Experimental Study Of In Vitro Chondrogenic Differentiation Of Human Bone Marrow-Derived Mesenchymal Stem Cells

Objectives To establish a method of isolating and culturing human bone marrow-derived mesenchymal stem cells(BMSCs) ; to investigate the feasibility of in vitro chondrogenic differentiation of BMSCs in monolayer culture as potential seeding cells in cartilage tissue engineering.Methods A volume of 6 ml bone marrow was collected from three volunteer donors respectively. MSCs were isolated from the Percoll fraction of mononuclear cells and cultured in the Dulbecco's Modified Eagle's Medium(DMEM)with low glucose containing 10% fetal bovine serum,flow cytometric analysis was performed to examine the expression of cell surface molecules on BMSCs. BMSCs from the third passage were grown in complete medium (A),inductive serum-free medium(B), inductive medium with 5% fetal bovine serum(C), inductive medium with 10% fetal bovine serum(D) respectively.The growth of the cultured BMSCs was observed.Immunohistochemistry and in situ hybridization were applied to detect the expression of collagen type Ⅱ.The synthetic proteoglycans were detected by toluidine blue staining.The proliferation of the cells was examined by 3H -TdR incorporation.Results 96% cells were alive after density gradient centrifugation.The adherent cells changed from a spindle-like appearance to a polygonal shape and expressed positive CD9,but negative CD34,CD38,CD45,CD61. After 7 days induction by inductive serum-free medium or inductive medium with 5% fetal bovine serum ,the cells were positively stained by toluidine blue, immunohistochemistry and in situ hybridization analysis proved strong type Ⅱ collagen expression.The sequences of the incorporation of 3H -TdR were D＞C＞A＞B.Conclusions The density gradient centrifugation is an effective method to isolate BMSCs from bone marrow aspirate,BMSCs cultured in vitro grew stably and were a homogenous population;10ng/ml TGF-β1 can induce the chondrogenic differentiation of the BMSCs,but it can not facilitate the proliferation of the cells in serum-free medium;the combination of TGF-β1 and fetal bovine serum in low concentration(5%) can not only facilitate the proliferation of BMSCs,but also induce the chondrogenic differentiation.