Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (alpha-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and alpha-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

Automated summary

During corneal wound healing, corneal keratocytes respond to multiple cues simultaneously, integrating both physical and soluble signals. The presence of adsorbed fibronectin initially activates keratocytes, resulting in changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (alpha-SMA). However, when simultaneous exposure to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB) occurs, keratocytes elongate and show reduced stress fibers and alpha-SMA expression. Furthermore, when plated on aligned collagen fibrils, keratocytes elongate in the direction of the fibrils in the presence of PDGF-BB.