Regeneration Of Corneal Endothelial Cells Research Articles

Prospective Assessment of Adjuvant Netarsudil Use in Patients Undergoing Descemet Stripping Only.

We sought to determine whether adjuvant use of netarsudil improves corneal clearance rate and regeneration of corneal endothelial cells in patients undergoing Descemet stripping only (DSO). We conducted a prospective assessment on the use of adjuvant netarsudil in 50 eyes from 25 patients undergoing DSO at Massachusetts Eye and Ear between May 2021 and May 2023. Our comparison group was a retrospective cohort of patients (23 eyes from 15 patients) who previously underwent DSO without the use of a postoperative rho-kinase inhibitor between September 2014 and March 2020. Use of netarsudil after DSO statistically significantly reduced time to corneal clearance, improved best corrected visual acuity, reduced pachymetric thickness, and increased central endothelial cell count (ECC) at 6 months postoperatively. Importantly, central ECC continued to increase beyond 12 months after DSO with central ECC still statistically significantly greater in eyes that received netarsudil than in eyes that received no netarsudil. A rho-kinase inhibitor, such as netarsudil, after DSO should be used if available to achieve the best corneal clearance, best corrected visual acuity, and ECC after surgery.

Research progress of corneal endothelial cell regeneration and replacement

AbstractCorneal endothelial cells (CECs) are crucial for the maintenance of corneal transparency and normal visual function. Corneal endothelial dysfunction can lead to corneal edema, opacity, and even blindness. Due to the limited proliferative capacity of human CECs and the global shortage of donor cornea, corneal endothelial regeneration and replacement always represent the most challenge in the basic research and clinical treatment of corneal diseases. Although there is a potential existence of corneal endothelial progenitor cells, the efficiency of Descemet stripping without endothelial keratoplasty remains controversial. In recent years, significant advancements have been made in the field of cultured endothelial cell regeneration and artificial material replacement. Here, we reviewed the current research and clinical progress of corneal endothelial cell regeneration and replacement, including the in vitro cultivation of primary human CECs, in vitro differentiation of stem cell‐derived CECs, tissue‐engineered corneal endothelium, and fabrication of artificial corneal endothelium. We also discussed the remaining questions regarding innovating clinical preventive and therapeutic strategies for corneal endothelial dysfunction.

Research advances in human corneal endothelial cell regeneration

Human corneal endothelial cells (HCECs) are the prerequisite for maintaining corneal transparency, but HCECs remain arrested at the G1 phase after embryonic development and can not proliferate and regenerate. Thus, the density of HCECs decreases spontaneously with corneal development. Systemic factors, primary corneal disease, refractive factors, glaucoma, inflammation, and trauma all can cause a massive loss of HCECs, lead to corneal edema and turbidity, and ultimately induce blindness. Currently, keratoplasty is the only effective treatment, but the scarcity of donor corneas and the limitation of corneal preservation technology restrict the availability of keratoplasty. Therefore, the most appealing way to tackle the tissue shortage problem is corneal endothelial cell regeneration. In recent years, not only the endogenous regeneration of HCECs mediated by surgery, drugs and gene therapy but also the exogenous regeneration of HCECs mediated by cell therapy have made fruitful progress. Although a number of regeneration strategies have entered the clinical trial stage, the wide clinical application of corneal endothelial regeneration is still far away. This review elaborates the basic research, clinical application and limitation of current strategies of corneal endothelial cell regeneration.

New Therapies for Corneal Endothelial Diseases: 2020 and Beyond.

Penetrating keratoplasty used to be the only surgical technique for the treatment of end-stage corneal endothelial diseases. Improvements in surgical techniques over the past decade have now firmly established endothelial keratoplasty as a safe and effective modality for the treatment of corneal endothelial diseases. However, there is a worldwide shortage of corneal tissue, with more than 50% of the world having no access to cadaveric tissue. Cell injection therapy and tissue-engineered endothelial keratoplasty may potentially offer comparable results as endothelial keratoplasty while maximizing the use of cadaveric donor corneal tissue. Descemet stripping only, Descemet membrane transplantation, and selective endothelial removal are novel therapeutic modalities that take this a step further by relying on endogenous corneal endothelial cell regeneration, instead of allogenic corneal endothelial cell transfer. Gene therapy modalities, including antisense oligonucleotides and clustered regularly interspaced short palindromic repeats-based gene editing, offer the holy grail of potentially suppressing the phenotypic expression of genetically determined corneal endothelial diseases at the asymptomatic stage. We now stand at the crossroads of exciting developments in medical technologies that will likely revolutionize the way we treat corneal endothelial diseases over the next 2 decades.

Potential Functional Restoration of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy by ROCK Inhibitor (Ripasudil)

Rho-associated kinase (ROCK) inhibitors have been successfully used as a rescue strategy in eyes that failed to clear after descemetorhexis without endothelial graft for treatment of Fuchs endothelial corneal dystrophy (FECD). The functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECD patients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECD patients and normal donors using exvivo tissue and invitro cellular models. Experimental study: laboratory investigation. This institutional study used endothelial cell-Descemet membrane lamellae from FECD patients (n= 450) undergoing Descemet membrane endothelial keratoplasty (FECD exvivo model), normal research-grade donor corneas (n= 30) after scraping off central endothelial cells (exvivo wound healing model), normal donor corneas (n= 20) without endothelial injury, and immortalized cell lines (n= 3) generated from FECD patients (FECD invitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30μM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls exvivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines invitro. These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECD patients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.

Ex vivo excimer laser ablation of cornea guttata and ROCK inhibitor-aided endothelial recolonization of ablated central cornea.

To determine whether excimer laser ablation of guttae is a viable strategy for removal of diseased tissue in Fuchs' endothelial corneal dystrophy (FECD) on excised human Descemet membranes and whether an excimer laser-created wound on healthy human corneas ex vivo is recolonized with corneal endothelial cells. Descemet membranes of FECD patients and corneal endothelium of normal human corneas were ablated ex vivo using an excimer laser licensed for glaucoma surgery. Specimens were kept in cell culture medium supplemented with 10μm of rho-kinase inhibitor ripasudil. Corneal endothelial cell regeneration was observed using light and electron scanning microscopy. Furthermore, the whole corneal samples were evaluated by haematoxylin/eosin staining and immunohistochemical analysis using antibodies against Na+ /K+ -ATPase. Guttae and corneal endothelium could be ablated with an excimer laser without total ultrastructural damage to theDescemet membrane or stroma. Nearly complete endothelial wound closure was accomplished after 26-38days in treated corneas. Light and electron scanning microscopy suggested the establishment of a layer of flat endothelial cells. Additionally, Na+ /K+ -ATPase expression could only be observed on the inner side of the Descemet membrane. Our proof of concept study demonstrated that excimer lasers can be used to ablate diseased tissue from excised FECD Descemet membranes ex vivo. Additionally, corneal endothelial cells recolonize a previously ablated endothelial area in healthy human corneas ex vivo under treatment with ripasudil. Thus, our results are the first experimental basis to further investigate the feasibility of an excimer laser ablation as a graftless FECD treatment option.

Characterization of surface modified glycerol/silk fibroin film for application to corneal endothelial cell regeneration

Corneal endothelial cells (CEnCs) play a fundamental role in maintaining the transparency of the cornea. CEnCs lose their full proliferating capacity when tissue damages occur. The loss in proliferation rate is associated with corneal edema and decrease in visual acuity, leading in severe cases, to blindness. In these situations, a corneal transplant is usually needed to restore the original tissue functions. Tissue engineering is an efficient alternative for the production of implantable films, which can regenerate the tissue functions regulating at the same time the immune-response.In this study, we proposed a stable and transparent film, composed of silk fibroin modified with glycerol (G/SF), as a potential substrate for corneal endothelial cells regeneration. Our results confirmed that G/SF films have a uniform structure, rougher surface and lower thickness respect to the SF film. In vitro tests show that G/SF films can induce a slight increase in CEnCs initial adhesion and proliferation rate if compared with the SF film. Morphology and gene expression evaluations demonstrated that the bioactive effects of silk fibroin were not affected by the presence of glycerol. For this reason, the G/SF films are suitable as CEnCs carrier and promising for the corneal damages treatments.

Topical Rho-Associated Kinase Inhibitor, Y27632, Accelerates Corneal Endothelial Regeneration in a Canine Cryoinjury Model.

Corneal endothelial cell regeneration varies by species, with nonhuman primates (NHPs) and rabbits displaying low and high proliferative capacities, respectively. Recent studies report that topical application of rho-associated kinase (ROCK) inhibitors accelerates corneal endothelial wound healing in animal models and human patients with endothelial dysfunction. This study determines the regenerative capacity of canine corneal endothelial cells in vivo and their response to a topical ROCK inhibitor, Y27632, after transcorneal freezing. Right eyes of 6 beagles underwent transcorneal freezing; 10 mM ROCK inhibitor Y27632 or vehicle control was applied topically to both eyes at least 4 times daily for 56 days. Endothelial cell density was evaluated by in vivo confocal microscopy, and corneal thickness was measured by Fourier-domain optical coherence tomography (FD-OCT) and ultrasound pachymetry. Transcorneal freezing induced severe central corneal edema in dogs, with restoration of transparency occurring within 4 weeks. Y27632 significantly decreased corneal thickness by FD-OCT and ultrasound pachymetry in the acute phase and significantly increased endothelial cell density at days 28 and 42 post-cryoinjury, suggesting faster restoration of endothelial cell recovery. Canine corneal endothelial function recovers at a similar rate as NHPs but more slowly than rabbits after cryoinjury. Faster corneal endothelial wound healing was observed by in vivo confocal microscopy and FD-OCT in dogs treated with Y27632 versus vehicle controls. Thus, a canine cryoinjury model may be a useful alternative to NHPs in detecting a response to therapies directed at endothelial regeneration.

Functionalized silk fibroin film scaffold using β-Carotene for cornea endothelial cell regeneration.

Design of corneal endothelium substitute is important for replacement of cadaveric cornea tissue. Our previous study has shown the suitability of silk fibroin (SF) as a biomaterial for cornea scaffold. In this study, we used β-Carotene (β-C) to enhance the regeneration of corneal endothelial cells (CEnCs) and maintain CEnC specific function. The fabricated film scaffolds showed desired transparency and hydrophilic properties which are crucial factors for vision recovery. The cell viability, phenotype and gene expression was examined using MTT assay, immunofluorescence and reverse transcription polymerase chain reactions. Compared with pristine SF scaffold, proper amount of β-C incorporated with SF scaffolds showed higher initial cell attachment, cell viability and mRNA expression. The results indicate that the fabricated SF film scaffold incorporated with small amount of β-C might be the suitable alternative corneal endothelium substitute for transplantation.

Descemet\u2019s Membrane Supports Corneal Endothelial Cell Regeneration in Rabbits

Descemet’s membrane (DM) helps maintain phenotype and function of corneal endothelial cells under physiological conditions, while little is known about the function of DM in corneal endothelial wound healing process. In the current study, we performed in vivo rabbit corneal endothelial cell (CEC) injury via CEC scraping, in which DM remained intact after CECs removal, or via DM stripping, in which DM was removed together with CECs. We found rabbit corneas in the CEC scraping group healed with transparency restoration, while there was posterior fibrosis tissue formation in the corneas after DM stripping on day 14. Following CEC scraping on day 3, cells that had migrated toward the central cornea underwent a transient fibrotic endothelial-mesenchymal transition (EMT) which was reversed back to an endothelial phenotype on day 14. However, in the corneas injured via DM stripping, most of the cells in the posterior fibrosis tissue did not originate from the corneal endothelium, and they maintained fibroblastic phenotype on day 14. We concluded that corneal endothelial wound healing in rabbits has different outcomes depending upon the presence or absence of Descemet’s membrane. Descemet’s membrane supports corneal endothelial cell regeneration in rabbits after endothelial injury.

Collagen type I-PLGA film as an efficient substratum for corneal endothelial cells regeneration

Surface modulations of desired biological construct design for regenerative medical therapy is considered to be highly crucial for cell growth and the subsequent regeneration of biologically competent tissues. In this study, we fabricated stable, transparent, collagen type-I-coated PLGA films (Col I–PLGA) as a potential substratum for the regeneration of corneal endothelial cells. Morphological and structural properties were analysed by FE–SEM, AFM, FTIR, contact angle, etc., and in vitro biocompatibility of the Col I–PLGA films was further tested in primary rabbit corneal endothelial cells (rCEnCs) as models. Compared with bare PLGA films, the Col I–PLGA films displayed the requisite surface roughness, with higher Ra (nm) values, transparency, good hydrophilicity, stability and water uptake. Next, cultured rCEnCs on Col I–PLGA films showed the characteristic polygonal shape of rCEnCs with enhanced initial attachment, proliferation and expression of mRNAs. Collectively, these results indicate that Col I–PLGA can be employed as a suitable alternative for high-quality corneal tissue expansion and transplantation. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibition of Matrix Metalloproteinase Activity Reverses Corneal Endothelial-Mesenchymal Transition

Exvivo culture or regeneration of corneal endothelial cells often is subjected to gradual endothelial-mesenchymal transition and loss of function. Here, we found that during exvivo culture, bovine corneal endothelial cells underwent endothelial-mesenchymal transition and had an up-regulated expression and activity of matrix metalloproteinases. Inhibition of matrix metalloproteinase activity in confluent bovine corneal endothelial cells decreased the level of endothelial-mesenchymal transition regulators: snail and slug. The phosphorylation and degradation of the key Wnt signaling pathway modulator active β-catenin also were accelerated with the broad-spectrum matrix metalloproteinase inhibitor Marimastat, which may result from decreased N-cadherin shedding and increased intact N-cadherin molecules on the cell membrane. Intracameral injection of Marimastat also suppressed basic fibroblast growth factor-induced endothelial-mesenchymal transition in a rat corneal endothelium cryo-injury model and significantly diminished the corneal edema. Our study indicated that inhibition of matrix metalloproteinase activity can reverse endothelial-mesenchymal transition and preserve the function of corneal endothelial cells both during exvivo culture and invivo. This may offer a potential therapeutic target in regenerative medicine for the treatment of corneal endothelial dysfunctions.

Silk fibroin film as an efficient carrier for corneal endothelial cells regeneration

Tissue engineered corneas are a promising alternative to the limited donor corneas for transplantation. Here, we fabricated a transparent silk fibroin film with varied sericin content — low sericin silk fibroin film (LSS) and high sericin silk fibroin film (HSS) — to evaluate its ability to serve as an efficient carrier for corneal endothelial cells. Both LSS and HSS were prepared from Bombyx mori containing different percentages of sericin −6.25% and 9.83%, respectively. Tissue culture polystyrene was used as a reference substratum. In vitro studies, such as a morphological evaluation, initial attachment, expression of mRNAs and proteins (ZO-1 and Na+/K+-ATPase), were performed using rabbit primary corneal endothelial cells as models on silk fibroin films. Results showed that the corneal endothelial cells grown on as-fabricated LSS and HSS were well-maintained, indicating no effect of the difference in sericin content in the films. However, the HSS displayed better initial cell attachment due to the adhesive properties of sericin. These results demonstrated that rabbit corneal endothelial cells grew well on a silk substratum although the percentage of sericin showed no significant difference.

Fabrication of transparent silk fibroin film for the regeneration of corneal endothelial cells; preliminary study

Dystrophies related to the corneal endothelium result in edema, loss of transparency, and finally, blindness. Currently, the only treatment of patients with these corneal endothelial dystrophies is transplantation. However, the limited availability of donor corneas is a main obstacle to transplanting healthy endothelia. In this study, we have chosen silk from the silkworm to make a substratum for corneal endothelial cells (CEnCs). This approach offers the possibility of overcoming the issue of limited supply of donor corneas. Two types of transparent silk films with different sericin contents have been prepared by boiling for 30 min and 1 h, respectively, as delivery vehicles for application on the corneal endothelium. The contents of sericin for high-sericin silk (HSS) and low-sericin silk (LSS) are 9.83% and 6.25%, respectively. Physical and chemical properties were analyzed by contact angle, transparency, tensile strength, FTIR, and DSC. Proliferation assay by MTT was conducted to confirm the viability of the CEnCs. These results demonstrate that the fabricated transparent silk films have sufficient physical and chemical properties as a possible carrier for CEnCs.

Effect of corticosteroids on healing of the corneal endothelium in cats.

Anterior segment surgery is frequently complicated by damage to the corneal endothelium. We examined the effects of corticosteroids, which are widely used for the suppression of postoperative inflammation, on the process of endothelial cell regeneration. The effect of corticosteroids on healing of the corneal endothelium was examined in 10 domestic cats. In both eyes a circular area, 8 mm in diameter, was scraped off at the center of the corneal endothelium without damaging Descemet's membrane. Immediately after scraping, as well as 2 and 5 days later, each animal received a unilateral retrobulbar injection of betamethasone sodium phosphate (2 mg). The other eye served as a control and received a retrobulbar injection of the vehicle only. Evaluation of the corneal endothelium 2, 5 and 7 days after the trauma revealed that relative to the control contralateral eyes, the corticosteroid-treated eyes exhibited a higher mean coefficient of variation of the corneal endothelium cell area, fewer hexagonal cells, a larger number of polygonal cells with 3, 4 7 and 8 cellular facets, thinner corneas and less inflammation. These findings suggest that corticosteroids unfavorably affect the regeneration of corneal endothelial cells after injury. As corticosteroids appear to have both positive and adverse effects on corneal function after trauma, they should be used with caution.

Penetrating Keratoplasty in the Cat: A Clinically Applicable Model

A series of 28 consecutive penetrating keratoplasties were performed on adult cats. Donor corneas (n = 14) were maintained in culture medium for 14-24 hours prior to transplantation. Rotational autografts (n = 7) were used to control for cell loss caused by culture maintenance as well as for the effects of surgery. Additional homografts (n = 7) were transplanted following removal of the corneal endothelium to study the extent of host corneal endothelial cell regeneration. Pre- and post-operative endothelial cell counts of the homografts made from specular micrographs demonstrated an average cell loss of 30% one month following surgery. A similar 30% average cell loss was present in the rotational autografts. Clinically, both homografts and autografts remained clear and were near normal in thickness. Homografts lacking endothelium exhibited persistent, severe edema that correlated with the inability of the host corneal endothelium to resurface the graft. Clinical and morphologic evidence of mild homograft rejection was observed in 15% of the animals that received normal homografts. Corneal endothelial cell loss following penetrating keratoplasty in the cat approximates that observed following the same procedure in the human. Additionally, regenerative capacity of the corneal endothelium in the cat, like that of the human, is limited. These features suggest that this cooperative, hardy animal is an excellent model in which to study many aspects of corneal transplantation that have direct application to the treatment of human corneal disease.