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 loss^4,5. Conventional therapies—including lubricants, antibiotic prophylaxis, and autologous serum eye drops—often provide only partial benefit and do not adequately promote stromal repair in moderate-to-severe defects^6,7,8.

To address these limitations, biomaterial-based therapies have emerged as promising alternatives due to their ability to protect the wound, support epithelial repair, and restore stromal tissue^{9,10,11,12,13,14}. Hydrogels are particularly attractive because of their biocompatibility, optical transparency, tunable mechanical behavior, and ability to incorporate therapeutic agents. Both natural and synthetic hydrogels, including gelatin^15,16,17, collagen^18,19, chitosan^20,21, polyethylene glycol (PEG)^22,23 and hyaluronic acid^18,24 have demonstrated potential in promoting corneal wound healing. Among these, gelatin-based hydrogels are especially valuable due to their collagen-derived composition and intrinsic cell-adhesion motifs.

Photo-crosslinkable hydrogels offer an additional advantage by enabling on-demand solidification directly on the ocular surface, enabling precise placement and improved mechanical stability without sutures. Riboflavin-mediated crosslinking, originally developed for keratoconus treatment, has recently been adapted for hydrogel photopolymerization due to its excellent biocompatibility, ophthalmic safety, and ability to generate reactive oxygen species that induce crosslinking under visible or UV light^25,26. Blue light (≈445 nm) is particularly advantageous for corneal applications, enabling efficient crosslinking while avoiding UV-associated risks^27,28,29.

This article presents the fabrication, enrichment, and surgical application of a photo-crosslinkable hydrogel composed of 5% (w/v) gelatin and 0.01% (w/v) riboflavin phosphate (RFP). A detailed protocol is provided for in vitro hydrogel preparation, including optional incorporation of human amniotic membrane extract (HAMe) or autologous serum (AS), as well as in vivo application in a rabbit stromal keratectomy model. HAMe is known to exert anti-inflammatory, anti-fibrotic, and pro-regenerative effects on the ocular surface^{30,31,32,33,34,35}, while autologous serum provides tear-like concentrations of vitamin A, EGF, fibronectin, and other growth factors essential for epithelial regeneration^36,37,38. Together, these elements support re-epithelialization, reduce inflammation, and potentially prevent stromal degradation^39,40.

While the described approach provides controlled photopolymerization and customizable bioactivity, its applicability is currently limited to superficial and mid-stromal defects and requires line-of-sight exposure to blue light, which may restrict use in opaque or deeply infiltrated tissues. By integrating hydrogel preparation and enrichment, ophthalmic handling, and postoperative care, including partial lateral tarsorrhaphy, this paper offers a reproducible approach that supports corneal repair, facilitates translation of light‑activated hydrogel technologies to clinical practice, and contributes to the development of standardized platforms for corneal regenerative research.

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).

1. Hydrogel synthesis

1. Gelatin–riboflavin precursor solutions (5% and 10% w/v)
   ​NOTE: Work under dim light or use amber bottles or bottles wrapped in aluminum foil to protect the solution from light exposure. Perform all steps under sterile conditions in a laminar flow hood when the hydrogel is intended for biological use.
   1. Weigh the required amounts of gelatin and riboflavin phosphate (RFP): for 10 mL of 5% (w/v) + 0.01% (w/v) RFP precursor, weigh 500 mg of gelatin and 1 mg of RFP. For 30 mL of 10% (w/v) + 0.02% (w/v) RFP stock, weigh 3 g of gelatin and 6 mg of RFP.
   2. Add the gelatin and RFP to the corresponding volume of deionized water (≈90% of the final volume). Stir gently until the powder is fully dispersed.
   3. Adjust the pH to 7.0 using 1 M NaOH, adding the base dropwise while monitoring the pH. Record the volume of NaOH used and the final adjusted volume. After pH adjustment, bring the solutions to their final volume with deionized water.
   4. Dissolve and homogenize the solution at 80 °C under magnetic stirring at 200 rpm for 30 min.
      NOTE: Handle hot solutions (>70–80 °C) using heat‑resistant gloves.
   5. Allow the solutions to cool to 30–40 °C and filter them through 0.22 µm polyethersulfone (PES) hydrophilic membrane filters into sterile amber bottles.
      NOTE: Warm viscous solutions gently (≈ 40–50 °C) before filtration if required; avoid overheating.
   6. Label the bottles with the composition, date, and operator initials. Store the filtered hydrogel solutions at 4 °C until use, ensuring that the storage period does not exceed 2 weeks from the time of preparation.
2. Supplemented gelatin hydrogels (final volume: 10 mL)
   1. Prepare autologous serum (AS) or human amniotic membrane extract (HAMe) following procedures described in sections 4 and 5. Keep extracts sterile and chilled until use.
   2. For each 10 mL of enriched hydrogel, mix 5 mL of the 10% (w/v) gelatin + 0.02% (w/v) RFP stock, and 5 mL of the corresponding biological extract (AS or HAMe).
      NOTE: Warm viscous solutions gently (≈ 40–50 °C) before filtration if required; avoid overheating.
   3. Filter the warm solution through a 0.22 µm PES hydrophilic membrane filter into sterile amber bottles.
   4. Label and store the hydrogels at 4 °C until use. Ensure that the storage period does not exceed 2 weeks from the time of preparation.
      ​NOTE: Dispose of all waste according to institutional biosafety regulations.

2. Hydrogel application in vitro

1. Prepare the hydrogel formulations as described in Section 1 and keep them at 32 °C prior to application to maintain adequate viscosity.
2. Using sterile technique, pipette the required volume of hydrogel into the target well, mold, dish, or substrate (Figure 1A).
3. Position the Led-C curing lamp 2 cm above the hydrogel surface. Initiate in situ crosslinking by exposing the hydrogel to blue light (λ = 420–480 nm) and a light output of 850-1000 mW/cm² for a total duration of 2 min. Deliver the illumination as six pulses of 20 s, inserting 5–10 s pauses between pulses to prevent local heating (Figure 1B).
4. Confirm complete gelation visually and by gently touching the surface with a sterile instrument to ensure the formation of a solid, manipulable hydrogel (Figure 1C,D).
5. Use the crosslinked hydrogel immediately or store at 4 °C under sterile conditions appropriate to the downstream application.
   ​NOTE: Maintain aseptic technique throughout the procedure. Avoid direct exposure of skin or eyes to blue‑light irradiation.

3. Procurement of human amniotic membrane (HAM)

NOTE: Only use placentas from seronegative donors who have provided informed consent and meet institutional and ethical requirements.

1. Donor eligibility and consent
   1. Confirm that each donor has been screened and tested negative for HIV types 1 and 2, hepatitis B and C, and syphilis.
   2. Obtain written informed consent from each donor before surgery.
   3. Obtain human placentas immediately after elective cesarean deliveries of healthy donors.
2. Collection and initial processing
   1. Collect each placenta in a sterile stainless-steel container immediately after childbirth.
   2. In the operating theater, rinse the placenta extensively with sterile saline solution to remove blood clots. Identify the amnion—the innermost transparent membrane lining the amniotic cavity—and carefully separate it from the chorion by gentle peeling toward the placental edge using sterile forceps.
      NOTE: Handle the membranes carefully to avoid tearing.
   3. After complete separation, attach sterile nitrocellulose filter pieces to the HAM stromal surface to facilitate manipulation and standardize its division. Divide the HAM into three defined regions (Figure 2A,B): distal amnion: 10 cm-wide section, medial amnion: 10 cm-wide section, proximal amnion: section extending to the placental edge.
3. Transport
   1. Place each HAM fragment into a sterile bottle, immersing it in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1.25 µg/mL amphotericin B, 50 µg/mL penicillin-streptomycin, and 50 µg/mL neomycin.
   2. Clearly label each bottle with donor code, date, and the region of origin (distal, medial, or proximal).
   3. Transport the samples immediately to the laboratory under clean and aseptic refrigerated conditions (4 °C).
      NOTE: Transport the membranes in sealed, triple packaging compliant with UN 3373 (Biological Substance, Category B), in accordance with institutional biosafety regulations governing human tissue.
4. Laboratory processing
   ​NOTE: Perform all subsequent steps in a Class II biological safety cabinet under sterile conditions.
   1. Cut each distal, medial, and proximal HAM fragment into squares of 4.0–4.5 × 4.0–4.5 cm using sterile scissors.
   2. Immerse the fragments in antibiotic-supplemented DMEM already mentioned and gently brush with sterile clamps to remove blood residues. Wash in DMEM containing 50 µg/mL penicillin-streptomycin for 2 x 5 min.
   3. Wash in DMEM without any antibiotics for 2 x 5 min.
   4. Wash these selected fragments in sterile phosphate-buffered saline (PBS).
   5. Transfer each fragment to a 5 mL sterile tube without any culture medium. Freeze the samples directly at –80 °C until further use.

4. Human amniotic membrane extract (HAMe) preparation

NOTE: Perform all steps under aseptic and cold conditions to minimize protein degradation and contamination.

1. Retrieve the tubes containing human amniotic membrane (HAM) fragments stored at −80 °C. Immerse the tubes in liquid nitrogen for 5 min to harden the tissue.
2. Transfer each frozen fragment to a pre-cooled mortar (−80 °C) and crush it thoroughly using a pestle until a soft, homogeneous powder is obtained.
3. Weigh the powdered sample immediately on a balance plate to minimize thawing. Transfer the weighed powder into a 5 mL tube and resuspend it in 3 mL of PBS supplemented with 5 µL/mg protease inhibitor cocktail.
   NOTE: Keep all tubes on ice during processing to preserve protein integrity.
4. Sonicate each sample for 20 min at 90% amplitude using the sonicator and 1.5 mm diameter probe, applying 15 s on / 15 s off cycles to prevent overheating.
5. Centrifuge the lysates at 3,000 × g for 10 min at 4 °C. Carefully collect the supernatant without disturbing the pellet.
6. Filter the supernatant through a 0.22 µm PES hydrophilic membrane filter using sterile conditions. Aliquot the filtered HAMe into 0.2 mL microtubes. Store the aliquots at −80 °C until further use.
   ​NOTE: Ensure that the storage period does not exceed 6 months from the time of preparation.

5. Extraction of blood-derived autologous serum from rabbits

NOTE: Perform all procedures for in vivo assays in New Zealand White rabbits (≈2 kg) under approval from the Animal Research Ethics Committee of the corresponding institution and in compliance with institutional guidelines.

1. Anaesthetize the rabbit locally with lidocaine (topical or subcutaneous) at the venipuncture site.
2. Collect blood from the central ear artery into 4.5 mL blood collection tubes containing a polymer gel separator (Figure 3A,B). Allow the blood to clot for 2 h at RT without agitation (Figure 3C).
3. Centrifuge the samples at 1,000 × g for 15 min at RT. Collect the supernatant (serum), which constitutes the rabbit autologous serum (AS) (Figure 3D).
4. Filter the serum through a 0.22 µm PES hydrophilic membrane filter. Aliquot into sterile 0.2 mL tubes and store at −80 °C until use.
   ​NOTE: Ensure that the storage period does not exceed 6 months from the time of preparation.

6. In vivo surgical procedure

NOTE: Perform all the experimental procedures in accordance with the Animal Research Ethics Committee of the corresponding institution and comply with the Tenets of the Declaration of Helsinki and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Preoperative preparation
   ​NOTE: Employ New Zealand White rabbits (≈2 kg) as the animal model.
   1. Prior to surgery, administer general anesthesia via intramuscular injection into the thigh of the animals using: Ketamine at 1 mL/kg (50 mg/mL) and Xylazine at 0.3 mL/kg (20 mg/mL). Confirm the absence of the corneal and foot reflex before proceeding.
   2. Place the animal in a lateral decubitus position on a sterile surgical platform and disinfect the periocular region with povidone-iodine solution. Place an eyelid speculum on the operative eye to maintain continuous exposure of the ocular surface during the procedure.
2. Anterior stromal keratectomy
   1. Center a 6.5 mm diameter vacuum trephine on the rabbit cornea (Figure 4A). Apply suction to stabilize the device and rotate the trephine three-quarter turns, achieving an approximate stromal depth of 187 µm.
   2. Perform an anterior stromal keratectomy within the trephined area using a crescent blade, removing the epithelial and anterior stromal layers until a smooth wound bed is obtained (Figure 4B).
3. In vivo hydrogel application
   1. Fill the corneal defect with 50 µL of the corresponding hydrogel formulation, preheated to 32 °C to ensure optimal viscosity (Figure 4C).
   2. Initiate in situ crosslinking by illuminating the corneal surface with blue light (λ= 420–480 nm) for 2 min. Deliver six 20 s pulses with 5–10 s pauses between pulses to limit local heating (Figure 4D).
4. Lateral partial tarsorrhaphy
   1. Identify the nasal one‑third of the upper and lower eyelid margins to allow medial exposure for observation and treatment.
   2. Place the first suture (use 1–2 sutures depending on eyelid length and stability needed) (Figure 4E).
      1. Grasp the lateral upper eyelid margin with forceps. Pass the needle through the grey line / eyelid margin from the skin side to the conjunctival side, taking a full-thickness bite but avoiding deep penetration into the tarsal plate if possible.
      2. Pass the needle through the corresponding point of the lower eyelid margin from the conjunctival side to the skin side to create apposition of the lid margins.
      3. Tie the suture with a secure surgeon’s knot and two square knots, leaving the knot on the skin surface positioned slightly to the outer side of the eyelid margin rather than directly over it.
      4. Place a second suture approximately 3–4 mm medial to the first one if additional stability is required, using the same full‑thickness U‑shaped suture or a simple interrupted stitch.
      5. Trim suture tails to 3–4 mm to reduce irritation, leaving enough length for later removal.
      6. Confirm that the eyelid apposition protects the treated cornea while allowing access to the medial and temporal conjunctiva/cornea for inspection and eye-drops treatment. Ensure that the tarsorrhaphy is not overly tight and that the animal’s eyelids are not ischemic (Figure 4F).
5. Postoperative monitoring and care
   1. Administer buprenorphine at 0.05 mg/kg via subcutaneous injection before keratectomy and every 12 h for 72 h to provide analgesia.
   2. Instill 3 mg/mL Tobramycin eye drops twice daily until complete wound closure to prevent infection.
   3. Apply one drop of 0.2% hyaluronic acid (HA) artificial tears to all eyes, including controls, four times daily to maintain corneal hydration.
   4. Inspect the tarsorrhaphy at least twice daily for signs of suture loosening, infection, local tissue necrosis, or undue tension. Keep the animal under observation for normal behavior, feeding and signs of ocular discomfort.
   5. At 72 h post procedure, cut the suture knot and gently withdraw each suture with forceps, taking care to avoid dragging contaminants across the conjunctival surface.
   6. Inspect the eyelid margins for proper healing and the corneal surface for epithelial integrity. Continue ocular treatment as required until complete epithelial closure is observed.
   7. Monitor and record ocular healing and clinical signs from day 3 to day 7, assessing epithelial closure, opacity, and neovascularization using slit-lamp microscopy and fluorescein staining.
      ​NOTE: If any sign of ischemia, wound dehiscence, or severe irritation appears, remove the suture(s) immediately and manage accordingly.
6. Draize acute ocular irritation assessment
   1. Restrain the rabbit gently using a towel/blanket wrap. Examine the iris and conjunctiva first under white light.
   2. Score conjunctival redness, chemosis (edema), and discharge according to the Draize scale (see Table 1 and Table 2).
   3. Record all six Draize parameters on the data sheet. Calculate the ocular irritation index (OII) as the sum of component scores; OII ranges from 0 to 110, where higher values indicate stronger irritation.
   4. Classify each eye according to Kay–Calandra categories based on the OII; record the category in the dataset.
7. Photographic documentation of wound healing
   1. Instill fluorescein solution(5 µL) into the inferior conjunctival sac.
   2. Illuminate the eye with blue light and assess corneal opacity (density and area) and the extent of fluorescein staining.
   3. Set the camera with manual white balance adjusted, optical zoom disabled, and position approximately 3 cm from the eye to capture all images using identical parameters across sessions. Capture images after fluorescein instillation with and without blue light illumination to delineate epithelial defects. Capture images at day 0 (pre‑tarsorrhaphy) and daily from day 3 (following removal of the tarsorrhaphy) until complete epithelial closure.
   4. Analyze the images with ImageJ software and quantify the area of fluorescein-stained regions.
      ​NOTE: Photographic documentation on days 1 and 2 is typically not feasible due to the partial tarsorrhaphy. Indicate that images from days 1 and 2 are missing from the dataset and report any adverse events (e.g., incidental corneal abrasion).
8. Euthanasia and tissue collection
   1. At day 21 (after the evaluation period of both eyes), anaesthetize animals deeply using ketamine (2 mL/kg) and xylazine (0.6 mL/kg) administered intramuscularly.
   2. Euthanize by intravenous injection of 10 mL saturated potassium chloride (KCl) into the marginal ear vein.
   3. Confirm death by the onset of rigor mortis and absence of vital reflexes before proceeding to tissue harvesting.

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