Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, 700-8525, Japan.
Bone. 2012 Apr;50(4):842-52. doi: 10.1016/j.bone.2012.01.021. Epub 2012 Feb 1.
Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca(2+) acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca(2+) dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca(2+) imaging system that allows the dynamic intracellular Ca(2+) concentration (Ca(2+)) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic Ca(2+) oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca(2+) stores, abolished these Ca(2+) responses in both osteoblasts and osteocytes, indicating that Ca(2+) release from intracellular stores plays a key role in the Ca(2+) oscillations of these bone cells in intact bone explants. Another possible Ca(2+) transient system to be considered is gap junctional communication through which Ca(2+) and other messenger molecules move, at least in part, across cell-cell junctions; therefore, we also investigated the role of gap junctions in the maintenance of the autonomic Ca(2+) oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18α-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of Ca(2+) oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous Ca(2+) oscillations that required the release of intracellular Ca(2+) stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed Ca(2+) oscillations of bone cells.
骨细胞形成一个复杂的三维网络，其中包括嵌入矿化细胞外基质中的成骨细胞和骨细胞。Ca(2+)作为一种普遍存在的第二信使，在各种生理细胞过程中传递信号，并将信号传递到细胞内部和细胞之间。然而，活骨中骨细胞的细胞内 Ca(2+)动力学尚未得到评估。在本研究中，我们开发了一种新的离体活 Ca(2+)成像系统，允许观察完整鸡颅骨外植体的动态细胞内 Ca(2+)浓度(Ca(2+))反应，而不会破坏骨网络。我们的活细胞成像分析首次揭示，成骨细胞和骨细胞都表现出重复的和自主的Ca(2+)振荡。内质网抑制剂 thapsigargin 诱导细胞内 Ca(2+)储存耗尽，可消除成骨细胞和骨细胞中的这些Ca(2+)反应，表明细胞内储存的 Ca(2+)释放在完整骨外植体中这些骨细胞的Ca(2+)振荡中起关键作用。另一个需要考虑的可能的Ca(2+)瞬变系统是缝隙连接通讯，通过该通讯，Ca(2+)和其他信使分子至少部分地穿过细胞-细胞连接移动；因此，我们还研究了缝隙连接在维持完整骨中观察到的自主Ca(2+)振荡中的作用。用三种不同的缝隙连接抑制剂 18α-甘草次酸、油酰胺和辛醇处理，显著降低了反应性骨细胞的比例，表明缝隙连接对于骨细胞中Ca(2+)振荡的维持很重要，但对成骨细胞的维持作用较小。总之，我们发现完整骨外植体中的骨细胞表现出自主的Ca(2+)振荡，这需要释放细胞内 Ca(2+)储存。此外，骨细胞通过缝隙连接特异性调节这些振荡，从而维持观察到的骨细胞Ca(2+)振荡。
