Effects of Topography and Extracellular Matrix Composition on Focal Adhesion Patterning in Human Corneal Fibroblasts

Corneal fibroblasts adhere to the extracellular matrix via integrin-containing focal adhesions (FAs). Although topographical cues are known to influence FA patterning in corneal fibroblasts, it is unclear how ECM composition, biophysical cues, and specific integrins modulate FA patterning in corneal fibroblasts. In this study, we cultured a human corneal fibroblast cell line (HTKs) on different ECM proteins and micropatterns of aligned collagen fibrils to determine the effects of ECM topography and composition on focal adhesion subcellular patterning. Using confocal imaging, we observed and quantified changes in FA and integrin patterning based on the underlying ECM type. More specifically, the presence of fibrillar topography as compared to monomeric collagen resulted in diminished FA number, area, and length. Using specific integrin blocking antibodies, we also demonstrate that HTKs use different integrin subunits to adhere to specific ECM coatings. For example, β₁ integrins are important in adhesion formation when corneal fibroblasts adhere to collagen, while α₅ integrin is important for the HTKs to adhere to fibronectin. Blocking of α₅ integrin did not completely inhibit cell spreading and FA patterning when cells adhered to fibronectin. These results suggest that there might be other fibronectin receptors that HTKs use in the absence of α₅ integrin. These results lay the foundation to understand the role of different integrin subunits in FA patterning. Through further experimentation using our developed platform, we envision that a better understanding of the integrins and their associated signaling could have implications for advanced in vitro and in vivo applications in cornea biology.