Transforming Growth Factor-β Stimulates Bone Matrix Apposition and Bone Cell Replication in Cultured Fetal Rat Calvariae*

Transforming growth factor-beta (TGF beta) stimulates the expression of extracellular matrix proteins and may be a local regulator of bone growth. The aims of this research were to localize the effect of TGF beta on bone matrix formation and to determine if this effect was dependent on increased cell replication, using histomorphometry and autoradiography of bone organ cultures. Half-calvariae of 21-day-old fetal rats were cultured with native or recombinant TGF beta 1 for 24 h and labeled either with 3Hproline for 0-24 or 24-48 h or with 3Hthymidine for the last 6 h of culture. Bones were fixed in glutaraldehyde, embedded in glycol methacrylate, and processed for autoradiography. Bone matrix formation was assessed as the matrix apposition rate per day and the percentage of 3Hproline-labeled bone surface. Cell replication was evaluated based on the number and percentage of 3Hthymidine labeled cells in the osteoblast cell zone, the osteoprogenitor cell zone, and the pericranial fibroblastic periosteum. Both native and recombinant TGF beta at 1-30 ng/ml increased bone matrix formation by 25-40% (P less than 0.05). At 30 ng/ml, TGF beta had a generalized mitogenic effect as cell replication increased by approximately 2-fold in all cell zones of the pericranial periosteum. TGF beta had specific effects on bone cell differentiation. The number of unlabeled cells lining the bone surface increased, and the number of osteoclasts on bone decreased. Inhibition of cell replication by hydroxyurea only partially blocked the stimulatory effect of TGF beta on bone matrix formation, suggesting that TGF beta may have independent effects on cell replication and differentiated bone cell function. In summary, TGF beta had a generalized mitogenic effect on the pericranial periosteum and specific stimulatory and inhibitory effects on bone cell differentiation and function.