As células-tronco mesenquimais derivados do tecido adiposo (ASC) apresentam potencial para uso em terapêuticas para reparação cardíaca e o modelo de suínos recapitula aspectos relevantes dos seres humanos. Nesta dissertação quisemos caracterizar ASC de porcos (pASC), após criopreservação e avaliar a responsividade destas células ao shear stress. Após descongelamento as pASC exibiram 90-95% de viabilidade, não apresentaram alterações morfológicas e nem na expressão de marcadores de superfície (CD29+ 99,74%±0,10; CD90+ 97,84%±1,32; CD44+ 99,39%±0,19, CD31- 1,75%±0,21; média±EPM, n=3). O tempo de dobramento médio foi de 63,51±16,46 horas (média±DP) e o dobramento populacional cumulativo aumentou constantemente até a passagem 10, com pequeno e gradual aumento na senescência (P5 3,25%±0,26 e P10 9,6%±0,29 SA-?-Gal). Além disso, as pASC responderam, in vitro, ao tratamento para diferenciação em adipócitos e osteócitos. Já a exposição ao SS (15 dyn/cm² por 48 hs) não induziu a expressão de marcadores endoteliais (CD31, VE-caderina e FLK-1), mas resultou no acúmulo de VEGF induzido por NO (15 dyn/cm² por 96 hs). Interessantemente, o SS induziu a fosforilação de ERK e AKT e a liberação de NO independente da magnitude do SS (1-30 dyn/cm², por 30 min). No entanto, longos períodos de estímulo (24-48 hs) e diferentes intensidades de shear stress induziram desigualmente a liberação de NO e VEGF (5 dyn/cm² maior do que 10 ou 15 dyn/cm²). Tomados em conjunto, nossos dados promovem evidências de que a viabilidade, morfologia, cinética de crescimento e resposta a estímulos químicos ou físicos de pASC não foram influenciados pela criopreservação. Além disso, a magnitude de SS aplicada a essas células afetou a liberação de NO e VEGF somente após longos períodos de exposição a esse estímulo / Adipose tissue-derived mesenchymal stem cells (ASC) offer potential regenerative therapeutic application for cardiac repair and the porcine model recapitulates key aspects relevant to humans. In this dissertation we wanted to establish the culture characteristics of pig ASC (pASC), especially after long-term cryopreservation and the effect of shear-stress (SS)-induced phenotypes. Upon thawing, pASC displayed 90-95% viability and no changes in morphological characteristics or in the expression of surface markers (CD29+ 99.74%±0.10; CD90+ 97.84%±1.32; CD44+ 99.39%±0.19, CD31- 1.75%±0.21; mean±SEM, n=3). Mean population doubling time was 63.51±16.46 hours (mean±SD) and cumulative population doubling increased constantly until passage 10 with a small and gradual increase in senescence (P5 3.25%±0.26 and P10 9.6%±0.29 SA--Gal staining). In addition, pASC in vitro responded to adipogenic and osteogenic chemical cues, whereas SS exposure (15 dyn/cm² up to 48 hours) failed to induce endothelial cell (EC) markers (CD31, VE-cadherin and FLK-1), but resulted in nitric oxide (NO)-induced VEGF accumulation (15 dyn/cm² up to 96 hours). Interestingly, SS-induced phosphorylation of ERK and AKT and the release of NO were independent of the magnitude of the stimulus (1-30 dyn/cm², up to 30minutes). In contrast, long term (24-48 hours) SS-induced NO and VEGF release under 5 dyn/cm² were higher than 10 or 15 dyn/cm2. Altogether, we provided evidence that pASC cell viability, morphology, growth characteristics and capacity to respond to chemical or physical cues were not influenced by cryopreservation. Moreover, the magnitude of the SS affected the NO and VEGF release in pASC only during long-term exposure to SS

Um novo método de modificação da superfície de nanopartículas de hidroxiapatita (HAP) por reação com cloreto de lauroíla foi desenvolvido, gerando a nanopartícula funcionalizada por laurato (HAP-CL). A superfície modificada da HAP foi confirmada por infravermelho, termogravimetria, ressonância magnética nuclear e análise elementar. Provamos por testes mecânicos a capacidade de criar compósitos com alto teor de HAP-CL em matrizes poliméricas de poli(L-acido láctico) (PLLA) e poli(succinato de isosorbídeo-b-L-lactídeo) (PLLA-co-PIS) sem perda significativa de propriedades mecânicas. Diferentes quantidades de HAP-CL, HAP enxertado com PLLA (PLLA-g-HAP) e HAP puro foram dispersadas em soluções de PLLA para formar fibras eletrofiadas. Para comparar a dispersão destas nanopartículas nas fibras e a sua morfologia, análise de microscopia eletrônica de varredura e de transmissão foram empregadas. A HAP-CL exibiu melhor dispersão na matriz polimérica do que o PLLA-g-HAP e HAP, e permitiu a produção de fibras com grande quantidade de HAP-CL (até 30% massa da fase mineral em relação à massa do polímero(mm/mp)), tanto para o PLLA como para o PLLA-co-PIS. Células tronco mesenquimais derivadas de polpa de dente foram cultivadas em fibras de PLLA com alto teor de HAP-CL, resultando em um aumento significativo da atividade de fosfatase alcalina (ALP), nos dias 14 e 21 (p <0,001) quando comparados com conteúdos mais baixos de HAP-CL, assim como um melhor processo de mineralização apresentado pelos teste de vermelho de alizarina depois de 21 dias (p <0,001). As células cultivadas nas fibras de PLLA-co-PIS contendo 30% de HAP-CL (mm/mp) apresentaram maior atividade de ALP após 21 dias (p <0,05), e melhor processo de mineralização, depois de 14 e 21 dias (p <0,05) do que as fibras de PLLA com 30% de HAP-CL (mm/mp). PLLA e PLLA-co-PIS, ambos contendo 30% de HAP-CL (mm/mp) induziram uma maior expressão de osteocalcina e osteopontina, dois marcadores de diferenciação osteoblástica, quando comparados ao PLLA e PLLA-co-PIS (controle). Finalmente, em experimentos in vivo, as fibras de PLLA-co-PIS contendo 30% de HAP-CL (mm/mp) apresentaram um desempenho superior no processo de neoformação óssea do que as fibras de PLLA com o mesmo conteúdo de nanopartículas. Em conclusão, os nossos resultados in vitro demonstraram que os suportes construídos de compósitos de PLLA-co-PIS contendo 30% de HAP-CL (mm/mp) se mostraram superiores tanto na adesão e proliferação quanto na diferenciação de células mesenquimais de polpa de dente em osteoblastos. Além disso, experimentos in vivo confirmaram estes resultados, demonstrando que estes nanocompósitos são excelentes modelos para implantes destinados a regeneração óssea. / A new method of surface modification of hydroxyapatite nanoparticles (HAP) by reaction with lauroyl chloride was developed, producing the laurate functionalized nanoparticle (HAP-CL). The surface modified HAP was confirmed by infrared, thermogravimetric analysis, nuclear magnetic resonance and elemental analysis. We proved by mechanical tests the ability to create composites with high HAP-CL content in poly(L-lactic acid) (PLLA) and poly(isosorbide succinate-b-L-lactide) (PLLA-co-PIS) polymeric matrixes without significant loss of mechanical properties. Different amounts of HAP-CL, HAP grafted with PLLA (PLLA-g-HAP) and HAP were dispersed in pure PLLA solutions to form nanofibers. To compare the dispersion of these nanoparticles in the fibers and their morphology, scanning and transmission electron microscopies were employed. HAP-CL showed better dispersion in the polymer matrix than the PLLA-g-HAP and HAP, and allowed fiber production with large amounts of HAP-CL (up to 30% mineral to polymer weight (wm/wp)) for both PLLA and PLLA-co-PIS. Mesenchymal stem cells derived from dental pulp were cultured in PLLA fibers with high levels of HAP-CL, resulting in a significant increase in alkaline phosphatase activity (ALP), on days 14 and 21 (p <0.001) as compared to those with lower content HAP-CL, as well as a better mineralization process shown by alizarin red test after 21 days (p <0.001). Cells grown in PLLA-co-PIS fibers containing 30% of HAP-CL showed higher ALP activity after 21 days (p <0.05) and a better mineralization process, after 14 and 21 days (p <0.05 ) than fibers of PLLA with 30% HAP-CL. PLLA and PLLA-co-PIS, both containing 30% of HAP-CL (wm/wp) induced a higher expression of osteocalcin and osteopontin, two osteoblast differentiation markers when compared with PLLA and PLLA-co-PIS (control). Finally, in the in vivo experiments, the PLLA-co-PIS fibers containing 30% HAP-CL (wm/wp) outperformed the process of bone formation than PLLA fibers with the same content of nanoparticles. In conclusion, our in vitro results demonstrated that scaffolds from composites of PLLA-co-PIS containing 30% HAP-CL (wm/wp) were superior both in adhesion and in the differentiation and proliferation of dental pulp stem cells in osteoblasts. Furthermore, in vivo experiments confirmed these results, demonstrating that these nanocomposites are excellent models for implants for bone regeneration

Na espécie equina, as enfermidades osteoarticulares causam prejuízo econômico e impacto negativo no desempenho atlético, devido aos danos causados na cartilagem articular. A regeneração da cartilagem hialina e a manutenção da integridade das estruturas que a compõe norteiam a busca do tratamento ideal. Neste contexto, este estudo foi delineado com o objetivo de investigar a presença de células-tronco mesenquimais (CTMs) no líquido sinovial (LS) e na membrana sinovial (MS) de equinos com articulações hígidas, com osteocondrite dissecante (OCD) e com osteoartrite (OA) e compará-las, visando estabelecer qual fonte celular possui melhor característica fenotípica e capacidade de diferenciação celular, mais especificamente, aquela que seja superior em relação à capacidade condrogênica. Foram utilizados equinos machos e fêmeas de diferentes idades, totalizando 97 articulações. O LS e MS foram coletados durante artroscopia e as células foram cultivadas, e avaliadas por citometria de fluxo com os anticorpos CD44, CD90, CD105, CD34; e por imunocitoquímica com os anticorpos nanog, oct4, PGP 9.5, lisozima, vimentina e citoqueratina. Adicionalmente, o potencial de diferenciação das células foi avaliado para as linhagens condrogênica, osteogênica e adipogênica. Foi realizado teste de tumorigenicidade em camundongos Balb-Cnu/nu, para comprovar aplicabilidade clínica, e posteriormente, as CTMs provenientes de LS de articulações hígidas foram aplicadas em articulações de equinos. A identidade das células foi comprovada durante o cultivo demonstrando características de adesão ao plástico e morfologia fibroblastóide. A média percentual das populações positivas para CD90 foi de 64,9% (LS-H), 48,3% (LS-OCD), 48,1% (LS-OA), 66,6% (MS-H), 40,2% (MS-OCD) e 40,3% (MS-OA). A porcentagem de células positivas para CD44 foi de 1,18% (LS-H), 3,98% (LS-OCD), 14,2% (LS-OA), 1,9% (MS-H), 2,17% (MS-OCD) 8,56% (MS-OA). Não foi observada expressão dos anticorpos CD34 e CD105. Na análise imunocitoquímica foi detectada expressão positiva para os anticorpos: lisozima, PGP 9.5, PCNA e vimentina, e negativa para nanog, oct4 e citoqueratina. A multipotência (osteogênica, condrogênica e adipogênica) das células foi confirmada através da coloração Alizarin Red para detecção de matriz de cálcio, Oil Red O para detecção de gotículas de gordura e azul de toluidina, alcian blue e hematoxilina eosina para detecção de matriz de proteoglicanos. Com relação aos resultados do teste tumorigênico, nenhum órgão dos camundongos foi afetado, assegurando a aplicabilidade das células estudadas. Ainda, as articulações de equinos tratadas, não apresentaram quaisquer sinais de reação inflamatória após aplicação de células alogênicas. Por fim, concluímos que, a fenotipagem positiva de CD44 e CD90 somada à capacidade de diferenciação nas linhagens osteogênica e condrogênica confirma a presença de CTMs nas populações celulares obtidas de LS e MS de equinos. Também foi observado que as células de LS provenientes de articulações hígidas, são as de melhor utilização clínica, uma vez que apresentaram maior expressão de CD90 e demonstraram melhor capacidade de diferenciação celular em relação às células derivadas de articulações enfermas. Além disso, possuem método mais fácil de colheita em relação à colheita de MS, visando futura terapia celular na rotina clínica / In the equine species, osteoarticular diseases cause significant economic losses and negative impact on equine athletic performance. The hyaline cartilage regeneration and the maintenance of integrity of its components guide the search for the ideal treatment. In this scenario, this study aimed to investigate the presence of mesenchymal stem cell (MSCs) in the synovial fluid (SF) and in the synovial membrane (SM) of healthy equine joints, osteoarthritic (OA) and osteochondritic joints (OCD), comparing their potential as cellular sources, according to their differentiation ability, in particular with superior chondrogenic potential and the phenotypic characteristics of the MSCs. Ninety-seven equine joints from males and females of different ages were used to harvest cells. SF and SM were obtained during arthroscopy and the cells SF and SM were cultured and assessed for CD90, CD44, CD105 and CD34 markers by flow cytometry, and nanog, oct4, PGP 9.5, lyzozyme, vimentin and cytokeratin were assessed by immunocytochemistry. Additionally, cells were evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. The tumorigenicity test was carried in Balb-C nu/nu mice, to verify the safety of cell sources and, later, mesenchymal stem cells harvested from healthy equine joints were injected into equine joints. The identity of these cells was confirmed during cell growth, through properties of plastic adhesion and fibroblastoid morphology. The mean percentage of CD90 positive cells was 64.9% (SF-H), 48.3% (SF-OCD), 48.1% (SF-OA), 66.6% (SM-H), 40.2% (SM- OCD) and 40.3% (SM-OA). The percentage of CD44 positive cells was 1.18 % (SF-H), 3.98% (SF-OCD), 14.2% (SF-OA), 1.9% (SM-H), 2.17% (SM-OCD) and 8.56% (SM-OA). The expression of CD34 and CD105 antibodies was not observed. Through immunocytochemical analysis, expression for lysozyme, PGP9.5, PCNA e vimentin antibodies was detected and negative expression for nanog, oct4 e cytokeratin was observed. The multipotent capacity of mesenchymal stromal cells for lineage differentiation (osteogenic, chondrogenoic and adipogenic) was confirmed with different staining techniques: Alizarin Red enabled detection of the calcium matrix, Oil Red O enabled the detection of fat droplets and Toluidin Blue, Alcian Blue and haematoxylin eosin enabled detection of proteoglycan matrix. Results of tumorigenic tests in mice showed no compromise of any internal organ, assuring applicability of the studied cells. Furthermore, equine joints treated with MSC harvested from healthy joints did not show any signs of an inflammatory reaction after injection of the allogeneic cells. The presence of cells with positive CD44 and CD90 phenotypes and with the ability to differentiate into osteogenic and chondrogenic lineages confirms the presence of MSCs in equine SF and SM. Cells obtained from healthy SF were more suitable for clinical application, for they presented higher CD90 expression and demonstrated greater differentiation capabilities, when compared to that of cells retrieved from compromised joints. In addition to that, SF derived cells are easier to obtain when compared to SM cells, aiming their future application clinical

INTRODUÇÃO: O objetivo deste estudo foi de avaliar a eficácia de um centrifugado osteogênico de medula óssea para estimular a consolidação de osteotomias da fíbula em coelhos. MÉTODOS: Este estudo experimental envolveu a utilização de dez coelhos machos adultos da raça Nova Zelândia albino. Realizou-se uma osteotomia transversa médio-diafisária da fíbula direita, seguida da adição local de uma esponja de colágeno absorvível embebida em um centrifugado osteogênico, obtido pela centrifugação de aspirado de medula óssea do osso ilíaco ipsilateral. A fíbula esquerda foi utilizada como controle, sendo feita a mesma osteotomia, porém neste caso adicionando-se somente a esponja de colágeno absorvível. O centrifugado de medula óssea elaborado em laboratório foi submetido à contagem do número de células nucleadas e a teste de viabilidade celular antes de ser administrada no local das osteotomias. Após quatro semanas os animais foram sacrificados para estudo dos calos ósseos formados. Os critérios de avaliação foram a mensuração da densidade mineral utilizando-se a densitometria óssea com DEXA, do volume do calo com tomografia computadorizada multi-slice e dos tecidos formados por meio de histomorfometria. RESULTADOS: O método utilizado para a centrifugação dos aspirados de medula óssea resultou em uma concentração média de células nucleadas três vezes maior que o número destas células nos aspirados originais, sem destruição celular significativa. A utilização deste centrifugado osteogênico resultou em um aumento médio na densidade mineral óssea dos calos de 40,3% e da quantidade relativa de tecido ósseo de 9,4%, sem aumento significativo nas quantidades relativas de cartilagem ou fibrose. Não houve aumento significativo no volume dos calos ósseos. CONCLUSÃO: A administração de centrifugado osteogênico de medula óssea utilizado neste estudo favoreceu a consolidação óssea de osteotomias experimentais em coelhos, observando-se uma melhora qualitativa do calo ósseo. / INTRODUTION: The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone-marrow aspirate to stimulate rabbit fibular osteotomies healing. METHOD: Ten white New Zeeland rabbits were used. A transverse middle-diaphysis fibular osteotomy was performed at the right fibula, where a collagen absorbable sponge embedded in the osteogenic centrifuged bone marrow aspirate was inserted. The left fibula was used as the control group, where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate. The centrifuged bone-marrow aspirate was arranged at the laboratory and submitted to nuclear cell count and cell viability test. The rabbits were killed at four weeks after surgery to evaluate bone callus formation. The results analysis was performed with DEXA bone densitometry to evaluate callus mineral mass, multislice computer tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation. RESULTS: The bone-marrow centrifugation technique increased the number of nucleated cells by three compared with the number of that cells in the original bone-marrow aspirates, without significant nucleated cell dead. The apply of centrifuged osteogenic bone-marrow aspirate resulted in an increased callus bone mineral mass by 40,3%, and increased relative rate of bone tissue formation by 9,4%, without increase the relative rate of cartilage or fibrous tissue. There was not increased callus volume. CONCLUSION: This study shows that the centrifuged osteogenic bone-marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits by qualitative improve of bone callus.

AA é uma doença grave caracterizada por pancitopenia e hipocelularidade medular, que pode levar a morte. Em casos severos os tratamentos são restritos ao transplante alogênico de MO e/ou uso de imunossupressores. Para isso, deve ser tipado o HLA entre doador e beneficiário. Com isso, as CTM são fontes celulares candidatas para este propósito, pois são praticamente não imunogênicas devido à sua ação parácrina de imunomodulação. Além disso, as CTM possuem um importante papel na hematopoiese. Para induzir AA utilizamos inicialmente ciclofosfamida (n=10) e radiação ionizante a 6 Gy (n=40), que foi o modelo experimental adotado. Após a radiação ocorreram três transplantes de CTIPDh com intervalo de 15 dias via IP. A resposta clínica do transplante foi monitorada pela avaliação da massa corpórea, hemograma e histopatologia da MO. O enxerto das CTIPDh na MO e seu efeito na hematopoiese, também foi verificado. Os resultados demonstraram que os animais irradiados e tratados com CTIPDh apresentaram benefícios clínicos em relação aos animais não tratados. Observamos enxertia das CTIPDh na MO e baço dos camundongos e recuperação dos componentes medulares em comparação aos animais não tratados. Assim, o transplante das CTIPDh são uma fonte terapêutica em potencial. / AA is a several disease characterized by peripheral pancytopenia and bone marrow hypoplasia, which can lead to death. In severe cases, treatments are limited to allogeneic BM transplant and/or immunosuppressants administration. For this purpose, the donor blood must be typed to identify their HLA. Thereby, MSC are appropriate candidates for therapeutic use, because they present low immunogenicity and provide immunomodulation effect in tissue repair. Furthermore, MSC have important role in supporting hematopoiesis. Thus, AA was induced using cyclophosphamide (n = 10) and 6 Gy ionizing radiation (n = 40), which caused BM ablation in mice. Thus ionizing radiation of AA model was selected. After radiation, the mice received three equal doses of hDPSC each 15-day via intraperitoneal injection. Clinical response was monitored by body mass, blood cells counting and histopathology of BM. The influence of hDPSC infusion on hematopoiesis and engraftment capacity of this cell was also investigated. Our data show the hDPSC transplantation in irradiated mice improves clinical conditions and the transplant intraperitoneal showed hDPSC grafting in BM and recovery of medullary components when compared to untreated group. The results indicate that hDPSC transplantation is a potential tool for cell-based therapeutics.

O cão é um excelente modelo pré-clínico para o estudo de doenças, testes farmacológicos e novas terapias para futura aplicação em humanos. Desta forma, estudamos o modelo canino como fonte de células tronco de anexos fetais, o líquido amniótico, alantóide e conteúdo vitelino. Uma vez que hoje, as células tronco apresentam uma esperança na cura de diversas doenças tanto nos cães, como no ser humano. Sendo assim, caracterizamos e estudamos o potencial de diferenciação dessas células, isoladas após a técnica de ovário salpingo histerectomia, de cadelas em campanhas de castração. Após o isolamento e a caracterização, somente foi estabelecida a cultura do líquido amniótico e alantóide. Para caracterizar as células, isoladas no intuito de comprovar que são células tronco verdadeiras, os seguintes Imunomarcadores foram usados, vimentina, nestina, citoqueratina-18 e oct-4, sendo os três primeiros positivos para células do líquido amniótico e alantóide. Induzimos a diferenciação dessas células para osso, cartilagem e gordura, utilizando protocolos previamente estabelecidos. As células tronco do líquido amniótico limitaram-se à diferenciação condrogênica e osteogênica enquanto que as células tronco do alantóide, limitaram-se a diferenciação condrogênica. Ao mesmo tempo, ambos os tipos celulares, não prosseguiram à diferenciação adipogênica. Surpreendentemente, o meio de diferenciação para gordura, induziu a mudança morfológica nestas células que passaram a apresentar a morfologia típica de células neuronais. Podemos concluir que provavelmente para diferenciação adipogênica, é preciso desenvolver outro meio de cultura, por outro lado, esse resultado sugere que devemos explorar o potencial neurogênico desses tipos celulares. / The dog is an excellent preclinical model for the study of diseases, pharmacological tests and new therapies for future application in humans. Thus, we studied the canine model as a source of stem cells from fetal membranes, amniotic fluid, allantois and fluid yolk. Since today, stem cells have a hope in curing various diseases in both dogs and humans. Therefore, we characterize and study the differentiation potential of these cells, isolated after the technique of ovarian salpingo hysterectomy. After the isolation and characterization, was only established the culture of amniotic and allantois fluid. To characterize the cells isolated in order to demonstrate that they are true stem cells, the following were used antibodies, vimentin, nestin, cytokeratin-18 and oct-4. The cells were reactive positively to vimentin, nestin, cytokeratin. We induced the differentiation of these cells osteogenic, chondrogenic, adipogenic, using previously established protocols. Stem cells from amniotic fluid were restricted to chondrogenic and osteogenic differentiation while the stem cells of the allantois, limited to chondrogenic differentiation. At the same time, both cell types, were not able to adipogenic differentiation. Surprisingly, the means of adipogenic differentiation, induced the typical morphology of neuronal cells. We can conclude that probably for adipogenic differentiation, we must develop other culture media, on the other hand, this result suggests that we should explore the neurogenic potential these cell types.

Le cartilage articulaire est composé de chondrocytes et d'une matrice extracellulaire organisés de manière stratifiée dans l'épaisseur du tissu. Ce tissu ne se régénère pas de manière efficace après une lésion. L'objectif de ce travail est de construire par pulvérisation des hydrogels à base d'alginate et de film multicouches de polyélectrolytes pour créer in vitro un néotissu pouvant combler des lésions de cartilage articulaire. La méthode a été validée en observant une bonne viabilité et une synthèse matricielle par les cellules, et de meilleures propriétés mécaniques des hydrogels pulvérisés à 0,9 bar par rapport au moulage. Après la pulvérisation de cellules souches mésenchymateuses, les résultats ont montré une bonne viabilité et une différenciation des cellules. Puis, des hydrogels bistratifiés ont été construits et cultivés jusqu'à 56 jours sans dissociation des couches et sans migration des cellules. Enfin, les hydrogels ont été fonctionnalisés en modifiant la composition des couches et en y appliquant des stimulations mécaniques. Les propriétés mécaniques des hydrogels varient en fonction de leur composition et sont meilleures pour ceux stratifiés. De plus, leur stimulation mécanique a permis de potentialiser l'effet du biomatériau sur la différenciation des cellules. En conclusion, cette étude montre que des cellules souches mésenchymateuses ensemencées dans un hydrogel bistratifié pulvérisé sont fonctionnelles en termes de différenciation chondrocytaire et de synthèse matricielle. Les propriétés mécaniques des hydrogels stratifiés ne sont pas altérées. De plus, la stimulation mécanique a potentialisé la différenciation des cellules / The articular cartilage is composed of chondrocytes and of a specific extracellular matrix which are organized depth-dependently. The tissue did not have an efficient self-renewal of defects. The purpose of this study is to build up layer-by-layer a stratified hydrogel by alternating gels and multilayers polyelectrolytes film spraying, in order to obtain a neotissu in vitro to fill lesions. First, the process was validated by observing a good cells viability and matrix synthesis, and stronger mechanical behaviors of sprayed hydrogels compared to molded one. Secondly, after their spraying, mesenchymal stem cells still have a good viability and their differentiation potential. Then, bistratified scaffolds were built up and cultured up to 56 days without layers dissociation and without cells migration between layers. Finally, scaffolds were functionalized by changing biomaterial composition and by applying mechanicals stimulations. Results show us not only that the composition influences the mechanical behavior of the hydrogel, but that the stratification did not affect it. Furthermore, mechanicals stimulations improve stem cells differentiation in function of biomaterials compositions. In conclusion, this study proves not only that we are able to build up stratified scaffold seeded with mesenchymal stem cells which still have their differentiation capability and synthesize matrix, but that mechanical behaviors are improved after the biomaterial spraying and not alter by the stratification. Moreover, mechanical stimulation applied to the scaffold improves the differentiation of mesenchymal stem cells to a chondrogenic phenotype

En ingénierie tissulaire, les biomatériaux, les cellules et l'induction de la différenciation, sont des facteurs à prendre en compte. L'objectif de cette étude est de connaitre l'effet de la nature des biomatériaux et leurs propriétés mécaniques sur la différenciation des cellules souches mésenchymateuses de la moelle osseuse. Dans un premier temps, nous avons étudié l'effet d'un biomatériau de nature protéique (le collagène de type I) supplémenté en microparticules d'hydroxyaptatite (HAP). Nous avons constaté que l'ajout d'HAP améliore les propriétés mécaniques de ce biomatériau et engage la différenciation des cellules vers des phénotypes ostéoarticulaires. Dans un deuxième temps, nous avons étudié l'effet d'un biomatériau à base d'alginate supplémenté par de l'acide hyaluronique ou des microparticules d'HAP, en utilisant un plan d'expériences pour choisir les matrices convenables pour l'étude biologique en fonction de leurs propriétés mécaniques. Nous avons constaté que les composants de ce biomatériau ont un effet sur l'élasticité de ce dernier et sur la différenciation des cellules souches mésenchymateuses. En conclusion, cette étude montre que les cellules souches mésenchymateuses sont sensibles à la composition du biomatériau et ses propriétés mécaniques / In tissue engineering, biomaterials, cells and the induction of cell differentiation are factors to be studied. The aim of this study is to know the effect of biomaterials composition and mechanical properties on the differentiation of mesenchymal stem cells from bone marrow. At first, we studied the effect of a protein biomaterial (collagen type I) supplemented with hydroxyaptatite (HAP) particles. We found that the addition of HAP improves the mechanical properties of the biomaterial and conditione cell differentiation towards osteoarticular lineages. In a second step, we studied the effect of biomaterial composed of alginate supplemented with hyaluronic acid or HAP particles, using an experimental design to select suitable matrices for biological study based on their mechanical properties. We found that the components of this biomaterial have an effect on elasticity of the latter and the differentiation of mesenchymal stem cells. In conclusion, this study shows that mesenchymal stem cells are sensitive to the composition of the biomaterial and its mechanical properties

Il existe plus de 100 formes de pathologies hépatiques causées par divers facteurs et touchant une grande quantité de personnes. Mais, le seul traitement pour les maladies du foie en phase terminale est la greffe du foie. Cependant, la greffe de foie échoue souvent à cause du déficit en donneurs hépatiques. Récemment, une nouvelle alternative innovante pour traiter les maladies du foie apparaît : les organes auto-construits. En ingénierie tissulaire du foie, la source de cellules, l’échafaudage décellularisé du foie et les bioréacteurs, sont des facteurs à prendre en compte. L’objectif de ce travail de thèse est d’étudier deux étapes nécessaires au développement d’un foie artificiel : les cellules et la décellularisation de l’organe. Tout d’abord, nous avons prélevé et caractérisé les cellules souches mésenchymateuses de la gelée de wharton (CSMs-GW) CSMs-GW et nous avons étudié leur potentiel de différenciation en hépatocytes. La deuxième étape du travail est consacrée à la décellularisation du foie. Nous avons obtenu des scaffolds acellulaires par la perfusion continue avec du SDS 1% et triton-X100 1%. En conclusion, cette étude montre la capacité de CSM-GW de se différencier en hépatocytes et la faisabilité de la décellularisation du foie. Ceci ouvre des perspectives intéressantes pour le développement d’un foie artificiel et le traitement des pathologies hépatiques / There are over 100 forms of liver diseases caused by various factors and affecting a lot of people. Unfortunately, the only treatment of a terminal liver disease is liver transplantation. However, liver transplantation often fails because of the deficit in human liver donors. Recently, a new innovative alternative for treating end-stage liver disease appears: self-built organ. In liver tissue engineering the source of cells, the decellularized liver scaffold and circular culture bioreactor, are essential factors to be taken into account. The objective of this thesis is to study two steps needed for the development of an artificial liver : cells and organ decellularization. In the first stage, we collected and characterize Wharton’s-Jelly mesenchymal stem cells (WJ-MSCs), and their differentiation potential into hepatocytes. In the second stage of the work, we developed a method for liver decellularization. We were able to get acellular scaffolds by continuous perfusion with 1% SDS and Triton X100 1%. In conclusion, this study shows the capability of WJ-MSC to be differentiated into hepatocytes and the feasibility to obtain acellular livers. That open perspectives toward the development of an artificial liver and the treatment of liver diseases

Le développement du séquençage ARN, ou RNAseq, a permis l'essor de la recherche intensive de biomarqueurs dans de nombreux domaines de la biologie. L’information complète du transcriptome contenue dans les données de sorties, permet à un bioinformaticien assidu de dépasser les connaissances actuelles et d’accéder, grâce à des pipelines informatiques avancés, à d’innombrables signatures d’intérêts inédites. Dans cette thèse nous mettons en avant que ces marqueurs potentiels, essentiellement explorés pour répondre à des problématiques clinique en conditions pathologiques, peuvent être utilisés pour affiner la caractérisation de types de cellules sans marqueurs strictement spécifiques. Nous nous sommes intéressés aux cellules souches mésenchymateuses (MSCs), un type de cellules souches adultes multipotentes, fortement utilisées en clinique mais ne possédant pas de marqueurs positifs strictement spécifiques.Notre étude se concentre sur la recherche des ARN longs non-codants non annotés. Ces ARNs, aussi nommés "lncRNA", constituent une classe émergente de transcrits encore peu explorée à ce jour. De plus, cette catégorie démontre une spécificité conditionnelle et tissulaire élevée. Nous avons élaboré un pipeline d’analyse RNAseq optimisé pour la reconstruction et la quantification de lncRNAs non annotés.En utilisant les données publiques de RNAseq, venant de différentes sources de MSCs et d'autres types de cellules, nous avons identifié de nouveaux lncRNA non annotés exprimés spécifiquement dans les MSCs.Nous avons développé pour ce projet Kmerator.jl, un outil qui permet de décomposer un transcrit en sous séquences spécifiques (k-mers) afin de chercher et quantifier plus rapidement la signature de nos candidats dans un grand nombre de données RNAseq. Kmerator a également été utilisé dans d'autres applications pour tester la qualité des données RNA-seq disponibles en accés public.Après validation de ces nouveaux biomarqueurs de MSCs par qPCR, nous avons eu recours à plusieurs outils informatiques pour prédire leurs fonctions potentielles. Enfin, nous avons analysé des données RNAseq « single-cell » pour aborder l’hétérogénéité d’expression au sein des populations MSCs. / The development of RNA sequencing, or RNAseq, have opened the path of intensive biomarkers research in many areas of biology. The complete information of the transcriptome contained in the output data, allows a bioinformatician to surpass the current knowledge and to access, thanks to advanced computer pipelines, to signatures of new interest. In this thesis, we are showing that these potential markers, classically used in clinical and pathological conditions, can be used to characterize cell types without extensive markers profile. We have studied mesenchymal stem cells, a type of adult multipotent stem cells, strongly used in clinics but without strickly specific positive markers. Our study mainly focuses on the search for non-annotated, long non-coding RNAs. These RNAs, also called "lncRNA", constitute an emerging class of transcripts and are still lightly explored.In addition, this category presents a highly tissue-related specificity. We have developed an optimized RNAseq pipeline for the reconstruction and quantification of non-annotated lncRNAs.Using public data from RNAseq, coming from different sources of MSC and other cell types, we have identified new non-annotated lncRNAs clearly and specifically expressed in MSCs. to complete this project, we developed Kmerator.jl, a bioinformatical tool that allows to decompose a transcript in k-mer, and select specific sub-sequences, in order to search and quantify at a faster rate the signature of our candidates in a large number of RNAseq dataset. After validation of these new biomarkers of MSCs by qPCR, we used several computer tools to predict their potential functions. Finally, we analyzed single-cell RNAseq data to address the heterogeneity of expression within MSC populations.