Treatment Of Corneal Blindness Research Articles

Clinical observation of autologous penetrating keratoplasty in the treatment of corneal blindness

Clinical observation of autologous penetrating keratoplasty in the treatment of corneal blindness

Comparative gene expression profiling reveals key pathways and genes different in skin epidermal stem cells and corneal epithelial cells.

Corneal epithelial cells (CECs) are required for corneal transparency and visual function, and corneal injuries may cause corneal blindness. Skin epidermal stem cells (SESCs), which share the same origin with CECs and have the potential of multi-directional differentiation are ideal seed cells for tissue engineered corneal construction to treat corneal blindness. This study aims to investigate critical genes and pathways that may modulate the transdifferentiation from SESCs to CECs. Isolated SESCs and CECs were used for gene expression analysis by microarray. GO and KEGG pathway of differently expressed genes (DEGs) were enriched using DAVID. The protein-protein interaction (PPI) network were then constructed using Cytoscape and highly interconnected module was subsequently isolated from the network by Molecular Complex Detection. Expression of the hub genes and other selected genes were then verified by qRT-PCR. We found 112 upregulated and 105 downregulated genes in CECs compared with SESCs. These DEGs were significantly enriched in focal adhesion, PI3K-Akt and TNF signaling pathway. Highly interconnected module of PPI network contains ten genes. Further regulatory network of these genes showed that ESR1 and SLC2A4 were hub genes. Our study identified gene expression in SESCs and CECs and suggested that several crucial genes and pathways may play critical roles in transdifferentiation from SESCs to CECs. It may help uncover molecular mechanisms and offer a foundation for promoting tissue-engineered cornea into clinical application.

Clinical outcomes for the treatment of corneal blindness using an epidescemetic keratoprosthesis

Clinical outcomes for the treatment of corneal blindness using an epidescemetic keratoprosthesis

Mimicking corneal stroma using keratocyte-loaded photopolymerizable methacrylated gelatin hydrogels.

Cell-laden methacrylated gelatin (GelMA) hydrogels with high (approximately 90%) transparency were prepared to mimic the natural form and function of corneal stroma. They were synthesized from GelMA with a methacrylation degree of 70% as determined by nuclear magnetic resonance. Hydrogels were strong enough to withstand handling. Stability studies showed that 87% of the GelMA hydrogels remained after 21days in phosphate buffered saline (PBS). Cell viability in the first 2days was over 90% for the human keratocytes loaded in the gels as determined with the live-dead analysis. Cells in the hydrogel elongated and connected to each other as observed by confocal laser scanning microscopy (CLSM) images and scanning electron microscope analysis after 3weeks in the culture medium and cells were seen to be distributed throughout the hydrogel bulk. Cells were found to synthesize collagen Types I and V, decorin, and biglycan (representative collagens and proteoglycans of human corneal stroma, respectively) showing that keratocytes maintained their functions and preserved their phenotypes in the hydrogels. Transparency of cell-loaded and cell-free hydrogels after 21days was found to be over 90% at all time points in the visible light range and was comparable to the transparency of the native cornea. The corneal stroma equivalent produced in this study that has cells entrapped in it leads to a product with homogenous distribution of cells. It was transparent at the very beginning and is expected to allow better vision than nontransparent substrates. It, therefore, has a significant potential to be used as an alternative to the current products used to treat corneal blindness.

Scaffold-free tissue engineering of functional corneal stromal tissue.

Blinding corneal scarring is predominately treated with allogeneic graft tissue; however, there is a worldwide shortage of donor tissue leaving millions in need of therapy. Human corneal stromal stem cells (CSSC) have been shown produce corneal tissue when cultured on nanofibre scaffolding, but this tissue cannot be readily separated from the scaffold. In this study, scaffold-free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs that can be transplanted in vivo without introducing the additional variables associated with exogenous scaffolding. CSSC were cultured on substrates with aligned microgrooves, which directed parallel cell alignment and matrix organization, similar to the organization of native corneal stromal lamella. CSSC produced sufficient matrix to allow manual separation of a tissue sheet from the grooved substrate. These constructs were cellular and collagenous tissue sheets, approximately 4μm thick and contained extracellular matrix molecules typical of corneal tissue including collagen types I and V and keratocan. Similar to the native corneal stroma, the engineered corneal tissues contained long parallel collagen fibrils with uniform diameter. After being transplanted into mouse corneal stromal pockets, the engineered corneal stromal tissues became transparent, and the human CSSCs continued to express human corneal stromal matrix molecules. Both in vitro and in vivo, these scaffold-free engineered constructs emulated stromal lamellae of native corneal stromal tissues. Scaffold-free engineered corneal stromal constructs represent a novel, potentially autologous, cell-generated, biomaterial with the potential for treating corneal blindness. Copyright © 2016 John Wiley & Sons, Ltd.

Human Turbinate-derived Mesenchymal Stem Cells Differentiated into Keratocyte Progenitor Cells

Objective: Though keratoplasty is used to treat corneal blindness, donor shortage, tendency of stimulated keratocyte transformed to fibroblast and immunological rejection are still big problems. As a solution, cornea tissue engineering using non-corneal tissue sourced cells become emerging issue. Thus, this study was designed to find novel material for keratoplasty. Methods: Human turbinate-derived mesenchymal stem cells (hTMSCs) were obtained from patients and cultured with differentiation medium for 14 days. The keratocyte markers, stem cell markers, early corneal stromal stem cell (CSSC) markers, were measured by real time-PCR. The MSC markers were detected by FACS. Results: After 14 days of differentiation medium exposure, hTMSCs expressed markers of keratocyte such as keratocan sulfate proteoglycan (KERA) and aldehydrogenase (ALDH). As the hTMSCs became keratocytes, the expression of embryonic ocular precursor markers ABCG2 and PAX6 decreased but were still measurable. Early CSSC markers including SIX2, SIX3, BMI expression was elevated after 7 d and reduced after 14 d of KDM treatment. The stem cell markers such as SOX2, Notch were decreased. After 14 d of differentiation, the hTMSCs expressed MSC markers CD73, CD90, and CD105, but not hematopoietic markers CD14, CD19, CD34, HLA-DR; these changes indicate development of a characteristic MSC phenotype. hTMSCs inhibited the tube-formation ability of human microvessel endothelial cells. hTMSCs derived from neural crest could differentiate into keratocyte progenitors. Conclusions: This study first reveals that the hTMSCs have potential to be differentiated into keratocyte progenitor- like cells. Use of hTMSCs derived from neural crest in cell-based therapeutics as source for corneal tissue engineering may overcome the problems of keratoplasty such as immunological rejection and limiting supply of human donor corneas.

Sequentially-crosslinked bioactive hydrogels as nano-patterned substrates with customizable stiffness and degradation for corneal tissue engineering applications.

Naturally-bioactive hydrogels like gelatin provide favorable properties for tissue-engineering but lack sufficient mechanical strength for use as implantable tissue engineering substrates. Complex fabrication or multi-component additives can improve material strength, but often compromises other properties. Studies have shown gelatin methacrylate (GelMA) as a bioactive hydrogel with diverse tissue growth applications. We hypothesize that, with suitable material modifications, GelMA could be employed for growth and implantation of tissue-engineered human corneal endothelial cell (HCEC) monolayer. Tissue-engineered HCEC monolayer could potentially be used to treat corneal blindness due to corneal endothelium dysfunction. Here, we exploited a sequential hybrid (physical followed by UV) crosslinking to create an improved material, named as GelMA+, with over 8-fold increase in mechanical strength as compared to regular GelMA. The presence of physical associations increased the subsequent UV-crosslinking efficiency resulting in robust materials able to withstand standard endothelium insertion surgical device loading. Favorable biodegradation kinetics were also measured invitro and invivo. We achieved hydrogels patterning with nano-scale resolution by use of oxygen impermeable stamps that overcome the limitations of PDMS based molding processes. Primary HCEC monolayers grown on GelMA+carrier patterned with pillars of optimal dimension demonstrated improved zona-occludin-1 expression, higher cell density and cell size homogeneity, which are indications of functionally-superior transplantable monolayers. The hybrid crosslinking and fabrication approach offers potential utility for development of implantable tissue-engineered cell-carrier constructs with enhanced bio-functional properties.

[Pay attention to the donor material supply for corneal transplantation].

Corneal transplantation is an important method in the treatment of corneal blindness. It is imperative to improve the treatment effectiveness of corneal disease and reduce the possibility of corneal blindness with the progress of corneal transplantation surgery, the construction and development of eye banks and the rational use of donor materials. This article reviews the component corneal transplantation technology promotion, eye bank construction and preparation of donor slices for component corneal transplantation surgery. (Chin J Ophthalmol, 2016, 52: 641-643).

Regenerative approaches for the cornea.

The cornea is the transparent front part of the eye that transmits light to the back of the eye to generate vision. Loss of corneal transparency, if irreversible, leads to severe vision loss or blindness. For decades, corneal transplantation using human donor corneas has been the only option for treating corneal blindness. Despite recent improvement in surgical techniques, donor cornea transplantation remains plagued by risks of suboptimal optical results and visual acuity, immune rejection and eventually graft failure. Furthermore, the demand for suitable donor corneas is increasing faster than the number of donors, leaving thousands of curable patients untreated worldwide. Here, we critically review the state of the art of biomaterials for corneal regeneration. However, the lessons learned from the use of the cornea as a disease model will allow for extension of the biomaterials and techniques for regeneration of more complex organs such as the heart.

Composite core-and-skirt collagen hydrogels with differential degradation for corneal therapeutic applications

Scarcity of donor tissue to treat corneal blindness and the need to deliver stem cells or pharmacologic agents to ensure corneal graft survival are major challenges. Here, new composite collagen-based hydrogels are developed as implants to restore corneal transparency while serving as a possible reservoir for cells and drugs. The composite hydrogels have a centrally transparent core and embedded peripheral skirt of adjustable transparency and degradability, with the skirt exhibiting faster degradation in vitro. Both core and skirt supported human epithelial cell populations in vitro and the skirt merged homogeneously with the core material to smoothly distribute a mechanical load in vitro. After in vivo transplantation in rabbit corneas over three months, composites maintained overall corneal shape and integrity, while skirt degradation could be tracked in vivo and non-invasively due to partial opacity. Skirt degradation was associated with partial collagen breakdown, thinning, and migration of host stromal cells and macrophages, while the central core maintained integrity and transparency as host cells migrated and nerves regenerated. ImpactThis study indicates the feasibility of a collagen-based composite hydrogel to maintain corneal stability and transparency while providing a degradable peripheral reservoir for cell or substance release.

HC-HA/PTX3 Purified From Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells to Maintain Quiescence of Limbal Epithelial Progenitor/Stem Cells.

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency.

Early Observation of Extracellular Matrix-Derived Hydrogels for Corneal Stroma Regeneration.

A lack of healthy transplantable tissue to treat corneal blindness has led researchers to investigate the development and application of different scaffold materials for corneal tissue engineering and regeneration. In this study, hydrogels fabricated from decellularized corneal extracellular matrix were developed as a new approach to corneal stromal tissue regeneration. Porcine corneas were decellularized using a technique that combined freeze-thaw cycles with a nuclease treatment. The corneas were then freeze-dried, milled, and digested in an acidic pepsin solution that was used to form a hydrogel after adjusting the pH and gelation temperature. The resultant corneal matrix hydrogels (CMHs) were seeded with human corneal stromal cells and cultured for several days. When compared to collagens hydrogels, CMHs had superior optical transparency, similar mechanical properties, and were better able to retain the stromal cells native keratocyte phenotype. The CMHs also supported cell viability and proliferation and contained sulfated glycosaminoglycan, a vital constituent, for maintaining corneal transparency. These results suggest that the CMHs could provide an exceptional biomaterial for corneal stroma regeneration.

Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory recommendations to limit the use of xenogenic materials.

Optimization of Ex Vivo Expansion of Limbal Epithelial Progenitors by Maintaining Native Niche Cells on Denuded Amniotic Membrane

Transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on epithelially denuded amniotic membrane (dAM) in supplemented hormonal epithelial medium (SHEM) is an alternative solution for treating corneal blindness due to limbal stem cell (SC) deficiency. Because the phenotype of limbal niche cells (NCs) is preserved better in serum-free modified embryonic stem cell (ESC) medium (MESCM) than SHEM, we question whether the aforementioned expansion protocol can be further optimized by maintaining limbal NCs using MESCM. Collagenase-isolated limbal clusters were cultured on dAM in SHEM or MESCM for 8 to 10 days. Epithelial outgrowth sheets removed by dispase were subjected to real-time quantitative polymerase chain reaction (qPCR) and immunostaining for expression of corneal epithelial markers (p63α, pax6, and K12) and NC markers (FLK-1, CD34, CD31, PDGFR-B, and α-SMA). A total of 1000 single cells were seeded on 6-well dish containing 3T3 feeder layers for 12 to 14 days before rhodamine B staining. Epithelial outgrowth in SHEM showed a significant loss of corneal SC and ESC markers when compared with freshly collagenase-isolated limbal clusters. Although the epithelial outgrowth was slower in MESCM, epithelial cell size was consistently smaller than that found in SHEM. Furthermore, MESCM maintained a significantly higher percentage of PCK-/ Vim+ cells and exhibited a significant upregulation of NC markers and corneal epithelial SC markers (K15, Bmi-1, and Msi-1) than SHEM. Furthermore, the number of purported holoclones was significantly promoted in MESCM than SHEM. These data collectively suggest that MESCM can be used to replace SHEM to further promote expansion of LEPC by maintaining limbal native NCs. Effective ex vivo expansion of limbal epithelial SC is a first and important step toward the success of treating corneal blindness caused by limbal stem cell deficiency and paves the way for future applications in regenerative medicine.

Corneal blindness: a global perspective.

Diseases affecting the cornea are a major cause of blindness worldwide, second only to cataract in overall importance. The epidemiology of corneal blindness is complicated and encompasses a wide variety of infectious and inflammatory eye diseses that cause corneal scarring, which ultimately leads to functional blindness. In addition, the prevalence of corneal disease varies from country to country and even from one population to another. While cataract is responsible for nearly 20 million of the 45 million blind people in the world, the next major cause is trachoma which blinds 4.9 million individuals, mainly as a result of corneal scarring and vascularization. Ocular trauma and corneal ulceration are significant causes of corneal blindness that are often underreported but may be responsible for 1.5-2.0 million new cases of monocular blindness every year. Causes of childhood blindness (about 1.5 million worldwide with 5 million visually disabled) include xerophthalmia (350,000 cases annually), ophthalmia neonatorum, and less frequently seen ocular diseases such as herpes simplex virus infections and vernal keratoconjunctivitis. Even though the control of onchocerciasis and leprosy are public health success stories, these diseases are still significant causes of blindness--affecting a quarter of a million individuals each. Traditional eye medicines have also been implicated as a major risk factor in the current epidemic of corneal ulceration in developing countries. Because of the difficulty of treating corneal blindness once it has occurred, public health prevention programmes are the most cost-effective means of decreasing the global burden of corneal blindness.