Low-intensity pulsed ultrasound induces apoptosis in osteoclasts: Fish scales are a suitable model for the analysis of bone metabolism by ultrasound

Abstract

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3 hours of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18 hours of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in the osteoclasts using the TUNEL (TdTmediated dUTP nick-end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3 hours of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure.Key words: LIPUS, osteoclasts, osteoblasts, fish scales, apoptosis, GeneChip analysis Abbreviations: ALP, alkaline phosphatase; Bcl-2, B-cell lymphoma 2; CTSK, Cathepsin K; Dlx5, distal less homeobox5; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; LIPUS, low-intensity pulsed ultrasound; OCN, osteocalcin; PMAIP, phorbol-12-myristate-13-acetateinduced protein 1; SAFHS, sonic accelerated fracture healing system; pNP, para-nitrophenol; TRAP, tartrate-resistant acid phosphatase; TUNEL, TdT-mediated dUTP nick end labelling; TNFRSF10A, tumor necrosis factor receptor superfamily, member a; Runx2a, runt related transcription factor 2a; Runx2b, runt related transcription factor 2b 3

Low-intensity pulsed ultrasound (LIPUS) prevents periprosthetic inflammatory loosening through FBXL2-TRAF6 ubiquitination pathway

“…used LIPUS on zebrafish scales (containing both osteoblasts and osteoclasts) for 20 min. Results showed that LIPUS can cause osteoclast apoptosis in their model34. The current study is different from Suzuki’s study.…”

A Treatment Combined Prussian Blue Nanoparticles With Low-intensity Pulsed Ultrasound Alleviates Cartilage Damage in Knee Osteoarthritis by Initiating PI3K/Akt/mTOR Pathway

“…Focused low-intensity pulsed ultrasound (FLIPUS) affected ECM production in KOA rabbits by decreasing chondrocyte apoptosis [46]. Conversely, LIPUS treatment promoted apoptosis and decreased the viability of endothelial cells, osteoclasts, and preadipocytes in human [47][48][49]. The opposite results may attribute to different parameters of LIPUS and different cells.…”

<b>Effects of low-intensity pulsed ultrasound on osteoclasts: Analysis with goldfish scales as a model of </b><b>bone </b>

“…Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated (21). Furthermore, the multinuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL for DNA breaks (21). Our previous report (21) indicates that LIPUS directly functions in osteoclasts and promotes apoptosis in osteoclasts shortly after exposure.…”

“…Therefore, the data of mammalian hard tissue support our present results. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated (21). Furthermore, the multinuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL for DNA breaks (21).…”

“…Furthermore, the multinuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL for DNA breaks (21). Our previous report (21) indicates that LIPUS directly functions in osteoclasts and promotes apoptosis in osteoclasts shortly after exposure. In the present study, the mRNA expression of RANK (osteoclastic marker) decreased after 3 h of incubation following LIPUS treatment.…”