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 fo r its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were us ed in this study. The results indicated that rhBMP-2 stimulated alkali ne phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in co nfluent cultures. The percent collagen synthesis also increased in a d ose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days aft er initiation. Cycloheximide (2 mu g/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzym e. Transforming growth factor-beta(1) (TGF-beta(1))-induced inhibition of alkaline phosphatase activity observed in confluent primary cultur es was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta(1) concentration. Also, rhBMP-2 produc ed a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enha nced 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-induc ed stimulation of alkaline phosphatase, we speculate that ascorbic aci d could amplify the osteoinductive effects of rhBMP-2 and thereby augm ent 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 phenotyp e in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta(1) may play a regulatory role in BMP-induc ed bone formation and ascorbic acid may potentiate the effects of rhBM P-2 in vivo.