Oral Biochemistry and Molecular Biology, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo, 060-8586, Japan.
Oral Diagnosis and Medicine, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan.
Sci Rep. 2021 May 13;11(1):10298. doi: 10.1038/s41598-021-89672-9.
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.
超声刺激是一种机械应力，低强度脉冲超声（LIPUS）设备已在临床上用于促进骨折愈合。然而，尚不清楚哪种骨骼细胞（特别是成骨细胞或骨细胞）对 LIPUS 刺激做出主要反应，以及它们如何促进骨折愈合。为了研究这个问题，我们利用了骨骼中缺乏骨细胞的斑马鱼和骨骼中有骨细胞的牙鲆作为体内模型。超声刺激可加速斑马鱼的骨折愈合，但不能加速牙鲆的骨折愈合。为了研究 LIPUS 刺激在成骨细胞中诱导的分子事件，我们对暴露于 LIPUS 的小鼠成骨细胞系进行了 RNA 测序。179 个基因对 LIPUS 刺激有反应，功能聚类分析从中鉴定出了几个与免疫、分泌和转录相关的分子特征。值得注意的是，在体内的斑马鱼中，LIPUS 也调节了分离出的大多数与转录相关的基因。然而，早期生长反应蛋白 1 和 2（Egr1、2）、JunB、叉头框 Q1（FoxQ1）和激活 T 细胞核因子 c1（NFATc1）的表达水平在牙鲆中不受 LIPUS 影响，这表明这些基因是通过成骨细胞介导的 LIPUS 依赖性骨折愈合的关键转录调控因子。因此，我们表明，嵌入骨骼中的成骨细胞是 LIPUS 诱导促进骨折愈合所必需的，通过对靶基因的转录控制，可能激活参与骨折愈合过程的邻近细胞。
