Sequential Coupling of Epidermal Growth Factor and Collagen to Polymers to Promote Corneal Epithelialization in vitro

Abstract

Purpose: : A method for sequentially coupling epidermal growth factor (EGF) and collagen type I to polymeric surfaces was explored for its potential to enhance the epithelialization of keratoprostheses.

Methods: : Poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) interpenetrating polymer network hydrogels and tissue culture polystyrene (TCPS) were explored as substrates for sequentially coupled EGF and collagen. Sequential coupling on PEG/PAA surfaces was carried out by photochemically coupling collagen type I to PEG/PAA using azide-active-ester crosslinking, followed by a second azide-active-ester crosslinking step to bind EGF to the collagen layer. Collagen-coated TCPS surfaces were used as positive controls by first depositing a collagen layer and then crosslinking EGF to this layer by azide-active-ester photochemistry. Primary corneal epithelial cells isolated from rabbit corneas were then grown on these surfaces in the absence of any EGF in the culture media. Cells were also grown on TCPS surfaces with EGF non-specifically adsorbed (but not covalently linked) to an underlying collagen coating as well as in the presence of solubilized EGF over a collagen coating. The cells were grown in culture and photographed in three high power fields for 1 week. Immunofluorescent staining of the marker for epithelial differentiation (cytokeratin 3/12) was visualized by fluorescent microscopy.

Results: : In the complete absence of EGF, cell growth remained sparse over 3 days on collagen-coated TCPS and PEG/PAA. In contrast, in the presence of both surface-coupled and solubilized EGF, the cells grew significantly better and more rapidly over 3 days on collagen-coated polymers. Simple adsorption of EGF to collagen was found to be insufficient to promote improved cell growth relative to positive controls. Both solubilized and surface-coupled EGF in combination with collagen coatings yielded epithelial cells that stained strongly for cytokeratin 3/12, indicating epithelial differentiation of the cultured cells. In the absence of EGF, epithelial cells exhibited minimal staining of cytokeratin 3/12.

Conclusions: : The results from these studies show that sequential coupling of EGF and collagen type I stimulates synergistic cellular growth with normal cellular differentiation. This method may be used to surface-modify a keratoprosthesis to create a biomimetic surface on which corneal epithelial and stromal cells can adhere and grow.

Keywords: cornea: epithelium • keratoprostheses • growth factors/growth factor receptors