Interaction of TGF-beta 1 and rhBMP-2 on human bone marrow stromal cells cultured in collagen gel matrix

Transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein -2 (BMP-2) are abundant proteins in the bone matrix. However, their interac tion in controlling osteoblast differentiation is not clearly understood. I n this study, HBMSCs were cultured in collagen gel matrix with different co ndition of exogenous rhBMP-2 and TGF-beta1 in order to determine the intera ction of BMP-2 and TGF-beta1 on human bone marrow stromal cells (HBMSCs) di fferentiation. The cultured cells were analyzed for cell proliferation, alk aline phophatase (ALP) activity and mineralization stainning with Von-Kossa . The cells treated with TGF-beta1 exhibited a higher late of cell growth t han those without. However, the cells cultured in collagen gel matrix showe d a lower rate of cell growth than the cells cultured in a monolayer. To in vestigate the effects of both cytokines on osteoblast differentiation, the cells were treated with 0, 1, 5, 10 ng/ml of TGF-beta1 for 2 days. This was followed by culturing with 0, 1, 5 and 10 ng/ml of TGF-beta1 and 100ng/ml of rhBMP-2 together for 3 days with the alkaline phosphatase (ALP) activity measured. The cells treated with 1ng/ml of TGF-beta1 responded efficiently to rhBMP-2 and expressed ALP activity with a level equivalent to that exhi bited by cells that were not treated with TGF-beta1. The cells treated with 5 and 10ng/ml of TGF-beta1 showed a dramatic decrease in ALP activity. The cells treated with 10ng/ml of TGF-beta1 followed by rhBMP-2 alone exhibite d an intermediate ALP activity. The cells treated with 100 ng/ml of rhBMP-2 demonstrated Von-Kossa positive solid deposits after 3 weeks,. while there were few Von-Kossa positive solid deposits when the cells treated with 10n g/ml of TGF-beta1. These results show that TGF-beta1 inhibits the effects o f rhBMP-2 on the osteoblast differentiation of HBMSCs in a dose dependant m anner. Furthermore, the effects of TGF-beta1 on HBMSCs are reversible. This suggest that TGF-beta1 and rhBMP-2 are coordinately controlled during the osteoblast differentiation of HMBSCs.