PROJECT SUMMARY Mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as morphogenesis, wound healing, and organization of bioengineered tissues. Our research focuses on how these interactions regulate corneal keratocyte behavior through development of culture models that mimic the 3-D tissue environment, and use of multi-dimensional imaging approaches in vitro, in situ, and in vivo. In the previous funding period, we used these tools to perform a comprehensive assessment of the differentiation and patterning of corneal keratocytes following photorefractive keratectomy (PRK) surgery in the rabbit. These studies provided novel insights into the nature of the transition between native stromal and fibrotic tissue, and how cells use the collagen lamellae as a template for tissue remodeling and regeneration. Subsequent studies combining superficial phototherapeutic keratectomy (PTK) and UV cross-linking (CXL) showed that CXL induces a disruption in normal cell patterning within the stroma, and appears to block the development of fibrosis on top of the stroma. These studies highlight the profound impact that changes in corneal structure and stiffness can have on overall corneal wound healing responses. However, the large size and overall symmetry of standard CXL procedures extends the normal time course of healing after PTK, and limits the scope of biomechanical insights that can be made. Using our in vitro 3D culture models, we also performed mechanistic studies on how cell spreading and collective migration are regulated in fibrin matrices, and identified key roles for fibronectin, 51 integrin, and local cell-induced matrix reorganization in this process. We also demonstrated for the first time, that inhibition of vimentin filament organization alters corneal fibroblast spreading, morphology and motility in 3-D matrices. Vimentin has been associated with myofibroblast transformation of corneal keratocytes in vivo, and recent studies in other systems suggest it can play a central role in regulating key aspects of cell mechanical behavior, including mechanosensing, polarization, and directional migration. Based on these and other published and pilot data, we now propose to: 1) Investigate the effects of tissue stiffness and anisotropy on corneal wound healing following PTK, by applying UV cross-linking in specific patterns determined from finite element modeling simulations, 2) Establish the time course of cell differentiation and cell/matrix patterning during the development and resolution (remodeling) of fibrosis following full thickness incisional injury, and determine the effects of modulating the wound boundary conditions and mechanical environment on these processes, and 3) Apply our established 3-D culture models and engineered 2-D substrates to investigate the role of vimentin in regulating corneal fibroblast differentiation, patterning and mechanical behavior. Given the general importance of ce...