AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bo...AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells(BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds(1) seeded with BMSCs;(2) wrapped with osteogenic matrix cell sheets; or(3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk. RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmentalor continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone nonunion. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness(mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.展开更多
We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation ...We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.展开更多
AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells(MSCs) using osteogenic matrix cell sheets(OMCSs).METHODS Rat MSCs were cultured in osteogenic media under one of four varyi...AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells(MSCs) using osteogenic matrix cell sheets(OMCSs).METHODS Rat MSCs were cultured in osteogenic media under one of four varying oxygen conditions: Normoxia(21% O_2) for 14 d(NN), normoxia for 7 d followed by hypoxia(5% O_2) for 7 d(NH), hypoxia for 7 d followed by normoxia for 7 d(HN), or hypoxia for 14 d(HH). Osteogenesis was evaluated by observing changes in cell morphology and calcium deposition, and by measuring osteocalcin secretion(ELISA) and calcium content. In vivo syngeneic transplantation using OMCSs and β-tricalcium phosphate discs, preconditioned under NN or HN conditions, was also evaluated by histology, calcium content measurements,and real-time quantitative PCR.RESULTS In the NN and HN groups, differentiated, cuboidal-shaped cells were readily observed, along with calcium deposits. In the HN group, the levels of secreted osteocalcin increased rapidly from day 10 as compared with the other groups, and plateaued at day 12(P < 0.05). At day 14, the HN group showed the highest amount of calcium deposition. In vivo, the HN group showed histologically prominent new bone formation, increased calcium deposition, and higher collagen type Ⅰ?messenger RNA expression as compared with the NN group.CONCLUSION The results of this study indicate that modifying oxygen tension is an effective method to enhance the osteogenic ability of MSCs used for OMCSs.展开更多
基金Supported by Grant-in-Aid for Young Scientists(KAKENHI)
文摘AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate(TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells(BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds(1) seeded with BMSCs;(2) wrapped with osteogenic matrix cell sheets; or(3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk. RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmentalor continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone nonunion. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness(mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.
文摘We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.
文摘AIM To establish a hypoxic environment for promoting osteogenesis in rat marrow stromal cells(MSCs) using osteogenic matrix cell sheets(OMCSs).METHODS Rat MSCs were cultured in osteogenic media under one of four varying oxygen conditions: Normoxia(21% O_2) for 14 d(NN), normoxia for 7 d followed by hypoxia(5% O_2) for 7 d(NH), hypoxia for 7 d followed by normoxia for 7 d(HN), or hypoxia for 14 d(HH). Osteogenesis was evaluated by observing changes in cell morphology and calcium deposition, and by measuring osteocalcin secretion(ELISA) and calcium content. In vivo syngeneic transplantation using OMCSs and β-tricalcium phosphate discs, preconditioned under NN or HN conditions, was also evaluated by histology, calcium content measurements,and real-time quantitative PCR.RESULTS In the NN and HN groups, differentiated, cuboidal-shaped cells were readily observed, along with calcium deposits. In the HN group, the levels of secreted osteocalcin increased rapidly from day 10 as compared with the other groups, and plateaued at day 12(P < 0.05). At day 14, the HN group showed the highest amount of calcium deposition. In vivo, the HN group showed histologically prominent new bone formation, increased calcium deposition, and higher collagen type Ⅰ?messenger RNA expression as compared with the NN group.CONCLUSION The results of this study indicate that modifying oxygen tension is an effective method to enhance the osteogenic ability of MSCs used for OMCSs.
