Study Of The ICA On The Differentiation Into Cartilage Of BMSCs

Objective:To establish a sustained and stable Bone marrow-derived mesenchymal stem cells(BMSCs) culture system which differentiat into cartilage cells phenotype in vitro.Observe the influence of Icariin(ICA) on the biological behavior of BMSCs to differentiate into cartilage.And preliminary study the possible mechanism and role of the ICA on the proliferation and differentiation into cartilage of BMSCs.Thus to optimize the two major factors of tissue engineering:the regulation of differentiation of seed cells,in order to provide a reference for improving the quality of the cartilage repair.Method:1.BMSCs were isolated and cultured from the 1-month-old New Zealand white rabbits through density-gradient centrifugation and cell attachment to tissue culture.2.Growth and ultromicrostructure of the BMSCs were observed by light-microscope and scan electron-microscope.The cell activities and cell cycle were observed with flow cytometry method.The grow curve of the BMSCs was described through MTT assay.3.BMSCs were induced and differentiate into osteoblasts and adipocytes by using specific induction culture medium.Then the cells were stained with Alkaline phosphatase(ALP) Calcified nodules,Oil red O,and analyse the result of all staining.4.The harvested BMSCs were randomly divided into dieffrent groups and been treated with ICA.The BMSCs were observed for proliferation through MTT assay.5.The third passage BMSCs were divided into ICA group,blank control group,classic induced group,and ICA+classic induced group,they were cultured respectively and intervene in the differentiation into cartilage.The typeⅡcollagen and Toluidine Blue staining were performed to reflect the chondrocyte function of different groups.6.Quantitative analysis was used to detect the secretion of Sulfated-glycosaminoglyca(S-GAG) during the differentiation into cartilage through DMB assay,and observed the classic liquid-induced interaction with ICA. Results:1.The BMSCs could be obtained through both density-gradient centrifugation and cell attachment methods,the bone marrow adherent in whole layer,Primary cultured BMSCs were adhered to plastic surface within 24 hours and reached confluence within 12-15days.2.The cell morphology change from spindle to polygon after been induced by specific induction cultural medium,BMSCs were successfully differentiated into osteoblasts,and adiposecytes;positive and strong reaction in ALP,calcified nodules,oil-red O staining.3.The ICA promote the proliferation of BMSCs.The best promotion effect of ICA was achieved when the concentration was 1×10^-5mol/L or 2×10^-6mol/L(P＜0.05).4.ICA in above concentrations can induced BMSCs differentiated into cartilage cells,their typeⅡcollagen and toluidine blue staining were negative,and no correlation with the concentration.5.The increase of the secretion of S-GAG showed ICA+classic induced groups were significantly higher than classic groups(P＜0.01).Conclusion:A perfect culture system of BMSCs was established in vitro.ICA could significantly promote the proliferation of BMSCs,ICA and classical cross-induced group can synergically promote cartilage differentiation of BMSCs.ICA could used in the regulation of BMSCs,thereby promote S-GAG and typeⅡcollagen secretion,it promoted the differentiation of cartilage tissue in the process of continuous sophisticated and improved the quality of cartilage repair.Summing up the above is the possible mechanism to affect bioactivity of chondrogenic phenotype Cells,and can reflect the related mechanism of Chinese medicine.