Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts.

Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0-10.0 g/cm(2)) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca(2+) pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca(2+) pathway.

The effects of low-intensity pulsed ultrasound exposure on gingival cells.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) has been used in fracture treatment to shorten the time needed for biologic wound healing. Clinical trials applying LIPUS in implant dentistry have reported accelerated soft-tissue healing and osseointegration. However, details of the clinical effects of LIPUS have not been well characterized. Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and angiogenesis in periodontal tissue. In this study, we focus on the effect of LIPUS on gingival epithelial cells and the role of CCN2/CTGF therein. METHODS: Gingival epithelial cells (GE1) were cultured in six-well cell-culture plates for 24 hours at 37°C with 5% CO(2), and then exposed to LIPUS for 15 minutes at 3-MHz frequency and 40-mW/cm(2) power. Total RNA was extracted after LIPUS exposure and analyzed by real-time polymerase chain reaction to detect CCN2/CTGF. Additionally, total protein from each sample after LIPUS exposure was immunoblotted with anti-CCN2/CTGF antibody. RESULTS: LIPUS exposure increased the mRNA level of CCN2/CTGF on exposure, and the level was significantly greater at 0 and 15 minutes after LIPUS exposure compared to the control. Western blotting analysis showed intense staining of CCN2/CTGF for 60 minutes after LIPUS exposure. The results demonstrate that LIPUS exposure accelerates soft-tissue healing by increasing CCN2/CTGF on exposure, in addition to its effects on bone formation. CONCLUSION: Our findings demonstrate that LIPUS exposure accelerates soft-tissue healing by increasing connective tissue growth factors via a mitogen-activated protein kinase signaling pathway on exposure.