Fabrication and Surgical Application of Enriched Photo-crosslinked Gelatin-Riboflavin Hydrogels for Corneal Wound...

This article presents a step‑by‑step protocol to fabricate, supplement, and surgically apply an in situ blue‑light-crosslinkable gelatin hydrogel for corneal stromal wound repair in rabbits. The hydrogel precursor comprises gelatin at 5% (w/v) and riboflavin phosphate at 0.01% (w/v), prepared under sterile and light‑protected conditions, sterile‑filtered, and warmed before use to achieve injectable viscosity. Optional incorporation of human amniotic membrane extract or rabbit autologous serum is detailed. On‑demand gelation is triggered on the ocular surface using blue light at λ = 420-480 nm for a total of 2 min. The in vivo method includes a reproducible anterior stromal keratectomy (trephine diameter 6.5 mm; depth approximately 187 µm), followed by hydrogel filling, light activation, and a partial lateral tarsorrhaphy to stabilize the treatment, favor wound healing, and standardize postoperative care. Representative in vitro outcomes evidence gel‑like viscoelastic behavior with shear‑thinning and rapid recovery, and high optical transmittance (> 90%) beyond 500 nm, indicating suitability for corneal use. In vivo, hydrogel‑based treatments support progressive epithelial closure, good ocular tolerance (low Draize scores at 3 and 7 days), and time‑dependent loss of visible hydrogel consistent with biodegradation and tissue replacement. Critical steps and pause points are highlighted to ensure reproducibility, including temperature control during dissolution and filtration, protection from light, and lamp positioning during crosslinking. The protocol enables suture‑free, precisely placed, light‑activated hydrogel stabilization, compatible with the addition of multiple bioactive supplements. It provides a practical platform for preclinical evaluation of next‑generation ophthalmic biomaterials and facilitates translation of photo‑crosslinkable hydrogel therapies for corneal wound repair.