Epidermal Growth Factor–Modified Polydimethylsiloxane for Keratoprostheses

Abstract

Abstract: : Purpose: Lack of corneal epithelial cell coverage over the anterior surface of a keratoprosthesis can lead to epithelial downgrowth and device extrusion. In this work, the effect of immobilized epidermal growth factor (EGF), a mitogen for epithelial cells, on cell growth over polydimethylsiloxane (PDMS) substrates is assessed. Methods: EGF was bound to PDMS substrates via polyethylene glycol (PEG) tethers by two methods. 1) Solution–first: EGF was first reacted with a bifunctional PEG in solution, then tethered to allyamine–modified PDMS. 2) Surface–first: PDMS was first modified with bifunctional PEG, and EGF was then reacted with the surface–bound PEG. The amount of bound EGF was varied by altering the EGF solution concentration. Human corneal epithelial cells were seeded onto EGF–modified and control surfaces and cultured in keratinocyte serum free medium with and without soluble EGF. Immunofluorescence was used to assess production of proteins including fibronectin, laminin and cytokeratin. Results: Modification by the solution–first method resulted in 46 to 91 ng/cm² of bound EGF. In this range, cells grew to confluence on the EGF–modified surfaces within two to three days of culture, even without soluble EGF in the medium, whereas cell coverage on unmodified PDMS surfaces remained incomplete at four days. Control surfaces with denatured EGF resulted in lower cell coverage at one and two days compared to native EGF, indicating that EGF is active at early time points in stimulating cell growth. Allylamine modification alone also improved cell growth over four days vs. unmodified PDMS. Positive staining for cytokeratin, fibronectin and laminin indicated the cells are differentiated and produce matrix proteins on the EGF–modified surfaces at significantly higher levels vs. control surfaces. Modification by the surface–first method resulted in 5 to 190 ng/cm² of bound EGF. Cell growth was biphasic, with highest coverage at 45 to 62 ng/cm² of EGF. Surface modification with PEG alone completely inhibited cell growth. Conclusions: Tethering of EGF to PDMS significantly improves growth of corneal epithelial cells. Effects depend on EGF concentration and underlying substrate modification.

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