Under homeostatic conditions, the cornea is avascular and contains few immune cells, but this changes rapidly following injury. Although the long-term consequences of corneal damage are well characterized, the earliest vascular and immune responses remain poorly understood. Here, we used a murine corneal alkali-burn model to examine limbal vascular activation and leukocyte recruitment immediately and at 2, 6, and 24 hours after injury. Limbal blood vessels underwent immediate dilation; however, vascular leakage into the corneal stroma occurred only in males. Lymphatic capillaries rapidly formed directed extensions toward the injury without significantly increasing their total vascular area, with males exhibiting longer extensions than females. Fluorescent dextran uptake provided evidence that these lymphatic vessels were functionally engaged in early tracer drainage. Despite pronounced vascular activation, early recruitment of neutrophils, monocytes, dendritic cells, macrophages, T cells, B cells, and natural killer cells remained limited. Thus, limbal blood and lymphatic vessels initiate the earliest response to corneal alkali injury before substantial leukocyte infiltration. These findings reveal sex-dependent differences in vascular permeability and lymphatic remodeling and identify the limbal vasculature as an early regulator of corneal inflammation and tissue repair.

Aims/Purpose: Corneal perforation is a medical emergency that can lead to blindness. Treatment options encompass cyanoacrylate or fibrin glue, both linked to side effects, including cytotoxicity. Gelatin methacryloyl‐based biomaterials offer an alternative to these adhesives [1, 2]. Achieving optical clarity and smooth surface integrity is crucial for the restoration of vision in treated corneal wounds.Methods: Currently, the delivery of corneal biomaterials utilizes standard syringe systems, which lack the accuracy to reconstruct the cornea's shape. Therefore, the use of a laser bioprinting technology, developed in the Boutopoulos lab [3], for achieving precise in‐situ corneal wound repair was examined. We used a photocrosslinkable ink comprising Gelatin Methacryloyl (GelMa), hydroxyethyl acrylate (HEA), and Lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate (LAP) as a photoinitiator.Results: Printability was optimized by generating nanoliter‐volume individual droplets using 230 μJ laser energy, a flow rate 20 microliter per minute, and a temperature of 37°C. Rheology, optical characterization, and bursting pressure measurements assessed its potential to seal corneal perforations. Results indicated a hydrogel storage modulus of 1.31 ± 0.31 KPa for printed LiQD cornea and a bursting pressure of 38 ± 6 mmHg when used to seal full thickness cornea perforation in cadaveric pig eyes. Optical clarity akin to the native cornea was observed (%light transmission: 93.12 ± 1.02 printed vs 92.61 ± 1.50). The OCT results showed that the LIST technique could potentially fill the corneal wounds and reconstruct the natural curvature of the wounded cornea.Conclusions: In conclusion, a precise printing system for delivering adhesive corneal regenerative biomaterials to wounds was developed and characterized.References Sharifi, S., et al., 2021. 6(11): p. 3947‐3961. Barroso, I.A., et al., 2022. 9(2). Ebrahimi Orimi, H., et al., Sci Rep, 2020. 10(1): p. 9730.

Biomaterials are helping in efforts to restore vision in the cornea. The entry of corneal biomaterials into clinical application and evaluation has also spurred rapid progress in efforts to restore other anterior segment tissues. It is impossible to discuss all the innovations in this expanding field, so this chapter gives a non-comprehensive overview. Finally, research considerations should include the regulatory and translational pathways in developing bioengineering corneal or anterior segment substitutes or implants.

Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious corneal blindness worldwide. Human donor corneal transplantation remains the primary treatment for scarred corneas resulting from herpes simplex keratitis (HSK), a severe inflammatory corneal disease caused by HSV-1 infection, despite a high risk of re-infection or immune rejection of the allografts. As possible alternatives to donor grafting for HSK, we developed cell-free, regeneration-stimulating corneal implants designed to work even under adverse inflammatory situations such as severe infections. The implants comprised short, fully synthetic collagen-like peptides conjugated to polyethylene glycol (CLP-PEG) and crosslinked using carbodiimide chemistry. Being cell-free, they lacked the cellular targets that an already activated immune system would encounter in these inflamed corneas. We tested the performance of these implants in guinea pig and rabbit models of HSK. Three different HSV-1 strains were used to create experimental HSK in rabbits and guinea pigs. There were no overall statistically significant species differences or species–strain differences in virus-induced mortality. At three months post-operation, all treated corneas showed tissue regeneration, but with haze or neovascularization. The initially cell-free CLP-PEG implants allowed for repopulation by ingrowing cells to regenerate neocorneal tissue, despite the inflammation. However, they did not prevent HSV-1 reactivation nor re-infection, as neovascularization and disorganization were observed within the neocorneas. A detailed histopathological examination revealed viral strain differences, but only KOS infection showed interspecies neovascularization differences. A more detailed examination with larger numbers of animals is merited to fully elucidate the effects of the different viral strains on rabbits versus guinea pigs.

The convenience, versatility, and biocompatibility of photocrosslinkable hydrogel precursors make them promising candidates for developing tissue engineering scaffolds. However, the current library of photosensitive materials is limited. This study reports, for the first time, the modification of quince seed mucilage (QS) with glycidyl methacrylate (GM), resulting in the synthesis of methacrylated QS (QSGM). The chemical composition and structure of QS were analyzed. The effects of reaction time, temperature, QS concentration, and GM/QS ratio on the degree of methacrylation, as well as the physicochemical, rheological, mechanical, and biological properties of the synthesized materials were explored. Chemical characterization using 1H NMR and FTIR confirmed the successful methacrylation of QS. Hydrogels fabricated from QSGMs at a 0.5 wt% concentration exhibited high swelling ratios of 320 to 580 g/g, and compressive strengths between 0.6 +/- 0.1 and 1.2 +/- 0.3 kPa. No significant changes in the rheological properties of hydrogel precursors were observed. Moreover, QSGM-based hydrogels supported cell encapsulation for 14 days with minimal cytotoxicity and immune cell activation. Finally, as a proof of concept, the potential use of QSGM for 3D printing was demonstrated. Overall, the results highlight the significant potential of QSGMs as a biomaterial of choice for soft tissue engineering applications.

Herpes simplex virus type 1 (HSV1) establishes latency in the trigeminal ganglia and reactivates to cause recurrent infections and severe complications, including herpes simplex keratitis (HSK), the leading cause of infectious corneal blindness in developed countries. We developed and characterized a targeted siRNA delivery system designed to suppress HSV1 by silencing the immediate-early gene ICP0, which encodes a protein essential for lytic infection and viral reactivation. To enhance neuronal accumulation, siRNA-loaded liposomes were decorated with nortriptyline (NTP), a ligand with high affinity for neuronal cells, including those in the trigeminal ganglia. The liposomes were optimized to exhibit nanoscale size, low polydispersity, neutral surface charge, high siRNA loading, spherical morphology, and long-term shelf stability. Studies in cell lines confirmed efficient internalization with good tolerability, and targeted liposomes showed enhanced binding to differentiated PC‑12 neuronal cells, competitively inhibited by free NTP, supporting their targeting specificity. Following retro-orbital administration in mice, NTP-targeted siHSV1 liposomes preferentially accumulated in the trigeminal ganglia. Antiviral efficacy was further assessed by topical application of siHSV1‑loaded liposomes within a biocompatible hydrogel in a murine HSV1 corneal perforation model of HSK. In the corneal limbus, targeted liposomes produced a significant reduction in viral load, whereas in the contralateral trigeminal ganglia, they significantly reduced viral load with a pronounced trend toward decreased ICP0 expression. These results demonstrate the feasibility of using NTP-mediated targeting ligand and support the potential of this delivery platform for treating latent HSV1, warranting further optimization for improved therapeutic efficacy.

Purpose: Corneal blindness is mainly treated by cornea transplantation. However, there is a shortage of cornea donors for surgeries in many countries. Even with accessible donors, patients suffering from severe pathologies causing inflammation may not benefit as they are at high risk for graft rejection. The Griffith laboratory is the first to use acellular human collagen implants to regenerate high‐risk corneas in a group of patients. For broader clinical applications, scalable collagen‐like peptide (CLP) synthetic hydrogels are being developed. It is important to identify design patterns to ensure the implants do not cause inflammation.Methods: The growth of stromal fibroblasts cultured on CLP hydrogels with and without RGD groups are compared, with collagen gels and tissue culture plastic as controls. Cellular growth and viability on the different substrates were compared using the AmarBlue assay. Next, TGF‐β was added to the media to stimulate fibroblast differentiation into myofibroblasts. The latter has been linked to corneal scarring. The proportion of resulting myofibroblasts was determined by immunostaining using anti‐smooth muscle actin (SMA) antibody. Stromal cells seeded onto the hydrogels were also stained against fibronectin to study its implication due to RGD. Furthermore, the CLP‐based hydrogel was tested in vivo on alkali burnt BALB/c mice to investigate their practical biocompatibility and efficacy.Results: α‐SMA expression was observed in cells grown on CLP‐RGD containing hydrogels but not on tissue culture plastic or CLP‐only control. There was a positive fibronectin expression by cells cultured on CLP‐RGD hydrogels, on 5% collagen hydrogels and on the issue culture control. The CLP‐RGD hydrogel is a smooth and soft gel with desirable water content. The in vivo test showed that there was no sign of inflammation or angiogenesis in the cornea after three months, which can prove the great biocompatibility of the prepared CLP‐based hydrogel.Conclusions: This project allowed for the testing of cell growth on RGD‐containing CLP hydrogels, which has not been done before. Early results suggest that RGD groups within CLPs induced myofibroblast differentiation and potential pro‐inflammatory responses.

Purpose: To develop a 3-dimensional corneal construct suitable for in vitro studies of disease conditions and therapies . Design: In vitro human corneal constructs were created using chemically crosslinked collagen and chondroitin sulfate extracellular matrix and seeded with 3 human corneal cell types (epithelial, stromal, and endothelial) together with neural cells. The neural cells were derived from hybrid neuroblastoma cells and the other cells used from immortalized human corneal cell lines. To check the feasibility and characterize the constructs, cytotoxicity, cell proliferation, histology, and protein expression studies were performed. Results: Optimized culture condition permitted synchronized viability across the cell types within the construct. The construct showed a typical appearance for different cellular layers, including healthy appearing, phenotypically differentiated neurons. The expected protein expression profiles for specific cell types within the construct were confirmed with western blotting. Conclusions: An in vitro corneal construct was successfully developed with maintenance of individual cell phenotypes with anatomically correct cellular loci. The construct may be useful in evaluation of specific corneal disorders and in developing different corneal disease models. Additionally, the construct can be used in evaluating drug targeting and/or penetration to individual corneal layers, testing novel therapeutics for corneal diseases, and potentially reducing the necessity for animals in corneal research at the early stages of investigation.

EDITORIAL article Front. Pharmacol., 31 January 2024Sec. Integrative and Regenerative Pharmacology Volume 15 - 2024 | https://doi.org/10.3389/fphar.2024.1369505

Pathophysiologic inflammation, e.g., from HSV-1 viral infection, can cause tissue destruction resulting in ulceration, perforation, and ultimately blindness. We developed an injectable Cornea-in-a-Syringe (CIS) sealant-filler to treat damaged corneas. CIS comprises linear carboxylated polymers of inflammation-suppressing 2-methacryloyloxyethyl phosphorylcholine, regeneration-promoting collagen-like peptide, and adhesive collagen-citrate glue. We also incorporated GF19, a modified anti-viral host defense peptide that blocked HSV-1 activity in vitro when released from silica nanoparticles (SiNP-GF19). CIS alone suppressed inflammation when tested in a surgically perforated and HSV-1-infected rabbit corneal model, allowing tissue and nerve regeneration. However, at six months post-operation, only regenerated neocorneas previously treated with CIS with SiNP-GF19 had structural and functional features approaching those of normal healthy corneas and were HSV-1 virus-free. We showed that composite injectable biomaterials can be designed to allow regeneration by modulating inflammation and blocking viral activity in an infected tissue. Future iterations could be optimized for clinical application.

Cornea blindness is still treated mainly by corneal transplantation, using donated allograft human corneas. However, a severe global shortfall leaves an estimated 12.7 million patients on waiting lists despite methods of using one cornea for two different partial‐thickness grafts. Even more individuals are not on waiting lists because they have no access to transplantation in their countries or regions. However, even if donor tissues were readily available, patients with inflamed, severely damaged corneas are at high risk of rejecting conventional transplantation. Various solutions have been proposed as alternatives to corneal allografting, including the development of keratoprostheses and stem cell grafting. We posited using cell‐free implants that mimic the extracellular matrix of the human cornea to recapitulate the environment during corneal development and stimulate the endogenous cells within the patient's eyes to affect regeneration. We used cell‐free corneal implants made from chemically crosslinked recombinant human collagen to promote in situ tissue regeneration in pathologic corneas, including high‐risk ones. We successfully developed and tested recombinant human solid collagen‐based implants that promoted stable regeneration in inflamed human patient corneas based on phosphorylcholine (MPC). However, solid corneas require a full operating theatre and staffing, which is costly and not accessible to more remote regions in Canada and globally. Burns and severe infections that can lead to perforations are medical emergencies that require immediate methods to seal the eye. In many centres, cyanoacrylate glue is used as a sealant, but this is toxic and kills surrounding cells, often necessitating follow‐on transplantation. We developed a synthetic, biocompatible, and adhesive liquid hydrogel (LiQD Cornea), which would replace these toxic cyanoacrylate glues. LiQD cornea is applied as a liquid but quickly adheres and gels within corneal tissue defects like a dental filling. It can be used to fill ulcers and other damaged areas. Then its similarity at a molecular level to a natural tissue framework promotes tissue regeneration, treating corneal perforations effectively without transplantation. Further, due to the synthetic nature of the material, risks associated with disease transmission are reduced compared to natural products. We see LiQD Cornea potentially used in outpatient clinics instead of operating theatres, maximizing practicality and minimizing health care costs.

Corneal HSV-1 infections are a leading cause of infectious blindness globally by triggering tissue damage due to the intense inflammation. HSV-1 infections are treated mainly with antiviral drugs that clear the infections but are inefficient as prophylactics. The body produces innate cationic host defence peptides (cHDP), such as the cathelicidin LL37. Various epithelia, including the corneal epithelium, express LL37. cHDPs can cause disintegration of pathogen membranes, stimulate chemokine production, and attract immune cells. Here, we selected GF17, a peptide containing the LL37 fragment with bioactivity but with minimal cytotoxicity, and added two cell-penetrating amino acids to enhance its activity. The resulting GF19 was relatively cell-friendly, inducing only partial activation of antigen presenting immune cells in vitro. We showed that HSV-1 spreads by tunneling nanotubes in cultured human corneal epithelial cells. GF19 given before infection was able to block infection, most likely by blocking viral entry. When cells were sequentially exposed to viruses and GF19, the infection was attenuated but not arrested, supporting the contention that the GF19 mode of action was to block viral entry. Encapsulation into silica nanoparticles allowed a more sustained release of GF19, enhancing its activity. GF19 is most likely suitable as a prevention rather than a virucidal treatment.

Bioinspired synthetic materials can be designed as reliable, cost-effective, and fully controlled alternatives to natural biomaterials for treating damaged tissues and organs. However, several hurdles need to be overcome for clinical translation, particularly for biomaterials gelled in situ. These include the potential toxicity of chemical crosslinkers used in the materials' assembly or breakdown products they generate and the challenges of fine-tuning the mechanical properties of the materials. Here, a minimalistic, adhesive soft material is developed by screening hundreds of potential formulations of self-assembling, custom-designed collagen-like peptide sequences for the in situ formation of tissue-bonding 3D hydrogels. Nine promising formulations for tissue repair are identified using a low-volume and rapid combinatory screening approach. It is shown that simply varying the ratio of the two key components promotes adhesion and fine-tunes the material's mechanical properties. The materials' skin and heart repair capabilities are assessed in vitro and clinically relevant animal models. The materials are also tested for corneal applications using ex vivo pig cornea models complemented by in vitro cell compatibility assays. Two components materials are presented that uses self-assembling, custom-designed collagen-like peptide sequences for on-the-spot formation of tissue-bonding 3D hydrogels. It is shown that simply varying the ratio of the components promotes adhesion and fine-tunes the material's mechanical properties. The materials' skin and heart repair capabilities are assessed& in vitro and clinically relevant animal models and using ex vivo pig cornea models. image

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The cornea is an ideal testing field for cell therapies. Its highly ordered structure, where specific cell populations are sequestered in different layers, together with its accessibility, has allowed the development of the first stem cell-based therapy approved by the European Medicine Agency. Today, different techniques have been proposed for autologous and allogeneic limbal and non-limbal cell transplantation. Cell replacement has also been attempted in cases of endothelial cell decompensation as it occurs in Fuchs dystrophy: injection of cultivated allogeneic endothelial cells is now in advanced phases of clinical development. Recently, stromal substitutes have been developed with excellent integration capability and transparency. Finally, cell-derived products, such as exosomes obtained from different sources, have been investigated for the treatment of severe corneal diseases with encouraging results. Optimization of the success rate of cell therapies obviously requires high-quality cultured cells/products, but the role of the surrounding microenvironment is equally important to allow engraftment of transplanted cells, to preserve their functions and, ultimately, lead to restoration of tissue integrity and transparency of the cornea.

Retinal degenerative diseases affect millions of people worldwide, and legal blindness is generally associated with the loss of cone photoreceptors located in the central region of the retina called the macula. Currently, there is no treatment to replace the macula. Addressing this unmet need, we employed control isogenic and hypoimmunogenic induced pluripotent stem cell lines to generate spontaneously polarized retinal sheets (RSs). RSs were enriched in retinal progenitor and cone precursor cells, which could differentiate into mature S- and M/L-cones in long-term cultures. Single-cell RNAseq analysis showed that RSs recapitulate the ontogeny of the developing human retina. Isolation of neural rosettes for sub-retinal transplantation effectively eliminated unwanted cells such as RPE cells. In a porcine model of chemically induced retinal degeneration, grafts integrated the host retina and formed a new, yet immature, photoreceptor layer. In one transplanted animal, functional and immunohistochemical assays suggest that grafts exhibited responsiveness to light stimuli and established putative synaptic connections with host bipolar neurons. This study underscores the potential and challenges of RSs for clinical applications.