Exogenous Nitric Oxide Up-regulates the Runx2 Via Bmp7 Overexpression to Increase the Osteoblast Matrix Production In Vitro

Abstract

Background: Nitric oxide (NO) is a signaling molecule that is required for the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). According to previous reports, high concentrations of sodium nitroprusside (SNP) inhibit the osteogenic differentiation of BMSCs, while its low concentration promotes this process. Objectives: The present investigation focused on evaluating the underlying mechanism of the osteogenic differentiation of BMSCs treated with low concentrations of SNP as an NO generating agent. Methods: The BMSCs after the 3rd passage was differentiated to osteoblasts when treated with 100 µM for 1 hour every 48 hours until 5, 10, 15, and 20 days of incubation. Then, the matrix production was estimated by quantitative alizarin red assay and calcium determination. The expression of different genes involved in osteogenic differentiation was statistically determined using the reverse transcriptase polymerase chain reaction. Finally, alkaline phosphatase activity was measured by a commercial kit. Results: The exogenous NO caused a significant (P<0.05) increase in the matrix production of differentiated BMSCs from day 5 to 20. The results showed the elevation of alkaline phosphatase activity and the up-regulation of its gene. Eventually, an increase was observed in the expression of a cascade of other genes such as osteonectin, Bmp7, Smad1, Runx2, and Raf1 in treated BMSCs. Conclusion: Overall, short-time treatment with a low concentration of exogenous NO increases the matrix production via gene up-regulation and protein production, which might open a new window in treating the low-density bone complication.