持続的な機械的刺激はRAW264.7細胞における破骨細胞分化を抑制する

mechanical stress

北海道歯学会

北海道歯学雑誌

Osteoclasts are bone-resorbing cells that play a pivotal role in bone remodeling. Our previous studies have been among the few in which the direct effects of mechanical stress on osteoclastogenesis were assessed. Our reports have shown inhibition of osteoclast fusion by cyclic mechanical stress. In this study, we examined the effect of continuous mechanical stress on osteoclastogenesis. We cultured RAW264.7 cells stimulated with the receptor activator of nuclear factor-κB ligand (RANKL) using a BioFlex plate. Osteoclasts were stimulated using a Flexcell tension system at elongation rates of 5 %, 10 %, and 15 % for two days. (This was 4 days after the culture was initiated.) On the second day a notable increase in the number of osteoclasts was found. The number of osteoclasts showed significant decreases compared with control osteoclasts after stimulation at elongation rates of 10 % and 15 %. Osteoclasts were also stimulated at an elongation rate of 10 % for 1, 3, 6, and 12 h each day for two days, and significant decreases in the numbers of osteoclasts were found for osteoclasts that were stimulated for 6 h and 12 h. Continuous mechanical stress inhibited osteoclastogenesis were found for cyclic mechanical stress. We counted the numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts that had 2-4 nuclei and 5 or more nuclei. We found that the number of osteoclasts with 2-4 nuclei was decreased by continuous mechanical stress. Measurement of the quantity of NO showed that the quantity increased in stimulated osteoclasts compared to that in control osteoclasts. Since the results suggested that NO was related to the decrease in the number of osteoclasts with 2-4 nuclei, we treated the cells with an NOS inhibitor (L-NMMA). Treatment with L-NMMA suppressed the decrease in number of osteoclasts with 2-4 nuclei. Measurement of mRNA expression by real-time PCR showed that the quantity of dendritic cell-specific transmembrane protein (DC-STAMP), which is one of the fusion factors for osteoclasts, was significantly decreased in stimulated osteoclasts compared with that in the control osteoclasts. However, there were no changes in inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). The results suggest that continuous mechanical stress is one of the factors that inhibit fusion of osteoclasts and that NO is involved in the inhibition.