Regulatory effect on osteoclast differentiation of soluble factors

Objectives: Mechanical stress is known to be important for regulating bone turnover. However, the detailed mechanisms are fully demonstrated. Therefore, we examined the effect of mechanical stress on osteoclast supportive cells using original compression model. Materials & Methods: Mouse osteoblast lineage cell, MC3T3-E1 cells were embedded in three-dimensional (3-D) gels and cultured with continuous compressive force (0-10.0 g/cm2) for 48 hr. Conditioned medium was collected and added to osteoclast progenitor RAW264.7 cells. Then the cells were incubated for indicated times in the presence of receptor activator of nuclear factor-ƒÛB ligand (RANKL). Osteoclast differentiation were detected by staining with tartrate resistant acid phosphatase (TRAP), and TRAP-positive multinucleated cells were considered to be osteoclast. NF-kB protein expression, phosphorylation and nuclear translocation were determined by western blots. The binding ability of conditioned medium to RANKL was measured using a quartz-crystal microbalance (QCM) technique. In some experiments, 3-D gels were supplemented with inhibitor of nencanonical Wnt signaling (KN93) prior to compressive force addition. Results: Conditioned media inhibited the differentiation of RAW264.7 cells into osteoclast via RANKL. Those inhibition were dose- and force-dependent manner. Conditioned media also inhibited the RANKL induced expression of TRAF6, phosphorylation of IkBa and nuclear translocation of p50 and p65. Conditioned medium demonstrated a high level of binding activity to RANKL and obstructed the binding of RANK to RANKL. This binding activity of conditioned media to RANKL were diminished when the 3-D gel was supplimented with KN93. Conclusion: These results suggest that mechanical stress enhanced the release of soluble factors from osteoblastic cells which binds to RANKL and strongly inhibits osteoclastgenesis through noncanonical Wnt signaling pathway.