a b s t r a c tLow-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dentistry. It has been shown that LIPUS induces the secretion of extracellular adenosine triphosphate (ATP), a key mediator of osteoblast response to mechanical stimuli. However, the detailed mechanism of LIPUS-induced osteogenesis has been elusive. In this study, we investigated the role of the P2X7 receptor in LIPUS-induced osteogenesis. LIPUS induced the release of extracellular ATP, differentiation of osteoblasts and osteogenesis via the P2X7 receptor, without affecting the activity of alkaline phosphatase (ALPase). These results suggest that LIPUS-induced extracellular ATP promotes bone formation via the osteoblast P2X7 receptor independently of ALPase.

Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)-induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3-E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF-induced extracellular ATP is released in osteoblasts, suggesting that TF-induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

Inflammatory cytokines, interleukin (IL)-1, IL-6, and TNF-α, are involved in inflammatory bone diseases such as rheumatoid osteoarthritis and periodontal disease. Particularly, periodontal disease, which destroys alveolar bone, is stimulated by lipopolysaccharide (LPS). Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dental treatments. However, the mechanism underlying effects of LIPUS on LPS-induced inflammatory cytokine are not well understood. We therefore aimed to investigate the role of LIPUS on LPS-induced IL-1α production. Mouse calvaria osteoblast-like cells MC3T3-E1 were incubated in the presence or absence of LPS (Porphyromonas gingivalis), and then stimulated with LIPUS for 30 min/day. To investigate the role of LIPUS, we determined the expression of IL-1α stimulated with LIPUS and treated with an angiotensin II receptor type 1 (AT1) antagonist, Losartan. We also investigate to clarify the pathway of LIPUS, we transfected siRNA silencing AT1 (siAT1) in MC3T3-E1. LIPUS inhibited mRNA and protein expression of LPS-induced IL-1α. LIPUS also reduced the nuclear translocation of NF-κB by LPS-induced IL-1α. Losartan and siAT1 blocked all the stimulatory effects of LIPUS on IL-1α production and IL-1α-mediated NF-κB translocation induced by LPS. Furthermore, PLCβ inhibitor U73122 recovered NF-κB translocation. These results suggest that LIPUS inhibits LPS-induced IL-1α via AT1-PLCβ in osteoblasts. We exhibit that these findings are in part of the signaling pathway of LIPUS on the anti-inflammatory effects of IL-1α expression.

These results indicate that the higher the dose of intermittent PTH administered, the greater the amount of bone formation beyond the skeletal envelop in the rat calvarium.

Periodontal disease is caused by inflammation induced by Porphyromonas gingivalis (P.g.) lipopolysaccharide (LPS) and involves expression of proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α, and receptor activator of nuclear factor kappa B ligand (RANKL), which are implicated in bone resorption. Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of bone fracture. However, the mechanisms by which LIPUS inhibits LPS-induced inflammatory cytokines are poorly understood. Therefore, we investigated the effects of LIPUS on LPS-induced expression of the proinflammatory cytokines IL-6 and RANKL. MC3T3-E1 cells were incubated in the presence or absence of P.g. LPS and then stimulated with LIPUS for 30 min/day for a maximum of 14 days. LPS increased mRNA and protein expressions of IL-6 and RANKL on day 14. In addition, mRNA expression of COX-2 LPS was higher after 3 and 7 days of LIPUS treatment. PGE was induced by LPS after 7 and 14 days of culture. LIPUS suppressed all stimulatory effects of LPS. These results suggest that LIPUS inhibits LPS-induced expression of inflammation cytokines by suppressing PGE production and might thus have potential applications in the treatment of periodontitis.