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

Abstract

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.

Altered mineralization of human osteoarthritic osteoblasts is attributable to abnormal type I collagen production

“…Indeed, although more abundant, this collagen matrix would not mineralize properly, which would lead to a less mineralized subchondral bone tissue. Third, the decrease in mineralization observed in vitro under basal conditions indicates that a cellular defect is responsible for this abnormal mineral deposition, a situation that is not fully corrected by the potent osteogenesis stimulator BMP-2 (42,43). The hypothesis that abnormal mineralization of OA osteoblasts could be linked with altered functioning of osteal tissue macrophages is also unlikely.…”

FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease

“…Some BMPs are also expressed in the dura, sutural mesenchyme, and the osteogenic front (Opperman 2000). BMP signaling promotes the differentiation and apoptosis of calvarial osteoblasts (Hay et al 1999Yamaguchi et al 2000) and may thus act in concert with FGFs to control calvarial growth and differentiation during intramembranous bone development (Fig. 4B;Opperman 2000;Marie et al 2002).…”

Syndecan-2 Affects the Basal and Chemotherapy-Induced Apoptosis in Osteosarcoma

“…Human osteosarcoma cell lines were MG63, U2OS, SaOS2, and OHS4. Immortalized osteoblastic cells (18) or primary cultures of osteoblasts isolated from nonpathologic human bone (19) served as normal controls. All cells were maintained in DMEM supplemented with 10% FCS and antibiotics (100 IU/mL penicillin and 100 Ag/mL streptomycin).…”