Biocompatibility of photoactivated collagen-riboflavin hydrogels for corneal regeneration

Abstract

Purpose : Corneal trauma and infections have high morbidity requiring urgent intervention and close follow-up. Novel techniques are needed to improve patient outcomes, decrease surgical burden, and prevent blindness. Here we study the in vitro and ex vivo biocompatibility and regenerative capacity of a collagen-riboflavin hydrogel crosslinked by UV light.

Methods : We have developed a hydrogel consisting of collagen type I and 0.25 mM riboflavin-5-phosphate photo-crosslinked by UV (365nm) light. Epithelial and stromal integration was assessed by admixing cultured stromal cells within the hydrogel pre-cursor solution, exposing the complex to UV light, and seeding epithelial cells on the formed hydrogels. Confocal microscopy was used to visualize adherence and identify immunohistochemical markers associated with normal morphology. Epithelial wound healing over the hydrogel was also tested in ex vivo rabbit corneas. A 5mm anterior keratectomy was performed and a photo-crosslinked collagen-riboflavin hydrogel was placed in the defect. The organ culture specimen was then incubated for 72 hours and analyzed with confocal microscopy. All procedures were conducted on physically crosslinked collagen hydrogels without photo-crosslinking.

Results : Microscopy of the in vitro tissue showed adherence of epithelial cells to the hydrogel-stromal interface. Immunohistochemical staining was positive for IL-1β and TNF-α in epithelial and stromal layers. The epithelial cells also expressed Ki67, CD44 receptors, ZO-1, and CK12. In the organ culture model, the hydrogel was visualized in the keratectomy wound with overlying re-epithelialization after 72 hours. The corneal epithelium demonstrated a multi-layered epithelium as well as ZO-1 staining. The photo-crosslinked hydrogel showed faster epithelial growth in the in vitro model and both faster and morphologically superior re-epithelialization in the organ culture model compared to the physically crosslinked hydrogel.

Conclusions : Collagen hydrogels photo-crosslinked using riboflavin and UV light support epithelial cell growth in vitro and in ex vivo organ culture. Upon immunohistochemical analysis, corneal epithelial cells in the in vitro model expressed markers of epithelial phenotype and, in organ culture, exhibited multi-layered morphology. The photo-crosslinked hydrogel was superior in its ability to support epithelial cell growth compared to the physically crosslinked hydrogel.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.