Fabrication and Surgical Application of Enriched Photo-crosslinked Gelatin–Riboflavin Hydrogels for Corneal Wound Repair in Rabbits

Summary

A reproducible protocol is presented for the preparation, enrichment, and application of an in situ blue‑light–crosslinkable gelatin–riboflavin hydrogel for corneal stromal wound repair in rabbits, including procedural steps for surgical handling, partial tarsorrhaphy, and postoperative care.

Abstract

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.

Introduction

The cornea is a transparent, avascular structure that accounts for approximately two-thirds of the eye’s refractive power, making its structural and functional integrity essential for vision¹. Due to its direct exposure to the external environment, the cornea is highly susceptible to trauma, infection, chemical injury, and postsurgical complications^2,3. Failure to rapidly and effectively restore epithelial integrity can lead to persistent epithelial defects (PEDs) and chronic corneal ulcers, ultimately resulting in stromal melting, perforation, scarring, and irreversible visual lo....

Protocol

Human placentas were obtained from donors with informed consent, in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of the University Hospital of Cruces (approval number CEIC E15/03). All animal experiments were approved by the Animal Research Ethics Committee of the University of the Basque Country (UPV/EHU) (approval number CEEA M20/2023/038) and complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were conducted at the UPV/EHU animal facility (SGIker), maintaining sterile technique throughout (Class II biosafety cabinet when handling biologicals).

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Results

Hydrogel synthesis and characterization
The hydrogel was synthesized and evaluated in vitro to determine its rheological and optical properties before and after photo-crosslinking. Shear rheology was used to characterize viscoelasticity, yielding, and flow behavior. Frequency sweeps showed G′ remained higher than G″ across all tested frequencies, indicating solid-like behavior (Figure 5A). Amplitude sweeps at 10 rad·s⁻¹ revealed a broad linear viscoel.......

Discussion

The present protocol provides a reproducible workflow for the preparation, loading, and application of a photo-crosslinkable gelatin–riboflavin hydrogel for corneal wound healing. Several in vitro steps are critical for achieving consistent gel quality and warrant emphasis because they are the most frequent sources of variability. First, maintaining the precursor solution at 80 °C during stirring ensures complete dissolution of gelatin and riboflavin phosphate, preventing particulate aggregates that c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amphotericin B	Gibco (Thermo Fisher Scientific)	15290018	Final concentration: 1.25 μg/mL
Autologous serum, rabbit (AS)	Prepared in-house	-	Prepared from whole blood; filtered through 0.22 μm PES
Blue LED curing lamp	Woodpecker Medical Instrument Co	LED.C model	Wavelength: λ=420–480 nm; Distance: 1–2 cm; Light output: 850-1000 mW/cm2
Buprenorphine (Buprecare)	Ecuphar	578950	Dose: 0.05 mg/kg, subcutaneous injection every 12 h for 72 h
Camera	Panasonic	LUMIX TZ35	Camera equipped with Leica optics
Cimarec Digital Stirring Hotplate	Thermo Fisher Scientific	SP131320-33	220-240 V, Max temperature: 540 °C
Crescent microsurgical blade	BVI Medical	373807	-
DMEM (Dulbecco’s Modified Eagle Medium)	Gibco (Thermo Fisher Scientific)	11966025	-
Eppendorf tubes (0.2 mL)	Merck KGaA	30124707	Kept cold for HAMe prep; aliquoting
Eppendorf tubes (5 mL)	Merck KGaA	30119606	Kept cold for HAMe prep; aliquoting
Fluorescein solution	Novartis	Colorcusi Fluotest	2% Fluorescein solution, 5 µl eye drops
Gibco Neomycin	Merck KGaA	N1142	Final concentration: 50 μg/mL
Gelatin powder	Sigma Aldrich	G1890	Dissolved at 80 °C; pH adjusted to 7.0
Hyaluronic acid artificial tears (0.2%)	Chem.-pharm. Fabrik GmbH	Hylo-Tear	-
ImageJ image processing software	National Institute of Mental Health	-	Developed by Wayne Rasband at the Research Services Branch
Ketamine (Ketolar)	Pfizer	631028.1	Dose: 35 mg/kg intramuscular injection
Lidocaine	Vet One	NDC 13985-222-04	Topical or subcutaneous injection
Penicillin–Streptomycin	Gibco (Thermo Fisher Scientific)	15140-122	Final concentration: 50 μg/mL
PES syringe filters, 0.22 μm0.22μm (Millex)	Merck KGaA	SLGP033RS	-
Phosphate-buffered saline (PBS)	Sigma Aldrich	P4417-100TAB	-
Protease inhibitor cocktail	Sigma Aldrich	P8340	Added at 5 μL per mg tissue
Povidone-iodine solution	Meda pharma	7167204	Preoperative disinfection
Riboflavin phosphate (RFP)	Sigma Aldrich	R0630000	Concentrations: 0.01% w/v (final); 0.02% w/v (stock)
Sonicator	Bandelein electronic	Sonoplus GM mini20	-
Sonicator probe	Bandelein electronic	MS 1.5	1.5 mm dimater probe, 63 mm length.
Tarsorrhaphy sutures (non-absorbable)	Alcon	8065308001	1–2 sutures as needed
Tobramycin ophthalmic solution (3 mg/mL)	Novartis	Tobrex	Dosing: twice daily until closure
Hessburg-Barron trephine, 6.5 mm diameter	Jedmed	21-8265	-
Vacutainer blood collection tubes with polymer gel separator	BD (Becton, Dickinson and Company)	367704	-
Xylazine solution	Laboratorios Calier SA	Xylagesic	Dose: 5 mg/kg intramuscular injection