Genes involved in osteogenic differentiation induced by low‑intensity pulsed ultrasound in goldfish scales

The teleost scale is a unique calcified tissue that contains osteoclasts, osteoblasts, osteocytes and the bone matrix, similar to mammalian bone. Here, the effects of low-intensity pulsed ultrasound (LIPUS) on osteoblasts and osteoclasts in goldfish scales were investigated. Scales were treated with LIPUS, which is equivalent to use under clinical conditions (30 mW/cm² for 20 min), then cultured at 15˚C. Alkaline phosphatase activity, a marker of osteoblasts, or tartrate-resistant acid phosphatase (TRAP) activity, a marker of osteoclasts was measured. The gene expression profile was examined using RNA-sequencing. Gene network and biological function analyses were performed using the Ingenuity^® Pathways Knowledge Base. A single exposure of LIPUS significantly increased ALP activity but did not affect TRAP activity. These data indicated that LIPUS induced osteoblastic activation in goldfish scales. Using RNA-sequencing, numerous genes that were significantly and differentially expressed 3, 6, and 24 h after LIPUS exposure were observed. Ingenuity^® pathway analysis demonstrated that three gene networks, GN-3h, GN-6h, and GN-24h, were obtained from upregulated genes at 3, 6 and 24 h culture, respectively, and included several genes associated with osteoblast differentiation, such as protein kinase D1, prostaglandin-endoperoxide synthase 2, TNFRSF11B (tumor necrosis factor receptor superfamily, member 11b) and WNT3A (Wnt family member 3A). A significant upregulation of expression levels of these genes in scales treated with LIPUS was confirmed by reverse transcription-quantitative polymerase chain reaction. These results contribute to elucidating the molecular mechanisms of osteoblast activation induced by LIPUS.