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）：本申请的广泛目标是了解渗透载荷在介导软骨细胞行为中的贡献中，在软骨细胞机械传导以及生理关节负荷中的培养研究中。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出改变渗透环境在骨关节炎（OA）中的作用。在此A1修订的建议中，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载荷反应中起着至关重要的作用。在这种情况下，细胞骨架可能对渗透载荷诱导的Ca2+变化有反应，或者渗透载荷引起的Ca2+变化可能对细胞骨架的初始组织有反应，或两者兼有反馈循环。报道的软骨细胞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。为了检验这一假设，我们提出了一些具体的目的，以启动我们对细胞骨架在介导对脂质基因表达的变化中发挥作用的作用的研究。特定目标1a。确定初始培养渗透压（慢性2小时暴露）对细胞骨架组织的影响（包括微丝的空间分布和稳态聚合物水平（MFS）和微管（MTS）（MTS），肌动蛋白和微管的总蛋白质含量，以及稳定微管的组织和含量）。特定目标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 v​​olume, 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.特定目标1C。重复对特定目标1B进行的重复实时[Ca2+] I研究，以调节细胞骨架组织或钙动员的药理剂预处理细胞。具体目标2。重复特定目标1对表面（SZC）和深区（DZC）软骨细胞。特定的目标3。在不同渗透压的培养基中进行2小时预孵育后，我们将使软骨细胞接到2小时的渗透载荷（可变幅度和频率），然后我们提出：特定的目标3A。对于SZC，MZC和DZC，在存在和不存在破坏钙动员的药理学剂的情况下量化细胞骨架组织。特定目标3B。在此期间测量Aggrecan基因表达。特定目标3C。确定使用药理学剂的策略调节细胞骨架组织或钙动员的药理学剂的策略，确定响应渗透载荷的响应于渗透载荷的响应于渗透载荷和肌动蛋白的细胞骨架组织。