間歇的な機械的刺激は RAW264.7 細胞において破骨細胞分化を抑制する

CD47

北海道歯学会

北海道歯学雑誌

Mechanical stress plays an important role in the remodeling of bone. In previous reports by this research team, we showed the suppression of osteoclast fusion by cyclic mechanical stress. In this study, we examined the effects of intermittent 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 10 % for 2 days from the fourth day of culture. In different treatments, mechanical stress was applied for 24 h, followed by bouts of continuous mechanical stress and no stress alternating at 1, 2, 3, 4, 6, or 12 h. In addition, we assayed osteoclast-related genetic mRNA expression using the real-time PCR method. We counted the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts with 2, 3, 4, 5, 6, or 7 nuclei, and those with more nuclei. The number of osteoclasts was lower after mechanical stress when compared with no mechanical stress. The number of total osteoclasts was significantly suppressed in the group stimulated every hour (1-h group) when compared with the group that was stimulated every 12 h (12-h group). Furthermore, we found that the number of osteoclasts with 2-4 nuclei was significantly reduced by intermittent continuous mechanical stress. The measurement of mRNA expression using real-time PCR showed the suppression of DC-STAMP, OC-STAMP, and CD47, which are fusion factors for osteoclasts. These significantly decreased in the 1-h group compared with that in 12-h group. CD47 participates in cell fusion with the mononuclear preosteoclasts among each other, and among the mononuclear preosteoclasts and those with 2-5 nuclei. Thus, the suppression of CD47 expression could reduce the number of osteoclasts with 2-4 nuclei. Furthermore, the total number of osteoclasts was reduced by the suppression of DC-STAMP and OC-STAMP expression. These results suggest that intermittent continuous mechanical stress is one factor that suppresses the fusion of osteoclasts, and that the cell fusion factor is controlled by the duration and frequency of intermittent stimulation by sustained extension.