Repairing Cartlige Defect With Tissue Engining Of BFGF And SOX9 Gene Transfected To The Bone Marrow Stromal Ce11s

This research was on bone marrow stromal cells(BMSCs)transfected by bFGF and SOX-9 gene with cell culture and gene engineering technique, were cultured on poly(lactic-co-glycolic)acid(PLGA) base with tissue engineering technique, and animal model with cartilage defect were prepared. The complex(BMSCs-PLGA )was planted into defect place of animal model. The effect of repair cartilage defect in vivo was evaluated by histology anatomy observation, collagenâ…¡immun ohistochemistry, etc.The research comprised three parts:Part A: Proliferation and identification of rabbit bone marrow stromal cells in vitroObjective To prepare seed cells for tissue engineering, bone marrow stromal cells(BMSCs)were isolated from rabbits marrow and cultured in vitro. Methods Some red bone marrow of rabbits were drew out and cultured by culture medium to harvest BMSCs.The proliferation and qualification of BMSCs was observed by lightmicroscope.MTT assay was used to drew the cell growth curve.Bone marrow stromal cells be freezed and resuscitatd in vitro. Results BMSCs were cultured,and proliferated well in the culture medium with active function which were qualified for monocyte by observation of lighemicroscope.A grouth curve was drawn by the results of MTT assay. BMSCs were cultivated and proliferated also well after it were freezed and resuscitated in vitro. Conclusion Bone marrow stromal cells could be cultured,differentiated,proliferated, freezed and resuscitated in vitro, it could possess of enough quantity and differentiation multipotential, so it could be suitable seed cell for tissue engineering.Part B Construction of eukaryotic expression vectors of bFGF and SOX-9 and transfection based on marrow stroma cells.Objective To investigate approach and possibililty of transfection on bFGF and SOX-9 gene into BMSCs. Method The eukaryotic expression vectors that were constructed by bFGF and SOX-9 cDNA were transfected into BMSCs by liposome induction mean. The transcription and expression of bFGF and SOX-9 gene in BMSCs were detected by morphology observation, immunohistochemistry, ELISA and RT-PCR means. The changes of biologic characterisctics of transfected MSCs were also observed. Results The stable overexpression of bFGF and SOX-9 proteins were detected in transfected BMSCs, and the differentiation towards cartilage of parts of BMSCs was observed. Conclution The stable expression of bFGF and SOX-9 gene in transfected BMSCs could induce these cells differentiate towards cartilage.Part C Transfected BMSCs were cultured on PLGA base with tissue engineering technique, and Animal experiment of repairing cartilage defect with complex of BMSCs-PLGAObjective To investigate possibility that BMSCs transfected by bFGF and SOX-9 gene could grow on PLGA base and possibility that tissue engineering method could repair cartilage defect in animal model. Method BMSCs transfected by bFGF and SOX-9 gene were cultured on PLGA base, and animal model with cartilage defect were prepared. The complex was planted into defect place of animal model. The effect of repair in vivo was evaluated by histology anatomy observation, collagenâ…¡immunohistochemistry, etc. Result BMSCs transfected by bFGF and SOX-9 gene grew on PLGA base well. The complex could repair the cartilage defect in animal model. Conclusion There were well consistence of biology between PLGA and the transfected BMSCs. The complex could repair the cartilage defect in animal model,could serve as a autograft substitute materia.