Assessment of Corneal Fibroblast Biomechanical Behavior

描述（由申请人提供）：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重塑。本研究的总体目标是确定在角膜成纤维细胞中调节这些关键过程的潜在生化和生物物理机制。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。利用这种创新方法获得的数据为亚细胞力的产生、基质重塑和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通过延伸假足产生局部牵引力诱导角膜成纤维细胞的扩散，而Rho通过沿着细胞体产生收缩力；这些影响

DESCRIPTION (provided by applicant): Cell-matrix mechanical interactions drive fundamental processes such as developmental morphogenesis, wound healing, and remodeling of bioengineered tissues. The overall goal of this research is to determine the underlying biochemical and biophysical mechanisms which regulate these critical processes in corneal fibroblasts. 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 of sub-cellular force generation, matrix remodeling, and the modulation of cell behavior by mechanical stress which together lead to the following Hypotheses: 1) Rac induces spreading of corneal fibroblasts via localized tractional force generation by extending pseudopodia, whereas Rho induces contractile force generati0n along the cell body; these effects are mediated by differences in sub-cellular regulation of myosin light chain phosphorylation, 2) corneal fibroblasts actively respond to increases or decreases in local matrix stress in order to maintain tensional homeostasis (constant tension); these responses are mediated by compensatory and reciprocal changes in Rho and Rac; and 3) the pattern and amount of permanent collagen matrix remodeling is maximal in stationary, contractile cells (high Rho and low Rac activity), and is enhanced by cell-cell mechanical communication at higher densities. 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) investigate the mechanical response of corneal fibroblasts to changes in ECM stress by inserting microneedles into the ECM next to isolated cells, in order to modulate matrix stiffness; and, 3) compare the pattern and amount of local collagen matrix remodeling by corneal fibroblasts at different cell densities and culture conditions. Accomplishing these Aims should provide a better understanding of how the fundamental processes of cell spreading, contraction and matrix remodeling are regulated. This is an important step towards our long-term objective of controlling the organization and mechanical behavior of corneal fibroblasts inside artificial matrices through the design of "smart" 3-D ECM scaffolds that incorporate both biochemical and mechanical cues.