Mechanical Stress Directly Induces Suppression of Osteoclast Differentiation in vitro

Objective: it is reported that mechanical stress to osteoblast and periodontal ligament cell suppresses osteoclast differentiation. However little has been known about a direct effect of mechanical stress to osteoclast differentiation. In this work, we investigated a role of mechanical stress to osteoclast differentiation treated with RANKL in RAW264.7 cells.

Methods: RAW cells, which were murine macrophage-like osteoclast precursors, were cultured with RANKL and mechanical stress according to Flexcell tension system (10% elongation at 30 cycles per min) for an indicated period. We counted the number of tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated osteoclasts, and measured mRNA by RT-PCR.

Results: There was a decrease in the number of osteoclasts under mechanical stress compared with the number under no mechanical stress. The number of large osteoclasts (8 nuclei or more) and the number of nuclei per osteoclast under mechanical stress also decreased. As the cells were cultured for a period of 4-7 days and/or for different periods of mechanical stress, osteoclast differentiation decreased with mechanical stress and increased after removing mechanical stress. Expression of mRNA for the osteoclast-specific genes, TRAP, matrix metalloproteinase-9, cathepsin-K and calcitonin receptor, decreased with mechanical stress and was associated to the number of osteoclasts. Inducible nitric oxide synthase mRNA, which inhibits osteoclast differentiation, increased with mechanical stress. In spite of the decrease in osteoclast number with mechanical stress, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) mRNA increased with mechanical stress. This results suggested that signal transduction to osteoclast differentiation suppressed downstream of NFATc1 with mechanical stress although NFATc1 transcription functioned properly.

Conclusion: These findings indicate that mechanical stress directly suppresses osteoclast differentiation, but as an increase of NFATc1 and NFATc2 with mechanical stress, suggesting that osteoclast differentiation is delayed.