| Objective:To investigate the bone marrow mesenchymal stem cells (BMSCs) of rabbit which are respectively induced to chondroblasts by TGF-β1and other factors for8d.11d,14d,17d and20d. proliferate in three-dimensional calcium alginate and grow into cartilage in vitro.Methods:The bone marrow was aspirated from the femur of New Zeal rabbits and BMSCs were isolated by gradient centrifugation. After cultivation, amplification, the3rd Passaged BMSCs were directed induced by the serum-free medium which contained TGF-β1, dexamethasone and other components to chondrocytes. The experimental groups were divided into A. B. C, D. E groups by the different induction time.Group A:induced for8d; group B:induced for11d:C:induced for14d;Group D:induced for17d:Group E: induced for20d. Compared with different groups’ chondroblasts by cell morphology.TB staining.type Ⅱ collagen immunohistochemistry. and Aggrecan (AG) content in induction medium.Composite the different groups’ chondroblast above with calcium alginate gel respectively,the cells density was1×107/ml.and continue to culture in vitro for14d. At the same time, observed the cells growth and proliferation in calcium alginate gel. detected collagen type II expression by immunohistochemistry. and used alcian blue colorimetric method to measue the content of each group’s AG.Comprehensively compared the the tissue engineered cartilages built by different experimental groups.Results:The BMSCs which were isolated by gradient centrifugation, had apparently transformed in morphology since induced8days.And had an obvious chondrocytes’ morphology in the14th day. The AG in induction medium could be detected at a low level in the4th day.and significantly elevated in the8th day. and maintain increased slowly to the20th day. In the14th day. the metachromatic particles could be found by TB staining.and the collagen type11immunohistochemistry was significantly positive in cell climbing slice. After composited with calcium alginate gel. each groups’ chondroblasts kept proliferating in the3D scaffolds gel, and became bounch of grapes kind by AO staining. The test of AG content showed that among each experimental group.and the positive control group the AG content is different (p<0.05).Group C and Group D were higher than the positive control group.and the highest was Group C.Conclusion:1. The BMSCs which were isolated by gradient centrifugation, almost had differentiated to chondrocytes after14days directed induced in vitro.2. Alginate gel has good biocompatibility to seed cells, and can be made using the injection form to implant: the chondroblasts which have induced for14days can proliferate and secrete cartilage extracellular matrix strongly in alginate gel.The injectable cartilage tissue engineering approach which take BMSCs as seed cells, and the calcium alginate as the scaffold is feasible. |
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