The team was co-led by May Griffith, a researcher at Maisonneuve-Rosemont Hospital Research Centre, which is affiliated with Université de Montréal and is part of the CIUSSS de l’Est-de-l’Île-de-Montréal.
The results of this multinational project have just been published in the journal Science Advances.
“Our work has led to an effective and accessible solution called LiQD Cornea to treat corneal perforations without the need for transplantation,” said Griffith.
She is also a full professor in the Department of Ophthalmology at Université de Montréal.
“This is good news for the many patients who are unable to undergo this operation due to a severe worldwide shortage of donor corneas,” she said.
“Until now, patients on the waiting list have had their perforated corneas sealed with a medical-grade super glue, but this is only a short-term solution because it is often poorly tolerated in the eye, making transplantation necessary.”
A synthetic, biocompatible and adhesive liquid hydrogel, LiQD Cornea, is applied as a liquid, but quickly adheres and gels within the corneal tissue.
The LiQD Cornea promotes tissue regeneration, thus treating corneal perforations without the need for transplantation.
Griffith praised the work of her trainees, Christopher McTiernan and Fiona Simpson, and her collaborators from around the world who have helped create a potentially revolutionary treatment to help people with vision loss avoid going blind.
“Vision is the sense that allows us to appreciate how the world around us looks,” said Griffith. “Allowing patients to retain this precious asset is what motivates our actions as researchers every day of the week.”
For Sylvain Lemieux, president and CEO of the CIUSSS de l’Est-de-l’Île-de-Montréal, “this innovative treatment in ophthalmology confirms the level of expertise of the Centre universitaire d’ophtalmologie de l’Université de Montréal (CUO) at the Maisonneuve-Rosemont Hospital (HMR).
“The HMR has one of the largest teams of ophthalmologists in Quebec and one of the best-equipped ophthalmology research laboratories in North America,” he said.
“The hard work of our scientists and clinicians contributes daily to best practices and knowledge development.
“The multiple therapeutic possibilities resulting from our fundamental research, particularly in regenerative medicine, benefit and give hope to people suffering from ophthalmological diseases not only in Quebec, but in the rest of the world,” he concluded.
The cornea is the transparent front surface of the eye that provides about two-thirds of the focusing power of the eye. Any permanent transparency loss from injury or disease can result in blindness. Currently, 23 million people globally have unilateral corneal blindness, while 4.9 million are bilaterally blind (1).
Transplantation with human donor corneas has been the mainstay for treating corneal blindness for a century. However, a global donor cornea shortage leaves 12.7 million on waiting lists, with only 1 in 70 patients treated (2).
Conditions requiring corneal transplantation include persistent ulceration leading to scarring or perforation after corneal infection, burns, autoimmune diseases, and physical trauma.
Corneal perforations are an emergency, and in many centers, the cornea is temporarily sealed using cyanoacrylate glue to maintain integrity and avoid losing the eye (3).
However, cyanoacrylate glue is toxic and can cause local irritation and inflammation. Its incomplete polymerization leaves behind toxic cyanoacrylate monomers, while its hydrolysis releases potentially toxic compounds like formaldehyde and alkyl cyanoacrylate (4).
These induce corneal scarring and vascularization. Patients generally require follow-up corneal transplantation. Despite these clear limitations, the use of cyanoacrylate glue to seal corneal perforations has remained the established emergency treatment for more than 50 years (5).
Other interventions include corneal suturing (6), tectonic corneal grafts (7), conjunctival flaps (8), multilayered amniotic membrane transplantation (9), soft “bandage” contact lenses (10), and tissue sealants.
Sealants examined include a variety of natural adhesives like fibrin, gelatin, chitosan, and alginate (11), as well as a number of synthetic polyethylene glycol (PEG) derivatives (12).
Most of these interventions, however, work only in a limited range of cases or require invasive surgery with possible limitations for future visual rehabilitation (13).
PEG-based sealants have shown promise in sealing perforating microincisions, but to the best of our knowledge, there is no study which has looked at their efficacy in sealing macroperforations.
Furthermore, PEG-based sealants typically require multicomponent mixing and suffer from short application windows. For example, ReSure (Ocular Therapeutix Inc.), which requires two-component mixing of PEG and a trilysine acetate solution, allows only a 20-s window for application upon initiation of polymerization (14).
A new bioadhesive, GelCORE, was recently reported as an alternative to cyanoacrylate glue for corneal tissue repair in partial-thickness corneal defects and corneal perforations. The authors used white light, with Eosin Y, triethanolamine (TEA), and N-vinylcaprolactam (VC) as initiators to gel a mixture of methacryloyl functionalized gelatin in situ (11).
The GelCORE report included a 14-day rabbit study in which a 50% thickness wound was repaired. However, because of the short duration of the study, long-term effects could not be evaluated.
The use of animal-derived gelatin has an associated risk of zoonotic disease transfer, and severe allergic reactions to both bovine and porcine gelatin in vaccines have been reported (15).
Photocrosslinking may also be problematic in the clinical setting. Patients with corneal inflammation are photophobic (light-sensitive) and may not be able to tolerate intense visible light application more than 4 min without retrobulbar or general anesthesia.
In a mechanism analogous to corneal cross-linking for keratoconus, the creation of free radicals in photocrosslinking may also be toxic to the corneal endothelium in thinned or perforated corneas (16).
Hyaluronic acid–based materials have also been tested as alternative bioadhesives in an in vitro organ setting using excised porcine eyes (17). This solution relied on hydrazone cross-linking of dopamine-modified hyaluronic acid (HA-DOPA), where dopamine supplied the tissue adhesive properties.
While successful in vitro, this material has not been evaluated in animal models. Neither GelCORE nor HA-DOPA was tested for repair of full-thickness corneal perforations, nor have they been examined as alternatives to donor corneal tissue for transplantation.
Over 10 years ago, our team members conducted a first-in-human clinical trial on cell-free, biosynthetic hydrogels made from recombinant human collagen type III (RHCIII). These hydrogels promoted stable corneal tissue and nerve regeneration, showing that they were immune-compatible alternatives to donor cornea transplantation in anterior lamellar keratoplasty (ALK) (18, 19).
Recently, we demonstrated that hydrogel implants derived from a short collagen-like peptide (CLP) conjugated to an inert, but mechanically robust, multifunctional PEG are functionally equivalent to the RHCIII-based implants when tested under preclinical conditions in mini-pigs (20).
The use of fully defined short synthetic peptides provides homogeneous materials that are easily modified and scaled up in comparison to their full-length analogs. In addition to being fully synthetic, the use of CLP-PEG collagen analogs circumvents the batch-to-batch heterogeneity seen with extracted proteins, as well as potential allergic reactions to xenogeneic proteins (21) and possible zoonotic disease transmission (22).
Despite being able to promote regeneration, these solid implants require an operating theater for implantation, involving costs for a full surgical team. Realistically, to reach the enormous numbers of patients awaiting transplantation, most of them living in low to middle income countries, we need a drastic paradigm change.
To date, vaccines have been vastly successful both in cost and delivery, with every person receiving a vaccine delivered in a syringe. By analogy, in dentistry, when someone has a cavity in a tooth, the pathologic tissue is removed, and the tooth is filled.
A similar paradigm is likely needed to tackle this important global issue, where the pathologic tissue is replaced by a regeneration-stimulating liquid corneal replacement, LiQD Cornea, in a syringe that gels in situ. Previously, we reported that CLP-PEG polymerizes in situ and can form a seal in experimental in vitro models of corneal perforation when supported by an ab interno patch (23).
In this study, we introduce the LiQD Cornea, a new injectable hydrogel matrix with adhesive properties. We examined the potential efficacy of our LiQD Cornea comprising CLP-PEG-fibrinogen as a sealant/filler of full-thickness corneal perforations and an alternative to lamellar corneal transplantation that potentially allow treatments to be carried out in an ophthalmologist’s office.
Physical and mechanical characterization
The CLP-PEG-fibrinogen LiQD Cornea formed a porous hydrogel upon gelation in the presence of thrombin and a nontoxic cross-linker, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (fig. S1). LiQD Cornea hydrogel samples showed a refractive index of 1.354 ± 0.037, consistent with human corneas and physical and chemical properties consistent with previous generations of RHCIII and CLP-PEG hydrogels (Table 1) (18, 20).
In the visible spectrum (400 to 800 nm), LiQD Cornea samples transmitted between 93 and 99% of incident light. The transmission of light in the ultraviolet (UV) region decreased to a low value of 19% in the UV-C spectral region. Bursting pressure testing using ex vivo porcine corneas showed that the LiQD Cornea formulation, although less robust than cyanoacrylate or fibrin sealant, nevertheless withstood 170 mmHg of pressure. This was a 7.7-fold increase over the average 11 to 21 mmHg of intraocular pressure within the human eyeball (Table 1).
Table 1
Optical, physical, and mechanical properties of LiQD Cornea hydrogels.
| Tensile strength (MPa) | Modulus (MPa) | Viscosity (Pa.s) | Transmission (%) | Refractive index | Water content (%) | Collagenase (mg/min) | Td (°C) |
| 0.02 | 0.16 | 31.7 ± 27.6 | 19–93% (UV) 93–99% (Visible) | 1.354 ± 0.037 | 91.2 ± 2.3 | 7.3 × 10−7 ± 6.1× 10−7 | 64 ± 8.5 |
| Material | Average bursting pressure (mmHg)* | Representative image of sealed ex vivo perforation model† | |||||
| Cyanoacrylate glue | >300 |  | |||||
| Fibrin sealant | 259 ± 14.5 |  | |||||
| LiQD Cornea | 170 ± 16.9 |  |
*Maximum pressure measured by the pressure transducer is 300 mmHg.
†Photographs of ex vivo porcine corneas, which were mounted in an artificial anterior chamber and perforated according to the described model and sealed/filled with the corresponding material. Red arrows highlight the interface of the applied material and the perforated cornea.
DISCUSSION
To address the severe shortfall of donor tissue in the treatment of corneal blindness, it is imperative that novel alternatives to corneal transplantation and perforation repair are developed.
While a number of techniques and materials are currently available to treat corneal defects and perforations, many of them involve complex procedures and use materials with poor biocompatibility, mechanical mismatch, and an inability to support regeneration. Ideally, any newly developed method should be easy to apply in a clinical setting, readily fill corneal defects, and seal perforations.
At the same time, it should support tissue regeneration, limiting the need for further surgical intervention and follow-up corneal transplantation.
Our results showed that LiQD Cornea behaved as an injectable liquid at temperatures above 37°C, gelling as it cools down. In situ gelation of the LiQD Cornea in animal corneas took 5 min at body temperature, after initiation with DMTMM, a nontoxic cross-linker (23).
Most patients with corneal perforations have inflamed eyes and are photophobic. Unlike light-activated systems, LiQD Cornea did not require a dedicated light source for curing. Without the requirement for light activation, no anesthetic will be needed in future clinical application to render the exposure to an intense light source for cross-linking tolerable.
In addition, photoinitiated cross-linking has been reported to have possible phototoxic effects on the corneal endothelial cells (16). Considering that the initial perforation in pathologic corneas would also affect the health of the endothelium, it would be prudent not to further deplete the local population of endothelial cells.
The incorporation of an approved surgical fibrin sealant permitted adhesion of the LiQD Cornea during in situ gelation. Corneal perforations in ex vivo corneas were completely sealed in situ with a bursting pressure of 170 mmHg, which is several-fold higher than normal intraocular pressures of 11 to 21 mmHg (29).
HCECs grew readily on the LiQD Cornea hydrogels. The BMDC study indicated that the LiQD Cornea did not activate dendritic cells unlike the positive control, LPS, which is a well-established activator of dendritic cells. As the LiQD Cornea formulation does not activate dendritic cells, the risk of graft rejection due to activation of CD4+ and CD8+ T cells is reduced (30).
The BMDM assay indicated that naïve BMDMs cultured in the presence of LiQD Cornea hydrogels primarily matured into an M2 phenotype that is associated with tolerogenic activity (31). These results taken together demonstrated that the LiQD Cornea formed a seal that will withstand the pressures encountered within the eye and will be fully biocompatible and immune compatible.
Injection of the LiQD Cornea into full-thickness corneal perforations in rabbits confirmed the ability to seal the wound gape. The completeness of the seal was validated by the addition of a postsurgical air bubble. The bubble was present up to 2 days after surgery, indicating that the material had created a complete seal that did not allow the leakage of air. The rabbit histology showed that the patch was completely reepithelialized. However, the 28-day duration of the study did not provide time for full stromal, endothelium, and nerve regeneration.
The Göttingen mini-pigs used were genetically coherent or homogenous. Hence, grafts from one animal to another were considered syngeneic, i.e., they were sufficiently identical and immunologically compatible to allow for transplantation.
The 12-month in vivo pig study confirmed that LiQD Cornea allowed regeneration of the corneal epithelium, stroma, and nerves. Even if the material does not achieve the desired perfectly smooth surface directly after application, OCT results showed that the corneal thickness and curvature were restored to those matching the syngeneic grafts and unoperated controls (fig. S2B).
The primary difference in the clinical performance of the LiQD Cornea and the syngeneic grafts was increased haze in the surgical site between 3 to 6 months after operation, during the period of rapid keratocyte in-growth into the cell-free matrix. Syngeneic grafts were already populated with donor cells, so no rapid in-growth of host cells was expected.
However, at 1 year after surgery, the haze was reduced to a low grade in three of four LiQD Cornea recipients, while the syngeneic graft outline was still visible in the corneas.
Neovascularization had accompanied the haze, as we had previously reported for solid CLP-PEG implants during the rapid cell population of cell-free grafts (20). However, as observed in previous solid implant studies (32, 33), the vessels receded over the 12-month observation period as haze cleared in three of four animals. While this small amount of vascularization and haze is not ideal, it is unlikely to lead to immune rejection.
LiQD Cornea does not activate dendritic cells in vitro and is acellular and repopulated by the host cells, unlike traditional corneal transplants that bring with them allogeneic cells (25) whose surface proteins can trigger immune reactions.
Vascularization could increase the risk of rejection of subsequent allografts (34), but LiQD Cornea is designed to regenerate the eye wall without the need for subsequent transplantation, avoiding problems with induced irregular astigmatism, rejection, and lack of access to transplant donor material. Where corneal perforations involve the central visual axis, at minimum, LiQD Cornea aims to restore eye wall integrity as a viable pathway for future rehabilitation. In our pig model, steroid medication was only administered for 5 days. In a clinical setting, it may be possible to modulate neovascularization during healing through application of topical steroids for a longer period.
We also found that LiQD corneas had lower expression of mature type I collagen in the cornea. This is in keeping with the fact that the LiQD Cornea matrix had no collagen, and hence, all collagen found at 12 months after surgery was due to active remodeling of the gel, in comparison to syngeneic grafts, which had a complete extracellular matrix at the time of grafting.
When considering the relative performance of the LiQD Cornea and the syngeneic grafts, it is important to note that the syngeneic grafts are likely less inflammatory than a standard clinical allograft, because of the genetic homogeneity of Göttingen mini-pigs.
As previously reported for CLP-PEG (20), the LiQD Cornea also induced the production of copious amounts of EVs that included exosomes, in comparison to the syngeneic grafts, and the lack of EVs in the untreated, healthy controls. We currently hypothesize that the presence of the EVs is linked to the production of new extracellular matrix in the surgical site, as the reduced collagen content in the LiQD Cornea suggests that the new tissue is still undergoing extracellular matrix protein secretion to restore the matrix at 12 months.
Overall, LiQD Cornea performed equivalently to syngeneic grafts, indicating a possible role as an alternative to conventional donor corneal transplantation for conditions treatable by lamellar transplantation.
However, as noted, it took the surgeon an average of two attempts to achieve the desired curvature, indicating that an appropriate point-of-care delivery device (fig. S3) is needed for clinical application.
The self-assembling, fully defined, synthetic collagen-like LiQD Cornea is considerably less costly than human recombinant collagen and reduces any risk of allergy or immune rejection associated with xenogeneic materials. In situ gelation potentially allows for clinical application in an outpatient clinic instead of an operating theater, thereby maximizing practicality while minimizing health care costs.
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