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The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na + /K + -ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All authors have read the journal's policy on conflicts of interest and authorship agreement., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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.

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 (<1 µm), highly transparent (>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., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 °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 μm, whereas DM measured 25.93 ± 0.26 μm (p < .0001). Light transmission was 90% for both LC and DM. On a collagenase challenge, LC and amniotic membrane were digested after 13 hr, whereas DM was digested after 17 hr. The surface area of focal adhesions for cells grown on coated LCs was at least double that compared with other conditions, whereas tight junctions, ion pumps, and hexagonal morphology were well maintained when endothelial cells were cultured on LCs. In conclusion, LCs demonstrate excellent scaffolding properties for tissue engineering and sustain the cell phenotype and can be considered a suitable substrate for ocular tissue engineering or as a template for future scaffolds., (© 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons 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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)