Study Of The Rabbit Bone Mesenchymal Stem Cells Cultured In Vitro On Chitosan Scaffolds

objective: To isolate rMSCs from the bone marrow of the rabbit via density gradient centrifugation and indentify them by the capability of multi-potential differentiation.To explore the characteristics of mesenchymal stem cells ( rMSCs ) derived from rabbit bone marrow which cultured on three-dimensional scaffold of chitosan.And under certain conditions,induce rMSCs to differentiate towards the tissue of nucleus pulposus in order to discuss the feasibility of nucleus pulposus tissue-engineering regeneration.Metliods:The MSCs were isolated from the rabbit bone marrow by density gradient centrifuge and adhesive property and then subcultured to the third passage.The rMSCs were seeded on the chitosan scaffolds and induced into nucleus pulposus tissue by adding transforming growth factorβl ( TGF-βl ) , dexamethasone, ITS( insulin\transferrin\ sodium selenite\ascorbic acid)on the scaffolds.Medium was changed and collected every 7 days for analysis of glycosaminoglycan content. At the end of the every culture period, scaffolds were processed for histology or for analysis of their proteoglycan, collagen. The cell proliferation on different conditions was detected with MTT automated colormentric microassay. All analyses were performed in duplicate, and each analytical condition was repeated four times using different cell preparations.Results:The cells separated by density gradient centrifuge and adhesive property owned the characteristics of rMSCs and the third passage had a good condition on the scaffolds of chitosan.The differentiation of rMSCs toward nucleus pulposus tissue was verified by the positive result of collagentⅡon the scaffolds by immunohistochemistry. The cell viability in scaffolds was better. the rMSCs with directed inducing showd the character of the nucleus pulposus while the ones that without directed inducing did not.Conclusions: The MSCs can be successfully separated by density gradient centrifuge and adhesive property.Under special conditions,the rMSCs which on the scaffolds of chitosan can differentiate toward the tissue of nucleus pulposus.