The optimal dose of recombinant human osteogenic protein-1 enhances differentiation of mouse osteoblast-like cells: an in vitro study.

ABSTRACT

OBJECTIVE: There is no certain conclusion on the effect of recombinant human Osteogenic Protein-1 (OP-1, BMP-7) on the proliferation of the osteoblast-like cell line, MC3T3-E1. Furthermore, the optimal dose of rhOP-1 on cell differentiation still needs to be elucidated. This investigation aims to delineate the biofunctional characteristics of rhOP-1 in inducing osteoblastogenesis of MC3T3-E1 through in vitro time-course and dose-response studies. DESIGN: MC3T3-E1 cells were cultured for 1, 4, 7 days with the addition of different rhOP-1 concentrations (0, 10, 20, 50, 100, 200, 400 ng/ml), and cell proliferation and cell differentiation were examined. RESULTS: MC3T3-E1 cell proliferation was stimulated by rhOP-1 in a dose-dependent manner (0-400 ng/ml) on day 1, whereas on day 4 and 7, it was still stimulated at low concentrations (10, 20, 50 ng/ml) but inhibited at high ones (200, 400 ng/ml). The alkaline phosphatase (ALP) activity, osteocalcin (OC) production, collagen deposition and extracellular matrix mineralization were dramatically elevated by rhOP-1 treatment, as a function of culture time and rhOP-1 concentration, and all of them reached a plateau at the concentration of 200 ng/ml. Real-time quantitative RT-PCR results showed Runx2, AKP-2, OC and Nog mRNA expressions increased in a dose- and time-dependent manner, and their expressions were significantly higher at high rhOP-1 concentrations than that of low ones. No significant differences were found between the effects of 200 ng/ml rhOP-1 and 400 ng/ml rhOP-1 on the differentiation of MC3T3-E1 cells, except the expression of Nog mRNA, whose expression level was much higher at 400 ng/ml than that at 200 ng/ml. CONCLUSIONS: These results suggest that cell proliferation of MC3T3-E1 is depended on culture time and rhOP-1 concentration, rhOP-1 could stimulate the differentiation of MC3T3-E1 cells and the optimal concentration could be 200 ng/ml.