[Corneal wound healing-Pharmacological treatment]. / Wundheilung der Kornea ­ Pharmakologische Therapie.

Physiological wound healing of the cornea is a complex process and involves numerous multifactorial tissue processes. A proper wound healing, especially without the formation of light-scattering scars, is essential to preserve the integrity and function of the cornea. Misdirected wound healing is of vast clinical relevance as it can lead to corneal fibrosis and the loss of optical transparency with subsequent reduction of visual acuity, up to blindness. In addition to the understanding of the pathophysiological mechanisms, the knowledge of therapeutic concepts and options for treating corneal wound healing disorders and fibrosis is essential to counteract a permanent damage of the cornea as early as possible. Nowadays, various pharmacological and surgical options are available for treatment. The decision, appropriate selection and indication for the optimal treatment depend primarily on the genesis and clinical appearance of the corneal wound, fibrosis or scar. The treatment of wound healing disorders ranges from the use of topical therapy and supportive measures up to tissue replacement procedures. As long as the mechanical stability of the cornea is intact and wound healing processes are still ongoing, a pharmacological modulation is reasonable, which is discussed in this article.

The application of a 4D-printed chitosan-based stem cell carrier for the repair of corneal alkali burns.

BACKGROUND: Corneal alkali burns can lead to ulceration, perforation, and even corneal blindness due to epithelial defects and extensive cell necrosis, resulting in poor healing outcomes. Previous studies have found that chitosan-based in situ hydrogel loaded with limbal epithelium stem cells (LESCs) has a certain reparative effect on corneal alkali burns. However, the inconsistent pore sizes of the carriers and low cell loading rates have resulted in suboptimal repair outcomes. In this study, 4D bioprinting technology was used to prepare a chitosan-based thermosensitive gel carrier (4D-CTH) with uniform pore size and adjustable shape to improve the transfer capacity of LESCs. METHODS: Prepare solutions of chitosan acetate, carboxymethyl chitosan, and ß-glycerophosphate sodium at specific concentrations, and mix them in certain proportions to create a pore-size uniform scaffold using 4D bioprinting technology. Extract and culture rat LESCs (rLESCs) in vitro, perform immunofluorescence experiments to observe the positivity rate of deltaNp63 cells for cell identification. Conduct a series of experiments to validate the cell compatibility of 4D-CTH, including CCK-8 assay to assess cell toxicity, scratch assay to evaluate the effect of 4D-CTH on rLESCs migration, and Calcein-AM/PI cell staining experiment to examine the impact of 4D-CTH on rLESCs proliferation and morphology. Establish a severe alkali burn model in rat corneas, transplant rLESCs onto the injured cornea using 4D-CTH, periodically observe corneal opacity and neovascularization using a slit lamp, and evaluate epithelial healing by fluorescein sodium staining. Assess the therapeutic effect 4D-CTH-loaded rLESCs on corneal alkali burn through histological evaluation of corneal tissue paraffin sections stained with hematoxylin and eosin, as well as immunofluorescence staining of frozen sections. RESULTS: Using the 4D-CTH, rLESCs were transferred to the alkali burn wounds of rats. Compared with the traditional treatment group (chitosan in situ hydrogel encapsulating rLESCs), the 4D-CTH-rLESC group had significantly higher repair efficiency of corneal injury, such as lower corneal opacity score (1.2 ± 0.4472 vs 0.4 ± 0.5477, p < 0.05) and neovascularization score (5.5 ± 1.118 vs 2.6 ± 0.9618, p < 0.01), and significantly higher corneal epithelial wound healing rate (72.09 ± 3.568% vs 86.60 ± 5.004%, p < 0.01). CONCLUSION: In summary, the corneas of the 4D-CTH-rLESC treatment group were similar to the normal corneas and had a complete corneal structure. These findings suggested that LESCs encapsulated by 4D-CTH significantly accelerated corneal wound healing after alkali burn and can be considered as a rapid and effective method for treating epithelial defects.

Biomimetic Corneal Stroma for Scarless Corneal Wound Healing via Structural Restoration and Microenvironment Modulation.

Corneal injury-induced stromal scarring causes the most common subtype of corneal blindness, and there is an unmet need to promote scarless corneal wound healing. Herein, a biomimetic corneal stroma with immunomodulatory properties is bioengineered for scarless corneal defect repair. First, a fully defined serum-free system is established to derive stromal keratocytes (hAESC-SKs) from a current Good Manufacturing Practice (cGMP)-grade human amniotic epithelial stem cells (hAESCs), and RNA-seq is used to validate the phenotypic transition. Moreover, hAESC-SKs are shown to possess robust immunomodulatory properties in addition to the keratocyte phenotype. Inspired by the corneal stromal extracellular matrix (ECM), a photocurable gelatin-based hydrogel is fabricated to serve as a scaffold for hAESC-SKs for bioengineering of a biomimetic corneal stroma. The rabbit corneal defect model is used to confirm that this biomimetic corneal stroma rapidly restores the corneal structure, and effectively reshapes the tissue microenvironment via proteoglycan secretion to promote transparency and inhibition of the inflammatory cascade to alleviate fibrosis, which synergistically reduces scar formation by ≈75% in addition to promoting wound healing. Overall, the strategy proposed here provides a promising solution for scarless corneal defect repair.

Smart coating by thermo-sensitive Pluronic F-127 for enhanced corneal healing via delivery of biological macromolecule progranulin.

As a leading cause of vision impairment and blindness, corneal alkali burns lead to long-term visual deterioration or even permanent visual impairment while effective treatment strategies remain a challenge. Herein, a thermo-sensitive hydrogel with the combination of multi-functional protein progranulin (PGRN), a biological macromolecule consisting of several hundred amino acids and possessing a high molecular weight, is efficiently prepared through a convenient stirring and mixing at the low temperature. The hydrogel can be easily administrated to the ocular surface contacting with the cornea, which can be immediately transformed into gel-like state due to the thermo-responsive behavior, realizing a site-specific coating to isolate further external stimulation. The smart coating not only exhibits excellent transparency and biocompatibility, but also presents a constant delivery of PGRN, creating a nutritious and supportive micro-environment for the ocular surface. The results show that the prepared functional hydrogel can efficiently suppress inflammation, accelerate re-epithelization, and intriguingly enhance axonal regeneration via modulation of multiple signaling pathways, indicating the novel designed HydrogelPGRN is a promising therapy option for serious corneal injury.

Intrastromal versus subconjunctival injection of mesenchymal stem/stromal cells for promoting corneal repair.

PURPOSE: Different approaches to delivery of mesenchymal stem/stromal cells (MSCs) for ameliorating corneal injuries have been investigated. This study was aimed to compare the efficacy of intrastromal and subconjunctival injection of human bone marrow-derived MSCs (hBM-MSCs) in a corneal epithelial injury model. METHODS: Twenty-four C57BL/6J mice underwent total corneal and limbal epithelial debridement. Then, the mice were divided into three different groups: (1) intrastromal hBM-MSCs injection, (2) subconjunctival hBM-MSCs injection, and (3) injection of frozen medium as a control. Mice were monitored by slit lamp and underwent anterior segment optical coherence tomography (ASOCT). Following euthanasia, the corneas were further evaluated by histology and immunostaining. RESULTS: hBM-MSC injection successfully healed epithelial defects regardless of the delivery route (P < 0.001). However, intrastromal injection was superior to subconjunctival injection in reducing defect area (P = 0.001). Intrastromal injection of hBM-MSCs also significantly reduced corneal opacity and neovascularization and improved ASOCT parameters compared to subconjunctival injection or no treatment (P < 0.001, P = 0.003, and P < 0.001, respectively). Although both of the treatment groups were positive for CK12 and had reduced levels of MUC5AC compared to the control, CK12 staining was stronger in the intrastromal group compared to the subconjunctival group. Also, persistency of MSCs was confirmed by in vivo (up to 2 weeks) and in vitro assessments (up to 4 weeks). CONCLUSIONS: Although the injection of hBM-MSC using both intrastromal and subconjunctival methods improve wound healing and reduce neovascularization and opacity, the intrastromal approach is superior in terms of corneal healing.

Reducing Sialylation Enhances Electrotaxis of Corneal Epithelial Cells.

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing.

Application of Noggin-Coated Electrospun Scaffold in Corneal Wound Healing.

Purpose: The objective of this study is to develop and characterize electrospun corneal bandage infused with Noggin protein and evaluate its therapeutic potential in the treatment of superficial nonhealing corneal ulceration. Methods: Electrospun nanofibrous scaffolds were created with different blend ratios of polycaprolactone and gelatin and coated with different concentrations of Noggin protein. Morphologic, mechanical, degradation, and surface chemistry of the developed scaffold was assessed. Biocompatibility of the developed scaffold with corneal epithelial cells was evaluated by looking at cell viability, proliferation, and immunostaining. In vitro wound healing in the presence of Noggin-coated scaffold was evaluated by measuring wound closure rate after scratch. Results: Uniform nanofibrous scaffolds coated with Noggin were constructed through optimization of electrospinning parameters and demonstrated mechanical properties better than or similar to commercially available contact lenses used in corneal wound healing. In the presence of Noggin-coated scaffold, corneal epithelial cells showed higher proliferation and wound-healing rate. Conclusions: This Noggin-coated electrospun scaffold represents a step toward, expanding treatment options for patients with indolent corneal ulcers. Translational Relevance: In this study, the feasibility of Noggin-coated electrospun scaffold as a therapeutic for indolent corneal ulcer was evaluated. This study also provides a better perspective for understanding electrospun scaffolds as a tunable platform to infuse topical therapeutics and use as a corneal bandage.

Plasma fibrin membranes loaded with bone marrow mesenchymal stem cells and corneal epithelial cells promote corneal injury healing via attenuating inflammation and fibrosis after corneal burns.

The shortage of corneal donors has prompted the development of tissue-engineered corneal grafts as an alternative solution. Currently, amniotic membranes with good biocompatibility are widely used as scaffolds for loading stem cells in the treatment of corneal injury. However, this approach has its limitations. In this study, BMSCs were induced to differentiate into corneal epithelial cells via direct contact co-culture, and platelet-poor plasma was used to prepare fibrin gels, which were compressed to remove excess liquid and then lyophilized to obtain plasma fibrin membranes (PFMs). A tissue-engineered corneal implant with PFMs as a scaffold loaded with BMSCs and corneal epithelial cells was designed and obtained. Scanning electron microscopy showed that PFMs have a uniformly distributed microporous surface that facilitates cell attachment and nutrient transport. The rheological results showed that the freeze-dried and rehydrated PFMs were more rigid than fresh membranes, which makes it easier to use them for transplantation after cell loading. The experimental results of a rat alkali burn cornea injury model showed that PFMs effectively reduced the inflammatory reaction, inhibited fibrosis, and accelerated the healing of corneal wounds. It was also found that some of the BMSCs were successfully implanted into the corneal injury site in rats and differentiated into corneal epithelial cells. These results demonstrate the potential of tissue-engineered corneal implants using BMSCs and corneal epithelial cells and PFMs as scaffolds as a new treatment option for corneal injury.

Intra-stromal injection of honey-treated keratocytes as a cell-based therapy for experimental corneal laceration.

OBJECTIVES: This study aimed to investigate the potential of honey-supplemented medium (HSM) for expanding corneal keratocytes and its transplantation in a model of corneal laceration. METHODS: Keratocytes were cultured in 1 % HSM- or 10 % fetal bovine serum (FBS)-supplemented medium for 24 h. The effect of HSM on keratocyte proliferation was evaluated using the MTT assay. The relative expression of Lum, Kera, and ALDH3A1, known markers of native keratocytes, was quantified by real-time PCR. The safety and efficacy of HSM-treated keratocyte intrastromal injection in a rabbit model of corneal laceration were also evaluated. RESULTS: The MTT assay showed that HSM treatment did not significantly affect cell viability compared to FBS-supplemented medium (84.71 ± 2.38 vs. 100.08 ± 10.92, respectively; p=0.076). Moreover, HSM-treated keratocytes had significantly increased expression of Lum, Kera, and ALDH3A1 compared to cells treated with FBS, while the expression of the proliferation biomarker Thy-1 did not significantly differ between the two treatments. Intrastromal injection of HSM-treated keratocytes in the laceration animal model was safe and uneventful, resulting in less stromal inflammation and neovascularization, and consequently, better final architecture with less residual haze compared to the group injected with FBS-treated keratocytes. CONCLUSIONS: These findings suggest that honey is a suitable supplement for keratocyte treatment and corneal cell therapy. The use of HSM may have potential applications in the treatment of corneal injuries and diseases.

Snowflake-inspired and blink-driven flexible piezoelectric contact lenses for effective corneal injury repair.

The cornea is a tissue susceptible to various injuries and traumas with a complicated cascade repair process, in which conserving its integrity and clarity is critical to restoring visual function. Enhancing the endogenous electric field is recognized as an effective method of accelerating corneal injury repair. However, current equipment limitations and implementation complexities hinder its widespread adoption. Here, we propose a snowflake-inspired, blink-driven flexible piezoelectric contact lens that can convert mechanical blink motions into a unidirectional pulsed electric field for direct application to moderate corneal injury repair. The device is validated on mouse and rabbit models with different relative corneal alkali burn ratios to modulate the microenvironment, alleviate stromal fibrosis, promote orderly epithelial arrangement and differentiation, and restore corneal clarity. Within an 8-day intervention, the corneal clarity of mice and rabbits improves by more than 50%, and the repair rate of mouse and rabbit corneas increases by over 52%. Mechanistically, the device intervention is advantageous in blocking growth factors' signaling pathways specifically involved in stromal fibrosis whilst preserving and harnessing the signaling pathways required for indispensable epithelial metabolism. This work put forward an efficient and orderly corneal therapeutic technology utilizing artificial endogenous-strengthened signals generated by spontaneous body activities.

Molecular and Cellular Mechanisms of Corneal Scarring and Advances in Therapy.

On the basis of WHO global blindness data, it may be stated that 23 million people globally suffer from unilateral corneal blindness, while 4 [...].

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

Mesenchymal Stem Cell Exosomes as Immunomodulatory Therapy for Corneal Scarring.

Corneal scarring is a leading cause of worldwide blindness. Human mesenchymal stem cells (MSC) have been reported to promote corneal wound healing through secreted exosomes. This study investigated the wound healing and immunomodulatory effects of MSC-derived exosomes (MSC-exo) in corneal injury through an established rat model of corneal scarring. After induction of corneal scarring by irregular phototherapeutic keratectomy (irrPTK), MSC exosome preparations (MSC-exo) or PBS vehicle as controls were applied to the injured rat corneas for five days. The animals were assessed for corneal clarity using a validated slit-lamp haze grading score. Stromal haze intensity was quantified using in-vivo confocal microscopy imaging. Corneal vascularization, fibrosis, variations in macrophage phenotypes, and inflammatory cytokines were evaluated using immunohistochemistry techniques and enzyme-linked immunosorbent assays (ELISA) of the excised corneas. Compared to the PBS control group, MSC-exo treatment group had faster epithelial wound closure (0.041), lower corneal haze score (p = 0.002), and reduced haze intensity (p = 0.004) throughout the follow-up period. Attenuation of corneal vascularisation based on CD31 and LYVE-1 staining and reduced fibrosis as measured by fibronectin and collagen 3A1 staining was also observed in the MSC-exo group. MSC-exo treated corneas also displayed a regenerative immune phenotype characterized by a higher infiltration of CD163+, CD206+ M2 macrophages over CD80+, CD86+ M1 macrophages (p = 0.023), reduced levels of pro-inflammatory IL-1ß, IL-8, and TNF-α, and increased levels of anti-inflammatory IL-10. In conclusion, topical MSC-exo could alleviate corneal insults by promoting wound closure and reducing scar development, possibly through anti-angiogenesis and immunomodulation towards a regenerative and anti-inflammatory phenotype.

Subconjunctival Administration of Mesenchymal Stem Cells Alleviates Ocular Inflammation in a Murine Model of Corneal Alkali Burn.

Corneal alkali burns cause extensive damage not only to the cornea but also to the intraocular tissues. As an anti-inflammatory therapy, subconjunctival administration of mesenchymal stem cells (MSCs) for corneal protection after corneal alkali burn has been explored. Little evidence demonstrates the potential of subconjunctival MSCs delivery in protecting the post-burn intraocular tissues. This study aimed to evaluate the therapeutic efficacy of subconjunctival injection of human placental (hP)-MSCs in protecting against ocular destruction after the burn. hP-MSCs were subconjunctivally administered to C57/BL mice after corneal alkali burn. Western blot of iNOS and CD206 was performed to determine the M1 and M2 macrophage infiltration in the cornea. Infiltration of inflammatory cells in the anterior uvea and retina was analyzed by flow cytometry. The TUNEL assay or Western blot of Bax and Bcl2 was used to evaluate the anti-apoptotic effects of MSCs. MSCs could effectively facilitate cornea repair by suppressing inflammatory cytokines IL-1ß, MCP-1, and MMP9, and polarizing CD206 positive M2 macrophages. Anterior uveal and retinal inflammatory cytokines expression and inflammatory cell infiltration were inhibited in the MSC-treated group. Reduced TUNEL positive staining and Bax/Bcl2 ratio indicated the anti-apoptosis of MSCs. MSC-conditioned medium promoted human corneal epithelial cell proliferation and regulated LPS-stimulated inflammation in RAW 264.7 macrophages, confirming the trophic and immunoregulatory effects of MSCs. Our findings demonstrate that subconjunctival administration of MSCs exerted anti-inflammatory and anti-apoptotic effects in the cornea, anterior uvea, and retina after corneal alkali burn. This strategy may provide a new direction for preventing post-event complications after corneal alkali burn.

[Corneal epithelial biomechanics: Resistance to stress and role in healing and remodeling]. / Biomécanique de l'épithélium cornéen : résistance au stress et implications dans la cicatrisation et le remodelage.

The corneal epithelium is one of the first tissue barriers of the eye against the environment. In recent years, many studies provided better knowledge of its healing, its behavior and its essential role in the optical system of the eye. At the crossroads of basic science and clinical medicine, the study of the mechanical stresses applied to the cornea makes it possible to learn the behavior of epithelial cells and better understand ocular surface disease. We describe herein the current knowledge about the adhesion systems of the corneal epithelium and their resistance to mechanical stress. We will also describe the involvement of these mechanisms in corneal healing and their role in epithelial dynamics. Adhesion molecules of the epithelial cells, especially hemidesmosomes, allow the tissue cohesion required to maintain the integrity of the corneal epithelium against the shearing forces of the eyelids as well as external forces. Their regeneration after a corneal injury is mandatory for the restoration of a healthy epithelium. Mechanotransduction plays a significant role in regulating epithelial cell behavior, and the study of the epithelium's response to mechanical forces helps to better understand the evolution of epithelial profiles after refractive surgery. A better understanding of corneal epithelial biomechanics could also help improve future therapies, particularly in the field of tissue engineering.

From bench to clinic: Emerging therapies for corneal scarring.

Corneal diseases are one of the leading causes of moderate-to-severe visual impairment and blindness worldwide, after glaucoma, cataract, and retinal disease in overall importance. Given its tendency to affect people at a younger age than other blinding conditions such as cataract and glaucoma, corneal scarring poses a huge burden both on the individuals and society. Furthermore, corneal scarring and fibrosis disproportionately affects people in poorer and remote areas, making it a significant ophthalmic public health problem. Traditional medical strategies, such as topical corticosteroids, are not effective in preventing fibrosis or scars. Corneal transplantation, the only effective sight-restoring treatment for corneal scars, is curbed by challenges including a severe shortage of tissue, graft rejection, secondary conditions, cultural barriers, the lack of well-trained surgeons, operating rooms, and well-equipped infrastructures. Thanks to tremendous research efforts, emerging therapeutic options including gene therapy, protein therapy, cell therapy and novel molecules are in development to prevent the progression of corneal scarring and compliment the surgical options currently available for treating established corneal scars in clinics. In this article, we summarise the most relevant preclinical and clinical studies on emerging therapies for corneal scarring in recent years, showing how these approaches may prevent scarring in its early development.

Human corneal stromal stem cells express anti-fibrotic microRNA-29a and 381-5p - A robust cell selection tool for stem cell therapy of corneal scarring.

INTRODUCTION: Corneal blindness due to scarring is treated with corneal transplantation. However, a global problem is the donor material shortage. Preclinical and clinical studies have shown that cell-based therapy using corneal stromal stem cells (CSSCs) suppresses corneal scarring, potentially mediated by specific microRNAs transported in extracellular vesicles (EVs). However, not every CSSC batch from donors achieves similar anti-scarring effects. OBJECTIVES: To examine miRNA profiles in EVs from human CSSCs showing "healing" versus "non-healing" effects on corneal scarring and to design a tool to select CSSCs with strong healing potency for clinical applications. METHODS: Small RNAs from CSSC-EVs were extracted for Nanostring nCounter Human miRNA v3 assay. MicroRNAs expressed > 20 folds in "healing" EVs (P < 0.05) were subject to enriched gene ontology (GO) term analysis. MiRNA groups with predictive regulation on inflammatory and fibrotic signalling were studied by mimic transfection to (1) mouse macrophages (RAW264.7) for M1 phenotype assay; (2) human corneal keratocytes for cytokine-induced fibrosis, and (3) human CSSCs for corneal scar prevention in vivo. The expression of miR-29a was screened in additional CSSC batches and the anti-scarring effect of cells was validated in mouse corneal wounds. RESULTS: Twenty-one miRNAs were significantly expressed in "healing" CSSC-EVs and 9 miRNA groups were predicted to associate with inflammatory and fibrotic responses, and tissue regeneration (P <10-6). Overexpression of miR-29a and 381-5p significantly prevented M1 phenotype transition in RAW264.7 cells after lipopolysaccharide treatment, suppressed transforming growth factor ß1-induced fibrosis marker expression in keratocytes, and reduced scarring after corneal injury. High miR-29a expression in EV fractions distinguished human CSSCs with strong healing potency, which inhibited corneal scarring in vivo. CONCLUSION: We characterized the anti-inflammatory and fibrotic roles of miR-29a and 381-5p in CSSCs, contributing to scar prevention. MiR-29a expression in EVs distinguished CSSCs with anti-scarring quality, identifying good quality cells for a scarless corneal healing.