Osteocyte Signaling Within Mineralized Lacuna-Canaliculi Microenvironment

矿化腔隙-小管微环境中的骨细胞信号传导

基本信息

批准号：
10240448
负责人：
Pranav Soman
金额：
$ 16.01万
依托单位：
SYRACUSE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10240448
关键词：
3D Print Ablation Biochemical Biocompatible Materials Body Fluids Bone Diseases Bone Matrix Calcium Signaling Cell Line Cells Cellular biology Characteristics Confocal Microscopy Dendritic Cells Diffusion Disease Evaluation Fluorescence Microscopy Functional disorder Gap Junctions Gases Gelatin Goals Homeostasis Hybrids Hydrogels Hypoxia Incubators Individual Lasers Location Measures Mechanical Stimulation Mechanics Methacrylates Methods Microfluidic Microchips Microfluidics Minerals Modeling Morphology Mus Nutrient Nutrient Canals Optics Orthopedic Procedures Osteocytes Osteon Pathology Patients Periodicity Physiological Play Printing Property Refractory Risk Signal Transduction Speed Structure Technology Testing Time Time Study Work base bone bone cell calcification cell injury design high risk hormonal signals in vitro Model in vivo inhibitor/antagonist insight mechanical force mechanotransduction new technology novel therapeutics preservation shear stress skeletal skeletal abnormality therapeutic target

项目摘要

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.

概括尽管人们普遍认为骨细胞通过感知、整合和调节骨稳态来调节骨稳态。转导机械和激素信号，骨细胞内动态信号的表征由于骨细胞嵌入骨基质内，网络一直具有挑战性。矿化腔隙小管 (MLC) 结构允许感测机械力和传导通过间隙连接和可溶性生化信号的分泌交换来传递信号。以空间梯度调节脉管系统和埋藏骨细胞之间必需营养素的获取对骨细胞信号传导的新认识对于开发新的治疗方法是必要的。为此，这项工作的目标是开发一种新的体外模型。这不仅可以模拟体内的 MLC 结构，还可以促进信号动力学的研究骨细胞网络受到有针对性的机械刺激或细胞损伤的假设，“营养物质”。骨细胞遇到的梯度是矿化腔隙-小管 (MLC) 结构的函数，该结构在转向调节其信号传播动态”，将使用三个特定目标进行测试，目标 1 将使用混合目标。激光打印 (HLP) 平台可开发模拟 MLC 结构的微流控芯片目标 2 将确定生成骨细胞网络的实验条件。在 MLC 芯片中使用小鼠 MLO-Y4 骨细胞细胞系 Aim 3 将表征增殖特性。骨细胞网络内的钙信号传导（幅度、范围、速度、不应期、尖峰同步）在有针对性的机械刺激、细胞损伤、细胞间连接消融或存在总之，(i) MLC 结构诱导的梯度营养的单独和组合效应。访问 (ii) 矿化基质、(iii) 环境缺氧和 (iv) 单细胞操作，了解钙信号传导从长远来看，该模型可以为骨细胞机械传导提供新的见解。扩展到患者特异性细胞，以筛选针对与相关骨骼病理学相关的治疗方法骨细胞功能障碍。