Osteocyte Signaling Within Mineralized Lacuna-Canaliculi Microenvironment

摘要 尽管人们普遍认为，骨细胞通过感知、整合和 传递机械和激素信号，骨细胞内动态信号的特征 由于其嵌入在骨基质中的位置，网络一直具有挑战性。骨细胞驻留在 矿化的腔隙-管(MLC)结构，允许感测机械力并将其传递给 通过缝隙连接和可溶性生化信号的分泌交换来传递信号。MLC结构 在空间梯度中调节血管和埋藏的骨细胞之间必需营养物质的获取 举止。对骨细胞信号转导的新认识对于开发新的治疗方法是必要的 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，而且有助于研究细胞内的信号动力学 骨细胞在靶向机械刺激或细胞损伤时形成网络。假设，“营养素 骨细胞遇到的倾斜度是矿化陷窝-管状(MLC)结构的函数，在 TURN调节他们的信号传播动态“，将使用三个具体目标进行测试。AIM 1将使用混合动力车 激光打印(HLP)平台开发一种模拟MLC结构的微流控芯片 营养物质的梯度传输特性。目标2将确定生成骨细胞网络的实验条件 在MLC芯片内使用小鼠MLO-Y4骨细胞系。目标3将描述传播特征 钙信号(幅度、幅度、速度、不应期、峰同步)在骨细胞网络中的作用 在有针对性的机械刺激、细胞损伤、细胞-细胞连接的消融或在 信号转导抑制剂。综上所述，(I)MLC结构诱导的梯度养分的单独效应和联合效应 (Ii)矿化基质，(Iii)环境低氧，和(Iv)单细胞操作，对钙信号的影响 动力学将为骨细胞的机械转导提供新的见解。从长远来看，这种模式可以 扩展到患者特定细胞以筛选针对与以下疾病相关的骨骼病理的治疗药物 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.