Corneal Repair Research Articles

Emerging tissue engineering strategies for the corneal regeneration.

Cornea as the outermost layer of the eye is at risk of various genetic and environmental diseases that can damage the cornea and impair vision. Corneal transplantation is among the most applicable surgical procedures for repairing the defected tissue. However, the scarcity of healthy tissue donations as well as transplantation failure has remained as the biggest challenges in confront of corneal grafting. Therefore, alternative approaches based on stem-cell transplantation and classic regenerative medicine have been developed for corneal regeneration. In this review, the application and limitation of the recently-used advanced approaches for regeneration of cornea are discussed. Additionally, other emerging powerful techniques such as 5D printing as a new branch of scaffold-based technologies for construction of tissues other than the cornea are highlighted and suggested as alternatives for corneal reconstruction. The introduced novel techniques may have great potential for clinical applications in corneal repair including disease modeling, 3D pattern scheming, and personalized medicine.

Evaluation of CRISPR/Cas9 mediated TGIF gene editing to inhibit corneal fibrosis in vitro

Corneal wound healing is influenced by many factors including transcriptional co-repressors and co-activators. Interactions of co-activators and co-repressors with Smads influence mechanistic loop facilitating transcription of alpha-smooth muscle actin (α-SMA), a key profibrotic gene, in corneal repair. The role of a transcriptional repressor, 5′TG3′-interacting factor (TGIF), in the regulation of α-SMA and myofibroblast formation in the cornea was shown previously by our group. This study tested a hypothesis if TGIF1 gene editing via CRISPR/Cas9 can ease myofibroblast formation in the cornea using an in vitro model. Primary human corneal stromal fibroblasts (hCSFs) generated from donor corneas received gene-editing plasmid facilitating loss (CRISPR/Cas9 knockout) or gain (CRISPR activation) of TGIF function by UltraCruz transfection reagent. Phase-contrast microscopy, immunoblotting, immunocytochemistry and quantitative polymerase chain reaction (qPCR) were used to measure levels of myofibroblast profibrotic genes (α-SMA, fibronectin, Collagen-I, and Collagen-IV) in hCSFs lacking or overexpressing TGIF1 after growing them in± transforming growth factor beta1 (TGF-β1) under serum-free conditions. The CRISPR-assisted TGIF1 activation (gain of function) in hCSFs demonstrated significantly decreased myofibroblast formation and messenger ribonucleic acid (mRNA) and protein levels of profibrotic genes. Conversely, CRISPR/Cas9-assisted TGIF knockdown (loss of function) in hCSFs demonstrated no significant change in the levels of myofibroblast formation or profibrotic genes under similar conditions. These results suggest that TGIF gene-editing approach can be employed to modulate the transcriptional activity of α-SMA in controlling pathological and promoting physiological wound healing in an injured cornea.

Collagen Film with Bionic Layered Structure and High Light Transmittance for Personalized Corneal Repair Fabricated by Controlled Solvent Evaporation Technique.

Corneal blindness is a common phenomenon, and corneal transplantation is an effective treatment for corneal defects. However, there is usually a mismatch between the corneal repair material and the degree of the patient’s corneal defect. Therefore, patients with different corneal defects need suitable corneal repair materials with a specific microstructure for personalized treatment. In this research, collagen films with bionic structures were fabricated through ethanol evaporation technique by regulating the volume ratios of collagen solution: ethanol = 10:0(Col)/9:1(CC91)/8:2(CC82)/CC73(CC73). Under various preparation conditions, the obtained collagen films contain layered structures of different density. SEM photos show that the CC73 film with a dense layer arrangement has a microstructure similar to that of the corneal epithelial layer, whereas the Col film has a loose layered structure similar to that of the corneal stroma layer. Four kinds of collagen films showed different optical properties and water absorption ability. A more ordered arrangement of internal layer structure leads to better mechanical properties of the collagen film. In view of this, we think that these collagen films with different microstructures and different interlayer spacing may have huge potential applications for personalized corneal repair.

An original amino acid formula favours in vitro corneal epithelial wound healing by promoting Fn1, ITGB1, and PGC-1α expression

Corneal disorders are frequent, involving most diabetic patients; among its manifestations, they include delayed wound healing. Since maintenance of mitochondrial homeostasis is fundamental for the cell, stimulation of mitochondrial biogenesis represents a unique therapeutic tool for preventing and treating disorders with a deficit in energy metabolism. We have recently demonstrated that a branched-chain amino acid (BCAA)-enriched mixture (BCAAem) supported mitochondrial biogenesis in cardiac and skeletal muscle, reduced liver damage caused by alcohol, and prevented the doxorubicin-dependent mitochondrial damage in cardiomyocytes. The present study aimed to investigate a new amino acid mixture, named six amino acids (6AA), to promote corneal epithelial wound healing by regulating mitochondrial biogenesis. A murine epithelium cell line (TKE2) exposed to this mixture showed increased mitochondrial biogenesis markers, fibronectin 1 (Fn1) and integrin beta 1 (ITGB1) involved in extracellular matrix synthesis and cell migration. Most importantly, the 6AA mixture completely restored the wound in scratch assays, confirming the potential of this new formula in eye disorders like keratopathy. Moreover, our results demonstrate for the first time that peroxisome proliferator-receptor γ coactivator 1 α (PGC-1α) is expressed in TKE2 cells, which controls mitochondrial function and corneal repair process. These results could be relevant for the treatment mainly focused on corneal re-epithelialisation.

Scaffold-Free Strategy Using a PEG-Dextran Aqueous Two-Phase-System for Corneal Tissue Repair.

Forming thin tissue constructs with minimal extracellular matrix surrounding them is important for tissue engineering applications. Here, we explore and optimize a strategy that enables rapid fabrication of scaffold-free corneal tissue constructs using the liquid-liquid interface of an aqueous two-phase system (ATPS) that is based on biocompatible polymers, dextran and polyethylene glycol. Intact tissue-like constructs, made of corneal epithelial or endothelial cells, can be formed on the interface between the two liquid phases of ATPS within hours and subsequently collected simply by removing the liquid phases. The formed corneal cell constructs express essential physiological markers and have preserved viability and proliferative ability in vitro. The corneal epithelial cell constructs are also able to re-epithelialize the corneal epithelial wound in vitro. The results suggest the promise of our reported strategy in corneal repair.

VEGF-A in Serum and Plasma Rich in Growth Factors (PRGF) Eye Drops.

Vascular endothelial growth factor (VEGF)-A, the most abundant subtype of the VEGF family in the eye, plays an important role in corneal homeostasis due to its ability to mediate corneal nerve repair. Repeated intravitreal anti-VEGF injections were shown to significantly reduce corneal nerve density, which might negatively affect corneal homeostasis and lead to a neuropathic dry eye disease. Currently, there are two effective modalities to treat dry eye while supplying VEGF to the ocular surface: serum eye drops (SED) and eye drops manufactured from plasma rich in growth factors (PRGF). The purpose of this study was to measure the VEGF-A concentration in SED and PRGF eye drops. Ten healthy volunteers donated blood on two separate occasions, 2 - 8 days apart. Thus, a total of 20 blood samples were processed to obtain both SED and PRGF. Concentrations of VEGF-A were quantified by a Simple Plex platform run in triplicate. The VEGF-A concentration in SED and PRGF was very similar between the two blood samples drawn from one individual donor but showed substantial interindividual variability. However, in all 20 samples, VEGF concentrations were substantially higher in SED samples (mean 238.7 ± 146.6 pg/mL) compared to PRGF samples (mean 67.4 ± 46.3 pg/mL). Based upon the analysis of variance (ANOVA) model for the measured concentrations with fixed effects for specimen (SED vs. PRGF) and subject, the mean difference between the SED and PRGF concentration was 168.1 pg/mL (95% confidence interval: [142.4, 193.9], p < 0.001). Our study showed that the VEGF concentration was higher in SED than in PRGF. This is an important finding, particularly for potential treatment of dry eye disease in patients with neuropathic eye disease, especially in patients that received repeated anti-VEGF intravitreal injections, or in patients with Sjögren's disease, where the level of VEGF in tears might be pathologically decreased. Hypothetically, VEGF might be needed to restore ocular surface homeostasis. Although growing evidence has shown that VEGF-A plays an important role in corneal homeostasis, only a randomized prospective clinical trial will show whether supplying VEGF-A to the ocular surface might successfully restore the corneal homeostasis and overcome the problem of corneal neuropathy in these patients. For such a trial, based on our results, an undiluted SED should be preferred over a PRGF due to the higher content of VEGF-A.

WNT16B enhances the proliferation and self-renewal of limbal epithelial cells via CXCR4/MEK/ERK signaling.

SummaryCulture of limbal epithelial cells (LECs) provides the principal source of transplanted limbal stem cells (LESCs) for treatment of limbal-stem-cell deficiency. Optimization of the culture conditions for in-vitro-expanded LECs will help to create a graft with an optimized quality and quantity of LESCs. This study aimed to investigate the effects of WNT16B on LECs and corneal wound healing and the underlying mechanism. Treatment with exogenous WNT16B increased the proliferative capacity and self-renewal of LECs in the cultures. We further revealed that C-X-C chemokine receptor type 4 (CXCR4) was vital for the effects of WNT16B, and activation of CXCR4/MEK/ERK signaling was pivotal in mediating the effects of WNT16B on LECs enriched for LESCs. The stimulatory effect of WNT16B on corneal epithelial repair was confirmed in a mouse corneal-wound-healing model. In summary, WNT16B enhances proliferation and self-renewal of LECs via the CXCR4/MEK/ERK signaling cascade and accelerates corneal-epithelial wound healing.

Photocurable antimicrobial silk-based hydrogels for corneal repair.

Corneal transplantation is the current gold standard treatment to restore visual acuity to patients with severe corneal diseases and injuries. Due to severe donor tissue shortage, efforts to develop a corneal equivalent have been made but the challenge remains unmet. Another issue of concern in ocular surgery is the difficult instillation and fast drainage of antibiotic ocular eye drops as bacterial infections can jeopardize implant success by delaying or impairing tissue healing. In this study, we developed antimicrobial silk‐based hydrogels that have the potential to be photoactivated in situ, fully adapting to the corneal injury shape. Gentamicin‐loaded methacrylated‐silk (SilkMA) hydrogels were prepared within minutes using low UV intensity (3 mW/cm2). SilkMA gels provided a Young's modulus between 21 and 79 kPa together with a light transmittance spectrum and water content (83%–90%) similar to the human cornea. Polymer concentration (15%–25%) was found to offer a tool for tailoring the physical properties of the hydrogels. We confirmed that the methacrylation did not affect the material's in vitro degradation and biocompatibility by observing fibroblast adhesion and proliferation. Importantly, agar diffusion tests showed that the synthesized hydrogels were able to inhibit Staphylococcus aureus and Pseudomonas aeruginosa growth for 72 h. These characteristics along with their injectability and viscoelasticity demonstrate the potential of SilkMA hydrogels to be applied in several soft tissue engineering fields. As such, for the first time we demonstrate the potential of photocurable antimicrobial SilkMA hydrogels as a novel biomaterial to facilitate corneal regeneration.

A Light-Curable and Tunable Extracellular Matrix Hydrogel for In Situ Suture-Free Corneal Repair.

Corneal injuries are a major cause of blindness worldwide. To restore corneal integrity and clarity, there is a need for regenerative bio-integrating materials for in-situ repair and replacement of corneal tissue. Here, we introduce Light-curable COrnea Matrix (LC-COMatrix), a tunable material derived from decellularized porcine cornea extracellular matrix containing un-denatured collagen and sulfated glycosaminoglycans. It is a functionalized hydrogel with proper swelling behavior, biodegradation, and viscosity that can be cross-linked in situ with visible light, providing significantly enhanced biomechanical strength, stability, and adhesiveness. Cross-linked LC-COMatrix strongly adheres to human corneas ex vivo and effectively closes full-thickness corneal perforations with tissue loss. Likewise, in vivo, LC-COMatrix seals large corneal perforations, replaces partial-corneal stromal defects and bio-integrates into the tissue in rabbit models. LC-COMatrix is a natural ready-to-apply bio-integrating adhesive that is representative of native corneal matrix with potential applications in corneal and ocular surgeries.

Drug-free contact lens based on quaternized chitosan and tannic acid for bacterial keratitis therapy and corneal repair

Bacterial keratitis (BK) and related inflammatory diseases causes irreversible damage to the corneal tissue. In this study, a novel polyacrylamide semi-interpenetrating network hydrogel including quaternized chitosan and tannic acid (PAM-QCS-TA) were used to construct a novel antibacterial and antioxidant contact lens. The obtained hydrogels showed high water content (>85%), swelling resistance, light transmittance (>90%) and adjustable mechanical property. Both quantitative and qualitative antibacterial experiments against Staphylococcus aureus and Escherichia coli (E. coli) indicated excellent sterilization function especially against E. coli (almost 100%). Due to the presence of tannin acid, it showed obvious antioxidant properties, which relieved oxidative stress and protect cells from reactive oxygen species-induced cytotoxicity. Animal experiments also indicated the shortened treatment time of BK (only 3 days) as well as the protection of eye tissue structure. Therefore, such drug-free antibacterial and antioxidant contact lens avoiding the development of drug resistance is a potential candidate in ocular infectious and inflammatory diseases treatment.

Multifunctional Baicalin-Modified Contact Lens for Preventing Infection, Regulating the Ocular Surface Microenvironment and Promoting Corneal Repair.

Corneal injury inevitably leads to disruption of the ocular surface microenvironment, which is closely associated with delayed epithelial cell repair and the development of infection. Recently, drug-loaded therapeutic contact lenses have emerged as a new approach to treating corneal injury due to their advantages of relieving pain, promoting corneal repair, and preventing infection. However, few therapeutic contact lenses could modulate the ocular surface’s inflammation and oxidative stress microenvironment. To address this, in this study, we covalently immobilized multifunctional baicalin (BCL), a flavon molecular with anti-inflammatory, anti-oxidative stress, and antibacterial capabilities, onto the surface of the contact lens. The BCL-modified contact lens showed excellent optical properties, powerful antibacterial properties, and non-toxicity to endothelial cells. Furthermore, the BCL-modified contact lens could significantly modulate the ocular surface microenvironment, including inhibition of macrophage aggregation and resistance to epithelium damage caused by oxidative stress. In animal models, BCL-modified corneal contact lens effectively promoted corneal epithelial cells repair. These excellent properties suggested that multifunctional BCL molecules had great application potential in the surface engineering of ophthalmic medical materials.

Characterization of Recruited Mononuclear Phagocytes following Corneal Chemical Injury.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12−/− mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6Chigh (proinflammatory) cells being most abundant at 1 day post-injury. Ly6Cint cells were highly expressed at 3 days post-injury and Ly6Cneg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c+ dendritic cells were abundant in corneas from Mmp12−/− mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

DsRNA Induced IFNβ-MMP13 Axis Drives Corneal Wound Healing.

PurposeCornea, the outermost transparent layer of the eye, is the first line of defense against external threats. Following injury, the wound healing response is crucial to corneal repair and regeneration, yet its underlying mechanism is poorly understood. Our study was designed to investigate the role of dsRNA and its regulatory network in corneal wound healing.MethodsA corneal wound healing model was established via the surgical removal of half of the corneal surface and adjoining limbus. RNase III was then used to clarify the role of dsRNA in corneal wound closure and RNA-seq was performed to investigate the mechanism of dsRNA in the healing process. Related gene expression was assessed using immunofluorescence staining, qPCR, and Western blot. Flow cytometry and scratch assay were used to analyze the proliferation and migration of limbal stem/progenitor cells (LSCs) in vitro and functional analysis of the target genes was completed using the corneal wound healing model.ResultsCorneal wound healing was delayed and impaired when the dsRNAs were removed or damaged following RNase III digestion. The dsRNAs released following corneal damage activate type I interferon (IFN-I) signaling, primarily IFNβ, via the corneal epithelium and neutralizing IFNβ or blocking IFN-I signaling delays corneal wound closure. Moreover, our data identified MMP13 as a downstream effector of IFNβ where its expression promotes LSC proliferation and enhances corneal epithelial reconstruction in vivo.ConclusionsThe dsRNA induced IFNβ-MMP13 axis plays a key role in corneal wound healing.

SUV39H1 regulates corneal epithelial wound healing via H3K9me3-mediated repression of p27

BackgroundCorneal epithelial wound healing (CEWH) is vital for maintaining the integrity and barrier function of the cornea. Although histone modifications mediating gene expression patterns is fundamental in some other tissues, it remains unclear whether these gene regulation patterns underlie CEWH. Suppressor of variegation 3-9 homolog 1 (SUV39H1) plays a vital role in mediating gene silencing via histone H3 trimethylation of lysine 9 (H3K9me3). This study aims to characterize the comprehensive signature of epigenetic modifiers and determine the role of SUV39H1 in CEWH.MethodsNanoString nCounter technology was used to detect the differentially expressed epigenetic modifiers during CEWH. Bioinformatic analyses were performed to reveal their involvement in this process. After knockdown of SUV39H1 with siRNA transfection, we determined the function of SUV39H1 on cell proliferation and migration in human corneal epithelial cells (HCECs) via MTS, EdU, and wound-healing assay, respectively. Flow cytometry analysis further confirmed the effect of SUV39H1 on the cell cycle of HCECs. Loss-of-function assays for SUV39H1 with siRNA injection or chaetocin assessed the role of SUV39H1 on CEWH in vivo. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting characterized the expression of SUV39H1 and its target genes. Chromatin immunoprecipitation assay was used to evaluate the distributions of H3K9me3 marks at the promoters of SUV39H1 target genes.ResultsWe first identified 92 differentially expressed epigenetic modifiers and revealed their involvement during CEWH. SUV39H1 was confirmed to be upregulated in response to corneal injury. Its downregulation significantly inhibited HCEC proliferation and retarded in vivo CEWH. Furthermore, knockdown of SUV39H1 upregulated the p27 expression level and reduced H3K9me3 marks at p27 promoter in HCECs. In addition, p27 was remarkably downregulated with elevated H3K9me3 marks at its promoter during in vivo CEWH.ConclusionsSUV39H1 plays a critical role in regulating corneal epithelial cell proliferation via H3K9me3-mediated suppression of p27 during CEWH. Our findings suggest that epigenetic modifiers such as SUV39H1 can be potential therapeutic approaches to accelerate corneal repair.

Advances in Regulatory Strategies of Differentiating Stem Cells towards Keratocytes.

Corneal injury is a commonly encountered clinical problem which led to vision loss and impairment that affects millions of people worldwide. Currently, the available treatment in clinical practice is corneal transplantation, which is limited by the accessibility of donors. Corneal tissue engineering appears to be a promising alternative for corneal repair. However, current experimental strategies of corneal tissue engineering are insufficient due to inadequate differentiation of stem cell into keratocytes and thus cannot be applied in clinical practice. In this review, we aim to clarify the role and effectiveness of both biochemical factors, physical regulation, and the combination of both to induce stem cells to differentiate into keratocytes. We will also propose novel perspectives of differentiation strategy that may help to improve the efficiency of corneal tissue engineering.

Clinical characteristics and functional outcome of pediatric ocular trauma in a third level reference hospital in Guadalajara, Mexico.

Ocular trauma is one of the leading causes of decreased visual acuity and monocular blindness in the pediatric population. Since its occurrence is potentially preventable, it is essential to understand its demographic characteristics and risk factors to alert and implement prevention and health promotion programs. We conducted a retrospective study in which we reviewed 187 clinical records of patients ≤ 15 years old with the diagnosis of severe ocular trauma admitted in 2017 to the Hospital Civil de Guadalajara. We analyzed demographic variables, circumstances of the event, type of treatment, and the evolution of visual acuity and complications. In total, 187 patients and 188 eyes were included; the average age was 6.99 ± 4.1 years. Children of 2 to 5 years of age (41%) were the most affected, and a higher occurrence was observed in males (73%). Open ocular trauma was the most common type of injury (72.7%) caused by sharp objects (45.4%). Most injuries occurred at home (78.1%), without adult supervision (48.1%), and during leisure time (74.9%). Most events were accidental (80.4%), although 12.3% were direct aggression. Surgery was required in 179 patients, in whom corneal injury repair (43.2%) was the most commonly used procedure. Final visual acuity was quantified in 132 eyes, and visual acuity < 20/200 was found in 18.1%. Loss of 4.2% of the eyeballs was recorded. The highest proportion of cases was identified in male patients ≤5 years old, unsupervised by an adult. Many eye injuries are preventable, so it is necessary to implement socio-educational programs in alliance with pediatric organizations that alert the severity of the problem and promote safe environments.

Corneal wounds repair emergencies during COVID‐19: 10/0 absorbable polyglactin sutures as a valuable strategy

AbstractPurposeThe aim of this study is to report the use of Vicryl 10‐0 (polyglactin 910) resorbable monofilament in corneal trauma during COVID‐19 pandemic: structural and functional results, and follow‐up adaptation during health crisis.MethodsDuring the COVID‐19 pandemic, nine patients were selected from the overall population of open or closed globe surgical emergencies (n = 37) in a french University Hospital. Selected patients were eligible for corneal sutures with Vicryl 10‐0, thus replacing the traditional 10‐0 nonabsorbable monofilament nylon suture. Postoperative treatment combined anti‐inflammatory drugs, topical antibiotics and lubricating drops. Outpatient visits were performed at D10, M2, M6 then every six month. Interim visits were performed if needed between D10 and M2 by teleconsultation. Patients received complete examination including different imaging. The main outcome was best corrected visual acuity. Secondary outcomes included average corneal astigmatism and the occurrence of complications.ResultsMean distance and near acuity improved from 0.711 ± 0.805 &amp; 0.556 ± 0.305 logMAR preoperatively to 0.056 ± 0.073 &amp; 0.333 ± 0.100 logMAR postoperatively (p = 0.032 and p = 0.043 respectively). Mean astigmatism decreased from 4.91 ± 4.21 diopters preoperatively to 0.99 ± 0.57 after suture resorption (p = 0.020). There were no serious adverse events or repeated surgery.ConclusionsPolyglactin 910 10‐0 could provide a maximum infectious risk reduction; a shorter topical treatment and antibiotic prophylaxis. The absorbable nature of the sutures simplifies logistics by reducing the number of early outpatient visits before M2. It avoids suture removal, especially for the pediatric population. Interim teleconsultation(s) on demand are interesting to evaluate the need for an additional face‐to‐face consultation. Vicryl 10‐0 sutures are ideally suited to the COVID‐19 pandemic logistical constraints, as a safe and effective alternative to nylon 10‐0 for the management of eligible corneal wounds.

Pars Plana Capsulectomy with Scissors and Micro Forceps: A Case Report of an Alternative Approach for Membranous Posterior Capsule Opacity

Pediatric eye injuries are the leading cause of unilateral blindness in childhood. Post-traumatic cataract development is common, and the risk of posterior capsule opacity occurrence is seen at a high rate. Nd-YAG laser capsulotomy or anterior vitrectomy may useful, but some patients develop thick posterior membranous capsule opacity and mentioned techniques may not be performed. A 6-year-old girl who had a corneal tear repair, traumatic cataract and thick posterior capsule membrane, which could not be cleared by Nd-YAG laser or vitrectomy, was removed with scissors and micro forceps through the pars plana vitrector entrances. Here, we present a case report as an alternative method of posterior capsulectomy.

Effects of Gelatin Methacrylate Hydrogel on Corneal Repair and Regeneration in Rats.

PurposeThis study investigates the repairing process of rat cornea after surgery of lamellar keratoplasty (LKP) and evaluates the effects of gelatin methacrylate (GelMA) hydrogel.MethodsIn the LKP group, the lamellar stroma matrixes of Sprague-Dawley rats were transplanted to enhanced green fluorescent protein rats, whereas those in the GelMA group were also embedded with a GelMA hydrogel during the corneal transplantation. Grafted eyes were harvested on days seven, 30, and 90. Hematoxylin and eosin staining, immunofluorescence staining, scanning electron microscopy, optical coherence tomography, and a slit-lamp microscope were used to study the process of corneal restoration and regeneration.ResultsA total of 42 rats were analyzed, including 18 rats in each of the experimental group and six rats in the control group. After three months, the infiltration degree of inflammatory cells differed between the LKP group and the GelMA group (P < 0.001). Moreover, in multiple comparisons in corneal thickness, significant difference was observed between the LKP group and the GelMA group. There was also divergence in the results between the LKP group and the control group (P < 0.001, P < 0.001). At the same time, the expression of α-smooth muscle actin (α-SMA) and transforming growth factor (TGF)-β1 varied distinctly between the LKP group and the GelMA group (P < 0.05, P < 0.001).ConclusionsSignificant differences were demonstrated between the LKP group and the GelMA group in inflammatory cell infiltration, corneal thickness, as well as the expression of α-SMA and TGF-β1. Those differences indicate the ability of GelMA hydrogel to support alleviation in corneal stroma fibrosis and show the influences of fibrosis in the dysfunction of corneal refractive power.Translational RelevanceOur research provides new ideas for the future development of LKP and tissue-engineered corneas.

Eph signaling is regulated by miRNA-210: Implications for corneal epithelial repair.

A distinct boundary exists between the progenitor cells in the basal limbal epithelium and the more differentiated corneal epithelial basal cells. We have shown that reciprocal expression patterns of EphA2 and Ephrin-A1 are likely to contribute to normal limbal-corneal epithelial compartmentalization as well as play a role in response to injury. How this signaling axis is regulated remains unclear. We have demonstrated that microRNAs (miRNAs) play critical roles in corneal epithelial wound healing and several miRNAs (e.g. miR-210) have been predicted to target ephrins. Previous expression profiling experiments demonstrated that miR-210 is prominently expressed in corneal epithelial cells. RNA-seq data acquired from miR-210-depleted HCECs showed up-regulation of genes involved in cellular migration. In addition, miR-210 is decreased after corneal injury while EphA2 is increased. Moreover, antago-210-treated HCECs markedly enhanced wound closure in a scratch wound assay. Antago-210 treatment resulted in increased EphA2 protein levels as well as pS897-EphA2, the pro-migratory form of EphA2. As expected, Ephrin-A1levels were reduced, while levels of a well-known target of miR-210, Ephrin-A3, were increased by antago-210 treatment. The increase in migration with antago-210 could be inhibited by Ephrin-A1 overexpression, Ephrin-A1-Fc treatment or siRNA depletion of EphA2. However, depletion of Ephrin-A3 did not have effects on the antago-210-induced increase in migration. In addition, Ephrin-A1 overexpression and siEphA2 dampened EGFR signaling, which is increased by antago-210. Our data clearly demonstrate a link between miR-210 and EphA2/Ephrin-A1signaling that regulates, in part, corneal epithelial migration. This interaction might potentially control the limbal-corneal epithelial boundary.