Novel approaches to anchoring therapeutic factors to corneal stroma to promote wound healing.

Abstract

Purpose : We hypothesized that corneal wound healing can be enhanced by anchoring growth factors and stem cells to the wound bed. We have sought to test this hypothesis in three ways: (1) by binding growth factors directly to the stromal wound bed, (2) through an injectable, in situ-forming, growth-factor-loaded collagen gel, and (3) by creating a living reservoir of growth factors through the encapsulation of human mesenchymal stem cells (hMSCs) within a carrier matrix on the ocular surface.

Methods : We have developed an array of biocompatible chemical techniques that enable (1) surface-coupling of growth factors, (2) crosslinking of a collagen gel loaded with growth factors, and (3) encapsulation of hMSCs within collagen gels, all directly onto corneal stromal collagen. These techniques include copper-free click chemistry through strain-promoted azide-alkyne cycloaddition (SPAAC), photoactivation of riboflavin using visible (blue) light, and in situ forming hydrogels through multi-arm succinimidyl chemistry.

Results : Growth-factor-loaded collagen gels made by SPAAC, visible light photoactivation of riboflavin, and succinimidyl chemistry all facilitated control over EGF release and degradability by collagenase. Growth factor binding by SPAAC and visible light photochemistry was demonstrated on both collagen-coated surfaces and debrided corneal stroma by an array of surface analytical techniques, including surface plasmon resonance, ELISA, and western blot. In vitro proliferation assays demonstrated enhanced corneal epithelial cell growth upon exposure to EGF-coupled surfaces. Pilot animal studies have shown that direct EGF binding to debrided corneal stroma using both SPAAC and visible light photochemistry were well-tolerated and accelerated initial wound healing. Collagen gels formed by all three chemical techniques maintained 90% viability of encapsulated keratocytes and hMSCs 72 hours after reaction and could be adhered to stromal collagen ex vivo.

Conclusions : Our results are consistent with our hypothesis that wound healing can be enhanced through the chemical anchoring of growth factors and stem cells. While further work is merited to elucidate the optimal conditions for each crosslinking strategy, copper-free click chemistry, visible light photochemistry, and succinimidyl chemistry all represent promising, biocompatible techniques for anchoringthese therapeutic factors to wounded tissue.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.