Osteocyte Signaling Within Mineralized Lacuna-Canaliculi Microenvironment

总结 尽管骨细胞通过感知、整合和调节骨稳态被广泛接受， 转导机械和激素信号，表征骨细胞内的动态信号 由于其嵌入骨基质中的位置，因此网络一直具有挑战性。骨细胞位于 矿化的腔隙-小管（MLC）结构允许感测机械力和转导， 通过间隙连接和可溶性生化信号分泌交换进行信号传导。MLC结构 以空间梯度调节血管系统和埋藏骨细胞之间必需营养物质的进入 方式对骨细胞信号传导的新认识将是开发新的治疗方法的必要条件。 涉及骨细胞功能障碍的疾病。为此，这项工作的目标是开发一种新的体外模型 这不仅将模拟体内类似MLC的结构，而且还有助于研究细胞内的信号动力学。 骨细胞网络在有针对性的机械刺激或细胞损伤。假设，“营养素 骨细胞遇到梯度是矿化腔隙-小管（MLC）结构的函数， 反过来调节其信号传播动态”，将使用三个具体目标进行测试。目标1将使用混合动力车 激光打印（HLP）平台，以开发模拟MLC结构的微流体芯片， 梯度养分输送特性目的2将确定实验条件，以产生骨细胞网络 在MLC芯片内使用小鼠MLO-Y 4骨细胞系。目标3将描述传播特性 骨细胞网络内钙信号传导（幅度、范围、速度、不应期、尖峰同步性） 在靶向机械刺激、细胞损伤、细胞-细胞连接消融时，或在存在 信号传导抑制剂总之，（i）MLC结构诱导的梯度营养素的单独和联合作用 访问（ii）矿化基质，（iii）环境缺氧，和（iv）单细胞操作，对钙信号传导 动力学将为骨细胞力学转导提供新的见解。从长远来看，这种模式可以 扩展到患者特异性细胞，以筛选靶向与以下疾病相关的骨骼病变的治疗剂： 骨细胞功能障碍

Summary Although it is widely accepted that osteocytes regulate bone homeostasis by sensing, integrating and transducing mechanical and hormonal signals, characterization of dynamic signaling within the osteocyte network has been challenging due to its location embedded within the bone matrix. Osteocytes reside within a mineralized lacunar-canalicular (MLC) structure allowing sensing of mechanical forces and transduction this signal through gap-junctions and secreted exchange of soluble biochemical signals. The MLC structure modulates access of essential nutrients between vasculature and entombed osteocytes in a spatially gradient manner. New understanding on osteocyte signaling will be necessary to develop new therapeutics for treating diseases that involve osteocyte dysfunction. To that end, the goal of this work is to develop a new in vitro model that will not only mimic the in vivo like MLC structure, but also facilitate the study of signaling dynamics within an osteocyte network upon targeted mechanical stimulation or cell damage. The hypothesis that, “the nutrient gradient that osteocyte encounter is a function of the mineralized lacunar-canalicular (MLC) structure, which in turn regulates their signal propagation dynamics”, will be tested using three specific aims. Aim 1 will use a Hybrid Laser Printing (HLP) platform to develop a microfluidic chip that mimics the MLC structure with associated gradient nutrient transport properties. Aim 2 will identify experimental conditions to generate osteocyte network within MLC chips using the mouse MLO-Y4 osteocyte cell line. Aim 3 will characterize propagation characteristics of calcium signaling (amplitude, range, velocity, refractory period, spike-synchrony) within osteocyte networks upon targeted mechanical stimulation, cell-damage, ablation of cell-cell connections, or in the presence of signaling inhibitors. In summary, individual and combined effects of (i) MLC structure-induced gradient nutrient access (ii) mineralized matrix, (iii) environmental hypoxia, and (iv) single cell manipulation, on calcium signaling dynamics will provide new insights into osteocyte mechanotransduction. In the long term, this model can be extended to patient-specific cells to screen therapeutics that target skeletal pathologies associated with osteocyte malfunctions.