Sutureless repair of corneal injuries using naturally derived bioadhesive hydrogels

Abstract

Corneal injuries are common causes of visual impairment worldwide. Accordingly, there is an unmet need for transparent biomaterials that have high adhesion, cohesion, and regenerative properties. Herein, we engineer a highly biocompatible and transparent bioadhesive for corneal reconstruction using a visible light cross-linkable, naturally derived polymer, GelCORE (gel for corneal regeneration). The physical properties of GelCORE could be finely tuned by changing prepolymer concentration and photocrosslinking time. GelCORE revealed higher tissue adhesion compared to commercial adhesives. Furthermore, in situ photopolymerization of GelCORE facilitated easy delivery to the cornea, allowing for bioadhesive curing precisely according to the required geometry of the defect. In vivo experiments, using a rabbit stromal defect model, showed that bioadhesive could effectively seal corneal defects and induce stromal regeneration and re-epithelialization. Overall, GelCORE has many advantages including low cost and ease of production and use. This makes GelCORE a promising bioadhesive for corneal repair.

Fig. 1. Synthesis, application, and in vitro characterization of GelCORE adhesive hydrogels.

(A) Schematic of the chemical reaction for GelCORE formation and photocrosslinking of the prepolymer solution with Eosin Y (photoinitiator), TEA (co-initiator), and VC (co-monomer). (B) Schematic diagram for the application of GelCORE for rapid and long-term repair of corneal injuries, which include (i) formation of stromal defect, (ii) application of the bioadhesive, (iii) regeneration of the epithelial layer, and (iv) stromal regeneration. (C) The prepolymer solution is injected into the corneal defect and exposed to visible light, forming (D) an adhesive GelCORE hydrogel. (E) Representative compressive stress-strain curves, (F) compressive moduli, and (G) elastic moduli of GelCORE adhesive hydrogels fabricated using 5%, 10%, and 20% (w/v) total polymer concentrations with varying photocrosslinking time points. (H) Water content of GelCORE adhesives produced by using 20% (w/v) polymer concentration and varying visible light exposure times at 37°C in DPBS over time. (I) In vitro degradation of 20% (w/v) GelCORE adhesive (4-min photocrosslinking time), in different concentrations of collagenase type II (Col II) solution in DPBS and 37°C over time. All hydrogels were polymerized by using 0.1 mM Eosin Y, 1.5% (w/v) TEA, and 1% (w/v) VC in distilled water. Data are reported as means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n ≥ 3).

Fig. 2. In vitro adhesion properties of GelCORE hydrogels using porcine skin and intestine as biological substrates.

(A) Schematic of the modified test for burst pressure measurements (ASTM F2392-04) and (B) average burst pressure of GelCORE adhesives (n ≥ 3) produced with varying polymer concentrations and photocrosslinking times, compared to two commercial adhesives including Evicel and CoSEAL. (C) Schematic of the modified test for lap shear strength measurements (ASTM F2255-05) and (D) average shear strengths of GelCORE adhesives (n ≥ 3) produced with varying polymer concentrations and photocrosslinking times, Evicel, and CoSEAL. (E) Schematic of the modified test for wound closure test (ASTM F2458-05) and (F) average adhesive strengths of GelCORE adhesives (n ≥ 3) produced with varying polymer concentrations and photocrosslinking times, compared to Evicel and CoSEAL. Data are means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (Photo credit: Ehsan Shirzaei Sani, UCLA)

Fig. 3. Ex vivo application and adhesion properties of GelCORE adhesives.

(A) Representative slit lamp photographs from rabbit eyes sealed by GelCORE bioadhesive, and (B) retention times of GelCORE bioadhesives, formed at various cross-linking times and prepolymer concentrations, on cornea tissues. (C) Representative optical coherence tomography images after ex vivo application of GelCORE adhesives to rabbit corneas at days 1, 14, and 28 after application. (D) Schematic of ex vivo burst pressure set up, including a syringe pump, a pressure sensor, and a recording system. (E) Average burst pressure of GelCORE adhesives formed by varying photocrosslinking time, compared to a commercially available ocular sealant, ReSure (control). Data are represented as means ± SD (**P < 0.01, ***P < 0.001, and ****P < 0.0001; n ≥ 4). [Photo credit: (A) Ahmad Kheirkhah, MEE, Harvard Medical School; (D) Ehsan Shirzaei Sani, UCLA]

Fig. 4. In vitro cytocompatibility of GelCORE bioadhesives.

Representative LIVE/DEAD images from corneal fibroblast cells seeded on (A) tissue culture well-plate, (B) GelCORE adhesives, and (C) ReSure sealant on days 1, 4, and 7 after seeding (scale bar, 100 μm). (D) Quantification of cell viability on GelCORE bioadhesives compared to tissue culture well plate and ReSure after 1, 4, and 7 days of culture. (E) Quantification of metabolic activity of corneal fibroblast cells seeded on control (tissue culture well plate), GelCORE hydrogels, and ReSure after 1, 4, and 7 days. Representative LIVE/DEAD images of corneal fibroblast cells grown on (F) tissue culture well plate and (G) GelCORE hydrogels based on a 2D scratch assay at 0, 1, and 3 days after scratching. (H) Quantification of relative cell densities migrated to the scratched area on GelCORE adhesives and control samples, at days 0, 1, 2, and 3. GelCORE hydrogels formed at 20% (w/v) final polymer concentration were used for 2D cell culture studies (photocrosslinking time, 4 min). Data are represented as means ± SD (**P < 0.01, ***P < 0.001, and ****P < 0.0001; n ≥ 3).

Fig. 5. In vivo application of GelCORE bioadhesives into corneal defects in rabbits.

(A and B) Representative images for creating a 50% depth corneal stromal defect on rabbit eye. (C) In situ application of GelCORE prepolymer solution into corneal defect. (D) Photocrosslinking and (E) formation of a transparent GelCORE adhesive hydrogel on corneal stromal defect. AS-OCT images (F) before and (G) after treatment with GelCORE. Seven days after application, the bioadhesive still had a smooth surface. GelCORE hydrogels were prepared by using 20% (w/v) total polymer concentration and 4-min light exposure time. [Photo credit: (A to E) Amir Sheikhi, UCLA]

Fig. 6. Corneal re-epithelialization after in vivo application of the bioadhesive to corneal defects in rabbit cornea.

(A) Representative slit lamp photographs and (B) cobalt blue with fluorescein staining after in vivo application of GelCORE adhesive to rabbit cornea at different time points. Progressive reduction in the size of corneal epithelial defect (green area in the central cornea) implicates epithelial migration over GelCORE. (Photo credit: Ahmad Kheirkhah, MEE, Harvard Medical School)

Fig. 7. Histological analysis after application of GelCORE bioadhesive in a rabbit corneal stromal defect model.

Representative hematoxylin and eosin histopathology images from (A) native rabbit corneas (without defect) and (B) rabbit cornea after application of GelCORE in a 50% depth stromal defect. Histological images for (C) untreated stromal defect (without bioadhesive) and (D) the defect treated with bioadhesive at day 14 after surgery (scale bars, 50 μm and 1 mm). (E) Thickness of stromal layer for the native cornea, GelCORE-treated, and untreated eyes at day 14 after surgery obtained from histological images. (F) Thickness of epithelial layer for native cornea and GelCORE-treated and untreated eyes at day 14 after surgery obtained from histological images. (G) Representative fluorescent immunohistochemical images (DAPI and CD45 marker) (i) from the area without defect and (ii) from corneal stromal defect treated with GelCORE bioadhesive at day 14 after surgery. GelCORE hydrogels were prepared at 20% (w/v) total polymer concentration and 4-min light exposure time. Data are represented as means ± SD (****P < 0.0001; n ≥ 3).