Assessment of Corneal Fibroblast Biomechanical Behavior

细胞-基质机械相互作用驱动基本过程，如发育形态发生、伤口愈合和生物工程组织的组织。本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，这将最终导致更有效的方法来指导其体内和体外的生物力学行为。在第一个资助期，我们开发了一个新的实验模型，用于直接研究三维纤维胶原基质内细胞-基质的机械相互作用。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对细胞行为的调节的潜在机制提供了新的见解，这些机制共同导致以下假设：1)Rac和RhoA活性之间的平衡在亚细胞水平上调节三维基质内角膜成纤维细胞的机械行为中起核心作用。这些作用是由细胞骨架和局灶黏附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)角膜成纤维细胞对局部基质应激（包括邻近细胞产生的应激）的增加或减少积极响应；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-细胞机械相互作用来增强。为了验证这些假设，我们提出了以下具体目标：1)利用我们的延时成像系统确定Rho和Rac在三维基质内调节角膜成纤维细胞的细胞骨架组织、力学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应力变化的动态力学响应；3)直接评估细胞诱导的三维胶原基质重组（胶原原纤维排列）过程，以及Rho和Rac在这一过程中的调节作用。这些研究将首次评估Rho和Rac在三维胶原基质中力产生和ECM重组的亚细胞模式中的作用。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提供独特的见解，这是发育生物学、伤口愈合和组织工程领域的关键过程。

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.