Bioactive peptides-incorporated photo-crosslinking hydrogel for suture-free repair of corneal injuries.

Epidermal growth factor (EGF) plays an important role in corneal epithelial migration and proliferation to improve the wound healing process. This study aimed to understand the role of NFκB in EGF-induced corneal epithelial wound healing through regulation of CTCF activity, which plays important roles in cell motility and migration to promote wound healing. The effect of NFκB p50 on corneal epithelial wound healing was investigated by comparing the eyes of wild-type and p50 knockout mice. We found that there was a significant retardation in corneal epithelial wound healing in the corneas of p50 knockout mice. Wound closure rates were measured in human corneal epithelial cells transfected with an NFκB activation-sensitive CTCF expression construct to demonstrate the effect of human CTCF expression under the control of EGF-induced NFκB activation on wound healing. EGF stimulation activated NFκB, which directly triggered the expression of the exogenous human CTCF in transfected cells and, subsequently, promoted human corneal epithelial cell motility, migration, and wound healing. Overexpression of CTCF in corneal epithelial cells and mouse corneas significantly enhanced the wound healing process. Furthermore, the effect of overexpressing NFκB p50 in corneal epithelial cells on the promotion of wound healing was abolished by knockdown of CTCF with CTCF-specific shRNA. Thus, a direct regulatory relationship between EGF-induced NFκB p50 and CTCF activation affecting corneal epithelial wound healing has been established, indicating that CTCF is, indeed, a NFκB p50-targeted and effective gene product in the core transcriptional network downstream from the growth factor-induced NFκB signaling pathway.

This study aimed to investigate the role of Gelam honey (GH) in accelerating reepithelialization of corneal abrasion. Corneal epithelial cells (CEC) isolated from New Zealand white rabbit corneas, were cultured and circular-shaped wounds were created onto them, representing the corneal abrasion model. These wounds were treated with basal (BM) and cornea media (CM) supplemented with GH. The percentage of wound closure was measured on day 0, 3, and 5. Expressions of cytokeratin 3 (CK3), cluster of differentiation 44 (CD 44), and connexin 43 (Cx43) were analyzed via qRT-PCR and immunocytochemistry. The results showed CEC cultured in GH-enriched media reepithelialized faster compared to control. Corneal abrasion treated with CM supplemented with GH closed completely on day 5. CK3, CD44, and Cx43 expressions correspond to the stages of reepithelialization. In conclusion, GH promotes the healing of the ex vivo corneal abrasion model. Further explorations of its potential as adjuvant therapy in treating corneal injuries are needed. PRACTICAL APPLICATIONS: Honey has been reported to have many medicinal properties including antibacterial, anti-inflammatory, and the ability to promote skin wound healing. However, the effects of honey on corneal wound healing have not been fully elucidated. In the present study, we aimed to determine the effects of Gelam honey (GH), well-known local honey obtained from the beehive of Gelam trees (Melaleuca spp.), on the ex vivo corneal abrasion model via cell migration study and analysis of genes and proteins during corneal epithelial wound healing. GH has proven to have accelerated effects on the corneal epithelial cell migration during the closure of the ex vivo corneal abrasion wound model. The expressions of the genes and proteins of the corneal epithelial wound healing markers were in accordance with the stages of healing. Therefore, GH has the potential to be developed as adjuvant therapy in the form of GH-based eye drop in treating corneal injuries.

ObjectiveThe repair of corneal epithelial injury is essential to maintain the cornea integrity and transparency, and the molecular regulation mechanism is still unclear. CALR promotes wound healing through a variety of biological effects. Therefore, this study explored effect and mechanism of CALR on corneal epithelial wound healing.MethodsThe model of repairing corneal epithelium injury in mice was established, and corneal epithelial tissues were collected from the model group and the control group. oe-CALR or sh-Wnt7a was transfected into HCE-2[50.B1] cells by Lipofectamine 2000 to over-express CALR or knock down Wnt7a in vitro. CALR mRNA expression was detected by RT-qPCR. CCK-8, clone formation assay, cell senescence, flow cytometry, wound healing and Transwell migration assays were used to detect the changes in proliferation, cell senescence, cell cycle and cell migration after transfection. CALR, Wnt7a and β-catenin proteins expression were detected by Western blot. Interaction between CALR and Wnt7a was detected by Co-immunoprecipitation.ResultsCALR expression was increased in mice corneal epithelial injury repair, suggesting that CALR might play vital role in this process. CALR overexpression promoted HCE-2[50.B1] proliferation and migration, inhibited cell senescence of HCE-2[50.B1], and relieved S phase block and increased the number of HCE-2[50] cells in G0/G1 phase. Wnt7a and CALR proteins expression were respectively detected in the protein complexes co-precipitated by anti-CALR antibody and anti-Flag antibody. The interaction between CALR and Wnt7a could activate the downstream β-catenin signaling pathway. Wnt7a knockdown attenuated the effect of CALR overexpression on HCE-2[50.B1] cells proliferation, senescence and migration.ConclusionCALR promotes proliferation and migration, inhibited senescence of HCE-2[50.B1] cells by Wnt7a, thus promoting corneal epithelial wound healing. This study will provide a theoretical basis for mechanism of CALR in corneal injury repair, and provide a new target for corneal injury clinical treatment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11033-025-10810-x.

Purpose Corneal epithelial cells play a vital role in the function of the cornea by forming a physical barrier to protect the eye from invasion by external pathogenic agents. A recent study showed that miR-155 promotes cutaneous wound healing. However, its function in corneal epithelial wound healing is unknown. The present study examined whether miR-155-5p reduces corneal epithelial permeability by remodeling epithelial tight junctions during corneal wound healing. Materials and Methods Rat corneal wounds were produced by removing the central corneal epithelium with a blunt scalpel blade under a dissecting microscope. One eye of each rat was treated with topical miR-155-5p, and the other eye was treated with topical agomir negative control for 3 days before and after corneal epithelial wounding. Corneal epithelial permeability was assessed by the macromolecular osmosis method. Expression of zona occludens 1 (ZO-1), occludin, and myosin light chain kinase (MLCK) and phosphorylation of myosin light chain (MLC) were detected by Western blot. Human corneal epithelial (HCE) cells were cultured in the upper chamber of Transwell filters, and transepithelial electrical resistance (TER) was measured using a voltohmmeter. The distribution of ZO-1 and occludin in HCE cells treated with miR-155-5p was determined by immunofluorescence. Results miR-155-5p significantly promoted the repair of corneal epithelial injury and reduced the permeability of the corneal epithelium. It significantly decreased expression of MLCK and phosphorylation of MLC and increased expression of the tight junction proteins ZO-1 and occludin in corneal epithelial cells during corneal wound healing. miR-155-5p significantly increased TER, decreased MLCK expression and MLC phosphorylation, increased ZO-1 and occludin expression, and promoted anchoring of tight junction proteins in the cell membrane and remodeling in HEC cells. Conclusions Our results suggest that miR-155-5p reduced corneal permeability and accelerated the recovery of corneal epithelial wounds by decreasing the expression of MLCK and phosphorylation of MLC and by remodeling tight junctions.

Insulin-like growth factors (IGFs) and either substance P (SP) or an SP-derived peptide (FGLM-amide) synergistically facilitate corneal epithelial wound healing in vitro and in vivo. The mechanism of this synergism and the clinical potential of these agents were further investigated by determination of the relevant functional domain of IGFs. The effects of IGF-derived peptides on corneal epithelial cell migration were evaluated with the rabbit cornea in an organ culture system. Corneal epithelial wound closure in vivo was also evaluated in rabbits after epithelial debridement with n-heptanol. In the presence of FGLM-amide, peptides corresponding to the C domain of IGF-1 or -2 significantly promoted corneal epithelial migration in vitro to an extent similar to that apparent with the full-length molecules. In contrast, peptides corresponding to the D domain of these growth factors had no such effect. Mutation of serine-34 in the C domain of IGF-1 to alanine abolished the synergistic effect with FGLM-amide on corneal epithelial migration. The C peptide of proinsulin did not affect corneal epithelial migration in the absence or presence of FGLM-amide. The administration of eye drops containing both the C-domain peptide of IGF-1 and FGLM-amide significantly promoted corneal epithelial wound closure in vivo. The C domain of IGF-1 or -2, for which no biological function has previously been identified, is essential for the synergistic effect of these growth factors with SP on corneal epithelial migration.

Photo-crosslinked hydrogels for tissue engineering of corneal epithelium

Objective: To study the safety of topical berberine solution in rabbit eyes and its effect on corneal epithelial repair in rabbit eyes. Methods: Experimental Study. Ninety-two Japanese rabbits were randomly divided into two groups by random number table method: the general group (32 rabbits, 64 eyes) and the corneal injury group (60 rabbits, 60 eyes).The general groups were further divided into 4 groups by random number table method, and each group has 8 rabbits (16 eyes). According to the administration of deionized water or 0.5, 1.0, 1.5 mg/ml berberine solution, they were divided into the general control group and the general A, B, and C group. Dosing with both eyes, each eye was given a single dose, and then it was given multiple times for 4 weeks after observation for 72h. After the corneal epithelium injury model made in the right eye of rabbits in the corneal injury groups, they were divided into a corneal injury control group and a corneal injury group A, B, and C according to the administration of deionized water or 0.5, 1.0, 1.5 mg/ml berberine solution. there were 5 rabbits (15 eyes) in each group, and the solutions were given continuously for 1 week. The rabbits in the general group were observed their behavioral changes, ocular surface and iris were scored by Draize eye irritation test scoring system. IOP was measured at different time points. Electroretinogram (ERG) was used to detect b-wave amplitude. In the corneal injury group, corneal epithelial defect repairment was observed at 1, 2, 3, 4, 5, 6, and 7 days after the corneal defect. Corneal histopathology observation after discontinuation of all rabbits. The pH value of rabbit tears was described by the paired t test, and the score of Draize eye irritation test were described by the rank-sum test. The analysis of variance and SNK-q were used for IOP, electroretinogram b-wave amplitude, corneal epithelial injury area and repair time. Results: No abnormal behavior was observed in the general group rabbits after single and multiple administration. There was no significant difference in the Draize eye irritation score among the general control group and the general group A, B, C at 1, 2, and 4 weeks of multiple administrations. Among them, the Draize eye irritation score of the general group C was 7 (0, 12), 6 (0, 10), 6 (0, 16) points (χ(2)=1.640, 0.265, 1.963, 1.381; P>0.05).There were no significant difference in IOP at different times among the general control group and the general group A, B, C at different times (F=0.065, 0.292, 0.015, 0.041; P>0.05). Before multiple administrations and after administration at 2, 4 weeks, the b-wave amplitudes of the general control group were (127.75±17.12), (129.18±15.83), (128.81±13.58) μV, and the general group A were (130.68).±18.75), (131.38±16.96), (130.62±12.18) μV,and the general group B were (128.00±16.74), (128.44±16.64), (129.06±16.16) μV, and the general group C were (131.81±19.37), (132.13±18.36), (129.94±12.60) μV. There was no statistically significant difference in b-wave amplitude in the groups at different times before and after administration (F=0.037, 0.011, 0.017, 0.702; P>0.05). There was no significant difference in the results of corneal histopathology among the general control group and the general group A, B, C. The area of corneal epithelial defect in each corneal injury group was statistically significant at different time (F=5.316, 25.864, 127.613; P<0.05). The corneal injury control group compared with the corneal injury group A, B, C, the corneal epithelial defect area in the corneal injury group C was significantly larger than the other three groups, with statistical differences (q=5.153, 10.313, 6.976; P<0.05). The repair time of corneal epithelial in control group and the group A,B,C of corneal injury were (83.0±1.85), (82.9±2.07), (83.7±2.09) and (101.6±2.20) h. The corneal epithelium defect repair time in group C was longer and the difference was statistically significant (F=301.437, P=0.000). Comparing the corneal injury control group and corneal injury group A and B, there was no statistical difference in the repair time of corneal epithelial defect (F=0.813, P=0.450). After repair, there was no significant difference in the pathological results of the corneal tissue between the corneal injury groups. Conclusions: Berberine solution in rabbit eyes with topical application was safety, and has no obvious toxic effect on the ocular surface and ERG of normal rabbits. 1.5 mg/ml berberine solution delayed the repair of experimental corneal epithelial defect, but had no effect on the integrity of corneal tissue after repair. (Chin J Ophthalmol, 2020, 56: 131-137).

Limbal stem cells are essential for continuous corneal regeneration and injury repair. METTL3-catalyzed N6-methyladenosine (m6A) mRNA modifications are involved in many biological processes and play a specific role in stem cell regeneration, while the role of m6A modifications in corneal injury repair remains unknown. In this study, we generated a limbal stem cell-specific METTL3 knockout mouse model and studied the role of m6A in repairing corneal injury caused by alkali burn. The results showed that METTL3 knockout in the limbal stem cells promotes the in vivo cell proliferation and migration, leading to the fast repair of corneal injury. In addition, m6A modification profiling identified stem cell regulatory factors AHNAK and DDIT4 as m6A targets. Our study reveals the essential functions of m6A RNA modification in regulating injury repair and provides novel insights for clinical therapy of corneal diseases.

Phytic acid-loaded polyvinyl alcohol hydrogel promotes wound healing of injured corneal epithelium through inhibiting ferroptosis

BackgroundTopical application of β-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of β-blocker eye drops on corneal epithelial wound healing.MethodsCorneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL β-blocker levobunolol, β1-adrenoceptor (β1AR)-specific antagonist atenolol or β2-adrenoceptor (β2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining.ResultsLevobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium.Conclusionβ-blocker eye drops impaired corneal wound healing by inhibiting the β2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective β1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage.

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo

Corneal scarring resulting from corneal injury presents a critical clinical challenge, exacerbated by the serious shortage of donor corneas and the potential risk of postoperative infections. Here, an EPPH Janus hydrogel with the bottom PPH layer is developed prepared through an in situ cross‐linking process between four‐arm polyethylene glycol (PEG) and negatively charged heparin, and the top layer formed by introducing ɛ‐poly‐L‐lysine (EPL) through a spray‐coating process. The PPH hydrogel exhibits excellent optical transparency, mechanical strength, bio‐adhesion, and biocompatibility. Most importantly, it electrostatically adsorbs cytokines secreted by the damaged corneal epithelium via its negatively charged heparin to inhibit the apoptosis and transdifferentiation of keratocytes, thus inhibiting corneal fibrosis. Moreover, the top EPL layer of EPPH hydrogel provides significant antibacterial properties and effectively prevents methicillin‐resistant S. aureus (MRSA) and Pseudomonas aeruginosa infections. The corneal defect model confirms the effectiveness of the EPPH hydrogel in preventing corneal fibrosis with an 84.8% reduction and alleviating the burden of postoperative care for infection. Thus, this work shows the great potential of versatile EPPH hydrogel with asymmetric anti‐fibrosis and anti‐infection functions as an artificial corneal graft, offering a possible alternative to corneal transplantation.

Chapter 10 - Hyaluronan and Associated Proteins in the Visual System