A "T.E.S.T." hydrogel bioadhesive assisted by corneal cross-linking for in situ sutureless corneal repair

Abstract

Corneal transplantation is an effective clinical treatment for corneal diseases, which, however, is limited by donor corneas. It is of great clinical value to develop bioadhesive corneal patches with functions of "Transparency" and "Epithelium & Stroma generation", as well as "Suturelessness" and "Toughness". To simultaneously meet the "T.E.S.T." requirements, a light-curable hydrogel is designed based on methacryloylated gelatin (GelMA), Pluronic F127 diacrylate (F127DA) & Aldehyded Pluronic F127 (AF127) co-assembled bi-functional micelles and collagen type I (COL I), combined with clinically applied corneal cross-linking (CXL) technology for repairing damaged cornea. The patch formed after 5 min of ultraviolet irradiation possesses transparent, highly tough, and strongly bio-adhesive performance. Multiple cross-linking makes the patch withstand deformation near 600% and exhibit a burst pressure larger than 400 mmHg, significantly higher than normal intraocular pressure (10-21 mmHg). Besides, the slower degradation than GelMA-F127DA&AF127 hydrogel without COL I makes hydrogel patch stable on stromal beds in vivo, supporting the regrowth of corneal epithelium and stroma. The hydrogel patch can replace deep corneal stromal defects and well bio-integrate into the corneal tissue in rabbit models within 4 weeks, showing great potential in surgeries for keratoconus and other corneal diseases by combining with CXL.

Keywords: AF127, Aldehyded Pluronic F127; AS-OCT, Anterior Segment Optical Coherence Tomography; Bioadhesives; CCK-8, Cell Counting Kit-8; COL I, Collagen Type I; CXL; CXL, Corneal Cross-linking; Corneal patch; DLS, Dynamic Light Scattering; DMEM, Dulbecco's Modified Eagle's Medium; ECM, Extracellular Matrix; F127DA, Pluronic F127 diacrylate; FBS, Fetal Bovine Serum; GelMA, Methacryloylated Gelatin; H&E, Hematoxylin and Eosin; IHC, Immunohistochemistry; IOP, Intraocular Pressure; PBS, Phosphate-buffered Saline; RF, Riboflavin-5-phosphate; ROS, Reactive Oxygen Species; SD, Standard Deviation; Sutureless repair; TEM, Transmission Electron Microscopy; Tough hydrogel; UV, Ultraviolet; α-SMA, Alpha Smooth Muscle Actin.

Graphical abstract

Scheme 1

Fabrication and application, as well as networks illustration of light-curable adhesive corneal hydrogel patch.

Fig. 1

Synthesis, micelle fabrication, and hydrogel fabrication. A. Synthesis of GelMA. B. Synthesis of F127DA and AF127. C. TEM images of F127DA micelle, AF127 micelle and co-assembled F127DA&AF127 micelle (bar scale: 20 nm, F127DA:AF127 = 1:1). D. Average particle size statistics. E. General transparency observation of precursor solution of GelMA, micelles and their mixture. F. Light transmission over the visible light spectrum, and light transmission at 450 nm. G. General transparency observation of hydrogels after UV irradiation. H. Light transmission of hydrogels at 450 nm.

Fig. 2

Compression and tensile tests on hydrogels. A. General observation of GelMA hydrogel and GelMA with co-assembled micelles hydrogel under compression. B. Stress-stain curves of hydrogels during compression testes. C. Compressive moduli of hydrogels. D. General observation of GelMA hydrogel, GelMA with co-assembled micelles hydrogel and GelMA with F127DA micelles hydrogel under stretching. E. Stress-strain curves of hydrogels during tensile testes. F. Elongation at break of hydrogels. G. Fracture stress of hydrogels. H. Toughness of hydrogels.

Fig. 3

Effect of COL I on transparency and mechanical performance of hydrogel. A. General transparency observation of precursor solutions and gelled hydrogels. B. Light transmission of precursor solutions and hydrogels at 450 nm. C. G′ of G8F4 and G8F4C3 hydrogels monitored while curing with UV to record the gelatin kinetics of hydrogels. D. Stress-strain curves of hydrogels during tensile testes. E. General observation of G8F4C3 hydrogel under extreme compression and stretching. F. Loading-unloading curves of G8F4C3 hydrogel. G. Fracture stress of hydrogels. H. Elongation at break of hydrogels. I. Toughness of hydrogels. J. Tensile moduli of G8F4 and G8F4C3 hydrogels.

Fig. 4

Adhesive ability of hydrogel G8F4C3. A. General observation of hydrogel G8F4C3 adhered to in vitro porcine cornea defect. B. Hydrogel G8F4C3 adhered to penetrating porcine cornea defect for burst test. C. Burst test device and burst pressure. D. Scheme of lap shear test. E. Lap shear adhesion curve of hydrogels to cornea. F. Adhesion strength of hydrogels. G. Operation process of corneal transplantation in vivo to evaluate the adhesive ability of hydrogel G8F4C3. H. Slit lamp photographs and Anterior segment optical coherence tomography (AS-OCT) images after sutureless lamellar keratoplasty using G8F4C3 hydrogel at 0 d and 7 d.

Fig. 5

Swelling, degradation and viscosity of corneal hydrogels. A. Mass swelling ratio during incubation in PBS. B. Changes in weight of hydrogels incubated with collagenase. C,D. Rheological characterization of viscosity of hydrogel precursor solutions with shear rate change at 37 °C and 25 °C. E. Representative photos and statistics showing the effect of gravity on the behavior of hydrogel precursor solutions while applied on the defect area during operations.

Fig. 6

In vitro cytocompatibility of hydrogels. A. Live/dead staining of corneal fibroblasts cultured on the surface of G12, G8F4, and G8F4C3 hydrogels (Bar scale: 50 μm). B. Cellular viability. C. Corneal fibroblasts proliferated on hydrogels after 1, 3, 5, and 7 days. (*p < 0.05, **p < 0.01).

Fig. 7

In vivo implantation of the hydrogels into rabbit corneal defects. A. Operation process of in vivo implantation of hydrogel. B. Representative slit lamp images (left) and AS-OCT images (right) after defect creation without treatment and after hydrogel treatments using G8F4 and G8F4C3.

Fig. 8

Postoperative observation of the corneal defects without treatment. A. Representative slit-lamp images and AS-OCT images in black group without treatment at 3, 7, 14, and 28 days. B. H&E, Masson and α-SMA immunohistochemical staining of regenerated tissue in black group (Bar scale: 50 μm).

Fig. 9

Postoperative observation of the corneal defects with hydrogels treatment. A. Representative slit lamp images and AS-OCT images in G8F4 hydrogel treated group at 3, 7, 14 and 28 days. B. Representative slit lamp images and AS-OCT images in G8F4C3 hydrogel treated group at 3, 7, 14 and 28 days. C. H&E, Masson and α-SMA immunohistochemical staining of regenerated tissue in G8F4 hydrogel treated group and G8F4C3 hydrogel treated group (Bar scale: 50 μm).