Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Bone. 2012 Nov;51(5):860-7. doi: 10.1016/j.bone.2012.08.116. Epub 2012 Aug 17.
The mechanisms underlying the detection of critically loaded or micro-damaged regions of bone by bone cells are still a matter of debate. Our previous studies showed that calcium efflux originates from pre-failure regions of bone matrix and MC3T3-E1 osteoblasts respond to such efflux by an increase in the intracellular calcium concentration. The mechanisms by which the intracellular calcium concentration increases in response to an increase in the pericellular calcium concentration are unknown. Elevation of the intracellular calcium may occur via release from the internal calcium stores of the cell and/or via the membrane bound channels. The current study applied a wide range of pharmaceutical inhibitors to identify the calcium entry pathways involved in the process: internal calcium release from endoplasmic reticulum (ER, inhibited by thapsigargin and TMB-8), calcium receptor (CaSR, inhibited by calhex), stretch-activated calcium channel (SACC, inhibited by gadolinium), voltage-gated calcium channels (VGCC, inhibited by nifedipine, verapamil, neomycin, and ω-conotoxin), and calcium-induced-calcium-release channel (CICRC, inhibited by ryanodine and dantrolene). These inhibitors were screened for their effectiveness to block intracellular calcium increase by using a concentration gradient induced calcium efflux model which mimics calcium diffusion from the basal aspect of cells. The inhibitor(s) which reduced the intracellular calcium response was further tested on osteoblasts seeded on mechanically loaded notched cortical bone wafers undergoing damage. The results showed that only neomycin reduced the intracellular calcium response in osteoblasts, by 27%, upon extracellular calcium stimulus induced by concentration gradient. The inhibitory effect of neomycin was more pronounced (75% reduction in maximum fluorescence) for osteoblasts seeded on notched cortical bone wafers loaded mechanically to damaging load levels. These results imply that the increase in intracellular calcium occurs by the entry of extracellular calcium ions through VGCCs which are sensitive to neomycin. N-type and P-type VGCCs are potential candidates because they are observed in osteoblasts and they are sensitive to neomycin. The calcium channels identified in this study provide new insight into mechanisms underlying the targeted repair process which is essential to bone adaptation.
细胞通过何种机制检测到骨骼中临界负荷或微损伤区域仍然存在争议。我们之前的研究表明，钙外流源于骨基质的早期破坏区域，MC3T3-E1 成骨细胞通过细胞内钙离子浓度的增加来对此类钙外流做出反应。细胞内钙离子浓度增加以响应细胞外钙浓度增加的机制尚不清楚。细胞内钙离子的升高可能通过细胞内钙库的释放和/或通过膜结合通道发生。本研究应用了广泛的药物抑制剂来鉴定参与该过程的钙内流途径：内质网（ER）内钙释放（被 thapsigargin 和 TMB-8 抑制）、钙受体（CaSR，被 calhex 抑制）、张力激活钙通道（SACC，被 gadolinium 抑制）、电压门控钙通道（VGCC，被 nifedipine、verapamil、neomycin 和 ω-conotoxin 抑制）和钙诱导钙释放通道（CICRC，被 ryanodine 和 dantrolene 抑制）。通过使用模拟钙从细胞基底侧面扩散的浓度梯度诱导钙外流模型，筛选这些抑制剂以阻断细胞内钙增加的有效性。进一步在机械加载有缺口皮质骨片上接种的成骨细胞上测试减少细胞内钙反应的抑制剂，这些骨片正在发生损伤。结果表明，只有新霉素在浓度梯度诱导的细胞外钙刺激下降低了成骨细胞的细胞内钙反应，降低了 27%。在机械加载至损伤负荷水平的缺口皮质骨片上接种的成骨细胞中，新霉素的抑制作用更为明显（最大荧光减少 75%）。这些结果表明，细胞内钙的增加是通过细胞外钙离子通过对新霉素敏感的 VGCC 进入细胞内发生的。N 型和 P 型 VGCC 是潜在的候选者，因为它们在成骨细胞中被观察到，并且对新霉素敏感。本研究中鉴定的钙通道为骨适应所必需的靶向修复过程的潜在机制提供了新的见解。
