Chondrocyte Mechanotransduction Using Microfluidics

使用微流体进行软骨细胞机械转导

• 批准号: 7472336
• 负责人: Clark T. Hung
• 金额: $ 30.33万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-06 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7472336
• 关键词: Actins; Basic Science; Behavior; Biochemical; Calcium; Cartilage; Cell Size; Cell physiology; Cells; Charge; Chondrocytes; Chronic; Culture Media; Cultured Cells; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; Data; Degenerative polyarthritis; Depth; Doctor of Philosophy; Environment; Equilibrium; Extracellular Matrix; Feedback; Frequencies; Gene Expression; Goals; Hour; Hydrostatic Pressure; In Situ; Joints; Laboratories; Light; Maintenance; Measures; Mechanics; Mediating; Microfilaments; Microfluidics; Microtubules; Motivation; Osmolar Concentration; Personal Satisfaction; Physical environment; Physiological; Play; Polymers; Property; Proteins; Proteoglycan; Rate; Reporting; Research Personnel; Role; Signal Transduction; Spatial Distribution; Stimulus; System; Testing; Time; Tissues; Tubulin; aggrecan; base; density; extracellular; insight; novel; programs; release of sequestered calcium ion into cytoplasm; response

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.

描述（由申请人提供）：本申请的主要目标是了解渗透负荷在软骨细胞机械转导的培养研究以及生理关节负荷中介导软骨细胞行为中的贡献。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会揭示渗透环境的变化在骨关节炎 (OA) 中所起的作用。在这个 A1 修订提案中，根据我们的初步数据，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发挥着关键作用。在这种情况下，细胞骨架可能对渗透负荷诱导的 Ca2+ 变化做出响应，或者渗透负荷诱导的 Ca2+ 变化可能对细胞骨架的初始组织做出响应，或者两者都可以在反馈回路中起作用。所报告的软骨细胞细胞骨架蛋白含量之间的区域差异，以及正常软骨细胞和 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力。为了检验这一假设，我们提出了几个具体目标来启动我们对细胞骨架在介导聚集蛋白聚糖基因表达变化中所起的作用的研究。具体目标 1a。确定初始培养物渗透压（长期暴露 2 小时）对细胞骨架组织的影响（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肌动蛋白和微管蛋白的组织和含量）细胞内稳定的微管）。具体目标 1b。测量中区软骨细胞 (MZC) 的细胞内钙 ([Ca2+]i) 实时渗透负荷（峰值大小、响应细胞百分比）和细胞大小变化（平衡体积、变化率和材料特性）引起的反应在不同渗透压的介质中预处理 2 小时后达到 10 分钟的渗透负荷（不同的大小和频率）。具体目标 1c。对用调节细胞骨架组织或钙动员的药物预处理的细胞重复特定目标 1b 的选定实时 [Ca2+]i 研究。具体目标 2. 对浅层 (SZC) 和深层 (DZC) 软骨细胞重复具体目标 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（不同大小和频率），然后我们提出：具体目标 3a。对于 SZC、MZC 和 DZC，在存在和不存在破坏钙动员的药物制剂的情况下量化细胞骨架组织。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。使用调节细胞骨架组织或钙动员的药物策略，确定响应渗透负荷的聚集蛋白聚糖基因表达是否依赖于 [Ca2+]i 以及肌动蛋白和微管的细胞骨架组织。