Study Of The Directed Inducing Cartilage Cells From Rat Bone Mesenchymal Stem Cells On Scaffolds And Cartilage Tissue-engineering Regeneration In Nude Mice

Objective: To explore and the characteristics of mesenchymal stem cells(MSCs) derived from rat bone marrow and compare with the difference of the directed inducing between scaffolds and planning condition ,and the feasibility of cartilage tissue-engineering regeneration. Methods: The MSCs were isolated from rat bone marrow by density gradient centrifuge and adhesive property, and immunohistochemistry was applied to detect the expression of SH3. The MSCs were preserved after they were suspensioned in 70%DMEM, 20%FBS , 10%DMSO, then induced into chondrogenic differentiation by adding transforming growth factor- β₁ (TGF- β₁) ,dexamethasone transferring and insulin on collage sponge and plane condition. Immunohistochemisstry and in situ hybridization were applied to detect the expression of collagen type II on the scaffolds and plane conditions.The cell proliferation on different conditions was detected with MTT automated colormentric microassay and cell cycle on different conditions was detected by flow cytometry(FCM). The MSCs and collagen sponge ,calcium alginate were mixed to form a comlex and planted under the skin of nude mice. Histochemical or immunohistochemistry were studied on the 4th week and 8th week.Results: The cells separated by density gradient centrifuge and adhesive property owned the characteristics of MSCs, and was verified by the positive result of SH3.The cryopreserved MSCs maintain high live rate of 85% in P1,P5. The differentiation of MSCs toward chondrogenic phenotype was verified by the positive result of collagen type II on the scaffolds and plane condition by immunohistochemisstry and in situ hybridization. The cell viability in scaffolds was better than that in plane conditions. There were chondrcytes and cartilage lacuna on the 4th week,and chondrocytes and the collagen secreted by them became more on the 8th week .But ,the MSCs without directed inducing didn't show the character of the chondrocyte.Conclusions: The MSCs can be successfully separated by density gradient centrifuge and adhesive property. The cryopreserved methods is adapt to MSC. Using directed inducing cartilage from bone MSCs combined with collage sponge to construction tissue-engineered cartilage is feasible and better than plane directed inducing. Directed inducing MSCs-alginate complex and MSCs-collagen sponge complex can form cartilage in nude mice.