Nitric oxide stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells

Background: Infant dura mater plays a critical role in calvarial development. This investigation examines the expression of nitric oxide synthase isoforms in the craniofacial skeleton and the influence of nitric oxide signaling on the growth and differentiation of fetal dural and calvarial bone cells. Methods: Sections of fetal and adult calvaria were evaluated for endothelial and inducible nitric oxide synthase expression by immunohistochemistry. Primary fetal (E18) murine dural cell and calvarial osteoblast cultures were treated with 1 mu M or 10 mu M DETA-NONOate, a nitric oxide donor compound, or 1 mM N-G-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase inhibitor. Controls were left untreated. Cell proliferation was measured at 48 hours, and mRNA transcripts for Runx2, alkaline phosphatase, and osteopontin were measured by reverse transcription and quantitative real-time polymerase chain reaction at 2 to 18 days. Experiments were performed in triplicate. Results: Fetal, but not adult, dural cells express endothelial nitric oxide synthase. DETA-NONOate stimulated osteoblast mitogenesis by 16 percent (p < 0.05) but did not affect proliferation of dural cells. L-NMMA inhibited proliferation of dural cells and calvarial osteoblasts by 35 percent (p < 0.01) and 17 percent (p = 0.05), respectively. Exogenous nitric oxide increased dural cell transcription of Runx2, alkaline phosphatase (p = 0.03), and osteopontin (p = 0.09) and calvarial osteoblast transcription of Runx2 (p = 0.02) and osteopontin (p < 0.01). Fetal calvarial osteoblasts and dural cells treated with L-NMMA demonstrated reduced transcription of Runx2 and alkaline phosphatase (p < 0.05). Conclusions: Fetal dural cells and calvarial osteoblasts express endothelial nitric oxide synthase. Nitric oxide enhances proliferation and differentiation of fetal dural cells and calvarial osteoblasts. These results suggest that endothelial nitric oxide synthase-derived nitric oxide may play an important role in development of the fetal craniofacial skeleton.