Transforming growth factor-beta inhibition of mineralization by neonatal rat osteoblasts in monolayer and collagen gel culture

Summary The latent form of transforming growth factor-beta (TGF-β) is a component of the extracellular matrix of bone. The active form, when locally injected in vivo, stimulates both inflammation and ectopic bone formation. The present study was undertaken to determine if TGF-β also stimulated mineralization by isolated rat calvarial osteoblasts cultured in collagen gels. Gels were used because they should mimic in vivo conditions better than classical monolayer culture. Compared to cells in monolayers, osteoblasts cultured in collagen gels exhibited slower growth, but higher alkaline phosphatase activity and mineral deposition. Cultured cells also synthesized the osteoblast-specific marker, osteocalcin. The increase in osteocalcin in cell layers was parallel to the increase in mineral deposition. In the presence of TGF-β, neither cell growth nor alkaline phosphatase activity increased. Instead, a small decrease occurred in both parameters when compared to untreated cultures. Accumulation of collagen, the major component of the extracellular matrix where mineralization occurs, was similar in untreated and TGF-β1-treated cultures. However, 8 pM TGF-β1 dramatically suppressed mineral deposition in both types of cultures. Despite TGF-β1 stimulating a fourfold increase in lactic acid, the consequent increase in culture medium acidity did not account for the inhibitory effects of TGF-β1 on mineralization. These results demonstrate that collagen gel culture is an improved technique over conventional monolayer culture for demonstrating differentiated osteoblast function and sensitivity to TGF-β1. TGF-β1, at a concentration that has little effect on cell growth, alkaline phosphatase activity, or collagen accumulation, is a potent inhibitor of mineralization. The mechanism by which TGF-β1 inhibits mineralization remains to be determined.