L’objectif de ce travail est de caractériser les effets antifibrotiques et angiogéniques de la FVS et d’évaluer l’impact du contexte sclérodermique sur ces propriétés angiogéniques. D’autre part, d’identifier une stratégie de monitoring biologique de cette thérapie applicable au contexte sclérodermique.Le premier volet a été de déterminer si la FVS injectée localement peut limiter les processus de fibrose in vivo. L’injection de FVS réalisée précocement ou tardivement s'accompagne d'une réduction significative de la surface de fibrose au profit des surfaces musculaires dans un modèle porcin d’incontinence urinaire. Le deuxième volet a été de rechercher si le contexte autologue de la sclérodermie systémique affecte les propriétés angiogéniques de la FVS. Cette étude a montré une légère altération de la capacité angiogénique sur des tests de Matrigel Plug in vivo associé à une signature transcriptomique de la FVS de patients sclérodermiques. Le troisième volet a été d’identifier une stratégie de monitoring biologique utile pour évaluer objectivement l’impact de nouvelles thérapies sur la vasculopathie associée à la sclérodermie systémique. L’élévation des PECs et de la Fractalkine prédisent de manière indépendante le score de sévérité de la maladie et la gravité de la fibrose pulmonaire. Ce travail a ainsi permis de progresser dans le développement d’une thérapie cellulaire innovante pour limiter la vasculopathie ischemique et la fibrose à l’origine du handicap des mains chez les patients sclérodermique et de monitorer son efficacité dans le futur. / The aim of this work is to characterize the antifibrotic and angiogenic effects of FVS and to evaluate the impact of the scleroderma context on these angiogenic properties. On the other hand, to identify a strategy of biological monitoring of this therapy applicable to the sclerodermic context.The first step was to determine whether locally injected FVS can limit fibrosis in vivo. The injection of SVF performed early or late is accompanied by a significant reduction in the area of fibrosis in favor of muscle surfaces in a porcine model of urinary incontinence. The second component was to investigate whether the autologous context of systemic scleroderma affects the angiogenic properties of SVF. This study showed a slight alteration of angiogenic capacity on in vivo Matrigel Plug assays associated with a transcriptomic signature of SVF of scleroderma patients. The third component was to identify a biological monitoring strategy that could be used to objectively evaluate the impact of new therapies on vasculopathy associated with systemic sclerosis. The elevation of EPCs and Fractalkine independently predict the severity score of the disease and the severity of pulmonary fibrosis.This work has made it possible to progress in the development of an innovative cell therapy to limit the ischemic vasculopathy and the fibrosis causing hand handicap in scleroderma patients and to monitor its effectiveness in the future.

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No. of bitstreams: 2 Dissertação - Alexandre Lamaro Cardoso - 2015.pdf: 3044296 bytes, checksum: a1b0257c1b7a1f083429fbd5076a9887 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-05-22 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Stromal vascular fraction and irradiation with low level laser are resources potencially applicable in the treatment of burn injuries. Objectives This study aimed to: (i) analyze the macroscopic, microscopic and morphometric effects of stromal vascular fraction application compared to conventional care in 3rd degree burn injuries in rats; (ii) analyze the macroscopic, microscopic and morphometric effects of stromal vascular fraction containing adipose derived stem cells application associated with low level laser compared to conventional care in 3rd degree burn injuries in rats; (iii) whether the macroscopic, microscopic and morphometric effects of stromal vascular fraction containing adipose derived stem cells application associated with low level laser irradiation are dependent on the irradiation dose applied. Method: This project was approved by the Ethics Committee on the use of animals at UFG, protocol number 035/2013. All animals received injections of stromal vascular fraction prepared from adipose tissue extracted from a animal donor and occlusive dressing. The animals were divided into 5 groups: control (CT), stromal vascular fraction (SVF), stromal vascular fraction plus Laser at 27mW of power (SVFL27), stromal vascular fraction plus Laser at 53mW of power (SVFL53) and stromal vascular fraction plus Laser at 76mW of power (SVFL76).The animals were followed ove days 3, 7 and 30 for the evaluation of macroscopic, microscopic and morphometric aspects. For statistical analysis we used the parametric "t test". Results: The morphometric analysis showed a reduction in wound area in SVFL27 group compared to the others, but without statistically significant difference. In SVFL76 group was observed inhibition of wound contraction compared to SVFL27 and CT groups (p <0,05). Was observed on day 3 significantly increased (p <0.05) infiltration of polymorphonuclear, fibrin formation and in the number of fibroblasts in all treated groups compared to CT. On the 7th day the mononuclear infiltrate, angiogenesis, collagen and number of fibroblasts was significantly higher in all treated groups compared to CT (p <0.05). The SVFL27 group also showed a higher number of fibroblasts compared to groups SVF, SVFL53 and SVFL76. At 30 days significantly increased reepitalization and collagen deposition were observed in all treated groups compared to CT (p <0.05). The SVFL53 group also showed higher polymorphonuclear infiltrate compared to other groups (p <0.05). Conclusion: Isolated application of SFV or in combination with low level Laser gives better results than conventional care in burn injury in rats and the effects of the combination of the two features are dose / Fração estromal vascular e irradiação com Laser de baixa potência são recursos potencialmente aplicáveis no tratamento de lesões por queimadura. O presente estudo teve por objetivo: (i) analisar os efeitos macroscópicos, microscópicos e morfométricos da aplicação de fração estromal vascular contendo células-tronco derivadas de tecido adiposo comparada ao cuidado convencional em lesões por queimadura de 3º grau em ratos; (ii) analisar os efeitos macroscópicos, microscópicos e morfométricos da aplicação de fração estromal vascular contendo células-tronco derivadas de tecido adiposo associada ao Laser de baixa potência comparada ao cuidado convencional em lesões por queimadura de 3º grau em ratos; (iii) analisar se os efeitos macroscópicos, microscópicos e morfométricos da aplicação de fração estromal vascular associada ao Laser de baixa potência são dependentes da dose de irradiação aplicada. Método: Este projeto foi aprovado pelo Comitê de Ética no uso de animais da UFG, protocolo número 035/2013. Todos os animais receberam injeções de fração estromal vascular preparada a partir do tecido adiposo extraído de animal doador e curativo oclusivo. Os animais foram distribuídos em 5 grupos: controle (CT), fração estromal vascular (FEV), fração estromal vascular mais Laser a 27mW de potência (FEVL27), fração estromal vascular mais Laser a 53mW de potência (FEVL53) e fração estromal vascular mais Laser a 76mW de potência (FEVL76). Foram acompanhados ao longo dos dias 3, 7 e trinta para a avaliação dos aspectos macroscópicos, microscópicos e morfométricos. Para análise estatística foi utilizado o teste Kruskal-Wallis. Resultados: a análise morfométrica mostrou redução da área da ferida mais acentuada no grupo FEVL27 comparado ao demais, porém, sem diferença estatisticamente significante. No grupo FEVL76 foi observada inibição da contração da ferida comparada aos grupos FEVL27 e CT. Foi observado no 3º dia aumento significativo (p <0,05) do infiltrado polimorfonuclear, formação de fibrina e número de fibroblastos em todos os grupos tratados comparados ao CT. No 7º dia o infiltrado mononuclear, angiogênese, colágeno e número de fibroblastos foi significativamente maior em todos os grupos tratados comparados ao CT (p <0,05). O grupo FEVL27 também apresentou maior número de fibroblastos comparado aos grupos FEV, FEVL53 e FEVL76. Aos 30 dias aumento significativo da reepitalização e deposição de colágeno foram observados em todos os grupos tratados comparados ao CT (p <0,05). O grupo FEVL53 também apresentou maior infiltrado polimorfonuclear comparado aos demais grupos (p <0,05). Conclusão: aplicação isolada de FEV ou em associação com Laser de baixa potência confere melhores resultados que o cuidado convencional em lesões por queimadura em ratos e os efeitos da associação dos dois recursos são dose dependentes.

L’ingénierie tissulaire représente un nouvel outil en chirurgie colorectale pour la prévention et le traitementdes fistules et pour la substitution sphinctérienne et rectale après chirurgie d’exérèse. Ce travail comprend :1) La sélection de la composante matricielle : in vivo, en remplacement d’un défect de paroi colique, 2matrices ont été comparées chez 16 lapins : groupe A, matrice de sous-muqueuse intestinale de porcdécellularisée (SIS, BioDesign®), référente; groupe B, matrice de chitosane, innovante. Les animaux étaientsacrifiés à 4 et 8 semaines. A 8 semaines, une régénération épithéliale plus précoce, un meilleur contrôle dela réponse inflammatoire avec un rapport de fibrose plus faible et l’obtention de quelques îlots de cellulesmusculaires lisses sont obtenus dans le groupe B.2) La confection d’un gel de délivrance cellulaire: in vitro, fibrine et chitosane ont été combinés selondifférentes formulations pour sélectionner un gel composite fibrine-chitosane, caractérisé par des testsmécaniques, de viabilité et de prolifération cellulaires et l’étude de son ultrastructure.3) Leur combinaison : in vivo, en remplacement d’un défect de paroi colique, nous avons comparé chez 20porcs la matrice acellulaire de chitosane à la matrice cellularisée par la fraction stromale vasculaireautologue, isolée et contenue dans le gel composite fibrine-chitosane. A 8 semaines, un recouvrementmuqueux complet de la zone implantée était observé dans les 2 groupes avec une régénération ad integrumde la paroi colique, y compris des cellules musculaires lisses confirmées par immunohistochimie, et unrapport de fibrose significativement plus faible dans le groupe cellularisé (15% vs. 50%, p=0,01). Enfin, unematrice de chitosane circonférentielle cellularisée a remplacé un défect colique de 2 cm de longueur chez 3porcs avec succès.Avec les critères de jugement tels que faisabilité technique, comportement matriciel et qualité de larégénération tissulaire, le chitosane présente un intérêt majeur pour la régénération tissulaire colorectale. / Tissue engineering is a new tool in colorectal surgery for the prevention and treatment of fistula and rectalsphincter substitution after surgery for resection. This work includes:1) The selection of the matrix component : in vivo, 2 matrices were compared in 16 rabbits: Group A, matrixof decellularized swine intestinal submucosa (SIS, BioDesign ®), or reference matrix; Group B, a three layersmatrix of Chitosan hydrogel, or new matrix. The animals were sacrificed at 4 and 8 weeks. At 8 weeks, earlierepithelial regeneration, better control of the inflammatory response with a lower fibrosis report and obtainingof some islets of smooth muscle cells are obtained in the B group.2) The conception of an optimal delivering cells system: in vitro, fibrin and Chitosan were combined accordingto different formulations for Select a composite gel fibrin-Chitosan, characterized by mechanical tests, viabilityand cellular proliferation and the study of its ultrastructure.3) Their combination: vivo, in lieu of a colonic wall defect, we compared, in 20 pigs, acellular Chtiosan matrixto Chitosan matrix cellularized by autologous stromal vascular fraction isolated and contained in the gelcomposite Chitosan-fibrin. At 8 weeks, a full mucosal recovery was observed in the 2 groups with recovery adintegrum of the colonic walls, including smooth muscle cells confirmed by immunohistochemistery. Thefiborsis ratio was significantly lower in the cellularized group (15% vs. 50%, p = 0, 01). Finally, a cellularizedmatrix of circumferential Chitosan has successfully implanted to replace a colonic defect of 2 cm in length in3 pigs success. With end points such as technical feasibility, matrix behavior and quality of tissueregeneration, Chitosan has a major interest for the Colorectal tissue regeneration.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Alternatives have been proposed to accelerate the tissue repair process, foremost among them the use of stem cells (SC). For implantation of these cells are needed biodegradable carriers, which allow better adhesion, provide a microenvironment suitable for adaptation and cell implantation site. The Platelet Rich Plasma (PRP) is an autologous source and cost of growth factors (GFs), which concentrates platelets and GFs released by them, accelerating bone formation and improving the quality of newly formed bone. It may be a carrier suitable for implantation of SC, therefore, not induce immune responses, and contains factors that induce osteoblastic differentiation. The goal of this work was to evaluate by clinical, biochemical, radiological and histological findings, the use of PRP associated with mononuclear stem cells (MoSC) in the cortical bone healing in dogs. Also proposed to elaborate a PRP protocol efficient in dogs by means of a small volume of whole blood standardize the use of two force plates simultaneously in orthostatic pattern, and comparing this kind with the use of a platform. To evaluate the effectiveness of this association, was used 30 adult dogs, female, mixed breed, weighing between 5 and 10 kg, separated by random draw in six treatments. Was made a failure elliptical 1,0x0,4cm medial cortical diaphyseal tibia of each animal, being completed in accordance with the proposed treatment: solution of sodium chloride 0,9% (G1), PRP (G2), the total fraction of mononuclear cells TFMC originated of bone marrow (G3), stromal vascular fraction - SVF derived of adipose tissue (G4), TFCM associated with PRP (G5) and SVF associated with PRP (G6). In the preoperative period was collected blood for preparation laboratory protocol of PRP, bone marrow collection and adipose tissue, and processing of fractions of MoSC. Were evaluated daily clinical degrees of lameness, radiological, girth before, and biomechanics in the immediate postoperative period, at 7, 14, 21, 30, 37 and 45 days, and biopsies at 15 and 45 days. In biopsy of 15 days, the failure was enlarged to 1,5x0,4cm and treatment redone, which is currently regarded as the immediate postoperative period in subsequent evaluations. The protocol of the PRP showed efficiency made, focusing on average 7,29 ± 3,21 times the original number of platelets from whole blood, from 7,5 ml of blood. The use of two force platforms simultaneously, was successful, without difference between using one or two platforms. In the X-ray analysis and biomechanical G2, G3, G5 and G6 groups were more effective in bone healing and support of the limb, followed by G4, and after G1. Histological evaluation showed higher degree of advancement scar G5 and G6, G4 in sequence and finally G1. Concluding that association MoSC and PRP is effective, being responsible for an early neoangiogenesis, accelerated osteogenesis, with better quality of bone formation, osteoblast density and intense. The groups G5 and G6 are the best adjuvant treatment of bone healing in dogs, followed by G2, after G3, G4 then, and lastly G1. / Alternativas têm sido propostas para acelerar o processo reparativo tecidual, destacando-se entre elas o uso das células-tronco (CT). Para o implante destas células são necessários carreadores biodegradáveis, que permitem melhor adesão, fornecem um microambiente adequado, e favorecem a adaptação celular no local de implantação. O Plasma rico em plaquetas (PRP) é uma fonte autógena e de baixo custo de fatores de crescimento (FCs), que concentra as plaquetas e os FCs liberados por elas, acelerando a formação óssea e melhorando a qualidade do osso neoformado. Pode ser um carreador adequado para o implante das CT, pois, não induz resposta imune, além de conter fatores indutores da diferenciação osteoblástica. O objetivo deste trabalho foi avaliar por estudos clínicos, biomecânicos, radiográficos e histológicos, o uso do PRP associado às células-tronco mononucleares (CTMo) na cicatrização óssea cortical em cães. Também se objetivou elaborar um protocolo de PRP eficiente em cães, por meio de um pequeno volume de sangue total, padronizar o uso de duas plataformas de força, simultaneamente, em padrão ortostático, nesta espécie e comparar com o uso de uma plataforma. Para avaliar a efetividade dessa associação, foram utilizados 30 cães adultos, fêmeas, sem raça definida, pesando entre 5 e 10kg, separados por sorteio aleatório em seis tratamentos. Foi confeccionada uma falha elíptica de 1,0x0,4cm na cortical medial diafisária da tíbia direita de cada animal, sendo preenchida de acordo com o tratamento proposto: solução de cloreto de sódio a 0,9% (G1), PRP (G2), fração total das células mononucleares - FTCM originadas da medula óssea (G3), fração vascular estromal - FVE derivada do tecido adiposo (G4), FTCM associada ao PRP (G5) e FVE associada ao PRP (G6). No período préoperatório foi realizada coleta de sangue, protocolo laboratorial para confecção do PRP, coleta de medula óssea e tecido adiposo, e processamento das frações das CTMo. Realizou-se avaliação clínica diária, dos graus de claudicação, radiográfica, perimetria da coxa antes, e biomecânica no pós-operatório imediato, aos 7, 14, 21, 30, 37 e 45 dias, e biópsias aos 15 e 45 dias. Na biópsia de 15 dias, a falha foi ampliada para 1,5x0,4cm e o tratamento refeito, sendo este momento considerado como o pós-operatório imediato nas avaliações subsequentes. O protocolo de PRP confeccionado demonstrou eficiência, concentrando em média, 7,29 ± 3,21 vezes o número inicial de plaquetas do sangue total, a partir de 7,5ml de sangue. O uso de duas plataformas de força, simultaneamente, obteve sucesso, não havendo diferença entre usar uma ou duas plataformas. Nas análises radiográficas e biomecânicas G2, G3, G5 e G6 foram os grupos mais efetivos na cicatrização óssea e no apoio do membro operado, seguidos de G4, e após G1. Na avaliação histológica e imuno-histoquímica observou-se maior grau de adiantamento cicatricial em G5 e G6, em sequência G4 e por fim G1. Concluí-se que associação CTMo e PRP é eficiente, sendo responsável por uma neoangiogênese precoce, osteogênese acelerada, com melhor qualidade do osso neoformado, e intensa densidade osteoblástica. Os grupos G5 e G6 são os melhores tratamentos adjuvantes da cicatrização óssea em cães, seguidos de G2, após G3, depois G4, e por último G1.

La transplantation rénale, thérapie de choix de l'insuffisance rénale chronique terminale, est limitée par une pénurie d'organes. Les greffons issus de donneurs décédés par arrêt cardiaque (DDAC) peuvent contribuer à pallier à cette pénurie au prix de stratégies thérapeutiques visant à améliorer l'issue de la transplantation. Les cellules souches mésenchymateuses (MSC) de l'organisme adulte ont des propriétés de sécrétion, d'immunomodulation et de différenciation intéressantes dans ce contexte.L'objectif de ce travail est d'évaluer, dans un modèle pré-clinique porcin de DDAC, le potentiel thérapeutique de deux populations cellulaires d'intérêt : les MSC issues du liquide amniotique (AFSC) et les cellules de la fraction vasculaire stromale du tissu adipeux (SVF). Les AFSC porcines injectées dans l'artère rénale 7 jours post-greffe, en raison de leur sensibilité à une séquence d'hypoxie réoxygénation (HR) in vitro, accélèrent la reprise de fonction et réduisent l'extension des lésions chroniques du greffon et sont détectées dans le rein 24h après injection. La SVF porcine, phénotypiquement proche de celle de l'Homme, est moins sensible à cette séquence d'HR et peut être injectée dans l'artère du greffon à sa reperfusion sans perturbation du flux sanguin rénal, avec une rétention des cellules dans le rein 24h post injection.Ce travail met en évidence le rôle bénéfique des AFSC dans la réparation des lésions ischémie-reperfusion des greffons issus des DDAC, ainsi que la faisabilité de l'injection de la SVF dans l'artère rénale après transplantation, et ouvre des pistes pour l'optimisation les protocoles d'administration de produits de thérapie cellulaire en transplantation. / Kidney transplantation is the best therapeutic option for end stage chronic kidney failure, but is limited by transplant shortage. Use of transplants from deceased after cardiac death donors (DCD) could represent an additional graft source, but there is a need for developing new therapeutic approaches like cell therapy to increase their recovery. Mesenchymal stem cells (MSC) potentially extracted from many adult tissues have interesting paracrine, immune-modulating, and differentiation properties in this context. This work aims to assess, in a preclinical porcine model DCD donor, the therapeutic potential of two cell populations of interest: amniotic fluid derived MSC (AFSC) and cells from stromal vascular fraction of adipose tissue (SVF). Delayed injection of AFSC 7 days following kidney transplantation because of their sensitivity to a specific Hypoxia Reoxygenation (HR) sequence in vitro, accelerates graft function recovery and limits chronic injuries to the transplanted organ. Cells are detectable into the transplanted kidney 24h after injection. Porcine SVF is phenotypically similar to human. Injected in renal artery simultaneously with organ reperfusion because of its resistance to the HR sequence, porcine SVF does not disturb renal blood flow and allow cell-retention within the organ 24h after injection. This work highlights the protective effect of AFSC against ischemia reperfusion lesions in grafts from DCD donors and the feasibility of SVF injection directly into the renal artery of the graft following kidney transplantation in DCD conditions. Moreover it opens new lines for optimizing injection protocols of cellular products in kidney transplantation.