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Effects of TGF-β1 and serum on the proliferation and chondrogenic differentiation of human bone marrow stromal cells

Objective To observe the effects of TGF-β1 and serum on the proliferation and chondrogenic differentiation of human bone marrow stromal cells (BMSCs) cultured in vitro. Methods A volume of 6 ml bone marrow was collected from 3 volunteer donors respectively. BMSCs 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. BMSCs from the third passage were grown in complete medium (Group A), inductive medium with 5%fetal bovine serum (Group B), and inductive medium with 10%fetal bovine serum (Group C) 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 proliferation of the cells was examined by 3H-TdR incorporation. Results 96%of the cells were alive after density gradient centrifugation. The adherent cells changed from a spindle-like appearance to a polygonal shape. After 7 days'induction by inductive medium with 5%fetal bovine serum, immunohistochemistry and in situ hybridization analysis proved strong type Ⅱcollagen expression. The sequences of the incorporation of 3H-TdR were C B A. Conclusions The density gradient centrifugation is an effective method to isolate BMSCs from bone marrow aspirate, for the BMSCs cultured in vitro grow stably. 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.