Comparison of the regenerative potential of different functionalized gelatin-based hydrogels as fillers of rabbit corneal wounds

Abstract

Objectives: Persistent epithelial defects (PEDs) and chronic corneal ulcers are lesions resistant to treatment for over 2 weeks, risking inadequate healing, reduced sensitivity, and corneal lysis or perforation. This study evaluates the regenerative potential of functionalized gelatin-based hydrogels for treating rabbit corneal wounds as a non-surgical alternative. Methods: Thirty female New Zealand white rabbits underwent anterior stromal keratectomy and were assigned to five groups: control (0.2% HA artificial tears) and four hydrogel treatment groups. Hydrogels included non-functionalized gelatin-RFP (H) and functionalized versions with infliximab (H-Ab), autologous serum (H-AS), and human amniotic membrane extracts (H-HAMe). Crosslinking was performed in situ with blue light. Corneas were evaluated at 7 and 21 days for re-epithelialization, fibrosis, and inflammation using histology, qPCR and immunohistochemistry, focusing on markers of proliferation (Ki67), differentiation (CK3), stemness (PAX6, p63, CD44), adhesion (integrin β4), and fibrosis (α-SMA). Results: All treatments supported re-epithelialization by day 7 and restored barrier function (ZO-1), with H-AS achieving the fastest closure. Expression of the adhesion marker integrin β4 improved over time across all groups. Hydrogel formulations promoted limbal activation (PAX6, CD44), with H-AS and H-HAMe showing elevated p63 expression at day 7. All hydrogels reduced fibrosis (α-SMA), though extracellular matrix organization varied. H-Ab and H-HAMe reduced inflammation (IL-1β), while H-AS showed minimal irritability. Conclusion: Functionalized gelatin-RFP hydrogels promote re-epithelialization, reduce fibrosis and inflammation, and restore ocular integrity, offering a promising solution for corneal wound repair.