Chondrocyte Mechanotransduction Using Microfluidics

描述(申请人提供)：本申请的主要目的是了解渗透负荷在软骨细胞力学转导培养研究中以及在生理关节负荷中对调节软骨细胞行为的贡献。虽然这项基础科学研究旨在提供有关软骨细胞机械转导的新信息，但我们预计一些发现可能有助于阐明渗透环境的变化在骨关节炎(OA)中所起的作用。在这个A1修订方案中，根据我们的初步数据，我们假设细胞骨架在调节关节软骨细胞的渗透负荷反应中起关键作用。在这种情况下，细胞骨架可能对渗透负荷引起的钙变化做出反应，或者渗透负荷引起的钙变化可能对细胞骨架的初始组织做出反应，或者两者都可能在反馈环中起作用。已报道的软骨细胞细胞骨架蛋白含量的地带性差异，以及正常软骨细胞和骨关节炎软骨细胞之间的细胞骨架差异，为我们的假设提供了额外的动机。为了验证这一假设，我们提出了几个具体的目标，以启动我们对细胞骨架在调节aggrecan基因表达变化中所起作用的研究。具体目标1a。确定初始培养渗透压(慢性2小时暴露)对细胞骨架组织(包括微丝(MFS)和微管(MTS)的空间分布和稳态聚合物水平，肌动蛋白和微管蛋白的总蛋白含量，以及稳定的微管的组织和含量)的影响。具体目标1b。在不同渗透压的培养液中预培养2小时后，测量实时渗透负荷对中层软骨细胞(MZCs)细胞内钙离子浓度([Ca~(2+)]i)(峰值、反应细胞百分率)和细胞大小变化(平衡体积、变化率和材料特性)对渗透负荷10min的反应(可变幅度和频率)。具体目标1c。在用调节细胞骨架组织或钙动员的药理学试剂预处理的细胞上，重复特定目标1b的选定实时[钙]i研究。特异靶2。对浅层(SZC)和深层(DZC)软骨细胞重复特异靶1。特定目的3.在不同渗透压的培养液中预孵育2小时后，我们将软骨细胞置于2小时的渗透负荷(不同的幅度和频率)，然后我们提出：特定的目标3a。对于SZC、MZC和DZC，在存在和不存在干扰钙动员的药物的情况下，量化细胞骨架组织。具体目标3b。在这段时间后测量聚集素基因的表达。具体目标3c。使用调节细胞骨架组织或钙动员的药物的策略，确定在渗透负荷下aggrecan基因的表达是否依赖于[Ca+]i以及肌动蛋白和微管的细胞骨架组织。

DESCRIPTION (provided by applicant): The broad goal of this application is to gain an understanding of the contribution of osmotic loading in mediating chondrocyte behavior in culture studies of chondrocyte mechanotransduction as well as in physiologic joint loading. While this basic science study is intended to provide new information regarding chondrocyte mechanotransduction, we anticipate that some findings may shed light on the role that changes to the osmotic environment play in osteoarthritis (OA). In this A1 revised proposal, based on our preliminary data, we hypothesize that the cytoskeleton plays a critical role in mediating the osmotic loading response of articular chondrocytes. In this scenario, the cytoskeleton may be responsive to osmotic loading-induced Ca2+ changes, or the osmotic loading-induced Ca2+ changes may be responsive to the initial organization of the cytoskeleton, or both, may be operative in a feedback loop. Reported zonal differences between the cytoskeletal protein content of chondrocytes, as well as cytoskeletal differences between normal and OA chondrocytes, provide added motivation for our hypothesis. To test this hypothesis, we propose several specific aims to initiate our studies of the role that the cytoskeleton plays in mediating changes to aggrecan gene expression. Specific Aim 1a. Determine the effect of initial culture osmolarity (chronic 2 hour exposure) on cytoskeletal organization (including spatial distribution and steady-state polymer levels of microfilaments (MFs) and microtubules (MTs), total protein content of actin and tubulin, and organization and content of stable microtubules) in the cell. Specific Aim 1b. Measure the response induced by real-time osmotic loading in intracellular calcium ([Ca2+]i) (peak magnitude, percentage of responding cells) and cell size change (equilibrium volume, rate of change and material properties) of middle zone chondrocytes (MZCs) to 10 min of osmotic loading (variable magnitude and frequency) following a 2 hr pretreatment in medium of varying osmolarities. Specific Aim 1c. Repeat selected real-time [Ca2+]i studies of Specific Aim 1b on cells pretreated with pharmacological agents that modulate cytoskeletal organization or calcium mobilization. Specific Aim 2. Repeat Specific Aim 1 on superficial (SZC) and deep zone (DZC) chondrocytes. Specific Aim 3. Following a 2 hour preincubation in culture medium of varying osmolarities, we will subject chondrocytes to 2 hours of osmotic loading (variable magnitude and frequency) and we then propose: Specific Aim 3a. For SZC, MZC, and DZCs, quantify cytoskeletal organization in the presence and absence of pharmacological agents that disrupt calcium mobilization. Specific Aim 3b. Measure aggrecan gene expression after this period. Specific Aim 3c. Determine if aggrecan gene expression in response to osmotic loading is dependent on [Ca2+]i and cytoskeletal organization of actin and microtubules using a strategy of pharmacological agents that modulate cytoskeletal organization or calcium mobilization.