Corneal Repair Research Articles

Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface.

BackgroundThe limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue.MethodsSphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue.ResultsSpheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation.ConclusionThese observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

Iris-claw versus posterior chamber fixation intraocular lens implantation in pediatric traumatic cataract

AimThis study aims to compare visual outcomes and complications of iris-fixated (claw) intraocular lens (IFIOL) implantation with those of posterior chamber intraocular lens (PCIOL) implantation in children with traumatic cataract. Settings and designRetrospective observational clinical audit. Materials and methodsA total of 50 pediatric traumatic cataract cases that underwent lens removal and IOL implantation (IFIOL or PCIOL) with or without corneal or corneoscleral tear repair between January 2009 and December 2013 were analyzed. After meeting the eligibility criteria, their pre- and postoperative visual outcomes and complication rates were recorded. Data were analyzed descriptively. ResultsOut of 50 children, IFIOL and PCIOL implantations were performed in one eye of each of 25 children. Their mean age was 11 ± 4 years (range 4–18 years). Primary (cataract removal with lens implantation) and secondary (corneal tear repair followed by cataract removal with lens implantation) procedures were performed in 19 (76%) and six (24%) children in the IFIOL group and in 21 (84%) and four (16%) children in the PCIOL group, respectively. There was an improvement in best corrected visual acuity postimplantation in both the IFIOL and the PCIOL group, and no significant difference in the logarithm of the minimum angle of resolution of best corrected visual acuity was observed between the two groups over 36 months. Only three eyes in the IFIOL group developed complications: one eye developed secondary glaucoma, one disenclavation of IOL haptic, and one cystoid macular edema. ConclusionBoth IFIOL and PCIOL implantations have good visual outcomes and minimal postoperative complications; therefore, IFIOL can be used as an alternative to PCIOL in children with traumatic cataract with inadequate capsular support.

Magnetic Nanoparticles as a Potential Vehicle for Corneal Endothelium Repair.

The corneal endothelium is paramount to the health and function of the cornea as damage to this cell layer can lead to corneal edema, opacification, and ultimately vision loss. Transplantation of the corneal endothelium is associated with numerous limitations, including graft rejection, thus an alternative therapeutic treatment is needed to restore endothelial layer integrity. We hypothesize that a nanotechnology-based approach using superparamagnetic iron oxide nanoparticles (SPIONPs) can ultimately be used to guide corneal endothelial cells (CECs) to injured areas via an external magnetic force without changing their morphology or viability. In this feasibility study we examined the effects of SPIONPs on the morphology and viability of bovine CECs in the presence of a magnetic force. The CECs were exposed to increasing SPIONP concentrations and the viability and cytoskeletal structure assessed over 3 days via metabolic analysis and rhodamine phalloidin staining. Significant differences (p < .05) in the metabolic activity of the CECs (100 × 10(6) SPIONP/cell) occurred in the presence of magnetic force versus those with no magnetic force. No differences were observed in the cytoskeleton of CECs in the presence or absence of magnetic force for all SPIONP concentrations. These SPIONPs will next be evaluated with human CECs for future applications.

Pluripotent Stem Cells and Other Innovative Strategies for the Treatment of Ocular Surface Diseases.

The cornea provides two thirds of the refractive power of the eye and protection against insults such as infection and injury. The outermost tissue of the cornea is renewed by stem cells located in the limbus. Depletion or destruction of these stem cells may lead to blinding limbal stem cell deficiency (LSCD) that concerns millions of patients around the world. Innovative strategies based on adult stem cell therapies have been developed in the recent years but they are still facing numerous unresolved issues, and the long term results can be deceiving. Today there is a clear need to improve these therapies, and/or to develop new approaches for the treatment of LSCD. Here, we review the current cell-based therapies used for the treatment of ocular diseases, and discuss the potential of pluripotent stem cells (embryonic and induced pluripotent stem cells) in corneal repair. As the secretion of paracrine factors is known to have a crucial role in maintaining stem cell homeostasis and in wound repair, we also consider the therapeutic potential of a promising novel pathway, the exosomes. Exosomes are nano-sized vesicles that have the ability to transfer RNAs and proteins to recipient cells, and several studies demonstrated their role in cell protection and wound healing. Exosomes could circumvent the hurdles of stem-cell based approaches, and they could become a strong candidate as an alternative therapy for ocular surface diseases.

A cell-based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair.

The goal of this study was to develop and validate a simple but quantitative cell-based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell-based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2. Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7-dimethoxy-3-phenylquinoxaline) and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), and four potential fibrosis inhibitors: 5-iodotubercidin (4-amino-5-iodo-7-(β-D-ribofuranosyl)-pyrrolo(2,3-d)pyrimidine), anisomycin, DRB (5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT-PCR and immunohistochemistry. Three "hits" were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.

A double network strategy to improve epithelization of a poly(2-hydroxyethyl methacrylate) hydrogel for corneal repair application

This paper presents a novel double network scaffold and its preparation methods, in which a cell-affinitive hydrogel was made by poly(2-hydroxyethyl methacrylate) and modified gelatin.

Expressão de metaloproteinases de matriz e PCNA em úlceras de córnea profundas, induzidas em coelhos, tratadas com plasma rico em plaquetas

O objetivo deste estudo foi avaliar a influência do plasma rico (PRP) e pobre (PPP) em plaquetas na proliferação celular e expressão de metaloproteinases de matriz (MMPs), durante a reparação de úlceras corneais profundas. Foram utilizadas 45 coelhas, distribuídas em 3 grupos (G) experimentais (n=15), designados como grupos PRP (GR), PPP (GP) e Controle (GC), de acordo com o tratamento. Todos os animais foram submetidos à indução cirúrgica unilateral de úlcera corneal. No GR e GP, o sangue autólogo foi centrifugado, utilizando-se protocolo padronizado, e foram confeccionados os colírios de PRP e PPP, e instilados cinco vezes ao dia. No GC, foi utilizado colírio lubrificante. Cada grupo foi subdividido (n=5), segundo o momento final de avaliação, sendo 4 (M4), 7 (M7) e 30 dias (M30). As córneas dos animais foram processadas para avaliação morfológica e imuno-histoquímica para PCNA, MMP1, MMP2, MMP9, MT1-MMP e TIMP1. No M4, os níveis de MMP2 foram maiores no GP e GR, sendo que, no M7, esse comportamento foi observado apenas no GP. No M30, no GR, verificou-se maior número de células epiteliais e marcação para MMP1 que o GP. No GR, a proliferação celular foi maior no M4 que nos demais momentos, e a marcação para MMP2 foi maior no M4 que no M30. O PRP estimula a proliferação celular na fase inicial (M4) do tratamento quando comparado aos demais momentos, diferentemente dos demais tratamentos. O uso de colírios de plasma rico e pobre em plaquetas influencia a expressão de metaloproteinases de matriz envolvidas no processo de reparação corneal.

The prospective randomized controlled study of primary pterygium surgery

Objective To study two different methods in treatment of primary pterygium surgery on corneal epithelial wound repair and the influence on the recurrence rate. Methods Used prospective controlled study, according to a number table 128 patients with 128 eyes were randomly divided into control group (64 cases), and observation group (64 cases). The two groups of patients underwent pterygium excision and limbal stem cell transplantation.The observation group (64 eyes) received 0.2 mg/mL mitomycin C of mitomycin-C(MMC) adjuvant therapy.Corneal epithelial wound repair speed was observed.The patients were followed up for 6-12 months, average 8.5months, the recurrence rate of pterygium was observed in the two groups. Results The grafts were smooth, and the conjunctival flap healed well in the two groups.The postoperative corneal epithelial healing time of the control group and the observation group were (2.13±0.37)d, (2.87±0.41)d, there was statistically significant difference (t=4.91, P 0.05). Conclusion There was no difference in recurrence rate between two kinds of pterygium surgical method, but the combined MMC can cause corneal epithelial healing delay. Key words: Pterygium; Limbal stem cell; Transplantation; Mitomycin C

Sphere-forming cells from peripheral cornea demonstrate a wound-healing response to injury.

The cornea is the initial refractive interface of the eye. Its transparency is critical for clear vision and is maintained by stem cells which also act to repair injury inflicted by external insults, such as chemical and thermal burns. Damage to the epithelium compromises its clarity and can reduce or eliminate the stem cell population, diminishing the ability for self-repair. This condition has been termed "limbal stem cell deficiency"; severe cases can lead to corneal blindness. Sphere-forming cells isolated from peripheral cornea are a potential source of stem and progenitor cells for corneal repair. When provided with appropriate substrate, these spheres have the ability to adhere and for cells to migrate outwards akin to that of their natural environment. Direct compression injury and remote scratch injury experiments were conducted on the sphere cells to gauge their wound healing capacity. Measures of proliferation, differentiation, and migration were assessed by immunohistochemical detection of EdU incorporation, α-smooth muscle actin expression and confocal image analysis, respectively. Both modes of injury were observed to draw responses from the spheres indicating wound healing processes. Direct wounding induced a rapid, but transient increase in expression of α-SMA, a marker of corneal myofibroblasts, followed by a proliferative and increasing migratory response. The spheres were observed to respond to remote injury as entire units, with no directional response seen for targeted repair over the scratch injury area. These results give strength to the future use of these peripheral corneal spheres as transplantable units for the regeneration of corneal tissue.

Silk fibroin composite membranes for application in corneal regeneration

ABSTRACTThe ordered microstructure of the corneal stroma determines the transparency of the cornea. The difficulty of constructing three‐dimensional corneal tissue mainly lies in the reconstruction of the corneal stroma. This article reports propionamide/silk fibroin composite membrane materials for use in corneal regeneration. X‐ray diffraction is used to explore the structure of the composite fibroin membrane. Propionamide acts as a crosslinking agent and inhibits the formation of larger crystal grains controlling the crystallization process. Corneal stromal cells are seeded on sterilized composite films. Propionamide/fibroin membranes with different blending proportions exhibit stable transparency and good cell compatibility. The results demonstrate that composite fibroin membranes are suitable potential materials for use in corneal stromal cell proliferation and repair. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42407.

Collagen based film with well epithelial and stromal regeneration as corneal repair materials: Improving mechanical property by crosslinking with citric acid.

Corneal disease can lead to vision loss. It has become the second greatest cause of blindness in the world, and keratoplasty is considered as an effective treatment method. This paper presents the crosslinked collagen (Col)-citric acid (CA) films developed by making use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The results showed that the Col-CA films had necessary optical performance, water content. The collagenase resistance of CA crosslinked films was superior to that of EDC crosslinked films. And CA5 film (Col:CA:EDC:NHS=60:3:10:10) had the best mechanical properties. Cell experiments showed that CA5 film was non-cytotoxic and human corneal epithelial cells could proliferate well on the films. Lamellar keratoplasty showed that the CA5 film could be sutured in the rabbit eyes and was epithelialized completely in about 10 days, and the transparency was restored quickly in 30±5 days. No inflammation and corneal neovascularization were observed at 6 months. Corneal stroma had been repaired; stromal cells and neo-stroma could be seen in the area of operation from the hematoxylin-eosin stained histologic sections and anterior segment optical coherence tomography images. These results indicated that Col-CA films were highly promising biomaterials that could be used in corneal tissue engineering and a variety of other tissue engineering applications.

708. Treatment of Inherited Eye Defects By Systemic Hematopoietic Stem Cell Transplantation

Corneal disease is a significant cause of blindness worldwide, largely due to the low availability of donor corneas and the risks associated with transplantation, such as immune rejection, cataract, and glaucoma. Stem cell transplantation represents an attractive alternative for corneal repair because of their capacity to differentiate into corneal cells. Limbal epithelial stem (LEST) cells, which reside within the corneo-limbus, have been used for more than a decade to repair corneal epithelium, and mesenchymal stem cells (MSCs) have been shown to migrate and promote corneal healing and even differentiate into epithelial/keratocyte-like cells. However, risks of immune rejection with LEST cells, and heterogeneity in repair mechanisms with MSCs have led to discrepancies in treatment outcomes. Alternatively, very little is known about the potential for hematopoietic stem cells (HSCs) in eye repair. HSCs have attractive features; they are multipotent cells capable of self-renewal and easily mobilized from bone marrow into the circulatory system. Here we report that engraftment of HSPCs can treat corneal defects in a mouse model of a degenerative hereditary metabolic disorder, cystinosis. Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin, resulting in cystine crystal accumulation in tissues. In eyes, a primary affected organ, crystals accumulate within the cornea causing photophobia and eventually blindness, if untreated. Using the mouse model of cystinosis, Ctns-/- mice, which develops similar eye anomalies than patients, we showed that systemic HSC transplantation led to the long-term preservation of the eyes. Effective therapy, however, was dependent on achieving relatively high level of donor-derived blood cell engraftment (more than 50%). Abundant GFP-expressing bone marrow-derived cells were detected within the cornea but also in the sclera, ciliary body, retina, choroid, and lens. In addition, HSCs engraftment led to substantial decrease in corneal cystine crystals, restoration of normal corneal thickness and intraocular pressure, and improved vision after one-year post-transplantation. Finally, we showed that HSCs mainly differentiated into MHC class II+ macrophages, which transferred cystinosin-bearing lysosomes via tunneling nanotubes to disease cells. This work is the first demonstration that HSCs can rescue corneal defects, which brings forth a new potential therapeutic strategy for treating ocular pathologies.

A Novel Method Using Quantum Dots for Testing the Barrier Function of Cultured Epithelial Cell Sheets.

The corneal epithelium provides a barrier to protect the deeper structures of the eye from any particles or pathogens. Cultured epithelial cell sheets are used in transplantation surgery for corneal repair or regeneration. The purpose of this study was to develop a novel method using fluorescent quantum dot nanoparticles for validating the quality and barrier function of cultured epithelial cell sheets. Human function epithelial cell sheets, cultured from oral mucosal or corneal limbal cells, were incubated in either normal calcium-containing medium or medium containing no calcium with a calcium chelator. Also contained in the media were suspensions of two different sizes of quantum dots. Following incubation, analysis of quantum dot penetration was carried out using confocal microscopy. In contrast to the cell sheets incubated in calcium-containing medium, removal of extracellular calcium resulted in the disruption of tight junctions, compromising the cell sheet's barrier function. This caused a reduction in transepithelial electrical resistance and deeper, more ubiquitous penetration of the quantum dots into the paracellular space and interior of the cell sheet. This method provides easy to interpret qualitative and quantitative data on the functionality of a cell sheet's tight junctions, as well as nanoscale and microscale structural information on its surface and interior morphology, and any localized areas of damage or abnormality. This novel technique could be used as part of the validation system for cultured epithelial cell sheets for use in transplantation.

Corneal Matrix Repair Carrier with Composite Silk Protein Membrane

Corneal transplantation is the only effective way to repair the damaged corneal tissue and solve the problem of insufficient donor cornea and immune rejection. Biocompatibility and stable transparent are necessary conditions of corneal stromal cells carrier. In this paper, the acetamide/silk (AC/SF) composite membranes are studied to be applied in corneal repair material. AC/SF membranes with different blending proportions had stable transparency, good cell compatibility. X-ray diffraction was used to investigate the structure of the composite films. The acetamide inhibited the formation of large crystalline particles, changed the crystal structure of silk fibroin and made the random coil structure convert to Silk I or Silk II. Therefore, acetamide was not only crosslinking agent but also crystallization inhibitor. The corneal stromal cells were seeded on sterilized composite membranes. After 5 h, the adhesion rate of stromal cells was more than 90%, cell could proliferate regularly on the composite membrane. There was no obvious difference in contrast to control plate. These results demonstrated that the composite membrane could promote corneal stromal cell proliferation.

Concise reviews: can mesenchymal stromal cells differentiate into corneal cells? A systematic review of published data.

The majority of stem cell therapies for corneal repair are based upon the use of progenitor cells isolated from corneal tissue, but a growing body of literature suggests a role for mesenchymal stromal cells (MSC) isolated from noncorneal tissues. While the mechanism of MSC action seems likely to involve their immuno-modulatory properties, claims have emerged of MSC transdifferentiation into corneal cells. Substantial differences in methodology and experimental outcomes, however, have prompted us to perform a systematic review of the published data. Key questions used in our analysis included: the choice of markers used to assess corneal cell phenotype, the techniques used to detect these markers, adequate reporting of controls, and tracking of MSC when studied in vivo. Our search of the literature revealed 28 papers published since 2006, with half appearing since 2012. MSC cultures established from bone marrow and adipose tissue have been best studied (22 papers). Critically, only 11 studies used appropriate markers of corneal cell phenotype, along with necessary controls. Ten out of these eleven papers, however, contained positive evidence of corneal cell marker expression by MSC. The clearest evidence is observed with respect to expression of markers for corneal stromal cells by MSC. In comparison, the evidence for MSC conversion into either corneal epithelial cells or corneal endothelial cells is often inconsistent or inconclusive. Our analysis clarifies this emerging body of literature and provides guidance for future studies of MSC differentiation within the cornea as well as other tissues.

Keratoglobus: An experience at a tertiary eye care center in India

Context:This study was carried out as a part of an internal audit and is the largest series of patients having keratoglobus, published in the literature. Poor visual acuity of the patients indicates the blinding nature of the disease.Aims:We report our experience with patients having keratoglobus at a tertiary eye care center in India.Settings and Design:Retrospective study.Materials and Methods:We analyzed adults and pediatric patients (<16 years) with keratoglobus, seen during 2008–2012. The age, gender, consanguinity, presenting ocular signs, ocular and systemic associations, visual acuity, corneal topography, and surgeries were documented.Results:Forty-eight patients (mean age 22 ± 15 years, 31 males) having keratoglobus were analyzed. 21 patients (42 eyes) were <16 years. Twelve eyes (16 events) had positive history of trauma. The presenting clinical signs were corneal scars/scars of tear repair (15 eyes), hydrops, healed and acute (14 eyes) and corneal or globe rupture (9 eyes). Best-corrected visual acuity was >20/40 in 6/42 (14.3%) pediatric eyes and 15/53 (28.30%) adults. Visual acuity ranging from counting of fingers to no light perception was noted in 20/53 (37.74%) adults and 21/42 (50%) pediatric patients; 13/20 (65%) with blue sclera and 8/22 eyes (36.37%) without blue sclera. Vernal keratoconjunctivitis was present in one pediatric patient. Choroidal osteoma, retinitis pigmentosa, and retinal detachment were present in adults. Surgeries performed were corneal tear repair (5 eyes), tissue adhesive application (2 eyes), descematopexy (4 eyes) and penetrating keratoplasty (PK - 8 eyes: Three had post-PK glaucoma, graft failure-one eye, 4 patients wore scleral lens - prosthetic replacement of the ocular surface ecosystem).Conclusions:About 50% of pediatric eyes (65% having blue sclera) had no functional vision. Trivial trauma was responsible for corneal rupture indicating need for protective glasses. About 50% patients had post-PK glaucoma though grafts were clear.

Clinical evaluation of rigid gas permeable contact lenses and visual outcome after repaired corneal laceration.

To evaluate the clinical value of rigid gas permeable contact lenses (RGPCLs) in patients with traumatic corneal scarring and address implications of primary corneal repair. Eighteen subjects with a history of corneal laceration were fit with RGPCLs. Scar locations were divided into two zones; each patient was examined using Pentacam. Entering data included uncorrected visual acuity (UCVA), spectacle-corrected visual acuity (SVA), time between injury and RGPCL fitting, location and size of scar, and amount of corneal astigmatism. Follow-up data included RGPCL visual acuity (RGPCLVA), RGPCL-related complications, and dropout characteristics. Visual acuity values were converted to logMAR for analysis. No serious complications occurred. The average time between suture removal and RGPCL fitting was 5.7±5.5 months. Average corneal astigmatism was -3.44±2.09 diopters. One subject had developed corneal ectasia. RGPCLVA was more than 0.1 in three subjects: one experienced primary corneal repair complications, and two subjects (<10 years) developed amblyopia. In both zones, the difference in RGPCLVA outcome between zone I and zone II was not statistically significant (F=0.060, P=0.809). The difference between SVA in zones I and II was found to be statistically significant (F=6.131, P=0.026), as were the differences between SVA and RGPCLVA (F=8.598, P=0.010). The scar size had no significant influence on RGPCLVA, SVA, or UCVA. Four participants (22.2%) were successfully fit. Dropout characteristics included ocular discomfort, inconvenience, parental apprehension, and low motivation. Rigid gas permeable contact lens is an ideal method for evaluating visual potential in patients with traumatic corneal astigmatism. Pentacam examinations of those patients with poor RGPCLVA can help an ophthalmologist find and understand existing problems in suture techniques.

The role of toll-like receptor 4 in corneal epithelial wound healing.

We evaluated the role of Toll-like receptor 4 (TLR4) in corneal epithelial wound healing. The expression of TLR4 during in vivo corneal epithelial wound healing was examined by immunostaining in mice. The expression and activation of TLR4 was studied in primary or telomerase-immortalized human corneal epithelial cells (HCEC). Scratch assay was performed to evaluate in vitro wound closure using live time-lapse microscopy. Transwell migration assay and Ki67 immunostaining were done to evaluate migration and proliferation, respectively. Lipopolysaccharide (LPS) was used to activate TLR4, whereas CLI-095 was used for its inhibition. The expression of inflammatory cytokines was determined by RT-PCR and ELISA. The activation of p42/44 and p38 was determined by immunoblotting. In the murine model, TLR4 immunostaining was noted prominently in the epithelium 8 hours after wounding. There was a 4-fold increase in the expression of TLR4 6 hours after in vitro scratch wounding (P < 0.001). Confocal microscopy confirmed the membrane localization of TLR4/MD2 complex. There was a significant increase in migration, proliferation, and wound closure in HCEC treated with LPS (P < 0.05), while there was significant decrease with TLR4 inhibition (P < 0.05). Addition of LPS to wounded HCEC resulted in a significant increase in the expression of IL-6, TNF-α, CXCL8/IL8, and CCL5/RANTES at the mRNA and protein levels. Likewise, LPS increased the activation of p42/44 and p38 in wounded HCEC. These results suggest that epithelial wounding induces the expression of functional TLR4. Toll-like receptor 4 signaling appears to contribute to early corneal epithelial wound repair by enhancing migration and proliferation.

Clinical efficacy of sodium hyaluronate and bFGF eye drops in dry eye and eyes after LASIK

Objective To observe the efficiency and safety of 0.1% sodium hyaluronate(SH)eye drops and basic fibroblast growth factor(bFGF) eye drops in simplex dry eye and dry eye after LASIK. Design Randomized controlled study. Participants 70 cases with simplex dry eye(group Ⅰ) and 40 post-LASIK cases(group Ⅱ) from January 2012 to August 2012. Method There were two subgroups in group Ⅰ and group Ⅱ respectively. 0.1% SH(A) and bFGF(B) eye drops were used randomly, four times a day for 14 days in group Ⅰ and 30 days in group Ⅱ. Changes of symptoms and signs were observed at different times. Dry eye symptoms including asthenopia, foreign body sensation, eye dry, burning sensation, swelling sensation, ophthalmodynia, conjunctiva congestion and photophobia were scored using questionnaire survey. Main Outcome Measures Score of dry eye symptoms, tear break-up time(BUT), Schirmer TestⅠ,corneal fluorescein staining(FL), symptoms changes of self-evaluation. Results Ocular symptoms and signs improved significantly after treatment in both subgroups of group Ⅰ(P0.05). 0.1% SH eye drops was better than bFGF eye drops in improving asthenopia(P=0.006) and ophthalmodynia(P=0.040) at 14 days after treatment(P0.05). SH was better to improve BUT(P=0.055) and Schirmer TestⅠ(P=0.573),and bFGF was better to reduce corneal fluorescein staining(P=0.375), but the different was not significant. Significant change of symptoms in patients using SH eye drops and bFGF eye drops were 94.3% and 78.6%, respectively. In group Ⅱ, there were symptoms in 1and 3 days after LASIK, and improved in 10 and 30 days post-operation. SH eye drops was better than bFGF eye drops in improving foreign body sensation(P=0.004), burning sensation(P0.001), swelling sensation(P=0.005) and conjunctiva congestion(P=0.007). BUT and Schirmer TestⅠ decreased in 10 days after LASIK, but increased in 30 days, than that of 10 days after operation(groupⅡA BUT:P=0.142, Schirmer TestⅠ: P=0.053; groupⅡB BUT: P=0.006, Schirmer TestⅠ: P=0.010). Corneal fluorescein staining aggravated in 10 day after LASIK, and improved in 30 days, with significant differences(groupⅡA P=0.005, groupⅡB P=0.079). There was no significant difference between groupⅡA and groupⅡB in changes of dry eye signs. No side-effect was found during observation. Conclusion SH and bFGF eye drops can facilitate corneal epithelial repair, improve tear film stability, and alleviate various symptoms in simplex dry eye and post-LASIK patients. SH eye drops is better than bFGF eye drops in improving certain symptoms including asthenopia, foreign body sensation, burning sensation, swelling sensation, conjunctiva congestion and ophthalmodynia.

ABCB5 is a limbal stem cell gene required for corneal development and repair.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.