Abstract
Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. However, it remains unclear which skeletal cells, in particular osteocytes or osteoblasts, primarily respond to LIPUS stimulation and how they contribute to fracture healing. To examine this, we utilized medaka, whose bone lacks osteocytes, and zebrafish, whose bone has osteocytes, as in vivo models. Fracture healing was accelerated by ultrasound stimulation in zebrafish, but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocytes, we performed RNA sequencing of a murine osteocytic cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation, and functional cluster analysis identified among them several molecular signatures related to immunity, secretion, and transcription. Notably, most of the isolated transcription-related genes were also modulated by LIPUS in vivo in zebrafish. However, expression levels of early growth response protein 1 and 2 (Egr1, 2), JunB, forkhead box Q1 (FoxQ1), and nuclear factor of activated T cells c1 (NFATc1) were not altered by LIPUS in medaka, suggesting that these genes are key transcriptional regulators of LIPUS-dependent fracture healing via osteocytes. We therefore show that bone-embedded osteocytes are necessary for LIPUS-induced promotion of fracture healing via transcriptional control of target genes, which presumably activates neighboring cells involved in fracture healing processes.
Highlights
Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing
Inflammation stage, soft callus formation stage, hard callus formation stage, and remodeling stage are indicated by arrows (Red arrows: zebrafish, Blue arrows: Medaka). (d) The body weight and total length of experimental group of medaka and zebrafish before the LIPUS application (n = 3–5). (e) Upper: Days required for complete fracture healing in LIPUS-treated and untreated medaka and zebrafish
Li et al reported that LIPUS-stimulated murine long bone osteocyte Y4 (MLO-Y4) cells secreted prostaglandin E2 (PGE2) and NO into culture media, and the differentiation of osteoblastic cells cultured in this media were changed s ignificantly[9]
Summary
Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. Generally supplied by gravity and exercise, is effective at inducing bone remodeling and maintaining bone mass due to the regulating biological functions of bone cells[5, 6], it seems reasonable to assume that LIPUS stimulation promotes fracture healing through control of bone cells Both bone-forming osteoblasts and mechanosensitive osteocytes are capable of responding to LIPUS, and the synergistic action of these cells is likely important for the efficacy of LIPUS treatment[7]. Li et al reported that LIPUS-stimulated murine long bone osteocyte Y4 (MLO-Y4) cells secreted prostaglandin E2 (PGE2) and NO into culture media, and the differentiation of osteoblastic cells cultured in this media were changed s ignificantly[9] Another method to apply mechanical stress, fluid-flow-induced shear stress, increases PGE2 release and cyclooxygenase 2 (Cox2) mRNA expression in osteocytic MLO-Y4 cells, and PGE2 is involved in the upregulation of connexin 43-based gap junctions in MLO-Y4 c ells[13]. It has not been confirmed whether osteocytes are necessary and valuable for LIPUS-mediated fracture healing with an in vivo study
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