Primary Experimental Study On In Vitro Construction Of Tissue-engineered Osteochondral Composite Using A Combination Of Biomimetic Scaffold Col-cs/ NHAC-PLA And Bone Mesenchymal Stem Cells

[Objective]1. To isolate, expand and identify porcine BMSCs.2. To construct recombinant lentivirus vectors of TGF-β1 gene and transfect BMSCs. providing efficient and sustained cells for bone and cartilage tissue engineering.3. To prepare Col-CS/ nHAC-PLA scaffold and examine its biocompatibility.[Methods]1. Isolation, expansion and identification of porcine BMSCs:Mononuclear cells were isolated from bone marrow by density gradient centrifugation, and then BMSCs were extracted by adherence characteristics and expanded by subculture. BMSCs were identified by flow cytometry to determine the purification of BMSCs.2. To construct recombinant lentivirus vectors of TGF-β1 gene and transfect BMSCs: The TGF-β1 cDNA were extracted and packed into lentivirus vector, and positive clones were identified by PCR and gene sequencing. BMSCs were then transfected by recombinant lentivirus vectors. The effects of transfection were checked by western blot, RT-PCR, immunocytochemistry and Elisa.3. The biocompatibility of Col-CS/ nHAC-PLA scaffold: According to Biological evaluation of medical devices, acute systemic toxicity tests, intracutaneous test, pyrogen test, hemolysis test, cytotoxicity test and bone implant test were executed.[Results]1. BMSCs were successfully isolated and expanded, and flow cytometry showed that the positive rates of specific molecular surface markers of BMSCs were 91.2%of CD90, 93.5%of CD105 and 4.4%of CD45.2. TGF-β1 cDNA was successfully packed into lentivirus vectors. RT-PCR showed TGF-β1 gene had successfully transfected into BMSCs. TGF-β1 protein expression could be detected by western blot, immunocytochemistry and Elisa.3. The biocompatibility of Col-CS/ nHAC-PLA scaffold: All results conformed to biological evaluation of medical devices.[Conclusions]1. High purity BMSCs could be achieved by combining density gradient centrifugation with suitable culture.2. TGF-β1 gene was successfully packed into lentivirus vectors. TGF-β1 gene was transfected into BMSCs by lentivirus vectors, and long term expression of TGF-β1 protein was observed.3. Col-CS/ nHAC-PLA scaffold was successfully prepared and possesses excellent biocompatibility.