Restore Corneal Transparency Research Articles

A double-crosslinked nanocellulose-reinforced dexamethasone-loaded collagen hydrogel for corneal application and sustained anti-inflammatory activity

In cases of blinding disease or trauma, hydrogels have been proposed as scaffolds for corneal regeneration and vehicles for ocular drug delivery. Restoration of corneal transparency, augmenting a thin cornea and postoperative drug delivery are particularly challenging in resource-limited regions where drug availability and patient compliance may be suboptimal. Here, we report a bioengineered hydrogel based on porcine skin collagen as an alternative to human donor corneal tissue for applications where long-term stability of the hydrogel is required. The hydrogel is reinforced with cellulose nanofibers extracted from the Ciona intestinalis sea invertebrate followed by double chemical and photochemical crosslinking. The hydrogel is additionally loaded with dexamethasone to provide sustained anti-inflammatory activity. The reinforced double-crosslinked hydrogel after drug loading maintained high optical transparency with significantly improved mechanical characteristics compared to non-reinforced hydrogels, while supporting a gradual sustained drug release for 60 days in vitro. Dexamethasone, after exposure to crosslinking and sterilization procedures used in hydrogel production, inhibited tube formation and cell migration of TNFα-stimulated vascular endothelial cells. The drug-loaded hydrogels suppressed key pro-inflammatory cytokines CCL2 and CXCL5 in TNFα-stimulated human corneal epithelial cells. Eight weeks after intra-stromal implantation in the cornea of 12 New-Zealand white rabbits subjected to an inflammatory suture stimulus, the dexamethasone-releasing hydrogels suppressed TNFα, MMP-9, and leukocyte and fibroblast cell invasion, resulting in reduced corneal haze, sustained corneal thickness and stromal morphology, and reduced overall vessel invasion. This collagen-nanocellulose double-crosslinked hydrogel can be implanted to treat corneal stromal disease while suppressing inflammation and maintaining transparency after corneal transplantation. Statement of significanceTo treat blinding diseases, hydrogel scaffolds have been proposed to facilitate corneal restoration and ocular drug delivery. Here, we improve on a clinically tested collagen-based scaffold to improve mechanical robustness and enzymatic resistance by incorporating sustainably sourced nanocellulose and dual chemical-photochemical crosslinking to reinforce the scaffold, while simultaneously achieving sustained release of an incorporated anti-inflammatory drug, dexamethasone. Evaluated in the context of a corneal disease model with inflammation, the drug-releasing nanocellulose-reinforced collagen scaffold maintained the cornea's transparency and resisted degradation while suppressing inflammation postoperatively. This biomaterial could therefore potentially be applied in a wider range of sight-threatening diseases, overcoming suboptimal administration of postoperative medications to maintain hydrogel integrity and good vision.

Clinical experience in rehabilitation of patients with Fuchs corneal dystrophy and cataract by ultrasonic phacoemulsification and descemetorhexis

BACKGROUND: The potential for peripheral corneal endothelial capabilities is a novel, poorly understood direction. The comprehensive development of various modifications of endothelial keratoplasty goes hand in hand with a lack of donor material. This explains the continuous search for safe tissue-preserving techniques.
 AIM: The aim of this study is to evaluate the clinical and functional results of treatment of patients with Fuchs corneal dystrophy and cataract using a combination of cataract ultrasound phacoemulsification and descemetorhexis.
 MATERIALS AND METHODS: The study included 3 patients (3 eyes), 3 women aged 65 to 78 (on average 73.6 7.5 years). The follow-up period in the postoperative period was 36 months. The inclusion criteria were: patient complaints about glare, blurred vision and cloudy vision in the morning; the location of the guttae in the central 5 mm zone, central corneal stromal edema less than 610 microns, the inability to count endothelial cells in the center, endothelial cells density on the periphery in the upper quadrant more than 1400 cells/mm2. All patients underwent phacoemulsification of cataract with IOL implantation and subsequent descemetorhexis in the 4 mm zone.
 RESULTS: At 1 month an increase in the central corneal thickness and a slight increase in uncorrected visual acuity (UCVA) and in best corrected visual acuity (BCVA). By the 3rd month, positive dynamics was present: an increase in UCVA and BCVA in all cases, a decrease in the central corneal thickness (559 20 m), resorption of stromal edema, increase in corneal transparency, possibility to calculate the endothelial cells density in the center (866 46 cells/mm2). At 36 months, the BCVA was 0.6, 0.4 and 0.5, respectively.
 CONCLUSIONS: Restoration of corneal transparency was achieved in 100% of cases (3 out of 3). An analysis of the clinical results of the descemetorhexis + phacoemulsification + IOL operation demonstrated an increase in visual acuity, an increase in corneal transparency and a decrease in stromal edema in patients with initial Fuchs corneal dystrophy and cataract. The possibility of not using donor material for the treatment of Fuchs corneal dystrophy is a promising trend. Further accumulation of material is required.

Restoration of corneal transparency in a patient with corneal scarring using Mesenchyme Stem Cells.

Restoration of corneal transparency in a patient with corneal scarring using Mesenchyme Stem Cells.

Growth factors and mechano-regulated reciprocal crosstalk with extracellular matrix tune the keratocyte–fibroblast/myofibroblast transition

Improper healing of the cornea after injury, infections or surgery can lead to corneal scar formation, which is associated with the transition of resident corneal keratocytes into activated fibroblasts and myofibroblasts (K–F/M). Myofibroblasts can create an extracellular matrix (ECM) niche in which fibrosis is promoted and perpetuated, resulting in progressive tissue opacification and vision loss. As a reversion back to quiescent keratocytes is essential to restore corneal transparency after injury, we characterized how growth factors with demonstrated profibrotic effects (PDGF, FGF, FBS, TGFβ1) induce the K–F/M transition, and whether their withdrawal can revert it. Indeed, the upregulated expression of αSMA and the associated changes in cytoskeletal architecture correlated with increases in cell contractility, fibronectin (Fn) and collagen matrix density and Fn fiber strain, as revealed by 2D cell culture, nanopillar cellular force mapping and a FRET-labeled Fn tension probe. Substrate mechanosensing drove a more complete K–F/M transition reversal following growth factor withdrawal on nanopillar arrays than on planar glass substrates. Using decellularized ECM scaffolds, we demonstrated that the K–F/M transition was inhibited in keratocytes reseeded onto myofibroblast-assembled, and/or collagen-1-rich ECM. This supports the presence of a myofibroblast-derived ECM niche that contains cues favoring tissue homeostasis rather than fibrosis.

Poly(l-Histidine)-Mediated On-Demand Therapeutic Delivery of Roughened Ceria Nanocages for Treatment of Chemical Eye Injury.

Development of topical bioactive formulations capable of overcoming the low bioavailability of conventional eye drops is critically important for efficient management of ocular chemical burns. Herein, a nanomedicine strategy is presented to harness the surface roughness-controlled ceria nanocages (SRCNs) and poly(l-histidine) surface coatings for triggering multiple bioactive roles of intrinsically therapeutic nanocarriers and promoting transport across corneal epithelial barriers as well as achieving on-demand release of dual drugs [acetylcholine chloride (ACh) and SB431542] at the lesion site. Specifically, the high surface roughness helps improve cellular uptake and therapeutic activity of SRCNs while exerting a negligible impact on good ocular biocompatibility of the nanomaterials. Moreover, the high poly(l-histidine) coating amount can endow the SRCNs with an ≈24-fold enhancement in corneal penetration and an effective smart release of ACh and SB431542 in response to endogenous pH changes caused by tissue injury/inflammation. In a rat model of alkali burn, topical single-dose nanoformulation can efficaciously reduce corneal wound areas (19-fold improvement as compared to a marketed eye drops), attenuate ≈93% abnormal blood vessels, and restore corneal transparency to almost normal at 4 days post-administration, suggesting great promise for designing multifunctional metallic nanotherapeutics for ocular pharmacology and tissue regenerative medicine.

Role of aquaporins in corneal healing post chemical injury

Aquaporins (AQPs) are transmembrane water channel proteins that regulate the movement of water through the plasma membrane in various tissues including cornea. The cornea is avascular and has specialized microcirculatory mechanisms for homeostasis. AQPs regulate corneal hydration and transparency for normal vision. Currently, there are 13 known isoforms of AQPs that can be subclassified as orthodox AQPs, aquaglyceroporins (AQGPs), or supraquaporins (SAQPs)/unorthodox AQPs. AQPs are implicated in keratocyte function, inflammation, edema, angiogenesis, microvessel proliferation, and the wound-healing process in the cornea. AQPs play an important role in wound healing by facilitating the movement of corneal stromal keratocytes by squeezing through tight stromal matrix and narrow extracellular spaces to the wound site. Deficiency of AQPs can cause reduced concentration of hepatocyte growth factor (HGF) leading to reduced epithelial proliferation, reduced/impaired keratocyte migration, reduced number of keratocytes in the injury site, delayed and abnormal wound healing process. Dysregulated AQPs cause dysfunction in osmolar homeostasis as well as wound healing mechanisms. The cornea is a transparent avascular tissue that constitutes the anterior aspect of the outer covering of the eye and aids in two-thirds of visual light refraction. Being the outermost layer of the eye, the cornea is prone to injury. Of the 13 AQP isoforms, AQP1 is expressed in the stromal keratocytes and endothelial cells, and AQP3 and AQP5 are expressed in epithelial cells in the human cornea. AQPs can facilitate wound healing through aid in cellular migration, proliferation, migration, extracellular matrix (ECM) remodeling and autophagy mechanism. Corneal wound healing post-chemical injury requires an integrative and coordinated activity of the epithelium, stromal keratocytes, endothelium, ECM, and a battery of cytokines and growth factors to restore corneal transparency. If the chemical injury is mild, the cornea will heal with normal clarity, but severe injuries can lead to partial and/or permanent loss of corneal functions. Currently, the role of AQPs in corneal wound healing is poorly understood in the context of chemical injury. This review discusses the current literature and the role of AQPs in corneal homeostasis, wound repair, and potential therapeutic target for acute and chronic corneal injuries.

Recent Advancements in Molecular Therapeutics for Corneal Scar Treatment

The process of corneal wound healing is complex and induces scar formation. Corneal scarring is a leading cause of blindness worldwide. The fibrotic healing of a major ocular wound disrupts the highly organized fibrillar collagen arrangement of the corneal stroma, rendering it opaque. The process of regaining this organized extracellular matrix (ECM) arrangement of the stromal layer to restore corneal transparency is complicated. The surface retention capacity of ocular drugs is poor, and there is a large gap between suitable corneal donors and clinical requirements. Therefore, a more efficient way of treating corneal scarring is needed. The eight major classes of interventions targeted as therapeutic tools for healing scarred corneas include those based on exosomes, targeted gene therapy, microRNAs, recombinant viral vectors, histone deacetylase inhibitors, bioactive molecules, growth factors, and nanotechnology. This review highlights the recent advancements in molecular therapeutics to restore a cornea without scarring. It also provides a scope to overcome the limitations of present studies and perform robust clinical research using these strategies.

Human SMILE-Derived Stromal Lenticule Scaffold for Regenerative Therapy: Review and Perspectives.

A transparent cornea is paramount for vision. Corneal opacity is one of the leading causes of blindness. Although conventional corneal transplantation has been successful in recovering patients’ vision, the outcomes are challenged by a global lack of donor tissue availability. Bioengineered corneal tissues are gaining momentum as a new source for corneal wound healing and scar management. Extracellular matrix (ECM)-scaffold-based engineering offers a new perspective on corneal regenerative medicine. Ultrathin stromal laminar tissues obtained from lenticule-based refractive correction procedures, such as SMall Incision Lenticule Extraction (SMILE), are an accessible and novel source of collagen-rich ECM scaffolds with high mechanical strength, biocompatibility, and transparency. After customization (including decellularization), these lenticules can serve as an acellular scaffold niche to repopulate cells, including stromal keratocytes and stem cells, with functional phenotypes. The intrastromal transplantation of these cell/tissue composites can regenerate native-like corneal stromal tissue and restore corneal transparency. This review highlights the current status of ECM-scaffold-based engineering with cells, along with the development of drug and growth factor delivery systems, and elucidates the potential uses of stromal lenticule scaffolds in regenerative therapeutics.

Combined Therapy Using Human Corneal Stromal Stem Cells and Quiescent Keratocytes to Prevent Corneal Scarring after Injury.

Corneal blindness due to scarring is conventionally treated by corneal transplantation, but the shortage of donor materials has been a major issue affecting the global success of treatment. Pre-clinical and clinical studies have shown that cell-based therapies using either corneal stromal stem cells (CSSC) or corneal stromal keratocytes (CSK) suppress corneal scarring at lower levels. Further treatments or strategies are required to improve the treatment efficacy. This study examined a combined cell-based treatment using CSSC and CSK in a mouse model of anterior stromal injury. We hypothesize that the immuno-regulatory nature of CSSC is effective to control tissue inflammation and delay the onset of fibrosis, and a subsequent intrastromal CSK treatment deposited collagens and stromal specific proteoglycans to recover a native stromal matrix. Using optimized cell doses, our results showed that the effect of CSSC treatment for suppressing corneal opacities was augmented by an additional intrastromal CSK injection, resulting in better corneal clarity. These in vivo effects were substantiated by a further downregulated expression of stromal fibrosis genes and the restoration of stromal fibrillar organization and regularity. Hence, a combined treatment of CSSC and CSK could achieve a higher clinical efficacy and restore corneal transparency, when compared to a single CSSC treatment.

Development and evaluation of the effectiveness of photodynamic therapy in inflammatory diseases of the ocular surface

Relevance. In the treatment of inflammatory diseases of the ocular surface, in order to achieve high efficiency in the shortest possible time, it is necessary for the therapy to be comprehensive. Today, in addition to traditional treatment methods, new technologies based on the use of low-energy lasers, which have high biological activity, are spreading.
 Aim. To improve the treatment results of the inflammatory diseases of the ocular surface by implementing photodynamic therapy (PDT) in experiment and clinic, using domestic equipment and elaborating optimal energetic parameters of laser irradiation.
 Methods. Objects of research: 130 nonlinear sexually mature rats weighing 150 grams contained in standard vivarium conditions and 110 eyes of patients with inflammatory eye diseases. Carrying out the investigation, experiments on animals, clinical and functional examination results of patients, and statistical analysis were taken into consideration.
 Results. A methodology for complex treatment of inflammatory diseases of the ocular surface was developed (with inclusion of PDT by the laser therapy device Vostok), based on experimental data and confirmed by clinical and functional ocular indices. PDT helps to increase the effectiveness of treatment, which is manifested by accelerated tissue regeneration, restoration of corneal transparency, increase of visual function in 86.7%, and reduction of treatment terms to 6 days.
 Conclusions. Anti-inflammatory and regenerative effectiveness of the proposed complex treatment of inflammatory diseases of the ocular surface with the PDT use was established: improvement in the clinical picture of conjunctivitis and keratitis was revealed, manifested by increase in corneal epithelization.

Xeno-free approach for the expansion of human adipose derived mesenchymal stem cells for ocular therapies

To restore corneal transparency and vision loss after an injury on the ocular surface, the use of human stem cells from different origins has been recently proposed. Mesenchymal stem cells (MSCs) seem to be an appropriate adult source of autologous stem cells due to their accessibility, high proliferation rate, and multipotent capacity. In this work, we developed a simple culture system to prepare a graft based on a fibrin membrane seeded with human MSCs. A commercial kit, PRGF Endoret®, was used to prepare both, the growth factors used as culture media supplement and the fibrin membrane grafts. Adipose-derived MSCs (Ad-MSCs) were expanded, characterised by flow cytometry and their multilineage differentiation potential confirmed by inducing adipogenesis, osteogenesis and chondrogenesis. Ad-MSCs seeded on the fibrin membranes were grafted onto athymic mice showing good biocompatibility with no adverse reactions observed during the follow up period. These findings support the assumption that a system in which all the biological components (cells, grow factors and carrier) are autologous, could potentially be used for future ex vivo expansion of Ad-MSCs to treat ocular conditions such as an inflammatory milieu, traumatic scars and loss of the regenerative capacity of the corneal epithelium that compromise the quality of vision.

Keratolenticular adhesion removal for type 2 Peters anomaly: a case report

BackgroundType 2 Peters anomaly is a rare anterior segment disorder characterized by central corneal leukoma with keratolenticular adhesion and cataract. Performing cataract surgery without corneal tissue transplantation in patients of type 2 Peters anomaly is extremely rare and challenging. We present a case of type 2 Peters anomaly treated by peeling off the adhesion without penetrating keratoplasty (PKP), in which restoration of corneal transparency is observed.Case presentationAn 11-month-old female infant of type 2 Peters anomaly presented with bilateral corneal opacity with distinct demarcation, keratolenticular adhesion and cataract, which was first noted at the age of 3 months. By peeling off the adhesion from corneal endothelium combined with lensectomy and vitrectomy, there was a gradual reduction in corneal opacity and improvement in visual acuity after surgery over a 2-year period. Her visual acuity had improved from light perception preoperatively to 20/50 at the latest follow-up. No sight-threatening postoperative complications were noted.ConclusionIt is safe and effective to peel off the keratolenticular adhesion in patients of type 2 Peters anomaly presented with distinctly demarcated corneal opacity.

Inverted Posterior Femto-Keratoplasty: Quality of the Surface of the Corneal Section and Preliminary Clinical Outcomes

Purpose. To evaluate the quality of the surface of an ultra-thin donor transplant prepared from the endothelial surface of the cornea using a femtosecond laser and to demonstrate the preliminary clinical results. Patients and Methods. 4 eyes were operated: 3 with Fuch`s endothelial dystrophy and 1 with pseudophakic bullous keratopathy. All patients were treated with DSEK with an ultrathin graft prepared by Alcon Wavelight FS 200 femtosecond laser (Germany). Before and after surgery UCVA, BSCVA, astigmatism, ECD were measured. EC death, graft thickness and CCT were evaluated at 12 months` observation. Atomic force microscopy was used for examination of 10 samples. Control group was 5 corneal flaps obtained by mechanical microkeratome (Moria SLK-2, France). Main group — 5 corneal flaps, obtained by femtosecond laser. Nonparametric Mann-Whitney test was used for statistical analysis. Coefficient of reliability p < 0.05 was considered to be significant. Results. RMS value of femto-laser group samples was 18.6 ± 7.8 um. RMS of microkeratome group samples was 22.3 ± 18.3 um. Statistical analysis did not reveal significant differences between the values of the studied parameter in these groups (p > 0.05). Transparent engraftment was observed in all cases, no corneal edema was identified. BSCVA ranged from 0.2 to 0.6, which was associated with the presence of concomitant pathology. Astigmatism was 1.35 ± 1.0 D. ECD = 1526 ± 434 cells/mm2. EC loss = 48.0 ± 12.8 %. Graft thickness in the central zone was 78.0 ± 18.1 µm. Center-Edge Index — 0.84 ± 0.12. CCT = 600 ± 31 um. No postoperative complications were found. Conclusion. The developed settings allowed to obtain high-quality ultra-thin graft with a sufficiently uniform surface without a risk of perforation. Preliminary clinical results showed the method potential for restoration of corneal transparency. ECD loss corresponded to the one achievable by DSAEK.

Hyaluronidase pharmacological properties and clinical application in ophthalmology

Corneal diseases are the fourth leading cause of blindness in the world, accounting for approximately 5 % of cases. Existing methods of treatment in more than 30 % of cases do not have a full therapeutic effect and when the process is stopped, they end with a persistent violation of corneal transparency, a decrease or complete loss of visual functions. The lack of effective means that evidence-based restore corneal transparency determines the relevance of the search for modern drugs and ways to deliver them, the possibilities of enhancing the therapeutic effect. The possibilities of using hyaluronidase preparations in ophthalmology are considered based on the study of its biological and pharmacological properties. Enzyme catalyzes the breakdown of acid mucopolysaccharides including hyaluronic acid by cleavage of the glycosidic bond β(1→4), the biological effect is determined by the molecular weight of the resulting fragments: high-molecular fragments have antiangiogenic properties, increased ability to bind fibrinogen, anti-inflammatory and immunosuppressive effects, and low-molecular fragments have pro-inflammatory activity and promote angiogenesis. The barrier function of the cornea is provided by the features of its anatomical structure, while the features of its regeneration with the formation of turbidity are accompanied by overexpression and migration to the stroma of cytokines TGF-β and PDGF, activation of myofibroblasts and the formation of a fibroproliferative response. The high anti-inflammatory, immunomodulatory, regenerative and antifibrotic activity of hyaluronidase, the possibility of its effect on a complex pathophysiological cascade of destructive processes and minimization of the scarring process stimulate more extensive experimental and clinical studies on the development of new methods of treating ophthalmic diseases using hyaluronidase drugs.

Corneal transparency restoration by gene therapy

AbstractLoss of corneal transparency after trauma, injury or infection results in blindness. Compromised wound healing because of excessive production of myofibroblasts has been identified a major mechanism for the loss of corneal transparency. We tested a hypothesis that de‐differentiation of corneal myofibroblast into precursor corneal stromal fibroblast by silencing of MyoD (basic helix‐loop‐helix) gene will restore corneal transparency by inhibiting alpha smooth muscle actin (αSMA) transcriptional machinery responsible for myofibroblast production. To test the hypothesis, donor human corneas, human corneal stromal fibroblasts (CSFs) and myofibroblasts (CMFs), MyoD/scrambled‐RNAi plasmids, nanoparticles, immunofluorescence, western blotting, qPCR, 2D/3D confocal microscopy, and slot blots were used. The fibrotic human corneas showed significantly greater MyoD expression compared to the normal human cornea (p < 0.05). Likewise, CMFs showed markedly increased MyoD compared to the CSFs in vitro (p < 0.01). The MyoD‐RNAi or scrambled‐RNAi delivery into CSFs and CMFs did not alter their appearance, viability, or proliferation in normal culture conditions. MyoD‐RNAi delivery into CMFs led to phenotype similar to CSFs in a time‐dependent manner. Furthermore, MyoD‐RNAi silencing in CMFs demonstrated significantly deceased αSMA mRNA and protein levels in a time‐dependent manner (p < 0.001). MyoD gene silencing is a novel approach for the restoration of corneal transparency by promoting de‐differentiation of opaque corneal myofibroblasts to transparent corneal keratocytes.

Therapeutic efficacy of different routes of mesenchymal stem cell administration in corneal injury

PurposeCorneal injuries are associated with significant impairment in vision. Mesenchymal stem cells (MSCs) have been shown to limit inflammation and promote tissue repair at the ocular surface. Here, we evaluate the efficacies of different modes of MSC delivery (topical, subconjunctival, intraperitoneal [IP] and intravenous [IV]) to promote tissue repair and restore corneal transparency in a murine model of corneal injury. MethodsMSCs were purified from the bone marrow of C57BL/6 mice and expanded using plastic adherence in vitro. Corneal injury was created using an Algerbrush, and 0.5 × 106 MSCs/mouse were administered via topical, subconjunctival, IP or IV routes. Qdot-labeled MSCs were employed to determine the effect of route of administration on corneal and conjunctival MSC frequencies. Corneal opacity scores were calculated using ImageJ. Expression of inflammatory cytokines was quantified by qPCR, and infiltration of CD45+ cells was evaluated by flow cytometry. ResultsSubconjunctival or IV administration results in increased frequencies of MSCs in ocular surface tissues following corneal injury, relative to topical or intraperitoneal delivery. Subconjunctival or IV administration reduces: (i) corneal opacity, (ii) tissue fibrosis as quantified by α-Sma expression, (iii) the expression of inflammatory cytokines (Il-1β and Tnf-α) and (iv) CD45+ inflammatory cell infiltration relative to untreated injured control animals. Administration via subconjunctival or IV routes was observed to accelerate corneal repair by restoring tissue architecture and epithelial integrity. ConclusionsOur data suggest that subconjunctival or IV delivery of MSCs has superior therapeutic efficacy compared to topical or IP delivery following corneal injury.

Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy

PurposeCorneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation.MethodsPrimary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining.ResultsMagnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study.ConclusionsOur results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.

Injection of Cultured Cells with a ROCK Inhibitor for Bullous Keratopathy

BackgroundCorneal endothelial cell (CEC) disorders, such as Fuchs’s endothelial corneal dystrophy, induce abnormal corneal hydration and result in corneal haziness and vision loss known as bullous keratopathy. We investigated whether injection of cultured human CECs supplemented with a rho-associated protein kinase (ROCK) inhibitor into the anterior chamber could increase CEC density.MethodsWe performed an uncontrolled, single-group study involving 11 persons who had received a diagnosis of bullous keratopathy and had no detectable CECs. Human CECs were cultured from a donor cornea; a total of 1×106 passaged cells were supplemented with a ROCK inhibitor (final volume, 300 μl) and injected into the anterior chamber of the eye that was selected for treatment. After the procedure, patients were placed in a prone position for 3 hours. The primary outcome was restoration of corneal transparency, with a CEC density of more than 500 cells per square millimeter at the central cornea at 24 weeks after cell injection. Secondary outcomes were a corneal thickness of less than 630 μm and an improvement in best corrected visual acuity equivalent to two lines or more on a Landolt C eye chart at 24 weeks after cell injection.ResultsAt 24 weeks after cell injection, we recorded a CEC density of more than 500 cells per square millimeter (range, 947 to 2833) in 11 of the 11 treated eyes (100%; 95% confidence interval [CI], 72 to 100), of which 10 had a CEC density exceeding 1000 cells per square millimeter. A corneal thickness of less than 630 μm (range, 489 to 640) was attained in 10 of the 11 treated eyes (91%; 95% CI, 59 to 100), and an improvement in best corrected visual acuity of two lines or more was recorded in 9 of the 11 treated eyes (82%; 95% CI, 48 to 98).ConclusionsInjection of human CECs supplemented with a ROCK inhibitor was followed by an increase in CEC density after 24 weeks in 11 persons with bullous keratopathy. (Funded by the Japan Agency for Medical Research and Development and others; UMIN number, UMIN000012534.)

Feasibility of cell-based therapy combined with descemetorhexis for treating Fuchs endothelial corneal dystrophy in rabbit model.

Corneal transparency is maintained by the corneal endothelium through its pump and barrier function. Severe corneal endothelial damage results in dysregulation of water flow and eventually causes corneal haziness and deterioration of visual function. In 2013, we initiated clinical research of cell-based therapy for treating corneal decompensation. In that study, we removed an 8-mm diameter section of damaged corneal endothelium without removing Descemet’s membrane (the basement membrane of the corneal endothelium) and then injected cultured human corneal endothelial cells (CECs) into the anterior chamber. However, Descemet’s membrane exhibits clinically abnormal structural features [i.e., multiple collagenous excrescences (guttae) and thickening] in patients with Fuchs endothelial corneal dystrophy (FECD) and the advanced cornea guttae adversely affects the quality of vision, even in patients without corneal edema. The turnover time of cornea guttae is also not certain. Therefore, we used a rabbit model to evaluate the feasibility of Descemet’s membrane removal in the optical zone only, by performing a small 4-mm diameter descemetorhexis prior to CEC injection. We showed that the corneal endothelium is regenerated both on the corneal stroma (the area of Descemet’s membrane removal) and on the intact peripheral Descemet’s membrane, based on the expression of function-related markers and the restoration of corneal transparency. Recovery of the corneal transparency and central corneal thickness was delayed in areas of Descemet’s membrane removal, but the cell density of the regenerated corneal endothelium and the thickness of the central corneal did not differ between the areas with and without residual Descemet’s membrane at 14 days after CEC injection. Here, we demonstrate that removal of a pathological Descemet’s membrane by a small descemetorhexis is a feasible procedure for use in combination with cell-based therapy. The current strategy might be beneficial for improving visual quality after CEC injection as a treatment for FECD.

Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities.

The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.