Chondrocyte Mechanotransduction Using Microfluidics

描述（由申请人提供）：本申请的广泛目标是了解在软骨细胞机械转导的培养研究以及生理关节负荷中渗透负荷在介导软骨细胞行为中的作用。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，一些发现可能会揭示渗透环境变化在骨关节炎（OA）中的作用。在这个A1修订提案中，基于我们的初步数据，我们假设细胞骨架在介导关节软骨细胞的渗透负荷反应中起着关键作用。在这种情况下，细胞骨架可以响应于渗透负荷诱导的Ca2+变化，或者渗透负荷诱导的Ca2+变化可以响应于细胞骨架的初始组织，或者两者都可以在反馈回路中起作用。报道的软骨细胞的细胞骨架蛋白含量之间的区域差异，以及正常和OA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，启动我们的研究的作用，细胞骨架介导的变化聚集蛋白聚糖基因的表达。具体目标1a。确定初始培养物渗透压（慢性暴露2小时）对细胞骨架组织（包括微丝（MF）和微管（MT）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基中预处理2小时后，测量由中间区软骨细胞（MZC）的细胞内钙（[Ca2+]i）（峰值大小，响应细胞的百分比）和细胞大小变化（平衡体积，变化速率和材料性质）对10分钟渗透负荷（可变大小和频率）的实时渗透负荷诱导的响应。具体目标1C。在用调节细胞骨架组织或钙动员的药物预处理的细胞上重复特定目的1b的选定实时[Ca2+]i研究。具体目标2。在浅表（SZC）和深层（DZC）软骨细胞上重复特定目标1。具体目标3。在不同渗透压的培养基中预孵育2小时后，我们将对软骨细胞进行2小时的渗透负荷（可变幅度和频率），然后我们提出：特定目标3a。对于SZC、MZC和DZC，在存在和不存在破坏钙动员的药理学试剂的情况下定量细胞骨架组织。具体目标3b.在此期间后测量聚集蛋白聚糖基因表达。具体目标3c。确定聚集蛋白聚糖基因表达对渗透负荷的反应是否依赖于[Ca2+]i和肌动蛋白和微管的细胞骨架组织，使用调节细胞骨架组织或钙动员的药理学试剂的策略。

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.