Experimental Study On The Repair Of Articular Cartilage Defects With Bone Marrow Derived Mesenchymal Stem Cells In Rabbits

Objectives To establish a method for isolating mesenchymal stem cells from rabbit bone marrow aspirate; to induce chondrogenic differentiation of BMSCs in vitro; to observe the tissue implanted into full-thickness defects of articular cartilage with the induced BMSCs.Methods Bone marrow aspirate of New Zealand white rabbits was density gradient centrifuged and cultured in plastic culture bottles to isolate BMSCs. BMSCs were cultured and multiplied in vitro, TGF-β1 was used to induce chondrogenic differentiation of BMSCs at high density culture (1×106/ml) and low density culture (1×104/ml). The induced cells were embedded in alginate and implanted into articular cartilage defects of rabbit knees, in the control groups, the defects were filled with alginateonly or left untreated, the repair tissue were examed grossly, histologically and electron microscoply at the end of 6 weeks and 12 weeks.Results 95% cells were alive after density gradient centrifugation, BMSCs had a similar spindle-like morphology, the cells cultured in vitro experienced latent phase , logarithmic growth phase and plateau phase , the average double time is 32.5 hours; BMSCs in high density were chondrogenesis induced by TGF-1, verified by positive results of immunohistochemistry , in situ hybridization and special staining; in the experimental group, the defects were filled with hyaline-like cartilage at the end of 6 weeks, the cartilage and the subchodral bone were remodeled at the end of 12 weeks after the operation, the expression of type II collagen in the repair tissue was verified byimmunohistochemistry and in situ hybridization. In the control groups, the defects were filled with fibrous tissue. There is an apparent difference between the experimental group and the two control groups with Wakitani histological grading system.Conclusions The combination of density gradientcentrifugation and adhere culture is an effective method to isolate BMSCs from bone marrow aspirate, BMSCs cultured in vitro show stable growth, rapid proliferation and may be used in tissue engineering; TCF-1 can induce the chondrogenesis differentiation of high density cultured BMSCs, the cell density is important in the chondrogenesis differentiation of BMSCs ; BMSCs embedded in alginate can promote the repair of articular cartilage defects, it may be a promising method for clinical treatment of articular cartilage injures.