Osteocytes as main responders to Low Intensity Pulsed Ultrasound for the treatment of fracture healing

Skeletal tissue is known to respond to mechanical stress. Ultrasound stimulation is one of mechanical stress and low intensity pulsed ultrasound (LIPUS) devices have been clinically utilized to promote the fracture healing. However, it is still not clear which skeletal cells, especially osteocytes or osteoblasts, mainly respond to LIPUS stimulation and how they contribute to fracture healing. To examine that, we utilized medaka known to have bones without embedded osteocytes and zebrafish known to have bones with embedded osteocytes as an in vivo model. Interestingly, fracture healing was accelerated by ultrasound stimulation in zebrafish but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocyte, we performed RNA-sequencing with the murine long bone osteocyte Y4 (MLO-Y4) cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation and functional cluster analysis defined that several molecular signatures related in immunity, secretion and transcription. Especially, most of isolated transcription related genes were modulated by LIPUS also in vivo experiments using zebrafish. Target genes analysis showed that inflammatory response and bone formation in fracture healing could be transcriptionally regulated in osteocytes by LIPUS stimulation. Among these transcription genes, early growth response (Egr) 1,2, JunB, Forkhead box Q1 (FoxQ1) and nuclear factor of activated T cells (NFAT) c1 were not altered by LIPUS in medaka unlike zebrafish suggesting that these genes would be key transcriptional regulator of LIPUS-dependent fracture healing through osteocytes. In this study, we revealed bone-equipped osteocytes is necessary for LIPUS-induced promotion of fracture healing through the transcriptional control of target genes presumably to activate neighboring cells involved in fracture healing event. Examining the effects of low-intensity pulsed ultrasound (LIPUS) stimulation on MLO-Y4 cells.