Seeing clearly: Using protein nanofibers to promote orderly corneal wound healing

Abstract

The study evaluates the ability of electrospun protein nanofibers to display topog. and biochem. cues to support epithelial closure, foster fibroblast recruitment and mitigate myofibroblast phenotype. Gelatin electrospun nanofibers were used to present integrin binding sites and mech. cues. By control of the electrode geometry, we prep. nanofiber mats with three types of orientation: isotropic, radial, and uniaxial. Rates of epithelial and fibroblast cell migration in vitro, measured using a mock wound healing assay, showed a result for epithelial cells. While fibroblasts (and a variety of cells in the literature) migrate rapidly on oriented nanofibers, corneal epithelial cells migrate faster on isotropic nanofibers than on aligned nanofibers or planar controls. Of particular importance, prodn. of smooth muscle actin (αSMA, green in image) by myofibroblasts (TGF-β transformed fibroblasts) was reduced in cells cultured on oriented nanofiber substrates. Nanofiber diam. over the range 100-220 nm does not measurably affect migration or αSMA expression. The mol. mechanism by which myofibroblasts respond to topog. cues was explored using siRNA transfected cells with knockdown of kinases assocd. with integrin mediated response (FAK and Raf-1) and transcriptional regulators assocd. with mechanotransduction (YAP and TAZ). We were surprised that the response does not involve integrin-mediated transduction; it involves YAP and TAZ (indicative of mechanostranduction). Preliminary in vivo expts. in mice and rabbits showed reepithelialization occurs as quickly over nanofiber scaffolds as it does over the native corneal stroma. The population of inflammatory cells was reduced in wound beds treated with nanofiber scaffolds relative to untreated controls. Oriented nanofiber scaffolds do not elicit an inflammatory response and have an anti-inflammatory effect. Oriented nanofiber substrates are well suited for corneal wound applications due to their transparency, non-cytotoxicity and ability to modulate the myofibroblast phenotype.