A. Chaudhari et al., RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2 STIMULATES DIFFERENTIATION IN PRIMARY CULTURES OF FETAL-RAT CALVARIAL OSTEOBLASTS, Molecular and cellular biochemistry, 167(1-2), 1997, pp. 31-39
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.