TRANSFORMING GROWTH-FACTOR-BETA INHIBITION OF MINERALIZATION BY NEONATAL RAT OSTEOBLASTS IN MONOLAYER AND COLLAGEN GEL CULTURE

The latent form of transforming growth factor-beta (TGF-beta) is a com ponent of the extracellular matrix of bone. The active form, when loca lly injected in vivo, stimulates both inflammation and ectopic bone fo rmation. The present study was undertaken to determine if TGF-beta als o stimulated mineralization by isolated rat calvarial osteoblasts cult ured in collagen gels. Gels were used because they should mimic in viv o conditions better than classical monolayer culture, Compared to cell s in monolayers, osteoblasts cultured in collagen gels exhibited slowe r growth: but higher alkaline phosphatase activity and mineral deposit ion. Cultured cells also synthesized the osteoblast-specific marker, o steocalcin. The increase in osteocalcin in cell layers was parallel to the increase in mineral deposition. In the presence of TGF-beta, neit her cell growth nor alkaline phosphatase activity increased. Instead, a small decrease occurred in both parameters when compared to untreate d cultures. Accumulation of collagen, the major component of the extra cellular matrix where mineralization occurs, was similar in untreated and TGF-beta 1-treated cultures. However, 8 pM TGF-beta 1 dramatically suppressed mineral deposition in both types of cultures. Despite TGF- beta 1 stimulating a fourfold increase in lactic acid, the consequent increase in culture medium acidity did not account for the inhibitory effects of TGF-beta 1 on mineralization. These results demonstrate tha t collagen gel culture is an improved technique over conventional mono layer culture for demonstrating differentiated osteoblast function and sensitivity to TGF-beta 1. TGF-beta 1, at a concentration that has li ttle effect on cell growth, alkaline phosphatase activity, or collagen accumulation, is a potent inhibitor of mineralization. The mechanism by which TGF-beta 1 inhibits mineralization remains to be determined.