Dissertations / Theses on the topic 'Corneal transplantation'

Cornea is the front transparent window of the eye which is responsible for optimal and clear vision. Transparency of this tissue is highly inevitable and cannot be compromised. Human cornea is made up of multiple layers out of which the posterior layer ‘endothelium’ is responsible for the transparency of the cornea. Endothelium is a monolayer of cells that allow the ions and solutes to transport from aqueous humour to the cornea and back which in turn maintains the transparency of the cornea by preserving the homeostasis between the anterior and posterior cornea. Earlier, it was observed that the endothelium had non regenerating capability however; recent studies have shown that these cells could be proliferated in vitro. Currently, the only method of treatment is the replacement of the diseased endothelium with the healthy donor endothelium. Penetrating keratoplasty which transplants a full thickness cornea was the only solution a decade ago. However, with the new advancements in the field of corneal transplants, specific surgical techniques like DMEK and DSAEK which replace only a part of the cornea have been identified. DSAEK replaces a part of the stroma along with the Descemet’s membrane and endothelium whereas DMEK only replaces the Descemet’s membrane and the endothelium and does not involve stroma. The results in terms of visual rehabilitation and outcomes have been found to be advantageous in these specific surgical procedures. However, DMEK is more challenging then DSAEK as DMEK is not yet a widespread technique, associated with steep learning curves and difficult donor tissue preparation. Despite DMEK is a challenging procedure it is becoming more popular because of the significant advantages in term of faster visual recovery, less postoperative astigmatism and reduced risk of transplant rejection, as compared to the other EK procedures. DMEK has several advantages in terms of rehabilitation rate and post-operative visual outcomes and therefore it is necessary to further refine this technique for a higher uptake of such surgeries and also considering that this is the only possible treatment for treating the patients suffering from endothelial dysfunctions. Although the corneal transplantation is well advanced, due to a limited supply of donor corneas for the transplantation purposes, alternative approaches like culturing corneal endothelium in vitro play an important role. Culturing the endothelium is not the only problem in EK but transplanting a 20 micron thick graft inside the recipient eye is another challenge. Moreover, the donor availability for culturing the corneal endothelium is less, making this strategy further more complicated. The thesis is therefore structured to highlight two significantly important issues in current scenario of endothelial keratoplasty, 1) posterior corneal transplantation or EK which is the on-going method of treatment for EK and 2) Human corneal endothelial cell culture which is the future of EK. Chapter 1 is an introduction to the world of eye banking, its current nature and development in the modern world and as a support to the surgeons not only in terms of new techniques but also devices for selective surgeries. It also highlights the preservation of the corneal tissues which is an important element in the field of eye banking. Eye banks play a significant role in the field of corneal transplants as they collect the human corneas and process them for transplantation. The corneas that are rejected for transplantation can be used for research and therefore development of eye banking and its research can change the field of corneal transplantation. Chapter 2 introduces the field of corneal cell culture and current techniques that are followed for culturing and possible transplantation of the cultured cells. To understand the reason and requirement of tissue engineering, it is important to study the human cornea, its extracellular matrix and its behaviour in different media. The biomechanical behaviour of the thin tissue i.e. the DM in different conditions becomes a relevant part of this study for future engineering which is studied in chapter 3. It is also important to standardize the currently available treatment options to reduce the burden of endothelial compromised patients in the future and avoid damages or tissue wastage that is currently occurring in the surgical theatres by providing standardized tissues in validated preservation medium which is studied in chapter 4. DMEK promises to become a more popular technique for the replacement of unhealthy corneal endothelium as it shows advantages like early rehabilitation rate and visual outcomes. Chapter 5 highlights the importance of new technique in rolling the DMEK tissue for easy insertion and unfolding in the recipient eye compared to the currently used technique with endothelium rolled in opposite direction. Presently, the DMEK tissues are either prepared in the surgical theatre or are stripped in the eye bank and shipped to the surgeons. However, there is no standardized procedure that could help validate a graft before surgery and provide a ready-to-use graft to the surgeons. Chapter 6 describes about a new technique of pre-loading a graft in a commercially available IOL cartridge which can be used as a preservation, transportation and transplantation device. This technique will further reduce graft wastage and will provide the surgeons a pre-validated graft further reducing the overall time in the surgical theatre and related costs. Thus different approaches for standardizing the DMEK technique were studied in the first phase of the thesis. HCECs are currently being cultured using young donor corneas. There are two major issues, firstly, the availability of the young donor corneas is less compared to the old donor corneas and secondly, there is no standard method of culturing the HCECs obtained so far. Therefore, to reduce the global tissue demand, there is a strong need to culture the HCECs from the old donor corneas which are less proliferative and less robust in nature but with high availability of the donor source. Chapter 7 is a study on isolation of HCECs and further culture of these cells from old donor corneas. Once the protocol was obtained, a full length study was performed with high sample size to prove the consistency of this technique which is highlighted in chapter 8. Meanwhile it was also noted that cells from old donors can be cultured using ROCK inhibitor in combination with Hyaluronic Acid (HA). HA induces mechanical force to the cells attaching them forcefully on the base and allows a higher proliferation of old donor cells which was studied in chapter 9. The second part of the thesis therefore investigates the culturing technique of HCECs from old donor corneas. However, once the cells are cultured, another challenge is to transplant them in the anterior chamber of the eye. This can be performed using two strategies, first, to implant the cells as suspension in the anterior chamber which is already been proposed, but the clinical evidence is still not confirmed yet, and second, to develop a carrier to transport the cultured cells. In chapter 10, we identified fish scales as a great source of collagen and therefore have investigated it as a potential scaffold to be used for HCECs culture and transplant in the future. It is also important to understand the regulations that govern the scientific studies and its use for clinical applications. Therefore, we also identified rHSA as a source to replace FCS for preserving human corneas in chapter 11. This will also help to create a synthetic media that could be used for GMP purposes for HCECs culture in the future. In conclusion, it was observed that pre-loading the tissues with endothelium-flapped inwards and preserved in dextran based medium could be a potential solution for providing a validated and standardized DMEK graft for the treatment of current endothelial dysfunction. Eye banks play a major role in the development of these surgical techniques and related devices which will change the face of corneal transplantation in the future. Alternatives like HCECs culture has a potential for the treatment of endothelial disorders and carriers like FSS could be used for culturing and transplanting these cells. However, the efficacy of these cells will only be validated after the clinical study. Considering the regulatory issues, synthetic medium would help both, the eye banks for preserving the corneas and its new products like pre-loaded DMEK and for cell culture in the future.
La cornea è quel tessuto trasparente che riveste la superficie anteriore dell'occhio, e che consente di avere una visione ottimale e chiara. La trasparenza di questo tessuto è fondamentale e non può essere compromessa. La cornea umana è costituita da più strati,tra cui lo strato posteriore o “endotelio” è responsabile della trasparenza della cornea. L’ endotelio è un monostrato di cellule che permettono agli ioni ed ai soluti di essere trasportati dall’ umor acqueo alla cornea e viceversa, e che a sua volta mantiene la trasparenza della cornea conservando l'omeostasi tra la cornea anteriore e posteriore. L’endotelio non possiede capacità rigenerative. Attualmente, l'unico metodo di trattamento è la sostituzione dell'endotelio danneggiato con l'endotelio di un donatore sano. La cheratoplastica perforante, che prevede trapianti di cornea a tutto spessore,rappresentava l'unica soluzione terapeuticafino ad un decennio fa. Tuttavia, con i nuovi progressi nel campo dei trapianti di cornea, sono state identificate specifiche tecniche chirurgiche, come DMEK e DSAEK, che sostituiscono solo una parte (o uno strato) della cornea. Sono I risultati ottenuti, in termini di riabilitazione visiva, si sono rivelati vantaggiosi grazie all’utilizzo di queste procedure chirurgiche specifiche. Tuttavia, la DMEK è più impegnativarispetto alla DSAEK in quanto non è ancora completamente standardizzata. La DMEK ha diversi vantaggi in termini di tasso di riabilitazione e risultati visivi post-operatori e quindi è necessario standardizzare questa tecnica per una maggiore diffusione di tali interventi e anche considerando che questo è l'unico trattamento possibile per la cura di pazienti affetti da disfunzioni endoteliali. Sebbene il trapianto di cornea sia in fase avanzata, a causa di una quantità limitata di cornee da donatori ai fini di trapianto, approcci alternativi come la coltura di endotelio corneale in vitro svolgono un ruolo importante. La coltura di endotelio non è l'unico problema nel trapianto di endotelio (EK)dal momento che trapiantare un innesto di 20 micron di spessore all'interno dell'occhio destinatario rappresenta una sfida ulteriore. Inoltre, la disponibilità dei donatori per la coltura di endotelio corneale è inferiore, rendendo questa strategia ulteriormente più complicata. La tesi è quindi strutturata in modo da mettere in evidenza due questioni molto importanti nell’ attuale scenario della cheratoplastica endoteliale, 1) trapianto di cornea posteriore o EK, che è l'attuale metodo di trattamento per la cheratoplastica endoteliale e 2) coltura delle cellule endoteliali della cornea umana, che rappresenta il futuro della cheratoplastica endoteliale. Il Capitolo 1 è un'introduzione sul mondo dell’ Eye Banking, sulle sue caratteristiche attuali, sullo sviluppo nel mondo moderno e sul supporto per i chirurghi, non solo in termini di nuove tecniche, ma anche di dispositivi per interventi selettivi. Si evidenzia anche la conservazione dei tessuti corneali, che è un elemento importante nel campo dell’Eye Banking. Le banche degli occhi svolgono un ruolo significativo nel settore dei trapianti di cornea, dal momento cheraccolgono le cornee umane e le analizzano per ilsuccessivo trapianto. Le cornee non idonee per il trapianto possono essere utilizzate per la ricerca e quindi lo sviluppo dell’Eye Bankinge la ricerca possono influenzare il campo del trapianto di cornea. Il Capitolo 2 introduce l’argomento delle colture cellulari corneali e le tecniche attuali che sono utilizzate per la coltura ed il trapianto di cellule coltivate. Per capire il motivo e l'esigenza dell’ingegnerizzazione dei tessuti, è importante studiare la cornea umana, la sua matrice extracellulare ed il suo comportamento in diversi mezzi di coltura. Il comportamento biomeccanico di un tessuto sottile (DM) in condizioni diverse rappresenta una parte rilevante di questo studio per la futura ingegnerizzazione,che viene descritta nel Capitolo 3. E’ inoltre importante standardizzare il trattamento attualmente disponibile allo scopo di ridurre in futuro l’onere di pazienti con endotelio compromesso ed evitare danni o sprechi di tessuto, che attualmente avvengono nelle sale chirurgiche, fornendo tessuti standardizzati in terreni di conservazione validati, come descritto nel Capitolo 4. La DMEK è considerata il futuro della cheratoplastica endoteliale, dal momento che presenta vantaggi quali la velocità dei tempi di riabilitazione ed i risultati visivi. Il Capitolo 5 mette in evidenza l'importanza della nuova tecnica che consiste nell’arrotolare il tessuto DMEK per consentire un facile inserimento per poi dispiegarlo nell'occhio ricevente, rispetto alla tecnica attualmente utilizzata con endotelio arrotolato in senso opposto. Attualmente, i tessuti DMEK sono o preparati in sala operatoria o allestiti in Banca degli Occhi e spediti ai chirurghi. Tuttavia, non vi è alcuna procedura standardizzata che possa contribuire ad ottenereun lembo endoteliale validato prima dell'intervento e fornire un innesto ready-to-use ai chirurghi. Il Capitolo 6 descrive una nuova tecnica di pre-caricamento di un lembo endoteliale in una cartuccia IOL disponibile in commercio che può essere utilizzato come dispositivo di conservazione, trasporto e trapianto. Questa tecnica consentirà di ridurre ulteriormente gli sprechi nei trapianti e fornirà ai chirurghi un innesto pre-convalidato,riducendo ulteriormente il tempo complessivo in sala operatoria edi relativi costi. Quindi nella prima fase della tesi, sono stati analizzati i diversi approcci per standardizzare la tecnica DMEK. Le HCECs sono attualmente coltivate usando cornee di donatori giovani. Ci sono due aspetti importanti, in primo luogo la disponibilità di tessuti di donatori giovani è minore rispetto a quella di donatori anziani, ed in secondo luogo non vi è, ad oggi, alcun metodo standardizzato di coltura delle HCECs. Pertanto, per ridurre la domanda di tessuti a livello mondiale, vi è una forte necessità di coltivare leHCECsderivanti da cornee di donatori anziani, che sono meno proliferative e meno resistenti in natura, ma per le quali vi è una elevata disponibilità della fonte donatrice. Il Capitolo 7 descrivelo studio sull'isolamento delle HCECs e la successiva coltura di tali cellule ottenute da cornee di donatori anziani. Una volta stabilito il protocollo, è stato eseguito uno studio completocon un alto campionamento, per dimostrare la coerenza di questa tecnica,come evidenziato nel Capitolo 8. Nel frattempo si è anche osservato che le cellule da donatori anziani possono essere coltivate utilizzando l’inibitore ROCK in combinazione con acido ialuronico (HA). HA induce una forza meccanica alle cellule per far sì che siano saldamente attaccate alla base e consentire così una maggiore proliferazione,come descritto nel Capitolo 9. La seconda parte della tesi indaga quindi la tecnica di coltura delle HCECs da cornee di donatori anziani. Tuttavia, una volta che le cellule sono coltivate, un'altra sfida è trapiantarle nella camera anteriore dell'occhio. Ciò può essere eseguito utilizzando due strategie: la prima è quella di ad impiantare le cellule in forma di sospensione nella camera anteriore, tecnica che è già stata proposta, ma che non ha ancora fornito un’evidenza clinica; la secondaè quella di sviluppare un substrato per il trasporto delle cellule coltivate. Nel Capitolo 10, si identifica la colla di pesce (FSS)come una grande fonte di collagene e quindi come un potenziale scaffold da utilizzare per la cultura HCECs e successivo trapianto. E’ inoltre importante capire le norme che regolano gli studi scientifici ed il loro uso nelle applicazioni cliniche. Pertanto, nel Capitolo 11, viene descritta l’identificazione dell’ rHSA come sostitutodell’ FCS per la conservazione di cornee umane. Questo contribuirà anche a creare un terreno di coltura sintetico che potrebbe essere utilizzato per la cultura HCECs in condizioni GMP in futuro. In conclusione, si è osservato che il pre-caricamento di tessuti con endotelio rivolto verso l'interno e conservati in un terreno con destrano, potrebbe rappresentare una possibile soluzione per fornire un lembo per DMEK validato e standardizzato per il trattamento delle disfunzioni endoteliali. Le banche degli occhi svolgono un ruolo importante nello sviluppo di queste tecniche chirurgiche e relativi dispositivi, che potranno cambiarele modalità del trapianto di cornea in futuro. Una tecnica alternativa come la coltura di HCECs ha in sèil potenziale per il trattamento di disturbi endoteliali e substrati come FSS potrebbero essere utilizzati per la coltura edil trapianto di queste cellule. Tuttavia, l'efficacia di queste cellule potrà essere validata solo dopo uno studio clinico. Considerando le questioni regolatorie, il terreno sintetico potrebbe aiutare le banche degli occhi sia per la conservazione delle cornee e dei i nuovi prodotti come DMEK pre-caricati sia, in futuro, per le colture.

Corneal transplantation is performed to restore vision or to relieve pain in patients with damaged or diseased corneas. However, approximately 40% of corneal allografts fail after 10 years. The most common cause of graft failure is irreversible immunological rejection, primarily mediated by CD4+ T cells, despite the topical application of glucocorticosteroids. The aim of this project was to investigate the anatomic site of antigen presentation during corneal transplantation in the rat, by using a lentiviral vector to express an anti-CD4 antibody fragment at potential sites of antigen presentation, including the donor corneal endothelium, the anterior segment of the eye and the cervical lymph nodes. Dual-gene lentiviral vectors were constructed by inserting the 2A self-processing sequence between two transgenes. This allowed expression of two transgenes within a single open reading frame. In vitro characterisation of the dual-gene vectors was performed in cell culture experiments, which showed that transgenic proteins were expressed at lower levels from dual-gene vectors compared to the expression from single-gene vectors and expression was lowest when the transgene was situated downstream of the 2A self-processing sequence. To locate the anatomic site of antigen presentation during corneal transplantation in rats, a lentiviral vector carrying an anti-CD4 antibody fragment was delivered to the corneal endothelium either immediately prior to corneal transplantation by ex vivo transduction of the donor corneas, or 5 days prior to corneal transplantation by anterior chamber injection into both the recipient and the donor rats. A separate group of recipient rats received intranodal injections of the lentiviral vector carrying an anti-CD4 antibody fragment into the cervical lymph nodes 2 days prior to corneal transplantation. Another group of rats underwent bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Corneal allografts were scored daily for opacity, inflammation and neovascularisation. Expression of the anti-CD4 antibody fragment from transduced tissues was detected using flow cytometry and polymerase chain reaction. Modest, but significant prolongation of corneal allograft survival was experienced by rats that received ex vivo transduction of the donor corneas with a lentiviral vector carrying an anti-CD4 antibody fragment immediately prior to corneal transplantation, but all grafts did eventually reject. Anterior chamber injection of the lentiviral vector carrying the anti-CD4 antibody fragment 5 days prior to corneal transplantation into both recipient and donor eyes did not prolong allograft survival. Intranodal injection of a lentiviral vector carrying an anti-CD4 antibody fragment did not prolong the survival of the corneal allografts, nor did bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Neither expression of the anti-CD4 antibody fragment in the cervical lymph nodes nor the removal of these nodes was able to prolong corneal allograft survival in rats, suggesting that T cell sensitisation could potentially occur elsewhere in the body. However, expression of the anti-CD4 antibody fragment from the donor corneal endothelium was able to prolong corneal allograft survival, suggesting that some antigen presentation might occur within the anterior segment of the eye. Based on the findings described in this thesis and those of others, I propose that antigen presentation in the rat occurs within anterior segment of the eye and within the secondary lymphoid tissues such as the cervical lymph nodes, and that inhibiting antigen presentation at one of these sites will delay graft rejection. However, to completely abolish antigen presentation during corneal transplantation in the rat, I hypothesise that antigen presentation within both the anterior segment of the eye and within the secondary lymphoid tissues must be inhibited.

Corneal transplantation is the oldest, most common and usually the most successful type of solid tissue allograft. The acceptancc of corneal allografts compared to other categories of allografts is called immune privilege. However, some conditions rob the corneal allograft of its immune privilege and promote rejection, which remains the leading cause of corneal graft failure. The precise immune mechanism underlying graft failure is incompletely understood. While differences in human leukocyte antigen (HLA) molecules between donor and host contribute to the alloreactivity driving the donor-antihost response, the cytokine milieu consisting of molecules that promote and regulate the alloresponse after transplantation is also critical. Single nucleotide polymorph isms (SNPs) in the promoter and coding regions of cytokine genes are associated with differential levels of expression and therefore play an important role in transplantation immunology. Cytokines are integral components of an inflammatory response and there are several potential sources of cytokine release within the cornea and the anterior chamber. This project is a candidate gene association study, focusing on genes known to be involved in ocular immune privilege and the compromise thereof, in corneal transplant recipients at increased risk of rejection (i.e., 'high risk corneal transplantation). In view of their central roles in initiation and suppression of the inflammatory response, tumour necrosis factor alpha (Tufa), interleukin - IO (IL• I 0), interleukin- 17 ( IL-1 7), thrombospondin•1 (TSP-I), vascular endothelial growth factor-A (VEG F-A) and the glucocorticoid cortisol were investigated. All transplants had three-year follow-up and results were analysed by PHASE (maximum-likelihood) analysis is to determine haplotype frequencies. Significant association between two extended TNF-u haplotypes and corneal graft outcome was found: TCTGGA was associated with a decreased risk of cornea] graft failure (n=384, RR 0.04, Cl 002-0.671, P <0.05, Pc <0.05) and TCTAGA was associated with increased risk of failure (n=384, RH. 3.59, Cl 3.21-4.03, P <0.05, Pc <0.05). In addition, a significant association was observed between the 3-1ocus TSP-] haplotype ACA, and an increased risk of corn ca I graft failure (n==359, OR 2.27, 95% Cl 1.65-3.13, P<0.05, P, <0.05).

Modulation of corneal transplant rejection using gene therapy shows promise in experimental models but the most appropriate vector for gene transfer is yet to be determined. The overarching aim of the thesis was to evaluate the potential of a lentiviral vector for use in human corneal transplantation. Specific aims were: (i) to assess the ability of an HIV-1-based lentiviral vector to mediate expression of the enhanced yellow fluorescent protein (eYFP), and a model secreted protein interleukin-10 (IL10), in ovine and human corneal endothelium; and (ii) to examine the influence of lentivirus-mediated IL10 expression on the survival of ovine corneal allografts. Four lentiviral vectors expressing eYFP under the control of different promoters, were tested: the simian virus type-40 (SV40) early promoter, the phosphoglycerate kinase (PGK) promoter, the elongation factor-1alpha (EF) promoter, and the cytomegalovirus (CMV) promoter. Two lentiviral vectors expressing IL10 were tested: one containing the SV40 promoter and another containing a steroid-inducible promoter (GRE5). Lentivirus-mediated expression in transduced ovine and human corneal endothelium was assessed by fluorescence microscopy, real-time quantitative RT-PCR and ELISA, following alterations of transduction period duration (2–24 hr) and vector dose, as well as in the presence or absence of polybrene or dexamethasone (GRE5 vector). It was also compared to expression mediated by adenoviral vectors. Orthotopic transplantation of ex vivo transduced donor corneas was performed in outbred sheep. Allografts were reviewed daily for vascularisation and signs of immunological rejection. Lentivirus-mediated eYFP expression was delayed in ovine corneal endothelium compared to human. However, in both species the final transduction rate was greater than 80% and expression was stable for at least 14 d in vitro. Lentivirus-mediated expression in ovine and human corneal endothelium was higher with the viral promoters in comparison to the mammalian promoters. A 24 h transduction of ovine corneal endothelium with the lentiviral vector encoding IL10 resulted in expression levels which were increasing after 15 d of organ culture but logarithmically lower than those achieved by adenovirus. Shortening the lentiviral transduction period to 2 h led to a reduction in expression, but the addition of polybrene (40 micrograms / ml) to the transduction mixture restored expression to levels comparable to those attained after a 24 h transduction period. Lentivirus-mediated IL10 expression was higher and more rapid in human corneal endothelium compared to ovine corneas. Dexamethasone-responsive transgene expression was observed in both ovine and human corneal endothelium using the lentiviral vector containing the GRE5 promoter. Lentivirus-mediated expression in ovine corneal endothelium was stable for 28 d in vivo. A modest prolongation of ovine corneal allograft survival (median of 7 d) was achieved by transduction of donor corneas for 2–3 h with the lentivirus expressing IL10. Attempts to increase the expression of IL10 by the addition of polybrene (40 micrograms / ml) to the transduction mixture, resulted in a toxic effect on corneal allografts which abrogated the beneficial effect of IL10. The lentiviral vector shows potential for the stable expression of therapeutic transgenes in human corneal transplantation. However, the mechanisms underlying the species-specific differences in HIV-1-mediated transgene expression will need to be elucidated and overcome if the ovine preclinical model is to provide justification for a clinical trial.

This thesis aims to place corneal allograft rejection in the context of general transplantation immunology, examine the role of lymphocyte subsets in the rejection process and consider the potential application of monoclonal antibody therapy in clinical corneal graft rejection. The literature relating to the current clinical practice of corneal grafting, with particular reference to corneal allograft rejection, is reviewed in chapter 1 to present the extent of the problem. Chapter 2 then reviews the mechanisms of allograft rejection from the literature of transplantation immunology, much of which has arisen from studies of kidney, heart, pancreatic islets and liver in animal models. The materials and methods are described in detail in chapter 3, and only the relevant experimental design is detailed in the Materials and Methods sections of the succeeding chapters. The experimental mouse model of transplanting corneal tissue into the renal subcapsular is evaluated in chapter 4, demonstrating that isografts survive indefinitely whereas allografts are rejected typically by 30 days. Pretransplant sensitisation decreased allograft survival time to 10 days. Immunohistochemistry demonstrated the presence of CD4⁺ and CD8⁺ lymphocytes and macrophages at the rejection site. Heterotopic corneal graft recipients were then treated with various monoclonal antibody regimes. Chapter 5 demonstrates that allograft survival can be increased by either anti-CD4 or anti-CD8 therapy, providing near total depletion of the respective lymphocyte subset is achieved. Xenograft rejection is shown to depend on mainly CD4⁺ lymphocytes in chapter 6, with no benefit being found of depleting the CD8⁺ subset in addition. A mild immunosuppressive effect of anti-Vβ8 monoclonal antibody is demonstrated and discussed in chapter 7. The final chapter discusses these results in the light of recent, related work in other transplant systems, and presents a case for a trial of intracameral pan-T-cell monoclonal antibody treatment.

About 25% of corneal grafts will fail within five years (mainly due to T-cell-mediated rejection) and the ex-vivo delivery of immunomodulatory genes to the cornea is one attractive method to improve survival rates. Early efforts focused on the use of viral vectors, whose toxicity and immunogenicity remain formidable barriers. Here, we developed an arginine-rich oligopeptide featuring a novel tri-block design. Each block served a specific purpose – DNA binding, endosomal escape and cell membrane penetration – and was systematically optimised. The peptides could effectively bind and protect DNA from physical and enzymatic degradation. Since the corneal endothelium is an important target for therapy, mouse corneal endothelial cells (MCEC) were chosen for initial in-vitro testing. The peptides depended heavily on cell surface heparan sulfate and utilised multiple endocytosis pathways to gain entry into MCEC. Importantly, they mediated indolamine 2,3-dioxygenase (IDO) mRNA and protein expression efficiently in both MCEC and murine corneas. IDO catalyses the biodegradation of tryptophan which T-lymphocytes sensitively require for growth. Hence, IDO over-expression in the cornea can prolong allograft survival by locally suppressing T-cell activity. We demonstrated that the IDO expressed was biologically active and could suppress the growth of CD4 T-cells in a proliferation assay. Immunohistochemistry further determined that IDO expression was localised to the corneal endothelium and epithelium. Corneal transplantations were then performed using a fully MHC-mismatched mouse model. However, the survival of IDO-transfected grafts was only marginally prolonged relative to GFP-transfected corneas and was shortened compared to untreated corneas. Toxicity, as evidenced by propidium iodide staining, was suspected to shorten graft survival following peptide-mediated transfection. Efforts to modulate toxicity by reducing the transfection period in the presence of chloroquine also failed to prolong graft survival. Future work should therefore focus on reducing toxicity while improving the transfection efficiency of this peptide carrier.

Orientador: Jose Paulo Cabral de Vasconcellos
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-25T23:02:33Z (GMT). No. of bitstreams: 1 Okanobo_Andre_M.pdf: 2897473 bytes, checksum: 5bad5928ff037c8396c72781f5829cc1 (MD5) Previous issue date: 2014
Resumo: Avaliação da qualidade de Vida dos Pacientes Antes e Após Realização de Transplante Penetrante de Córnea. OBJETIVO Verificar o impacto do transplante de córnea sobre a qualidade de vida dos indivíduos submetidos à cirurgia no serviço de Oftalmologia HC-UNICAMP no período de outubro de 2005 a outubro de 2006. METODO: O estudo foi aprovado pelo comitê de ética e pesquisa da FCM-UNICAMP. Foram inclusos indivíduos com indicação de transplante de córnea do ambulatório de Doenças Externas da Oftalmologia do HC UNICAMP no período de outubro de 2005 a outubro de 2006 e que aceitaram em participar do estudo. Foram incluídos 32 indivíduos. Os critérios de exclusão foram transplante tectônico, indivíduos com déficit visual importante causado por outra afecção ocular além das alterações corneanas. Realizou-se exame oftalmológico assim como aspectos demográficos e história ocular dos pacientes incluídos no estudo. Métodos objetivos como claridade do transplante e melhor acuidade visual corrigida e método subjetivo, a qualidade de vida, através do questionário SF-36 adaptado. RESULTADOS: Houve diferença significativa de satisfação entre gêneros com maior escore no sexo masculino (p = 0.0319). No método objetivo como a acuidade visual corrigida teve aumento significativo do olho transplantado (p<0.0001); A AV média antes do transplante era de 0,98 ± 0,1 logMAR e após 0,48 ± 0.38 logMAR. Quanto o escore de qualidade de vida (SF-36), método subjetivo, após o transplante houve um aumento significativo (p<0.0001). O escore do SF-36 antes do transplante foi de 49,11% ± 19,28 (média ± DP) e após o transplante de 71, 98% ± 24,28 (média ± DP) com uma melhora de 22, 87% (p<0.0001).Os indivíduos mais satisfeitos foram aqueles que tiveram a pontuação mais alta no SF-36 (r = 0.60; P = 0.0002). A satisfação parece não estar correlacionada com a melhora da AVCC no olho transplantado (r = 0.3186; P = 0.0755). Ocorreu uma fraca correlação entre a melhora da AVCC no olho transplantado e a melhora do SF-36 (r=0.28382; p=0.1154). Não houve diferença significativa do SF-36 nos indivíduos com visão baixa nos dois olhos quando comparado com os pacientes com visão boa em um dos olhos (p=0.2998 ) CONCLUSÃO: O transplante penetrante de córnea proporcionou melhora na pontuação dos métodos subjetivo e objetivos. Os indivíduos com maior satisfação foram melhor avaliados através do método subjetivo (SF-36). Não houve diferença significativa na qualidade de vida dos indivíduos com pior acuidade visual no olho contralateral quando comparado com os indivíduos com boa visão no olho contralateral, mas análise de um maior número de indivíduos é necessária
Abstract: Purpose: To assess and measures the patient satisfaction before and after penetrating keratoplasty. Methods: The study of approved by the ethics committee of FCM-UNICAMP. Data were collected from 32 patients who underwent penetrating keratoplasty (PK) between October 2005 and October 2006. Demographic, ocular history, objective treatment outcome measures such as clarity of the graft and best-corrected visual acuity (BCVA) of both eyes were collected prospectively. In addition to assessment of quality of life were obtained by an interview before the PK and after at least one year of postoperative. Results: The average age of subjects was 42 ± 22 (mean ± SD) years and 68.75 % were women. On average, men were most satisfied. BCVA was 0,98 ± 0,1 logMAR before and 0,48 ± 0.38 logMAR after PK showing a significant improvement on BCVA (P < 0.0001). SF-36 score was 49.11% ± 19.28% before and 71.98% ± 24.28% after PK showing a significant improvement after the PK (p< 0.0001). The most satisfied patients where those who had a better score on SF-36 (r = 0.60; p = 0.0002). Satisfaction seems not to be correlated to improvement in BCVA at the transplanted eye (r = 0.3186; p = 0.0755). There was weak correlation between improvement in BCVA on the transplanted eye and improvement of SF-36 score (r = 0.28382; p=0.1154). There was no significant improvement in SF-36 in patients with worse BCVA in the other eye (p = 0.2998). Conclusions: Penetrating keratoplasty has a positive effect on objective and subjective outcome measures. Patient satisfaction is better predicted by subjective outcomes. There is no significant difference in quality of life in patients with worse BCVA in the contralateral eye when compared to eye with good BCVA in the contralateral eye, but analysis of a larger number of patients is necessary to confirm that
Mestrado
Oftalmologia
Mestre em Ciências Médicas

Les agressions graves de la surface oculaire peuvent se compliquer d'ulcérations cornéennes épithéliales, de néovascularisation, d'opacification et d'inflammation chroniques, échappant à tout traitement médical. Les progrès dans la compréhension de la physiologie du renouvèlement de l'épithélium et du stroma cornéen ont permis d'introduire une approche thérapeutique, la greffe de cellules souches. L'objectif de notre étude est d'apporter une source de cellules souches cornéennes par thérapie cellulaire afin de restituer une fonction épithéliale et stromale permettant l'obtention d'une cornée claire chez l'animal. Les cellules souches épithéliales limbiques (LSC) et les cellules stromales limbiques (SSC) ont été isolées dans un milieu exempt de produits animaux tout en gardant leur caractère indifférencié. L'effet régénérateur des SSC a été étudié in vivo dans un modèle de fibrose cornéenne chez la souris. Les SSC ont été injectées dans le stroma cornéen et ont permis la restitution d'un stroma organisé et d'une cornée transparente. Le projet a aussi consisté à élaborer et mettre en forme une matrice à base de collagène I afin de favoriser la culture concomitante de tous les types cellulaires présents dans la cornée tout en préservant organisation, transparence et propriétés mécaniques. La connaissance des conditions physico-chimique du collagène a permis de produire des matrices transparentes qui ont pu être colonisées par les SSC et les LSC. Notre étude ouvre de nouvelles perspectives dans le traitement des pathologies provocant des opacités cornéennes afin de faire face au manque de dons de cornée
Cornea, the outer part of the eye, features high transparency crucial for good vision. The maintenance of a healthy cornea is linked to the presence of corneal stem cells in the limbus (peripheral region of the cornea). Several diseases can lead to its opacification requiring transplantation of donor tissue to restore vision. Although corneal transplantation has achieved clinical success, there is a shortage of donor corneas worldwide. To solve this problem, cell therapy and tissue-engineered artificial cornea are promising approaches that could eventually outperform current treatment. Corneal epithelial stem cells (LSC) and stromal stem cells (SSC) were isolated in feeder free and xeno free medium. The therapeutic effect of SSC was investigated in vivo by their injection in mouse cornea treated with liquid nitrogen. SSC have the ability to restore the extracellular matrix organization and corneal transparency. Transparent collagen I fibrillated matrices have been synthesized and were evaluated for fibril organization, transparency, mechanical properties and their ability to be repopulated by corneal stromal stem cells and to support limbal epithelial stem cell growth. We show that the matrices were organized and transparent. SSC and LSC were able to repopulate and to grow into the collagen matrix. These cells present a potential for stem cell-based treatment of corneal blindness

La queratoplàstia endotelial de la membrana de Descemet (DMEK) és la tècnica d'elecció actual per al tractament de l’edema corneal irreversible. Després que Melles la desenvolupés el 2006, Muraine va proposar el 2013 una tècnica alternativa per a la dissecció i implantació de l’empelt. Aquesta tècnica aportava dues novetats: la hidrodissecció amb trepanació parcial i curvatura corneal invertida del teixit donant i el plegament de l’empelt amb l'endoteli a la seva cara interna, que afavoria la protecció de l’endoteli i la tendència natural de l’empelt a desplegar-se a la cambra anterior del receptor. L’objectiu d’aquesta tesi doctoral és comparar la tècnica de Muraine amb la tècnica estàndard analitzant-ne la densitat de cèl·lules endotelials (DCE) i agudesa visual (AV) postoperatòries, els temps quirúrgics i les complicacions intraoperatòries i postoperatòries. Es va dur a terme un estudi de cohorts prospectiu observacional multicèntric en la pràctica clínica habitual a l’Hospital Universitari MútuaTerrassa i l’Institut de Microcirurgia Ocular. El seguiment postoperatori va ser de sis mesos, amb controls com a mínim l’endemà, al cap d’una setmana i al cap d’un, tres i sis mesos de la cirurgia. Es van incloure 27 ulls de 20 pacients al grup de la tècnica Estàndard i 42 ulls de 40 pacients al grup intervingut amb la tècnica Muraine. La DCE als sis mesos va ser de 1488 (1337-1679) cèl·lules/mm2 al grup Estàndard i de 1170 (734-1614) cèl·lules/mm2 al grup Muraine (P=0.10). L’AV mitja als sis mesos va ser de 0.89 al grup Estàndard i 0.79 al grup Muraine, en l’escala decimal (P=0.19). Al voltant del 80% van assolir una AV de 0.5 o més i el 50-70%, 0.8 o més. Es va observar que la DCE i el percentatge de pèrdua de DCE al mes de la cirurgia eren equivalents als de la tècnica estàndard. El percentatge de pèrdua de DCE als sis mesos va ser superior amb la tècnica de Muraine, si bé la DCE va ser clínicament comparable. L’AV assolida als sis mesos va ser equivalent. La tècnica de Muraine va ser tan segura com la tècnica estàndard per a l’obtenció de l’empelt. La incidència de complicacions intraoperatòries amb l’empelt preparat amb la tècnica de Muraine en ulls amb facoemulsificació no complicada no va ser estadísticament superior. La dissecció de l’empelt amb la tècnica de Muraine va ser més lenta. El desplegament, per contra, va ser lleugerament més ràpid. Totes dues tècniques van tenir una taxa elevada de supervivència de l’empelt. La complicació postoperatòria més freqüent en ambdós grups va ser l’edema macular quístic. Els empelts dissecats amb la tècnica de Muraine van tenir una major incidència de necessitat de reinsuflació.
Descemet’s membrane endothelial keratoplasty (DMEK) is the current gold standard treatment for irreversible corneal oedema. After Melles developed this technique in 2006, Muraine proposed in 2013 an alternative technique for the dissection and implantation of the graft. Its main contributions were: hidrodissecting the graft from a partially trephined, inverted donor tissue, and folding the graft over the endothelial side, which favoured the protection of endothelial cells and the graft’s natural tendency to unfold in the receptor’s anterior chamber. The purpose of this doctoral thesis is to compare Muraine’s technique to the Standard through analysis of the postoperative endothelial cell density (ECD) and visual acuity (VA), surgical time, and intraoperative and postoperative complications. An observational, multicentric, prospective, cohorts trial was carried out in Hospital Universitari MútuaTerrassa and Institut de Microcirurgia Ocular in a daily praxis basis. There were follow-up controls over the six months following the surgery, at least at day one, first week and first, third and sixth months. Twenty-seven eyes from 20 patients were included in the Standard technique group. Forty-two eyes from 40 patients were included in the Muraine’s technique group. The ECD at six months was 1488 (1337-1679) cells/mm2 for the Standard group and 1170 (734-1614) cells/mm2 for Muraine’s group. The mean VA at six months was 0.89 for the Standard group and 0.79 for Muraine’s group, in the decimal scale (P=0.19). Around 80% of the eyes reached a VA of 0.5 or higher and 50-70%, 0.8 or higher. The ECD and the percentage of ECD loss with Muraine’s technique at the first month after surgery were equivalent to the Standard technique’s. The percentage of ECD loss at six months was higher with Muraine’s technique, although the ECD was clinically comparable. The VA achieved at six months was equivalent. Muraine’s technique was as safe as the Standard technique for the graft dissection. The incidence of intraoperative complications among the eyes with uncomplicated phacoemulsification was not statistically higher with Muraine’s technique. The graft dissection with Muraine’s technique was slower. Conversely, the unfolding was slightly faster. Both techniques had a high graft survival rate. The most frequent postoperative complication in both groups was cystoid macular oedema. The grafts dissected with Muraine’s technique had a higher incidence of need for rebubbling.

Corneal transplants are a safe and effective treatment for corneal disease, but are hampered by the limited supply and quality of donor tissue. The goal of this project was therefore to evaluate the use of membranes prepared from silk protein as a scaffold on which to grow corneal tissue substitutes in the laboratory from corneal endothelial cells.

Transplantation of ex-vivo cultured limbal epithelial cells (LEC) is an established treatment for total limbal stem cell deficiency. However, this therapy has preceded the scientific understanding of limbal epithelial stem cells (LESC), the LESC niche and the biological mechanism that underpins it. This body of work tested the hypothesis that the LESC niche has a specialised structure and that an understanding of this may lead to improvements in outcomes and understanding of this therapy. The corneal limbus was investigated using 3D confocal microscopy, scanning electron microscopy, wholemount immunofluorescence and in-vivo confocal microscopy. This produced the most comprehensive study of limbal architecture performed to date. The structure of the LESC niche was identified and the effect of age and disease examined. Next, a clinical study of ex-vivo expansion and transplantation of LEC was performed in patients with LESC deficiency. A defined set of objective outcome measures enabled a baseline standard for outcomes of this therapy to be described. Future improvements to this therapy can be assessed using these techniques. Human amniotic membrane (HAM) is the leading candidate for a surrogate LESC niche. The method of HAM processing was investigated and found to be critical to achieve this. A method of significantly improving the yield of LESC in culture on HAM was identified. It remains to be seen whether this will translate into improved clinical outcomes. Finally, a disconnection exists between published data indicating a good clinical outcome and data demonstrating the survival of transplanted cells. This work assessed and demonstrated the feasibility of using quantum dot nanocrystal labelling of transplanted LESC to track cells post transplantation. It is concluded that translation of these findings to modify and improve current treatment protocols may result in improvements in outcomes for patients with LESC deficiency undergoing this therapy.

Objetivo: Avaliar a função visual de pacientes submetidos a transplante lamelar anterior profundo (DALK) utilizando a dissecção com tunelizador manual e fio. Métodos: Foram incluídos 33 pacientes com ceratocone que apresentavam BCVA <= 0,60 LogMar, miopia e astigmatismo entre 8,00 e 10,00D, K central médio > 53,00D, ausência de cicatrizes, espessura corneana mínima entre 300 e 400 um. Foi feita avaliação oftalmológica completa no pré e no pós-operatório de 6 a 8 meses. Estas avaliações foram complementadas com exame topográfico da córnea, microscopia especular para avaliação da densidade das células endoteliais corneanas, aberrometria corneana e exame de tomografia de coerência óptica do segmento anterior. As variáveis BCVA, UCBA e os valores totais das aberrações corneanas de alta ordem foram correlacionadas com a espessura do leito estromal residual. Resultados: Os pacientes submetidos à DALK apresentaram BCVA de 0,68 ± 0,27 LogMar o que representa BCVA superior a 20/40 em 60% da amostra analisada. Não foram observadas micro ou macroperfurações. Houve diminuição na contagem endotelial de 2702,87 ± 548,87 células por mm2 para 2282,10 ± 525,66 células por mm2 . A dissecção do estroma profundo com o fio facilitou a remoção de tecido estromal posterior, fato corroborado com o achado de que o leito residual estromal aferido foi de 49,18 ± 18,36 ?m na região central e foi inferior a 80 ?m em grande parte dos pacientes estudados. No que se refere à regularidade da dissecção, observou-se tendência a valores mais elevados de espessura residual na periferia (60,09 ± 17,70 ?m). Não houve correlação da BCVA, UCVA e do total de aberrações de alta ordem da córnea com a espessura do leito estromal residual. Conclusão: A apreciação dos resultados desse estudo mostrou que com a técnica utilizada para realização de DALK em portadores de ceratocone obteve-se resultados topográficos e funcionais semelhantes a outras técnicas consagradas pela literatura. A facilidade na dissecção do estroma profundo, a regularidade da dissecção e a presença de baixíssimo índice de conversão para transplantes penetrantes são encorajadores
Objective: Evaluate the visual function of patients undergoing deep anterior lamellar keratoplasty (DALK) using a manual spatula and a wire dissection. Methods: Thirty three keratoconus patients were included, meeting the following inclusion criteria: BCVA logMAR <=0,60, myopia and astigmatism between 8.00 and 10,00D, K central average > 53.00D, no corneal scars and minimal corneal thickness between 300 and 400 um. Complete ocular evaluation was performed preoperatively and postoperatively in 6-8 months. These assessments were supplemented by topographical survey of the cornea, specular microscopy to evaluate the density of corneal endothelial cells, corneal wavefront analysis and examination of optical coherence tomography of the anterior segment (Visante). The BCVA variables, UCVA and the total amounts of corneal higher-order aberrations were correlated with the the residual stromal bed thickness. Results: Patients that undergone to DALK with the described technique presented a BCVA of 0.68 ± 0.27 logMAR which represents a BCVA of more than 20/40 at 60% of the analyzed sample. There were no micro or macroperforations. We observed a small decrease in the endothelial cell count from 2702.87 ± 548.87 cells per mm2 to 2282.10 ± 525.66 cells per mm2. The dissection of the deep stroma with a wire facilitated the posterior stromal tissue removal, thus the measured stromal bed thickness was 49.18 ± 18.36 ?m in the central region and less than 80 ?m in the majority of the studied patients. As regards the dissection regularity, we showed a tendency to higher values of residual thickness at the periphery (60.09 ± 17.70 ?m). There was no correlation of the BCVA, UCVA and total corneal higher-order aberrations with the residual stromal bed thickness. Conclusion: The assessment of the study data showed that the described technique achieved a topographical and functional result similar to other techniques consecrated by literature. The shallow learning curve, the ease to perform the dissection of the deep stroma, the postoperative stromal regularity and the presence of very low conversion rate for penetrating keratoplasty are encouraging

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-20T17:53:34Z No. of bitstreams: 1 flaviabatistabarbosadesa.pdf: 1013858 bytes, checksum: 1e5c85e290deeb77a83c4367e5c61db1 (MD5)
Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T11:27:01Z (GMT) No. of bitstreams: 1 flaviabatistabarbosadesa.pdf: 1013858 bytes, checksum: 1e5c85e290deeb77a83c4367e5c61db1 (MD5)
Made available in DSpace on 2016-07-02T11:27:01Z (GMT). No. of bitstreams: 1 flaviabatistabarbosadesa.pdf: 1013858 bytes, checksum: 1e5c85e290deeb77a83c4367e5c61db1 (MD5) Previous issue date: 2012-03-26
Objetivo: Analisar os fatores que influenciam o processo de doação de córneas entre os potenciais doadores de um Hospital Público e de um Hospital Privado de Juiz de Fora. Métodos: estudo seccional do tipo descritivo. Foram rastreados os óbitos ocorridos em um Hospital Público e um Hospital Privado de Juiz de Fora no período de 01 janeiro de 2010 a 31 de dezembro de 2010, identificados os potenciais doadores de córneas, verificado se os óbitos foram notificados à Central de Notificação, Captação e Distribuição de Órgãos (CNCDO) da Zona da Mata de Juiz de Fora e se esta notificação resultou ou não em doação. Caso não tenha ocorrido doação, foi justificada a causa da não efetivação da mesma, e caso tenha ocorrido, foi analisado o destino da córnea captada (transplante ou perda). Os dados foram processados e discutidos utilizando-se a análise estatística feita através do Programa SPSS (Statistical Package for the Social Sciences), versão 13.0. Resultados: A população em estudo se caracteriza por serem em sua maioria pacientes do sexo masculino (53,1%), com idade entre 2 a 80 anos (69,1%), com causas mortis que não contraindicaram o transplante de córneas (59,1%). Dos 863 óbitos, 138 (16%) não foram notificados à CNCDO-Zona da Mata e destes, 54 eram potenciais doadores. Do total de óbitos, 210 (24,3%) foram classificados como potenciais doadores. O percentual de captação efetiva dos casos entrevistados foi de 34,9%. Foram realizadas 30 doações de córneas (19,2%). Observou-se que das 60 córneas captadas (30 doadores), 75,0% (45) foram descartadas. A não efetivação da doação foi justificada pela recusa familiar em 58 casos (7,0%), pelos problemas logísticos ou estruturais em 125 casos (15,0%) e pelas contraindicações médicas registradas na CNCDO-Zona da Mata em 650 casos (78,0%). Conclusão: Da população estudada, 24,3% eram potenciais doadores de córneas. A contraindicação médica foi a maior justificativa para a não efetivação da doação de córneas, correspondendo a 78,0% das causas. Considerando que apenas 25% das córneas captadas foram transplantadas, conclui-se que há uma grande desigualdade entre o número de doadores potenciais e o número de doadores reais para o transplante de córneas, pois muito se perde durante todo o processo de doação.
Objective: To analyze factors that influence the process of cornea donation among potential donors for a Public Hospital and a Private Hospital in Juiz de Fora. Methods: A descriptive cross sectional study. We traced the deaths occurred in a Public Hospital and a Private Hospital in Juiz de Fora in the period from January 1, 2010 to December 31, 2010, identified the potential donors of corneas, checked whether the deaths were reported to the Central Notification, Procurement and Distribution organs (CNCDO) from Zona of Mata in Juiz de Fora and whether or not this notification resulted in donation. If there were no donation, it was justified the cause of not being done, and if it has occurred, we analyzed the fate of the captured cornea (transplantation or loss). The data were analyzed and discussed using the statistical analysis performed using the SPSS (Statistical Package for Social Sciences) version 13.0. Results: The mortality profile of the institutions under study is characterized by being mostly male (53.1%), aged from 2 to 80 years (69.1%), with cause of death that did not prevent corneal transplantation to be done (59.1%). Of the 863 deaths, 138 (16%) were not notified to CNCDO-Zona of Mata and of these, 54 were potential donors. Of the total deaths, 210 (24.3%) were classified as potential donors. The percentage of effective capture of the cases surveyed was 34.9%. 30 donations of corneas were performed (19.2%). It was observed that from 60 corneal grafts (30 donors), 75.0% (45) were discarded. The effectiveness of the donation was not justified by family refusal in 58 cases (7.0%), by logistical or structural problems in 125 cases (15.0%) and recorded in the medical contraindications CNCDO-Zona da Mata in 650 cases (78.0 %). Conclusion: The study population, 24.3% were potential donors of corneas. The medical contraindication was the biggest reason for not effecting the donation of corneas, corresponding to 78.0% of the causes. Whereas only 25% of corneal grafts were transplanted, it is concluded that a considerable difference between the number of potential donors and the number of actual donors for transplantation of corneas, since much is lost during the donation process.

Objetivo: Analisar a influência da espessura corneana central na acuidade visual (AV) corrigida após transplante de córnea endotelial lamelar profundo (TCELP). Métodos: Foram estudados de forma prospectiva 155 olhos de 127 pacientes portadores de ceratopatia bolhosa ou distrofia endotelial de Fuchs no sexto mês de pós-operatório do TCELP, entre março de 2000 e março de 2005. Foram excluídos pacientes com outras alterações oculares que justificassem baixa AV. Todos os pacientes foram submetidos à avaliação oftálmica, quando foram determinadas AV corrigida, por meio de exame refratométrico, e espessura corneana central, através da paquimetria ultra-sônica. As técnicas usada foram previamente descritas. Os olhos foram agrupados de acordo com as medidas de AV: grupo I (20/20 - 20/30), grupo II (20/40 - 20/50), grupo III (20/60 - 20/80), grupo IV (20/100 - 20/400). Para correlação com paquimetria e análise estatística, as medidas de AV foram convertidas da tabela de Snellen para a tabela logarítmica (logMAR). Foram criadas variáveis categóricas para expressar status de faixa de normalidade de espessura corneana (entre 495 e 651 ?m), usando como pontos de corte valores encontrados na literatura. Resultados: A média, o desvio padrão e a variação da paquimetria foi: grupo I (n=38) 571 ±80 um, 408 a 784 um; grupo II (n=79) 598 ±80 um, 437 a 816 um; grupo III (n=30) 605 ±99 um, 454 a 945 um e grupo IV (n=8) 607 ±120 ?m, 410 a 781 ?m. Analisando o resultado da AV e a porcentagem de casos com espessura corneana acima de 651 um, foi observada associação linear significativa (P=0,037; ?2 de tendência linear) entre o aumento da paquimetria e a piora da AV. Analisando a associação entre os grupos de AV e a porcentagem de casos com espessura corneana abaixo da faixa de normalidade (<495 um), não foi encontrada significância estatística (P=0,92; x2 de Pearson). Quando analisado o resultado visual do grupo I em relação ao resultado dos grupos II+III+IV em conjunto, observou-se que somente 13% dos casos do grupo I e 30% dos casos dos demais grupos apresentaram espessura corneana maior do que 651 ?m. Essa correlação demonstrou significância estatística limítrofe (P=0,066; x2 de Pearson com correção de Yates). Conclusão: Observou-se associação linear significativa entre piora da AV corrigida e aumento da espessura corneana central. Quando analisados somente casos com paquimetria abaixo da faixa de normalidade, não foi observada associação significativa entre piora da AV corrigida e espessura corneana central
Purpose: To analyze the influence of central corneal thickness in the corrected visual acuity (VA) after deep lamellar endothelial corneal keratoplasty (DLEK). Methods: Retrospective study of 155 eyes of 127 patients 6 months post-op DLEK between March 2000 and March 2005. These patients had been previously diagnosed with either bullous keratopathy or Fuch\'s endothelial dystrophy. Patients with other ophthalmic conditions that could cause loss of vision were excluded. All patients underwent ophthalmic evaluation to determine corrected VA by means of refraction and central corneal thickness by means of ultrasonic pachymetry. Eyes were grouped according to visual acuity into 4 groups: I (20/20 - 20/30), II (20/40 - 20/50), III (20/60 - 20/80), IV (20/100 - 20/400). For statistical analysis and corelation with pachymetry, VA measurements were converted to logMAR. Categorical variables were created to express normal range corneal thickness status (from 495 to 651 um) using values published on the literature. Results: Mean and standart deviation pachymetry values were: group I (n=38) 571 ±80 ?m, ranging from 408 to 784 um; group II (n=79) 598 ±80 um, ranging from 437 to 816 ?m; group III (n=30) 605 ±99 um, ranging from 454 to 945 um and group IV (n=8) 607 ±120 um, ranging from 410 to 781 ?m. Analyzing the VA results and the percentage of cases with corneal thickness above 651 um, a significant linear correlation between higher pachymetry and worse VA was observed (P=0.037; linear trend). Analyzing the association between the different groups and the percentage of cases with corneal thickness bellow 495 um, there was no statistical significance (P=0.92; Pearson\'s x2). When analyzing the visual results of group I compared to groups II+III+IV together, it was observed that only 13% of group I cases and 30% of cases from the other groups presented corneal thickness greater then 651 um. This correlation showed borderline statistical significance (P=0.066; Pearson\'s x2 with Yates\' correction). Conclusions: A significant linear correlation between increased corneal thickness and worse VA was observed. When analyzing only cases bellow normal pachymetry, there was no correlation between corneal thickness and worse VA

Introdução: As doenças da córnea são responsáveis por cerca de 5% da cegueira reversível no mundo e o transplante de córnea (TC) é importante para o tratamento dessas enfermidades. A partir de fontes de dados oficiais e públicas, foram analisados o progresso e as dificuldades relacionados ao TC no Brasil nos últimos 16 anos, bem como desigualdades regionais, gastos do Sistema Único de Saúde (SUS) e indicadores de qualidade dos bancos de tecido ocular (BTOs). Métodos: Estudo retrospectivo e analítico com dados sobre TCs e BTOs no Brasil, no período de janeiro de 2001 a dezembro de 2016, divulgados pelo Sistema Nacional de Transplantes (SNT), pela Associação Brasileira de Transplante de Órgãos (ABTO) e pela Agência Nacional de Vigilância Sanitária (ANVISA). Para verificação de existência de tendência, comparação de médias entre as regiões e verificação da diferença de médias, foram utilizados o teste de Cochran-Armitage, a Análise de Variância e as comparações múltiplas de Duncan, respectivamente. Em todos os testes foi utilizado um nível de significância de 5%. Resultados: No Brasil, houve aumento: de 2,4 vezes no número de TCs (de 6.193 - 35,2 pmp para 14.641 - 71,0 pmp - p < 0,001); de 50,7% na eficácia do atendimento à demanda populacional por TCs (de 35,3% para 53,2% - p < 0,001); de 27,8% no número de globos e córneas in situ doados (de 24.608 - 127,1 pmp para 31.450 - 152,6 pmp - p < 0,001); de 31,7% nas córneas preservadas (de 21.012 para 27.674); de 2,4 vezes no gasto financeiro total com TCs (de R$ 9.179.688 para R$ 22.060.973); e de 2,2 vezes no gasto unitário com TC (de R$ 716 para R$ 1.603). A fila de espera para TC reduziu em 45,4% (de 23.549 - 123,0 pmp para 12.865 - 62,4 pmp - p < 0,001). As duas principais causas para a não doação foram as contraindicações médicas (média de 42,5%) e a recusa familiar (média de 36,6%). As principais causas de descarte de córneas foram a sorologia positiva para hepatite B (média de 33%), validade tecidual (média de 30,9%) e qualidade imprópria do tecido (16,8%). A Eficácia na Preservação de Córnea (EPC), o Coeficiente de Descarte de Córnea (CDC) e a Eficácia no Fornecimento de Córnea para Transplante (ECT) foram em média 88%, 37% e 63% ao longo dos anos, respectivamente. Os melhores índices foram apresentados nas regiões Sul, Centro-Oeste e Sudeste e os piores no Norte e Nordeste. Conclusão: No Brasil, o pequeno número de doações e a grande taxa de descarte de córneas são as principais dificuldades ao adequado atendimento à demanda populacional por TCs. Contudo, o país aumentou a capacidade de transplantar córneas e reduziu as filas de espera em 16 anos
Introduction: Corneal diseases account for about 5% of reversible blindness in the world and Corneal Transplantation (CT) is important for the treatment of these diseases. From official and public data sources, the progress and difficulties related to CT in Brazil in the last 16 years were analyzed, as well as regional inequalities, expenses for the Unified Health System (SUS) and quality indicators of ocular tissue banks (OTBs). Methods: A retrospective and analytical study with data on CT and OTB in Brazil, from January 2001 to December 2016, published by the National Transplantation System (SNT), the Brazilian Organ Transplantation Association (ABTO) and the National Surveillance Agency Sanitary (ANVISA). The Cochran-Armitage test, the Analysis of Variance and the Duncan\'s multiple comparisons were used to verify the existence of trend, comparison of means between regions and verification of the mean difference, respectively. A significance level of 5% was used in all tests. Results: In Brazil, there was an increase: of 2.4 times in the number of CTs (from 6,193 - 35.2 pmp to 14,641 - 71.0 pmp - p < 0.001); of 50.7% in the efficacy of meeting the population demand for CTs (from 35.3% to 53.2% - p < 0.001); of 27.8% in the number of donated globes and corneas in situ (from 24,608 - 127.1 pmp to 31,450 - 152.6 pmp - p < 0.001); of 31.7% in preserved corneas (from 21,012 to 27,674); of 2.4 times in the total finance expense with CTs (from R$ 9,179,688 to R$ 22,060,973); and 2.2 times the unit expense with CT (from R$ 716 to R$ 1,603). The waiting list for CT decreased by 45.4% (from 23,549 - 123.0 pmp to 12,865 - 62.4 pmp - p < 0.001). The two main causes for non-donation were medical contraindications (mean of 42.5%) and family refusal (mean of 36.6%). The main causes of corneal discard were positive serology for hepatitis B (mean of 33%), tissue validity (mean of 30.9%) and inadequate tissue quality (16.8%). Efficacy in Corneal Preservation (EPC), Corneal Discarding Coefficient (CDC) and Efficacy Supply of Corneas for Transplantation (ECT) averaged 88%, 37%, and 63% over the years, respectively. The best indexes were presented in the South, Midwest and Southeast regions and the worst in the North and Northeast. Conclusion: In Brazil, the small number of donations and the high rate of discard of corneas are the main difficulties to the adequate attendance to the population demand by CTs. However, the country increased the ability to transplant corneas and reduced waiting lists in 16 years

Corneal dystrophies are commonly referred to as an congenital condition. Surgical complications are usually worse then the primary dysfunction and patients need to go through large surgical process. Only 25 % of the patients reach driving licence vision after a surgery. The aim of this study was to investigate if the application of human embryonic stem cells (hESc) could replace epithelial cells of the human cornea. Corneal markers such as cytokeratins CK3, CK15, CK19 and Pax - 6 were analysed by immunohistochemistry. HES - Cellect was used as indication of  stem cell potential of the transplanted cells. hESc transplanted onto the cornea could be seen to attach and expand dominantly  towards Bowman’s membrane. Human embryonic stem cells in culture were relatively positive for markers, contradictionally stem cells  in the epithelial trails lost their stem differentiation  potential  and appeared to be negative for all markers used in these trails. Optimization of stem cells differentiation into epithelial which may in the future may gives us the ability to perform clinical applications with successful outcome.

Made available in DSpace on 2015-10-06T13:03:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-23. Added 1 bitstream(s) on 2015-10-06T13:18:55Z : No. of bitstreams: 1 000847355.pdf: 1190450 bytes, checksum: 198ab4a47eec622beaa61096061775a2 (MD5)
Introdução: A córnea é uma túnica transparente responsável por 60% do poder refrativo do olho. Segundo a Organização Mundial da Saúde (OMS), doenças que afetam a transparência da córnea são responsáveis por 5,1% do total de cerca de 45 milhões de cegos. O Brasil é o país que mais realiza transplantes de córnea na América Latina. A taxa de perda endotelial gira em torno de 0,6/ano em pessoas normais. Essa taxa é acelerada após cirurgias oculares ou glaucoma. Essa perda, após o transplante penetrante devido trauma inicial, reação imune, glaucoma secundário, interação celular entre o doador e receptor, seria de 7,8%/ano nos primeiros cinco anos e 4,2%/ano após 10 anos. Alguns estudos sugerem perda endotelial maior no Descemet's stripping automated endothelial keratoplasty/Ceratoplastia endothelial automatizada stripping de Descemet (DSAEK) (34% após seis meses) do que no transplante penetrante (11%) enquanto outros mostram que a perda endotelial após um ano é maior no transplante penetrante. A sobrevida do enxerto em ambas as técnicas foram semelhantes e o resultado óptico superior na técnica DSAEK. Há quase um século o transplante penetrante tem sido a técnica cirúrgica de escolha no manejo de alterações corneais. Apesar das vantagens de procedimentos lamelares, como menor risco de complicações intra-oculares e rejeição do enxerto, o transplante penetrante ainda mostra ser a técnica de preferência da maioria dos transplantadores mundiais. Entretanto, nos últimos anos, várias técnicas de ceratoplastia lamelar têm sido desenvolvidas, modificadas ou melhoradas, principalmente técnicas para substituição da porção posterior para correção da ceratopatia bolhosa. A escolha de ceratopatia bolhosa pós facectomia foi por esta ser a principal causa de transplantes relacionados a disfunções endoteliais. Objetivo: Avaliar a eficá cia e a seguranç a d a ceratoplastia endotelial quando...
Introduction: The cornea is a transparent organ responsible for 60% of the refractive power of the eye. According to the World Health Organization (WHO), diseases affecting the transparency of the cornea are responsible for 5.1% of total of about 45 million blind. Brazil is the country that performs corneal transplants in Latin America. Endothelial loss rate is around 0.6 / year in normal people. This rate is accelerated after eye surgery or glaucoma. This loss after penetrating keratoplasty because initial trauma, immune reaction, secondary glaucoma, cell interaction between donor and recipient, it would be 7.8% / year in the first five years and 4.2% / year after 10 years. Some studies suggest a greater endothelial cell loss in DSAEK (34% after six months) than in penetrating keratoplasty (11%) while others show that endothelial cell loss after one year is higher in penetrating keratoplasty. Graft survival in both techniques were similar and higher optical result in DSAEK technique. For nearly a century the penetrating keratoplasty has been surgical technique of choice in the management of corneal changes. Despite the advantages of lamellar procedures such as intraocular lower risk of complications and graft rejection further shows penetrating keratoplasty is the preferred technique of most of the world transplant. However, in recent years, several lamellar keratoplasty techniques have been developed, modified or improved, especially techniques for replacement of the posterior portion for correction of bullous keratopathy. Objective: To evaluate the efficacy and safety in endothelial keratoplasty when compared to penetrating keratoplasty for the improvement of visual acuity in aphakic and pseudophakic bullous keratopathy. Methods: Systematic review of randomized controlled trials (RCTs) and / or quasirandomized studies that assessed endothelial keratoplasty versus penetrating keratoplasty in adults diagnosed with aphakic or ...

In dieser Arbeit wurden Methoden entwickelt und verglichen, um aus Corneoskleralringen langzeitorgankonservierter Hornhäute und intakten, kryokonservierten Amnionmembranen Limbusepithel-Amnion-Transplantate herzustellen. Als erfolgreichste Kultivierungsmethode stellte sich hierbei signifikant die Explantat-Technik mit nach unten gerichtetem Limbusepithel heraus. Hier konnte eine Auswachsrate von 42 % erzielt werden. Es wurde weiterhin gezeigt, dass das ausgewachsene, mehrschichtige Limbusepithel proliferationsfähige TACs (Transient Amplifying Cells) enthält. Weiterhin konnten mittels Regressionsanalyse signifikante Zusammenhänge zwischen Spenderalter, Post-mortem-Zeit, Organkultur-Dauer und der Auswachsrate beschrieben werden. Kurzgefasst wurde die Vermutung bestätigt, dass jede Verlängerung der unterschiedlichen Zeiten eine Verringerung der Auswachsrate zur Folge hat. Die hergestellten Limbusepithel-Amnion-Transplantate könnten für Patienten mit Limbusstammzellinsuffizienz unterschiedlicher Genese verwendet werden.

Orientador: Newton Kara-Jose
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-14T21:13:45Z (GMT). No. of bitstreams: 1 Santos_MarcioAlexandreAraujoFlorenciodos_D.pdf: 1714788 bytes, checksum: 4df269b178244131a0b3d831ea27e272 (MD5) Previous issue date: 2009
Resumo: Os objetivos deste estudo foram identificar o perfil epidemiológico dos familiares responsáveis pela autorização ou recusa à doação de córneas de seus parentes falecidos no Hospital de Clínicas da Universidade Estadual de Campinas; suas razões para autorizar ou não a doação; e razões para ser ou não doador de córneas. O estudo foi desenvolvido na Faculdade de Ciências Médicas, no Hospital de Clínicas da Universidade Estadual de Campinas - Unicamp, Campinas, São Paulo, Brasil, entre dezembro de 2005 e dezembro de 2006. O desenho adotado nesta pesquisa foi um estudo transversal do tipo analítico e descritivo, realizado através de questionário testado em estudo piloto. A amostra populacional desse estudo foi calculada baseado em teste estatístico para estudo descritivo e variável qualitativa. Foram realizadas 184 entrevistas e os resultados sugeriram que os familiares que eram solteiros, divorciados ou separados; católicos ou sem religião; que possuíam conhecimento que sua religião era a favor da doação; que possuíam atitudes altruísticas; que possuíam algum grau de conhecimento sobre doação e que obtinham informação sobre doação pela TV ou rádio, autorizaram mais a doação das córneas de seu parente falecido. As razões citadas para autorizar a doação de córneas foram: solidariedade às famílias que esperam por uma córnea; respeito ao desejo em doar do falecido; atitude altruística de ajudar ao próximo; conforto ao momento de dor que a família vivia. E as razões citadas contra a doação foram: respeito ao desejo em não doar do parente falecido; desconfiança na utilização da córnea; demora para captar a córnea e razões religiosas. O conhecimento desse perfil e dessas razões poderá levar a um melhor entendimento da recusa familiar e assim, planejar medidas mais efetivas para aumentar a doação de córneas.
Abstract: The objectives of this study was to identify the epidemiological profile of the family responsible for granting or refusing the donation of corneas of their deceased relatives in the Clinical Hospital of State University of Campinas and identify their reasons for granting or not giving, and reasons to be or not donor corneas. The study was conducted at the College of medical Sciences of Clinical Hospital of state University of Campinas, São Paulo, Brazil, between December 2005 and December 2006. The design adopted in this research study was a cross-sectional analytical and descriptive study through a questionnaire tested in a pilot study. The sample population of this study was calculated based on statistical test for a descriptive and qualitative variable. One hundred eighty four interviews were conducted and the results suggested that family members who were single, divorced or separated, Catholic or no religion, who had knowledge that their religion was in a favor donation, which had altruistic attitudes, which had some degree of knowledge about cornea donation and they obtained information about donation by TV or radio were more significant to authorize the donation of corneas of their deceased relative. Reasons cited to authorize the donation of corneas were: solidarity, the deceased wanted to donate, altruistic attitude to the next, comfort the moment of pain that the family lived. And the reasons cited against the donation were the deceased relative did not want to be a donor, do not have confidence in the use of the cornea, the delay to capture the cornea and religion reasons. Knowledge of this profile and theses reasons may lead to a better understanding of family refusal and so, plan the most effective measures to increase the donation of corneas.
Doutorado
Oftalmologia
Doutor em Ciências Médicas

Orientador: Regina Paolucci El Dib
Coorientador: Amélia Kamegasawa
Banca: Eliane Jorge
Banca: Tais W.
Resumo: Introdução: A córnea é uma túnica transparente responsável por 60% do poder refrativo do olho. Segundo a Organização Mundial da Saúde (OMS), doenças que afetam a transparência da córnea são responsáveis por 5,1% do total de cerca de 45 milhões de cegos. O Brasil é o país que mais realiza transplantes de córnea na América Latina. A taxa de perda endotelial gira em torno de 0,6/ano em pessoas normais. Essa taxa é acelerada após cirurgias oculares ou glaucoma. Essa perda, após o transplante penetrante devido trauma inicial, reação imune, glaucoma secundário, interação celular entre o doador e receptor, seria de 7,8%/ano nos primeiros cinco anos e 4,2%/ano após 10 anos. Alguns estudos sugerem perda endotelial maior no Descemet's stripping automated endothelial keratoplasty/Ceratoplastia endothelial automatizada "stripping" de Descemet (DSAEK) (34% após seis meses) do que no transplante penetrante (11%) enquanto outros mostram que a perda endotelial após um ano é maior no transplante penetrante. A sobrevida do enxerto em ambas as técnicas foram semelhantes e o resultado óptico superior na técnica DSAEK. Há quase um século o transplante penetrante tem sido a técnica cirúrgica de escolha no manejo de alterações corneais. Apesar das vantagens de procedimentos lamelares, como menor risco de complicações intra-oculares e rejeição do enxerto, o transplante penetrante ainda mostra ser a técnica de preferência da maioria dos transplantadores mundiais. Entretanto, nos últimos anos, várias técnicas de ceratoplastia lamelar têm sido desenvolvidas, modificadas ou melhoradas, principalmente técnicas para substituição da porção posterior para correção da ceratopatia bolhosa. A escolha de ceratopatia bolhosa pós facectomia foi por esta ser a principal causa de transplantes relacionados a disfunções endoteliais. Objetivo: Avaliar a eficá cia e a seguranç a d a ceratoplastia endotelial quando...
Abstract: Introduction: The cornea is a transparent organ responsible for 60% of the refractive power of the eye. According to the World Health Organization (WHO), diseases affecting the transparency of the cornea are responsible for 5.1% of total of about 45 million blind. Brazil is the country that performs corneal transplants in Latin America. Endothelial loss rate is around 0.6 / year in normal people. This rate is accelerated after eye surgery or glaucoma. This loss after penetrating keratoplasty because initial trauma, immune reaction, secondary glaucoma, cell interaction between donor and recipient, it would be 7.8% / year in the first five years and 4.2% / year after 10 years. Some studies suggest a greater endothelial cell loss in DSAEK (34% after six months) than in penetrating keratoplasty (11%) while others show that endothelial cell loss after one year is higher in penetrating keratoplasty. Graft survival in both techniques were similar and higher optical result in DSAEK technique. For nearly a century the penetrating keratoplasty has been surgical technique of choice in the management of corneal changes. Despite the advantages of lamellar procedures such as intraocular lower risk of complications and graft rejection further shows penetrating keratoplasty is the preferred technique of most of the world transplant. However, in recent years, several lamellar keratoplasty techniques have been developed, modified or improved, especially techniques for replacement of the posterior portion for correction of bullous keratopathy. Objective: To evaluate the efficacy and safety in endothelial keratoplasty when compared to penetrating keratoplasty for the improvement of visual acuity in aphakic and pseudophakic bullous keratopathy. Methods: Systematic review of randomized controlled trials (RCTs) and / or quasirandomized studies that assessed endothelial keratoplasty versus penetrating keratoplasty in adults diagnosed with aphakic or ...
Mestre

Orientador: Newton Kara-Jose
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-13T21:12:46Z (GMT). No. of bitstreams: 1 Costa_DacioCarvalho_D.pdf: 11382661 bytes, checksum: a5c3e2c591c002085753bfe5397e34f3 (MD5) Previous issue date: 2009
Resumo: Objetivo: Comparar a eficácia da injeção subconjuntival de 20 mg de triancinolona associada a prednisolona 1% tópica com a injeção intravenosa de 500 mg de metilprednisolona associada a prednisolona 1% tópica no tratamento da rejeição endotelial de transplante de córnea. Métodos: Estudo caso-controle realizado no Hospital das Clínicas da UNICAMP. Os pacientes submetidos a transplante penetrante de córnea que apresentaram primeiro episódio de rejeição endotelial com até 15 dias do início dos sintomas durante o período de novembro de 2005 a outubro de 2006 foram tratados com injeção subconjuntival de 20 mg de acetonido de triancinolona associado a acetato de prednisolona 1% tópico. Estes pacientes foram pareados por idade e diagnóstico com pacientes submetidos a tratamento com injeção intravenosa de 500 mg de succinato sódico de metilprednisolona associado a acetato de prednisolona 1% tópico e analisados quanto à capacidade de reversão do episódio de rejeição, pressão intraocular aos 30 dias e acuidade visual ao final de 1 ano. Resultados: 16 pacientes foram tratados com 20 mg de triancinolona subconjuntival e prednisolona 1% tópica durante o período de recrutamento e foram pareados com 16 pacientes tratados com 500 mg de metilprednisolona intravenosa e prednisolona 1% tópica. Ao final de 1 ano, o grupo tratado com triancinolona obteve melhores resultados do que o grupo tratado com metilprednisolona (p=0,025), obtendo 15 pacientes com córnea transparente enquanto o grupo tratado com metilprednisolona obteve 10 pacientes. 3 pacientes do grupo tratado com triancinolona apresentaram segundo episódio de rejeição durante o seguimento e foram retratados com sucesso enquanto no grupo da metilprednisolona, 4 pacientes apresentaram segunda rejeição, com 2 pacientes apresentando falência com o retratamento e 2 obtendo sucesso. A pressão intraocular subiu nos dois grupos (p=0,002) após 30 dias, porém não houve diferença entre os grupos (p=0,433). A acuidade visual melhorou após 1 ano em ambos os grupos (p=0,049) e o grupo tratado com triancinolona obteve melhor acuidade visual (p=0,002). Conclusão: A injeção subconjuntival de 20 mg de triancinolona combinada com prednisolona 1% tópica mostrou-se mais eficaz em reverter episódios de rejeição de transplante de córnea neste estudo caso-controle do que a aplicação intravenosa de 500 mg de metilprednisolona. Estudos adicionais necessitam ser realizados para verificar a segurança e eficácia deste tratamento em grandes populações
Abstract: Purpose: To compare the efficacy of 20 mg subconjunctival triamcinolone in association with topical prednisolone 1% to 500 mg intravenous methylprednisolone in association with topical prednisolone 1% in the treatment of cornea endothelial graft rejection. Methods: Case-control study carried out at State University of Campinas Hospital. Patients submitted to penetrating keratoplasty that presented first episode of corneal endothelial rejection within 15 days of symptoms onset between November 2005 and October 2006 were treated with 20 mg subconjunctival injection of triamcinolone acetate in association with topical prednisolone acetate 1%. These patients were matched for age and diagnosis to patients that were submitted to a single 500 mg intravenous injection of methylprednisolone sodium succinate in association with topical prednisolone acetate 1% and analyzed regarding the reversion of the rejection episode, intraocular pressure at day 30 and visual acuity at the end of 1 year. Results: 16 patients were treated with 20 mg subconjunctival triamcinolone and topical prednisolone 1% during the period of recruitment and were matched to 16 patients treated with 500 mg intravenous methylprednisolone and topical prednisolone 1%. At the end of 1 year, the group treated with triamcinolone had a better outcome than the group treated with methylprednisolone (p=0.025), having 15 patients with clear grafts as the group treated with methylprednisolone had 10 patients. 3 patients from the group treated with triamcinolone had new rejection episodes during follow-up and were retreated successfully as in the group treated with methylprednisolone 4 patients had a new rejection episode, with 2 progressing to failure and 2 to success with retreatment. Intraocular pressure rose in both groups (p=0.002) at day 30 but there were no statistically significant differences between the groups (p=0.433). Visual acuity improved after 1 year in both groups (p=0.049) and the group treated with triamcinolone had better visual acuities (p=0.002). Conclusions: 20 mg subconjunctival injection of triamcinolone acetonide associated with topical prednisolone acetate 1% showed to be more effective than 500 mg intravenous methylprednisolone associated with prednisolone acetate 1% in this case-control study. Further studies need to be accomplished to verify its safety and effectiveness in larger populations
Doutorado
Oftalmologia
Doutor em Ciências Médicas

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

The situation of corneal transplants in Brazil resembles that of other countries, where it is observed an insufficient offer of cornea to meet the need of carrying out transplantations. This study aims to characterize cornea donors and patients who underwent transplantation, evaluating epidemiological data, indications from surgery, presence of rejection and length of waiting list. Method: This is an observational, transversal and descriptive study, where data were collected from all donors and receptors charts, filed at the Notification, Catchment and Distribution Center of Sergipe, from January 2000 to April 2009, and these data were summarized as average and standard deviation or medians and percentiles for the quantitative variables, while for categorical variables it were used simple frequency, percentages and trust interval for 95% when necessary. Results: It was observed a total of 272 donors (544 corneas), from which 415 corneas were recovered for transplantation, and that was the total amount of receptors surveyed. The average donors age was 39.4 ± 15.9 years, with male predominance (68%) and the most frequent cause of death was trauma (56%). There was a recovery of 76.2% from the donated corneas, 7.7% among the total donations tested positive for infectious diseases, which prompted its discard. Optsol® was the solution most used for preservation (56%). Among cornea receptors there was a predominance of transplant indication for bullous keratopathy (37.3%), followed by scarring (23%) and keratoconus (18.3%). The average receptors age was 51.4 ± 25.0 years, 51% males and 49% females. The percentage of rejection was 8.7%. The median waiting time for transplantation was 9.9, months, the 25 percentile was 2.2 months and the 75 percentile was 38.6 months. Conclusion: Cornea donors were predominantly young males, victims of trauma, mostly resulting in good quality of the cornea with a high recovery of these donations. The rejection level was considered low. Waiting time for transplantation was considered high, and there has been a large increase in the waiting list, that has tripled over almost 10 years. The main indication for transplant surgery was pseudophakic bullous keratopathy.
A situação dos transplantes de córnea no Brasil se assemelha a de outros países, onde se observa uma oferta de córnea insuficiente para suprir a necessidade de realização dos transplantes. O presente estudo tem como Objetivo: a caracterização dos doadores de córnea e dos pacientes que se submeteram ao transplantes, avaliando dados epidemiológicos, indicações da cirurgia, presença de rejeição, tempo de lista de espera. Método: Trata-se de um estudo observacional, transversal, descritivo, onde foram coletados dados dos prontuários de todos os doadores e receptores de córneas, arquivados na Central de Notificação, Captação e Distribuição de Órgãos de Sergipe, no período de janeiro de 2000 a abril de 2009, sendo esses dados sumarizados como média e desvio padrão ou medianas e percentis para as variáveis quantitativas, enquanto para as categóricas utilizaram-se freqüência simples, percentagens e intervalo de confiança para 95%, quando necessário. Resultados: Foi observado um total de 272 doadores (544 córneas), com um número total de 415 córneas aproveitadas para transplante, e esse foi o número total de receptores pesquisados. A média de idade dos doadores foi de 39,4 ± 15,9 anos, com predomínio do sexo masculino com 68% (185/272) e a causa morte mais freqüente foi o trauma com 56% (152/272). Houve um aproveitamento de 79% (429/544) das córneas doadas, e apenas 415 transplantes foram elegíveis para a análise no presente estudo, de acordo com os critérios de inclusão e exclusão. Verificou-se uma positividade para doenças infecciosas entre o total das doações de 7,7% (44/544), que motivou o descarte das mesmas, além de 13% (70/544), descartadas por baixa qualidade do tecido. A solução mais utilizada para preservação foi o Optsol® (56%). Entre os receptores de córneas houve um predomínio da indicação de transplantes pela ceratopatia bolhosa (37,3%), seguida do leucoma (23,%) e posteriormente o ceratocone (18,3%). A idade média dos receptores foi de 51,4 ± 25,0 anos, sendo o percentual de 51% para o sexo masculino e 49% para o sexo feminino. O percentual de rejeição foi de 8,7%. Quanto ao tempo de espera pelo transplante, a mediana foi 9,9, meses, o percentil 25 foi 2,2 meses e o percentil 75 foi 38,6 meses., além de observa-se um aumento do número de pacientes na fila de espera, que passou de 89 em 2000, a 325, ao final de 2009. Conclusão: Os doadores de córnea foram predominantemente homens, jovens, vítimas de trauma, em sua maioria, implicando em boa qualidade da córnea, com um alto aproveitamento destas doações. O nível de rejeição foi considerado baixo o tempo de espera pelo transplante foi considerado alto, além de se verificar um grande aumento da fila de espera, que triplicou ao longo de quase 10 anos. A principal indicação para a cirurgia de transplante foi a ceratopatia bolhosa do pseudofácico,.

The series of studies comprising this thesis was developed to answer a number of key inter-related questions in regard to eye banking and corneal transplantation in New Zealand. The source and management of donor tissue procured by the New Zealand National Eye Bank (NZNEB) was analysed. Significant trends were identified with respect to donor demographics, donor procurement source, improved donor tissue processing and storage, decreased biological contamination, and increased utilization of corneal tissue. Current trends and ethnicity differences in indications for penetrating keratoplasty (PKP) were investigated. Keratoconus was identified as the most common indication for PKP in New Zealand, accounting for a significantly higher proportion of PKPs than other published reports. Keratoconus was the most common indication for PKP throughout all ethnicity groups and was particularly common in the Maori and Polynesian populations. Significant trends were identified including an increase in the number of PKPs for regraft and Fuchs’ endothelial dystrophy and a decrease for aphakic or pseudophakic bullous keratopathy and viral keratitis. Survival and visual outcome following PKP in New Zealand was investigated using univariate and multivariate analysis. Several independent risk factors were identified that influenced outcome of PKP. Active inflammation at PKP, pre-existing vascularisation, pre-operative glaucoma, small or large graft size, intra-operative complications, episodes of reversible rejection and a pre-operative diagnosis of regraft, trauma or infection resulted in a significantly decreased survival rate. Advancing recipient age, active inflammation at the time of PKP, pre-existing vascularisation, pre-operative glaucoma, episodes of reversible rejection, bullous keratopathy, trauma and non-infective keratitis were associated with poor visual outcome. Patient characteristics, indications, surgical details, and outcome of paediatric keratoplasty were analysed. Acquired non-traumatic indications accounted for the majority of paediatric keratoplasties in New Zealand. This study highlighted keratoconus as a particularly common indication for paediatric keratoplasty when compared to other countries. Survival and visual outcome was better for acquired compared to congenital indications. The effects of corneal parameters on the measurement of endothelial cell density (ECD) in the normal eye were analysed. Corneal thickness appears to be negatively correlated to ECD in the normal cornea for all age groups. Corneal diameter is correlated to ECD measurement in children but not in adults. Corneal curvature was not significantly correlated to ECD measurement, but this needs further investigation. Confocal microscopy and slit scanning topography were used to analyze endothelial morphology and function in the short and long term following PKP. The results of this study are in concordance with other published reports that have identified an accelerated loss of endothelial cells and more rapid development of abnormal endothelial cells in transplanted corneas compared to normal corneas.

La dystrophie cornéenne endothéliale de Fuchs (FECD, pour l’abréviation du terme anglais « Fuchs endothelial corneal dystrophy ») est une maladie de l'endothélium cornéen. Sa pathogenèse est mal connue. Aucun traitement médical n’est efficace. Le seul traitement existant est chirurgical et consiste dans le remplacement de l’endothélium pathologique par un endothélium sain provenant de cornées de la Banque des yeux. Le traitement chirurgical, en revanche, comporte 10% de rejet immunologique. Des modèles expérimentaux sont donc nécessaires afin de mieux comprendre cette maladie ainsi que pour le développement de traitements alternatifs. Le but général de cette thèse est de développer un modèle expérimental de la FECD en utilisant le génie tissulaire. Ceci a été réalisé en trois étapes. 1) Tout d'abord, l'endothélium cornéen a été reconstruit par génie tissulaire en utilisant des cellules endothéliales en culture, provenant de patients atteints de FECD. Ce modèle a ensuite été caractérisé in vitro. Brièvement, les cellules endothéliales cornéennes FECD ont été isolées à partir de membranes de Descemet prélevées lors de greffes de cornée. Les cellules au deuxième ou troisième passages ont ensuite été ensemencées sur une cornée humaine préalablement décellularisée. Suivant 2 semaines de culture, les endothélia cornéens reconstruits FECD (n = 6) ont été évalués à l'aide d'histologie, de microscopie électronique à transmission et d’immunomarquages de différentes protéines. Les endothélia cornéens reconstruits FECD ont formé une monocouche de cellules polygonales bien adhérées à la membrane de Descemet. Les immunomarquages ont démontré la présence des protéines importantes pour la fonctionnalité de l’endothélium cornéen telles que Na+-K+/ATPase α1 et Na+/HCO3-, ainsi qu’une expression faible et uniforme de la protéine clusterine. 2) Deux techniques chirurgicales (DSAEK ; pour « Descemet stripping automated endothelial keratoplasty » et la kératoplastie pénétrante) ont été comparées pour la transplantation cornéenne dans le modèle animal félin. Les paramètres comparés incluaient les défis chirurgicaux et les résultats cliniques. La technique « DSAEK » a été difficile à effectuer dans le modèle félin. Une formation rapide de fibrine a été observée dans tous les cas DSAEK (n = 5). 3) Finalement, la fonctionnalité in vivo des endothélia cornéens reconstruits FECD a été évaluée (n = 7). Les évaluations in vivo comprenaient la transparence, la pachymétrie et la tomographie par cohérence optique. Les évaluations post-mortem incluaient la morphométrie des cellules endothéliales, la microscopie électronique à transmission et des immunomarquage de protéines liées à la fonctionnalité. Après la transplantation, la pachymétrie a progressivement diminué et la transparence a progressivement augmenté. Sept jours après la transplantation, 6 des 7 greffes étaient claires. La microscopie électronique à transmission a montré la présence de matériel fibrillaire sous-endothélial dans toutes les greffes d’endothelia reconstruits FECD. Les endothélia reconstruits exprimaient aussi des protéines Na+-K+/ATPase et Na+/HCO3-. En résumé, cette thèse démontre que les cellules endothéliales de la cornée à un stade avancé FECD peuvent être utilisées pour reconstruire un endothélium cornéen par génie tissulaire. La kératoplastie pénétrante a été démontrée comme étant la procédure la plus appropriée pour transplanter ces tissus reconstruits dans l’œil du modèle animal félin. La restauration de l'épaisseur cornéenne et de la transparence démontrent que les greffons reconstruits FECD sont fonctionnels in vivo. Ces nouveaux modèles FECD démontrent une réhabilitation des cellules FECD, permettant d’utiliser le génie tissulaire pour reconstruire des endothelia fonctionnels à partir de cellules dystrophiques. Les applications potentielles sont nombreuses, y compris des études physiopathologiques et pharmacologiques.
Fuchs endothelial corneal dystrophy (FECD) is a primary disease of the corneal endothelium. Its pathogenesis is poorly understood. No medical treatment is effective. Surgical treatment (the only available treatment) carries 10% of immunogenic rejection. Experimental models are needed in order to better understand the disease and to investigate potential autologous treatments (to prevent immunogenic rejection). The overall goal of this thesis is to develop an experimental model for FECD using tissue engineering. This was achieved in three steps. 1) An in vitro tissue-engineered FECD model was created and characterized. Briefly, Descemet’s membranes from patients with late-stage FECD undergoing Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) were used to isolate and culture FECD endothelial cells. Second or third-passaged FECD endothelial cells were seeded on a previously decellularized human cornea. After 2 weeks in culture, TE-FECD corneas (n=6) were assessed using histology, transmission electron microscopy (TEM) and immunofluorescence labeling of various proteins. TE-FECD endothelium yielded a monolayer of polygonal cells well adhered to Descemet’s membrane. The TE-FECD corneal endothelium expressed the function-related proteins Na+-K+/ATPase α1 and Na+/HCO3-. Clusterin expression was faint and uniform. 2) In order to determine the best surgical procedure to transplant the TE-FECD corneas in the feline model, a DSAEK procedure was evaluated and compared to penetrating keratoplasty technique. DSAEK assessments included surgical challenges and clinical outcomes. DSAEK technique was challenging to perform in the feline model. Rapid fibrin formation was observed in all DSAEK cases (n=5). 3) The in vivo functionality of the TE-FECD corneas was assessed. TE-FECD corneas were grafted in the feline model (n=7) using penetrating keratoplasty procedure and observed for seven days. In vivo assessments included transparency, pachymetry, optical coherence tomography, endothelial cell morphometry, TEM and immunostaining of function-related proteins. After transplantation, pachymetry gradually decreased and transparency gradually increased. Seven days after transplantation, 6 out of 7 grafts were clear. Post-mortem TEM showed subendothelial loose fibrillar material deposition in all TE-FECD grafts. The TE grafted endothelium expressed Na+-K+/ATPase and Na+/HCO3-. This thesis demonstrates that endothelial cells from late-stage FECD corneas can be used to engineer a corneal endothelium. Compared to DSEAK, penetrating keratoplasty is a more appropriate procedure for corneal transplantation in the feline model, since the DSAEK procedure in the feline model presently yields inconsistent clinical results. Restoration of corneal thickness and transparency demonstrates that the TE-FECD grafts are functional in vivo. This novel FECD living model suggests a potential role of tissue engineering for FECD cell rehabilitation. Potential applications are numerous, including pathophysiological and pharmacological studies.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: O objectivo deste estudo é determinar as recentes mudanças nas indicações para transplantes de córnea e no uso de tecido corneano em Coimbra.Métodos: os dados relativos a todos os transplantes realizados no Centro Hospitalar e Universitário de Coimbra (CHUC) entre 2011 e 2016 foram colhidos e compilados no Banco de Olhos dos CHUC. Analisámos retrospectivamente a idade, género, diagnóstico primário e técnica de transplante utilizada. Resultados: Ao longo dos 6 anos considerados, foram realizados 711 transplantes de córnea. A indicação para transplante mais frequente foi o re-transplante, responsável por 207 (29.2%) procedimentos, seguido da queratopatia bolhosa com 125 (17.6%) casos e queratocone, com 118 (16.6%) casos. Não foram encontradas alterações estatisticamente significativas nas indicações entre os 6 anos do estudo. No total, foram realizadas 506 (71.3%) queratoplastias penetrantes (PK), 129 (18.2%) Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) e 64 (9.0%) Deep Anterior Lamellar Keratoplasty (DALK). Ao longo dos anos, observámos uma diminuição estatisticamente significativa dos números de PK, acompanhada de um aumento da realização de DALK e DSAEK.Conclusões: os registos do banco de olhos dão-nos a capacidade de avaliar a evolução dos transplantes de córnea. As indicações permaneceram estáveis ao longo da janela temporal considerada. Comparando com outros estudos, apresentamos mais re-transplantes, menos queratocones (particularmente comparando com estudos mais antigos), e percentagens semelhantes de queratopatia bolhosa e distrofias da córnea. Em termos de técnica cirúrgica, este estudo contribui para evidenciar a popularidade crescente de queratoplastias lamelares em oposição a PK. Em conclusão, as indicações e técnicas para transplante de córnea continuam em evolução rápida, e merecem investigação continuada de modo a optimizar as actividades de bancos de olhos e centros de transplantação.
Introduction: The aim of this study is to determine the recent trends in corneal transplant indications and corneal tissue use in Coimbra. Methods: data concerning all corneal transplantation procedures performed at Centro Hospitalar e Universitário de Coimbra (CHUC) between 2011 and 2016 were collected and stored at the CHUC Eye Bank. We retrospectively analysed recipient age, gender, primary diagnosis and transplantation technique.Results: Across the 6 years considered, 711 corneal transplants were reviewed for analysis. The most frequent indication for corneal transplantation was regraft, which accounted for 207 (29.2%) of all procedures, followed by bullous keratopathy, with 125 cases (17.6%) and keratoconus, with 118 cases (16.6%). No statistically significant shift in indications for grafting was identified over the 6-year period (p = 0.70). All years accounted, penetrating keratoplasty (PK) accounted for 506 procedures (71.3%), Descemet’s stripping automated endothelial keratoplasty (DSAEK) for 129 (18.2%), and deep anterior lamellar keratoplasty (DALK) for 64 (9.0%). Over the 6 years, we observed a statistically significant decline in the numbers of PK, accompanied by an increase in DSAEK and DALK. Conclusions: Eye bank registries provide an effective means to evaluate corneal transplantation evolution. Transplant indications have remained stable across the time frame considered. Compared with other series, we report more repeat grafts, less keratoconus (particularly when comparing with older studies) and similar percentages of bullous keratopathy and corneal dystrophies. In terms of surgical technique, this study provides further evidence of the increasing popularity of lamellar keratoplasties, in opposition to PK. In conclusion, the indications and techniques for corneal transplantation continue to evolve rapidly, and merit continued investigation to optimize the activities of eye banks and transplant centres.

Introduction : Malgré leur état non-prolifératif in vivo, les cellules endothéliales cornéennes (CEC) peuvent être amplifiées in vitro. Leur transplantation subséquente par injection intracamérale pourrait surmonter la pénurie de tissus associée à l’allo-greffe traditionnelle – l’unique traitement définitif disponible pour les endothéliopathies cornéennes. Objectif : Évaluer la fonctionnalité d’un endothélium cornéen reconstitué par injection de CEC dans la chambre antérieure du félin. Méthodes : Les yeux droits de 16 animaux ont été opérés. Huit ont été désendothélialisés centralement avec injection de 2x10e5 (n=4) ou 1x10e6 (n=4) CEC félines supplémentées avec Y-27632 et marquées avec SP-DiOC18(3). Deux ont été désendothélialisés complètement et injectés avec 1x10e6 CEC et Y-27632. Six contrôles ont été désendothélialisés centralement (n=3) ou complètement (n=3) et injectés avec Y-27632 sans CEC. La performance clinique, l’intégrité anatomique, le phénotype fonctionnel et l’expression de SP-DiOC18(3) du nouvel endothélium ont été étudiés. Résultats : Les cornées greffées avec 2x10e5 CEC et les contrôles désendothélialisés centralement ont réussi le mieux cliniquement. Les contrôles désendothélialisés complètement sont restés opaques. L’histopathologie a révélé une monocouche endothéliale fonctionnelle dans les cornées greffées avec 2x10e5 CEC et les contrôles désendothélialisés centralement, une multicouche endothéliale non-fonctionnelle dans les cornées désendothélialisées centralement et greffées avec 1x10e6 CEC, et un endothélium fibrotique non-fonctionnel dans les cornées désendothélialisées complètement. L’expression de SP-DiOC18(3) était rare dans les greffes. Conclusion : La thérapie par injection cellulaire a reconstitué un endothélium partiellement fonctionnel, auquel les CEC injectées n’ont contribué que peu. L’injection de Y-27632 sans CEC a reconstitué l’endothélium le plus sain. Des études additionnelles investiguant l’effet thérapeutique de Y-27632 seul sont justifiées.
Introduction : Despite their growth arrest in vivo, corneal endothelial cells (CEC) can be amplified in vitro. Their subsequent transplantation by cell-injection therapy could overcome the tissue scarcity associated with traditional allo-transplantation, which is the only currently available treatment for irreversible corneal endothelial failure. Objective : To evaluate the functionality of a corneal endothelium reconstituted by cell- injection therapy in the feline. Methods: The right eyes of 16 animals underwent surgery. Eight underwent central endothelial scraping and injection with 2x10e5 (n=4) or 1x10e6 (n=4) feline CEC supplemented with Y-27632 and labeled with SP-DiOC18(3). After total scraping, two eyes were injected with 1x10e6 labeled CEC and Y-27632. The central (n=3) or entire (n=3) endothelium was scraped in six controls followed by Y-27632 injection without CEC. Outcomes included clinical performance, anatomical integrity, functional phenotype and SP-DiOC18(3) expression of the new endothelium. Results: Corneas grafted with 2x10e5 CEC and centrally scraped controls performed the best clinically. Entirely scraped controls remained hazy and thick. Histopathology revealed a confluent, functional endothelial monolayer in corneas grafted with 2x10e5 CEC and centrally scraped controls, a non-uniform, non-functional endothelial multilayer in centrally scraped corneas grafted with 1x10e6 CEC, and a non-functional fibrotic endothelium in entirely scraped grafts and controls. SP-DiOC18(3) was scarce in grafts and absent in controls. Conclusion : Cell-injection therapy reconstituted an incompletely functional endothelium, to which injected CEC contributed little. Y-27632 injection without CEC reconstituted the healthiest endothelium. Further studies investigating the therapeutic effect of Y-27632 alone are warranted.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: Os transplantes de córnea de alto risco apresentam taxas de rejeição mais elevadas e precoces devido à perda do “privilégio imunológico” habitualmente presente em doentes submetidos a transplante de córnea. Não havendo atualmente consenso sobre qual o melhor esquema imunossupressor a utilizar nestes casos, avaliámos o impacto do tacrolimus tópico na sobrevida do transplante, quando adicionado ao esquema terapêutico previamente utilizado no Serviço de Oftalmologia do Centro Hospitalar e Universitário de Coimbra.Métodos: Analisámos de forma observacional e retrospetiva o pós-operatório do transplante de córnea em doentes considerados de alto risco submetidos a esquema imunossupressor com recurso ao tacrolimus pomada oftálmica 0.2mg/g 2id. Os doentes foram classificados como alto risco caso tivessem sido submetidos a transplante de córnea prévio e apresentassem dois ou mais quadrantes do estroma da córnea com neovascularização. A informação foi obtida através dos registos clínicos relativos às consultas de pré e pós-operatório. O grupo de controlo foi constituído pelo mesmo grupo de doentes, considerando os dados relativos ao transplante de córnea realizado previamente ao transplante em estudo, no qual não foi adicionado tacrolimus ao esquema de imunossupressão. Desta forma, a comparação foi feita entre o transplante anterior, sem adição de tacrolimus, e o transplante atual (com tacrolimus) no mesmo grupo de doentes.Resultados: A amostra foi constituída por 16 doentes, 8 do género masculino e 8 do género feminino, sendo a idade média de 62,94 ± 18,50 anos (média ± desvio-padrão). Durante um follow up de 20,00 ± 9,83 meses (mediana ± desvio-padrão), cerca de 18,8% dos doentes em estudo apresentaram rejeição do transplante, comparativamente a 100% dos doentes no grupo controlo, durante o follow up de 14,00 ± 50,33 meses (mediana ± desvio-padrão). Discussão: Estudos anteriores revelam taxas de sobrevida dos transplantes de córnea de alto risco inferiores a 35% aos 10 anos e sem melhoria destes valores ao longo das últimas décadas. A nossa análise revelou uma melhoria da sobrevida dos transplantes de córnea de alto risco caso com a adição do tacrolimus tópico pomada oftálmica ao esquema terapêutico convencional, baseado em corticosteroides tópicos.Conclusão: O tacrolimus tópico revelou-se seguro e eficaz no prolongamento da sobrevida do transplante em doentes de alto risco.
Purpose: High-risk corneal grafts have high levels of rejection because they lose their “immunological privilege”. Nowadays there is no consensus of which immunosuppression should be used, so we studied how topical tacrolimus ointment could help prevent rejection in these cases, when added to the previous immunosuppression regimen on the Ophthalmological Department of Centro Hospitalar e Universitário de Coimbra.Methods: We retrospectively analyzed the post-operative time in high-risk patients who used an immunosuppression regimen with topical tacrolimus ointment 0.2mg/g twice a day. The information was gathered from the clinical files. Patients were considered high risk if they had history of previous graft rejection and at least two quadrants of stromal neovascularitization. The controls were previous grafts in the same group of patients.Results: We studied 16 patients, 8 males and 8 females, with a media age of 62,94 ± 18,50 years (media ± standard deviation). During a follow-up of 20.00 ± 9.83 months (median ± standard deviation), about 18,8% of patients in the study group experienced transplant rejection, compared to 100% of patients in the control group. The median follow-up of the control group (previous transplant without tacrolimus) was 14,00 ± 50.33 months (median ± standard deviation).Discussion: Previous studies showed survival rates less than 35% at 10 years, with no improvement of these values during the last decades. Our study showed that topical tacrolimus ointment increases the survival rate of the graft if added to the previous topical steroid regimen.Conclusion: Topical tacrolimus ointment is safe and effective in prolonging graft survival in high-risk patients.

皮膚是人體最大的器官，具有多種功能，其中最重要的功能之一就是作為身體內部和外界環境之間的的保護屏障。完整地修復這一保護屏障是創傷癒合和組織再生領域的一個重要內容。本論文探討了人類臍帶被覆上皮細胞 (cord lining epithelial cells, CLECs)作為一種幹細胞來源，可用于表皮重建的潛能.
本論文的第二章對CLECs的體外分離和增殖進行了詳細地描述。這一類細胞具有較長的染色體端粒，較高的增殖潛能和傳代能力。同時，它們表達上皮幹細胞和多能性幹細胞的標誌性表面抗原。它們還具有多種分化潛能，包括成脂、成骨和成軟骨。然而當皮下異種移植後，它們並不會形成畸胎瘤。
本論文的第三章對CLECs的免疫特性進行了評估。結果顯示CLECs不但具有低免疫原性，還具有免疫調節功能。它們表達典型性的一型主要組織相容性複合體(MHC class I)，即人白細胞ABC抗原(HLA-ABC)，但不表達典型性的二型主要組織相容性複合體(MHC class II)，即人白細胞DR抗原(HLA-DR)。它們同時還表達非典型性的MHC class I, 包括人白細胞G抗原和人白細胞E 抗原(HLA-G和HLA-E), 但不表達共激分子(CD40, CD80和CD86)。此外，體外檢測還發現它們表達適度的促炎/抗炎細胞因子和大量的生長因子.
本論文的第四章對CLECs在表皮重建應用中的潛能進行了考察。結果顯示無論在體外器官培養還是異種移植動物模型中，CLECs都能形成分層的上皮結構，與用表皮細胞構建的分層上皮結構相類似。而且在CLECs構建的皮膚替代物中證實了有表皮分化標誌性抗原的表達。
結論：本論文證明了CLECs具有幹細胞樣特性但無致瘤性，具有低免疫原性和表皮分化的可塑性。研究結果支持CLECs在創傷癒合和皮膚再生領域的臨床應用可行性.
The skin is the largest organ in the body and has multiple functions. One of the most important functions is to serve as a protective barrier between the internal and external environments of the body. Restoration of the integrity of this protective barrier is an essential aspect of wound healing and tissue regeneration. In this thesis, the potential of human umbilical cord lining epithelial cells (CLECs) as a source of stem cells with appropriate differentiation capacity for epidermal reconstitution has been explored.
The isolation and propagation of CLECs from human umbilical cord lining epithelium were described in Chapter II. The cells presented a long telomere length and had high proliferative potential and passaging capability. They were also shown to display both epithelial and pluripotent stem cell markers. They were capable of multipotent differentiation, including adipogenesis, osteogenesis and chondrogenesis. However, they didn’t form teratoma after subcutaneous xenotransplantation until 12 weeks.
The immune properties of CLECs in vitro were assessed in Chapter III. The cells were shown to have low immunogenicity but high immunosuppressive function. They expressed classical major histocompatibility complex (MHC) class I antigens (HLA-ABC), but not MHC class II antigen (HLA-DR). They also expressed non-classical MHC class I antigens (HLA-G and HLA-E), but lacked the expression of the co-stimulatory molecules (CD40, CD80 and CD86). Moreover, they expressed moderate pro/anti-inflammatory cytokines and multiple growth factors both in cell supernatants and cell lysates.
The potential of CLECs for epidermal reconstitution was investigated in Chapter IV. In both organotypic culture and xenotransplantation model, CLECs were capable of generating a stratified epithelial structure, which is similar to that constructed by using keratinocytes. Furthermore, the expression of epidermal differentiation markers was verified in CLEC-constructed skin substitutes.
In conclusion, the stem cell-like properties of CLECs have been demonstrated in the present study. In addition to the lack of tumorigenicity, CLECs also have low immunogenicity and significant plasticity in epidermal differentiation. The findings support the potential clinical application of CLECs in wound healing and skin regeneration.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Cai, Yijun.
"October 2012."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 114-129).
Abstract also in Chinese.
Abstrac --- p.i
Table of Contents --- p.v
Abbreviations --- p.vii
List of Figures --- p.viii
List of Tables --- p.x
Chapter Chapter I --- Introduction --- p.1
Skin --- p.3
Wound healing --- p.6
Wound regeneration and repair --- p.6
Recent history of wound treatment --- p.9
Skin substitutes --- p.11
Stem cells for wound treatment --- p.14
Stem cells overview --- p.15
Adult stem cells --- p.16
Fetal stem cells --- p.18
Amniotic membrane derived stem cells --- p.19
Umbilical cord stem cells --- p.22
Hypothesis and Specific aims --- p.24
Chapter Chapter II --- The Isolation and Characterization of the Stem Cell-like Properties of Human Umbilical Cord Lining Epithelial Cells --- p.28
Introduction --- p.28
Materials and methods --- p.30
Results --- p.47
Discussion --- p.62
Conclusion --- p.67
Chapter Chapter III --- The assessment of the Immune Properties of Human Umbilical Cord Lining Epithelial Cells --- p.69
Introduction --- p.69
Materials and methods --- p.72
Results --- p.75
Discussion --- p.83
Conclusion --- p.88
Chapter Chapter IV --- The Investigation of the Potential of Human Umbilical Cord Lining Epithelial Cells for the Epidermal Reconstitution --- p.89
Introduction --- p.89
Materials and methods --- p.91
Results --- p.94
Discussion --- p.101
Conclusion --- p.104
Chapter Chapter V --- Summary and Future Plan --- p.105
Summary --- p.105
Future plan --- p.108
Acknowledgements --- p.113
References --- p.114
Appendix --- p.130

博士
長庚大學
臨床醫學研究所
101
Recent advances in stem cell (SC) biology have led to novel application of stem cell-based therapies. Second to hematopoietic SCs, epithelial SCs are among the most studied populations. The primary source of corneal epithelium is considered to be a population of SCs, residing in the limbal region and giving rise to transient amplifying cells. Not only do SCs ensure that the corneal epithelium undergoes continual self-renewal, they are also responsible for repair and regeneration of the corneal epithelial tissue. Unfortunately, there are still limitations for use of adult SCs. First, there are no definitive markers for the identification of adult SCs in general and corneal SCs in particular. Second, ex vivo systems for manipulating SCs are still ill-defined. Finally, it remains unclear about the safety, efficacy, and phenotype of the ex vivo cultivated epithelial cell sheets after transplantation into the human body. Limbal SC deficiency of any cause may result in poor corneal epithelialization, persistent epithelial defects, corneal vascularization, corneal scarring, i.e. conjunctivalization of the cornea, leading to decreased vision, ocular discomfort and pain, and an unstable ocular surface. The past two decades have witnessed considerable progress in the treatment of such ocular surface diseases. Ex vivo cultivated corneal epithelial SC transplantation, an advanced form of conventional limbal transplantation, is now considered one surgical modality of choice. Autologous in nature for the proto-type surgical method though, for bilateral total limbal deficiency, it is important to realize that without the use of immunosuppressives, allogeneic cultivated corneal epithelial cells are easily rejected. On the other hand, definitive evidence for the long-term persistence of transplanted SCs is not yet provided. Recently, autologous cultivated oral mucosal epithelial transplantation (COMET) was developed in Japan and is now recognized as an innovation in cell therapy. However, compared with corneal epithelial cells, the inferior anti-angiogenic effect and epithelial barrier function of oral mucosal epithelial cells (OMECs) may result in less satisfactory epithelial transparency and regularity, thus limiting the visual outcome after COMET, which is the main disadvantage of this technique. In the first part of this study, we executed phase I clinical trial of COMET. Since April 2006, COMET was performed in the acute (n = 6) and chronic stage (n = 7) of corneal burns. The results show the clinical potential of COMET to promote epithelialization and reduce inflammation in acute burns, to reconstruct the conjucntival and corneal surface in chronic burns, and to improve visual acuity after subsequent ocular surface reconstruction procedures. Free from rejection and repeatable, COMET may be considered an alternative treatment or bridge therapy for the management of severe corneal burns. In the second part of this study, adopting conventional histopathology and immunofluorescent confocal microscopy, we investigated differentiation-related and putative SC markers in corneas after COMET as well as oral mucosa and cultivated OMECs for COMET. Between 10 and 22 months after COMET, corneal tissues were obtained after penetrating keratoplasty (PKP, n = 1) in and after conjunctivolimbal autografting (CLAU, n = 3). The results confirm the notion that keratin 8, normally expressed by simple epithelia, is expressed by both normal corneal and conjunctival epithelia, but not by normal OMECs unless in malignancy. Importantly, small and compact p75-positive cells in the basal epithelium are likely to contain SCs and are suggestive of long-term existence of OMECs in corneas after transplantation. In the third part of this study, following the similar protocol, we investigated angiogenesis-related factors in corneas after COMET in another six patients. Between 9 and 26 months after COMET, corneal tissues were obtained after PKP (n = 3) and CLAU (n = 3). The results support clinical speculation that low levels or lack of anti-angiogenic activity of cultivated OMECs result in corneal neovascularization after COMET. Specifically, soluble fms-like tyrosine-1 (sFlt-1), tissue inhibitor of metalloprotease-3 (TIMP-3), and thrombospondin-1 (TSP-1) probably play a pivotal role in determining heterogeneous angiogenic activity of transplanted OMECs in corneas after COMET. Through the phenotypic analysis of the aforementioned clinical trial, we have justified the rationale for clinical use of cultivated epithelial transplantation. We have also paved a way to further improve the clinical practicability of this innovative procedure.

In dieser Arbeit wurden Methoden entwickelt und verglichen, um aus Corneoskleralringen langzeitorgankonservierter Hornhäute und intakten, kryokonservierten Amnionmembranen Limbusepithel-Amnion-Transplantate herzustellen. Als erfolgreichste Kultivierungsmethode stellte sich hierbei signifikant die Explantat-Technik mit nach unten gerichtetem Limbusepithel heraus. Hier konnte eine Auswachsrate von 42 % erzielt werden. Es wurde weiterhin gezeigt, dass das ausgewachsene, mehrschichtige Limbusepithel proliferationsfähige TACs (Transient Amplifying Cells) enthält. Weiterhin konnten mittels Regressionsanalyse signifikante Zusammenhänge zwischen Spenderalter, Post-mortem-Zeit, Organkultur-Dauer und der Auswachsrate beschrieben werden. Kurzgefasst wurde die Vermutung bestätigt, dass jede Verlängerung der unterschiedlichen Zeiten eine Verringerung der Auswachsrate zur Folge hat. Die hergestellten Limbusepithel-Amnion-Transplantate könnten für Patienten mit Limbusstammzellinsuffizienz unterschiedlicher Genese verwendet werden.

碩士
高雄醫學大學
職業安全衛生研究所
97
Chemical burn is the absolute ocular emergency. It is the most severe injuries to the organ of vision with a very high percentage of unfavorable outcomes. It often causes extensive damage and results in permanent visual impairment. Recurrent epithelial erosions, symblepharon, corneal scar, corneal ulceration, severe stromal inflammation, and corneal neovascularization are common clinical complications of alkali burn .We studied the effect of cultured human adipose tissue-derived stem cells on regeneration of rabbit cornea after alkaline chemical burn. Human adipose tissue–derived stem cells are served as the source of donor material. The frozen stem cells were defreezing and sub-cultured up to 80% with Keratinocyte -SFM (Invitrogen ) and supplements of EGF , BFE( Bovine pituitary extraction), 10 %FBS ( FBS 50 cc / SFM 500 cc),GM solution 100 ul / 500 cc SFM. The cell suspension was cultured in 25 cm2 culture flasks at 37oC and 5% CO2. Culture medium was replaced by 80-90 % every 2 days. The study was performed on 8 rabbits (2-2.5 kg) with alkaline burns of the cornea. NaOH-impregnated disks (impregnated for 5 minutes, size as 7 mm in diameter) were placed into strictly central cornea area for 40 sec. After removal of the disks, the eyes were washed with 20 ml saline for 30 sec. All the procedures were taken under general anesthesia (5% ketamine plus rompune 1 to 2 mg/kg IM) and local analgesia of the cornea (1% Proparacaine Hydrochloride). Immediately after the chemical burn, experimental animals received a single subconjunctival injection of stem cell suspension (1.3×105 cells/ 0.2 ml ml). Controls were injected with 0.2 ml saline. Topical treatment with gentamycin oint (twice per day) was applied for preventing secondary infections. The eyes were eviscerated after sacrifice on days 30. Histological studies were performed on paraffin sections stained with hematoxylin and eosin. Real-time PCR are performed to detect the surface markers of P 63, E-Catherin, Beta-catenin, and Connexin 43. We evaluate the corneal functions via the following criteria, corneal opacity assessments, Real-time PCR and Histology, to prove the corneal regeneration effects of human adipose tissue - derived stem cells. The average grading of experimental group is graded as 1-2 while the controlled group is graded as 4.This means the stem cells offer more cell renewal processes than the controlled group. Real-time for beta- catena, connexin43 (Cx43), E-Catherin, and P63 are checked, which represent as cell membrane, cell membrane, non-neural epithelium and corneal stratified epithelium individually. Positive correlation of beta- catenin, connexin43 (Cx43), E-Catherin means good cell renewal for damage repair of corneal epithelium related to chemical burn. P63 shows non-significant change. Histologically, there are 5-6 cell layers at epithelium for the experimental group and 2-3 cell layers for the controlled group. Except the 8th rabbits with poor section of histology, 6 experimental groups show 5-6 cell layers and 7 control group show 2-3 cell layers over the epithelia . Transplantation of cultured human adipose tissue derived stem cells on rabbits corneal chemical burn promotes cell renewal and damage repair. Corneal transparency, Real-time PCR, epithelium cell layers are evaluated and proved as positive for wound healing.

Experimental models of a transfer of stem cells for therapeutic purposes Abstract Stem cell therapy currently represents a standard procedure of treating a wide variety of hereditary diseases and serious injuries. Development of the most suitable way of transfer of stem cells into the patient body remains very important question concerning this type of therapy. In our experiments we used nanofiber scaffolds for stem cell cultivation and their subsequent transfer. These nanofibers were prepared by the original needleless electrospun NanospiderTM technology. Allogeneic cornea or skin graft were transplanted from B6 mice to BALB/c mice. The grafts were covered by a nanofibrous scaffold with cultivated stem cells. Stem cells were stained by an imunofluorescent dye to enable us to monitore their migration from nanofibers into tissues and consequent distribution in the body and characterize changes of this distribution in the time. The methods of ELISA and PCR were used to confirm that mesenchymal stem cells support the production of antiinflammatory cytokines IL-4 and IL-10 and contribute to inhibition of production of proinflammatory cytokines IL-1, IFNγ and inducible nitric oxide synthase. We confirmed an important beneficial role of nanofiber scaffolds in transplantation of mesenchymal stem cells. Nanofiber...

Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...