Ex vivo real-time observation of Ca2+ signaling in living bone in response to shear stress applied on the bone surface
Ex vivo real-time observation of Ca2+ signaling in living bone in response to shear stress applied on the bone surface
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DOI：
10.1016/j.bone.2012.12.002
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发表时间：
2013-03-01
期刊：
影响因子：
4.1
通讯作者：
Yamashiro, Takashi
中科院分区：
文献类型：
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作者：
Ishihara, Yoshihito;Sugawara, Yasuyo;Yamashiro, Takashi
骨细胞通过产生各种生物信号来响应机械刺激，其中最早的事件之一是细胞内钙（[Ca2+](i)）动员。我们最近开发的体外活[Ca2+](i)成像系统显示，完整骨外植体中的骨细胞表现出自主的[Ca2+](i)振荡，并且骨细胞通过间隙连接特异性地调节这些振荡。然而，[Ca2+](i)信号网络在机械转导中的行为和连通性尚未在完整骨中进行研究。我们在此介绍了一种新的流体流动平台，用于探测活的完整骨中的细胞信号网络，该平台允许在骨外植体表面应用毛细管驱动的流动，同时对[Ca2+](i)变化进行实时荧光监测。在水流的作用下，成骨细胞和骨细胞中应答细胞的百分比增加，同时外植体中c-fos表达上调。然而，峰值相对荧光强度增强不明显。18 α - ga是一种可逆的间隙连接抑制剂，可以显著阻断骨细胞的[Ca2+](i)，反应性，但对成骨细胞没有任何重大影响。相反，这种治疗显著降低了成骨细胞和骨细胞的血流激活振荡反应频率。当被Gd3+阻断时，拉伸激活的膜通道在血流诱导的[Ca2+](i)反应中受到的影响较小。这些发现表明，通过间隙连接介导的细胞间通讯和半通道，流诱导的机械刺激伴随着成骨细胞和骨细胞的自主[Ca2+](i)振荡的激活。尽管骨在体内如何感知机械刺激仍有待阐明，但目前的研究表明，通过增强间隙连接和半通道介导的[Ca2+](i)动员的细胞-细胞信号传导可能作为机械转导的早期信号事件参与。(C) 2012爱思唯尔公司版权所有。
Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca2+](i)) mobilization. Our recently developed ex vivo live [Ca2+](i) imaging system revealed that bone cells in intact bone explants showed autonomous [Ca2+](i) oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca2+](i) signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca2+](i) changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 alpha-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca2+](i), responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd3+, is less affected in the flow-induced [Ca2+](i) response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca2+](i) oscillations in both osteoblasts and osteocytes via gap junction-mediated cell-cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca2+](i) mobilization could be involved as an early signaling event in mechanotransduction. (C) 2012 Elsevier Inc. All rights reserved.