Assessment of Corneal Fibroblast Biomechanical Behavior

角膜成纤维细胞生物力学行为的评估

• 批准号: 7171790
• 负责人: W MATTHEW PETROLL
• 金额: $ 34.08万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171790
• 关键词: 3-Dimensional; ATP phosphohydrolase; Actinin; Adhesives; Behavior; Biomechanics; Biomedical Engineering; Biophysical Process; Cell Density; Cell physiology; Cells; Collagen; Collagen Fibril; Confocal Microscopy; Cornea; Cytoskeletal Modeling; Data; Development; Developmental Biology; Elements; Equilibrium; Exertion; Experimental Models; Extracellular Matrix; Fibrillar Collagen; Fibroblasts; Focal Adhesions; Funding; Generations; Goals; Growth Factor; Image; Image Analysis; In Vitro; Lasers; Lead; Measures; Mechanical Stress; Mechanics; Mediating; Modeling; Molecular; Monitor; Morphogenesis; Myosin Light Chain Kinase; Myosin Light Chains; Myosin Type II; Pathway interactions; Pattern; Phosphorylation; Platelet-Derived Growth Factor; Play; Process; Proteins; Regulation; Relaxation; Research; Rho-associated kinase; Role; Stress; Stretching; System; Testing; Time; Tissue Engineering; Tissues; Wound Healing; ZYX gene; base; cell behavior; digital imaging; driving force; in vivo; innovation; insight; lysophosphatidic acid; migration; myosin phosphatase; response; rho; time use

DESCRIPTION: Cell-matrix mechanical interactions drive fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. The overall goal of this research is to determine the underlying cellular and molecular mechanisms that regulate these critical biophysical processes in corneal fibroblasts, which should ultimately lead to more effective approaches to directing their biomechanical behavior in vivo and in vitro. In the first funding period, we developed a new experimental model for directly investigating cell-matrix mechanical interactions inside 3-D fibrillar collagen matrices. Data obtained using this innovative approach has provided new insights into potential mechanisms regulating sub-cellular force generation, matrix reorganization, as well as the modulation of cell behavior by mechanical stress which together lead to the following Hypotheses: 1) The balance between Rac and RhoA activity plays a central role in regulating the mechanical behavior of corneal fibroblasts inside 3-D matrices at the subcellular level. These effects are mediated by dynamic changes in cytoskeletal and focal adhesion organization, as well as differential regulation of myosin light chain (MLC) phosphorylation, 2) Corneal fibroblasts actively respond to increases or decreases in local matrix stress (including that produced by neighboring cells); these responses are mediated by compensatory and reciprocal changes in Rho and Rac activation, and 3) The pattern and amount of collagen matrix reorganization can be modulated by altering the balance between Rho and Rac activity, and is enhanced by cell-cell mechanical interactions. To test these hypotheses, we propose the following Specific Aims: 1) Determine the role of Rho and Rac in regulating cytoskeletal organization, mechanical behavior, and sub-cellular force generation by corneal fibroblasts inside 3-D matrices using our time-lapse imaging system, 2) Measure the dynamic mechanical response of corneal fibroblasts to changes in ECM stress; and, 3) Directly assess the process of cell-induced 3-D collagen matrix reorganization (alignment of collagen fibrils), and the roles of Rho and Rac in regulating this process. These studies will be the first to assess the roles of Rho and Rac on the subcellular pattern of force generation and ECM reorganization within 3-D collagen matrices. Overall, this research should provide unique insights into the mechanisms controlling corneal fibroblast migration, contraction, and matrix reorganization, critical processes in the fields of developmental biology, wound healing and tissue engineering.

描述：细胞-基质机械相互作用驱动基本过程，例如发育形态发生、伤口愈合和生物工程组织的组织。这项研究的总体目标是确定调节角膜成纤维细胞中这些关键生物物理过程的潜在细胞和分子机制，这最终应导致更有效的方法来指导其体内和体外生物力学行为。在第一个资助期间，我们开发了一种新的实验模型，用于直接研究 3D 纤维状胶原基质内的细胞-基质机械相互作用。 使用这种创新方法获得的数据为调节亚细胞力产生、基质重组以及机械应力调节细胞行为的潜在机制提供了新的见解，这些机制共同得出以下假设：1）Rac 和 RhoA 之间的平衡活性在亚细胞水平调节 3D 基质内角膜成纤维细胞的机械行为中发挥着核心作用。这些效应是由细胞骨架和粘着斑组织的动态变化以及肌球蛋白轻链 (MLC) 磷酸化的差异调节介导的，2) 角膜成纤维细胞主动响应局部基质应力（包括邻近细胞产生的应力）的增加或减少;这些反应是由 Rho 和 Rac 激活的补偿性和相互变化介导的，3) 胶原基质重组的模式和数量可以通过改变 Rho 和 Rac 活性之间的平衡来调节，并通过细胞与细胞的机械相互作用得到增强。为了检验这些假设，我们提出以下具体目标：1) 使用我们的延时成像系统确定 Rho 和 Rac 在调节 3D 矩阵内角膜成纤维细胞的细胞骨架组织、机械行为和亚细胞力产生中的作用, 2) 测量角膜成纤维细胞对ECM应力变化的动态机械反应； 3) 直接评估细胞诱导的 3-D 胶原基质重组（胶原原纤维排列）的过程，以及 Rho 和 Rac 在调节该过程中的作用。这些研究将首次评估 Rho 和 Rac 对 3-D 胶原基质内力生成和 ECM 重组的亚细胞模式的作用。总的来说，这项研究应该为控制角膜成纤维细胞迁移、收缩和基质重组的机制、发育生物学、伤口愈合和组织工程领域的关键过程提供独特的见解。