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The cornea is an important barrier to consider when developing ophthalmic formulations, but proper modeling of this multilayered tissue remains a challenge. This is due to the varying properties associated with each layer in addition to the dynamics of the tear film. Hence, the most representative models to date rely on animals. Animal models, however, differ from humans in several aspects and are subject to ethical limitations. Consequently, in vitro approaches are being developed to address these issues. This review focuses on the barrier properties of the cornea and evaluates the most advanced three-dimensional cultures of human corneal equivalents in literature. Their application potential is subsequently assessed and discussed in the context of preclinical testing along with our perspective toward the future. Impact statement Most ocular drugs are applied topically, with the transcorneal pathway as the main administration route. Animal corneas are currently the only advanced models available, contributing to the drug attrition rate. Anatomical and physiological interspecies differences might account for a poor translatability of preclinical results to clinical trials, urging researchers to devise better corneal equivalents. This review elaborates on the emerging generation of three-dimensional in vitro models, which comprises spheroids, organoids, and organs-on-chips, which can serve as a stepping stone for advancements in this field.

Currently, the treatment of corneal diseases caused by damage to the corneal endothelium requires a donor cornea. Because of their limited availability (1 donor cornea for 70 patients in need), researchers are investigating alternative approaches that are independent of donor tissue. One of them includes the development of a tissue engineered scaffold onto which corneal endothelial cells are seeded. In order to function as a suitable substrate, some of its essential properties including thickness, permeability, transparency and mechanical strength should meet certain demands. Additionally, the membrane should be biocompatible and allow the formation of a functional endothelium on the surface. Many materials have already been investigated in this regard including natural, semi-synthetic and synthetic polymers. In the current review, we present an overview of their characteristics and provide a critical view on the methods exploited for material characterization. Next, also the suitability of scaffolds to serve their purpose is discussed along with an overview of natural tissues (e.g. amniotic membrane and lens capsule) previously investigated for this application. Eventually, we propose a consistent approach to be exploited ideally for membrane characterization in future research. This will allow a scientifically sound comparison of materials and membranes investigated by different research groups, hence benefitting research towards the creation of a suitable/optimal tissue engineered endothelial graft.

Currently, there are very few well-established treatments to stimulate corneal endothe-lial cell regeneration in vivo as a cure for corneal endothelial dysfunctions. The most frequently performed intervention for a damaged or dysfunctional corneal endothe-lium nowadays is corneal endothelial keratoplasty, also known as lamellar corneal transplantation surgery. Newer medical therapies are emerging and are targeting the regeneration of the corneal endothelium, helping the patients regain their vision with-out the need for donor tissue. Alternatives to donor tissues are needed as the aging population requiring transplants, has further exacerbated the pressure on the corneal eye banking system. Significant ongoing research efforts in the field of corneal regen-erative medicine have been made to elucidate the underlying pathways and effector proteins involved in corneal endothelial regeneration. However, the literature offers little guidance and selective attention to the question of how to fully exploit these pathways. The purpose of this paper is to provide an overview of wound healing characteristics from a biochemical level in the lab to the regenerative features seen in the clinic. Studying the pathways involved in corneal wound healing together with their key effector proteins, can help explain the effect on the proliferation and migration capac-ity of the corneal endothelial cells. (Translational Research 2022; 248:111-127)

Corneal neurotization is a promising surgical approach for the treatment of moderate to severe neurotrophic keratopathy. This technique aims to restore corneal sensation by transferring healthy nerves, either directly or via a conduit, to the anesthetic cornea. This review provides a report on the current state of development, evidence, and experience in the field. We summarize the data available from clinical reports and case series, placing an emphasis on the diversity of the surgical techniques reported. While these data are encouraging, they also highlight the need for a consensus in reporting outcomes and highlight how the next step will involve validating putative outcome parameters when researching and reporting corneal neurotization surgery.

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:19 ispartof: location:Switzerland status: published

Pterygium is a multifaceted pathology that displays apparent conflicting characteristics: benign (e.g., self-limiting and superficial), bad (e.g., proliferative and potentially recurrent) and ugly (e.g., signs of preneoplastic transformation). The natural successive question is: why are we lacking reports showing that pterygium lesions become life-threatening through metastasis, especially since pterygium has considerable similarities with UV-related malignancies on the molecular level? In this review, we consider how our pathophysiological understanding of the benign pterygium pathology overlaps with ocular surface squamous neoplasia and skin cancer. The three UV-related disorders share the same initial insult (i.e., UV radiation) and responsive repair mechanisms to the ensuing (in)direct DNA damage. Their downstream apoptotic regulators and other cellular adaptations are remarkably alike. However, a complicating factor in understanding the fine line between the self-limiting nature of pterygium and the malignant transformation in other UV-related diseases is the prominent ambiguity in the pathological evaluation of pterygium biopsies. Features of preneoplastic transformation (i.e., dysplasia) are used to define normal cellular reactions (i.e., atypia and metaplasia) and vice versa. A uniform grading system could help in unraveling the true nature of this ancient disease and potentially help in identifying the earliest intervention point possible regarding the cellular switch that drives a cell’s fate towards cancer.

The cornea is an important barrier to consider when developing ophthalmic formulations, but proper modeling of this multilayered tissue remains a challenge. This is due to the varying properties associated with each layer in addition to the dynamics of the tear film. Hence, the most representative models to date rely on animals. Animal models, however, differ from humans in several aspects and are subject to ethical limitations. Consequently

Corneal blindness is the fourth leading cause of visual impairment. Of specific interest is blindness due to a dysfunctional corneal endothelium which can only be treated by transplanting healthy tissue from a deceased donor. Unfortunately, corneal supply does not meet the demand with only one donor for every 70 patients. Therefore, there is a huge interest in tissue engineering of grafts consisting of an ultra-thin scaffold seeded with cultured endothelial cells. The present research describes the fabrication of such artificial Descemet membranes based on the combination of a biodegradable amorphous polyester (poly (d,l-lactic acid)) and crosslinkable gelatins. Four different crosslinkable gelatin derivatives are compared in terms of processing, membrane quality, and function, as well as biological performance in the presence of corneal endothelial cells. The membranes are fabricated through multi-step spincoating, including a sacrificial layer to allow for straightforward membrane detachment after production. As a consequence, ultrathin (90%), semi-permeable membranes could be obtained with high biological potential. The membranes supported the characteristic morphology and correct phenotype of corneal endothelial cells while exhibiting similar proliferation rates as the positive control. As a consequence, the proposed membranes prove to be a promising synthetic alternative to donor tissue.

Ex vivo grown human corneal endothelial cells (HCEnC) are a new emerging treatment option to treat visually impaired patients aimed at alleviating the current global donor shortage. Expanding HCEnC is still challenging, and obtaining cells in sufficient quantities is a limiting factor. It is already known that conditioned medium obtained from bone marrow mesenchymal stem cells can stimulate the proliferation of endothelial cells. The aim of this study was to take this work a step further to identify some of the underlying factors responsible. We confirmed the stimulatory effect of the mesenchymal stem cell secretome seen previously and separated the exosomes from the soluble proteins using size exclusion chromatography. We demonstrated the presence of exosomes and soluble proteins in the early and late fractions, respectively, with transmission electron microscopy and protein assays. Proliferation studies demonstrated that growth stimulation could be reproduced with the later protein-rich fractions but not with the exosome-rich fraction. Antibody assays revealed the presence of the secreted proteins EGF, IGFBP2, and IGFBP6 in protein-high fractions, but the growth enhancement was not seen with purified protein formulations. In conclusion, we confirmed the stimulatory effect of stem cell-conditioned medium and have determined that the effect was attributable to the proteins rather than to the exosomes. We were not able to reproduce the growth stimulation, however, with the pure recombinant protein candidates tested. Specific identification of the underlying proteins using proteomics could render a bioactive protein that can be used for ex vivo expansion of cells or as an in vivo drug to treat early corneal endothelial damage.

The idea of transplanting a sheet of laboratory-grown corneal endothelium dates back to 1978; however, the ideal scaffold is still lacking. We hypothesized that human crystalline lens capsules (LCs) could qualify as a scaffold and aimed to characterize the properties of this material for endothelial tissue engineering. LCs were isolated from donor eyes, stored at -80 degrees C, and decellularized with water and trypsin-EDTA. The decellularization was investigated by nuclear staining and counting and the capsule thickness was determined by optical coherence tomography and compared with Descemet's membrane (DM). Transparency was examined by spectrometry, and collagenase degradation was performed to evaluate its resistance to degradation. Cell-scaffold interaction was assessed by measuring focal adhesions surface area on LC and plastic. Finally, primary corneal endothelial cells were grown on LCs to validate the phenotype. Trypsin-EDTA decellularized most effectively, removing 99% of cells. The mean LC thickness was 35.76 +/- 0.43 mu m, whereas DM measured 25.93 +/- 0.26 mu m (p

Purpose. To investigate the biocompatibility of fish scale-derived scaffolds (FSS) with primary human corneal endothelial cells (HCEnCs). Methods. HCEnCs were isolated from 30 donor corneas in a donor-matched study and plated in precoated Lab-Tek slides (n=15) and FSS (n=15). Cell morphology, proliferation/migration, and glucose uptake were studied (n=30). Hoechst, ethidium homodimer, and calcein AM (HEC) staining was performed to determine viability and toxicity (n=6). The cell surface area was calculated based on calcein AM staining. HCEnCs were stained for ZO-1 (n=6) to detect tight junctions and to measure cell morphology; Ki-67 (n=6) to measure proliferating cells; and vinculin to quantify focal adhesions (n=6). The formation of de novo extracellular matrix was analyzed using histology (n=6). Results. HCEnCs attach and grow faster on Lab-Tek slides compared to the undulating topography of the FSS. At day 11, HCEnCs on Lab-Tek slide grew 100% confluent, while FSS was only 65% confluent (p=0.0883), with no significant difference in glucose uptake between the two (p=0.5181) (2.2 μg/mL in Lab-Tek versus 2.05 μg/mL in FSS). HEC staining showed no toxicity. The surface area of the cells in Lab-Tek was 409.1 μm2 compared to 452.2 μm2 on FSS, which was not significant (p=0.5325). ZO-1 showed the presence of tight junctions in both conditions; however, hexagonality was higher (74% in Lab-Tek versus 45% in FSS; p=0.0006) with significantly less polymorphic cells on Lab-Tek slides (8% in Lab-Tek versus 16% in FSS; p=0.0041). Proliferative cells were detected in both conditions (4.6% in Lab-Tek versus 4.2% in FSS; p=0.5922). Vinculin expression was marginally higher in HCEnCs cultured on Lab-Tek (234 versus 199 focal adhesions; p=0.0507). Histological analysis did not show the formation of a basement membrane. Conclusions. HCEnCs cultured on precoated FSS form a monolayer, displaying correct morphology, cytocompatibility, and absence of toxicity. FSS needs further modification in terms of structure and surface chemistry before considering it as a potential carrier for cultured HCEnCs.

Corneal endothelial dysfunction is one of the leading causes of corneal edema and visual impairment, requiring corneal endothelial transplantation. The treatments are limited, however, by both logistics and a global donor shortage. As a result, corneal researchers are striving to develop tissue-engineered constructs as an alternative. Recently, the clinical results of the first patients treated using a novel corneal endothelial cell therapy were reported, and it is likely many more will follow shortly. As we move from lab to clinic, it is crucial that we establish accurate and robust methods of proving the cellular identity of these products, both in genotype and phenotype. In this review, we summarized all of the markers and techniques that have been reported during the development of corneal endothelial cell therapies over the past decade. The results show the most frequently used markers were very general, namely Na+/K+ ATPase and zonula occludens-1 (ZO-1). While these markers are expressed in nearly every epithelial cell, it is the hexagonal morphology that points to cells being corneal endothelium in nature. Only 11% of articles aimed at discovering novel markers, while 30% were already developing cell therapies. Finally, we discuss the potential of functional testing of cell products to demonstrate potency in parallel with identity markers. With this review, we would like to highlight that, while this is an exciting era in corneal endothelial cell therapies, there is still no accepted consensus on a unique endothelial marker panel. We must ask the question of whether or not we are getting ahead of ourselves and whether we need to refocus on basic science rather than enter clinics prematurely.

Corneal blindness is traditionally treated by transplantation of a donor cornea, or in severe cases by implantation of an artificial cornea or keratoprosthesis. Due to severe donor shortages and the risks of complications that come with artificial corneas, tissue engineering in ophthalmology has become more focused on regenerative strategies using biocompatible materials either with or without cells. The stroma makes up the bulk of the corneal thickness and mainly consists of a tightly interwoven network of collagen type I, making it notoriously difficult to recreate in a laboratory setting. Despite the challenges that come with corneal stromal tissue engineering, there has recently been enormous progress in this field. A large number of research groups are working towards developing the ideal biomimetic, cytocompatible and transplantable stromal replacement. Here we provide an overview of the approaches directed towards tissue engineering the corneal stroma, from classical collagen gels, films and sponges to less traditional components such as silk, fish scales, gelatin and polymers. The perfect stromal replacement has yet to be identified and future research should be directed at combined approaches, in order to not only host native stromal cells but also restore functionality. Statement of Significance In the field of tissue engineering and regenerative medicine in ophthalmology the focus has shifted towards a common goal: to restore the corneal stroma and thereby provide a new treatment option for patients who are currently blind due to corneal opacification. Currently the waiting lists for corneal transplantation include more than 10 million patients, due to severe donor shortages. Alternatives to the transplantation of a donor cornea include the use of artificial cornea, but these are by no means biomimetic and therefore do not provide good outcomes. In recent years a lot of work has gone into the development of tissue engineered scaffolds and other biomaterials suitable to replace the native stromal tissue. Looking at all the different approaches separately is a daunting task and up until now there was no review article in which every approach is discussed. This review does include all approaches, from classical tissue engineering with collagen to the use of various alternative biomaterials and even fish scales. Therefore, this review can serve as a reference work for those starting in the field and but also to stimulate collaborative efforts in the future. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd.

Human corneal endothelium has long been thought to be a nonmitotic cell layer with no endogenous reparative potential. Pathologies that damage endothelial function result in corneal decompensation and, if untreated, blindness. The mainstay of treatment involves partial or complete corneal replacement, amounting to 40% of all corneal transplants performed worldwide. We summarize the case reports describing complications postoperatively in the form of (sub)total graft detachment and those resulting in postoperative bare stroma. Complications during cataract and glaucoma surgeries leading to an uncovered posterior cornea are also included. We discuss the newer treatment strategies that are alternatives for current Descemet membrane endothelial keratoplasty and Descemet stripping automated endothelial keratoplasty, including partial grafts and stripping of the diseased cell layer. In more than half of the cases reviewed, corneal transparency returned despite incomplete or no corneal endothelial cell transplantation. We question the existing paradigm concerning corneal endothelial wound healing in vivo. The data support further clinical study to determine the safety of simple descemethorexis in central endothelial pathologies, such as Fuchs endothelial corneal dystrophy, where presence of healthy peripheral cells may allow successful corneal recompensation without the need for donor cells. (C) 2017 The Authors. Published by Elsevier Inc.

The ability to see our surroundings is possibly the most invaluable of senses in human life. Each year hundreds of thousands of new cases of visual impairment occur, mostly caused by trauma to the eye and subsequent scarring but also due to rare immune disorders. Loss of vision leads to a significant reduction in the quality of life and unfortunately many of the conditions are notoriously difficult to treat. Recently a number of new approaches have been explored, including the use of stem cell technologies as a treatment for different types of visual impairment. This chapter gives an overview of the role of stem cells in corneal wound repair and in the regeneration of corneal tissues. First, corneal development and important characteristics with regards to the anatomy and histology will be discussed. Second, the cornea sources different stem cell types, each having specific characteristics that are used to distinguish between them. Third, injuries to the cornea elicit a number of responses for which specific treatments are required. Corneal stem cells posses wound healing capacities which make them suitable candidates to enhance current treatments. The last parts of this chapter will focus on clinical applications of stem cells in the cornea and currently used methods for corneal reconstruction through traditional and tissue engineering approaches. Alternative stem cell sources will be exemplified with regards to the potential applications concerning corneal repair. To conclude this chapter, the future prospects of stem cell applications in corneal regeneration and wound repair will be discussed.

Purpose : Corneal endothelial transplantation is limited by a donor shortage, making transplant lists long. As hypothesized in 1978, tissue-engineering could overcome this hurdle by growing corneal endothelial cells (CEnCs) on transplantable scaffolds. Despite the global interest, no tissue-engineered graft has made it yet to the clinic in part due to challenging properties scaffolds must fulfil. This study investigates the use of Poly-DL-lactic (PDLLA) membranes as scaffolds to aid in production of corneal endothelial grafts. Methods : Membranes were synthesized by spincoating a 10 w/v% solution of gelatin type A on glass as a sacrificial layer (Fig 1A). PDLLA was spincoated on top and plasma treated for surface activation. Gelatin B derivatives, gelatine-methacrylamide (gel-MOD) and gel-MOD-2-aminoethyl methacrylate (gel-MOD-AEMA) were analyzed using Nuclear Magnetic Resonance (NMR) spectroscopy, spincoated on the PDLLA and UV crosslinked (Fig 1B). The membranes were characterized with x-ray photoemission spectroscopy (XPS), optical profilometry (OP) to determine thickness and glucose diffusion. Also, cytocompatibility was investigated using primary and immortalized CEnCs followed by immunocytochemistry. Results : NMR spectra confirmed the modification of gel-MOD(-AEMA). The presence of stable gelatin layers during the synthesis was confirmed using XPS. The developed membranes exhibited thicknesses ranging from 1.1 - 2.6 µm as obtained via OP and the PDLLA showed a diffusion glucose coefficient of 0.0161cm/s. Finally, the membranes were seeded with primary and immortalized CEnCs and exhibited the characteristic hexagonal shape. Immunocytochemistry showed expression of Na+/K+ ATPase and ZO-1 along the cell membranes of primary and immortalized CEnCs cultured on both culture plastic and gel-MOD(-AEMA) membranes. Conclusions : In this study, membranes were constructed using PDLLA for mechanical strength and functionalized gelatins to mimic the extracellular matrix. The membranes displayed glucose permeability and because of the ultrathin dimensions they qualify as scaffolds for endothelial keratoplasty. Cultured endothelial cells expressed hallmark proteins representing their pumping and barrier function. Therefore, these novel ultrathin membranes are potentially a suitable scaffold for corneal endothelial tissue engineering required to alleviate the donor shortage. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.