ABSTRACT
Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane
ABSTRACT
PURPOSE: To evaluate the long-term outcome of HLA-matched lr-CLAL for bilateral ocular surface disorders. METHODS: A retrospective, non-comparative interventional case series study of 39 eyes of 32 patients with bilateral surface disorders and clinical diagnosis of limbal stem cell deficiency who underwent HLA-matched lr-CLAL. Visual acuity (VA), ambulatory vision (> 20/200) and ocular surface stability were evaluated as main outcomes. Donor limbus was obtained from a sibling or a parent of the patient, after an appropriate Class I and II HLA match. RESULTS: One year after surgery, VA improved in 46.2 percent, ambulatory vision was achieved in 48.7 percent and a stable corneal surface was achieved in 84.6 percent of the eyes. At the final follow-up (mean, 48.7 ± 30.6 months), 66.6 percent of the eyes that had gained VA one year after surgery maintained an improved VA (p=0.28), 94.7 percent of eyes that had achieved ambulatory vision one year after surgery maintained 20/200 or better (p<0.001) and 93.9 percent still had a stable corneal surface (p=0.043) at the final follow-up. CONCLUSIONS: HLA-matched lr-CLAL can be an adequate method of treatment for bilateral ocular surface disorders, with a reasonable percentage of success of long-term results.
OBJETIVO: Avaliar os resultados em longo prazo do transplante alogênico inter vivo de limbo conjuntival para doenças bilaterais da superfície ocular com compatibilidade HLA doador-receptor. MÉTODOS: Estudo retrospectivo, não comparativo e intervencionista de 39 olhos de 32 pacientes submetidos a transplante alogênico de limbo com compatibilidade HLA e diagnóstico de deficiência límbica. Foram analisados como desfechos principais acuidade visual, visão ambulatorial (> 20/200) e estabilidade da superfície ocular. Limbo doador foi obtido de parentes do paciente após estudo de compatibilidade HLA classe I e II. RESULTADOS: Com um ano de pós-operatório, a acuidade visual melhorou em 46,2 por cento, visão ambulatorial foi atingida em 48,7 por cento e estabilidade da superfície corneana em 84,6 por cento dos pacientes. Ao final do seguimento (média, 48,7 ± 30,6 meses), 66,6 por cento dos olhos que haviam ganho acuidade visual um ano após a cirurgia mantiveram esta melhora (p=0,28), 94,7 por cento dos olhos que haviam alcançado visão ambulatorial um ano após a cirurgia mantiveram visão de 20/200 ou melhor (p<0,001) e 93,9 por cento ainda tinham superfície corneana estável (p=0,043) ao final do seguimento. CONCLUSÕES: O transplante alogênico inter vivo de limbo conjuntival com compatibilidade HLA revelou-se método adequado no tratamento de doenças bilaterais da superfície ocular, com uma porcentagem significativa de sucesso a longo-prazo.