Effects of bone morphogenetic protein-2 on human neonatal calvaria cell differentiation.

Bone morphogenetic proteins (BMPs) are factors that promote osteoblastic cell differentiation and osteogenesis. It is unknown whether BMPs may act on human osteoblastic cells by increasing immature cell growth and/or differentiation. We investigated the short- and long-term effects of recombinant human (rh)BMP-2 on cell growth and osteoblast phenotype in a new model of human neonatal pre-osteoblastic calvaria cells (HNC). In short-term culture, rhBMP-2 (20-100 ng/ml) inhibited DNA synthesis and increased alkaline phosphatase (ALP) activity without affecting osteocalcin (OC) production. When cultured for 3 weeks in the presence of ascorbic acid and inorganic phosphate to induce cell differentiation, HNC cells initially proliferated, type 1 collagen mRNA and protein levels rose, and then decreased, whereas OC mRNA and protein levels, and calcium accumulation into the extracellular matrix increased at 2 to 3 weeks. A transient treatment with rhBMP-2 (50 ng/ml) for 1 to 7 days which affected immature HNC cells, decreased cell growth, increased ALP activity and mRNA, and induced cells to express ALP, osteopontin, and OC at 7 days, as shown by immunocytochemistry. At 2 to 3 weeks, matrix mineralization was markedly increased despite cessation of treatment, and although OC and Col 1 mRNA and protein levels were not changed. A continuous treatment with rhBMP-2 for 3 weeks which affected immature and mature cells reduced cell growth, increased ALP activity and mRNA at 1 week and increased OC mRNA and protein levels and calcium content in the matrix at 3 weeks, indicating complete osteoblast differentiation. These results indicate that the differentiating effects of BMP-2 on human neonatal calvaria are dependent on duration of exposure. Although long-term exposure led to complete differentiation of OC-synthesizing osteoblasts, the primary effect of rhBMP-2 was to promote osteoblast marker expression in immature cells, which was sufficient to induce optimal matrix mineralization independently of cell growth and type 1 collagen expression.