Large magnitude of force leads to NO-mediated cell shrinkage in single osteocytes implying an initial apoptotic response.

Damage accumulation in the bone under continuous daily loading causes local mechanical overloading known to induce osteocyte apoptosis, which promotes bone resorption to repair bone damage. However, only a few studies have investigated the mechanism of apoptosis in mechanically overloaded osteocytes. As mechanically stimulated osteocytes produce nitric oxide (NO), which triggers apoptosis in various cell types, we aimed to elucidate the mechanism underlying apoptosis in mechanically overloaded osteocytes, focusing on intracellular NO. To investigate the effects of force magnitude on apoptosis and intracellular NO production, we isolated osteocytes from DMP1-EGFP mice and subjected them to quantitative local forces via fibronectin-coated micro beads targeting integrin on the cell surface using a magnetic tweezer. Cell shrinkage was microscopically examined, and intracellular NO production was visualized using DAR-4 M. Mechanical stimulation revealed relationships between force magnitude, apoptosis, and intracellular NO production. The application of a smaller force resulted in no significant cell shrinkage or intracellular NO production; however, a larger force caused a rapid increase in intracellular NO production followed by cell shrinkage. Besides, intracellular NOS (NO synthase) inhibition and NO donation revealed the pro-apoptotic roles of NO in osteocytes. L-NAME (NOS inhibitor)-treated cells displayed no significant shrinkage under a larger force, whereas SNP (NO donor)-treated cells showed cell shrinkage and Annexin V fluorescence, indicating apoptosis. Collectively, our study demonstrates that larger force leads to NO production-mediated osteocyte shrinkage, implying an initial apoptotic response and highlighting the importance of NO production in bone damage.