Recombinant human bone morphogenetic protein-2 stimulates differentiation in primary cultures of fetal rat calvarial osteoblasts

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 mu g/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta(1) (TGF-beta(1))-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta(1) concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 mu g/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta(1) may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.