Response of Osteoblasts and Osteoclasts to Low-Intensity Pulsed Ultrasound: In Vitro and In Vivo Analyses Using Goldfish Scales

Abstract

Objectives: Low-intensity pulsed ultrasound (LIPUS), which is known as a noninvasive therapy, promotes the repair of bone fractures. Until now, studies of LIPUS have focused on the proliferation and the differentiation of osteoblasts. However, the effects of LIPUS on osteoclasts are still not fully understood. Therefore, we examined the effects of LIPUS on osteoclastogenesis using goldfish scales with both osteoclasts and osteoblasts as a model of bone. Materials and Methods: Ultrasound was generated by the Sonic Accelerated Fracture Healing System (SAFHS 4000J; Teijin Pharma, Ltd). Scales were collected from goldfish under anesthesia; they were then treated with LIPUS for 20 minutes, incubated at 15°C for 3, 6, 12, and 24 hours in L-15 medium, and their mRNA expression was measured. Next, the influence of LIPUS on scale regeneration was investigated to confirm the results obtained from an in vitro experiment. Results and Discussion: After 6 hours of incubation following LIPUS treatment, RANKL mRNA increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 hours of incubation, and the mRNA expression of functional genes was significantly upregulated after 12 hours of incubation. However, the mRNA expression of osteoprotegerin, which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 hours of incubation. Thus, we determined that LIPUS moderately activates osteoclasts via RANK/RANKL signaling and may induce bone formation. Next, we performed an in vivo experiment with goldfish. After 2 weeks of daily LIPUS exposure, the regeneration rate of the LIPUS-treated scales was significantly higher than that of control scales. These data support the hypothesis that LIPUS moderately activates osteoclasts and induces bone formation.