the attachment and growth of MSCs and exhibits good biocompatibility with MSCs. BNNTs show high protein adsorption ability, promote the proliferation of MSCs and increase the secretion of total protein by MSCs. Especially, BNNTs enhance the alkaline phosphatase (ALP) activity as an early marker of osteoblasts, ALP/total protein and osteocalcin (OCN) as a late marker of osteogenic differentiation, which shows that BNNTs can enhance osteogenesis of MSCs. The release of trace boron and the stress on cells exerted by BNNTs with a fiber structure may account for the enhanced differentiation of MSCs into osteoblasts. Therefore BNNTs are potentially useful for bone regeneration in orthopedic applications.

  4. Osteogenic Activity of Locally Applied Small Molecule Drugs in a Rat Femur Defect Model

    Directory of Open Access Journals (Sweden)

    Jessica A. Cottrell

    2010-01-01

    Full Text Available The long-term success of arthroplastic joints is dependent on the stabilization of the implant within the skeletal site. Movement of the arthroplastic implant within the bone can stimulate osteolysis, and therefore methods which promote rigid fixation or bone growth are expected to enhance implant stability and the long-term success of joint arthroplasty. In the present study, we used a simple bilateral bone defect model to analyze the osteogenic activity of three small-molecule drug implants via microcomputerized tomography (micro-CT and histomorphometry. In this study, we show that local delivery of alendronate, but not lovastatin or omeprazole, led to significant new bone formation at the defect site. Since alendronate impedes osteoclast-development, it is theorized that alendronate treatment results in a net increase in bone formation by preventing osteoclast mediated remodeling of the newly formed bone and upregulating osteoblasts.

  5. Histone methyltransferases and demethylases:regulators in balancing osteogenic and adipogenic differentiation of mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Peng Deng; Qian-Ming Chen; Christine Hong; Cun-Yu Wang

    2015-01-01

    Mesenchymal stem cells (MSCs) are characterized by their self-renewing capacity and differentiation potential into multiple tissues. Thus, management of the differentiation capacities of MSCs is important for MSC-based regenerative medicine, such as craniofacial bone regeneration, and in new treatments for metabolic bone diseases, such as osteoporosis. In recent years, histone modification has been a growing topic in the field of MSC lineage specification, in which the Su(var)3–9, enhancer-of-zeste, trithorax (SET) domain-containing family and the Jumonji C (JmjC) domain-containing family represent the major histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs), respectively. In this review, we summarize the current understanding of the epigenetic mechanisms by which SET domain-containing KMTs and JmjC domain-containing KDMs balance the osteogenic and adipogenic differentiation of MSCs.

  6. Porous polylactic acid-silica hybrids: preparation, characterization, and study of mesenchymal stem cell osteogenic differentiation.

    Science.gov (United States)

    Pandis, Christos; Trujillo, Sara; Matos, Joana; Madeira, Sara; Ródenas-Rochina, Joaquín; Kripotou, Sotiria; Kyritsis, Apostolos; Mano, João F; Gómez Ribelles, José Luis

    2015-02-01

    A novel approach to reinforce polymer porous membranes is presented. In the prepared hybrid materials, the inorganic phase of silica is synthesized in-situ and inside the pores of aminolyzed polylactic acid (PLA) membranes by sol-gel reactions using tetraethylorthosilicate (TEOS) and glycidoxypropyltrimethoxysilane (GPTMS) as precursors. The hybrid materials present a porous structure with a silica layer covering the walls of the pores while GPTMS serves also as coupling agent between the organic and inorganic phase. The adjustment of silica precursors ratio allows the modulation of the thermomechanical properties. Culture of mesenchymal stem cells on these supports in osteogenic medium shows the expression of characteristic osteoblastic markers and the mineralization of the extracellular matrix.

  7. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    Isotope Labelling with Amino acids in Culture) membrane protein fractions of the hMSC under undifferentiated as well as differentiated conditions. Our stem cell lab has developed a human MSC cell line over-expressing the hTERT subunit of the telomerase gene, enabling stable long term in vitro culture...... devoid of cell senescence as encountered in primary cultures. The cell line used in this study has been shown capable of forming bone in vivo and osteogenic differentiation in vitro. We quantified a total number of 972 proteins of which 80 % fall within the categories of: transmembrane, membrane......  Mesenchymal stem cells (MSCs) (or marrow stromal cells) represent a population of cells located in the bone marrow that are capable of differentiation into several mesodermal cell types including osteoblasts. MSCs cannot presently be isolated based on protein markers, as no specific markers exist...

  8. Surface hydrophilicity of PLGA fibers governs in vitro mineralization and osteogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Minnah; Arora, Aditya; Katti, Dhirendra S., E-mail: dsk@iitk.ac.in

    2014-12-01

    Interfacial properties of biomaterials play an important role in governing their interaction with biological microenvironments. This work investigates the role of surface hydrophilicity of electrospun poly(lactide-co-glycolide) (PLGA) fibers in determining their biological response. For this, PLGA is blended with varying amounts of Pluronic®F-108 and electrospun to fabricate microfibers with varying surface hydrophilicity. The results of mineralization study in simulated body fluid (SBF) demonstrate a significant enhancement in mineralization with an increase in surface hydrophilicity. While presence of serum proteins in SBF reduces absolute mineral content, mineralization continues to be higher on samples with higher surface hydrophilicity. The results from in vitro cell culture studies demonstrate a marked improvement in mesenchymal stem cell —adhesion, elongation, proliferation, infiltration, osteogenic differentiation and matrix mineralization on hydrophilized fibers. Therefore, hydrophilized PLGA fibers are advantageous both in terms of mineralization and elicitation of favorable cell response. Since most of the polymeric materials being used in orthopedics are hydrophobic in nature, the results from this study have strong implications in the future design of interfaces of such hydrophobic materials. In addition, the work proposes a facile method for the modification of electrospun fibers of hydrophobic polymers by blending with a poloxamer for improved bone tissue regeneration. - Highlights: • Surface hydrophilicity of PLGA modulated by blending with Pluronic F-108. • Hydrophilized fibers support better in vitro mineralization. • Mineralization trends retained in the presence of adsorbed serum proteins. • Hydrophilized fibers promote better cell adhesion and proliferation. • Hydrophilized fibers also enable better osteogenic differentiation.

  9. Mechanical properties and osteogenic potential of hydroxyapatite-PLGA-collagen biomaterial for bone regeneration.

    Science.gov (United States)

    Bhuiyan, Didarul B; Middleton, John C; Tannenbaum, Rina; Wick, Timothy M

    2016-08-01

    A bone graft is a complicated structure that provides mechanical support and biological signals that regulate bone growth, reconstruction, and repair. A single-component material is inadequate to provide a suitable combination of structural support and biological stimuli to promote bone regeneration. Multicomponent composite biomaterials lack adequate bonding among the components to prevent phase separation after implantation. We have previously developed a novel multistep polymerization and fabrication process to construct a nano-hydroxyapatite-poly(D,L-lactide-co-glycolide)-collagen biomaterial (abbreviated nHAP-PLGA-collagen) with the components covalently bonded to each other. In the present study, the mechanical properties and osteogenic potential of nHAP-PLGA-collagen are characterized to assess the material's suitability to support bone regeneration. nHAP-PLGA-collagen films exhibit tensile strength very close to that of human cancellous bone. Human mesenchymal stem cells (hMSCs) are viable on 2D nHAP-PLGA-collagen films with a sevenfold increase in cell population after 7 days of culture. Over 5 weeks of culture, hMSCs deposit matrix and mineral consistent with osteogenic differentiation and bone formation. As a result of matrix deposition, nHAP-PLGA-collagen films cultured with hMSCs exhibit 48% higher tensile strength and fivefold higher moduli compared to nHAP-PLGA-collagen films without cells. More interestingly, secretion of matrix and minerals by differentiated hMSCs cultured on the nHAP-PLGA-collagen films for 5 weeks mitigates the loss of mechanical strength that accompanies PLGA hydrolysis. PMID:27120980

  10. Osteogenic activity of cyclodextrin-encapsulated doxycycline in a calcium phosphate PCL and PLGA composite.

    Science.gov (United States)

    Trajano, V C C; Costa, K J R; Lanza, C R M; Sinisterra, R D; Cortés, M E

    2016-07-01

    Composites of biodegradable polymers and calcium phosphate are bioactive and flexible, and have been proposed for use in tissue engineering and bone regeneration. When associated with the broad-spectrum antibiotic doxycycline (DOX), they could favor antimicrobial action and enhance the action of osteogenic composites. Composites of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and a bioceramic of biphasic calcium phosphate Osteosynt® (BCP) were loaded with DOX encapsulated in β-cyclodextrin (βCD) and were evaluated for effects on osteoblastic cell cultures. The DOX/βCD composite was prepared with a double mixing method. Osteoblast viability was assessed with methyl tetrazolium (MTT) assays after 1day, 7day, and 14days of composite exposure; alkaline phosphatase (AP) activity and collagen production were evaluated after 7days and 14days, and mineral nodule formation after 14days. Composite structures were evaluated by scanning electron microscopy (SEM). Osteoblasts exposed to the composite containing 25μg/mL DOX/βCD had increased cell proliferation (p<0.05) compared to control osteoblast cultures at all experimental time points, reaching a maximum in the second week. AP activity and collagen secretion levels were also elevated in osteoblasts exposed to the DOX/βCD composite (p<0.05 vs. controls) and reached a maximum after 14days. These results were corroborated by Von Kossa test results, which showed strong formation of mineralization nodules during the same time period. SEM of the composite material revealed a surface topography with pore sizes suitable for growing osteoblasts. Together, these results suggest that osteoblasts are viable, proliferative, and osteogenic in the presence of a DOX/βCD-containing BCP ceramic composite. PMID:27127066

  11. Enhanced gene delivery by chitosan-disulfide-conjugated LMW-PEI for facilitating osteogenic differentiation.

    Science.gov (United States)

    Zhao, Xiaoli; Li, Zhaoyang; Pan, Haobo; Liu, Wenguang; Lv, Minmin; Leung, Frankie; Lu, William W

    2013-05-01

    Chitosan-disulfide-conjugated LMW-PEI (CS-ss-PEI) was designed to combine the biocompatibility of chitosan and the gene delivery ability of polyethylenimine (PEI) using bio-reducible disulfide for bone morphogenetic protein (BMP2) gene delivery in mediating osteogenic differentiation. It was prepared by conjugating low molecular weight PEI (LMW-PEI) to chitosan through oxidization of thiols introduced for the formation of disulfide linkage. The structure, molecular weight and buffer capacity were characterized by Fourier transform infrared (FTIR), light scattering and acid-base titration, respectively. The reduction in molecular weight of CS-ss-PEI by the reducing agent indicated its bio-reducible property. With the increment in the LMW-PEI component, the copolymer showed increased DNA binding ability and formed denser nanocomplexes. CS-ss-PEI exhibited low cytotoxicity in COS-1, HepG2 and 293T cells over the different weight ratios. The transfection efficiency of CS-ss-PEI4 was significantly higher than that of PEI 25k and comparable with Lipofectamine in mediating luciferase expression. Its application for BMP2 gene delivery was confirmed in C2C12 cells by BMP2 expression. For inducing in vitro osteogenic differentiation, CS-ss-PEI4 mediated BMP2 gene delivery showed a stronger effect in MG-63 osteoblast cells and stem cells in terms of alkaline phosphatase activity and mineralization compared with PEI25k and Lipofectamine. This study provides a potential gene delivery system for orthopedic-related disease. PMID:23395816

  12. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  13. Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.

    Science.gov (United States)

    Cameron, Kate; Travers, Paul; Chander, Chaman; Buckland, Tom; Campion, Charlie; Noble, Brendon

    2013-01-01

    Insufficient, underactive, or inappropriate osteoblast function results in serious clinical conditions such as osteoporosis, osteogenesis imperfecta and fracture nonunion and therefore the control of osteogenesis is a medical priority. In vitro mesenchymal stem cells (MSCs) can be directed to form osteoblasts through the addition of soluble factors such as β-glycerophosphate, ascorbic acid, and dexamethasone; however this is unlikely to be practical in the clinical setting. An alternative approach would be to use a scaffold or matrix engineered to provide cues for differentiation without the need for soluble factors. Here we describe studies using Silicate-substituted calcium phosphate (Si-CaP) and unmodified hydroxyapatite (HA) to test whether these materials are capable of promoting osteogenic differentiation of MSCs in the absence of soluble factors. Si-CaP supported attachment and proliferation of MSCs and induced osteogenesis to a greater extent than HA, as evidenced through upregulation of the osteoblast-related genes: Runx2 (1.2 fold), Col1a1 (2 fold), Pth1r (1.5 fold), and Bglap (1.7 fold) Dmp1 (1.1 fold), respectively. Osteogenic-associated proteins, alkaline phosphatase (1.4 fold), RUNX2, COL1A1, and BGLAP, were also upregulated and there was an increased production of mineralized bone matrix (1.75 fold), as detected by the Von Kossa Assay. These data indicate that inorganic substrates are capable of directing the differentiation programme of stem cells in the absence of known chemical drivers and therefore may provide the basis for bone repair in the clinical setting.

  14. Osteogenic efficiency of in situ gelling poloxamine systems with and without bone morphogenetic protein-2

    Directory of Open Access Journals (Sweden)

    A Rey-Rico

    2011-04-01

    Full Text Available In situ gelling solutions for minimally invasive local application of bone growth factors are attracting increasing attention as efficient and patient-friendly alternative to bone grafts and solid scaffolds for repairing bone defects. Poloxamines, i.e., X-shaped poly(ethylene oxide-poly(propylene oxide block copolymers with an ethylenediamine core (Tetronic®, were evaluated both as an active osteogenic component and as a vehicle for rhBMP-2 injectable implants. After cytotoxicity screening of various poloxamine varieties, Tetronic 908, 1107, 1301 and 1307 solutions were chosen as the most cytocompatible and their sol-to-gel transitions were rheologically characterized. Viscoelastic gels, formed at 37 ºC, sustained protein release under physiological-like conditions. Formulations of rhBMP-2 led to differentiation of mesenchymal stem cells to osteoblasts, quantified as alkaline phosphatase activity with a maximum at day 7, and to mineralized nodules. Interestingly, poloxamine solely gels led to an initial proliferation of the mesenchymal stem cells (first week, followed by differentiation to osteoblasts (second to third week. Histochemical analysis revealed that Tetronic 908 is only osteoinductive; Tetronic 1107 is mostly osteoinductive, although its use leads to a minor differentiation to adipocytes; Tetronic 1307, solely or loaded with rhBMP-2, causes differentiation of both osteoblasts and adipocytes. Enhanced expression levels of CBFA-1 and collagen type I were observed for Tetronic 908, 1107 and 1307, both solely and combined with rhBMP-2. The intrinsic osteogenic activity of poloxamines (not observed for Pluronic F127 offers novel perspectives for bone regeneration using minimally invasive procedures (i.e., injectable scaffolds and overcoming the safety and the cost/effectiveness concerns associated with large scale clinical use of recombinant growth factors.

  15. Cordycepin prevents oxidative stress-induced inhibition of osteogenesis.

    Science.gov (United States)

    Wang, Feng; Yin, Peipei; Lu, Ye; Zhou, Zubin; Jiang, Chaolai; Liu, Yingjie; Yu, Xiaowei

    2015-11-01

    Oxidative stress is known to be involved in impairment of osteogenesis and age-related osteoporosis. Cordycepin is one of the major bioactive components of Cordyceps militaris that has been shown to exert antioxidant and anti-inflammatory activities. However, there are few reports available regarding the effects of cordycepin on osteogenesis and the underlying mechanism. In this study, we investigated the potential osteoprotective effects of cordycepin and its mechanism systematically using both in vitro model as well as in vivo mouse models. We discovered that hydrogen peroxide (H2O2)-induced inhibition of osteogenesis which was rescued by cordycepin treatment in human bone marrow mesenchymal stem cells (BM-MSCs). Cordycepin exerted its protective effects partially by increasing or decreasing expression of osteogenic and osteoclastogenesis marker genes. Treatment with cordycepin increased Wnt-related genes' expression whereas supplementation of Wnt pathway inhibitor reversed its protective effects. In addition, administration of cordycepin promoted osteogenic differentiation of BM-MSCs by reducing oxidative stress in both ovariectomized and aged animal models. Taken together, these results support the protective effects of cordycepin on oxidative stress induced inhibition of osteogenesis by activation of Wnt pathway. PMID:26462178

  16. Curcumin increases rat mesenchymal stem cell osteoblast differentiation but inhibits adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Qiaoli Gu

    2012-01-01

    Full Text Available Background: Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric and has effects on bone health and fat formation. The bone marrow mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into osteoblasts and adipocytes. Osteoblast differentiation of MSCs can be a result of upregulation of heme oxygenase (HO-1 expression. Curcumin can potently induce HO-1 expression. Objective: The present study describes the effects of curcumin on rat MSC (rMSCs differentiation into osteoblasts and adipocytes. Materials and Methods: Rat bone marrow MSCs were isolated and treated with or without curcumin. Osteoblast differentiation was confirmed and determined by alkaline phosphatase (ALP activity, mineralized nodule formation, the expression of Runx2 (runt-related transcription factor 2 and osteocalcin. Adipocyte differentiation was determined by Oil red O staining and the expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2 and CCAAT/enhancer-binding protein (C/EBP α. Results: Curcumin increased ALP activity and osteoblast-specific mRNA expression of Runx2 and osteocalcin when rMSCs were cultured in osteogenic medium. In contrast, curcumin decreased adipocyte differentiation and inhibited adipocyte-specific mRNA expression of PPARγ2 and C/EBPα when rMSCs were cultured in adipogenic medium. HO-1 expression was increased during osteogenic differentiation of rMSCs. Conclusions: These findings demonstrate that curcumin can promote osteogenic differentiation of rMSCs and inhibit adipocyte formation. The effect of curcumin on osteogenic differentiation of rMSCs is correlated with HO-1 expression.

  17. Osteogenic protein-1 increases the fixation of implants grafted with morcellised bone allograft and ProOsteon bone substitute: an experimental study in dogs

    DEFF Research Database (Denmark)

    Baad-Hansen, Thomas Einer; Overgaard, S; Lind, M;

    2007-01-01

    weeks osteogenic protein-1 increased bone formation and the energy absorption of implants grafted with allograft and ProOsteon. A composite of allograft, ProOsteon and osteogenic protein-1 was comparable, but not superior to, allograft used on its own. ProOsteon alone cannot be recommended as a......Impacted bone allograft is often used in revision joint replacement. Hydroxyapatite granules have been suggested as a substitute or to enhance morcellised bone allograft. We hypothesised that adding osteogenic protein-1 to a composite of bone allograft and non-resorbable hydroxyapatite granules...... surrounded by a concentric 3 mm gap. These gaps were randomly allocated to four different procedures in each dog: 1) bone allograft used on its own; 2) ProOsteon used on its own; 3) allograft and ProOsteon used together; or 4) allograft and ProOsteon with the addition of osteogenic protein-1. After three...

  18. Dose effect of tumor necrosis factor-alpha on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds.

    Science.gov (United States)

    Mountziaris, Paschalia M; Tzouanas, Stephanie N; Mikos, Antonios G

    2010-03-01

    This study presents a first step in the development of a bone tissue engineering strategy to trigger enhanced osteogenesis by modulating inflammation. This work focused on characterizing the effects of the concentration of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on osteogenic differentiation of mesenchymal stem cells (MSCs) grown in a 3D culture system. MSC osteogenic differentiation is typically achieved in vitro through a combination of osteogenic supplements that include the anti-inflammatory corticosteroid dexamethasone. Although simple, the use of dexamethasone is not clinically realistic, and also hampers in vitro studies of the role of inflammatory mediators in wound healing. In this study, MSCs were pre-treated with dexamethasone to induce osteogenic differentiation, and then cultured in biodegradable electrospun poly(epsilon-caprolactone) (PCL) scaffolds, which supported continued MSC osteogenic differentiation in the absence of dexamethasone. Continuous delivery of 0.1 ng/mL of recombinant rat TNF-alpha suppressed osteogenic differentiation of rat MSCs over 16 days, which was likely the result of residual dexamethasone antagonizing TNF-alpha signaling. Continuous delivery of a higher dose, 5 ng/mL TNF-alpha, stimulated osteogenic differentiation for a few days, and 50 ng/mL TNF-alpha resulted in significant mineralized matrix deposition over the course of the study. These findings suggest that the pro-inflammatory cytokine TNF-alpha stimulates osteogenic differentiation of MSCs, an effect that can be blocked by the presence of anti-inflammatory agents like dexamethasone, with significant implications on the interplay between inflammation and tissue regeneration.

  19. Tissue characteristics of high- and low-incidence plutonium-induced osteogenic sarcoma sites in life-span beagles

    International Nuclear Information System (INIS)

    On the basis of information gathered from the 239Pu life-span study in beagles at the University of Utah, the tissue features were found to be characteristic of high-incidence bone-tumor sites compared to low-incidence sites included more hematopoietic tissues in the bone marrow; greater trabecular bone mass; greater bone remodeling rates; greater mineral apposition rates; greater density and activity of bone surface cells; greater density of putative bone-cell precursors; greater initial uptake of plutonium on bone surfaces; and greater marrow vascular volumes and a venous sinusoidal bed. Although most of these studies are not yet complete, the information being collected should contribute to our understanding of the mechanisms of radiation-induced osteogenic sarcomas. This should aid in predicting the types and characteristics of osseous tissues where radiation-induced osteogenic sarcomas may arise in humans. 25 refs., 4 figs., 3 tabs

  20. Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers

    Institute of Scientific and Technical Information of China (English)

    Ruth Alvarez; Hye-Lim Lee; Cun-Yu Wang; Christine Hong

    2015-01-01

    Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations:CD51/CD140a, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24%of PDLCs were CD511/CD140a1, 0.8%were CD2711, and 2.4%were STRO-11/CD1461. Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD2711 DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

  1. Osteogenic potential of in situ TiO{sub 2} nanowire surfaces formed by thermal oxidation of titanium alloy substrate

    Energy Technology Data Exchange (ETDEWEB)

    Tan, A.W. [Department of Biomedical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia); Ismail, R.; Chua, K.H. [Department of Physiology, Faculty of Medicine, National University of Malaysia, 50300 Kuala Lumpur (Malaysia); Ahmad, R. [Department of Mechanical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia); Akbar, S.A. [Department of Materials Science and Engineering, The Ohio State University, Columbus, OH 43210 (United States); Pingguan-Murphy, B., E-mail: bpingguan@um.edu.my [Department of Biomedical Engineering, University of Malaya, 50603 Kuala Lumpur (Malaysia)

    2014-11-30

    Highlights: • In situ titanium dioxide (TiO{sub 2}) nanowire surface structures were fabricated on Ti-6Al-4V substrate using thermal oxidation. • Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression of primary human osteoblasts were examined on the TiO{sub 2} nanowire surfaces. • TiO{sub 2} nanowire surfaces showed enhanced osteogenic potential as compared to the planar surface. - Abstract: Titanium dioxide (TiO{sub 2}) nanowire surface structures were fabricated in situ by a thermal oxidation process, and their ability to enhance the osteogenic potential of primary osteoblasts was investigated. Human osteoblasts were isolated from nasal bone and cultured on a TiO{sub 2} nanowires coated substrate to assess its in vitro cellular interaction. Bare featureless Ti-6Al-4V substrate was used as a control surface. Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression were examined on the TiO{sub 2} nanowire surfaces as compared to the control surfaces after 2 weeks of culturing. Cell adhesion and cell proliferation were assayed by field emission scanning electron microscope (FESEM) and Alamar Blue reduction assay, respectively. The nanowire surfaces promoted better cell adhesion and spreading than the control surface, as well as leading to higher cell proliferation. Our results showed that osteoblasts grown onto the TiO{sub 2} nanowire surfaces displayed significantly higher production levels of alkaline phosphatase (ALP), extracellular (ECM) mineralization and genes expression of runt-related transcription factor (Runx2), bone sialoprotein (BSP), ostoepontin (OPN) and osteocalcin (OCN) compared to the control surfaces. This suggests the potential use of such surface modification on Ti-6Al-4V substrates as a promising means to improve the osteointegration of titanium based implants.

  2. Solitary osseous metastasis of rectal carcinoma masquerading as osteogenic sarcoma on post-chemotherapy imaging: A case report

    Energy Technology Data Exchange (ETDEWEB)

    Udare, Amar; Sable, Nilesh; Kumar, Rajiv; Thakur, Meenakshi; Juvekar, Shashikant [Tata Memorial Hospital, Mumbai (India)

    2015-02-15

    Solitary metastases from colorectal carcinoma in the absence of hepatic or pulmonary metastases are rare. These can have a diverse imaging appearance, particularly after chemotherapy. It is important identify patients with solitary skeletal metastases, as they have a better prognosis than those with multiple skeletal or visceral metastases. We describe an unusual case of a solitary metastasis to the femur in a case of colon carcinoma that went undiagnosed and later presented with imaging features of osteogenic sarcoma.

  3. In Vitro Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells Seeded on Carboxymethyl Cellulose-Hydroxyapatite Hybrid Hydrogel.

    Directory of Open Access Journals (Sweden)

    Gabriella eTeti

    2015-10-01

    Full Text Available Stem cells from human dental pulp have been considered as an alternative source of adult stem cells in tissue engineering because of their potential to differentiate into multiple cell lineages.Recently, polysaccharide based hydrogels have become especially attractive as matrices for the repair and regeneration of a wide variety of tissues and organs. The incorporation of inorganic minerals as hydroxyapatite nanoparticles can modulate the performance of the scaffolds with potential applications in tissue engineering. The aim of this study was to verify the osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs cultured on a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Human DPSCs were seeded on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel and on carboxymethyl cellulose hydrogel for 1, 3, 5, 7, 14 and 21 days. Cell viability assay and ultramorphological analysis were carried out to evaluate biocompatibility and cell adhesion. Real Time PCR was carried out to demonstrate the expression of osteogenic and odontogenic markers. Results showed a good adhesion and viability in cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel, while a low adhesion and viability was observed in cells cultured on carboxymethyl cellulose hydrogel. Real Time PCR data demonstrated a temporal up-regulation of osteogenic and odontogenic markers in dental pulp stem cells cultured on carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. In conclusion, our in vitro data confirms the ability of DPSCs to differentiate toward osteogenic and odontogenic lineages in presence of a carboxymethyl cellulose—hydroxyapatite hybrid hydrogel. Taken together, our results provide evidence that DPSCs and carboxymethyl cellulose—hydroxyapatite hybrid hydrogel could be considered promising candidates for dental pulp complex and periodontal tissue engineering.

  4. A bioceramic with enhanced osteogenic properties to regulate the function of osteoblastic and osteocalastic cells for bone tissue regeneration.

    Science.gov (United States)

    Roohani-Esfahani, Seyed-Iman; No, Young Jung; Lu, Zufu; Ng, Pei Ying; Chen, Yongjuan; Shi, Jeffrey; Pavlos, Nathan J; Zreiqat, Hala

    2016-01-01

    Bioceramics for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast and osteoclast cells. Osteogenic properties of the material are essential for rapid bone regeneration and new bone formation. The aim of this study was to develop a silicate-based ceramic, gehlenite (GLN, Ca2Al2SiO7), and characterise its physiochemical, biocompatibility and osteogenic properties. A pure GLN powder was synthesised by a facile reactive sintering method and compacted to disc-shaped specimens. The sintering behaviour and degradation of the GLN discs in various buffer solutions were fully characterised. The cytotoxicity of GLN was evaluated by direct and indirect methods. In the indirect method, primary human osteoblast cells (HOBs) were exposed to diluted extracts (100, 50, 25, 12.5 and 6.25 mg ml(-1)) of fine GLN particles in culture medium. The results showed that the extracts did not cause any cytotoxic effect on the HOBs with the number of cells increasing significantly from day 1 to day 7. GLN-supported HOB attachment and proliferation, and significantly enhanced osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein) were compared with biphasic calcium phosphate groups (BCP, a mixture of hydroxyapatite (60wt.%) and β-tricalcium phosphate(40wt.%)). We also demonstrated that in addition to supporting HOB attachment and proliferation, GLN promoted the formation of tartrate-acid resistance phosphatase (TRAP) positive multinucleated osteoclastic cells (OCs) derived from mouse bone marrow cells. Results also demonstrated the ability of GLN to support the polarisation of OCs, a prerequisite for their functional resorptive activity which is mainly influenced by the composition and degradability of biomaterials. Overall, the developed GLN is a prospective candidate to be used in bone regeneration applications due its effective osteogenic properties and biocompatibility. PMID

  5. Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Yi-Chun Kuo

    Full Text Available Surface plasmon resonance (SPR biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs. OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

  6. Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells.

    Science.gov (United States)

    Kuo, Yi-Chun; Ho, Jennifer H; Yen, Ta-Jen; Chen, How-Foo; Lee, Oscar Kuang-Sheng

    2011-01-01

    Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

  7. Osteogenic potential of human calcitonin gene-related peptide alpha gene-modified bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    WANG Yi-sheng; WANG Ya-han; ZHAO Guo-qiang; LI Yue-bai

    2011-01-01

    Background Most of the basic and clinical studies of osteonecrosis of the femoral head (ONFH) are restricted to bone tissues only,whereas various systems are involved in the onset and development of ONFH,including nervous system.Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism,and plays key roles in the growth,repair and reconstruction of bone.Calcitonin gene-related peptide (CGRP),which is secreted by peptidergic nerve,is the main mediator of bone metabolism.It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Additionally,it enhances the osteoblast mass and the rate of osteoblast formation,and reduces the bone resorption by acting on osteoblasts and osteoclasts.Hence,we aimed to construct recombinant retrovirus vector pLNCX2-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs (BMSCs/pLNCX2-hCGRPα) after virus infection.Methods The constructed recombinant retrovirus vector pLNCX2-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000.Virus was collected for BMSCs infection.The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting,respectively.The cell proliferation was determined by methyl thiazoleterazolium (MTT) assay.The osteogenic potential of BMSCs was evaluated by alkaline phosphatase (ALP) activity.Results Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX2-hCGRPα cells.These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs (P <0.05).Conclusion BMSCs/pLNCX2-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.

  8. Mineralization Content Alters Osteogenic Responses of Bone Marrow Stromal Cells on Hydroxyapatite/Polycaprolactone Composite Nanofiber Scaffolds

    OpenAIRE

    Popat, Ketul C.; Timothy T. Ruckh; Weaver, Justin R.; Carroll, Derek A.

    2012-01-01

    Synthetic tissue scaffolds have a high potential impact for patients experiencing osteogenesis imperfecta. Using electrospinning, tissue scaffolds composed of hydroxyapatite/polycaprolactone (HAp/PCL) composite nanofibers were fabricated with two different HAp concentrations—1% and 10% of the solid scaffold weight. After physico-chemical scaffold characterization, rat bone marrow stromal cells were cultured on the composite scaffolds in maintenance medium and then in osteogenic medi...

  9. Thermally labile components of aqueous humor potently induce osteogenic potential in adipose-derived mesenchymal stem cells.

    Science.gov (United States)

    Morgan, Joshua T; Kwon, Heung Sun; Wood, Joshua A; Borjesson, Dori L; Tomarev, Stanislav I; Murphy, Christopher J; Russell, Paul

    2015-06-01

    Adipose-derived mesenchymal stem cells (ASCs) hold promise for use in cell-based therapies. Their intrinsic anti-inflammatory properties are potentially useful for treatments of inflammatory conditions such as uveitis, while their ability to differentiate along multiple cell lineages suggests use in regenerating damaged or degenerated tissue. However, how ASCs will respond to the intraocular environment is poorly studied. We have recently reported that aqueous humor (AH), the fluid that nourishes the anterior segment of the eye, potently increases alkaline phosphatase (ALP) activity of ASCs, indicating osteogenic differentiation. Here, we expand on our previous findings to better define the nature of this response. To this end, we cultured ASCs in the presence of 0, 5, 10, and 20% AH and assayed them for ALP activity. We found ALP activity correlates with increasing AH concentrations from 5 to 20%, and that longer treatments result in increased ALP activity. By using serum free media and pretreating AH with dextran-coated charcoal, we found that serum and charcoal-adsorbable AH components augment but are not required for this response. Further, by heat-treating the AH, we established that thermally labile components are required for the osteogenic response. Finally, we showed myocilin, a protein present in AH, could induce ALP activity in ASCs. However, this was to a lesser extent than untreated 5% AH, and myocilin could only partially rescue the effect after heat treatment, documenting there were additional thermally labile constituents of AH involved in the osteogenic response. Our work adds to the understanding of the induction of ALP in ASCs following exposure to AH, providing important insight in how ASCs will be influenced by the ocular environment. In conclusion, increased osteogenic potential upon exposure to AH represents a potential challenge to developing ASC cell-based therapies directed at the eye.

  10. Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts

    OpenAIRE

    Siddiqui, Sahabjada; Arshad, Mohammad

    2014-01-01

    Background Osteoporosis is one of the prevalent diseases in ageing populations. Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder. Punica granatum (PG) represent a potent fruit-bearing medicinal herb which exerted valuable anti-osteoporotic activities. The present study was carried out to validate the in vitro osteogenic effects of the PG seed extract in primary calvarial osteoblast cultures harvested from neonatal ...

  11. Single-Layer Graphene Enhances the Osteogenic Differentiation of Human Mesenchymal Stem Cells In Vitro and In Vivo.

    Science.gov (United States)

    Liu, Yunsong; Chen, Tong; Du, Feng; Gu, Ming; Zhang, Ping; Zhang, Xiao; Liu, Jianzhang; Lv, Longwei; Xiong, Chunyang; Zhou, Yongsheng

    2016-06-01

    In recent years, although several studies have demonstrated the potential of graphene-coated substrates in promoting attachment, proliferation and differentiation of osteoblasts and mesenchymal stem cells (MSCs), the effects of single-layer graphene on the osteogenic differentiation of human MSCs (hMSCs) remains unclear, especially in vivo. In this study, we transferred chemical vapor deposition (CVD) grown single-layer graphene to glass slides and observed its effects on adhesion, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs) in vitro. Then, in vivo, we incubated hASCs and hBMMSCs on single-layer graphene-coated smooth titanium (Ti) disks before implanting them into the back subcutaneous area of nude mice. We found that single-layer graphene accelerated cell adhesion to the substrate without influencing cell proliferation of hMSCs. Moreover, we present the first study that explores the epigenetic role of single-layer graphene in determining stem cell fate. By utilizing epigenetic approaches, we reveal that single-layer graphene promotes osteogenic differentiation of hMSCs both in vitro and in vivo, potentially by upregulating methylation of H3K4 at the promoter regions of osteogenesis-associated genes. Overall, our results highlight the potential of this material in implants and injured tissues in clinical applications. PMID:27319220

  12. Osteogenic differentiation of stem cells from human exfoliated deciduous teeth on poly(ε-caprolactone) nanofibers containing strontium phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Su, Wen-Ta, E-mail: f10549@ntut.edu.tw [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Wu, Pai-Shuen [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan (China); Huang, Te-Yang [Department of Orthopedic Surgery, Mackay Memorial Hospital, Taipei, Taiwan (China)

    2015-01-01

    Mimicking the architecture of the extracellular matrix is an effective strategy for tissue engineering. Composite nanofibers similar to natural bone structure can be prepared via an electrospinning technique and used in biomedical applications. Stem cells from human exfoliated deciduous teeth (SHEDs) can differentiate into multiple cell lineages, such as cells that are alternative sources of stem cells for tissue engineering. Strontium has important functions in bone remodeling; for example, this element can simulate bone formation and decrease bone resorption. Incorporating strontium phosphate into nanofibers provides a potential material for bone tissue engineering. This study investigated the potential of poly(ε-caprolactone) (PCL) nanofibers coated or blended with strontium phosphate for the osteogenic differentiation of SHEDs. Cellular morphology and MTT assay revealed that nanofibers effectively support cellular attachment, spreading, and proliferation. Strontium-loaded PCL nanofibers exhibited higher expressions of collagen type I, alkaline phosphatase, biomineralization, and bone-related genes than pure PCL nanofibers during the osteogenic differentiation of SHEDs. This study demonstrated that strontium can be an effective inducer of osteogenesis for SHEDs. Understanding the function of bioceramics (such as strontium) is useful in designing and developing strategies for bone tissue engineering. - Highlights: • SHEDs have been considered as alternative sources of adult stem cells in tissue engineering. • Strontium phosphate into nanofibers provides a potential material for bone tissue engineering. • Nanofibers coated or blended with strontium phosphate for the osteogenic differentiation of SHEDs.

  13. Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds

    Science.gov (United States)

    di Luca, Andrea; Ostrowska, Barbara; Lorenzo-Moldero, Ivan; Lepedda, Antonio; Swieszkowski, Wojcech; van Blitterswijk, Clemens; Moroni, Lorenzo

    2016-03-01

    Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.

  14. Osteogenic Capacity of Human Adipose-Derived Stem Cells is Preserved Following Triggering of Shape Memory Scaffolds.

    Science.gov (United States)

    Tseng, Ling-Fang; Wang, Jing; Baker, Richard M; Wang, Guirong; Mather, Patrick T; Henderson, James H

    2016-08-01

    Recent advances in shape memory polymers have enabled the study of programmable, shape-changing, cytocompatible tissue engineering scaffolds. For treatment of bone defects, scaffolds with shape memory functionality have been studied for their potential for minimally invasive delivery, conformal fitting to defect margins, and defect stabilization. However, the extent to which the osteogenic differentiation capacity of stem cells resident in shape memory scaffolds is preserved following programmed shape change has not yet been determined. As a result, the feasibility of shape memory polymer scaffolds being employed in stem cell-based treatment strategies remains unclear. To test the hypothesis that stem cell osteogenic differentiation can be preserved during and following triggering of programmed architectural changes in shape memory polymer scaffolds, human adipose-derived stem cells were seeded in shape memory polymer foam scaffolds or in shape memory polymer fibrous scaffolds programmed to expand or contract, respectively, when warmed to body temperature. Osteogenic differentiation in shape-changing and control scaffolds was compared using mineral deposition, protein production, and gene expression assays. For both shape-changing and control scaffolds, qualitatively and quantitatively comparable amounts of mineral deposition were observed; comparable levels of alkaline phosphatase activity were measured; and no significant differences in the expression of genetic markers of osteogenesis were detected. These findings support the feasibility of employing shape memory in scaffolds for stem cell-based therapies for bone repair.

  15. Single-Layer Graphene Enhances the Osteogenic Differentiation of Human Mesenchymal Stem Cells In Vitro and In Vivo.

    Science.gov (United States)

    Liu, Yunsong; Chen, Tong; Du, Feng; Gu, Ming; Zhang, Ping; Zhang, Xiao; Liu, Jianzhang; Lv, Longwei; Xiong, Chunyang; Zhou, Yongsheng

    2016-06-01

    In recent years, although several studies have demonstrated the potential of graphene-coated substrates in promoting attachment, proliferation and differentiation of osteoblasts and mesenchymal stem cells (MSCs), the effects of single-layer graphene on the osteogenic differentiation of human MSCs (hMSCs) remains unclear, especially in vivo. In this study, we transferred chemical vapor deposition (CVD) grown single-layer graphene to glass slides and observed its effects on adhesion, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBMMSCs) in vitro. Then, in vivo, we incubated hASCs and hBMMSCs on single-layer graphene-coated smooth titanium (Ti) disks before implanting them into the back subcutaneous area of nude mice. We found that single-layer graphene accelerated cell adhesion to the substrate without influencing cell proliferation of hMSCs. Moreover, we present the first study that explores the epigenetic role of single-layer graphene in determining stem cell fate. By utilizing epigenetic approaches, we reveal that single-layer graphene promotes osteogenic differentiation of hMSCs both in vitro and in vivo, potentially by upregulating methylation of H3K4 at the promoter regions of osteogenesis-associated genes. Overall, our results highlight the potential of this material in implants and injured tissues in clinical applications.

  16. Differentiations and Functional State of Osteogenic Cells in Conditions of Microgravity

    Science.gov (United States)

    Onishchenko, Ganna; Rodionova, Natalia; Markevich, Ganna; Markevich, Ganna

    The space flight factors (space radiation, magnetic fields etc.) affect considerably the state of bone tissue, leading to the development of osteoporosis and osteopenia in the bone skeleton. Many aspects of reactions of bone tissue cells still remain unclear until now. With the use of electron microscopy and autoradiography with 3H-thymidine we studied the samples gathered from the femoral bone epiphyses and metaphyses of rats flown on board American Spacelab -2 and in experiments with modeling of microgravity ("tail suspension" method). In our work the main attention is focused on studying the ultrastructure and metabolism of osteogenetic cells. The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis: rough endoplasmic reticulum (RER), Golgy complex (GC), as well as the state of mitochondria and cell nucleus. As compared with a control, the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms. We can judge of this from the reduction volume of RER, GC, mitochondria in osteoblasts. RER loses architectonics typical for osteoblasts and, as against the control, is represented by short narrow canaliculi distributed throughout the cy-toplasm; some canals disintegrate. GC is slightly pronounced, mitochondria become smaller in size and acquire an optically dark matrix. These phenomena are supposed to be associated with the desorganization of microtubules and microfilaments in the cells under microgravity condi-tions. The number of degrading and apoptotic cells increases in the population of osteoblasts. The dynamics of labeled cells following various intervals after 3H-thymidine injection testifies to a delay in the rates of osteoblasts' differentiation and their transformation to osteocytes in the experiment animals. A lower 3H-glycine uptake by the osteogenic cells and bone matrix as compared with a control is

  17. Sympathetic denervation-induced MSC mobilization in distraction osteogenesis associates with inhibition of MSC migration and osteogenesis by norepinephrine/adrb3.

    Directory of Open Access Journals (Sweden)

    Zhaojie Du

    Full Text Available The sympathetic nervous system regulates bone formation and resorption under physiological conditions. However, it is still unclear how the sympathetic nerves affect stem cell migration and differentiation in bone regeneration. Distraction osteogenesis is an ideal model of bone regeneration due to its special nature as a self-engineering tissue. In this study, a rat model of mandibular distraction osteogenesis with transection of cervical sympathetic trunk was used to demonstrate that sympathetic denervation can deplete norepinephrine (NE in distraction-induced bone callus, down-regulate β3-adrenergic receptor (adrb3 in bone marrow mesenchymal stem cells (MSCs, and promote MSC migration from perivascular regions to bone-forming units. An in vitro Transwell assay was here used to demonstrate that NE can inhibit stroma-derived factor-1 (SDF-1-induced MSC migration and expression of the migration-related gene matrix metalloproteinase-2 (MMP-2 and downregulate that of the anti-migration gene tissue inhibitor of metalloproteinase-3 (TIMP-3. Knockdown of adrb3 using siRNA abolishes inhibition of MSC migration. An in vitro osteogenic assay was used to show that NE can inhibit the formation of MSC bone nodules and expression of the osteogenic marker genes alkaline phosphatase (ALP, osteocalcin (OCN, and runt-related transcription factor-2 (RUNX2, but knockdown of adrb3 by siRNA can abolish such inhibition of the osteogenic differentiation of MSCs. It is here concluded that sympathetic denervation-induced MSC mobilization in rat mandibular distraction osteogenesis is associated with inhibition of MSC migration and osteogenic differentiation by NE/adrb3 in vitro. These findings may facilitate understanding of the relationship of MSC mobilization and sympathetic nervous system across a wide spectrum of tissue regeneration processes.

  18. Sympathetic denervation-induced MSC mobilization in distraction osteogenesis associates with inhibition of MSC migration and osteogenesis by norepinephrine/adrb3.

    Science.gov (United States)

    Du, Zhaojie; Wang, Lei; Zhao, Yinghua; Cao, Jian; Wang, Tao; Liu, Peng; Zhang, Yabo; Yang, Xinjie; Cheng, Xiaobing; Liu, Baolin; Lei, Delin

    2014-01-01

    The sympathetic nervous system regulates bone formation and resorption under physiological conditions. However, it is still unclear how the sympathetic nerves affect stem cell migration and differentiation in bone regeneration. Distraction osteogenesis is an ideal model of bone regeneration due to its special nature as a self-engineering tissue. In this study, a rat model of mandibular distraction osteogenesis with transection of cervical sympathetic trunk was used to demonstrate that sympathetic denervation can deplete norepinephrine (NE) in distraction-induced bone callus, down-regulate β3-adrenergic receptor (adrb3) in bone marrow mesenchymal stem cells (MSCs), and promote MSC migration from perivascular regions to bone-forming units. An in vitro Transwell assay was here used to demonstrate that NE can inhibit stroma-derived factor-1 (SDF-1)-induced MSC migration and expression of the migration-related gene matrix metalloproteinase-2 (MMP-2) and downregulate that of the anti-migration gene tissue inhibitor of metalloproteinase-3 (TIMP-3). Knockdown of adrb3 using siRNA abolishes inhibition of MSC migration. An in vitro osteogenic assay was used to show that NE can inhibit the formation of MSC bone nodules and expression of the osteogenic marker genes alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor-2 (RUNX2), but knockdown of adrb3 by siRNA can abolish such inhibition of the osteogenic differentiation of MSCs. It is here concluded that sympathetic denervation-induced MSC mobilization in rat mandibular distraction osteogenesis is associated with inhibition of MSC migration and osteogenic differentiation by NE/adrb3 in vitro. These findings may facilitate understanding of the relationship of MSC mobilization and sympathetic nervous system across a wide spectrum of tissue regeneration processes. PMID:25144690

  19. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells’ osteogenic differentiation

    Directory of Open Access Journals (Sweden)

    Tai IC

    2015-09-01

    Full Text Available I-Chun Tai,1–3 Yao-Hsien Wang,3 Chung-Hwan Chen,3,4 Shu-Chun Chuang,3 Je-Ken Chang,3–5 Mei-Ling Ho1–3,6 1Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 2Department of Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 3Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; 4Department of Orthopedics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; 5Department of Orthopedics, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan; 6Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung, Taiwan Abstract: Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an

  20. Protocols for in vitro Differentiation of Human Mesenchymal Stem Cells into Osteogenic, Chondrogenic and Adipogenic Lineages.

    Science.gov (United States)

    Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Musarò, Paola; Turco, Valentina; Gnecchi, Massimiliano

    2016-01-01

    Mesenchymal stem cells (MSC) possess high plasticity and the potential to differentiate into several different cell types; this characteristic has implications for cell therapy and reparative biotechnologies. MSC have been originally isolated from the bone marrow (BM-MSC), but they have been found also in other tissues such as adipose tissue, cord blood, synovium, skeletal muscle, and lung. MSC are able to differentiate in vitro and in vivo into several cell types such as bone, osteocytes, chondrocytes, adipocytes, and skeletal myocytes, just to name a few.During the last two decades, an increasing number of studies have proven the therapeutic potential of MSC for the treatment of neurodegenerative diseases, spinal cord and brain injuries, cardiovascular diseases, diabetes mellitus, and diseases of the skeleton. Their immuno-privileged profile allows both autologous and allogeneic use. For all these reasons, the scientific appeal of MSC is constantly on the rise.The identity of MSC is currently based on three main criteria: plastic-adherence capacity, defined epitope profile, and capacity to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. Here, we describe standard protocols for the differentiation of BM-MSC into the osteogenic, chondrogenic, and adipogenic lineages. PMID:27236670

  1. Ground reaction forces and osteogenic index of the sport of cyclocross.

    Science.gov (United States)

    Tolly, Brian; Chumanov, Elizabeth; Brooks, Alison

    2014-01-01

    Weight-bearing activity has been shown to increase bone mineral density. Our purpose was to measure vertical ground reaction forces (GRFs) during cyclocross-specific activities and compute their osteogenic index (OI). Twenty-five healthy cyclocross athletes participated. GRF was measured using pressure-sensitive insoles during seated and standing cycling and four cyclocross-specific activities: barrier flat, barrier uphill, uphill run-up, downhill run-up. Peak and mean GRF values, according to bodyweight, were determined for each activity. OI was computed using peak GRF and number of loading cycles. GRF and OI were compared across activities using repeated-measures ANOVA. Number of loading cycles per activity was 6(1) for barrier flat, 8(1) barrier uphill, 7(1) uphill run-up, 12(3) downhill run-up. All activities had significantly (P < 0.01) higher peak GRF, mean GRF values and OI when compared to both seated and standing cycling. The barrier flat condition (P < 0.01) had highest peak (2.9 times bodyweight) and mean GRF values (2.3 times bodyweight). Downhill run-up (P < 0.01) had the highest OI (6.5). GRF generated during the barrier flat activity is similar in magnitude to reported GRFs during running and hopping. Because cyclocross involves weight bearing components, it may be more beneficial to bone health than seated road cycling.

  2. Surface hydrophilicity of PLGA fibers governs in vitro mineralization and osteogenic differentiation.

    Science.gov (United States)

    Thomas, Minnah; Arora, Aditya; Katti, Dhirendra S

    2014-12-01

    Interfacial properties of biomaterials play an important role in governing their interaction with biological microenvironments. This work investigates the role of surface hydrophilicity of electrospun poly(lactide-co-glycolide) (PLGA) fibers in determining their biological response. For this, PLGA is blended with varying amounts of Pluronic®F-108 and electrospun to fabricate microfibers with varying surface hydrophilicity. The results of mineralization study in simulated body fluid (SBF) demonstrate a significant enhancement in mineralization with an increase in surface hydrophilicity. While presence of serum proteins in SBF reduces absolute mineral content, mineralization continues to be higher on samples with higher surface hydrophilicity. The results from in vitro cell culture studies demonstrate a marked improvement in mesenchymal stem cell-adhesion, elongation, proliferation, infiltration, osteogenic differentiation and matrix mineralization on hydrophilized fibers. Therefore, hydrophilized PLGA fibers are advantageous both in terms of mineralization and elicitation of favorable cell response. Since most of the polymeric materials being used in orthopedics are hydrophobic in nature, the results from this study have strong implications in the future design of interfaces of such hydrophobic materials. In addition, the work proposes a facile method for the modification of electrospun fibers of hydrophobic polymers by blending with a poloxamer for improved bone tissue regeneration.

  3. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Alessandra Pisciotta

    Full Text Available Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS. FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

  4. Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation

    Science.gov (United States)

    Correia, Clara R.; Pirraco, Rogério P.; Cerqueira, Mariana T.; Marques, Alexandra P.; Reis, Rui L.; Mano, João F.

    2016-02-01

    A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

  5. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

    DEFF Research Database (Denmark)

    Harkness, Linda M; Mahmood, Amer; Ditzel, Nicholas;

    2011-01-01

    The derivation of osteogenic cells from human embryonic stem cells (hESC) has been hampered by the absence of easy and reproducible protocols. hESC grown in feeder-free conditions, often show a sub population of fibroblast-like, stromal cells growing between the colonies. Thus, we examined...... the possibility that these cells represent a population of stromal (mesenchymal) stem cells (hESC-stromal). Two in house derived hES cell lines (Odense3 and KMEB3) as well as an externally derived cell line (Hues8) were transitioned to feeder-free conditions. A sub population of fibroblast-like cells established...... between the hESC colonies were isolated by selective adherence to hyaluronic acid-coated plates (100μg/ml) and were characterized using a combination of FACS analysis and staining. The cells were CD44(+), CD29(+), CD73(+), CD166(+), CD146(+), and CD105(+); and, Oct4(-), CD34(-), CD45(-) and CXCR4(-). When...

  6. Incorporation of osteogenic and angiogenic small interfering RNAs into chitosan sponge for bone tissue engineering.

    Science.gov (United States)

    Jia, Sen; Yang, Xinjie; Song, Wen; Wang, Lei; Fang, Kaixiu; Hu, Zhiqiang; Yang, Zihui; Shan, Chun; Lei, Delin; Lu, Bin

    2014-01-01

    Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs), ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1) and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1), which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and angiogenic siRNAs may be used as a scaffold for bone regeneration. The dual siRNA concept may also be useful in the biofunctionalization of other materials. PMID:25429217

  7. Novel Implant Coating Agent Promotes Gene Expression of Osteogenic Markers in Rats during Early Osseointegration

    Directory of Open Access Journals (Sweden)

    Kostas Bougas

    2012-01-01

    Full Text Available The aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants. Forty-five rats distributed in three equally sized groups were provided with one control (turned and one test (laminin-1-coated implant and were sacrificed after 3, 7, and 21 days. Real-time reverse-transcriptase polymerase chain reaction was performed for osteoblast markers (alkaline phosphatase, runt-related transcription factor 2, osteocalcin, type I collagen, and bone morphogenic protein 2, osteoclast markers (cathepsin K and tartrate-resistant acid phosphatase, inflammation markers (tumor necrosis factor α, interleukin 1β and interleukin 10, and integrin β1. Bone implant contact (BIC and bone area (BA were assessed and compared to the gene expression. After 3 days, the expression of bone markers was higher for the control group. After 7 days, the expression of integrin β1 and osteogenic markers was enhanced for the test group, while cathepsin K and inflammation markers were down-regulated. No significant differences in BIC or BA were detected between test and control at any time point. As a conclusion, implant coating with laminin-1 altered gene expression in the bone-implant interface. However, traditional evaluation methods, as histomorphometry, were not adequately sensitive to detect such changes due to the short follow-up time.

  8. Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds.

    Science.gov (United States)

    Morelli, Sabrina; Salerno, Simona; Holopainen, Jani; Ritala, Mikko; De Bartolo, Loredana

    2015-06-20

    The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation.

  9. TGF-beta 1 Gene-Activated Matrices Regulated the Osteogenic Differentiation of BMSCs

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Poly (lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol)(PLGA-[ASP-PEG]) scaffold materials were linked with a novel nonviral vector (K)16GRGDSPC through cross linker Sulfo-LC-SPDP to construct a new type of nonviral gene transfer system. Eukaryotic expressing vector containing transforming growth factor beta 1 (pcDNA3-TGFβ1) was encapsulated by the system. Bone marrow stromal cells (BMSCs) obtained from rabbit were cultured on PLGA-[ASP-PEG] modified by (K)16GRGDSPC and TGF-β1 gene and PLGA-[ASP-PEG] modified by (K)16GRGDSPC and empty vector pcDNA3 as control.The expressions of osteogenic makers of the BMSCs cultured on the TGF-β1 gene-activated scaffold materials were found significantly higher than those of the control group (P<0.05). A brand-new way was provided for regulating seed cells to directionally differentiate into osteoblasts for bone defect restoration in bone tissue engineering.

  10. Hexosamine-Induced TGF-β Signaling and Osteogenic Differentiation of Dental Pulp Stem Cells Are Dependent on N-Acetylglucosaminyltransferase V

    Directory of Open Access Journals (Sweden)

    Yi-Jane Chen

    2015-01-01

    Full Text Available Glycans of cell surface glycoproteins are involved in the regulation of cell migration, growth, and differentiation. N-acetyl-glucosaminyltransferase V (GnT-V transfers N-acetyl-d-glucosamine to form β1,6-branched N-glycans, thus playing a crucial role in the biosynthesis of glycoproteins. This study reveals the distinct expression of GnT-V in STRO-1 and CD-146 double-positive dental pulp stem cells (DPSCs. Furthermore, we investigated three types of hexosamines and their N-acetyl derivatives for possible effects on the osteogenic differentiation potential of DPSCs. Our results showed that exogenous d-glucosamine (GlcN, N-acetyl-d-glucosamine (GlcNAc, d-mannosamine (ManN, and acetyl-d-mannosamine (ManNAc promoted DPSCs’ early osteogenic differentiation in the absence of osteogenic supplements, but d-galactosamine (GalN or N-acetyl-galactosamine (GalNAc did not. Effects include the increased level of TGF-β receptor type I, activation of TGF-β signaling, and increased mRNA expression of osteogenic differentiation marker genes. The hexosamine-treated DPSCs showed an increased mineralized matrix deposition in the presence of osteogenic supplements. Moreover, the level of TGF-β receptor type I and early osteogenic differentiation were abolished in the DPSCs transfected with siRNA for GnT-V knockdown. These results suggest that GnT-V plays a critical role in the hexosamine-induced activation of TGF-β signaling and subsequent osteogenic differentiation of DPSCs.

  11. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO{sub 2} coating with magnesium impregnation

    Energy Technology Data Exchange (ETDEWEB)

    Cecchinato, Francesca, E-mail: francesca.cecchinato@mah.se [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Karlsson, Johan [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Ferroni, Letizia; Gardin, Chiara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Galli, Silvia; Wennerberg, Ann [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Zavan, Barbara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Andersson, Martin [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Jimbo, Ryo [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki (Japan)

    2015-07-01

    The aim of this study was to evaluate the osteogenic response of human adipose-derived stromal cells (ADScs) to mesoporous titania (TiO{sub 2}) coatings produced with evaporation-induced self-assembly method (EISA) and loaded with magnesium. Our emphasis with the magnesium release functionality was to modulate progenitor cell osteogenic differentiation under standard culture conditions. Osteogenic properties of the coatings were assessed for stromal cells by means of scanning electron microscopy (SEM) imaging, colorimetric mitochondrial viability assay (MTT), colorimetric alkaline phosphates activity (ALP) assay and real time RT-polymerase chain reaction (PCR). Using atomic force microscopy (AFM) it was shown that the surface expansion area (S{sub dr}) was strongly enhanced by the presence of magnesium. From MTT results it was shown that ADSc viability was significantly increased on mesoporous surfaces compared to the non-porous one at a longer cell culture time. However, no differences were observed between the magnesium impregnated and non-impregnated surfaces. The alkaline phosphatase activity confirmed that ADSc started to differentiate into the osteogenic phenotype after 2 weeks of culturing. The gene expression profile at 2 weeks of cell growth showed that such coatings were capable to incorporate specific osteogenic markers inside their interconnected nano-pores and, at 3 weeks, ADSc differentiated into osteoblasts. Interestingly, magnesium significantly promoted the osteopontin gene expression, which is an essential gene for the early biomaterial–cell osteogenic interaction. - Highlights: • The magnesium loading presents a transitory effect on mesoporous TiO{sub 2} surface topography • The mesoporous structure promotes cellular attachment and spreading • The mesoporous structure activates osteogenesis of mesenchymal stem cells in absence of osteogenic promoters • The physical adsorbed magnesium is suggested to be involved in the expression of

  12. Cytokines TNF-α, IL-6, IL-17F, and IL-4 Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells

    Directory of Open Access Journals (Sweden)

    Angela P. Bastidas-Coral

    2016-01-01

    Full Text Available During the initial stages of bone repair, proinflammatory cytokines are released within the injury site, quickly followed by a shift to anti-inflammatory cytokines. The effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of mesenchymal stem cells is controversial. Here, we investigated the effect of the proinflammatory cytokines TNF-α, IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 on proliferation and osteogenic differentiation of human adipose stem cells (hASCs. hASCs were treated with TNF-α, IL-6, IL-8, IL-17F, or IL-4 (10 ng/mL for 72 h mimicking bone repair. TNF-α reduced collagen type I gene expression but increased hASC proliferation and ALP activity. IL-6 also strongly enhanced ALP activity (18-fold, as well as bone nodule formation by hASCs. IL-8 did not affect proliferation or osteogenic gene expression but reduced bone nodule formation. IL-17F decreased hASC proliferation but enhanced ALP activity. IL-4 enhanced osteocalcin gene expression and ALP activity but reduced RUNX2 gene expression and bone nodule formation. In conclusion, all cytokines studied have both enhancing and reducing effects on osteogenic differentiation of hASCs, even when applied for 72 h only. Some cytokines, specifically IL-6, may be suitable to induce osteogenic differentiation of mesenchymal stem cells as a strategy for enhancing bone repair.

  13. Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model

    Science.gov (United States)

    Ke, Ke; Li, Qi; Yang, Xiaofeng; Xie, Zhijian; Wang, Yu; Shi, Jue; Chi, Linfeng; Xu, Weijian; Hu, Lingling; Shi, Huali

    2016-01-01

    Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol—3 kinase/AKT signaling pathway. PMID:27756897

  14. Incorporation of Fucoidan in β-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage

    Science.gov (United States)

    Puvaneswary, Subramaniam; Raghavendran, Hanumantharao Balaji; Talebian, Sepehr; Murali, Malliga Raman; A Mahmod, Suhaeb; Singh, Simmrat; Kamarul, Tunku

    2016-01-01

    In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology. PMID:27068453

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Cytokines TNF-α, IL-6, IL-17F, and IL-4 Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells

    Science.gov (United States)

    Bravenboer, Nathalie

    2016-01-01

    During the initial stages of bone repair, proinflammatory cytokines are released within the injury site, quickly followed by a shift to anti-inflammatory cytokines. The effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of mesenchymal stem cells is controversial. Here, we investigated the effect of the proinflammatory cytokines TNF-α, IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 on proliferation and osteogenic differentiation of human adipose stem cells (hASCs). hASCs were treated with TNF-α, IL-6, IL-8, IL-17F, or IL-4 (10 ng/mL) for 72 h mimicking bone repair. TNF-α reduced collagen type I gene expression but increased hASC proliferation and ALP activity. IL-6 also strongly enhanced ALP activity (18-fold), as well as bone nodule formation by hASCs. IL-8 did not affect proliferation or osteogenic gene expression but reduced bone nodule formation. IL-17F decreased hASC proliferation but enhanced ALP activity. IL-4 enhanced osteocalcin gene expression and ALP activity but reduced RUNX2 gene expression and bone nodule formation. In conclusion, all cytokines studied have both enhancing and reducing effects on osteogenic differentiation of hASCs, even when applied for 72 h only. Some cytokines, specifically IL-6, may be suitable to induce osteogenic differentiation of mesenchymal stem cells as a strategy for enhancing bone repair.

  17. Mesenchymal Stem Cells Ageing: Targeting the "Purinome" to Promote Osteogenic Differentiation and Bone Repair.

    Science.gov (United States)

    Noronha-Matos, J B; Correia-de-Sá, P

    2016-09-01

    Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852

  18. Daily low-intensity pulsed ultrasound-mediated osteogenic differentiation in rat osteoblasts

    Institute of Scientific and Technical Information of China (English)

    Akito Suzuki; Tadahiro Takayama; Naoto Suzuki; Michitomo Sato; Takeshi Fukuda; Koichi Ito

    2009-01-01

    There were few studies investigating the effects of the mechanical stimulation provided by daily low-intensity pulsed ultrasound (LIPUS) treatment. LIPUS is known to accelerate bone mineralization and regeneration; however, the precise cellular mechanism is unclear. Our purpose was to determine how daily LIPUS treat-ment affected cell viability, alkaline phosphatase activity, osteogenesis-related gene expression, and mineralized nodule formation in osteoblasts. The typical osteoblastic cell line ROS 17/2.8 cells were cul-tured in the absence or presence of LIPUS stimulation. Daily LIPUS treatments (1.5 MHz; 20 min) were admi-nistered at an intensity of 30 mW/cm2 for 14 days. Expression of osteogenesis-related genes was examined at mRNA levels using real-time polymerase chain reac-tion and at protein levels using western blotting analy-sis. LIPUS stimulation did not affect the rate of cell viability. Alkaline phosphatase activity was increased after 10 days of culture with daily LIPUS stimulation. LIPUS significantly increased the expression of mRNAs encoding Runx2, Msx2, DIx5, osterix, bone sialoprotein,and bone morphogenetic protein-2, whereas it signifi-cantly reduced the expression of mRNA encoding the transcription factor AJ18. Mineralized nodule for-mation was markedly increased on Day 14 of LIPUS stimulation. LIPUS stimulation directly affected osteo-genic cells, leading to mineralized nodule formation. LIPUS is likely to have a fundamental influence on key functional activities of osteoblasts in alveolar bone.

  19. Effects of Tumor Necrosis Factor-alpha on the Osteogenic Differentiation of Mouse Bone Marrow-derived Mesenchymal Stem Cells%肿瘤坏死因子-α对小鼠骨髓间充质干细胞成骨分化影响研究

    Institute of Scientific and Technical Information of China (English)

    何琳琳; 兰飞; 薛小平

    2011-01-01

    Objective: To investigate the effects of tumor necrosis factor-alpha (TNF-α) on the osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods: BM-MSCs were cultured in osteogenic media (OS) with TNF-α using BM-MSCs in OS without TNF-α as a control group.Alkaline phosphatase staining was tested on the 7 days and Alizarin Red S (ARS) staining and quantification were tested on the 21 days. On the 7 days, mRNA were extracted from one set of the cells and Runx2 and Osterix mRNA expression were analysed by real-time RT-PCR , another set of the cells were lysed and Runx2 and Osterix protein expression were analysed by SDS-PAGE. Results: TNF-α inhibits alkaline phosphatase activity. TNF-α inhibits mineralized nodules formation. TNF-α inhibits Runx2 and Osterix mRNA and protein expression.Conclusion: TNF-α inhibition of the osteogenic differentiation of mouse BM-MSCs, in part, is through suppressing Runx2 and Osterix mRNA and protein expression which are the two key transcription factors of osteogenic differentiation.%目的:探讨肿瘤坏死因子-α(Tumor Necrosis Factor alpha,TNF-α)对小鼠骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells,BM-MSCs)成骨分化的影响.方法:以在促成骨分化培养基(Osteogenic media,OS)中培养的小鼠BM-MSCs作为对照,用含有TNF-α的OS处理小鼠BM-MSCs.第7天进行碱性磷酸酶染色,或培养第21天进行茜素红染色和定量分析.第7天时用一份细胞提取得到的mRNA通过实时定量RT-PCR测定Runx2和Osterix的mRNA表达,用另一份细胞得到的细胞裂解液通过SDS-PAGE来测定Runx2和Osterix的蛋白质表达.结果:TNF-α抑制碱性磷酸酶活性;TNF-α抑制矿化骨节的形成;TNF-α抑制Runx2和Osterix的mRNA和蛋白质表达.结论:TNF-α对小鼠BM-MSCs成骨分化的抑制,可能部分是通过抑制成骨分化中两个关键的转录因子Runx2和Osterix的mRNA和蛋白质表达来实现的.

  20. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

    Science.gov (United States)

    Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

    2015-01-01

    An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces. PMID:26214191

  1. Surface functionalization of nanoporous alumina with bone morphogenetic protein 2 for inducing osteogenic differentiation of mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yuanhui; Ju, Yang; Morita, Yasuyuki; Xu, Baiyao [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Song, Guanbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-04-01

    Many studies have demonstrated the possibility to regulate cellular behavior by manipulating the specific characteristics of biomaterials including the physical features and chemical properties. To investigate the synergistic effect of chemical factors and surface topography on the growth behavior of mesenchymal stem cells (MSCs), bone morphorgenic protein 2 (BMP2) was immobilized onto porous alumina substrates with different pore sizes. The BMP2-immobilized alumina substrates were characterized with scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Growth behavior and osteogenic differentiation of MSCs cultured on the different substrates were investigated. Cell adhesion and morphological changes were observed with SEM, and the results showed that the BMP2-immobilized alumina substrate was able to promote adhesion and spreading of MSCs. MTT assay and immunofluorescence staining of integrin β1 revealed that the BMP2-immobilized alumina substrates were favorable for cell growth. To evaluate the differentiation of MSCs, osteoblastic differentiation markers, such as alkaline phosphatase (ALP) activity and mineralization, were investigated. Compared with those of untreated alumina substrates, significantly higher ALP activities and mineralization were detected in cells cultured on BMP2-immobilized alumina substrates. The results suggested that surface functionalization of nanoporous alumina substrates with BMP2 was beneficial for cell growth and osteogenic differentiation. With the approach of immobilizing growth factors onto material substrates, it provided a new insight to exploit novel biofunctional materials for tissue engineering. - Highlights: • BMP2 was immobilized onto nanoporous alumina substrates with different pore sizes. • BMP2-immobilized substrates were able to promote adhesion and spreading of MSCs. • BMP2-immobilized substrates were favorable for cell growth of MSCs. • BMP2-immobilized substrates promoted osteogenic

  2. Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Wang, Yuli; Yin, Ying; Jiang, Fei; Chen, Ning

    2015-02-01

    Human amnion mesenchymal stem cells (HAMSCs) can be obtained from human amniotic membrane, a highly abundant and readily available tissue. HAMSC sources present fewer ethical issues, have low immunogenicity, anti-inflammatory properties, considerable advantageous characteristics, and are considered an attractive potential treatment material in the field of regenerative medicine. We used a co-culture system to determine whether HAMSCs could promote osteogenesis in human bone marrow mesenchymal stem cells (HBMSCs). We isolated HAMSCs from discarded amnion samples and collected them using pancreatin/collagenase digestion. We cultured HAMSCs and HBMSCSs in basal medium. Activity of alkaline phosphatase (ALP), an early osteogenesis marker, was increased in the co-culture system compared to the control single cultures, which we also confirmed by ALP staining. We used immunofluorescence testing to investigate the effects of co-culturing with HAMSCs on HBMSC proliferation, which revealed that the co-culturing enhanced EdU expression in HBMSCs. Western blotting and quantitative real-time PCR indicated that co-culturing promoted osteogenesis in HBMSCs. Furthermore, Alizarin red S staining revealed that extracellular matrix calcium levels in mineralized nodule formation produced by the co-cultures were higher than that in the controls. Using the same co-culture system, we further observed the effects of HAMSCs on osteogenic differentiation in primary osteoblasts by Western blotting, which better addressed the mechanism for HAMSCs in bone regeneration. The results showed HAMSCs are osteogenic and not only play a role in promoting HBMSC proliferation and osteogenic differentiation but also in osteoblasts, laying the foundation for new regenerative medicine methods.

  3. Improvement of in vitro physicochemical properties and osteogenic activity of calcium sulfate cement for bone repair by dicalcium silicate

    International Nuclear Information System (INIS)

    Highlights: • Dicalcium silicate can improve osteogenic activity of calcium sulfate cement. • The higher the calcium sulfate content, the shorter the setting time in the composite cement. • The results were useful for designing calcium-based cement with optimal properties. -- Abstract: An ideal bone graft substitute should have the same speed of degradation as formation of new bone tissue. To improve the properties of calcium sulfate hemihydrate (CSH) featured for its rapid resorption, a low degradation material of dicalcium silicate (DCS) was added to the CSH cement. This study examined the effect of DCS (20, 40, 60 and 80 wt%) on the in vitro physicochemical properties and osteogenic activities of the calcium-based composite cements. The diametral tensile strength, porosity and weight loss of the composite cements were evaluated before and after soaking in a simulated body fluid (SBF). The osteogenic activities, such as proliferation, differentiation and mineralization, of human mesenchymal stem cells (hMSCs) seeded on cement surfaces were also examined. As a result, the greater the DCS amount, the higher the setting time was in the cement. Before soaking in SBF, the diametral tensile strength of the composite cements was decreased due to the introduction of DCS. On 180-day soaking, the composite cements containing 20, 40, 60 and 80 wt% DCS lost 80%, 69%, 61% and 44% in strength, respectively. Regarding in vitro bioactivity, the DCS-rich cements were covered with clusters of apatite spherulites after soaking for 7 days, while there was no formation of apatite spherulites on the CSH-rich cement surfaces. The presence of DCS could reduce the degradation of the CSH cements, as evidenced in the results of weight loss and porosity. More importantly, DCS may promote effectively the cell proliferation, proliferation and mineralization. The combination of osteogenesis of DCS and degradation of CSH made the calcium-based composite cements an attractive choice for

  4. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

    Science.gov (United States)

    Yan, Xueying; Ehnert, Sabrina; Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A; Pelisek, Jaroslav; Nussler, Andreas K

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  5. 5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

    Directory of Open Access Journals (Sweden)

    Xueying Yan

    Full Text Available The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a as compared to Ad-MSCs isolated from younger donors (<45 a. 5-hydroxymethylcytosine (5 hmC and 5-methylcytonsine (5 mC distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors.

  6. 5-Azacytidine Improves the Osteogenic Differentiation Potential of Aged Human Adipose-Derived Mesenchymal Stem Cells by DNA Demethylation

    Science.gov (United States)

    Culmes, Mihaela; Bachmann, Anastasia; Seeliger, Claudine; Schyschka, Lilianna; Wang, Zhiyong; Rahmanian-Schwarz, Afshin; Stöckle, Ulrich; De Sousa, Paul A.; Pelisek, Jaroslav; Nussler, Andreas K.

    2014-01-01

    The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors. PMID:24603866

  7. A genome wide analysis of alternative splicing events during the osteogenic differentiation of human cartilage endplate-derived stem cells.

    Science.gov (United States)

    Shang, Jin; Wang, Honggang; Fan, Xin; Shangguan, Lei; Liu, Huan

    2016-08-01

    Low back pain is a prevalent disease, which leads to suffering and disabilities in a vast number of individuals. Degenerative disc diseases are usually the underlying causes of low back pain. However, the pathogenesis of degenerative disc diseases is highly complex and difficult to determine. Current therapies for degenerative disc diseases are various. In particular, cell-based therapies have proven to be effective and promising. Our research group has previously isolated and identified the cartilage endplate‑derived stem cells. In addition, alternative splicing is a sophisticated regulatory mechanism, which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. The present study continued to investigate alternative splicing events in osteogenic differentiation of cartilage endplate‑derived stem cells. An Affymetrix Human Transcriptome Array 2.0 was used to detect splicing changes between the control and differentiated samples. Additionally, molecular function and pathway analysis were also performed. Following rigorous bioinformatics analysis of the data, 3,802 alternatively spliced genes were identified, and 10 of these were selected for validation by reverse transcription‑polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis also revealed numerous enriched GO terms and signaling pathways. To the best of our knowledge, the present study is the first to investigate alternative splicing mechanisms in osteogenic differentiation of stem cells on a genome‑wide scale. The illumination of molecular mechanisms of stem cell osteogenic differentiation may assist the development novel bioengineered methods to treat degenerative disc diseases.

  8. A genome wide analysis of alternative splicing events during the osteogenic differentiation of human cartilage endplate-derived stem cells.

    Science.gov (United States)

    Shang, Jin; Wang, Honggang; Fan, Xin; Shangguan, Lei; Liu, Huan

    2016-08-01

    Low back pain is a prevalent disease, which leads to suffering and disabilities in a vast number of individuals. Degenerative disc diseases are usually the underlying causes of low back pain. However, the pathogenesis of degenerative disc diseases is highly complex and difficult to determine. Current therapies for degenerative disc diseases are various. In particular, cell-based therapies have proven to be effective and promising. Our research group has previously isolated and identified the cartilage endplate‑derived stem cells. In addition, alternative splicing is a sophisticated regulatory mechanism, which greatly increases cellular complexity and phenotypic diversity of eukaryotic organisms. The present study continued to investigate alternative splicing events in osteogenic differentiation of cartilage endplate‑derived stem cells. An Affymetrix Human Transcriptome Array 2.0 was used to detect splicing changes between the control and differentiated samples. Additionally, molecular function and pathway analysis were also performed. Following rigorous bioinformatics analysis of the data, 3,802 alternatively spliced genes were identified, and 10 of these were selected for validation by reverse transcription‑polymerase chain reaction. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis also revealed numerous enriched GO terms and signaling pathways. To the best of our knowledge, the present study is the first to investigate alternative splicing mechanisms in osteogenic differentiation of stem cells on a genome‑wide scale. The illumination of molecular mechanisms of stem cell osteogenic differentiation may assist the development novel bioengineered methods to treat degenerative disc diseases. PMID:27278552

  9. Effects of immunosuppressants, FK506 and cyclosporin A, on the osteogenic differentiation of rat mesenchymal stem cells

    OpenAIRE

    Byun, Yu-Kyung; Kim, Kyoung-Hwa; Kim, Su-Hwan; Kim, Young-Sung; Koo, Ki-Tae; Kim, Tai-Il; Seol, Yang-Jo; Ku, Young; Rhyu, In-Chul; Lee, Yong-Moo

    2012-01-01

    Purpose The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done ...

  10. Shear stress induced by an interstitial level of slow flow increases the osteogenic differentiation of mesenchymal stem cells through TAZ activation.

    Directory of Open Access Journals (Sweden)

    Kyung Min Kim

    Full Text Available Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif, a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

  11. The collagen I mimetic peptide DGEA enhances an osteogenic phenotype in mesenchymal stem cells when presented from cell-encapsulating hydrogels.

    Science.gov (United States)

    Mehta, Manav; Madl, Christopher M; Lee, Shimwoo; Duda, Georg N; Mooney, David J

    2015-11-01

    Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.

  12. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    Energy Technology Data Exchange (ETDEWEB)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L. [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal); Gomes, Manuela E., E-mail: megomes@dep.uminho.pt [3Bs Research Group–Biomaterials, Biodegradables and Biomimetics, University of Minho, Guimarães (Portugal); ICVS/3Bs–PT Government Associate Laboratory, Braga/Guimarães (Portugal)

    2015-11-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages. - Highlights: • Cellular viability was not negatively influenced by the nanoparticles. • Chondrogenic medium influences more the synthesis of cartilage-like ECM than MNPs. • Synergetic effect among

  13. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    International Nuclear Information System (INIS)

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages. - Highlights: • Cellular viability was not negatively influenced by the nanoparticles. • Chondrogenic medium influences more the synthesis of cartilage-like ECM than MNPs. • Synergetic effect among

  14. Increased Osteogenic Differentiation of Periodontal Ligament Stem Cells on Polydopamine Film Occurs via Activation of Integrin and PI3K Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Jeong Seok Lee

    2014-11-01

    Full Text Available Background/Aims: Mussel-inspired polydopamine (PDA is known to be an effective bioadhesive and bioactive material for controlling stem cell fate, which is important in stem cell-based regenerative medicine; however, the effect of PDA on osteogenic differentiation of periodontal ligament stem cells (PDLSCs is not fully understood. In this study, we investigated the osteoinductive effect of PDA on PDLSCs and examined how this phenomenon is encouraged. Methods: Osteogenic induction of PDLSCs was established by culturing cells on PDA film or on an uncoated polystyrene surface as a control. Osteogenic differentiation of PDLSCs was assessed by measurement of intracellular calcium levels and alkaline phosphatase (ALP activity as well as by evaluation of protein expression of osteocalcin (OCN, osterix (OSX, and runt-related transcription factor 2 (RUNX2. Results: The PDLSCs cultured on PDA film showed higher osteogenic activity than those on the control surface. Moreover, PDLSCs on PDA film expressed increased levels of the integrin adhesion receptors integrin α5 and β1 compared to control cells. Expression of one isoform of the intracellular signaling protein phosphatidylinositol-3-kinase (PI3K, p110γ, was increased in PDLSCs on PDA film in a PDA dose-dependent manner. This signaling protein was found to interact with integrin β1, demonstrating integrin-linked PI3K activation in response to PDA. Finally, the blockage of PI3K reduced the PDA-induced osteogenic activity of PDLSCs. Conclusion: our findings suggest that the bioadhesive PDA stimulates osteogenic differentiation of PDLSCs via activation of the integrin α5/β1 and PI3K signaling pathways.

  15. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    DEFF Research Database (Denmark)

    Wu, Kaimin; Xu, Jie; Liu, Mingzhe;

    2013-01-01

    osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection...... formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing...

  16. 高糖及糖基化终末产物对人脂肪干细胞成骨分化能力的影响%Effects of high glucose and advanced glycation end-products on osteogenic differentiation of human adipose-derived stromal cells in vitro

    Institute of Scientific and Technical Information of China (English)

    李冬松; 李叔强; 蔡波; 王苹; 冯卫; 刘建国

    2011-01-01

    BACKGROUND: Bone metabolism disorder happens in diabetic environment, bone defects in which are difficult to repair. Study addressing osteogenic property of adipose-derived stroma cells (ADSCs) in diabetic environment provides theoretical basis for its application in certain environment.OBJECTIVE: To explore the effects of high glucose (HG) and advanced glycation end-products (AGEs) on osteogenic capacity of human ADSCs. METHODS: 100 mg/L AGEs and 27.5 mmol/L HG were used to simulate in vitro diabetic environment and intervened ADSCs osteogenic differentiation. The cells were divided into 4 groups, with 6 samples in each group. The expression of type Ⅰ collagen was examined by fluorescent immunofluorescence at 21 days after osteogenic induction. The number of calcification nodes was counted under contrast phase microscopy at 14, 21 and 28 days. RESULTS AND CONCLUSION: Fluorescent quantitation scan showed that the type Ⅰ collagen amount of the AGEs+HG treated group was 2.76 times lower than that of the control group. AGEs+HG reduced the number of ADSCs calcification nodes compared with the control, HG, and AGEs groups, the differences were statistical significant (P < 0.01). AGEs and HG exposure inhibit the cognate osteogenic differentiation of ADSCs, which suggest that AGEs and HG are unfavorable factors that reduce ADSCs osteogenic ability.%背景:因糖尿病条件下骨质代谢存在紊乱,对这类骨缺损的修复具有挑战性,研究糖尿病环境下脂肪干细胞的成骨特性将为其在特定环境下的应用提供理论基础.目的:观察高糖、糖基化终末产物对人脂肪干细胞成骨分化能力的影响.方法:选取27.5 mmol/L高糖、100 mg/L糖基化终末产物体外模拟糖尿病环境,干预人脂肪干细胞成骨分化;实验分为4组,每组设立6个样本.通过荧光染色检测脂肪干细胞诱导成骨21 d时的Ⅰ型胶原表达量,矿化结节染色观测各组中等量脂肪干细胞在14,21,28 d时矿化结节

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

    Institute of Scientific and Technical Information of China (English)

    TIAN Xiao-bin; SUN Li; YANG Shu-hua; ZHANG Yu-kun; HU Ru-yin; FU De-hao

    2008-01-01

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

  18. Preparation and evaluation of biodegradable films containing the potent osteogenic compound BFB0261 for localized delivery.

    Science.gov (United States)

    Umeki, Nobuo; Sato, Takayuki; Harada, Masahiro; Takeda, Junko; Saito, Shuji; Iwao, Yasunori; Itai, Shigeru

    2011-02-14

    To achieve sustained release of 3-ethyl-4-(4-methylisoxazol-5-yl)-5-(methylthio) thiophene-2-carboxamide (BFB0261), a new potent osteogenic compound for treating bone disorders, we prepared film formulations containing BFB0261 and the following newly synthesized biodegradable polymers by a solvent casting technique: poly(D,L-lactic acid) (PLA), poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(D,L-lactic acid)-block-poly(ethylene glycol) (PLA-PEG), and poly(D,L-lactic acid-co-trimethylene carbonate) (PLA-TMC) polymers or copolymers. Powder X-ray diffractometry (PXRD), differential thermal analysis (DTA), scanning electron microscopy (SEM), and tensile testing were performed to examine the physicochemical properties of these films. Almost all the films exhibited a smooth and homogeneous surface, as observed by SEM. In addition, PXRD and DTA revealed that BFB0261 existed in an amorphous state in the films. The in vitro release of BFB0261 from PLA100 (M(w): 251 kDa), PLAPEG9604H (PLA/PEG ratio: 96:4; M(w): 181 kDa), PLAPEG8515H (PLA/PEG ratio: 85:15; M(w): 51.5 kDa), or PLAPEG8020 (PLA/PEG ratio: 80:20; M(w): 33.7 kDa) films followed zero-order kinetics with slow release up to 12 weeks following incubation. Although release of BFB0261 from PLA-TMC films followed first-order kinetics, sustained release of BFB0261 for 12 weeks was still observed for PLATMC8416 (PLA/TMC ratio: 84:16; M(w): 170 kDa) films. Furthermore, when the BFB0261-loaded films constructed from various polymers were implanted subcutaneously on rat backs, the PLAPEG8515H and PLATMC8416 films were capable of achieving sustained release of BFB0261 at the administrated site for 12 weeks. Therefore, the present data indicate that films constructed from PLAPEG8515H or PLATMC8416 may be applicable to bone or tissue engineering. PMID:21047548

  19. Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

    Directory of Open Access Journals (Sweden)

    Liskova J

    2015-01-01

    Full Text Available Jana Liskova,1 Oleg Babchenko,2 Marian Varga,2 Alexander Kromka,2 Daniel Hadraba,1 Zdenek Svindrych,1 Zuzana Burdikova,1 Lucie Bacakova1 1Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract: Nanocrystalline diamond (NCD films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination or oxygen atoms (O-termination. Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix

  20. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells.

    Science.gov (United States)

    Wu, Xiaonan; Miao, Leiying; Yao, Yingfang; Wu, Wenlei; Liu, Yu; Chen, Xiaofeng; Sun, Weibin

    2014-01-01

    Periodontal repair is a complex process in which regeneration of alveolar bone is a vital component. The aim of this study was to develop a biodegradable scaffold with good biocompatibility and osteoinductive ability. Two types of composite fibrous scaffolds were produced by electrospinning, ie, type I collagen/poly(ε-caprolactone) (COL/PCL) and type I collagen/poly(ε-caprolactone)/nanoscale hydroxyapatite (COL/PCL/nHA) with an average fiber diameter of about 377 nm. After a simulated body fluid (SBF) immersion test, the COL/PCL/nHA-SBF scaffold developed a rough surface because of the calcium phosphate deposited on the fibers, suggesting that the presence of nHA promoted the mineralization potential of the scaffold. Energy dispersive X-ray spectroscopy clearly showed the calcium and phosphorus content in the COL/PCL/nHA and COL/PCL/nHA-SBF scaffolds, confirming the findings of nHA and calcium phosphate precipitation on scanning electron micrographs. Water contact analysis revealed that nHA could improve the hydrophilic nature of the COL/PCL/nHA-SBF scaffold. The morphology of periodontal ligament cells cultured on COL/PCL-SBF and COL/PCL/nHA-SBF was evaluated by scanning electron microscopy. The results showed that cells adhered to either type of scaffold and were slightly spindle-shaped in the beginning, then extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has

  1. Incorporation of osteogenic and angiogenic small interfering RNAs into chitosan sponge for bone tissue engineering

    Directory of Open Access Journals (Sweden)

    Jia S

    2014-11-01

    Full Text Available Sen Jia,1,* Xinjie Yang,1,* Wen Song,2,* Lei Wang,1 Kaixiu Fang,3 Zhiqiang Hu,1,4 Zihui Yang,1 Chun Shan,1 Delin Lei,1 Bin Lu1 1Department of Oral and Maxillofacial Surgery, 2Department of Prosthetic Dentistry, 3Department of Implant Dentistry, School of Stomatology, State Key Laboratory of Military Stomatology, Fourth Military Medical University, Xi’an People’s Republic of China; 4Department of Otorhinolaryngology, No 113 Hospital of People’s Liberation Army, Ningbo, People’s Republic of China *These authors contributed to this paper equally and are considered to be joint first authors Abstract: Engineered bone substitutes are being extensively explored in response to growing demand. However, the angiogenesis that occurs during bone formation is often overlooked in scaffold design. In this novel study, we incorporated two small interfering RNAs (siRNAs, ie, small interfering RNA targets casein kinase 2 interaction protein 1 (siCkip-1 and small interfering RNA targets soluble VEGF receptor 1 (siFlt-1, which can promote osteogenesis and angiogenesis, into a chitosan sponge. This scaffold could maintain siRNAs for over 2 weeks in neutral phosphate-buffered saline and degraded rapidly in the presence of lysozyme. The chitosan sponge with siCkip-1 and siFlt-1 in vitro bioactivity was investigated using mesenchymal stem cells. Target genes were significantly suppressed, and osteocalcin, alkaline phosphatase, and vascular endothelial growth factor were significantly upregulated. Alizarin Red staining revealed that mineralization of the extracellular matrix was markedly enhanced by dual transfection. Further analysis by immunofluorescence confirmed that the siRNA-modified scaffold simultaneously improved the expression of osteocalcin and von Willebrand factor. In vivo testing in a skull critical-size defect model showed marked bone regeneration in rats treated with siCkip-1 and siFlt-1. In conclusion, chitosan sponge containing osteogenic and

  2. Sustained release of TGFbeta3 from PLGA microspheres and its effect on early osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Moioli, Eduardo K; Hong, Liu; Guardado, Jesse; Clark, Paul A; Mao, Jeremy J

    2006-03-01

    Despite the widespread role of transforming growth factor-beta3 (TGFbeta3) in wound healing and tissue regeneration, its long-term controlled release has not been demonstrated. Here, we report microencapsulation of TGFbeta3 in poly-d-l-lactic-co-glycolic acid (PLGA) microspheres and determine its bioactivity. The release profiles of PLGA-encapsulated TGFbeta3 with 50:50 and 75:25 PLA:PGA ratios differed throughout the experimental period. To compare sterilization modalities of microspheres, bFGF was encapsulated in 50:50 PLGA microspheres and subjected to ethylene oxide (EO) gas, radio-frequency glow discharge (RFGD), or ultraviolet (UV) light. The release of bFGF was significantly attenuated by UV light, but not significantly altered by either EO or RFGD. To verify its bioactivity, TGFbeta3 (1.35 ng/mL) was control-released to the culture of human mesenchymal stem cells (hMSC) under induced osteogenic differentiation. Alkaline phosphatase staining intensity was markedly reduced 1 week after exposing hMSC-derived osteogenic cells to TGFbeta3. This was confirmed by lower alkaline phosphatase activity (2.25 +/- 0.57 mU/mL/ng DNA) than controls (TGFbeta3- free) at 5.8 +/- 0.9 mU/mL/ng DNA (p 0.05). These findings provide baseline data for potential uses of microencapsulated TGFbeta3 in wound healing and tissue-engineering applications.

  3. Sustained release of bone morphogenetic protein 2 via coacervate improves the osteogenic potential of muscle-derived stem cells.

    Science.gov (United States)

    Li, Hongshuai; Johnson, Noah Ray; Usas, Arvydas; Lu, Aiping; Poddar, Minakshi; Wang, Yadong; Huard, Johnny

    2013-09-01

    Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle by a modified preplate technique exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. MDSCs retrovirally transduced to express bone morphogenetic proteins (BMPs) can differentiate into osteocytes and chondrocytes and enhance bone and articular cartilage repair in vivo, a feature that is not observed with nontransduced MDSCs. These results emphasize that MDSCs require prolonged exposure to BMPs to undergo osteogenic and chondrogenic differentiation. A sustained BMP protein delivery approach provides a viable and potentially more clinically translatable alternative to genetic manipulation of the cells. A unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD), was used to bind, protect, and sustain the release of bone morphogenetic protein-2 (BMP2) in a temporally and spatially controlled manner. Prolonged exposure to BMP2 released by the PEAD:heparin delivery system promoted the differentiation of MDSCs to an osteogenic lineage in vitro and induced the formation of viable bone at an ectopic site in vivo. This new strategy represents an alternative approach for bone repair mediated by MDSCs while bypassing the need for gene therapy.

  4. In vitro performance of 13-93 bioactive glass fiber and trabecular scaffolds with MLO-A5 osteogenic cells.

    Science.gov (United States)

    Modglin, Vernon C; Brown, Roger F; Fu, Qiang; Rahaman, Mohamed N; Jung, Steven B; Day, Delbert E

    2012-10-01

    This in vitro study was performed to evaluate the ability of two types of porous bioactive glass scaffolds to support the growth and differentiation of an established osteogenic cell line. The two scaffold types tested included 13-93 glass fiber and trabecular-like scaffolds seeded with murine MLO-A5 cells and cultured for intervals of 2 to 12 days. Culture in MTT-containing medium showed metabolically active cells both on the surface and within the interior of the scaffolds. Scanning electron microscopy revealed well-attached cells on both types of scaffolds with a continual increase in cell density over a 6-day period. Protein measurements also showed a linear increase in cell density during the incubation. Activity of alkaline phosphatase, a key indicator of osteoblast differentiation, increased about 10-fold during the 6-day incubation with both scaffold types. The addition of mineralization media to MLO-A5 seeded scaffolds triggered extensive formation of alizarin red-positive mineralized extracellular material, additional evidence of cell differentiation and completion of the final step of bone formation on the constructs. Collectively, the results indicate that the 13-93 glass fiber and trabecular scaffolds promote the attachment, growth, and differentiation of MLO-A5 osteogenic cells and could potentially be used for bone tissue engineering applications. PMID:22528984

  5. Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

    Science.gov (United States)

    Chen, Hongfeng; Titushkin, Igor; Stroscio, Michael; Cho, Michael

    2007-02-15

    Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

  6. Sustained release of TGFbeta3 from PLGA microspheres and its effect on early osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Moioli, Eduardo K; Hong, Liu; Guardado, Jesse; Clark, Paul A; Mao, Jeremy J

    2006-03-01

    Despite the widespread role of transforming growth factor-beta3 (TGFbeta3) in wound healing and tissue regeneration, its long-term controlled release has not been demonstrated. Here, we report microencapsulation of TGFbeta3 in poly-d-l-lactic-co-glycolic acid (PLGA) microspheres and determine its bioactivity. The release profiles of PLGA-encapsulated TGFbeta3 with 50:50 and 75:25 PLA:PGA ratios differed throughout the experimental period. To compare sterilization modalities of microspheres, bFGF was encapsulated in 50:50 PLGA microspheres and subjected to ethylene oxide (EO) gas, radio-frequency glow discharge (RFGD), or ultraviolet (UV) light. The release of bFGF was significantly attenuated by UV light, but not significantly altered by either EO or RFGD. To verify its bioactivity, TGFbeta3 (1.35 ng/mL) was control-released to the culture of human mesenchymal stem cells (hMSC) under induced osteogenic differentiation. Alkaline phosphatase staining intensity was markedly reduced 1 week after exposing hMSC-derived osteogenic cells to TGFbeta3. This was confirmed by lower alkaline phosphatase activity (2.25 +/- 0.57 mU/mL/ng DNA) than controls (TGFbeta3- free) at 5.8 +/- 0.9 mU/mL/ng DNA (p 0.05). These findings provide baseline data for potential uses of microencapsulated TGFbeta3 in wound healing and tissue-engineering applications. PMID:16579687

  7. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jing, E-mail: pengjingtd@163.com [Airport College, Civil Aviation University of China, Tianjin 300300 (China); Zhang, Xinming, E-mail: xinmingmail@163.com [Tianjin Product Quality Inspection Technology Research Institute, Tianjin 300384 (China); School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Liu, Yunde [School of Medical Laboratory, Tianjin Medical University, Tianjin 300203 (China); Yang, Xianjin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China)

    2015-11-01

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression.

  8. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    International Nuclear Information System (INIS)

    Research highlights: → High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. → Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. → Rbpj upregulated promoter activities of Runx2 and Ose2. → Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  9. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shengchao [Department of Preventive Dentistry, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, 710032 Xi' an (China); Kawashima, Nobuyuki, E-mail: kawashima.n.endo@tmd.ac.jp [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Sakamoto, Kei; Katsube, Ken-ichi [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Umezawa, Akihiro [Department of Reproductive Biology and Pathology, National Institute for Child Health and Development, 2-10-4 Ohkura, Setagaya-ku, Tokyo 157-8535 (Japan); Suda, Hideaki [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); GCOE Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2010-09-10

    Research highlights: {yields} High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. {yields} Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. {yields} Rbpj upregulated promoter activities of Runx2 and Ose2. {yields} Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  10. 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.

  11. Nanomaterials enhance osteogenic differentiation of human mesenchymal stem cells similar to a short peptide of BMP-7

    Directory of Open Access Journals (Sweden)

    Lock J

    2011-11-01

    Full Text Available Jaclyn Lock, Huinan Liu Department of Bioengineering, University of California, Riverside, CA, USA Background: Nanomaterials have unique advantages in controlling stem cell function due to their biomimetic characteristics and special biological and mechanical properties. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. Methods: This in vitro study investigated the effects of nano-hydroxyapatite, nano-hydroxyapatite-polylactide-co-glycolide (PLGA composites, and a bone morphogenetic protein (BMP-7-derived short peptide (DIF-7c on osteogenic differentiation of human mesenchymal stem cells (MSC. The peptide was chemically functionalized onto nano-hydroxyapatite, incorporated into a nanophase hydroxyapatite-PLGA composite or PLGA control, or directly injected into culture media. Results: Unlike the PLGA control, the nano-hydroxyapatite-PLGA composites promoted adhesion of human MSC. Importantly, nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites promoted osteogenic differentiation of human MSCs, comparable with direct injection of the DIF-7c peptide into culture media. Conclusion: Nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites provide a promising alternative in directing the adhesion and differentiation of human MSC. These nanocomposites should be studied further to clarify their effects on MSC functions and bone remodeling in vivo, eventually translating to clinical applications. Keywords: human mesenchymal stem cells, osteogenesis, stem cell differentiation, bone morphogenetic protein, peptide delivery, nanocomposites

  12. Osteogenic Potential of Cultured Bone Marrow Stromal CellsTransfected with Transforming Growth Factor β1 Gene in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To study the osteogenic potential of cultured bone marrow stromal cells (BMSCs) transfected with transforming growth factor β1 (TGF-β1) gene in vitro, cultured BMSCs were transfected with the complexes of pcDNA3-TGF-β1 and Lipofectamine Reagent in vitro. The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF-β1 gene could promote the osteogenic potential of cultured BMSCs.

  13. Titania nanotube delivery fetal bovine serum for enhancing MC3T3-E1 activity and osteogenic gene expression

    International Nuclear Information System (INIS)

    Titania nanotube (TNT) delivery of fetal bovine serum (FBS) was conducted on titanium (Ti) to enhance bone tissue repair. Scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) showed FBS increased the tube wall thickness and decreased the tube diameter. Attenuated total reflectance Fourier transform infrared further confirmed that FBS completely covered the TNT and changed the surface composition. Water contact angle tests showed TNT/FBS possessed hydrophilic properties. Compared to original Ti, the TNT/FBS group had more attached osteoblasts after 2 h and enhanced filopodia growth at 0.5 h. Significantly, more osteoblasts were also observed on TNT/FBS after 7 d culturing. FBS was released steadily from TNT; about 70% of FBS had been released at 3 d and 90% at 5 d, as shown by the bicinchoninic acid method. TNT/FBS also enhanced subsequent osteoblast differentiation and gene expression; the quantum real-time polymerase chain reaction test showed that TNT/FBS up-regulated alkaline phosphatase and osteocalcin gene expression at 7 d and 14 d. Therefore, TNT/FBS delivered sustained in situ nutrition and enhanced osteoblast activity and osteogenic gene expression. - Highlights: • Fetal Bovine Serum (FBS) was filled in titania nanotube (TNT) structures. • FBS provided sustained-release in situ nutrition for surface osteoblast growth. • TNT/FBS enhanced osteoblast activity and osteogenic gene expression

  14. Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

    Science.gov (United States)

    Vaes, Bart L T; Lute, Carolien; van der Woning, Sebastian P; Piek, Ester; Vermeer, Jenny; Blom, Henk J; Mathers, John C; Müller, Michael; de Groot, Lisette C P G M; Steegenga, Wilma T

    2010-02-01

    S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

  15. Perfusion Flow Enhances Osteogenic Gene Expression and the Infiltration of Osteoblasts and Endothelial Cells into Three-Dimensional Calcium Phosphate Scaffolds

    Directory of Open Access Journals (Sweden)

    Matthew J. Barron

    2012-01-01

    Full Text Available Maintaining cellular viability in vivo and in vitro is a critical issue in three-dimensional bone tissue engineering. While the use of osteoblast/endothelial cell cocultures on three-dimensional constructs has shown promise for increasing in vivo vascularization, in vitro maintenance of cellular viability remains problematic. This study used perfusion flow to increase osteogenic and angiogenic gene expression, decrease hypoxic gene expression, and increase cell and matrix coverage in osteoblast/endothelial cell co-cultures. Mouse osteoblast-like cells (MC3T3-E1 were cultured alone and in co-culture with mouse microvascular endothelial cells (EOMA on three-dimensional scaffolds for 1, 2, 7, and 14 days with or without perfusion flow. mRNA levels were determined for several osteogenic, angiogenic, and hypoxia-related genes, and histological analysis was performed. Perfusion flow downregulated hypoxia-related genes (HIF-1α, VEGF, and OPN at early timepoints, upregulated osteogenic genes (ALP and OCN at 7 days, and downregulated RUNX-2 and VEGF mRNA at 14 days in osteoblast monocultures. Perfusion flow increased cell number, coverage of the scaffold perimeter, and matrix area in the center of scaffolds at 14 days. Additionally, perfusion flow increased the length of endothelial cell aggregations within co-cultures. These suggest perfusion stimulated co-cultures provide a means of increasing osteogenic and angiogenic activity.

  16. Cytokines TNF-α, IL-6, IL-17F, and IL-4 differentially affect osteogenic differentiation of human adipose stem cells

    NARCIS (Netherlands)

    A.P. Bastidas-Coral; A.D. Bakker; B. Zandieh-Doulabi; C.J. Kleverlaan; N. Bravenboer; T. Forouzanfar; J. Klein-Nulend

    2016-01-01

    During the initial stages of bone repair, proinflammatory cytokines are released within the injury site, quickly followed by a shift to anti-inflammatory cytokines. The effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of mesenchymal stem cells is controversial. Here, we i

  17. Influence Factors on Osteogenic Potential of Mesenchymal Stem Cells%间充质干细胞成骨性能的影响因素

    Institute of Scientific and Technical Information of China (English)

    谢方南

    2012-01-01

    Mesenchymal stem cell (MSC) has broad prospects in the field of regenerative medicine and tissue engineering due to its highly self-renewal and multi-lineage differentiation ability. Although there are many researches about MSC osteogenic differentiation, its osteogenic efficiency is still very low both in vitro and in vivo. Therefore, how to improve the MSC osteogenic performance has become the focus of researchers. MSC osteogenic performance is influenced by many factors. In this review, the influence factors are discussed from four aspects; cell sources, approaches of isolation and purification, induction strategies and vascularization.%间充质干细胞(Mesenchymal stem cell,MSC)因高度的自我增殖与多向分化能力在再生医学和组织工程领域具有广阔的应用前景.目前,MSC成骨分化的研究很多,但体外、内的成骨效率仍然较低.因此,提高MSC的成骨性能成为关注热点.MSC成骨性能受多方面因素的影响,我们从细胞来源、分离纯化、诱导策略及血管化等4个方面对影响MSC成骨性能的因素进行综述.

  18. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

  19. The use of SHP-2 gene transduced bone marrow mesenchymal stem cells to promote osteogenic differentiation and bone defect repair in rat.

    Science.gov (United States)

    Fan, Dapeng; Liu, Shen; Jiang, Shichao; Li, Zhiwei; Mo, Xiumei; Ruan, Hongjiang; Zou, Gang-Ming; Fan, Cunyi

    2016-08-01

    Bone tissue engineering is a promising approach for bone regeneration, in which growth factors play an important role. The tyrosine phosphatase Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is essential for the differentiation, proliferation and metabolism of osteoblasts. However, SHP-2 has never been systematically studied for its effect in osteogenesis. We predicted that overexpression of SHP-2 could promote bone marrow-derived mesenchymal stem cell (BMSC)osteogenic differentiation and SHP-2 transduced BMSCs could enhance new bone formation, determined using the following study groups: (1) BMSCs transduced with SHP-2 and induced with osteoblast-inducing liquid (BMSCs/SHP-2/OL); (2) BMSCs transduced with SHP-2 (BMSCs/-SHP-2); (3) BMSCs induced with osteoblast-inducing liquid (BMSCs/OL) and (4) pure BMSCs. Cells were assessed for osteogenic differentiation by quantitative real-time polymerase chain reaction analysis, western blot analysis, alkaline phosphatase activity and alizarin red S staining. For in vivo assessment, cells were combined with beta-tricalcium phosphate scaffolds and transplanted into rat calvarial defects for 8 weeks. Following euthanasia, skull samples were explanted for osteogenic evaluation, including micro-computed tomography measurement, histology and immunohistochemistry staining. SHP-2 and upregulation of its gene promoted BMSC osteogenic differentiation and therefore represents a potential new therapeutic approach to bone repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1871-1881, 2016. PMID:26999642

  20. Combined Effects of Mechanical Strain and Hydroxyapatite/Collagen Composite on Osteogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yan Huang

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs represent a promising source for bone repair and regeneration. Recent lines of evidence have shown that appropriate strain could regulate the osteogenic differentiation of MSCs. Our previous study demonstrated that hydroxyapatite/collagen (HA/Col composite also played an important role in the osteogenic differentiation of MSCs. The aim of this study is to investigate the effects of mechanical strain and HA/Col composite on the osteogenic differentiation of rat bone marrow derived MSCs (rBMSCs in vitro. rBMSCs were treated with cyclic strain generated by a self-designed stretching device with or without the presence of HA/Col composite. Osteogenic differentiation levels were evaluated using reverse transcription polymerase chain reaction (RT-PCR, alkaline phosphatase spectrophotometry, and western blotting. The results demonstrated that mechanical strain combined with HA/Col composite could obviously induce the differentiation of rBMSCs into osteoblasts, which had a better effect than only mechanical strain or HA/Col composite treatment. This provides a new avenue for mechanistic studies of stem cell differentiation and a novel approach to obtain more committed differentiated cells.

  1. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

    Directory of Open Access Journals (Sweden)

    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  2. Electrospun fibrous scaffolds combined with nanoscale hydroxyapatite induce osteogenic differentiation of human periodontal ligament cells

    Directory of Open Access Journals (Sweden)

    Wu XN

    2014-08-01

    extended gradually with stretched filopodia, indicating an ability to fill the fiber pores. A Cell Counting Kit-8 assay showed that both scaffolds supported cell proliferation. However, real-time quantitative polymerase chain reaction analysis showed that expression of the bone-related markers, alkaline phosphatase and osteocalcin, was upregulated only on the COL/PCL/nHA-SBF scaffold, indicating that this scaffold had the ability to induce osteogenic differentiation of periodontal ligament cells. In this study, COL/PCL/nHA-SBF produced by electrospinning followed by biomimetic mineralization had combined electrospun fibers with nHA in it. This scaffold has good biocompatibility and osteoinductive ability as a result of the characteristics of nHA, so could be innovatively applied to periodontal tissue engineering as a potential scaffold. Keywords: nanoscale hydroxyapatite, electrospinning, periodontal ligament cells 

  3. Encapsulation of bone morphogenic protein-2 with Cbfa1-overexpressing osteogenic cells derived from human embryonic stem cells in hydrogel accelerates bone tissue regeneration.

    Science.gov (United States)

    Kim, Min Jung; Park, Ji Sun; Kim, Sinae; Moon, Sung-Hwan; Yang, Han Na; Park, Keun-Hong; Chung, Hyung-Min

    2011-08-01

    Bone tissue defects caused by trauma and disease are significant problems in orthopedic surgery. Human embryonic stem cells (hESCs) hold great promise for the treatment of bone tissue disease in regenerative medicine. In this study, we have established an effective method for the differentiation of osteogenic cells derived from hESCs using a lentiviral vector containing the transcription factor Cbfa1. Differentiation was initiated in embryoid body formation of Cbfa1-expressing hESCs, resulting in a highly purified population of osteogenic cells based on flow cytometric analysis. These cells also showed characteristics of osteogenic cells in vitro, as determined by reverse-transcription (RT)-polymerase chain reaction and immunocytochemistry using osteoblast-specific markers. We also evaluated the regenerative potential of Cbfa1-expressing cells derived from hESCs (hESC-CECs) compared with hESCs and the osteogenic effects of bone morphogenic protein-2 (BMP2) encapsulated in thermoreversible hydrogel in vivo. hESC-CECs were embedded in hydrogel constructs enriched with BMP2 to promote bone regeneration. We observed prominent mineralization and the formation of nodule-like structures using von Kossa and alizarin red S staining. In addition, the expression patterns of osteoblast-specific genes were verified by RT-polymerase chain reaction, and immunohistochemical analysis revealed that collagen type 1 and Cbfa1 were highly expressed in hESC-CECs compared with other cell types. Taken together, our results suggest that encapsulation of hESC-CECs with BMP2 in hydrogel constructs appears to be a promising method to enhance the in vitro osteoblastic differentiation and in vivo osteogenic activity of hESC-CECs.

  4. In vitro osteogenic induction of bone marrow stromal cells with encapsulated gene-modified bone marrow stromal cells and in vivo implantation for orbital bone repair.

    Science.gov (United States)

    Deng, Yuan; Zhou, Huifang; Yan, Chenxi; Wang, Yefei; Xiao, Caiwen; Gu, Ping; Fan, Xianqun

    2014-07-01

    Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with

  5. The effect of magnetic stimulation on the osteogenic and chondrogenic differentiation of human stem cells derived from the adipose tissue (hASCs)

    Science.gov (United States)

    Lima, João; Gonçalves, Ana I.; Rodrigues, Márcia T.; Reis, Rui L.; Gomes, Manuela E.

    2015-11-01

    The use of magnetic nanoparticles (MNPs) towards the musculoskeletal tissues has been the focus of many studies, regarding MNPs ability to promote and direct cellular stimulation and orient tissue responses. This is thought to be mainly achieved by mechano-responsive pathways, which can induce changes in cell behavior, including the processes of proliferation and differentiation, in response to external mechanical stimuli. Thus, the application of MNP-based strategies in tissue engineering may hold potential to propose novel solutions for cell therapy on bone and cartilage strategies to accomplish tissue regeneration. The present work aims at studying the influence of MNPs on the osteogenic and chondrogenic differentiation of human adipose derived stem cells (hASCs). MNPs were incorporated in hASCs and cultured in medium supplemented for osteogenic and chondrogenic differentiation. Cultures were maintained up to 28 days with/without an external magnetic stimulus provided by a magnetic bioreactor, to determine if the MNPs alone could affect the osteogenic or chondrogenic phenotype of the hASCs. Results indicate that the incorporation of MNPs does not negatively affect the viability nor the proliferation of hASCs. Furthermore, Alizarin Red staining evidences an enhancement in extracellular (ECM) mineralization under the influence of an external magnetic field. Although not as evident as for osteogenic differentiation, Toluidine blue and Safranin-O stainings also suggest the presence of a cartilage-like ECM with glycosaminoglycans and proteoglycans under the magnetic stimulus provided. Thus, MNPs incorporated in hASCs under the influence of an external magnetic field have the potential to induce differentiation towards the osteogenic and chondrogenic lineages.

  6. Flexible Yttrium-Stabilized Zirconia Nanofibers Offer Bioactive Cues for Osteogenic Differentiation of Human Mesenchymal Stromal Cells.

    Science.gov (United States)

    Cadafalch Gazquez, Gerard; Chen, Honglin; Veldhuis, Sjoerd A; Solmaz, Alim; Mota, Carlos; Boukamp, Bernard A; van Blitterswijk, Clemens A; Ten Elshof, Johan E; Moroni, Lorenzo

    2016-06-28

    Currently, the main drawback of ceramic scaffolds used in hard tissue regeneration is their low mechanical strength. Stabilized zirconia, especially the tetragonal 3% yttrium-stabilized zirconia (YSZ) phase, has been considered as a bioinert ceramic material with high mechanical strength. In the present work, flexible nanofibrous YSZ scaffolds were prepared by electrospinning. The obtained scaffolds showed remarkable flexibility at the macroscopic scale, while retaining their stiffness at the microscopic scale. The surface nanoroughness of the scaffolds could be tailored by varying the heat treatment method. Our results demonstrate that the osteogenic differentiation and mineralization of seeded human mesenchymal stromal cells were supported by the nanofibrous YSZ scaffolds, in contrast to the well-known bioinert behavior of bulk YSZ. These findings highlight that flexible ceramic scaffolds are an appealing alternative to the current brittle ceramics for bone tissue regeneration applications. PMID:27294434

  7. Labeling and imaging of human mesenchymal stem cells with quantum dot bioconjugates during proliferation and osteogenic differentiation in long term.

    Science.gov (United States)

    Shah, B; Clark, P; Stroscio, M; Mao, J

    2006-01-01

    Quantum dots (QDs) are semiconductor nanocrystals that serve as promising alternatives to organic dyes for cell labeling. Because of their unique spectral, physical and chemical properties, QDs are useful for concurrently monitoring several intercellular and intracellular interactions in live normal cells and cancer cells over periods ranging from less than a second to over several days (several divisions of cells). Here, peptide CGGGRGD is immobilized on CdSe-ZnS QDs coated with carboxyl groups by cross linking with amine groups. These conjugates are directed by the peptide to bind with selected integrins on the membrane of human Mesenchymal stem cells. Upon overnight incubation with optimal concentration, QDs effectively labeled all the cells. Here, we report long-term labeling of human bone-marrow-derived mesenchymal stem cells (hMSCs) with RGD-conjugated QDs during self replication and differentiation into osteogenic cell lineages.

  8. Ectopic osteogenic tissue formation by MC3T3-E1 cell-laden chitosan/hydroxyapatite composite scaffold.

    Science.gov (United States)

    Koç, Aysel; Elçin, Ayşe Eser; Elçin, Yaşar Murat

    2016-09-01

    This study evaluates the suitability of a macroporous three-dimensional chitosan/hydroxyapatite (CS/HA) composite as a bone tissue engineering scaffold using MC3T3-E1 cells. The CS/HA scaffold was produced by freeze-drying, and characterized by means of SEM and FTIR. In vitro findings demonstrated that CS/HA supported attachment and proliferation of cells, and stimulated extracellular matrix (ECM) production. Tissue biocompatibility and osteogenic capacity of the cell-laden constructs were evaluated in an ectopic Wistar rat model. In vivo results showed that the MC3T3-E1 cell-laden CS/HA was essentially histocompatible, promoted neovascularization and calcified matrix formation, and secreted osteoblast-specific protein. We conclude that the composite scaffold evaluated has potential for applications in bone regeneration. PMID:25968048

  9. Effect of chemotherapy on the retention of sup(99m)Tc-methylenediphosphonate by the osteogenic sarcoma of the mouse

    Energy Technology Data Exchange (ETDEWEB)

    Senekowitsch, R.; Kriegel, H.

    1980-08-01

    The cytostatic action of Adriblastin on the accumulation of sup(99m)Tc-MDP by a transplantable osteogenic sarcoma in the mouse was investigated. The quantitative determination of the activity concentration in the tumor showed a concentration value 8 days after the administration of the cytostatic which was nearly twice that found in the untreated animals. This could be clearly observed in scintigrams obtained with a gamma-camera using a pinhole-collimator. The histoautoradiography likewise showed a higher concentration of the radiopharmaceutical which can be attributed to higher ossification within the tumor due to the application of the cytostatic agent. Our results indicate that scintigraphy with sup(99m)Tc-MDP is a good follow-up method during treatment with cytostatics.

  10. Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels

    Science.gov (United States)

    2016-01-01

    For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites.

  11. Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels

    Science.gov (United States)

    2016-01-01

    For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites. PMID:27547223

  12. Transcriptomics comparison between porcine adipose and bone marrow mesenchymal stem cells during in vitro osteogenic and adipogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Elisa Monaco

    Full Text Available Bone-marrow mesenchymal stem cells (BMSC are considered the gold standard for use in tissue regeneration among mesenchymal stem cells (MSC. The abundance and ease of harvest make the adipose-derived stem cells (ASC an attractive alternative to BMSC. The aim of the present study was to compare the transcriptome of ASC and BMSC, respectively isolated from subcutaneous adipose tissue and femur of 3 adult pigs, during in vitro osteogenic and adipogenic differentiation for up to four weeks. At 0, 2, 7, and 21 days of differentiation RNA was extracted for microarray analysis. A False Discovery Rate ≤0.05 for overall interactions effect and P<0.001 between comparisons were used to determine differentially expressed genes (DEG. Ingenuity Pathway Analysis and DAVID performed the functional analysis of the DEG. Functional analysis of highest expressed genes in MSC and genes more expressed in MSC vs. fully differentiated tissues indicated low immunity and high angiogenic capacity. Only 64 genes were differentially expressed between ASC and BMSC before differentiation. The functional analysis uncovered a potential larger angiogenic, osteogenic, migration, and neurogenic capacity in BMSC and myogenic capacity in ASC. Less than 200 DEG were uncovered between ASC and BMSC during differentiation. Functional analysis also revealed an overall greater lipid metabolism in ASC, while BMSC had a greater cell growth and proliferation. The time course transcriptomic comparison between differentiation types uncovered <500 DEG necessary to determine cell fate. The functional analysis indicated that osteogenesis had a larger cell proliferation and cytoskeleton organization with a crucial role of G-proteins. Adipogenesis was driven by PPAR signaling and had greater angiogenesis, lipid metabolism, migration, and tumorigenesis capacity. Overall the data indicated that the transcriptome of the two MSC is relatively similar across the conditions studied. In addition

  13. Cementogenesis and the induction of periodontal tissue regeneration by the osteogenic proteins of the transforming growth factor-beta superfamily.

    Science.gov (United States)

    Ripamonti, U; Petit, J-C; Teare, J

    2009-04-01

    The antiquity and severity of periodontal diseases are demonstrated by the hard evidence of alveolar bone loss in gnathic remains of the Pliocene/Pleistocene deposits of the Bloubank Valley at Sterkfontein, Swartkrans and Kromdrai in South Africa. Extant Homo has characterized and cloned a superfamily of proteins which include the bone morphogenetic proteins that regulate tooth morphogenesis at different stages of development as temporally and spatially connected events. The induction of cementogenesis, periodontal ligament and alveolar bone regeneration are regulated by the co-ordinated expression of bone morphogenetic proteins. Naturally derived and recombinant human bone morphogenetic proteins induce periodontal tissue regeneration in mammals. Morphological analyses on undecalcified sections cut at 3-6 mum on a series of mandibular molar Class II and III furcation defects induced in the non-human primate Papio ursinus show the induction of cementogenesis. Sharpey's fibers nucleate as a series of composite collagen bundles within the cementoid matrix in close relation to embedded cementocytes. Osteogenic protein-1 and bone morphogenetic protein-2 possess a structure-activity profile, as shown by the morphology of tissue regeneration, preferentially cementogenic and osteogenic, respectively. In Papio ursinus, transforming growth factor-beta(3) also induces cementogenesis, with Sharpey's fibers inserting into newly formed alveolar bone. Capillary sprouting and invasion determine the sequential insertion and alignment of individual collagenic bundles. The addition of responding stem cells prepared by finely mincing fragments of autogenous rectus abdominis muscle significantly enhances the induction of periodontal tissue regeneration when combined with transforming growth factor-beta(3) implanted in Class II and III furcation defects of Papio ursinus. PMID:18842117

  14. Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

    Science.gov (United States)

    Granchi, Donatella; Ochoa, Gorka; Leonardi, Elisa; Devescovi, Valentina; Baglìo, Serena Rubina; Osaba, Lourdes; Baldini, Nicola; Ciapetti, Gabriela

    2010-06-01

    Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.

  15. Enhanced Osteogenic and Vasculogenic Differentiation Potential of Human Adipose Stem Cells on Biphasic Calcium Phosphate Scaffolds in Fibrin Gels.

    Science.gov (United States)

    van Esterik, Fransisca A S; Zandieh-Doulabi, Behrouz; Kleverlaan, Cornelis J; Klein-Nulend, Jenneke

    2016-01-01

    For bone tissue engineering synthetic biphasic calcium phosphate (BCP) with a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ratio of 60/40 (BCP60/40) is successfully clinically applied, but the high percentage of HA may hamper efficient scaffold remodelling. Whether BCP with a lower HA/β-TCP ratio (BCP20/80) is more desirable is still unclear. Vascular development is needed before osteogenesis can occur. We aimed to test the osteogenic and/or vasculogenic differentiation potential as well as degradation of composites consisting of human adipose stem cells (ASCs) seeded on BCP60/40 or BCP20/80 incorporated in fibrin gels that trigger neovascularization for bone regeneration. ASC attachment to BCP60/40 and BCP20/80 within 30 min was similar (>93%). After 11 days of culture BCP20/80-based composites showed increased alkaline phosphatase activity and DMP1 gene expression, but not RUNX2 and osteonectin expression, compared to BCP60/40-based composites. BCP20/80-based composites also showed enhanced expression of the vasculogenic markers CD31 and VEGF189, but not VEGF165 and endothelin-1. Collagen-1 and collagen-3 expression was similar in both composites. Fibrin degradation was increased in BCP20/80-based composites at day 7. In conclusion, BCP20/80-based composites showed enhanced osteogenic and vasculogenic differentiation potential compared to BCP60/40-based composites in vitro, suggesting that BCP20/80-based composites might be more promising for in vivo bone augmentation than BCP60/40-based composites. PMID:27547223

  16. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com; Guo, Zhengze, E-mail: zhzeguo@163.com; Li, Dehua, E-mail: lidehuafmmu@163.com

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  17. Low-level vibrations retain bone marrow's osteogenic potential and augment recovery of trabecular bone during reambulation.

    Directory of Open Access Journals (Sweden)

    Engin Ozcivici

    Full Text Available Mechanical disuse will bias bone marrow stromal cells towards adipogenesis, ultimately compromising the regenerative capacity of the stem cell pool and impeding the rapid and full recovery of bone morphology. Here, it was tested whether brief daily exposure to high-frequency, low-magnitude vibrations can preserve the marrow environment during disuse and enhance the initiation of tissue recovery upon reambulation. Male C57BL/6J mice were subjected to hindlimb unloading (HU, n = 24, HU interrupted by weight-bearing for 15 min/d (HU+SHAM, n = 24, HU interrupted by low-level whole body vibrations (0.2 g, 90 Hz for 15 min/d (HU+VIB, n = 24, or served as age-matched controls (AC, n = 24. Following 3 w of disuse, half of the mice in each group were released for 3 w of reambulation (RA, while the others were sacrificed. RA+VIB mice continued to receive vibrations for 15 min/d while RA+SHAM continued to receive sham loading. After disuse, HU+VIB mice had a 30% greater osteogenic marrow stromal cell population, 30% smaller osteoclast surface, 76% greater osteoblast surface but similar trabecular bone volume fraction compared to HU. After 3 w of reambulation, trabecular bone of RA+VIB mice had a 30% greater bone volume fraction, 51% greater marrow osteoprogenitor population, 83% greater osteoblast surfaces, 59% greater bone formation rates, and a 235% greater ratio of bone lining osteoblasts to marrow adipocytes than RA mice. A subsequent experiment indicated that receiving the mechanical intervention only during disuse, rather than only during reambulation, was more effective in altering trabecular morphology. These data indicate that the osteogenic potential of bone marrow cells is retained by low-magnitude vibrations during disuse, an attribute which may have contributed to an enhanced recovery of bone morphology during reambulation.

  18. Inhibition of mTOR and HIF pathways diminishes chondro-osteogenesis and cell proliferation in chondroblastoma.

    Science.gov (United States)

    Yang, Xiao; Yang, Zheng-jie; Liu, Feng-xiang; Zeng, Ke; Qian, Ming-quan; Chen, Gang; Shi, Lei; Zhu, Guo-xing

    2013-10-01

    Chondroblastoma (CBL) is a benign bone tumor occurring mostly in teenagers. Despite this, CBL can recur and metastasize after curettage, which may impede normal epiphysis. In search of a novel targeted therapy for CBL, we aimed at BMP-2, a factor critical for chondro-osteogenesis and chondrocyte proliferation. Two pathways upstream of BMP-2, the mTOR and HIF, were targeted with rapamycin (Rapa) and FM19G11 (FM), respectively. Using immunohistochemistry, we found BMP-2 was highly expressed in CBL tissues. CBL cells explanted and confirmed with higher BMP-2 level than normal cartilage. Protumorigenic effect of Rapa and FM on CBL cells were transduced via BMP-2. Combination of Rapa and FM conferred stronger inhibition of cell proliferation than either monotherapy and inhibited levels of chondro-osteogenic markers (Sox9, aggrecan, and type II collagen). To minimize the adverse effect of Rapa, we performed screening in essential amino acids and found leucine deprivation-sensitized CBL cells to Rapa. Combination treatment of low dose Rapa, FM, and leucine deprivation conferred compatible inhibitory effects on CBL cell proliferation, chondro-osteogenic potential, and tumorigenic capacity. We conclude that targeting BMP-2 using mTOR/HIF inhibition could potently curb the disease. Addition of low-leucine diet could lower the dose of rapamycin in chase for less toxicity.

  19. Inhibition of mTOR and HIF pathways diminishes chondro-osteogenesis and cell proliferation in chondroblastoma.

    Science.gov (United States)

    Yang, Xiao; Yang, Zheng-jie; Liu, Feng-xiang; Zeng, Ke; Qian, Ming-quan; Chen, Gang; Shi, Lei; Zhu, Guo-xing

    2013-10-01

    Chondroblastoma (CBL) is a benign bone tumor occurring mostly in teenagers. Despite this, CBL can recur and metastasize after curettage, which may impede normal epiphysis. In search of a novel targeted therapy for CBL, we aimed at BMP-2, a factor critical for chondro-osteogenesis and chondrocyte proliferation. Two pathways upstream of BMP-2, the mTOR and HIF, were targeted with rapamycin (Rapa) and FM19G11 (FM), respectively. Using immunohistochemistry, we found BMP-2 was highly expressed in CBL tissues. CBL cells explanted and confirmed with higher BMP-2 level than normal cartilage. Protumorigenic effect of Rapa and FM on CBL cells were transduced via BMP-2. Combination of Rapa and FM conferred stronger inhibition of cell proliferation than either monotherapy and inhibited levels of chondro-osteogenic markers (Sox9, aggrecan, and type II collagen). To minimize the adverse effect of Rapa, we performed screening in essential amino acids and found leucine deprivation-sensitized CBL cells to Rapa. Combination treatment of low dose Rapa, FM, and leucine deprivation conferred compatible inhibitory effects on CBL cell proliferation, chondro-osteogenic potential, and tumorigenic capacity. We conclude that targeting BMP-2 using mTOR/HIF inhibition could potently curb the disease. Addition of low-leucine diet could lower the dose of rapamycin in chase for less toxicity. PMID:23760978

  20. Is 1, 25-dihydroxyvitamin D3 an ideal substitute for dexamethasone for inducing osteogenic differentiation of human adipose tissue-derived stromal cells in vitro?

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yong-sheng; LIU Yun-song; TAN Jian-guo

    2006-01-01

    Background Human adipose tissue-derived stromal cells (hADSCs) can be induced to differentiate along anosteoblastic lineage under stimulation of dexamethasone (DEX). Recent studies, however, have questioned theefficacy of glucocorticoids such as DEX in mediating the osteogenesis process of skeletal progenitor cells andprocessed lipoaspirate cells. Is it possible to find a substitute for DEX? Therefore, this study was designed toinvestigate osteogenic capacity and regulating mechanisms for osteoblastic differentiation of hADSCs bycomparing osteogenic media (OM) containing either 1, 25-dihydroxyvitamin D3 (VD) or DEX and determine ifVD was an ideal substitute for DEX as an induction agent for the osteogenesis of hADSCs.Methods Osteogenic differentiation of hADSCs was induced by osteogenic medium (OM) containing either 10nmol/L VD or 100 nmol/L DEX. Differentiation of hADSCs into osteoblastic lineage was identified by alkalinephosphatase (ALP) staining, von Kossa staining, and reverse transcription-polymerase chain reaction assays formRNA expression of osteogenesis-related genes such as type Ⅰ collagen (COL Ⅰ), bone sialoprotein (BSP),osteocalcin (OC), bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, BMP-7, runt-related transcriptionfactor 2/core binding factor α1 (Runx2/Cbfal), osterix (Osx), and LIM mineralization protein-1 (LMP-1).Results von Kossa staining revealed that the differentiated cells induced by both VD and DEX weremineralized in vitro. They also expressed osteoblast-related markers, such as ALP, COL I, BSP, and OC.Runx2/Cbfal, Osx, BMP-6, and LMP-1 were upregulated during VD and DEX-induced hADSC osteoblasticdifferentiation, but BMP-4, BMP-7 were not. BMP-2 was only expressed in VD-induced differentiated cells.Conclusions VD or DEX-induced hADSCs differentiate toward the osteoblastic lineage in vitro. Runx2/Cbfa1,Osx, BMP-2, BMP-6, and LMP-1 are involved in regulating osteoblastic differentiation of hADSCs, but BMP-4,BMP-7 are not. VD, but not DEX

  1. Local transplantation of osteogenic pre-differentiated autologous adipose-derived mesenchymal stem cells may accelerate non-union fracture healing with limited pro-metastatic potency.

    Science.gov (United States)

    Han, Duanyang; Han, Na; Zhang, Peixun; Jiang, Baoguo

    2015-01-01

    Fracture non-union is a serious complication in orthopedic clinical practice. Mesenchymal stem cells are believed to play a vital role in fracture healing process. Among various origins of mesenchymal stem cell, adipose derived stem cells hold great promise especially in clinical milieu. However, the wide spread application of mesenchymal stem cell based therapy is impeded by the pro-metastasis nature of the mesenchymal stem cell itself. Based on the findings from previous studies, we hypothesize that local transplanted osteogenic pre-differentiatiated adipose stem cell may promote the non-union fracture healing. Moreover, the pre-differnetiation stem cells by down-regulating the expression of CCL5 and CCL2. This novel osteogenic pre-differnetiation technique may help clinical orthopedists to resolve the refractory non-union cases and shed new light on other stem cell based therapies to counteract to avoid the pro-metastasis nature of the mesenchymal stem cells. PMID:25785146

  2. Osteoprotegerin inhibits calcification of vascular smooth muscle cell via down regulation of the Notch1-RBP-Jκ/Msx2 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Shaoqiong Zhou

    Full Text Available OBJECTIVE: Vascular calcification is a common pathobiological process which occurs among the elder population and in patients with diabetes and chronic kidney disease. Osteoprotegerin, a secreted glycoprotein that regulates bone mass, has recently emerged as an important regulator of the development of vascular calcification. However, the mechanism is not fully understood. The purpose of this study is to explore novel signaling mechanisms of osteoprotegerin in the osteoblastic differentiation in rat aortic vascular smooth muscle cells (VSMCs. METHODS AND RESULTS: VSMCs were isolated from thoracic aorta of Sprague Dawley rats. Osteoblastic differentiation of VSMCs was induced by an osteogenic medium. We confirmed by Von Kossa staining and direct cellular calcium measurement that mineralization was significantly increased in VSMCs cultured in osteogenic medium; consistent with an enhanced alkaline phosphatase activity. This osteoblastic differentiation in VSMCs was significantly reduced by the addition of osteoprotegerin in a dose responsive manner. Moreover, we identified, by real-time qPCR and western blotting, that expression of Notch1 and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both the mRNA and protein levels, these effects were dose-dependently abolished by the treatment of osteoprotegerin. Furthermore, we identified that Msx2, a downstream target of the Notch1/RBP-Jκ signaling, was markedly down-regulated by the treatment of osteoprotegerin. CONCLUSION: Osteoprotegerin inhibits vascular calcification through the down regulation of the Notch1-RBP-Jκ signaling pathway.

  3. Comparative study of osteogenic potential of a composite scaffold incorporating either endogenous bone morphogenetic protein-2 or exogenous phytomolecule icaritin: an in vitro efficacy study.

    Science.gov (United States)

    Chen, S-H; Wang, X-L; Xie, X-H; Zheng, L-Z; Yao, D; Wang, D-P; Leng, Y; Zhang, G; Qin, L

    2012-08-01

    A local delivery system with sustained and efficient release of therapeutic agents from an appropriate carrier is desirable for orthopedic applications. Novel composite scaffolds made of poly (lactic-co-glycolic acid) with tricalcium phosphate (PLGA/TCP) were fabricated by an advanced low-temperature rapid prototyping technique, which incorporated either endogenous bone morphogenetic protein-2 (BMP-2) (PLGA/TCP/BMP-2) or phytomolecule icaritin (ICT) (PLGA/TCP/ICT) at low, middle and high doses. PLGA/TCP served as control. In vitro degradation, osteogenesis and release tests showed statistical differences among PLGA/TCP/ICT, PLGA/TCP and PLGA/TCP/BMP-2 groups, where PLGA/TCP/ICT had the desired slow release of bioactive icaritin in a dose-dependent manner, whereas there was almost no BMP-2 release from the PLGA/TCP/BMP-2 scaffolds. PLGA/TCP/ICT significantly increased more ALP activity, upregulated mRNA expression of osteogenic genes and enhanced calcium deposition and mineralization in rabbit bone marrow stem cells cultured on scaffolds compared with the other two groups. These results indicate the desired degradation rate, osteogenic capability and release property in PLGA/TCP/ICT composite scaffold, as icaritin preserved its bioactivity and structure after incorporation, while PLGA/TCP/BMP-2 did not show an initially expected osteogenic potential, owing to loss of the original bioactivity of BMP-2 during its incorporation and fabrication procedure. The results suggest that PLGA/TCP composite scaffolds incorporating osteogenic ICT might be a promising approach for bone tissue bioengineering and regeneration. PMID:22543006

  4. Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three dimensional scaffolds

    OpenAIRE

    Gomes, Manuela E.; Sikavitsas, V. I.; Behravesh, E.; Reis, R. L.; Mikos, Antonios G.

    2003-01-01

    This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, w...

  5. Comparison of osteogenic potentials of human rat BMP4 and BMP6 gene therapy using [E1-] and [E1-,E2b-] adenoviral vectors

    OpenAIRE

    Li, Hongwei; Li, Jin Zhong; D. Pittman, Debra; Amalfitano, Andy; Hankins, Gerald R.; Helm, Gregory A.

    2006-01-01

    Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP) first-generation adenoviral vectors (ADhBMPs) are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-genera...

  6. Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo

    Science.gov (United States)

    Liu, Yun-Song; Sun, Yu-chun; Wang, Yu-guang; Wang, Yong; Lyu, Pei-Jun

    2016-01-01

    Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects. PMID:27332814

  7. Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Xiao-Fei Wang

    Full Text Available Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs in three-dimensional (3D bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN. Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM compared with that in normal proliferation medium (PM. Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

  8. The Impact of Acute and Chronic Weight Restriction and Weight Regulation practices on Physiological, Osteogenic, Metabolic and Cognitive Function in Elite Jockeys

    OpenAIRE

    Dolan, Eimear

    2010-01-01

    Horse racing is a weight category sport. One of the key challenges facing jockeys is the pressure of “making weight” throughout the protracted racing season. Aim: The aim of this study was to examine the effect of a chronically weight restrictive lifestyle and acute weight loss practices on aspects of physiological, osteogenic, metabolic and cognitive function in jockeys. Methods: The primary aim was achieved through the completion of four related studies. Study One: The effect of a 4% reduct...

  9. Human serum is a suitable supplement for the osteogenic differentiation of human adipose-derived stem cells seeded on poly-3-hydroxibutyrate-co-3-hydroxyvalerate scaffolds.

    Science.gov (United States)

    de Paula, Ana Cláudia Chagas; Zonari, Alessandra Arcoverde Cavalcanti; Martins, Thaís Maria da Mata; Novikoff, Silviene; da Silva, Alexandra Rodrigues Pereira; Correlo, Vitor Manuel; Reis, Rui L; Gomes, Dawidson Assis; Goes, Alfredo Miranda

    2013-01-01

    Human adipose-derived stem cells (hASCs) are currently a point of focus for bone tissue engineering applications. However, the ex vivo expansion of stem cells before clinical application remains a challenge. Fetal bovine serum (FBS) is largely used as a medium supplement and exposes the recipient to infections and immunological reactions. In this study, we evaluated the osteogenic differentiation process of hASCs in poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) scaffolds with the osteogenic medium supplemented with pooled allogeneic human serum (aHS). The hASCs grown in the presence of FBS or aHS did not show remarkable differences in morphology or immunophenotype. The PHB-HV scaffolds, which were developed by the freeze-drying technique, showed an adequate porous structure and mechanical performance as observed by micro-computed tomography, scanning electron microscopy (SEM), and compression test. The three-dimensional structure was suitable for allowing cell colonization, which was revealed by SEM micrographs. Moreover, these scaffolds were not toxic to cells as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The differentiation capacity of hASCs seeded on scaffolds was confirmed by the reduction of the proliferation, the alkaline phosphatase (AP) activity, expression of osteogenic gene markers (AP, collagen type I, Runx2, and osteocalcin), and the expression of bone markers, such as osteopontin, osteocalcin, and collagen type I. The osteogenic capacity of hASCs seeded on PHB-HV scaffolds indicates that this scaffold is adequate for cell growth and differentiation and that aHS is a promising supplement for the in vitro expansion of hASCs. In conclusion, this strategy seems to be useful and safe for application in bone tissue engineering.

  10. Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells

    Directory of Open Access Journals (Sweden)

    Felipe Augusto Andre Ishiy

    2015-01-01

    Full Text Available Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED and MSC-like cells from iPSCs from SHED (iPS-SHED and from human dermal fibroblasts (iPS-FIB. MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.

  11. Multi-Composite Bioactive Osteogenic Sponges Featuring Mesenchymal Stem Cells, Platelet-Rich Plasma, Nanoporous Silicon Enclosures, and Peptide Amphiphiles for Rapid Bone Regeneration

    Directory of Open Access Journals (Sweden)

    Dongmei Fan

    2011-06-01

    Full Text Available A novel bioactive sponge was created with a composite of type I collagen sponges or porous poly(e-caprolactone (PCL scaffolds, platelet-rich plasma (PRP, BMP2-loaded nanoporous silicon enclosure (NSE microparticles, mineralizing peptide amphiphiles (PA, and mesenchymal stem cells (MSC. Primary MSC from cortical bone (CB  tissue proved to form more and larger colony units, as well as produce more mineral matrix under osteogenic differentiation, than MSC from bone marrow (BM. Coating pre-treatments were optimized for maximum cell adhesion and mineralization, while a PRP-based gel carrier was created to efficiently deliver and retain MSC and  microparticles within a porous scaffold while simultaneously promoting cell recruitment, proliferation, and angiogenesis. Components and composite sponges were evaluated for osteogenic differentiation in vitro. Osteogenic sponges were loaded with MSC, PRP, PA, and NSE and implanted subcutaneously in rats to evaluate the formation of bone tissue and angiogenesis in vivo. It was found that the combination of a collagen sponge with CB MSC, PRP, PA, and the BMP2-releasing NSE formed the most bone and was most vascularized by four weeks compared to analogous composites featuring BM MSC or PCL or lacking PRP, PA, and NSE. This study indicates that CB MSC should be considered as an alternative to marrow as a source of stem cells, while the PRP-PA cell and microparticle delivery system may be utilized for diverse tissue engineering applications.

  12. Gold nanoparticles promote osteogenic differentiation in human adipose-derived mesenchymal stem cells through the Wnt/β-catenin signaling pathway

    Directory of Open Access Journals (Sweden)

    Choi SY

    2015-07-01

    Full Text Available Seon Young Choi,1 Min Seok Song,1 Pan Dong Ryu,1 Anh Thu Ngoc Lam,2 Sang-Woo Joo,2 So Yeong Lee1 1Laboratory of Veterinary Pharmacology, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, 2Department of Chemistry, Soongsil University, Seoul, South Korea Abstract: Gold nanoparticles (AuNPs are attractive materials for use in biomedicine due to their physical properties. Increasing evidence suggests that several nanoparticles induce the differentiation of human mesenchymal stem cells into osteoblasts and adipocytes. In this study, we hypothesized that chitosan-conjugated AuNPs promote the osteogenic differentiation of human adipose-derived mesenchymal stem cells. For the evaluation of osteogenic differentiation, alizarin red staining, an alamarBlue® assay, and a quantitative real-time polymerase chain reaction analysis were performed. In order to examine specific signaling pathways, immunofluorescence and a western blotting assay were performed. Our results demonstrate that chitosan-conjugated AuNPs increase the deposition of calcium content and the expression of marker genes related to osteogenic differentiation in human adipose-derived mesenchymal stem cells at nontoxic concentrations. These results indicate that chitosan-conjugated AuNPs promote osteogenesis through the Wnt/β-catenin signaling pathway. Therefore, chitosan-conjugated AuNPs can be used as a reagent for promoting bone formation. Keywords: chitosan-conjugated gold nanoparticle, mineralization, nonphosphorylated beta-catenin

  13. A comparative study on in vitro osteogenic priming potential of electron spun scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for tissue engineering application.

    Science.gov (United States)

    Balaji Raghavendran, Hanumantha Rao; Puvaneswary, Subramaniam; Talebian, Sepehr; Murali, Malliga R; Raman Murali, Malliga; Naveen, Sangeetha V; Vasudevaraj Naveen, Sangeetha; Krishnamurithy, G; McKean, Robert; Kamarul, Tunku

    2014-01-01

    A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200-950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (PCol/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.

  14. Electrospun Gelatin/β-TCP Composite Nanofibers Enhance Osteogenic Differentiation of BMSCs and In Vivo Bone Formation by Activating Ca2+-Sensing Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Xuehui Zhang

    2015-01-01

    Full Text Available Calcium phosphate- (CaP- based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/β-tricalcium phosphate (TCP and confirmed their biological activity in vitro and bone regeneration in vivo. However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca2+-sensing receptor signaling.

  15. Balancing the Osteogenic and Antibacterial Properties of Titanium by Codoping of Mg and Ag: An in Vitro and in Vivo Study.

    Science.gov (United States)

    Zhao, Yaochao; Cao, Huiliang; Qin, Hui; Cheng, Tao; Qian, Shi; Cheng, Mengqi; Peng, Xiaochun; Wang, Jiaxin; Zhang, Yin; Jin, Guodong; Zhang, Xianlong; Liu, Xuanyong; Chu, Paul K

    2015-08-19

    To simultaneously enhance the osteogenic and antibacterial properties of titanium, we introduced magnesium (Mg), silver (Ag), or both by using the plasma immersion ion implantation (PIII) technique, producing three PIII sample groups, namely, Mg-doped titanium (Mg-PIII), Ag-doped titanium (Ag-PIII), and Mg and Ag codoped titanium (Mg/Ag-PIII). The in vitro antibacterial efficacy of Mg/Ag-PIII group was about 7-10% higher than that of Ag-PIII. In vitro and in vivo results demonstrated that osteogenic property of Mg/Ag PIII group was better than that of Ag-PIII or Mg-PIII. It was believed that the galvanic effects between Mg and Ag NPs played a key role in facilitating the release of Mg but reducing the release of silver, answering for the selective performances of the Mg/Ag-PIII group over bacterial and mammalian cells. This study demonstrated that the integration of multiple functional elements could be realized by the dual-source PIII technique, and in this case, the antibacterial properties and osteogenic property of titanium could be balanced.

  16. In vitro osteogenic induction of human marrow-derived mesenchymal stem cells by PCL fibrous scaffolds containing dexamethazone-loaded chitosan microspheres.

    Science.gov (United States)

    Omidvar, Noushin; Ganji, Fariba; Eslaminejad, Mohamadreza Baghaban

    2016-07-01

    This research reports the encapsulation of dexamethasone (Dex) within the chitosan microspheres (CSMs) embedded in a fibrous structure of poly(ɛ-caprolactone) (PCL) to provide a platform for osteogenic differentiation of human mesenchymal stem cells (hMSCs). Dex loaded CSMs were prepared by spray drying a mixture of chitosan and Dex. Then, they were electrospun with PCL solution to create a bilayer fibrous scaffold (PCL/CSMs-Dex). The CSMs act as good depots for sustained release of Dex over a period of 14 days, without noticeable burst release. This is mainly attributed to the core-shell structure of the final PCL/CSMs-Dex-matrix, which could prolong the release and eliminate the initial burst. The water contact angle of PCL scaffolds decreased from 141.4 ± 3.8 to 118.4 ± 7.6 in the presence of CSMs. Improved proliferation of hMSCs cultured on PCL/CSMs-Dex scaffolds was also evidenced. Furthermore, osteogenic assays showed an increase in alkaline phosphatase activity and mineral deposits. The expression of bone-specific genes also confirmed the osteogenic differentiation of cells cultured on these Dex-loaded core-shell structures. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1657-1667, 2016. PMID:26916786

  17. Comparison of osteogenic potentials of human rat BMP4 and BMP6 gene therapy using [E1-] and [E1-,E2b-] adenoviral vectors

    Directory of Open Access Journals (Sweden)

    Hongwei Li, Jin Zhong Li, Debra D. Pittman, Andy Amalfitano, Gerald R. Hankins, Gregory A. Helm

    2006-01-01

    Full Text Available Osteogenic potentials of some recombinant human bone morphogenetic protein (BMP first-generation adenoviral vectors (ADhBMPs are significantly limited in immunocompetent animals. It is unclear what role expression of viral proteins and foreign proteins transduced by adenoviral vectors play in the host immune response and in ectopic bone formation. In this study two sets of experiments were designed and performed. First, rat BMP6 cDNA were amplified, sequenced, and recombined in first-generation adenoviral vector (ADrBMP6. A comparison of human and rat BMP6 adenoviral vectors demonstrated identical osteogenic activities in both immunodeficient and immunocompetent rats. Second, the activities of recombinant human BMP6 in E1- (ADhBMP6 and [E1-,E2b-] ( [E1-,E2b-]ADGFP&hBMP6, and [E1-,E2b-]ADhBMP6 adenoviral vectors were compared in both in vitro and in vivo models. Similar activities of these two generations of BMP adenoviral vectors were found in all models. These results indicate that the amount of viral gene expression and the source of the BMP cDNA are not major factors in the interruption of osteogenic potentials of recombinant BMP6 adenoviral vectors in immunocompetent animals.

  18. Osteogenic and antimicrobial nanoparticulate calcium phosphate and poly-(D,L-lactide-co-glycolide) powders for the treatment of osteomyelitis

    Energy Technology Data Exchange (ETDEWEB)

    Uskoković, Vuk, E-mail: vuk21@yahoo.com [Therapeutic Micro and Nanotechnology Laboratory, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA (United States); Hoover, Charles [Department of Cell and Tissue Biology, University of California, San Francisco, CA (United States); Vukomanović, Marija [Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Belgrade (Serbia); Advanced Materials Department, Jožef Stefan Institute, Ljubljana (Slovenia); Uskoković, Dragan P. [Institute of Technical Sciences, Serbian Academy of Sciences and Arts, Belgrade (Serbia); Desai, Tejal A. [Therapeutic Micro and Nanotechnology Laboratory, Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA (United States)

    2013-08-01

    Development of a material for simultaneous sustained and localized delivery of antibiotics and induction of spontaneous regeneration of hard tissues affected by osteomyelitis stands for an important clinical need. In this work, a comparative analysis of the bacterial and osteoblastic cell response to two different nanoparticulate carriers of clindamycin, an antibiotic commonly prescribed in the treatment of bone infection, one composed of calcium phosphate and the other comprising poly-(D,L-lactide-co-glycolide)-coated calcium phosphate, was carried out. Three different non-cytotoxic phases of calcium phosphate, exhibiting dissolution and drug release profiles in the range of one week to two months to one year, respectively, were included in the analysis: monetite, amorphous calcium phosphate and hydroxyapatite. Spherical morphologies and narrow size distribution of both types of nanopowders were confirmed in transmission and scanning electron microscopic analyses. The antibiotic-containing powders exhibited sustained drug release contingent upon the degradation rate of the carrier. Assessment of the antibacterial performance of the antibiotic-encapsulated powders against Staphylococcus aureus, the most common pathogen isolated from infected bone, yielded satisfactory results both in broths and on blood agar plates for all the analyzed powders. In contrast, no cytotoxic behavior was detected upon the incubation of the antibiotic powders with the osteoblastic MC3T3-E1 cell line for up to three weeks. The cells were shown to engage in a close contact with the antibiotic-containing particles, irrespective of their internal or surface phase composition, polymeric or mineral. At the same time, both types of particles upregulated the expression of osteogenic markers osteocalcin, osteopontin, Runx2 and protocollagen type I, suggesting their ability to promote osteogenesis and enhance remineralization of the infected site in addition to eliminating the bacterial source of

  19. 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

  20. Ligustrazine Inhibits Cartilage Endplate Hypertrophy via Suppression of TGF-β1

    Science.gov (United States)

    Liu, Shufen; Zhao, Bizeng; Shi, Huipeng; Liang, Qianqian; Fu, Yishan; Yang, Zhu

    2016-01-01

    CEP hypertrophy is one of the characteristics of intervertebral disc degeneration (IDD). LIG exerts a protective effect on IDD in animal model. The effect of LIG on CEP hypertrophy is further investigated in the present study. Cells were isolated from hypertrophic samples obtained from patients during vertebral fusion surgery. Cellular proliferation and the expression of type I collagen (Col I) and TGF-β1 were tested. In the bipedal rats, the edges of the CEP and the sizes of noncartilaginous outgrowth, as well as the expression of osteogenic markers, Col1a, ALP, Runx2, and TGF-β1, were detected. Within two passages, the condensed hypertrophic CEP cells exhibited osteogenic capacity by bony-like nodules and ALP positive staining, along with increased expression of Col I and TGF-β1. LIG inhibited proliferation of CEP cells and downregulated the expression of Col I and TGF-β1 in vitro. Furthermore, LIG attenuated CEP hypertrophy on the lumbar spine of bipedal rats by reducing Col1a, ALP, Runx2, and TGF-β1 mRNA expression and TGF-β1 distribution in vivo. We concluded LIG exerted a preventive effect on CEP hypertrophy via suppression of TGF-β1 levels. This information could be used to develop alternative therapeutic methods to treat spinal CEP hypertrophy. PMID:27563332

  1. Posttreatment and retention outcomes with and without periodontally accelerated osteogenic orthodontics assessed using ABO objective grading system

    Directory of Open Access Journals (Sweden)

    Donald J Ferguson

    2016-01-01

    Full Text Available Background: The posttreatment and retention outcomes following nonextraction orthodontic therapy, with and without corticotomy, were assessed using the American Board of Orthodontists objective grading system (OGS. Purpose: The purpose was to determine if the course of retention was any different following alveolar decortication and augmentation bone grafting, i.e., periodontally accelerated osteogenic orthodontics (PAOO. Materials and Methods: Study casts and panoramic radiographs of patients with and without PAOO (28 subjects each were selected on the basis of the following: (1 comprehensive nonextraction orthodontic treatment using straight wire edgewise appliances for Class I crowding, (2 availability of immediate posttreatment records and retention records at least 1 year post de-bracketing, and (3 use of Hawley removable retainers with similar wearing instructions. Results: Independent and paired t-test statistical testing revealed the following: (1 Posttreatment orthodontic outcomes were the same, with or without corticotomy. (2 During retention, 5 of 8 ABO grading criteria improved for the sample without corticotomy, and 6 of 8 ABO grading criteria improved for the group with corticotomy. (3 Retention outcome scores were lower (better for alignment and marginal ridges in the corticotomy-facilitated group. (4 The total score was significantly lower (better for the corticotomy group at retention and the increment of total score change decreased (improved significantly more during retention following corticotomy. Conclusions: The retention phase was more favorable following corticotomy because the amount of OGS total score change demonstrated a significantly improved retention outcome following PAOO therapy.

  2. Mussel-inspired alginate gel promoting the osteogenic differentiation of mesenchymal stem cells and anti-infection.

    Science.gov (United States)

    Zhang, Shiwen; Xu, Kaige; Darabi, Mohammad Ali; Yuan, Quan; Xing, Malcolm

    2016-12-01

    Alginate hydrogels have been used in cell encapsulation for many years but a prevalent issue with pure alginates is that they are unable to provide enough bioactive properties to interact with mammalian cells. This paper discusses the modification of alginate with mussel-inspired dopamine for cell loading and anti-infection. Mouse bone marrow stem cells were immobilized into alginate and alginate-dopamine beads and fibers. Through live-dead and MTT assay, alginates modified by dopamine promoted cell viability and proliferation. In vitro cell differentiation results showed that such an alginate-dopamine gel can promote the osteogenic differentiation of mesenchymal stem cell after PCR and ALP assays. In addition to that, the adhesive prosperities of dopamine allowed for coating the surface of alginate-dopamine gel with silver nanoparticles, which provided the gel with significant antibacterial characteristics. Overall, these results demonstrate that a dopamine-modified alginate gel can be a great tool for cell encapsulation to promote cell proliferation and can be applied to bone regeneration, especially in contaminated bone defects. PMID:27612740

  3. Proliferation and osteogenic differentiation of human mesenchymal stem cells on zirconia and titanium with different surface topography.

    Science.gov (United States)

    Hirano, Tomoki; Sasaki, Hodaka; Honma, Shinya; Furuya, Yoshitaka; Miura, Tadashi; Yajima, Yasutomo; Yoshinari, Masao

    2015-01-01

    The purpose of this study was to elucidate behavior of human mesenchymal stem cells (hMSCs) on yttria stabilized tetragonal zirconia polycrystals (TZP) and commercial pure titanium (CpTi) with different surface topography. Mirror-polished (MS), sandblasted with 150-μm alumina (SB150) and SB150 acid-etched (SB150E) were prepared on TZP and CpTi. Proliferation, osteogenic differentiation of hMSCs was evaluated. The scanning electron microscopy showed that micro- and nano-topographies were created on both TZP and CpTi SB150E surfaces. The proliferation ability, ALP activity, expression of Runx2 on the both SB150E specimens was significantly higher than those on the other specimens. These results suggested that creation of micro- and nano-topographies on TZP and CpTi by blast and acid-etching may offer a promising method for enhancing the proliferation and differentiation of hMSCs in clinical application. PMID:26632237

  4. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, L. A. [University of Erlangen-Nuremberg Medical Center, Department of Plastic and Hand Surgery (Germany); Hild, N.; Mohn, D.; Stark, W. J. [ETH Zurich, Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering (Switzerland); Hoppe, A. [University of Erlangen-Nuremberg, Department of Materials Science and Engineering, Institute of Biomaterials (Germany); Gbureck, U. [University of Wuerzburg, Department for Functional Materials in Medicine and Dentistry (Germany); Horch, R. E.; Kneser, U. [University of Erlangen-Nuremberg Medical Center, Department of Plastic and Hand Surgery (Germany); Boccaccini, A. R., E-mail: aldo.boccaccini@ww.uni-erlangen.de [University of Erlangen-Nuremberg, Department of Materials Science and Engineering, Institute of Biomaterials (Germany)

    2013-07-15

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30-35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 {mu}g/cm Superscript-Two (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 {mu}g/cm Superscript-Two , Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes.

  5. Porous tantalum coatings prepared by vacuum plasma spraying enhance bmscs osteogenic differentiation and bone regeneration in vitro and in vivo.

    Science.gov (United States)

    Tang, Ze; Xie, Youtao; Yang, Fei; Huang, Yan; Wang, Chuandong; Dai, Kerong; Zheng, Xuebin; Zhang, Xiaoling

    2013-01-01

    Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration. PMID:23776648

  6. Accelerated Osteogenic Orthodontics™ for retreatment of a patient with diminished root length and absence of the maxillary central incisor

    Directory of Open Access Journals (Sweden)

    Armando Montesinos F

    2015-10-01

    Full Text Available An 18-year-old female patient visited a university orthodontics department with a chief complaint of an unesthetic appearance of her teeth, including a protruded upper central incisor and unsatisfactory results from previous orthodontic treatment. Pretreatment records showed a Class II skeletal and dental relation with proclined upper and lower incisors, replacement of an absent upper left central incisor with the left upper cuspid, presence of the upper left deciduous cuspid, mild crowding, and 4 mm of overbite and overjet. The panoramic radiograph showed shortened roots of multiple teeth. Accelerated Osteogenic Orthodontics™ (AOO™ was recommended as an approach to reduce the treatment time and the risk of further root shortening. Despite being more expensive and requiring a surgical procedure, this treatment option was very attractive to the patient. The overall treatment time was 14 months. Facial balance was improved, and good occlusal relationships were achieved from the functional and esthetic perspectives. In conclusion, surgically facilitated orthodontics (specifically, AOO™ is an efficient and safe therapeutic tool for treating or retreating orthodontic patients with diminished root length.

  7. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    International Nuclear Information System (INIS)

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30–35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 μg/cm² (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 μg/cm², Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes

  8. Modified basal osteotomy combined with osteogenic distraction (Hemi-Wing distraction) for correction of facial asymmetry: A new technique.

    Science.gov (United States)

    Muñoz, Ruben; Diaz, Alvaro; Golaszewski, Jose

    2014-01-01

    Complete restoration of facial asymmetry is always difficult to achieve. Facial asymmetry due to growth disturbances of the jaws almost requires orthognathic surgical correction, followed, in many cases, by soft tissue corrections. Mandibular hypoplasia is the earliest skeletal manifestation of Hemifacial microsomy and the clinical defect becomes worse with the time, due to asymmetric growth and secondary midface deformity accompanying. Despite correction of the occlusal plane, facial asymmetry can persist if the mandibular body differs in height. We designed a new technique for skeletal correction of the mandibular basal plane combined with orthognatic surgery that avoided the disadvantages and limitations of other techniques. A 20-year-old male patient with facial asymmetry due to Hemifacial microsomy Type I also requires preoperative orthodontic treatment to align and level their teeth. He showed a 2mm midline shift to the left in combination with a cross bite of the left side. We decide to do a vertical enlargement of the mandibular left border by mandibular Hemiwing osteotomy and unilateral split ramus osteotomy for dental lines alignment with 8 mm of advancement of the hemi - wing genioplasty. Modificated basal osteotomy combined with osteogenic distraction works better than the classic total basal osteotomy with autologous bone graft, if used for the correct indications. We advocate this technique for its efficacy, simplicity, and safety. This technique can be apply for correction of vertical and transverse discrepancies of the mandibular border and combined with sagittal ramus osteotomies for correction of asymmetrical dental lines and oclusal plane. PMID:25593870

  9. Altered MicroRNA Expression Profile in Exosomes during Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    Zhang, Shui-Jun; Zhao, Chen; Qiu, Bin-Song; Gu, Hai-Feng; Hong, Jian-Fei; Cao, Li; Chen, Yu; Xia, Bing; Bi, Qin; Wang, Ya-Ping

    2014-01-01

    The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs) culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (∼8.84%) could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05) when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221) were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation. PMID:25503309

  10. Altered microRNA expression profile in exosomes during osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Ji-Feng Xu

    Full Text Available The physiological role of microRNAs (miRNAs in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (∼8.84% could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05 when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221 were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation.

  11. 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....

  12. Ectopic expression of telomerase enhances osteopontin and osteocalcin expression during osteogenic differentiation of human mesenchymal stem cells from elder donors

    Directory of Open Access Journals (Sweden)

    Machado CB

    2009-01-01

    Full Text Available Age related bone loss is one of the most prevalent diseases in the elder population. The osteoblasts are the effectors cells of bone formation and regeneration. With the aging the osteoblasts become senescent reducing their ability to produce bone. Cellular replicative senescence is triggered by telomers shortening. Telomerase elongate the telomers length and maintain the cell proliferative capacity. Here, we demonstrated that the expression of human telomerase reverse transcriptase mediated by an adenovirus vector increases the levels of osteopontin and osteocalcin mRNA during the in vitro osteogenic differentiation of elderly human mesenchymal stem cells. Bone marrow human mesenchymal stem cells were obtained from old donors (>65 years and induced to differentiate into osteoblasts for 14 days. The levels of mRNA of human telomerase reverse transcriptase, osteopontin and osteocalcin during the differentiation were assessed by semi-quantitative PCR before and during the differentiation on days 7 and 14. Infected cells showed 1.5 fold increase in telomerase expression. Also telomerized cells exhibit 1.5 fold increase in osteopontin and 0.5 fold increase in osteocalcin expression compared to primary osteoblasts isolated from the same donors. The transformed cells were not able to form tumours in NUDE mice.

  13. Porous tantalum coatings prepared by vacuum plasma spraying enhance bmscs osteogenic differentiation and bone regeneration in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Ze Tang

    Full Text Available Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS, which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.

  14. Stem Cells Grown in Osteogenic Medium on PLGA, PLGA/HA, and Titanium Scaffolds for Surgical Applications

    Directory of Open Access Journals (Sweden)

    Annalia Asti

    2010-01-01

    Full Text Available Pluripotent adipose tissue-derived stem cells (hASCs can differentiate into various mesodermal cell types such as osteoblasts, chondroblasts, and myoblasts. We isolated hASCs from subcutaneous adipose tissue during orthopaedic surgery and induced the osteogenic differentiation for 28 days on three different synthetic scaffolds such as polylactide-co-glycolide (PLGA, polylactide-co-glycolide/hydroxyapatite (PLGA/HA, and trabecular titanium scaffolds (Ti6Al4V. Pore size can influence certain criteria such as cell attachment, infiltration, and vascularization. The aim of this study was to investigate the performance of PLGA and PLGA/HA scaffolds with a higher porosity, ranging between 75% and 84%, with respect to Ti scaffolds but with smaller pore size, seeded with hASCs to develop a model that could be used in the treatment of bone defects and fractures. Osteogenesis was assessed by ELISA quantitation of extracellular matrix protein expression, von Kossa staining, X-ray microanalysis, and scanning electron microscopy. The higher amount of protein matrix on the Ti scaffold with respect to PLGA and PLGA/HA leads to the conclusion that not only the type of material but the structure significantly affects cell proliferation.

  15. The osteogenic effects of swimming, jumping, and vibration on the protection of bone quality from disuse bone loss.

    Science.gov (United States)

    Falcai, M J; Zamarioli, A; Okubo, R; de Paula, F J A; Volpon, J B

    2015-06-01

    We assessed and compared the effects of swimming, jumping, and vibration therapies on the prevention of bone loss because of unloading. Eighty Wistar rats were randomly divided into eight groups: S, permanent hind limb-suspended rats; CON, control rats; S + Swim, unloading interrupted by swimming exercise; S + C(Swim), suspension interrupted by regular weight-bearing with the same duration as in the S + Swim protocol; S + Jump, unloading interrupted by jumping exercise; S + C(Jump), suspension interrupted for regular weight-bearing as in the S + Jump group; S + Vibr, unloading interrupted by vibration; and S + C(Vibr), suspension with interruptions for regular weight-bearing with the same protocol as that used for the S + Vibr rats. At the end of the experiment, the bone mineral density, bone strength, histomorphometric parameters, and serum levels of the bone markers were analyzed. The hind limb-suspended rats exhibited bone quality loss. In contrast, the trained rats showed a significant increase in bone mass, bone strength, bone formation, and serum levels of bone markers compared with the respective controls. Although we did not find a significant difference among the three physical exercises, the osteogenic effect of vibration was slightly lower than that of swimming and jumping. Thus, all physical exercises were efficient in preventing bone loss because of unloading and preserving bone quality. PMID:24779886

  16. Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Cen; Kong, Xiangdong [Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Zhang, Sheng-Min [Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074 (China); Lee, In-Seop, E-mail: inseop@yonsei.ac.kr [Bio-X Center, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018 (China); Institute of Natural Sciences, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2015-04-15

    Graphical abstract: - Highlights: • Mineral/OGP nanocomposite layers were synthesized biomimetically on Ti substrates. • Incorporated OGP affected the morphology and ultimate structure of mineral. • Incorporated OGP improved the MSCs adhesion, proliferation, and ALP activity. - Abstract: Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants.

  17. Accelerated Osteogenic Orthodontics™ for retreatment of a patient with diminished root length and absence of the maxillary central incisor.

    Science.gov (United States)

    Montesinos F, Armando; Linares T, Silvana; Pérez-Gasque B, Marisol

    2015-10-01

    An 18-year-old female patient visited a university orthodontics department with a chief complaint of an unesthetic appearance of her teeth, including a protruded upper central incisor and unsatisfactory results from previous orthodontic treatment. Pretreatment records showed a Class II skeletal and dental relation with proclined upper and lower incisors, replacement of an absent upper left central incisor with the left upper cuspid, presence of the upper left deciduous cuspid, mild crowding, and 4 mm of overbite and overjet. The panoramic radiograph showed shortened roots of multiple teeth. Accelerated Osteogenic Orthodontics™ (AOO™) was recommended as an approach to reduce the treatment time and the risk of further root shortening. Despite being more expensive and requiring a surgical procedure, this treatment option was very attractive to the patient. The overall treatment time was 14 months. Facial balance was improved, and good occlusal relationships were achieved from the functional and esthetic perspectives. In conclusion, surgically facilitated orthodontics (specifically, AOO™) is an efficient and safe therapeutic tool for treating or retreating orthodontic patients with diminished root length. PMID:26644760

  18. Evaluation of Osteoconductive and Osteogenic Potential of a Dentin-Based Bone Substitute Using a Calvarial Defect Model

    Directory of Open Access Journals (Sweden)

    Ibrahim Hussain

    2012-01-01

    Full Text Available The aim of this study was to assess the osteoconductive and osteogenic properties of processed bovine dentin using a robust rabbit calvarial defect model. In total, 16 New Zealand White rabbits were operated to create three circular defects in the calvaria. One defect was left unfilled, one filled with collected autogenous bone, and the third defect was filled with the dentin-based bone substitute. Following surgery and after a healing period of either 1 or 6 weeks, a CT scan was obtained. Following sacrificing, the tissues were processed for histological examination. The CT data showed the density in the area grafted with the dentin-based material was higher than the surrounding bone and the areas grafted with autologous bone after 1 week and 6 weeks of healing. The area left unfilled remained an empty defect after 1 week and 6 weeks. Histological examination of the defects filled with the dentin product after 6 weeks showed soft tissue encapsulation around the dentin particles. It can be concluded that the rabbit calvarial model used in this study is a robust model for the assessment of bone materials. Bovine dentin is a biostable material; however, it may not be suitable for repairing large 4-wall defects.

  19. Systematical Evaluation of Mechanically Strong 3D Printed Diluted magnesium Doping Wollastonite Scaffolds on Osteogenic Capacity in Rabbit Calvarial Defects

    Science.gov (United States)

    Sun, Miao; Liu, An; Shao, Huifeng; Yang, Xianyan; Ma, Chiyuan; Yan, Shigui; Liu, Yanming; He, Yong; Gou, Zhongru

    2016-01-01

    Wollastonite (CaSiO3; CSi) ceramic is a promising bioactive material for bone defect repair due to slightly fast degradation of its porous constructs in vivo. In our previous strategy some key features of CSi ceramic have been significantly improved by dilute magnesium doping for regulating mechanical properties and biodegradation. Here we demonstrate that 6 ~ 14% of Ca substituted by Mg in CSi (CSi-Mgx, x = 6, 10, 14) can enhance the mechanical strength (>40 MPa) but not compromise biological performances of the 3D printed porous scaffolds with open porosity of 60‒63%. The in vitro cell culture tests in vitro indicated that the dilute Mg doping into CSi was beneficial for ALP activity and high expression of osteogenic marker genes of MC3T3-E1 cells in the scaffolds. A good bone tissue regeneration response and elastoplastic response in mechanical strength in vivo were determined after implantation in rabbit calvarial defects for 6‒12 weeks. Particularly, the CSi-Mg10 and CSi-Mg14 scaffolds could enhance new bone regeneration with a significant increase of newly formed bone tissue (18 ~ 22%) compared to the pure CSi (~14%) at 12 weeks post-implantation. It is reasonable to consider that, therefore, such CSi-Mgx scaffolds possessing excellent strength and reasonable degradability are promising for bone reconstruction in thin-wall bone defects. PMID:27658481

  20. Preparation and investigation of polylactic acid, calcium carbonate and polyvinylalcohol nanofibrous scaffolds for osteogenic differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    A. Doustgani

    2016-04-01

    Full Text Available Objective(s: In this study, the effect of electrospun fiber orientation on proliferation and differentiation of mesenchymal stem cells (MSCs was evaluated. Materials and Methods: Aligned and random nanocomposite nanofibrous scaffolds were electrospun from polylactic acid (PLA, poly (vinyl alcohol (PVA and calcium carbonate nanoparticles (nCaP. The surface morphology of prepared nanofibrous scaffolds with and without cell was examined using scanning electron microscopy. Mechanical properties of electrospun nanofibrous scaffolds were determined with a  universal testing machine. The in vitro properties of fabricated scaffolds was also investigated by the MTT assay and alkaline phosphatase activity (ALP.Results: The average fiber diameter for aligned and random nanofibers were 82 ± 12 nm and 124 ± 25 nm, respectively. The mechanical testing indicated the higher tensile strength and elastic modulus of aligned nanofibers. MTT and ALP results showed that alignment of nanofiber increased the osteogenic differentiation of stem cells.Conclusion: Aligned nanofibrous nanocomposite scaffolds of PLA/nCaP/PVA could be an excellent substrate for MSCs and represents a potential bone-filling material.

  1. The synergistic induction of bone formation by the osteogenic proteins of the TGF-β supergene family.

    Science.gov (United States)

    Ripamonti, Ugo; Parak, Ruqayya; Klar, Roland M; Dickens, Caroline; Dix-Peek, Thérèse; Duarte, Raquel

    2016-10-01

    The momentum to compose this Leading Opinion on the synergistic induction of bone formation suddenly arose when a simple question was formulated during a discussion session on how to boost the often limited induction of bone formation seen in clinical contexts. Re-examination of morphological and molecular data available on the rapid induction of bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) shows that hTGF-β3 replicates the synergistic induction of bone formation as invocated by binary applications of hOP-1:hTGF-β1 at 20:1 by weight when implanted in heterotopic sites of the rectus abdominis muscle of the Chacma baboon, Papio ursinus. The rapid induction of bone formation in primates by hTGF-β3 may stem from bursts of cladistic evolution, now redundant in lower animal species but still activated in primates by relatively high doses of hTGF-β3. Contrary to rodents, lagomorphs and canines, the three mammalian TGF-β isoforms induce rapid and substantial bone formation when implanted in heterotopic rectus abdominis muscle sites of P. ursinus, with unprecedented regeneration of full thickness mandibular defects with rapid mineralization and corticalization. Provocatively, thus providing potential molecular and biological rationales for the apparent redundancy of osteogenic molecular signals in primates, binary applications of recombinant human osteogenic protein-1 (hOP-1) with low doses of hTGF-β1 and -β3, synergize to induce massive ossicles in heterotopic rectus abdominis, orthotopic calvarial and mandibular sites of P. ursinus. The synergistic binary application of homologous but molecularly different soluble molecular signals has indicated that per force several secreted molecular signals are required singly, synchronously and synergistically to induce optimal osteogenesis. The morphological hallmark of the synergistic induction of bone formation is the rapid differentiation of large osteoid seams enveloping

  2. Synthesis and characterization of nanosized calcium phosphates by flame spray pyrolysis, and their effect on osteogenic differentiation of stem cells

    International Nuclear Information System (INIS)

    The present study evaluates the synthesis of biocompatible osteoconductive and osteoinductive nano calcium phosphate (CaP) particles by industrially applied, aerosol-derived flame spray pyrolysis method for biomedical field. Calcium phosphate nanoparticles were produced in a range of calcium-to-phosphorus ratio, (1.20–2.19) in order to analyze the morphology and crystallinity changes, and to test the bioactivity of particles. The characterization results confirmed that nanometer-sized, spherical calcium phosphate particles were produced. The average primary particle size was determined as 23 nm by counting more than 500 particles in TEM pictures. XRD patterns, HRTEM, SAED, and SEM analyses revealed the amorphous nature of the as-prepared nano calcium phosphate particles at low Ca/P ratios. Increases in the specific surface area and crystallinity were observed with the increasing Ca/P ratio. TGA–DTA analysis showed that the thermally stable crystal phases formed after 700 °C. Cell culture studies were conducted with urine-derived stem cells that possess the characteristics of mesenchymal stem cells. Synthesized amorphous nanoparticles did not have cytotoxic effect at 5–50 μg/ml concentration range. Cells treated with the as-prepared nanoparticles had higher alkaline phosphatase (ALP) enzyme activity than control cells, indicating osteogenic differentiation of cells. A slight decrease in ALP activity of cells treated with two highest Ca:P ratios at 50 μg/ml concentration was observed at day 7. The findings suggest that calcium phosphate nanoparticles produced in this work have a potential to be used as biomaterials in biomedical applications

  3. Identification of long non-coding RNA involved in osteogenic differentiation from mesenchymal stem cells using RNA-Seq data.

    Science.gov (United States)

    Song, W Q; Gu, W Q; Qian, Y B; Ma, X; Mao, Y J; Liu, W J

    2015-01-01

    The aim of this study was to identify long non-coding RNA (lncRNA) associated with osteogenic differentiation from mesenchymal stem cells (MSCs) using high-throughput RNA sequencing (RNA-Seq) data. RNA-Seq dataset was obtained from the European Bioinformatics Institute (accession No. PRJEB4496), including two replicates each for immortalized mesenchymal stem cells iMSC#3 cultured in growth medium (GM) and differentiation medium (DM) for 28 days. The clean reads were aligned to a hg19 reference genome by Tophat and assembled by Cufflinks to identify the known and novel transcripts. RPKM values were calculated to screen for differentially expressed RNA. Novel lncRNA were screened based on various filter criteria. Subsequently, the underlying function of novel lncRNAs were predicted by functional annotation by ERPIN, a co-expression network was constructed by WGCNA and the KEGG pathway enriched by KOBAS. A total of 3171 RNA differentially expressed between the DM and GM groups (2597 mRNA and 574 lncRNA) were identified. Among the 574 differentially expressed lncRNA, 357 were known and 217 were novel lncRNA. Furthermore, 32 novel lncRNA were found to be miRNA precursors (including miR-689, miR-640, miR-601, and miR-544). A total of 14,275 co-expression relationships and 217 co-expression networks were obtained between novel lncRNA and mRNA. The differentially expressed lncRNA and mRNA were enriched into 6 significant pathways, including those for cancer, ECM-receptor interaction, and focal adhesion. Therefore, novel lncRNAwere identified and their underlying function predicted, which may provide the basis for future analyses of the role of lncRNA in osteoblastic differentiation.

  4. Target disruption of ribosomal protein pNO40 accelerates aging and impairs osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Lin, Yen-Ming; Wu, Chih-Ching; Chang, Yu-Chen; Wu, Chu-Han; Ho, Hsien Li; Hu, Ji Wei; Chang, Ren-Chi; Wang, Chung-Ta; Ouyang, Pin

    2016-01-22

    pNO40/PS1D, a novel nucleolar protein, has been characterized as a core protein of eukaryotic 60S ribosome and at least two splicing forms of pNO40 mRNAs with alternative starting sites have been identified. Through production of knockout (ko) mice with either exon 2 (△E2), exon 4 (△E4) or △E2+E4 targeted disruption we identified a cryptic splicing product occurring in the ko tissues examined which in general cannot be observed in regular RT-PCR detection of wild-type (wt) animals. Among ko animals, △E4 null embryos exhibited prominent senescence-associated β-galactosidase (SA-β-gal) staining, a marker for senescent cells, in notochord, forelimbs and heart while bone marrow-derived mesenchymal stem cells (MSCs) from △E4 null mice developed accelerated aging and osteogenic differentiation defects compared to those from wt and other isoform mutant mice. Examination of the causal relationship between pNO40 deficiency and MSC-accelerated aging revealed △E4 null disruption in MSCs elicits high levels of ROS and elevated expression levels of p16 and Rb but not p53. Further analysis with iTraq identified CYP1B1, a component of the cytochrome p450 system, as a potential molecule mediating ROS generation in pNO40 deficient MSCs. We herein established a mouse model of MSC aging through pNO40-targeted depletion and demonstrated the effects of loss of pNO40 on bone homeostasis.

  5. The in vitro and in vivo effects of the low molecular weight fucoidan on the bone osteogenic differentiation properties.

    Science.gov (United States)

    Hwang, Pai-An; Hung, Yu-Lan; Phan, Nam Nhut; Hieu, Bui-Thi-Ngoc; Chang, Po-Ming; Li, Kuan-Lun; Lin, Yen-Chang

    2016-08-01

    Osteoporosis has been reported as a hidden death factor in aged people. So far, prevention and treatment therapies for osteoporosis only slow down the progress but do not treat the disease. Fucoidan has been recognized its roles in anti-tumor, anti-inflammatory, anti-coagulant and antiviral activities. To date, low molecular weight (LMW) fucoidan role in bone loss disease has been not determined yet. Therefore, this study aims to figure out potential effects of LMW fucoidan in osteoporosis in vitro and in vivo. LMW fucoidan was extracted from fresh Sargassum hemiphyllum showing a significant increase in 7F2 cell viability to 150.33 ± 6.50 % relative to normal fucoidan (130.12 ± 5.74 %). The expression of level BMP-2, ALP, osteocalcin significantly increased with 2.28 ± 0.06, 2.18 ± 0.12 and 2.06 ± 0.07 fold, respectively. The RT-PCR assay showed that LMW fucoidan increased mRNA expression of BMP-2, ALP, osteocalcin, COL I, BSP and osteonectin. Furthermore, the bone density and bone ash weight were considerably boosted by the oral administration of 280 mg/kg LMW fucoidan and 100 mg/kg calcium carbonate in C57BL/6J female aged mice. The present finding indicated that LMW fucoidan triggered osteogenic differentiation in vitro, and had an anabolic effect on bone mineralization in vivo. Dietary intake of LMW fucoidan from S. hemiphyllum suggested playing a role in the enhancement of bone loss with increasing age. PMID:26271462

  6. Synthesis and characterization of nanosized calcium phosphates by flame spray pyrolysis, and their effect on osteogenic differentiation of stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ataol, Sibel; Tezcaner, Ayşen [Middle East Technical University, Department of Biomedical Engineering (Turkey); Duygulu, Ozgur [TUBITAK Marmara Research Center, Materials Institute (Turkey); Keskin, Dilek [Middle East Technical University, Department of Biomedical Engineering (Turkey); Machin, Nesrin E., E-mail: nesrinmachin@gmail.com [Kocaeli University, Department of Chemical Engineering (Turkey)

    2015-02-15

    The present study evaluates the synthesis of biocompatible osteoconductive and osteoinductive nano calcium phosphate (CaP) particles by industrially applied, aerosol-derived flame spray pyrolysis method for biomedical field. Calcium phosphate nanoparticles were produced in a range of calcium-to-phosphorus ratio, (1.20–2.19) in order to analyze the morphology and crystallinity changes, and to test the bioactivity of particles. The characterization results confirmed that nanometer-sized, spherical calcium phosphate particles were produced. The average primary particle size was determined as 23 nm by counting more than 500 particles in TEM pictures. XRD patterns, HRTEM, SAED, and SEM analyses revealed the amorphous nature of the as-prepared nano calcium phosphate particles at low Ca/P ratios. Increases in the specific surface area and crystallinity were observed with the increasing Ca/P ratio. TGA–DTA analysis showed that the thermally stable crystal phases formed after 700 °C. Cell culture studies were conducted with urine-derived stem cells that possess the characteristics of mesenchymal stem cells. Synthesized amorphous nanoparticles did not have cytotoxic effect at 5–50 μg/ml concentration range. Cells treated with the as-prepared nanoparticles had higher alkaline phosphatase (ALP) enzyme activity than control cells, indicating osteogenic differentiation of cells. A slight decrease in ALP activity of cells treated with two highest Ca:P ratios at 50 μg/ml concentration was observed at day 7. The findings suggest that calcium phosphate nanoparticles produced in this work have a potential to be used as biomaterials in biomedical applications.

  7. Effects of negative pressure wound therapy on mesenchymal stem cells proliferation and osteogenic differentiation in a fibrin matrix.

    Directory of Open Access Journals (Sweden)

    Jin Zhu

    Full Text Available Vacuum-assisted closure (VAC negative pressure wound therapy (NPWT has been proven to be an effective therapeutic method for the treatment of recalcitrant wounds. However, its role in bone healing remains to be unclear. Here, we investigated the effects of NPWT on rat periosteum-derived mesenchymal stem cells (P-MSCs proliferation and osteoblastic differentiation in a 3D fibrin matrix. P-MSCs underwent primary culture for three passages before being used to construct cell clots. The fibrin clots were incubated with NPWT under continuous suction at -125 mmHg in a subatmospheric perfusion bioreactor. Clots exposed to atmospheric pressure served as the static control. Compared to the control group, cell proliferation significantly increased in NPWT group after incubation for 3 days. There was no statistical difference in apoptosis rate between two groups. The ALP activity and mineralization of P-MSCs all increased under continuous suction. The expressions of collagen type 1 and transcription factor Cbfa-1 were higher at the 1-, 3-, and 7-day timepoints and the expressions of osteocalcin and integrin β5 were higher at the 3-, and 7-day timepoints in the NPWT group. These results indicate that a short time treatment with NPWT, applied with continuous suction at -125 mmHg, can enhance cellular proliferation of P-MSCs and induce the differentiation toward an osteogenic phenotype. The mechanotransduction molecule integrin β5 was found to be highly expressed after NPWT treatment, which indicates that NPWT may play a positive role in fracture healing through enhance bone formation and decrease bone resorption.

  8. Demethylation of IGFBP5 by Histone Demethylase KDM6B Promotes Mesenchymal Stem Cell-Mediated Periodontal Tissue Regeneration by Enhancing Osteogenic Differentiation and Anti-Inflammation Potentials.

    Science.gov (United States)

    Liu, Dayong; Wang, Yuejun; Jia, Zhi; Wang, Liping; Wang, Jinsong; Yang, Dongmei; Song, Jianqiu; Wang, Songlin; Fan, Zhipeng

    2015-08-01

    Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered a promising method for periodontitis treatment. The molecular mechanism underlying directed differentiation and anti-inflammatory actions remains unclear, thus limiting potential MSC application. We previously found that insulin-like growth factor binding protein 5 (IGFBP5) is highly expressed in dental tissue-derived MSCs compared with in non-dental tissue-derived MSCs. IGFBP5 is mainly involved in regulating biological activity of insulin-like growth factors, and its functions in human MSCs and tissue regeneration are unclear. In this study, we performed gain- and loss-of-function assays to test whether IGFBP5 could regulate the osteogenic differentiation and anti-inflammatory potential in MSCs. We found that IGFBP5 expression was upregulated upon osteogenic induction, and that IGFBP5 enhanced osteogenic differentiation in MSCs. We further showed that IGFBP5 prompted the anti-inflammation effect of MSCs via negative regulation of NFκB signaling. Depletion of the histone demethylase lysine (K)-specific demethylase 6B (KDM6B) downregulated IGFBP5 expression by increasing histone K27 methylation in the IGFBP5 promoter. Moreover, IGFBP5 expression in periodontal tissues was downregulated in individuals with periodontitis compared with in healthy people, and IGFBP5 enhanced MSC-mediated periodontal tissue regeneration and alleviated local inflammation in a swine model of periodontitis. In conclusion, our present results reveal a new function for IGFBP5, provide insight into the mechanism underlying the directed differentiation and anti-inflammation capacities of MSCs, and identify a potential target mediator for improving tissue regeneration.

  9. Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

    Science.gov (United States)

    Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

    2016-03-01

    Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering.

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

    Directory of Open Access Journals (Sweden)

    Ariadne Cristiane Cabral Cruz

    2012-12-01

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

  11. Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion

    Directory of Open Access Journals (Sweden)

    Song W

    2015-03-01

    Full Text Available Wen Song,1,* Xin Song,2,* Chuanxu Yang,2 Shan Gao,2 Lasse Hyldgaard Klausen,2 Yumei Zhang,1 Mingdong Dong,2 Jørgen Kjems21State Key Laboratory of Military Stomatology, Department of Prosthetic Dentistry, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 2Interdisciplinary Nanoscience Center (iNANO, Aarhus University, Aarhus, Denmark*These authors contributed equally to this workAbstract: Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA for sustained gene silencing in nearby cells, the layer-by-layer (LbL approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP. The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more

  12. Osteogenic differentiation of MC3T3-E1 cells on poly(L-lactide)/Fe{sub 3}O{sub 4} nanofibers with static magnetic field exposure

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qing [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun [Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China); Deng, Xuliang [Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081 (China); Yang, Xiaoping, E-mail: yangxp@mail.buct.edu.cn [State Key Laboratory of Organic–inorganic Composites, Beijing University of Chemical Technology, Beijing 100029 (China); Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029 (China)

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT–1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe{sub 3}O{sub 4} nanoparticles (NPs). The PLLA/Fe{sub 3}O{sub 4} composite nanofibers demonstrated homogeneous dispersion of Fe{sub 3}O{sub 4} NPs, and their magnetism depended on the contents of Fe{sub 3}O{sub 4} NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe{sub 3}O{sub 4} composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe{sub 3}O{sub 4} NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. - Highlights: • Magnetic nanofibers containing well-dispersed Fe{sub 3}O{sub 4} nanoparticles were produced. • Static magnetic field (SMF) was applied to perform the culture of osteoblasts. • Osteogenic differentiation was enhanced on magnetic substrate with exposure to SMF.

  13. Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway

    Directory of Open Access Journals (Sweden)

    Mao LX

    2015-11-01

    Full Text Available Lixia Mao,1,* Jiaqiang Liu,1,* Jinglei Zhao,1 Jiang Chang,2 Lunguo Xia,1 Lingyong Jiang,1 Xiuhui Wang,2 Kaili Lin,2,3 Bing Fang11Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Science, Top Priority Clinical Medical Center of Shanghai Municipal Commission of Health and Family Planning, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai Jiao Tong University, 2State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 3Shanghai Engineering Research Center of Tooth Restoration and Regeneration, School of Stomatology, Tongji University, Shanghai, People’s Republic of China*These authors contributed equally to this workAbstract: The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods] were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2, ALP, osteocalcin (OCN, cementum attachment protein (CAP, and cementum protein (CEMP as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor

  14. A novel technique for guided bone regeneration using platelet-rich plasma and osteogenic progenitor cells: Literature-based rationale and case report.

    Science.gov (United States)

    Kwon, TaeHyun; Grieco, Peter C; Levin, Liran; Intini, Giuseppe

    2016-03-01

    Achieving predictable guided bone regeneration in critical size defects for future endosseous dental implant therapy poses a great challenge to clinicians. A novel technique utilizing autogenous osteogenic progenitor cells, calcium sulfate activated platelet-rich plasma in addition to particulate allograft was successfully used to augment a severely deficient maxillary anterior edentulous ridge. After 6 months of healing, satisfactory radiographic and clinical bone gain was noted with significant increase in alveolar ridge width. Endosseous implants were placed and restored successfully. The techniques with underlying clinical and biologic rationales are presented and discussed in this report.

  15. Inhibition in multiclass classification

    OpenAIRE

    Huerta, Ramón; Vembu, Shankar; Amigó, José M.; Nowotny, Thomas; Elkan, Charles

    2012-01-01

    The role of inhibition is investigated in a multiclass support vector machine formalism inspired by the brain structure of insects. The so-called mushroom bodies have a set of output neurons, or classification functions, that compete with each other to encode a particular input. Strongly active output neurons depress or inhibit the remaining outputs without knowing which is correct or incorrect. Accordingly, we propose to use a classification function that embodies unselective inhibition and ...

  16. The transcriptional profile of mesenchymal stem cell populations in primary osteoporosis is distinct and shows overexpression of osteogenic inhibitors.

    Directory of Open Access Journals (Sweden)

    Peggy Benisch

    Full Text Available Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79-94 years old suffering from osteoporosis (hMSC-OP. In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1 and of genes involved in osteoclastogenesis (CSF1, PTH1R, but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of ∼30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP.Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L

  17. Titanium-enriched hydroxyapatite-gelatin scaffolds with osteogenically differentiated progenitor cell aggregates for calvaria bone regeneration.

    Science.gov (United States)

    Ferreira, João R; Padilla, Ricardo; Urkasemsin, Ganokon; Yoon, Kun; Goeckner, Kelly; Hu, Wei-Shou; Ko, Ching-Chang

    2013-08-01

    Adequate bony support is the key to re-establish both function and esthetics in the craniofacial region. Autologous bone grafting has been the gold standard for regeneration of problematic large bone defects. However, poor graft availability and donor-site complications have led to alternative bone tissue-engineering approaches combining osteoinductive biomaterials and three-dimensional cell aggregates in scaffolds or constructs. The goal of the present study was to generate novel cell aggregate-loaded macroporous scaffolds combining the osteoinductive properties of titanium dioxide (TiO2) with hydroxyapatite-gelatin nanocomposites (HAP-GEL) for regeneration of craniofacial defects. Here we investigated the in vivo applicability of macroporous (TiO2)-enriched HAP-GEL scaffolds with undifferentiated and osteogenically differentiated multipotent adult progenitor cell (MAPC and OD-MAPC, respectively) aggregates for calvaria bone regeneration. The silane-coated HAP-GEL with and without TiO2 additives were polymerized and molded to produce macroporous scaffolds. Aggregates of the rat MAPC were precultured, loaded into each scaffold, and implanted to rat calvaria critical-size defects to study bone regeneration. Bone autografts were used as positive controls and a poly(lactic-co-glycolic acid) (PLGA) scaffold for comparison purposes. Preimplanted scaffolds and calvaria bone from pig were tested for ultimate compressive strength with an Instron 4411(®) and for porosity with microcomputerized tomography (μCT). Osteointegration and newly formed bone (NFB) were assessed by μCT and nondecalcified histology, and quantified by calcium fluorescence labeling. Results showed that the macroporous TiO2-HAP-GEL scaffold had a comparable strength relative to the natural calvaria bone (13.8±4.5 MPa and 24.5±8.3 MPa, respectively). Porosity was 1.52±0.8 mm and 0.64±0.4 mm for TiO2-HAP-GEL and calvaria bone, respectively. At 8 and 12 weeks postimplantation into rat

  18. Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors

    Science.gov (United States)

    Bloise, Nora; Ceccarelli, Gabriele; Minzioni, Paolo; Vercellino, Marco; Benedetti, Laura; De Angelis, Maria Gabriella Cusella; Imbriani, Marcello; Visai, Livia

    2013-12-01

    Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm2. The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

  19. Production of polymeric micelles by microfluidic technology for combined drug delivery: application to osteogenic differentiation of human periodontal ligament mesenchymal stem cells (hPDLSCs).

    Science.gov (United States)

    Capretto, L; Mazzitelli, S; Colombo, G; Piva, R; Penolazzi, L; Vecchiatini, R; Zhang, X; Nastruzzi, C

    2013-01-20

    The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs. PMs were designed for the co-delivery of dexamethasone (Dex) and ascorbyl-palmitate (AP) to in vitro cultured human periodontal ligament mesenchymal stem cells (hPDLSCs) for the combined induction of osteogenic differentiation. Mixtures of block-copolymers and drugs in organic, water miscible solvent, were conveniently converted in PMs within microfluidic channel leveraging the fast mixing at the microscale. Our results demonstrated that the drugs can be efficiently co-encapsulated in PMs and that different production parameters can be adjusted in order to modulate the PM characteristics. The comparative analysis of PM produced by microfluidic and conventional procedures confirmed that the use of microfluidics platforms allowed the production of PMs in a robust manner with improved controllability, reproducibility, smaller size and polydispersity. Finally, the analysis of the effect of PMs, containing Dex and AP, on the osteogenic differentiation of hPDLSCs is reported. The data demonstrated the effectiveness and safety of PM treatment on hPDLSC. In conclusion, this report indicates that microfluidic approach represents an innovative and useful method for PM controlled preparation, warrant further evaluation as general methodology for the production of colloidal systems for the simultaneous drug delivery. PMID:22884778

  20. Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice.

    Science.gov (United States)

    Alaee, Farhang; Hong, Seung-Hyun; Dukas, Alex G; Pensak, Michael J; Rowe, David W; Lieberman, Jay R

    2014-09-01

    We evaluated the osteoprogenitor response to rhBMP-2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre-osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and α-smooth muscle actin (α-SMA-Cherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP-2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre-osteoblasts demonstrated a maximal response (% GFP + cells/TV) in the Col3.6GFPtopaz mice at day 7 (7.2% ± 6.0, p Col2.3GFP cells was seen at days 14 (8.04% ± 5.0) and 21 (8.31% ± 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes. PMID:24888702

  1. Bioactivity and osteogenic cell response of TiO{sub 2} nanotubes coupled with nanoscale calcium phosphate via ultrasonification-assisted electrochemical deposition

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jianyu, E-mail: chenjianyu-b2@163.com [Guanghua School of Stomatology, Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Zhang, Zhiguang, E-mail: 13580393430@163.com [Guanghua School of Stomatology, Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Ouyang, Jianglin; Chen, Xianshuai [Guangzhou Institute of Advanced Technology, Chinese Academy of Science, Guangzhou 511458 (China); Xu, Zhewu [Guanghua School of Stomatology, Hospital of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Sun, Xuetong [Guangzhou Institute of Advanced Technology, Chinese Academy of Science, Guangzhou 511458 (China)

    2014-06-01

    Ultrasonification-assisted electrochemical deposition was used to introduce nanoscale calcium phosphate (CaP) into well-ordered TiO{sub 2} nanotube arrays (NTA) fabricated by anodic oxidation. Field emission scanning electron microscopy, energy dispersive spectroscopy, X-ray diffraction, and a drop-shape analysis system were used to investigate the morphology, constituent components and hydrophilicity of the nanostructured CaP/NTA surface. Bioactivity and osteogenic cell response were also characterized by hydroxyapatite (HA) formation tests, protein adsorption tests, and in vitro preosteoblast culture experiments. Abundant acicular nanoscale calcium phosphate was successfully deposited on the inner and outer walls of the nanotubes. After immersion in simulated body fluid, increased hydroxyapatite formation was apparent on the surface of TiO{sub 2} nanotubes coupled with nanoscale CaP when compared to simple nanotube structures and polished titanium. The CaP/NTA surface also adsorbed a greater amount of protein after being exposed to bovine serum albumin solution. During cell culture experiments, the preosteoblasts exhibited enhanced cellular adhesion, proliferation, and differentiation on the CaP/NTA surfaces. The results demonstrate that the introduction of nanoscale calcium phosphate into self-organized TiO{sub 2} nanotubes via a straightforward ultrasonification-assisted deposition technique enhances the bioactivity and osteogenic cell response, owing to the combined effects of the nanostructured surface topography, chemical composition, and hydrophilicity.

  2. Novel Osteogenic Ti-6Al-4V Device For Restoration Of Dental Function In Patients With Large Bone Deficiencies: Design, Development And Implementation

    Science.gov (United States)

    Cohen, D. J.; Cheng, A.; Kahn, A.; Aviram, M.; Whitehead, A. J.; Hyzy, S. L.; Clohessy, R. M.; Boyan, B. D.; Schwartz, Z.

    2016-01-01

    Custom devices supporting bone regeneration and implant placement are needed for edentulous patients with large mandibular deficiencies where endosteal implantation is not possible. We developed a novel subperiosteal titanium-aluminum-vanadium bone onlay device produced by additive manufacturing (AM) and post-fabrication osteogenic micro-/nano-scale surface texture modification. Human osteoblasts produced osteogenic and angiogenic factors when grown on laser-sintered nano-/micro-textured surfaces compared to smooth surfaces. Surface-processed constructs caused higher bone-to-implant contact, vertical bone growth into disk pores (microCT and histomorphometry), and mechanical pull-out force at 5 and 10 w on rat calvaria compared to non surface-modified constructs, even when pre-treating the bone to stimulate osteogenesis. Surface-modified wrap-implants placed around rabbit tibias osseointegrated by 6 w. Finally, patient-specific constructs designed to support dental implants produced via AM and surface-processing were implanted on edentulous mandibular bone. 3 and 8 month post-operative images showed new bone formation and osseointegration of the device and indicated stability of the dental implants. PMID:26854193

  3. Metformin Decreases Reactive Oxygen Species, Enhances Osteogenic Properties of Adipose-Derived Multipotent Mesenchymal Stem Cells In Vitro, and Increases Bone Density In Vivo

    Directory of Open Access Journals (Sweden)

    Krzysztof Marycz

    2016-01-01

    Full Text Available Due to its pleiotropic effects, the commonly used drug metformin has gained renewed interest among medical researchers. While metformin is mainly used for the treatment of diabetes, recent studies suggest that it may have further application in anticancer and antiaging therapies. In this study, we investigated the proliferative potential, accumulation of oxidative stress factors, and osteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs isolated from mice treated with metformin for 8 weeks. Moreover, we investigated the influence of metformin supplementation on mice bone density and bone element composition. The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferative potential, generated a robust net of cytoskeletal projections, had reduced expression of markers associated with cellular senescence, and decreased amount of reactive oxygen species in comparison to control group. Furthermore, we demonstrated that these cells possessed greatest osteogenic differentiation potential, while their adipogenic differentiation ability was reduced. We also demonstrated that metformin supplementation increases bone density in vivo. Our result stands as a valuable source of data regarding the in vivo influence of metformin on ASCs and bone density and supports a role for metformin in regenerative medicine.

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

    Science.gov (United States)

    Claros, Silvia; Rico-Llanos, Gustavo A.; Becerra, José; Andrades, José A.

    2014-01-01

    Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo. PMID:24968268

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

    Directory of Open Access Journals (Sweden)

    Silvia Claros

    2014-06-01

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

  6. Metformin Decreases Reactive Oxygen Species, Enhances Osteogenic Properties of Adipose-Derived Multipotent Mesenchymal Stem Cells In Vitro, and Increases Bone Density In Vivo.

    Science.gov (United States)

    Marycz, Krzysztof; Tomaszewski, Krzysztof A; Kornicka, Katarzyna; Henry, Brandon Michael; Wroński, Sebastian; Tarasiuk, Jacek; Maredziak, Monika

    2016-01-01

    Due to its pleiotropic effects, the commonly used drug metformin has gained renewed interest among medical researchers. While metformin is mainly used for the treatment of diabetes, recent studies suggest that it may have further application in anticancer and antiaging therapies. In this study, we investigated the proliferative potential, accumulation of oxidative stress factors, and osteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs) isolated from mice treated with metformin for 8 weeks. Moreover, we investigated the influence of metformin supplementation on mice bone density and bone element composition. The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferative potential, generated a robust net of cytoskeletal projections, had reduced expression of markers associated with cellular senescence, and decreased amount of reactive oxygen species in comparison to control group. Furthermore, we demonstrated that these cells possessed greatest osteogenic differentiation potential, while their adipogenic differentiation ability was reduced. We also demonstrated that metformin supplementation increases bone density in vivo. Our result stands as a valuable source of data regarding the in vivo influence of metformin on ASCs and bone density and supports a role for metformin in regenerative medicine. PMID:27195075

  7. Effects of hydroxyapatite nanostructure on channel surface of porcine acellular dermal matrix scaffold on cell viability and osteogenic differentiation of human periodontal ligament stem cells

    Directory of Open Access Journals (Sweden)

    Ge S

    2013-05-01

    Full Text Available Shaohua Ge,1 Ning Zhao,1 Lu Wang,1 Hong Liu,2 Pishan Yang11Shandong Provincial Key Laboratory of Oral Biomedicine, Department of Periodontology, Shandong University; 2State Key Laboratory of Crystal Materials, Center of Bio and Micro/Nano Functional Materials, Shandong University, Jinan, People's Republic of ChinaAbstract: A new nanostructured hydroxyapatite-coated porcine acellular dermal matrix (HAp-PADM was fabricated by a biomimetic mineralization method. Human periodontal ligament stem cells were seeded on HAp-PADM and the effects of this scaffold on cell shape, cytoskeleton organization, cell viability, and osteogenic differentiation were examined. Periodontal ligament stem cells cultured on HAp-PADM exhibited different cell shape when compared with those on pure PADM. Moreover, HAp-PADM promoted cell viability and alkaline phosphatase activity significantly. Based on quantitative real-time polymerase chain reaction, the expression of bone-related markers runt-related transcription factor 2 (Runx2, osteopontin (OPN, and osteocalcin (OCN upregulated in the HAp-PADM scaffold. The enhancement of osteogenic differentiation of periodontal ligament stem cells on the HAp-PADM scaffold was proposed based on the research results. The results of this study highlight the micro-nano, two-level, three-dimensional HAp-PADM composite as a promising scaffold for periodontal tissue engineering.Keywords: hydroxyapatite, scaffold, nanostructure, proliferation, differentiation, tissue engineering

  8. Design of biomimetic and bioactive cold plasma-modified nanostructured scaffolds for enhanced osteogenic differentiation of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Wang, Mian; Cheng, Xiaoqian; Zhu, Wei; Holmes, Benjamin; Keidar, Michael; Zhang, Lijie Grace

    2014-03-01

    The objective of this study was to design a biomimetic and bioactive tissue-engineered bone construct via a cold atmospheric plasma (CAP) treatment for directed osteogenic differentiation of human bone morrow mesenchymal stem cells (MSCs). Porous nanocrystalline hydroxyapatite/chitosan scaffolds were fabricated via a lyophilization procedure. The nanostructured bone scaffolds were then treated with CAP to create a more favorable surface for cell attachment, proliferation, and differentiation. The CAP-modified scaffolds were characterized via scanning electron microscope, Raman spectrometer, contact angle analyzer, and white light interferometer. In addition, optimal CAP treatment conditions were determined. Our in vitro study shows that MSC adhesion and infiltration were significantly enhanced on CAP modified scaffolds. More importantly, it was demonstrated that CAP-modified nanostructured bone constructs can greatly promote total protein, collagen synthesis, and calcium deposition after 3 weeks of culture, thus making them a promising implantable scaffold for bone regeneration. Moreover, the fibronectin and vitronection adsorption experiments by enzyme-linked immunosorbent assay demonstrated that more adhesion-mediated protein adsorption on the CAP-treated scaffolds. Since the initial specific protein absorption on scaffold surfaces can lead to further recruitment as well as activation of favorable cell functions, it is suggested that our enhanced stem cell growth and osteogenic function may be related to more protein adsorption resulting from surface roughness and wettability modification. The CAP modification method used in this study provides a quick one-step process for cell-favorable tissue-engineered scaffold architecture remodeling and surface property alteration.

  9. Biocompatibility and enhanced osteogenic differentiation of human mesenchymal stem cells in response to surface engineered poly(D,L-lactic-co-glycolic acid) microparticles.

    Science.gov (United States)

    Rogers, Catherine M; Deehan, David J; Knuth, Callie A; Rose, Felicity R A J; Shakesheff, Kevin M; Oldershaw, Rachel A

    2014-11-01

    Tissue engineering strategies can be applied to enhancing osseous integration of soft tissue grafts during ligament reconstruction. Ligament rupture results in a hemarthrosis, an acute intra-articular bleed rich in osteogenic human mesenchymal stem cells (hMSCs). With the aim of identifying an appropriate biomaterial with which to combine hemarthrosis fluid-derived hMSCs (HF-hMSCs) for therapeutic application, this work has investigated the biocompatibility of microparticles manufactured from two forms of poly(D,L-lactic-co-glycolic acid) (PLGA), one synthesized with equal monomeric ratios of lactic acid to glycolic acid (PLGA 50:50) and the other with a higher proportion of lactic acid (PLGA 85:15) which confers a longer biodegradation time. The surfaces of both types of microparticles were functionalized by plasma polymerization with allylamine to increase hydrophilicity and promote cell attachment. HF-hMSCs attached to and spread along the surface of both forms of PLGA microparticle. The osteogenic response of HF-hMSCs was enhanced when cultured with PLGA compared with control cultures differentiated on tissue culture plastic and this was independent of the type of polymer used. We have demonstrated that surface engineered PLGA microparticles are an appropriate biomaterial for combining with HF-hMSCs and the selection of PLGA is relevant only when considering the biodegradation time for each biomedical application.

  10. Inhibition of bone morphogenetic protein signal transduction prevents the medial vascular calcification associated with matrix Gla protein deficiency.

    Directory of Open Access Journals (Sweden)

    Rajeev Malhotra

    Full Text Available Matrix Gla protein (MGP is reported to inhibit bone morphogenetic protein (BMP signal transduction. MGP deficiency is associated with medial calcification of the arterial wall, in a process that involves both osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs and mesenchymal transition of endothelial cells (EndMT. In this study, we investigated the contribution of BMP signal transduction to the medial calcification that develops in MGP-deficient mice.MGP-deficient mice (MGP(-/- were treated with one of two BMP signaling inhibitors, LDN-193189 or ALK3-Fc, beginning one day after birth. Aortic calcification was assessed in 28-day-old mice by measuring the uptake of a fluorescent bisphosphonate probe and by staining tissue sections with Alizarin red. Aortic calcification was 80% less in MGP(-/- mice treated with LDN-193189 or ALK3-Fc compared with vehicle-treated control animals (P<0.001 for both. LDN-193189-treated MGP(-/- mice survived longer than vehicle-treated MGP(-/- mice. Levels of phosphorylated Smad1/5 and Id1 mRNA (markers of BMP signaling did not differ in the aortas from MGP(-/- and wild-type mice. Markers of EndMT and osteogenesis were increased in MGP(-/- aortas, an effect that was prevented by LDN-193189. Calcification of isolated VSMCs was also inhibited by LDN-193189.Inhibition of BMP signaling leads to reduced vascular calcification and improved survival in MGP(-/- mice. The EndMT and osteogenic transdifferentiation associated with MGP deficiency is dependent upon BMP signaling. These results suggest that BMP signal transduction has critical roles in the development of vascular calcification in MGP-deficient mice.

  11. Osteogenic sarcoma : imaging advances

    International Nuclear Information System (INIS)

    The contents are classification of osteosarcoma, radiographic appearance, radionuclide imaging, PET - positron emission tomography scanning, arteriography, computed tomography, MRI imaging, response of chemotherapy (43 refs.)

  12. Novel daidzein analogs enhance osteogenic activity of bone marrow-derived mesenchymal stem cells and adipose-derived stromal/stem cells through estrogen receptor dependent and independent mechanisms

    Science.gov (United States)

    Osteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein anddaidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal s...

  13. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

    DEFF Research Database (Denmark)

    Jensen, Jonas; Tvedesøe, Claus; Rölfing, Jan Hendrik Duedal;

    2016-01-01

    INTRODUCTION: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. METHODS: BMSCs and DPSCs were extracted from the tibia bone mar...

  14. Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via Klotho upregulation.

    Science.gov (United States)

    Zhao, Yang; Zhao, Ming-Ming; Cai, Yan; Zheng, Ming-Fei; Sun, Wei-Liang; Zhang, Song-Yang; Kong, Wei; Gu, Jun; Wang, Xian; Xu, Ming-Jiang

    2015-10-01

    Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo. PMID:26061549

  15. Effect of silk fibroin/hydroxyapatite scaffold on the viability and osteogenic properties of adipose-derived stem cells under osteogenic induction%丝素蛋白/羟基磷灰石支架对成骨诱导脂肪干细胞活性及成骨性能的影响

    Institute of Scientific and Technical Information of China (English)

    刘浩; 褚亚伟; 丁涛; 程力; 朱浩明

    2015-01-01

    BACKGROUND:Adipose-derived stem cels under osteogenic induction can be combined with biodegradable silk fibroin/hydroxyapatite scaffold, which is expected to develop a new biocompatible and osteogenic bone fusion material. OBJECTIVE:To study the effect of silk fibroin/hydroxyapatite composite on the viability and osteogenic properties of adipose-derived stem cels after osteogenic induction. METHODS:Adipose-derived stem cels were obtained from rat’s fat tissue, then adherently cultured, proliferated and passaged in vitro. Passage 3 cels were cultured in conditioned medium for osteogenic induction, and then seeded onto silk fibroin/hydroxyapatite scaffold as experimental group. Adipose-derived stem cels cultured on the cover glasses at the same condition acted as control group. The celular morphology, proliferation and differentiation were assessed respectively by means of phase contrast microscope, MTT assay and alkaline phosphatase activity measurement. RESULTS AND CONCLUSION:After osteogenic induction, adipose-derived stem cels could adhere to the scaffold material and proliferate on the surface of silk fibroin/hydroxyapatite scaffold normaly. No significant difference was found in cel proliferation and alkaline phosphatase activity between the experimental and control groups (P > 0.05), suggesting the celular activity and function were not affected by the material. These findings indicate that silk fibroin/hydroxyapatite composite material has good cytocompatibility. Subject headings: Silk; Hydroxyapatites; Stem Cels; Adipose Tissue; Biocompatible Materials; Tissue Engineering.%背景:成骨诱导后的脂肪干细胞与可降解丝素蛋白/羟基磷灰石支架复合,可望研制出一种具有良好生物相容性及成骨性能的新型骨融合材料。目的:探讨丝素蛋白/羟基磷灰石支架对成骨诱导脂肪干细胞增殖活性及成骨性能的影响。方法:获取大鼠脂肪干细胞后体外贴壁培养、扩增,将第3代细

  16. Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion.

    Science.gov (United States)

    Song, Wen; Song, Xin; Yang, Chuanxu; Gao, Shan; Klausen, Lasse Hyldgaard; Zhang, Yumei; Dong, Mingdong; Kjems, Jørgen

    2015-01-01

    Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA) for sustained gene silencing in nearby cells, the layer-by-layer (LbL) approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA) nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP). The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more importantly, a gradual release of the film allowed the siRNA to be maintained on the titanium surface over approximately 1 week. In vitro transfection revealed that the LbL film-associated siRNA could consistently suppress GFP expression in H1299 without showing significant cytotoxicity. The LbL film loading with osteogenic siRNA could dramatically increase the osteogenic differentiation in MG63. In conclusion, LbL technology can potentially modify titanium

  17. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    Directory of Open Access Journals (Sweden)

    Wu K

    2013-05-01

    Full Text Available Kaimin Wu,1,* Jie Xu,2,* Mengyuan Liu,1 Wen Song,1 Jun Yan,1 Shan Gao,3 Lingzhou Zhao,2 Yumei Zhang1 1Department of Prosthetic Dentistry, 2Department of Periodontology and Oral Medicine, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 3The Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark; School of Stomatology, Tianjin Medical University, Tianjin, People’s Republic of China*Both authors contributed equally to this workAbstract: MicroRNA (miRNA regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall

  18. Prognostic significance of tumor size in child osteogenic sarcoma Significado prognóstico do tamanho tumoral no osteossarcoma infantil

    Directory of Open Access Journals (Sweden)

    Marcos Almeida Matos

    2002-09-01

    Full Text Available From 1995 to 1998, twenty seven pediatric patients were included in a prospective clinical study. Their age ranging from 8.9 to 17.5 years old (mean 13.5, 20 (74% were male and 7(26% female, with a follow-up going from 3 to 39 months (average of 17. Osteogenic sarcoma arises in Knee (55.5%, ankle (26%, shoulder (11.1%, pelvis (3.7% and hip (3.7%. Tumors less than 12cm were considered being small ones, while those bigger than 12cm were considered large. .All patients were submitted to clinical, radiological, histological evaluation and graded by Enneking(5 system (13 were IIB and 8 were IIIB; treated by chemotherapeutic agents, according to "Brazillian Protocol 91" and had a local surgical control. There were carried out 9 (42.9% radical surgical interventions and 12 (57.1% surgeries of limb preservation. According to lesion size, eight (38.1% out of 14 (67.9% patients with a tumor greater than 12cm died; in contrast, none out of 7 with tumor less than 12cm relapsed or died. Authors have concluded that tumor size can be a good factor for prognosis indication, being low cost, simple, easy to reproduce and specially useful for taking pre-treatmente decisions.Entre 1995 a 1998, vinte e sete pacientes foram incluídos neste estudo clínico prospectivo. A idade variou entre 8,9 e 17,5 anos (média de 13,5, 20 (74% eram do sexo masculino e 7 (26% do sexo feminino com seguimento entre 3 e 39 meses (média de 17. O osteossarcoma incidiu no joelho (55,5%, tornozelo (26%, ombro (11,1%0, pelve (3,7% e quadril (3,7%. Tumores menores que 12cm eram considerados pequenos e maiores que 12cm eram considerados grandes. Os pacientes foram submetidos a avaliação clínica, radiológica e histológica e classificados pelo sistema de Enneking(5 (13 eram IIB e 8 eram IIIB; foram tratados com agentes quimioterápicos de acordo com o protocolo brasileiro 91 e pelo controle cirúrgico local. Foram realizadas 9 (42,9% cirurgias radicais e 12 (57,1% cirurgias de preserva

  19. Methods of Telomerase Inhibition

    OpenAIRE

    Andrews, Lucy G.; Tollefsbol, Trygve O.

    2008-01-01

    Telomerase is central to cellular immortality and is a key component of most cancer cells although this enzyme is rarely expressed to significant levels in normal cells. Therefore, the inhibition of telomerase has garnered considerable attention as a possible anticancer approach. Many of the methods applied to telomerase inhibition focus on either of the two major components of the ribonucleoprotein holoenzyme, that is, the telomerase reverse transcriptase (TERT) catalytic subunit or the telo...

  20. A Halogen-Containing Stilbene Derivative from the Leaves of Cajanus cajan that Induces Osteogenic Differentiation of Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Cai, Jia-Zhong; Tang, Rong; Ye, Gui-Fu; Qiu, Sheng-Xiang; Zhang, Nen-Ling; Hu, Ying-Jie; Shen, Xiao-Ling

    2015-01-01

    A new natural halogen-containing stilbene derivative was isolated from the leaves of Cajanus cajan (L.) Millsp. and identified as 3-O-(3-chloro-2-hydroxyl-propanyl)-longistylin A by comprehensive spectroscopic and chemical analysis, and named cajanstilbene H (1). It is the first halogen-containing stilbene derivative found from plants. In human mesenchymal stem cells (hMSC) from bone marrow, 1 did not promote cell proliferation, but distinctly enhanced osteogenic differentiation of hMSC in time- and dose-dependent manners. In six human cancer cell lines, 1 showed a moderate inhibitory effect on cell proliferation, with IC50 values of 21.42-25.85 μmol·L(-1). PMID:26111172

  1. Intra-arterial infusion of radiosensitizer (BUdR) combined with hypofractionated irradiation and chemotherapy for primary treatment of osteogenic sarcoma

    International Nuclear Information System (INIS)

    Combined modality treatment was given in nine patients of osteogenic sarcoma wherein the tumor was unresectable because of location or amputation was refused. This alternative to massive surgery comprised hypofractionated irradiation, intra-arterial infusion of the radiosensitizer 5'-bromodeoxyuridine (BUdR) and adjuvant systemic chemotherapy. Local control was achieved in seven of the nine patients. Four survived, all without evidence of disease at 6, 7.1, 8.8, and 10.5 years after completion of irradiation. Pulmonary metastases developed in six patients - of whom one survives, following high-dose pulmonary irradiation and additional chemotherapy. Significant soft-tissue injury occurred in five patients. On the basis of our experience, the authors believe that new approaches using modifications of external beam irradiation with different fractionation schedules or better radiosensitizing compounds may hold promise for patients with non-resectable osteosarcoma

  2. Mitotic activity and delay in fixation of tumour tissue. The influence of delay in fixation on mitotic activity of a human osteogenic sarcoma grown in athymic nude mice.

    Science.gov (United States)

    Graem, N; Helweg-Larsen, K

    1979-09-01

    The purpose of the present investigation was to study the effect of delay in fixation on the mitotic activity in tumour tissue. A human osteogenic sarcoma, especially suitable for counting of mitoses, grown in athymic nude mice, was fixed with varying delay and the mitotic, prophase, metaphase and ana-telophase indices were determined. An almost exponential decline of the mitotic index was observed with a reduction to 49.4% and 15.0% after respectively 60 and 180 minutes. The proportional incidence of prophases, metaphases and ana-telophases changed so that a relative accummulation of advanced phases occured during the 180 minutes of observation. It is concluded that delay in fixation of a magnitude, which is not uncommon in routine surgical pathology, may allow the majority of mitoses to terminate, resulting in unreliable assessments of mitotic activity.

  3. Identification of Proteins with Potential Osteogenic Activity Present in the Water-Soluble Matrix Proteins from Crassostrea gigas Nacre Using a Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Daniel V. Oliveira

    2012-01-01

    Full Text Available Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM. We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2 were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

  4. A Halogen-Containing Stilbene Derivative from the Leaves of Cajanus cajan that Induces Osteogenic Differentiation of Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Jia-Zhong Cai

    2015-06-01

    Full Text Available A new natural halogen-containing stilbene derivative was isolated from the leaves of Cajanus cajan (L. Millsp. and identified as 3-O-(3-chloro-2-hydroxyl-propanyl-longistylin A by comprehensive spectroscopic and chemical analysis, and named cajanstilbene H (1. It is the first halogen-containing stilbene derivative found from plants. In human mesenchymal stem cells (hMSC from bone marrow, 1 did not promote cell proliferation, but distinctly enhanced osteogenic differentiation of hMSC in time- and dose-dependent manners. In six human cancer cell lines, 1 showed a moderate inhibitory effect on cell proliferation, with IC50 values of 21.42–25.85 μmol·L−1.

  5. A Comparative Evaluation of the Mechanical Properties of Two Calcium Phosphate/Collagen Composite Materials and Their Osteogenic Effects on Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2016-01-01

    Full Text Available Adipose-derived stem cells (ADSCs are ideal seed cells for use in bone tissue engineering and they have many advantages over other stem cells. In this study, two kinds of calcium phosphate/collagen composite scaffolds were prepared and their effects on the proliferation and osteogenic differentiation of ADSCs were investigated. The hydroxyapatite/β-tricalcium phosphate (HA/β-TCP composite scaffolds (HTPSs, which have an additional β-tricalcium phosphate, resulted in better proliferation of ADSCs and showed osteogenesis-promoting effects. Therefore, such composite scaffolds, in combination with ADSCs or on their own, would be promising for use in bone regeneration and potential clinical therapy for bone defects.

  6. Monolithic calcium phosphate/poly(lactic acid) composite versus calcium phosphate-coated poly(lactic acid) for support of osteogenic differentiation of human mesenchymal stromal cells.

    Science.gov (United States)

    Tahmasebi Birgani, Zeinab; van Blitterswijk, Clemens A; Habibovic, Pamela

    2016-03-01

    Calcium phosphates (CaPs), extensively used synthetic bone graft substitutes, are often combined with other materials with the aim to overcome issues related to poor mechanical properties of most CaP ceramics. Thin ceramic coatings on metallic implants and polymer-ceramic composites are examples of such hybrid materials. Both the properties of the CaP used and the method of incorporation into a hybrid structure are determinant for the bioactivity of the final construct. In the present study, a monolithic composite comprising nano-sized CaP and poly(lactic acid) (PLA) and a CaP-coated PLA were comparatively investigated for their ability to support proliferation and osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells (hMSCs). Both, the PLA/CaP composite, produced using physical mixing and extrusion and CaP-coated PLA, resulting from a biomimetic coating process at near-physiological conditions, supported proliferation of hMSCs with highest rates at PLA/CaP composite. Enzymatic alkaline phosphatase activity as well as the mRNA expression of bone morphogenetic protein-2, osteopontin and osteocalcin were higher on the composite and coated polymer as compared to the PLA control, while no significant differences were observed between the two methods of combining CaP and PLA. The results of this study confirmed the importance of CaP in osteogenic differentiation while the exact properties and the method of incorporation into the hybrid material played a less prominent role. PMID:26787486

  7. 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.

  8. Enzymatically cross-linked gelatin-phenol hydrogels with a broader stiffness range for osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Wang, Li-Shan; Du, Chan; Chung, Joo Eun; Kurisawa, Motoichi

    2012-05-01

    An injectable hydrogel system, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugates chemically cross-linked by an enzyme-mediated oxidation reaction, has been designed as a biodegradable scaffold for tissue engineering. In light of the role of substrate stiffness on cell differentiation, we herein report a newly improved Gtn hydrogel system with a broader range of stiffness control that uses Gtn-HPA-tyramine (Gtn-HPA-Tyr) conjugates to stimulate the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The Gtn-HPA-Tyr conjugate was successfully synthesized through a further conjugation of Tyr to Gtn-HPA conjugate by means of a general carbodiimide/active ester-mediated coupling reaction. Proton nuclear magnetic resonance and UV-visible measurements showed a higher total phenol content in the Gtn-HPA-Tyr conjugate than that content in the Gtn-HPA conjugate. The Gtn-HPA-Tyr hydrogels were formed by the oxidative coupling of phenol moieties catalyzed by hydrogen peroxide (H(2)O(2)) and horseradish peroxidase (HRP). Rheological studies revealed that a broader range of storage modulus (G') of Gtn-HPA-Tyr hydrogel (600-26,800 Pa) was achieved using different concentrations of H(2)O(2), while the G' of the predecessor Gtn-HPA hydrogels was limited to the range of 1000 to 13,500 Pa. The hMSCs on Gtn-HPA-Tyr hydrogel with G' greater than 20,000 showed significantly up-regulated expressions of osteocalcin and runt-related transcription factor 2 (RUNX2) on both the gene and protein level, with the presence of alkaline phosphatase, and the evidence of calcium accumulation. These studies with the Gtn-HPA-Tyr hydrogel with G' greater than 20,000 collectively suggest the stimulation of the hMSCs into osteogenic differentiation, while these same observations were not found with the Gtn-HPA hydrogel with a G' of 13,500.

  9. The effect of the coumarin-like derivative osthole on the osteogenic properties of human periodontal ligament and jaw bone marrow mesenchymal stem cell sheets.

    Science.gov (United States)

    Gao, Li-Na; An, Ying; Lei, Ming; Li, Bei; Yang, Hao; Lu, Hong; Chen, Fa-Ming; Jin, Yan

    2013-12-01

    Cell sheet engineering is a scaffold-free delivery concept that has been shown to improve mesenchymal stem cell-mediated regeneration of injured or pathologically damaged periodontal tissues in preclinical studies and several clinical trials. However, the best strategy for cell sheet production remains to be identified. The aim of this study was to investigate the biological effects of osthole, a coumarin-like derivative extracted from Chinese herbs, on the cell sheet formation and osteogenic properties of human periodontal ligament stem cells (PDLSCs) and jaw bone marrow mesenchymal stem cells (JBMMSCs). Patient-matched PDLSCs and JBMMSCs were isolated, and an appropriate concentration of osthole for cell culture was screened for both cell types in terms of cell proliferation and alkaline phosphatase (ALP) activity. Next, the best mode of osthole stimulation for inducing the formation of sheets by each cell type was selected by evaluating the amount of their extracellular matrix (ECM) protein production as well as osteogenic-related gene expression. Furthermore, both PDLSC and JBMMSC sheets obtained from each optimized technique were transplanted subcutaneously into nude mice to evaluate their capacity for ectopic bone regeneration. The results revealed that 10(-5) m/L osthole significantly enhanced the proliferation of both PDLSCs and JBMMSCs (P osthole groups (P > 0.05). In addition, 10(-5) m/L osthole was the best concentration to promote the ALP activities of both cells (P osthole throughout the entire culture stage (10 days) for PDLSCs or at the early stage (first 3 days) for JBMMSCs was the most effective osthole administration mode for cell sheet formation (P osthole-mediated PDLSC and JBMMSC sheets formed more new bone than those obtained without osthole intervention (P osthole stimulation may enhance ECM production and positively affect cell behavior in cell sheet engineering. PMID:24095254

  10. Plasma Surface Modification of Polyhedral Oligomeric Silsequioxane-Poly(carbonate-urea) Urethane with Allylamine Enhances the Response and Osteogenic Differentiation of Adipose-Derived Stem Cells.

    Science.gov (United States)

    Chaves, Camilo; Alshomer, Feras; Palgrave, Robert G; Kalaskar, Deepak M

    2016-07-27

    This study present amino functionalization of biocompatible polymer polyhedral oligomeric silsequioxane-poly(carbonate-urea) urethane (POSS-PCU) using plasma polymerization process to induce osteogenic differentiation of adipose derived stem cells (ADSCs). Optimization of plasma polymerization process was carried out keeping cell culture application in mind. Thus, samples were rigorously tested for retention of amino groups under both dry and wet conditions. Physio-chemical characterization was carried out using ninhydrin test, X-ray photon spectroscopy, scanning electron microscopy, and static water contact analysis. Results from physio chemical characterization shows that functionalization of the amino group is not stable under wet conditions and optimization of plasma process is required for stable bonding of amino groups to the POSS-PCU polymer. Optimized samples were later tested in vitro in short and long-term culture to study differentiation of ADSCs on amino modified samples. Short-term cell culture shows that initial cell attachment was significantly (p PCU) compared to unmodified POSS-PCU. NH2-POSS-PCU samples also facilitates osteogenic differentiation of ADSCs as confirmed by immunological staining of cells for extracellular markers such as collagen Type I and osteopontin. Quantification of total collagen and ALP activity also shows significant (p PCU samples compared to unmodified POSS-PCU. A pilot study also confirms that these optimized amino modified POSS-PCU samples can further be functionalized using bone inducing peptide such as KRSR using conventional wet chemistry. This further provides an opportunity for biofunctionalization of the polymer for various tissue specific applications. PMID:27384590

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

    International Nuclear Information System (INIS)

    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 β-TCP. The alkaline phosphatase (ALP), the calcium and phosphonium, the osteocalcin in media of the third passage RDFCs on biomaterial β-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 β-TCP by scanning electron microscopy. The ALP activity, the calcium and phosphoniums and the osteocalcin content of dexamethasone (10-8 M) or/and BMP-2 (100 ng ml-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+β-TCP+BMP-2+Dex group was significantly higher than that in the RDFCs+β-TCP+BMP-2 group. In contrast, β-TCP, RDFCs+β-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 β-TCP. β-TCP supported fast cellular adhesion, proliferation and differentiation of RDFCs. The feasibility of its application in periodontal tissue engineering was also proved.

  12. Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

    Science.gov (United States)

    Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, M L B; Mishra, Durga Prasad

    2015-12-22

    Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer. PMID:26621832

  13. Quorum sensing inhibition

    DEFF Research Database (Denmark)

    Persson, T.; Givskov, Michael Christian; Nielsen, J.

    2005-01-01

    /receptor transcriptional regulator in some clinically relevant Gram-negative bacteria. The present review contains all reported compound types that are currently known to inhibit the QS transcriptional regulator in Gram-negative bacteria. These compounds are sub-divided into two main groups, one comprising structural...

  14. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus;

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  15. Ethyl-2, 5-dihydroxybenzoate displays dual activity by promoting osteoblast differentiation and inhibiting osteoclast differentiation.

    Science.gov (United States)

    Kwon, Byeong-Ju; Lee, Mi Hee; Koo, Min-Ah; Kim, Min Sung; Seon, Gyeung Mi; Han, Jae-Jin; Park, Jong-Chul

    2016-03-11

    The interplay between bone-forming osteoblasts and bone-resorbing osteoclasts is essential for balanced bone remodeling. In this study, we evaluate the ability of ethyl-2, 5-dihyrdoxybenzoate (E-2, 5-DHB) to affect both osteoblast and osteoclast differentiation for bone regeneration. Osteogenic differentiation of human mesenchymal stem cells (hMSCs) was quantified by measuring alkaline phosphatase (ALP) activity and calcium deposition. To evaluate osteoclast differentiation, we investigated the effect of E-2, 5-DHB on RANKL-activated osteoclastogenesis in RAW 264.7 cells. E-2, 5-DHB enhanced ALP activity and inhibited RAW 264.7 cell osteoclastogenesis in vitro. To assess the in vivo activity of E-2, 5-DHB, hMSCs were delivered subcutaneosuly alone or in combination with E-2, 5-DHB in an alginate gel into the backs of nude-mice. Histological and immunohistochemical evaluation showed significantly higher calcium deposition in the E-2, 5-DHB group. Osteocalcin (OCN) was highly expressed in cells implanted in the gels containing E-2, 5-DHB. Our results suggest that E-2, 5-DHB can effectively enhance osteoblast differentiation and inhibit osteoclast differentiation both in vitro and in vivo. Understanding the dual function of E-2, 5-DHB on osteoblast and osteoclast differentiation will aid in future development of E-2, 5-DHB as a material for bone tissue engineering.

  16. Inhibition of Histone Acetylation as a Tool in Bone Tissue Engineering

    NARCIS (Netherlands)

    Boer, de Jan; Licht, Ruud; Bongers, Marloes; Klundert, van der Tessa; Arends, Roel; Blitterswijk, van Clemens

    2006-01-01

    Our approach to bone tissue engineering is the in vitro expansion and osteogenic differentiation of bone marrow–derived human mesenchymal stem cells (hMSCs) and their subsequent implantation on porous ceramic materials. Current osteogenic differentiation protocols use dexamethasone to initiate the o

  17. Plastics for corrosion inhibition

    CERN Document Server

    Goldade, Victor A; Makarevich, Anna V; Kestelman, Vladimir N

    2005-01-01

    The development of polymer composites containing inhibitors of metal corrosion is an important endeavour in modern materials science and technology. Corrosion inhibitors can be located in a polymer matrix in the solid, liquid or gaseous phase. This book details the thermodynamic principles for selecting these components, their compatibility and their effectiveness. The various mechanisms of metal protection – barrier, inhibiting and electromechanical – are considered, as are the conflicting requirements placed on the structure of the combined material. Two main classes of inhibited materials (structural and films/coatings) are described in detail. Examples are given of structural plastics used in friction units subjected to mechano-chemical wear and of polymer films/coatings for protecting metal objects against corrosion.

  18. Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification.

    Science.gov (United States)

    Kiffer-Moreira, Tina; Yadav, Manisha C; Zhu, Dongxing; Narisawa, Sonoko; Sheen, Campbell; Stec, Boguslaw; Cosford, Nicholas D; Dahl, Russell; Farquharson, Colin; Hoylaerts, Marc F; Macrae, Vicky E; Millán, José Luis

    2013-01-01

    Medial vascular calcification (MVC) is common in patients with chronic kidney disease, obesity, and aging. MVC is an actively regulated process that resembles skeletal mineralization, resulting from chondro-osteogenic transformation of vascular smooth muscle cells (VSMCs). Here, we used mineralizing murine VSMCs to study the expression of PHOSPHO1, a phosphatase that participates in the first step of matrix vesicles-mediated initiation of mineralization during endochondral ossification. Wild-type (WT) VSMCs cultured under calcifying conditions exhibited increased Phospho1 gene expression and Phospho1(-/-) VSMCs failed to mineralize in vitro. Using natural PHOSPHO1 substrates, potent and specific inhibitors of PHOSPHO1 were identified via high-throughput screening and mechanistic analysis and two of these inhibitors, designated MLS-0390838 and MLS-0263839, were selected for further analysis. Their effectiveness in preventing VSMC calcification by targeting PHOSPHO1 function was assessed, alone and in combination with a potent tissue-nonspecific alkaline phosphatase (TNAP) inhibitor MLS-0038949. PHOSPHO1 inhibition by MLS-0263839 in mineralizing WT cells (cultured with added inorganic phosphate) reduced calcification in culture to 41.8% ± 2.0% of control. Combined inhibition of PHOSPHO1 by MLS-0263839 and TNAP by MLS-0038949 significantly reduced calcification to 20.9% ± 0.74% of control. Furthermore, the dual inhibition strategy affected the expression of several mineralization-related enzymes while increasing expression of the smooth muscle cell marker Acta2. We conclude that PHOSPHO1 plays a critical role in VSMC mineralization and that "phosphatase inhibition" may be a useful therapeutic strategy to reduce MVC.

  19. Donor-matched mesenchymal stem cells from knee infrapatellar and subcutaneous adipose tissue of osteoarthritic donors display differential chondrogenic and osteogenic commitment

    Directory of Open Access Journals (Sweden)

    S Lopa

    2014-04-01

    Full Text Available Cell-based therapies have recently been proposed for the treatment of degenerative articular pathologies, such as early osteoarthritis, with an emphasis on autologous mesenchymal stem cells (MSCs, as an alternative to terminally differentiated cells. In this study, we performed a donor-matched comparison between infrapatellar fat pad MSCs (IFP-MSCs and knee subcutaneous adipose tissue stem cells (ASCs, as appealing candidates for cell-based therapies that are easily accessible during surgery. IFP-MSCs and ASCs were obtained from 25 osteoarthritic patients undergoing total knee replacement and compared for their immunophenotype and differentiative potential. Undifferentiated IFP-MSCs and ASCs displayed the same immunophenotype, typical of MSCs (CD13+/CD29+/CD44+/CD73+/CD90+/CD105+/CD166+/CD31-/CD45-. IFP-MSCs and ASCs showed similar adipogenic potential, though undifferentiated ASCs had higher LEP expression compared to IFP-MSCs (p < 0.01. Higher levels of calcified matrix (p < 0.05 and alkaline phosphatase (p < 0.05 in ASCs highlighted their superior osteogenic commitment compared to IFP-MSCs. Conversely, IFP-MSCs pellets showed greater amounts of glycosaminoglycans (p < 0.01 and superior expression of ACAN (p < 0.001, SOX9, COMP (p < 0.001 and COL2A1 (p < 0.05 compared to ASCs pellets, revealing a superior chondrogenic potential. This was also supported by lower COL10A1 (p < 0.05 and COL1A1 (p < 0.01 expression and lower alkaline phosphatase release (p < 0.05 by IFP-MSCs compared to ASCs. The observed dissimilarities between IFP-MSCs and ASCs show that, despite expressing similar surface markers, MSCs deriving from different fat depots in the same surgical site possess specific features. Furthermore, the in vitro peculiar commitment of IFP-MSCs and ASCs from osteoarthritic donors towards the chondrogenic or osteogenic lineage may suggest a preferential use for cartilage and bone cell-based treatments, respectively.

  20. PPARgamma inhibits osteogenesis via the down-regulation of the expression of COX-2 and iNOS in rats.

    Science.gov (United States)

    Lin, Tzu-Hung; Yang, Rong-Sen; Tang, Chih-Hsin; Lin, Chih-Peng; Fu, Wen-Mei

    2007-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, is considered as an anti-osteoblastic factor associated with adiposity and the elderly osteoporosis due to a defect in osteoblastogenesis. We have found that oral administration of PPARgamma activator rosiglitazone decreased tibia BMD and serum ALP but left serum calcium and osteoclast marker C-terminal telopeptide unaffected. In addition, we examined the inhibitory mechanisms of PPARgamma on the bone formation by using PPARgamma activators ciglitazone and 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2). Our data indicated that PPARgamma ligands decreased both mineralized bone nodules and alkaline phosphatase (ALP) activities in cultured primary osteoblasts. Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) was inhibited by ciglitizone and 15d-PGJ2. Furthermore, PPARgamma ligands inhibited NF-kappaB associated downstream COX-2 and iNOS osteogenic signaling. The ultrasound (US)-induced elevation of COX-2 and iNOS expression and nitric oxide (NO) production were attenuated in the presence of PPARgamma ligands. Furthermore, local administration of PPARgamma ligands into the metaphysis of rat tibia decreased the bone volume in secondary spongiosa. These results suggest that the activation of PPARgamma inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation. These data may reflect osteoporosis and less bone formation in the aging people and patients treated with thiazolidinediones. PMID:17669705

  1. Melatonin reversed tumor necrosis factor-alpha-inhibited osteogenesis of human mesenchymal stem cells by stabilizing SMAD1 protein.

    Science.gov (United States)

    Lian, Chengjie; Wu, Zizhao; Gao, Bo; Peng, Yan; Liang, Anjing; Xu, Caixia; Liu, Lei; Qiu, Xianjian; Huang, Junjun; Zhou, Hang; Cai, Yifeng; Su, Peiqiang; Huang, Dongsheng

    2016-10-01

    Tumor necrosis factor-alpha (TNFα) plays a pivotal role in inflammation-related osteoporosis through the promotion of bone resorption and suppression of bone formation. Numerous drugs have been produced to treat osteoporosis by inhibiting bone resorption, but they offer few benefits to bone formation, which is what is needed by patients with severe bone loss. Melatonin, which can exert both anti-inflammatory and pro-osteogenic effects, shows promise in overcoming TNFα-inhibited osteogenesis and deserves further research. This study demonstrated that melatonin rescued TNFα-inhibited osteogenesis of human mesenchymal stem cells and that the interactions between SMURF1 and SMAD1 mediated the crosstalk between melatonin signaling and TNFα signaling. Additionally, melatonin treatment was found to downregulate TNFα-induced SMURF1 expression and then decrease SMURF1-mediated ubiquitination and degradation of SMAD1 protein, leading to steady bone morphogenetic protein-SMAD1 signaling activity and restoration of TNFα-impaired osteogenesis. Thus, melatonin has prospects for treating osteoporosis caused by inflammatory factors due to its multifaceted functions on regulation of bone formation, bone resorption, and inflammation. Further studies will focus on unveiling the specific mechanisms by which melatonin downregulates SMURF1 expression and confirming the clinical therapeutic value of melatonin in the prevention and therapy of bone loss associated with inflammation. PMID:27265199

  2. Effect of an Experimental Direct Pulp-capping Material on the Properties and Osteogenic Differentiation of Human Dental Pulp Stem Cells

    Science.gov (United States)

    Yu, Fan; Dong, Yan; Yang, Yan-wei; Lin, Ping-ting; Yu, Hao-han; Sun, Xiang; Sun, Xue-fei; Zhou, Huan; Huang, Li; Chen, Ji-hua

    2016-01-01

    Effective pulp-capping materials must have antibacterial properties and induce dentin bridge formation; however, many current materials do not satisfy clinical requirements. Accordingly, the effects of an experiment pulp-capping material (Exp) composed of an antibacterial resin monomer (MAE-DB) and Portland cement (PC) on the viability, adhesion, migration, and differentiation of human dental pulp stem cells (hDPSCs) were examined. Based on a Cell Counting Kit-8 assay, hDPSCs exposed to Exp extracts showed limited viability at 24 and 48 h, but displayed comparable viability to the control at 72 h. hDPSC treatment with Exp extracts enhanced cellular adhesion and migration according to in vitro scratch wound healing and Transwell migration assays. Exp significantly upregulated the expression of osteogenesis-related genes. The hDPSCs cultured with Exp exhibited higher ALP activity and calcium deposition in vitro compared with the control group. The novel material showed comparable cytocompatibility to control cells and promoted the adhesion, migration, and osteogenic differentiation of hDPSCs, indicating excellent biocompatibility. This new direct pulp-capping material containing MAE-DB and PC shows promise as a potential alternative to conventional materials for direct pulp capping. PMID:27698421

  3. The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

    Science.gov (United States)

    Kao, Chia-Tze; Hsu, Tuan-Ti; Huang, Tsui-Hsien; Wu, Yu-Tin; Chen, Yi-Wen; Shie, Ming-You

    2016-02-01

    Angiogenesis plays an important role in determining the biostimulation of bone regeneration, in either new bone or blood vessel formation. Human umbilical cord cells (HUVECs) are important effector cells in angiogenesis and are indispensable for osteogenesis and for their heterogeneity and plasticity. However, there are very few studies about the effects of HUVECs on diode laser-stimulated/regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVECs on laser-stimulated osteogenesis. Several bone formation-related proteins were also significantly up-regulated by the diode laser stimulation, indicating that HUVECs may participate in diode laser-stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVECs were diode laser-treated, the osteogenesis differentiation of the hMSCs was significantly promoted, indicating the important role of HUVECs in diode laser-enhanced osteogenesis. Adequately activated HUVECs are vital for the success of diode laser-stimulated hard-tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair.

  4. Effects of Culture Substrate Made of Poly(N-isopropylacrylamide-co-acrylic acid) Microgels on Osteogenic Differentiation of Mesenchymal Stem Cells.

    Science.gov (United States)

    Dai, Zhuojun; Shu, Yinglan; Wan, Chao; Wu, Chi

    2016-01-01

    Poly(N-isopropylacrylamide) (PNIPAM)-based polymers and gels are widely known and studied for their thermoresponsive property. In the biomaterials category, they are regarded as a potential cell culture substrate, not only because of their biocompatibility, but also their special character of allowing controlled detachment of cells via temperature stimulus. Previous research about PNIPAM-based substrates mostly concentrated on their effects in cell adhesion and proliferation. In this study, however, we investigate the influence of the PNIPAM-based substrate on the differentiation capacity of stem cells. Especially, we choose P(NIPAM-AA) microgels as a culture dish coating and mesenchymal stem cells (MSCs) are cultured on top of the microgels. Interestingly, we find that the morphology of MSCs changes remarkably on a microgel-coated surface, from the original spindle form to a more stretched and elongated cell shape. Accompanied by the alternation in morphology, the expression of several osteogenesis-related genes is elevated even without inducing factors. In the presence of full osteogenic medium, MSCs on a microgel substrate show an enhancement in the expression level of osteopontin and alizarin red staining signals, indicating the physical property of substrate has a direct effect on MSCs differentiation. PMID:27618001

  5. Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sogo, Yu [National Institute of Advanced Industrial Science and Technology (AIST), Institute for Human Science and Biomedical Engineering, Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Ito, Atsuo [National Institute of Advanced Industrial Science and Technology (AIST), Institute for Human Science and Biomedical Engineering, Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Matsuno, Tomonori [Department of Oral and Maxillofacial Surgery, School of Life Dentistry at Tokyo, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159 (Japan); Oyane, Ayako [National Institute of Advanced Industrial Science and Technology (AIST), Nanotechnology Research Institute, Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Tamazawa, Gaku [Department of Oral and Maxillofacial Surgery, School of Life Dentistry at Tokyo, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159 (Japan); Satoh, Tazuko [Department of Oral and Maxillofacial Surgery, School of Life Dentistry at Tokyo, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159 (Japan); Yamazaki, Atsushi [Department of Resources and Environmental Engineering, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555 (Japan); Uchimura, Eiji [Department of Resources and Environmental Engineering, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555 (Japan); Ohno, Tadao [Department of Resources and Environmental Engineering, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555 (Japan)

    2007-06-01

    Fibronectin (Fn) and type I collagen (Col) were immobilized on a surface of a hydroxyapatite (HAP) ceramic by coprecipitation with calcium phosphate in a supersaturated calcium phosphate solution prepared by mixing clinically approved infusion fluids. These proteins and the calcium phosphate precipitate formed a composite surface layer. As a result, the proteins were immobilized firmly as not to be released completely for 3 d in a physiological salt solution. When human mesenchymal stem cells (hMSCs) were cultured on a HAP ceramic in a differentiation medium supplemented with dexamethasone, {beta}-glycerophosphate and ascorbic acid, hMSCs spread well within 1 h. The alkaline phosphatase (ALP) activity of hMSCs cultured on the Fn-calcium phosphate composite layer significantly increased compared with that of hMSCs cultured on the untreated HAP ceramic. On the other hand, Col did not increase the ALP activity of hMSCs and no synergy between Fn and Col was observed. Therefore, the Fn-calcium phosphate composite layer formed on the HAP is useful for the enhancement of the spreading and osteogenic differentiation of hMSCs in vitro.

  6. Bioreducible-Cationic Poly(amido amine)s for Enhanced Gene Delivery and Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Jeong, Hansaem; Lee, Eun-Seo; Jung, Giyoung; Park, Jungha; Jeong, Byeongmoon; Ryu, Kyung Ha; Hwang, Nathaniel S; Lee, Hyukjin

    2016-05-01

    The development of efficient and safe gene delivery carriers has been a major challenge in the clinical application of non-viral gene therapy. Herein, we report novel bioreducible poly(amido amine)s for the efficient delivery of genetic material such as plasmid DNA. A library of 34 different bioreducible polymer compounds was synthesized and screened to find lead materials for in vitro gene transfection. Our lead material (CBA-106) allows effortless polyplex formation with genetic materials by electrostatic interactions at the weight ratio of 1:5 (DNA/polymer). Polyplexes were further characterized by DLS and AFM analysis. Enhanced serum stability and bioreducibility under physiological conditions were confirmed, in addition to low cellular cytotoxicity. When compared with a commercially available gene delivery carrier (Lipofectamine 2000), CBA-1 06 shows comparable or even surpassing gene transfection efficiency. Furthermore, BMP-2 plasmids were efficiently delivered to tonsil-derived mesenchymal stem cells (TMSCs) for osteogenic commitment in vitro and in vivo. Taken together, our results clearly demonstrate the potential of novel bioreducible polymeric systems for gene delivery applications. We suggest that our system can provide a valuable platform for the broad application of gene regulation in cell therapy and regenerative medicine. PMID:27305823

  7. Development and utilization of a Walker 256 tumor-induced osteogenic small animal model for study of (Tc-99m)diphosphonate complexes

    International Nuclear Information System (INIS)

    The objective of this research was to develop and utilize a Walker 256 tumor induced osteogenic small animal model to study /sup 99m/Tc(NaBH4)HEDP complexes. A solid tumor was induced in muscles adjacent to the tibia of Fischer-344 rats by the implantation of Walker 256 carcinoma cells. Histopathological studies confirmed the induction of discrete osteogenesis on the periosteal surface of the tibia. The biodistribution of [/sup 99m/Tc]HMDP and [/sup 99m/Tc]MDP was determined in 18 tumor bearing animals and in the same number of normal animals. The results of the study were found to be comparable with clinical findings in humans. The model was proved to be valid for studying bone imaging agents. Seven /sup 99m/Tc(NaBH4)HEDP complexes were obtained from the separation of a reaction mixture by anion exchange HPLC. Two complexes were treated as a single entity. Six biodistribution studies of /sup 99m/Tc(NaBH4)HEDP complexes were conducted. Results indicated that each complex had a distinct biodistribution pattern

  8. The effect of newer anti-rheumatic drugs on osteogenic cell proliferation: an in-vitro study

    Directory of Open Access Journals (Sweden)

    Laing Patrick

    2009-05-01

    Full Text Available Abstract Background Disease modifying anti-rheumatic drugs (DMARDs may interfere with bone healing. Previous studies give conflicting advice regarding discontinuation of these drugs in the peri-operative setting. No consensus exists in current practice especially with the newer DMARDs such as Leflunomide, Etanercept, and Infliximab. The aim of this study was to assess the in-vitro effect of these drugs alone and in relevant clinical combinations on Osteoblast activity. Methods Osteoblasts were cultured from femoral heads obtained from five young otherwise healthy patients undergoing total hip replacement. The cells were cultured using techniques that have been previously described. A full factorial design was used to set up the experiment on samples obtained from the five donors. Normal therapeutic concentrations of the various DMARDs were added alone and in combination to the media. The cell proliferation was estimated after two weeks using spectrophotometric technique using Roche Cell proliferation Kit. Multilevel regression analysis was used to estimate which drugs or combination of drugs significantly affected cell proliferation. Results Infliximab and Leflunomide had an overall significant inhibitory effect (p Conclusion Our study indicates that in-vitro osteoblast proliferation can be inhibited by the presence of certain DMARDs. Combinations of drugs had an influence and could negate the action of a drug on osteoblast proliferation. The response to drugs may be donor-dependent.

  9. Beneficial bacteria inhibit cachexia.

    Science.gov (United States)

    Varian, Bernard J; Goureshetti, Sravya; Poutahidis, Theofilos; Lakritz, Jessica R; Levkovich, Tatiana; Kwok, Caitlin; Teliousis, Konstantinos; Ibrahim, Yassin M; Mirabal, Sheyla; Erdman, Susan E

    2016-03-15

    Muscle wasting, known as cachexia, is a debilitating condition associated with chronic inflammation such as during cancer. Beneficial microbes have been shown to optimize systemic inflammatory tone during good health; however, interactions between microbes and host immunity in the context of cachexia are incompletely understood. Here we use mouse models to test roles for bacteria in muscle wasting syndromes. We find that feeding of a human commensal microbe, Lactobacillus reuteri, to mice is sufficient to lower systemic indices of inflammation and inhibit cachexia. Further, the microbial muscle-building phenomenon extends to normal aging as wild type animals exhibited increased growth hormone levels and up-regulation of transcription factor Forkhead Box N1 [FoxN1] associated with thymus gland retention and longevity. Interestingly, mice with a defective FoxN1 gene (athymic nude) fail to inhibit sarcopenia after L. reuteri therapy, indicating a FoxN1-mediated mechanism. In conclusion, symbiotic bacteria may serve to stimulate FoxN1 and thymic functions that regulate inflammation, offering possible alternatives for cachexia prevention and novel insights into roles for microbiota in mammalian ontogeny and phylogeny. PMID:26933816

  10. 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...

  11. Chemical Inhibition of Autophagy

    DEFF Research Database (Denmark)

    Baek, Eric; Lin Kim, Che; Gyeom Kim, Mi;

    2016-01-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical...... autophagy inhibitors on the specific productivity (qp), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine...... significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp. The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical...

  12. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  13. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    International Nuclear Information System (INIS)

    Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

  14. Backward semantic inhibition in toddlers

    OpenAIRE

    Chow, J.; Aimola Davies, AM; Fuentes, LJ; Plunkett, KR

    2016-01-01

    Attention-switching is a crucial ability required in our everyday life, from toddlerhood to adulthood. In adults, shifting attention from one word (e.g., dog) to another (e.g., sea) results in backward semantic inhibition, i.e., the inhibition of the initial word (dog). This study examines whether attention-switching is accompanied by backward semantic inhibition in toddlers using the preferential looking paradigm. The findings demonstrate that a backward inhibitory mechanism operates during ...

  15. Two-layer membranes of calcium phosphate/collagen/PLGA nanofibres: in vitro biomineralisation and osteogenic differentiation of human mesenchymal stem cells

    Science.gov (United States)

    Hild, Nora; Schneider, Oliver D.; Mohn, Dirk; Luechinger, Norman A.; Koehler, Fabian M.; Hofmann, Sandra; Vetsch, Jolanda R.; Thimm, Benjamin W.; Müller, Ralph; Stark, Wendelin J.

    2011-02-01

    The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a-CaP/Col/PLGA compositions were synthesised by electrospinning to mimic the actual composition of bone tissue. Immersion in simulated body fluid and in cell culture medium resulted in the deposition of a hydroxyapatite layer. Incubation of hMSC for 4 weeks allowed for assessment of the proliferation and osteogenic differentiation of the cells on both sides of the double membrane. Confocal laser scanning microscopy was used to observe the proper adhesion of the cells. Calcium and collagen content was proven by Alizarin red S and Sirius red assays. Acute cytotoxic effects of the nanoparticles or the chemicals used in the scaffold preparation could be excluded based on viability assays (alamarBlue and alkaline phosphatase activity). The findings suggest possible application of such double membranes is in treatment of bone defects with complex geometries as wound dressing material.The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a

  16. Effects of low-level laser therapy on the expression of osteogenic genes related in the initial stages of bone defects in rats

    Science.gov (United States)

    Fernandes, Kelly Rossetti; Ribeiro, Daniel Araki; Rodrigues, Natália Camargo; Tim, Carla; Santos, Anderson Amaro; Parizotto, Nivaldo Antônio; de Araujo, Heloisa Selistre; Driusso, Patrícia; Rennó, Ana Claudia Muniz

    2013-03-01

    We evaluate the effects of low-level laser therapy (LLLT) on the histological modifications and temporal osteogenic genes expression during the initial phase of bone healing in a model of bone defect in rats. Sixty-four Wistar rats were divided into control and treated groups. Noncritical size bone defects were surgically created at the upper third of the tibia. Laser irradiation (Ga-Al-As laser 830 nm, 30 mW, 0.028 cm2, 1.071 W/cm2, 1 min and 34 s, 2.8 Joules, 100 J/cm2) was performed for 1, 2, 3, and 5 sessions. Histopathology revealed that treated animals presented higher inflammatory cells recruitment, especially 12 and 36 h postsurgery. Also, a better tissue organization at the site of the injury, with the presence of granulation tissue and new bone formation was observed on days three and five postsurgery in the treated animals. The quantitative real time polymerase chain reaction showed that LLLT produced a significantly increase in mRNA expression of Runx-2, 12 h and three days post-surgery, a significant upregulation of alkaline phosphatase mRNA expression after 36 h and three days post-surgery and a significant increase of osteocalcin mRNA expression after three and five days. We concluded that LLLT modulated the inflammatory process and accelerated bone repair, and this advanced repair pattern in the laser-treated groups may be related to the higher mRNA expression of genes presented by these animals.

  17. Effects of microgravity on bone marrow mesenchymal stem cells into osteogenic differentiation%微重力对骨髓间充质干细胞成骨分化的影响

    Institute of Scientific and Technical Information of China (English)

    张新昌; 韩标; 王强松; 李昊; 张西正

    2014-01-01

    骨髓间充质干细胞(BMSCs)是一种多能成体于细胞,是组织工程重要的种子细胞来源之一.微重力对BMSCs成骨分化具有抑制作用,可使骨量减少和骨微结构改变,从而导致骨质疏松症.这一