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      A Native-Like Corneal Construct Using Donor Corneal Stroma for Tissue Engineering

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          Abstract

          Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon Epikeratome system. The morphology, phenotype, regenerative capacity and transplantation potential were evaluated by hematoxylin eosin and immunofluorescent staining, a wound healing model, and the xeno-transplantation of the corneal constructs to nude mice. An optically transparent and stratified epithelium was rapidly generated on donor corneal stromal substrate and displayed native-like morphology and structure. The cells were polygonal in the basal layer and became flattened in superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium in vivo. The differentiation markers, keratin 3, involucrin and connexin 43, were expressed in full or superficial layers. Interestingly, certain basal cells were immunopositive to antibodies against limbal stem/progenitor cell markers ABCG2 and p63, which are usually negative in corneal epithelium in vivo. It suggests that this bioengineered corneal epithelium shared some characteristics of human limbal epithelium in vivo. This engineered epithelium was able to regenerate in 4 days following from a 4mm-diameter wound created by a filter paper soaked with 1 N NaOH. This corneal construct survived well after xeno-transplantation to the back of a nude mouse. The transplanted epithelium remained multilayer and became thicker with a phenotype similar to human corneal epithelium. Our findings demonstrate that natural corneal stroma is an optimal substrate for tissue bioengineering, and a native-like corneal construct has been created with epithelium containing limbal stem cells. This construct may have great potential for clinical use in corneal reconstruction.

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          Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells.

          Stevens-Johnson syndrome, ocular pemphigoid, and thermal or chemical burns can cause scarring and opacification of the cornea and loss of vision. Transplantation of epithelial cells from the limbus of the contralateral cornea can restore useful vision. However, this procedure requires a large limbal graft from the healthy eye and is not possible in patients who have bilateral lesions. We took specimens of limbal epithelial cells from the healthy contralateral eyes of six patients with severe unilateral corneal disease. The epithelial cells were cultured and expanded on amniotic membrane. The amniotic membrane, together with the sheet of limbal epithelial cells, was transplanted to the denuded corneal surface of the damaged eye after superficial keratectomy to remove fibrovascular ingrowth. The mean (+/-SD) follow-up period was 15+/-2 months. Complete reepithelialization of the corneal surface occurred within two to four days of transplantation in all six eyes receiving transplants. By one month, the ocular surface was covered with corneal epithelium, and the clarity of the cornea was improved. In five of the six eyes receiving transplants (83 percent), the mean visual acuity improved from 20/112 to 20/45. In one patient with a chemical burn who had total opacification of the cornea, the acuity improved from the ability to count fingers at 40 cm to 20/200. No patient had recurrent neovascularization or inflammation in the transplanted area during the follow-up period. Transplantation of autologous limbal epithelial cells cultured on amniotic membrane is a simple and effective method of reconstructing the corneal surface and restoring useful vision in patients with unilateral deficiency of limbal epithelial cells.
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            Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas.

            After n-heptanol removal of the total corneal epithelium and a limbal lamellar keratectomy, 23 rabbit eyes developed features of limbal stem cell deficiency including conjunctival epithelial ingrowth, vascularization and chronic inflammation. One month later, 10 control eyes received a total keratectomy, and 13 experimental eyes received additional transplantation of glycerin-preserved human amniotic membrane. In 3 months of follow-up, all control corneas were revascularized to the center with granuloma and retained a conjunctival epithelial phenotype. In contrast, five corneas in the experimental group became clear with either minimal or no vascularization; the rest had either mid peripheral (n = 5) or total (n = 3) vascularization and cloudier stroma. The success of corneal surface reconstruction correlated with the return of a cornea-like epithelial phenotype and the preservation of amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. These results suggest that measures taken to facilitate epithelialization without allowing host fibrovascular ingrowth onto the amniotic membrane might prove this procedure clinically useful for ocular surface reconstruction.
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              Identification and characterization of limbal stem cells.

              The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SC) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SC biology remains the ability to identify stem cells in situ and in vitro. Until recently, the identification of limbal stem cells mainly has been based on general properties of stem cells, e.g. lack of differentiation, prolonged label-retaining, indefinite capacity of proliferation exemplified by the clonogenic assay as well as their special role in corneal wound healing. During the last years, a number of molecular markers for the limbal SC compartment has been proposed, however, their role in distinguishing limbal SC from their early progeny is still under debate. Data reported from the literature combined with our own recent observations suggest, that the basal epithelial cells of the human limbus contain ABCG2, K19, vimentin, KGF-R, metallothionein, and integrin alpha9, but do not stain for K3/K12, Cx43, involucrin, P-cadherin, integrins alpha2, alpha6, and beta4, and nestin, when compared to the basal cells of the corneal epithelium. A relatively higher expression level in basal limbal cells was observed for p63, alpha-enolase, K5/14, and HGF-R, whereas there were no significant differences in staining intensity for beta-catenin, integrins alphav, beta1, beta2, and beta5, CD71, EGF-R, TGF-beta-RI, TGF-beta-RII, and TrkA between limbal and corneal basal epithelial cells. Therefore, a combination of differentiation-associated markers (e.g. K3/K12, Cx43, or involucrin) and putative SC-associated markers (e.g. ABCG2, K19, vimentin, or integrin alpha9) may provide a suitable tool for identification of human limbal SC. While most putative SC markers label the majority of limbal basal cells and, therefore, may not distinguish SC from progenitor cells, only ABCG2 was strictly confined to small clusters of basal cells in the limbal epithelium. At present, ABCG2 therefore appears to be the most useful cell surface marker for the identification and isolation of corneal epithelial SC. Moreover, the characteristics of the specific microenvironment of corneal SC, as provided by growth factor activity and basement membrane heterogeneity in the limbal area, could serve as additional tools for their selective enrichment and in vitro expansion for the purpose of ocular surface reconstruction.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                15 November 2012
                : 7
                : 11
                : e49571
                Affiliations
                [1 ]Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States of America
                [2 ]Department of Ophthalmology, the Affiliated Hospital of Qingdao University Medical College, Qingdao, China
                [3 ]Department of Ophthalmology, Chonnam National University Medical School and Hospital, Gwangju, South Korea
                University of Reading, United Kingdom
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: DQL SCP JL. Performed the experiments: DQL JL KCY LZ CSP RL PM. Analyzed the data: DQL JL KCY ZS SCP. Contributed reagents/materials/analysis tools: DQL CSP. Wrote the paper: JL DQL ZS.

                Article
                PONE-D-12-18303
                10.1371/journal.pone.0049571
                3499466
                23166715
                03bae523-0f92-40e9-ad6f-3e5b773a08d3
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 June 2012
                : 10 October 2012
                Page count
                Pages: 8
                Funding
                This study was supported by Department of Defense CDMRP PRMRP FY06 PR064719 (DQL), National Institutes of Health Grant EY11915 (SCP), Lions Foundation for Sight, Research to Prevent Blindness, Oshman Foundation, and William Stamps Farish Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Immunochemistry
                Biotechnology
                Bioengineering
                Biomedical Engineering
                Tissue Engineering
                Immunology
                Immunologic Techniques
                Immunofluorescence
                Model Organisms
                Animal Models
                Mouse
                Molecular Cell Biology
                Cellular Types
                Stem Cells
                Adult Stem Cells
                Gene Expression
                Engineering
                Bioengineering
                Biomedical Engineering
                Medicine
                Ophthalmology

                Uncategorized
                Uncategorized

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