Quantitation of rabbit corneal epithelial cell outgrowth on polymeric substrates in vitro

The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. To study the effects of polymer surface properties on corneal epithelial cell outgrowth, a quantitative in vitro cell outgrowth assay was done. Polymers with systematic variations in hydroxyl content were used as outgrowth substrates. These polymers were characterized by electron spectroscopy for chemical analysis for elemental surface composition and by captive air-bubble contact angle for surface wettability. Circular corneal buttons were punched from excised rabbit corneas, placed onto these substrates, and incubated in a hormonally enriched culture medium. Outgrowth of the epithelial cells was allowed to proceed onto the substrates for 4 days. The outgrowth areas were measured, and an outgrowth index was calculated for each substrate by comparing it with tissue culture polystyrene substrate. The highest outgrowth generally occurred on substrates with intermediate wettabilities (captive air-bubble contact angles of approximately 45-75 degree); it was less on substrates of lower or higher wettabilities. Protein coatings of albumin, immunoglobulin G (IgG), fibronectin, and culture medium were found to lower the wettabilities of native substrates. Albumin and IgG precoating were shown to reduce epithelial cell outgrowth; fibronectin precoating was shown to improve outgrowth on most substrates. These results suggest that epithelial cell outgrowth is influenced by both substrate and protein interactions.