Células tronco mesenquimais (CTm) apresentam potencial para tratamento da doença renal pela possibilidade de promover regeneração tecidual e recuperação funcional, possivelmente por seus efeitos parácrinos. Na última década, o líquido amniótico foi descrito como uma fonte promissora de extração e isolamento de CTm. Alguns estudos mostraram o efeito renoprotetor das CTm derivadas do líquido amniótico (CTmLA) na doença renal aguda e crônica, quando inoculadas precocemente. Entretanto, ainda não foi estudado o efeito da administração de CTmLA em modelo experimental de doença renal crônica (DRC) com a lesão já estabelecida, situação esta que reproduz melhor a apresentação clínica da doença nos pacientes. Assim, o objetivo do presente estudo foi analisar o efeito da inoculação de CTmLA na região subcapsular renal no modelo de DRC já estabelecido. As CTmLA foram obtidas de pacientes no segundo trimestre de gestação e isoladas através da sua capacidade de aderência ao plástico. A caracterização das CTm foi feita por citometria de fluxo e pela diferenciação celular in vitro. O modelo de DRC utilizado foi o de nefrectomia 5/6 (Nx) que, pela perda de massa renal, evolui com hipertensão arterial, proteinúria, glomeruloesclerose, fibrose intersticial e perda progressiva da função renal. Quinze dias após a indução do modelo, estas alterações já são marcantes e agravam-se com 30 dias. Foram realizados 2 protocolos experimentais: no protocolo I, os animais Nx com DRC estabelecida receberam dose única de CTmLA (5x105) na região subcapsular renal e foram acompanhados por 30 e 60 dias de experimento. No protocolo II, os animais Nx com DRC estabelecida receberam duas doses de CTmLA (5x105) na região subcapsular renal, no 15° e 30° dia após a nefrectomia 5/6, e foram acompanhados por 30 dias, totalizando 60 dias de experimento. Os animais foram subdivididos nos grupos: Sham, ratos submetidos à cirurgia fictícia; Sham+CTmLA, ratos submetidos à Sham que receberam CTmLA; Nx, ratos submetidos à nefrectomia 5/6; Nx+CTmLA, ratos Nx que receberam CTmLA. Para verificar a localização das CTmLA no tecido renal foi realizada a hibridização in situ para cromossomo XY. Foram realizadas análises dos parâmetros clínicos e laboratoriais, além de análise histológica, imunohistoquímica, PCR em tempo real e multiplex. Resultados: as CTmLA cultivadas mostraram grande capacidade de aderência, crescimento em colônia e de diferenciação em células osteogênicas, adipogênicas e condrogênicas. A análise por citometria mostrou-se positiva para CD29, CD44, CD90 e CD105, com uma pequena população de células de CD14, CD34, CD45 e CD117, confirmando a presença preponderante de CTm. Protocolo I: Após 30 dias, a inoculação de CTmLA, dose única, preveniu a elevação da pressão arterial, da proteinúria, da glomeruloesclerose, recuperando a expressão dos marcadores de podócitos, WT-1 e sinaptopodina. Entretanto, não houve efeito benéfico nos níveis de creatinina sérica e na fibrose intersticial, após 30 e 60 dias. O tratamento com CTmLA promoveu uma diminuição marcante do número de macrófagos e uma discreta queda dos leucócitos no infiltrado inflamatório renal, além da diminuição do número de miofibroblastos no interstício renal. Citocinas pró-inflamatórias foram encontradas em menor concentração no tecido renal dos animais que receberam CTmLA (IL-1beta, TNF-alfa, MCP-1 e RANTES). Não houve alteração significativa das citocinas Th1 e Th2, exceto por um aumento da IL-4 nos animais tratados com CTmLA. Os animais que foram acompanhados por 60 dias tiveram uma melhora da proteinúria, da glomeruloesclerose, diminuição do infiltrado de macrófagos e uma melhora da expressão de WT-1. Não foram observadas diferenças estatísticas nos parâmetros de creatinina sérica e fibrose intersticial, aos 30 e 60 dias. Protocolo II: Nos animais que receberam a segunda dose de CTmLA e foram acompanhados por 60 dias observou-se prevenção da elevação da pressão arterial e da proteinúria, além de uma marcante diminuição da fibrose intersticial. Em conclusão, o presente estudo mostrou, pela primeira vez, que a terapia com CTmLA foi capaz de induzir renoproteção nos animais com doença renal crônica estabelecida. O tratamento com CTmLA pode representar uma nova abordagem terapêutica bloqueando a progressão da doença renal crônica / Mesenchymal stem cells (mSC) represent therapeutic potential for the treatment of renal diseases, due to their ability to induce tissue regeneration and functional recovery. Human amniotic fluid stem cells (AFmSC) are a class of fetal, pluripotent stem cells, which present characteristics intermediate between embryonic and adult stem cells. These cells are characterized by the expression of mesenchymal stem cells markers. In addition, they have the ability to differentiate into lineages of all embryonic germ layers. They also show high proliferative rates, but do not induce tumor formation. Therefore, AFmSC are considered to be a very promising cell source and these characteristics have generated a great interest concerning their potential renoprotective effects. The aim of this study was to analyze the effects of AFmSC in an experimental model of chronic kidney disease, the 5/6 nephrectomy model (Nx), after the disease has been established, in order to more closely resemble the clinical settings in humans. AFmSC derived from second-trimester amniocentesis were isolated by plastic adhesion. After 4-7 passages, AFmSC characteristics were confirmed by flow cytometry and by their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages. Two experimental protocols were performed: In protocol I, rats underwent 5/6 nephrectomy (Nx) or sham surgery at day 0, received at day 15 a single dose of hAFmSC (5x105 cells) injected under the renal capsule and were studied at day 30 and 60 days. In protocol II, rats underwent Nx or sham surgery, and received at days 15 and 30, two doses of hAFmSC (5x105 cells) injected under the renal capsule, and were studied at day 60. In both protocols, the animals were subdivided into four groups: Sham, rats submitted to fictitious surgery; Sham+hAFmSC, Sham rats that received hAFmSC; Nx, rats submitted to nephrectomy 5/6; Nx+hAFmSC, Nx rats receiving hAFmSC. The hAFmSC were followed in the renal tissue by in situ hybridization for XY chromosome. In all the groups, clinical and histological parameters were analyzed by immunohistochemistry and real-time PCR. Results: AFmSC cultivated demonstrated an ability to adhere to plastic, to grow in colonies and to differentiate in osteogenic, adipogenic and chondrogenic cells. Quantitative analysis of cell markers by flow cytometry showed that isolated cells were positive for CD29, CD44, CD90 and CD105, with a small population of cells positive for CD14, CD34, CD45 and CD117, confirming a preponderant presence of mSC. Protocol I: After 30 days, the single dose of hAFmSC significantly reduced the blood pressure levels, proteinuria, glomerulosclerosis and improved the expression of podocytes markers, WT-1 and synaptopodin. A marked decrease on the number of macrophages and a discrete decrease of leucocyte infiltration, as well as a reduction of interstitial myofibroblasts was observed. Treatment with hAFmSC significantly reduced some proinflammatory cytokines (IL1beta, TNF-alpha, MCP-1 and RANTES). No significant difference in Th1 or Th2 cytokines was observed, except for IL-4 increase in Nx rats treated with hAFmSC. At 60 days of follow-up, Nx rats treated with hAFmSC presented reduced proteinuria, glomerulosclerosis and macrophages besides increase in WT-1 expression. No improvements were observed on serum creatinine and of interstitial fibrosis, after 30 and 60 days. Protocol II: Inoculation of two doses of hAFmSC in Nx rats improved blood pressure levels, proteinuria and interstitial fibrosis at day 60. In conclusion, the present study demonstrated, for the first time, that hAFmSC induced renoprotection in animals with established chronic kidney disease. Treatment with hAFmSC may represent a novel therapeutic approach for blocking the progression of chronic kidney disease

O uso de células-tronco mesenquimais (CTMs) na medicina regenerativa, principalmente quando associado ao sistema nervoso, requer alternativas em relação à via de aplicação. A associação da terapia celular com a nanotecnologia para uso em neurociências, desenvolvida nesse trabalho, é uma abordagem inovadora no Brasil. Dessa forma, as matrizes de nanofibras, produzidas pela técnica de electrospinning (ES), funcionam como suportes para a proliferação e diferenciação celular proporcionando uma alternativa para a reconstituição do tecido lesado. O processo de regeneração do tecido neural pode ser aperfeiçoado com a liberação controlada de fatores neurotróficos, através do uso dessas matrizes. Entre esses fatores, encontra-se o NGF (Nerve Growth Factor – fator de crescimento neural), o qual exerce um papel central no desenvolvimento, manutenção e sobrevivência dos neurônios. Além disso, características de superfície das matrizes, como o alinhamento de nanofibras, podem estimular a diferencição neural. O objetivo principal deste trabalho foi desenvolver matrizes de nanofibras alinhadas e não alinhadas com e sem o NGF incorporado, através da técnica ES de emulsão. Além disso, objetivou-se avaliar o comportamento celular, bem como a capacidade de diferenciação neural das CTMs, sobre as estruturas tridimensionais desenvolvidas. As CTMs foram extraídas da polpa de dentes decíduos esfoliados humanos. Quatro grupos de scaffolds foram desenvolvidos, caracterizados e avaliados: scaffolds com fibras randomizadas e com fibras alinhadas, sendo cada tipo com e sem o NGF incorporado. As análises físico-químicas realizadas foram morfologia, diâmetro das fibras e degradabilidade do biomaterial. Os parâmetros biológicos avaliados foram morfologia, adesão, viabilidade e proliferação celular, bem como a citotoxicidade frente ao biomaterial. A diferenciação neural foi quantificada através da expressão dos genes neurais nestina, β- III tubulina e NSE (enolase específica para neurônios). As matrizes de nanofibras produzidas mostraram-se satisfatórias para o cultivo de CTMs, mimetizando a estrutura física da matriz extracelular (MEC). Além disso, a técnica utilizada permitiu a obtenção de estruturas com nanofibras alinhadas e randomizadas. As CTMs cultivadas nas matrizes foram capazes de aderir e proliferar com vantagens para adesão nas matrizes alinhadas contendo o NGF, em relação às matrizes alinhadas controle. As estruturas produzidas não apresentaram características tóxicas permitindo que as CTMs mantivessem a viabilidade ao longo do tempo. A avaliação da diferenciação neural das CTMs indicou que todos os grupos de matrizes foram capazes de promover o aumento da expressão de genes neurais. Tal capacidade foi observada tanto para CTMs cultivadas sobre as matrizes com o meio controle quanto com o meio de indução neural. Esses achados mostram a possível influência das características químicas e topográficas providas pelos substratos produzidos. As características da matriz artificial permitem que as CTMs respondam adequadamente ao microambiente e expressem genes neurais, podendo auxiliar na regeneração tecidual quando aplicada em lesões do sistema nervoso. / The use of mesenchymal stem cells (MSCs) in regenerative medicine, particularly when associated with the nervous system, requires alternatives with respect to cell application methods. The association of cellular therapy with nanotechnology for use in neuroscience, developed with this work, is an innovative approach in Brazil. Scaffolds produced by electrospinning (ES) technique act as supports for cell proliferation and differentiation, providing an alternative to reconstitute the damaged tissue. The process of neural tissue regeneration can be improved through the controlled release of neurotrophic factors from the scaffolds. Among these factors, NGF (Nerve Growth Factor) plays a central role in the development, maintenance and survival of neurons. Furthermore, surface characteristics of nanofibers, such as alignment, can stimulate neural differentiation. The main objective of this study was to develop aligned nanofiber scaffolds and random nanofiber scaffolds with and without NGF incorporated through emulsion ES. In addition it was aimed to characterize the physico-chemical properties of the scaffolds, related to the extracellular matrix (ECM) and evaluate the cell behavior, as well as the neural differentiation on these three-dimensional devices. The MSCs were extracted from the dental pulp of human exfoliated deciduous teeth. Four groups of scaffolds were developed, characterized and evaluated: scaffolds with randomized fibers and with aligned fibers, each type with and without NGF incorporated. The physico-chemical analyzes performed were morphology, fiber diameter and degradability of the biomaterial. The biological parameters evaluated were cell morphology, adhesion, proliferation and viability, as well as cytotoxicity by the biomaterial. The neural differentiation was quantified by measuring gene expression for the neural genes nestin, β-III tubulin and NSE (neuron-specific enolase). The scaffolds produced demonstrated a satisfactory environment for MSC growth, mimicking the ECM physical structure. Furthermore, the technique allowed for the production of scaffolds with aligned and with randomized nanofibers. MSCs cultured on scaffolds were able to adhere and proliferate, with better adhesion performance on aligned nanofiber scaffolds with NGF incorporated, when compared to aligned nanofiber scaffolds control. The devices produced showed nontoxic characteristics permitting MSCs to maintain their viability over time. The evaluation of MSC neural differentiation indicated that all groups of scaffolds were able to upregulate neural genes expression. Such ability was observed for both MSCs cultured on scaffolds with control medium as on scaffolds under neural induction medium. These features provided by this artificial ECM permit proper MSC response to microenvironment, leading to neuronal genes expression, which could improve tissue regeneration when applied to nerve lesions.

Danos ao sistema nervoso central (SCN) resultam em perda de conexões axonais, das funções motoras e sensoriais. Uma das estratégias para seu reparo é o transplante de células-tronco mesenquimais (CTMs). Porém essa alternativa requer uma adequada via de aplicação. Nesse sentido, o uso de matrizes alinhadas pode ser usado para apoiar o crescimento e diferenciação das CTMs e, quando incorporadas com fatores de crescimento, otimizam o processo de regeneração tecidual. O objetivo desse trabalho foi avaliar a diferenciação neural das CTMs cultivadas sobre matrizes de nanofibras orientadas com o fator de crescimento epidermal (EGF) incorporado. Os scaffolds com fibras alinhadas foram produzidos por electrospinning de emulsão e avaliados conforme a sua morfologia, o diâmetro das nanofibras, a degradabilidade e a liberação do EGF. As CTMs utilizadas foram provenientes da polpa de dentes decíduos esfoliados humanos. Essas células foram cultivadas nos scaffolds e avaliadas conforme os testes biológicos: adesão, viabilidade, proliferação, citotoxicidade e diferenciação neural. Os scaffolds com fibras alinhadas controle (AC) e contendo o EGF (AE) apresentaram morfologia, diâmetro das nanofibras e tempo de degradação semelhantes. Com base no total de EGF presente na matriz AE, 90,14% foi liberado após 28 dias. O citoesqueleto e o núcleo das CTMs cultivadas nos scaffolds AC e AE estavam mais alongados e alinhados quando comparado com as CTMs cultivadas no poço de cultura (controle). As CTMs aderiram mais nas matrizes AE em relação às matrizes AC, porém a proliferação e viabilidade celular foram similares, exceto no tempo de 72 horas, o qual a viabilidade no grupo controle foi maior, em comparação aos demais grupos. Os scaffolds AC e AE não foram tóxicos para as CTMs. Em relação aos resultados da neuro-diferenciação, a expressão de nestina e neurofilamentos consideravelmente maior em todos os grupos analisados quando comparado ao grupo controle. A expressão de βIII-tubulina e GFAP foi maior em todos os grupos diferenciados quando comparada ao grupo controle. A maioria das CTMs cultivadas nas matrizes AC e AE, induzidas ou não à diferenciação neural, apresentaram correntes dependente de voltagem para sódio. O valor de condutância máxima foi maior para todos os grupos analisados quando comparado ao grupo controle onde as células não foram diferenciadas. Portanto, as matrizes com nanofibras orientadas induzem à diferenciação neural das CTMs em neurônios funcionais tanto na ausência como na presença de EGF incorporado. As matrizes AE ainda mostraram ser capazes de melhorar a adesão celular. Dessa forma, conclui-se que as matrizes de nanofibras estudadas são uma possível estratégia para otimização da regeneração de lesões neurológicas. / Damage to the central nervous system (CNS) results in loss of axonal connections and motor and sensory functions. One of the strategies for its repair is the transplantation of mesenchymal stem cells (MSCs). However, this requires a suitable application route. Accordingly, the use of scaffolds support the growth of MSCs and, when incorporated with growth factors, optimize the regeneration process. The purpose of this study was to evaluate the neural differentiation of MSCs cultured on nanofiber matrices oriented with epidermal growth factor (EGF) incorporated. Aligned scaffolds were produced by electrospinning emulsion and evaluated according to their degradation, the morphology and diameter of the nanofibers, and release of EGF from the nanofibers. MSCs used were from human exfoliated deciduous teeth (SHED). These cells were cultured on the scaffolds and evaluated according to biological tests: adhesion, viability, proliferation, cytotoxicity and neural differentiation. The aligned control scaffolds (AC) containing EGF (AE) presented similar morphology, diameter of nanofibers and degradation time. Based on the total EGF present in the scaffold AE, 90.14% was released after 28 days. The cytoskeleton and the core of the MSCs cultured on scaffolds AC and AE were more aligned and elongated when compared to the MSCs grown on plate wells (control). MSCs adhered more to matrices AE when compared to matrices AC, although proliferation and cell viability were similar, except after 72 hours. In this period, the viability of the control group was higher when compared to the rest of the groups. Scaffolds AC and AE were not toxic to MSCs. In regard to the results of neuro-differentiation, the expression of nestin and neurofilament was much higher in all groups than the control group. The expression of βIII tublin and GFAP was higher in all differentiated groups than the control group. Most of the MSCs grown in matrices AC and AE, induced or not to neural differentiation, showed voltage-dependent sodium currents. The maximum value of conductance of these groups was higher for the cells in all groupscompared to the control group, where the cells were not differentiated. Therefore, oriented nanofiber matrices induce neural differentiation of MSCs into functional neurons both in the absence and in the presence of incorporated EGF. The matrices AE also showed improved cell adhesion. Thus, these matrices are a possible strategy for optimizing the regeneration of neurologic lesions.

Les maladies cardiovasculaires constituent un problème de santé publique. Les pré et postconditionnement ischémiques mais aussi pharmacologiques constituent autant d‘avancées qui permettront l‘amélioration de la prise en charge des malades en situation d‘ischémie/reperfusion myocardique. Néanmoins, la morbi-mortalité des maladies cardiovasculaires reste importante et nécessite le développement de nouvelles techniques. Les premiers résultats de la thérapie cellulaire myocardique ont été décevants, et s‘il est désormais établi que l‘on ne peut régénérer le myocarde, les effets bénéfiques observés, notamment avec les cellules souches mésenchymateuses semblent en rapport avec un effet paracrine qui passe par l‘activation de la voie de signalisation PI3kinase/Akt sur un mécanisme comparable à celui du postconditionnement ischémique. Les médiateurs de cet effet sont vraisemblablement des facteurs de croissance comme le VEGF ou l‘IGF-1 même si un effet individuel direct de l‘une ou l‘autre de ces molécules n‘a pu être mis en évidence. La modulation de l‘activité de l‘ATP synthase mitochondriale est également une cible thérapeutique prometteuse. Cette enzyme inverse son activité et hydrolyse l‘ATP durant l‘ischémie, conduisant à dépléter le pool d‘ATP intracellulaire et accélérer la survenue de la mort cellulaire. De nouvelles molécules ayant un effet similaire à l‘IF1 permettent de bloquer cette inversion d‘activité de l‘ATP synthase, de préserver l‘ATP et donc d‘améliorer la survie cellulaire par un effet de type préconditionnement ischémique. Ces 2 techniques, très différentes mais non antinomiques, pourraient faire partie de l‘arsenal thérapeutique dans les années à venir / Cardiovascular diseases are a major problem of public health management. Ischemic and pharmacological pre and postconditioning should significantly improve the prognosis of patients suffering from myocardial ischemia/reperfusion. However, the morbi-mortality of these patients is still high and research must remain active. The first results of myocardial stem cell therapy show that we cannot regenerate myocardium but a recent meta-analysis reported positive effects that can be explained through a paracrine mechanism. Mesenchymal stem cells protect ischemic cardiomyocytes from reperfusion injury through a paracrine activation of the PI3kinase/Akt pathway in a similar way to ischemic postconditioning. The mediators of this protection could be growth factors such as VEGF or IGF-1 though we couldn’t demonstrate a direct effect of one or the other. Modulating the activity of the ATP synthase during ischemia is another promising therapeutic target. This enzyme reverses its activity and hydrolyses ATP when the supply in oxygen is impaired. This leads to the reduction of the cellular pool of ATP and accelerates cell death. We identified new small molecules with a similar effect to IF1 that can selectively inhibit the reverse activity of the ATP synthase, preserve ATP and thus increase cell survival in a preconditioning-like effect. These two different techniques could be part of the therapeutic arsenal against ischemia/reperfusion in the next decades.

La cellule répond aux contraintes physiques exercées par son environnement par un ensemble de mécanismes regroupés sous le terme de mécanotransduction. Ces processus font appel aux molécules impliquées dans l’adhésion cellulaire, au cytosquelette et au noyau. Ces contraintes environnementales, qu’elles soient liées à la rigidité du support, à sa topographie ou à la nature de sa chimie de surface, vont moduler la morphologie cellulaire et impacter le comportement de la cellule. Afin d’étudier cette influence du support, nous avons ensemencé des cellules souches mésenchymateuses (CSMs) de moelle osseuse (MO) issues d’une culture primaire sur des surfaces de mica vierges ou fonctionnalisées de façon homogène avec des molécules naturelles (la fibronectine FN et le peptide RGD cyclique) ou avec des multicouches de polyélectrolytes PEM (cinq cycles de Chitosan/PAA ou de Chitosan/PSS). Nous avons ensuite étudié la morphologie, la prolifération et la différenciation de ces cellules après 12 jours de culture. Il en résulte que les CSMs de MO adhèrent sur toutes les surfaces, traitées ou non, et bien que leur étalement soit moindre sur les surfaces vierges, elles adoptent une morphologie de type fibroblastique similaire à leur phénotype physiologique. Leur pourcentage de confluence varie significativement en fonction du traitement de surface utilisé. En effet la confluence maximale a été observée pour les surfaces greffées avec la FN (93.25 ± 2.75 %) alors que les surfaces traitées avec les PEM présentent des pourcentages de confluence bien plus faibles (61.00 ± 4.08 % pour le couple chitosan/PAA et 54.75 ± 1.75 % pour le couple Chitosan/PSS), s’expliquant principalement par une latence cellulaire en début de culture. Enfin, les cellules cultivées sur nos surfaces ne réagissent à aucune des trois colorations Oil Red O, Alcian Blue ou Alizarin Red S, suggérant une absence de différenciation dans les voies adipogénique, chondrogénique ou ostéogénique induite par ces surfaces. Ainsi, le contrôle de la chimie du support ne permet pas à lui seul un contrôle de la différenciation cellulaire. Cette étude ouvre la voie à l’étape suivante au cours de laquelle l’influence des supports à chimie et géométrie contrôlées. De même, la souche E.coli (bactérie pathogène) répond aux contraintes physiques et chimiques qui lui ont été imposées. Ces contraintes qu’elles soient liées à la topographie ou la nature de la chimie de surface font appel à des molécules naturelles impliquées dans le comportement des bactéries et leur morphologie en particulier sur leur taille. Pour étudier cet impact, nous avons mis en contact la souche E.coli E2146 avec des surfaces de mica vierges ou traitées de façon homogène ou patternée avec des molécules naturelles (la FN et le peptide RGD cyclique). Ensuite, nous avons étudié le taux de recouvrement et la taille des bactéries. Il en résulte que les bactéries adhèrent sur l’ensemble des surfaces bien que l’adhésion soit moindre sur les surfaces de mica vierges. Leur taux de recouvrement varie significativement pour une surface donnée. En effet, le taux de recouvrement et la taille maximaux sont observés sur des surfaces patternées greffées avec la FN, ce qui prouve leur efficacité et l’impact qu’elles ont sur le comportement de E.coli. Nous avons donc démontré dans ce travail de thèse l’influence des propriétés de surfaces sur la croissance de cellules vivantes telles que les cellules souches ou les bactéries. / The cell responds to the physical constraints exerted by its environment by a set of mechanisms grouped under the term of mechanotransduction. These processes involve the molecules involved in cell adhesion, the cytoskeleton and the nucleus. These environmental constraints, whether related to the rigidity of the support, to its topography or to the nature of its surface chemistry, will modulate the cellular morphology and impact the behavior of the cell. In order to study this influence of the support, we have seeded bone marrow mesenchymal stem cells from a primary culture on virgin mica surfaces or functionalized homogeneously with natural molecules (fibronectin and the cyclic RGD peptide) or with polyelectrolyte multilayers (five cycles of Chitosan/PAA or Chitosan/PSS). We then studied the morphology, proliferation and differentiation of these cells after 12 days of culture. As a result, bone marrow mesenchymal stem cells adhere to all surfaces, whether treated or not, and although they are less spread on virgin surfaces, they adopt a fibroblastic type morphology similar to their physiological phenotype. Their percentage of confluence varies significantly depending on the surface treatment used. Indeed the maximum confluence was observed for the surfaces grafted with fibronectin (93.25 ± 2.75%) whereas the surfaces treated with the polyelectrolyte multilayers have much lower confluence percentages (61.00 ± 4.08% for the chitosan/PAA couple) and 54.75 ± 1.75% for the Chitosan/PSS couple), mainly due to cell latency at the beginning of culture. Finally, cells cultured on our surfaces do not respond to any of the three Oil Red O, Alcian Blue or Alizarin Red S stains, suggesting a lack of differentiation in the adipogenic, chondrogenic or osteogenic pathways induced by these surfaces. Thus, the control of the support chemistry alone does not allow control of cell differentiation. This study paves the way for the next step in which the influence of controlled chemistry and geometry media will be studied. Similarly, the E. coli strain (pathogenic bacterium) responds to the physical and chemical constraints imposed on it. These constraints, whether related to the topography or the nature of surface chemistry, involve natural molecules involved in the behavior of bacteria and their morphology, in particular their size. To study this impact, we contacted E.coli strain E2146 with virgin mica surfaces or treated homogeneously or patterned with natural molecules (fibronectin and cyclic RGD peptide). Then we studied the recovery rate and the size of the bacteria. As a result, the bacteria adhere to all surfaces although adhesion is less on virgin mica surfaces. Their recovery rate varies significantly for a given area. Indeed, the recovery rate and the maximum size are observed on patterned surfaces grafted with fibronectin which proves their effectiveness and the impact they have on the behavior of E. coli. We have therefore demonstrated in this thesis the influence of surface properties on the growth of living cells such as stem cells or bacteria.

Le tissu adipeux, longtemps considéré comme réservoir énergétique et tissu de soutien mécanique a été promu au rang d'organe endocrine car sécréteur d'hormones. Il est utilisé depuis une trentaine d'année en chirurgie plastique cosmétique et réparatrice comme agent de comblement dans le cadre de lipoatrophie, de reconstruction mammaire ou de liftings. Ses effets régénérateurs observés sont attribués à la présence de cellules souches mésenchymateuses (CSM), les ASC (adipose-derived stem/stromal cells) assimilables aux CSM de la moelle osseuse. Leur auto renouvèlement et multipotentialité sont exploités en médecine régénérative. Nous nous sommes intéressés à la cicatrisation des plaies difficiles. Le premier article porte sur la sélection de différents dispositifs de prélèvement et d'extraction des ASC humaines à partir de lipoaspirat. Par rapport à la méthode de référence et quel que soit le dispositif, les cellules extraites ne montrent pas de résultats significativement différents en terme de rendement d'extraction et viabilité cellulaire, de capacité proliférative (clonogénicité, doublement de population et temps de doublement), de phénotype (marqueurs CD90, CD73, HLA-ABC CD45, CD14 et HLA-DR par cytométrie en flux), de stabilité génétique (caryotype et expression du gène codant pour hTERT) et de multipotentialité (différenciation adipo-, ostéo- et chondrogénique). Dans nos mains, le dispositif GID-SVF1 s'est montré le plus pratique tant au niveau du prélèvement que de son utilisation. Le second article démontre biocompatibilité et la biodisponibilité du produit fini (ASC dans acide hyaluronique) in vitro et sa tolérance chez la souris nude. Le troisième article confirme sa tolérance et établit son efficacité chez la souris nude sur de large plaie excisionnelle utilisant le modèle de Galiano. En effet, la cicatrisation complète est plus rapide pour les souris traitées avec les ASC (14 jours contre 21 jours pour les souris non traitées, p<0.001) avec une meilleure vascularisation du tissu cicatriciel (immunomarquage CD31, iontophorèse couplée au laser Doppler). Ce travail complète les prérequis aux essais cliniques en validant le prélèvement grâce à des dispositifs permettant la digestion des lipoaspirats et l'obtention de la SVF. L'amélioration significative de la cicatrisation dans notre modèle de plaie valide non seulement l'efficacité des ASC mais aussi leur véhicule / Adipose tissue, for a while considered as an energy reservoir and a mechanical support tissue, is nowadays recognized as an endocrine organ. It is used for more than thirty years in cosmetic and reconstructive plastic surgery as filler for lipodystrophy, breast reconstruction or lifting. Its regenerative potential is attributed to mesenchymal stem cells (MSC), the so-called ASC (Adipose-derived stem/stromal cells) similar to MSC from bone marrow. Their self-renewing capacity and their multupotency are exploited in regenerative medicine. We are interested in chronic wound healing. The first article deals with several devices for the harvesting of fat and the isolation of human ASC from lipoaspirates. When compared to the reference method and whatever the device, the extracted cells do not show any statistical differences in terms of cell yield and viability, proliferative capacity (clonogenicity, population doubling and doubling time), phenotype (CD90, CD73, HLA-ABC CD45, CD14 et HLA-DR using flow cytometry), genetic stability (karyotype and hTERT gene expression) and multipotentiality (adipo-, osteo- et chondrogenic differentiation). In our hands, GID SVF-1TM device was the most easy to handle for the harvest and also for its use. The second article demonstrates the biocompatibility and bioavailability of our final product (ASC in hyaluronic acid) in vitro and its tolerance in nude mice. The third article reinforces its tolerance and establishes its effectiveness in nude mice on large excisional wounds using Galiano model. Indeed, complete wound healing is more rapid in ASC-treated mice (14 days versus 21 days in untreated mice, p<0.001) with a better healed tissue vascularization (CD31 immunostaining, Dopplercoupled iontophoresis). This work fulfills the requirements for clinical trials in validating the harvest of lipoaspirate using devices that allow fat digestion and SVF extraction. The significant improvement of wound healing in our model validates not only ASC effectiveness but also their vehicle

INTRODUÇÃO O câncer é uma das principais causas de morte no mundo, sendo responsável por cerca de 8 milhões de mortes por ano, segundo dados da OMS. As mortes por câncer são provocadas por tumores que se originam em órgãos como pulmão, fígado, estômago, intestino, mama e esôfago. As células-tronco mesenquimais (CTM) foram identificadas em vários órgãos e estudos da sua interação com células tumorais têm apresentado resultados indicando ação inibitória sobre alguns tipos de tumores. Para explorar essa questão foram analisados os efeitos sobre células tumorais de carcinoma hepatocelular humano (HepG-2) do meio condicionado (MC) obtido do cultivo de CTM isoladas de tecido adiposo (TA), líquido amniótico (LA) e geleia de Wharton (GW). MÉTODOS Os MCs foram coletados após 24 horas de incubação das CTM sub-confluentes com ?-MEM contendo 20% de soro fetal bovino (SFB). Os MCs foram centrifugados e passados através de filtros de 0,22 ?M e armazenadas a -20 °C. O MC da própria célula HepG-2 foi utilizado como controle. Os efeitos dos MCs sobre a proliferação de células HepG-2 foram testados por ensaio MTT, em várias concentrações após 24 h de incubação. O ciclo celular de células HepG-2 tratadas com MC a 25%, 50% ou 75% foi analisado por citometria de fluxo (coloração IP) utilizando o software Modfit LT. A expressão dos genes bcl-2, bcl-6, CCND1 foi analisada por RT-PCR. A proliferação celular foi avaliada pela expressão das proteínas survivina, Bcl-2, PCNA e Ki-67 e pela quantificação de mitocôndrias com corante MitoTracker, assim como pelo potencial de membrana mitocondrial por corante JC-1 Mitoscreen utilizando equipamento de high content analysis. RESULTADOS Os meios condicionados de células-tronco de tecido adiposo (MC-TA) não alteraram a proliferação de células tumorais HepG-2 e os meios condicionados de células-tronco de líquido amniótico (MC-LA) e de geleia de Wharton (MC-GW) provocam aumento da proliferação, confirmada pela contagem de células com núcleos corados com Hoescht 33342. A análise de alterações do ciclo celular demonstrou que a exposição de células HepG-2 aos meios MC-LA diminuem as células na fase G0/G1 e aumentam na fase G2/M do ciclo celular. A expressão dos genes CCND1, Bcl2 e Bcl6 que estão relacionados à proliferação e morte celular não apresentaram alteração. A quantificação das proteínas PCNA e survivina não apresentou alteração sob efeito dos MC, porém a comparação direta entre células tratadas com MC-LA e MC-TA indicou a tendência das células tratadas com MC-LA proliferarem. O percentual de células HepG-2 expressando a proteína Ki-67 foi significativamente menor em relação ao controle quando tratadas com MC-TA, não apresentando diferenças quando tratadas com MC-LA e MC-GW. A contagem de mitocôndrias evidenciou aumento de mitocôndrias nas células HepG-2 tratadas com MC-LA e efeito não significativo dos tratamentos com MC-TA e MC-GW. A diferença do potencial de membrana mitocondrial por JC-1 apresentou aumento da polarização nas células HepG-2 cultivadas com MC-TA. CONCLUSÃO Os resultados confirmam que as células-tronco mesenquimais diferem de acordo com a sua origem tecidual em sua ação na proliferação das células HepG-2. Mais estudos são necessários para estabelecer a causa destas ações, que não parecem ser devido a mediadores mais comuns na proliferação e morte celular / INTRODUCTION Cancer is a leading cause of death worldwide, accounting for about 8 million deaths per year, according to WHO data. Cancer deaths are caused by tumors that originate in organs such as lung, liver, stomach, bowel, breast and esophagus. The mesenchymal stem cells (MSCs) have been identified in many organs and studies of their interaction with tumor cells have shown results indicating an inhibitory effect on some types of tumors. To explore this question the effects of the conditioned medium (CM) obtained from mesenchymal stem cell isolated from adipose tissue (AT), amniotic fluid (AF) and Wharton jelly (WJ) on tumor cells of human hepatocellular carcinoma (HepG-2) were analyzed. METHODS The MSC CM was collected after 24 hours incubation of sub confluent MSC with ?-MEM containing 20% fetal bovine serum (FBS). The MSC CM were centrifuged and passed through 0.22 ?M filter and stored at -20° C. The CM of HepG-2 cell itself was used as control. The effects of contrast media on proliferation of HepG-2 cells were tested by MTT assay at various concentrations after 24 h of incubation. The cell cycle HepG-2 cells treated with CM at 25%, 50% or 75% was analyzed by flow cytometry (PI staining) using Modfit software LT. The expression of the genes Bcl-2, Bcl-6, CCND1 was analyzed by RT-PCR. Cell proliferation was assessed by the expression of survivin, Bcl-2, Ki-67 and PCNA proteins, and the quantization of mitochondrial by MitoTracker dye, as well as the mitochondrial membrane potential by JC-1 Mitoscreen dye using high content analysis equipment. RESULTS The conditioned media of mesenchymal stem cells (MSC) from adipose tissue (AT-CM) did not alter the proliferation of tumor HepG-2 cells and conditioned media of MSC cells from amniotic fluid (AF-CM) and Wharton jelly (WJ-CM) caused increased proliferation, confirmed by counting cells with nuclei stained with Hoechst 33342. The cell cycle analysis showed that exposure of HepG-2 cells to AF-CM means decrease the cells in G0 / G1 cell cycle phase and increase in phase G2 / M. The expression of Bcl2, Bcl6 and CCND1 genes that are related to proliferation and cell death did not change. The quantization of PCNA and survivin protein did not change under the effect of conditioned media, but a direct comparison between cells treated with AF-CM and AT-CM indicated the tendency of cells treated with AF-CM proliferate. The percentage of HepG-2 cells expressing the protein Ki-67 was significantly lower than the control when treated with AT-CM and no differences when treated with AF-CM and WJ-CM. The counting of mitochondria showed increased mitochondria in HepG-2 cells treated with AF-CM and no significant effect of treatment with WJ-CM and AT-CM. The difference in mitochondrial membrane potential by JC-1 showed an increase in polarization in HepG-2 cells cultured with AT-CM. CONCLUSION The results confirm that mesenchymal stem cells differ according to their tissue origin in its action on the proliferation of HepG-2 cells. More studies are needed to establish the cause of these actions, which seem to be not related to the most common mediators in cell proliferation and death

A cartilagem articular, por sua natureza avascular, apresenta uma capacidade limitada de regeneração. Uma abordagem terapêutica para o tratamento de defeitos da cartilagem consiste na utilização de células ou tecidos aplicados ao local da lesão. O objetivo deste estudo foi avaliar o efeito da aplicação de centrifugado de medula óssea autóloga em lesões osteocondrais no joelho de coelhos, em comparação com um grupo controle de lesões osteocondrais sem preenchimento, analisando o comportamento histológico destes grupos em função do tempo. Foram utilizados doze coelhos da raça Nova Zelândia, albinos, machos, adultos, submetidos a uma lesão osteocondral, de 4 mm de diâmetro e 3 mm de profundidade, em ambos os joelhos, na região da tróclea femoral. Nos joelhos direitos, que constituíram o Grupo Estudo, o defeito osteocondral foi preenchido por um coágulo de células mesenquimais, obtidas por centrifugação de um aspirado da medula óssea e selado com cola de fibrina. Nos joelhos esquerdos, que constituíram o Grupo Controle, o defeito osteocondral não recebeu qualquer preenchimento. Os animais foram divididos em três grupos de quatro coelhos, estudados após oito, 16 e 24 semanas. Os resultados foram descritos com base em uma escala de pontuação histológica que avaliou a morfologia celular, a reconstrução do osso subcondral, o aspecto da matriz, o preenchimento do defeito, a regularidade da superfície e a conexão das margens. A análise estatística foi realizada pelo Teste t-student para dados pareados na comparação entre Grupo Estudo e Grupo Controle. Para as comparações através do fator temporal, utilizou-se o Teste ANOVA one way. Com 5% de confiança, rejeitou-se a hipótese de igualdade entre os Grupos Estudo e Controle. Notou-se uma distância decrescente entre os escores dos Grupos Estudo e Controle com o aumento do tempo, bem como uma tendência crescente do valor da escala para o Grupo Controle. Concluiu-se que a aplicação de centrifugado de medula óssea em defeitos osteocondrais no joelho de coelhos mostrou melhor resultado na avaliação histológica, em comparação ao Grupo Controle. Analisando a evolução dos grupos através do tempo, houve uma aproximação de seus escores histológicos, sobretudo pelo aumento observado no Grupo Controle / The articular cartilage, due to its avascular nature, presents a limited regeneration capacity. A therapeutical approach to the treatment of cartilage defects consists of the utilization of cells or tissues applied to the lesion site. The aim of this study was to evaluate the effect of applying autologous bone marrow centrifuged in osteochondral lesions in the knees of rabbits, compared to a control group of osteochondral lesions without any filling, analyzing the behavior of these groups in terms of time. Twelve adult albino male New Zealand rabbits were used being submitted to an osteochondral lesion of 4 mm in diameter and 3 mm deep in both knees, at the femoral trochlea area. On the right knees, which comprised the Study Group, the osteochondral defect was filled by a clot of mesenchymal cells, obtained by centrifugation of an aspirate from bone marrow and sealed with fibrin glue. On the left knees, which comprised the Control Group, the osteochondral defect did not get any filling. The animals were divided into 3 groups of 4 rabbits, and studied after eight, 16 and 24 weeks. The results were described based on a histological grading scale which took into account the cell morphology, the subchondral bone reconstruction, the matrix staining, the filling of the defect, the surface regularity and the bonding of the edges. The statistical analysis was made by the t-student Test for paired data in the comparison between the Study Group and the Control Group. For the comparisons made by the time factor, it was used the ANOVA Test one way. With 5% level of confidence, the hypothesis of equality between the Study and Control Groups was rejected. It was observed a decreasing distance between scores of the Study and Control Groups as time increased, as well as an increasing tendency of the scale value for the Control Group. It was concluded that the application of autologous bone marrow centrifuged in osteochondral defects in the knees of rabbits showed better result in histological evaluation, in comparison to the Control Group. By analyzing the evolution of the groups through time, there was an approach of their histological scores, especially by the increase observed in the Control Group

Les nanotopographies de surface présentant des dimensions comparables à celles des éléments de la matrice extracellulaire offrent la possibilité de réguler le comportement cellulaire. L’étude de l’impact de la nanotopographie sur la réponse cellulaire a été toujours limitée compte tenu des précisions limitées sur les géométries produites, en particulier sur les plus grandes surfaces. Des matériaux base silicium présentant des nanopiliers avec des géométries parfaitement contrôlées ont été fabriqués et leur impact sur la différentiation ostéogénique de cellules souches mésenchymateuses humaines (hMCSs) a été étudié. Des matériaux avec des nanopiliers de dimensions critiques comprises entre 40 et 200 nm et des écarts types inférieurs à 15% sur un wafer de silicium, ont été réalisés en profitant de la capacité d’auto-assemblage des copolymères à blocs. Pour mettre en évidence si des modifications de la chimie de la surface des nanopiliers pourraient favoriser la différenciation des MSCs, des peptides mimétiques ont été greffés sur les matériaux fabriqués. Un peptide connu pour sa capacité d’améliorer l'adhésion cellulaire (peptide RGD), un peptide synthétique capable d'améliorer l'ostéogenèse (peptide mimétique BMP-2) et une combinaison de ces deux peptides ont été immobilisés de manière covalente sur les matériaux silicium présentant des nanopiliers de différentes géométries (diamètre, espacement et hauteur).Les essais d'immunofluorescence et de réaction en chaîne de la polymérase quantitative (RT-qPCR) révèlent un impact des nanotopographies sur la différenciation ostéogénique des hMSCs. De plus, il a été constaté que la différenciation des cellules dépendait de l'âge du donneur. La fonctionnalisation de surface a permis une augmentation supplémentaire de l'expression des marqueurs ostéogéniques, en particulier lorsque le peptide RGD et le peptide mimétique BMP-2 sont co-immobilisés en surface. Cette étude met clairement en évidence l’impact de nanostructures avec différentes bioactivités sur la différentiation de MSCs. Ces matériaux pourront trouver leur place dans des cultures in vitro, dans l’élaboration de nouveaux biomatériaux osseux et dans de nouveaux produits d’ingénierie tissulaire. / Nanotopography with length scales of the order of extracellular matrix elements offers the possibility of regulating cell behavior. Investigation of the impact of nanotopography on cell response has been limited by inability to precisely control geometries, especially at high spatial resolutions, and across practically large areas. This work allowed the fabrication of well-controlled and periodic nanopillar arrays of silicon to investigate their impact on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Silicon nanopillar arrays with critical dimensions in the range of 40-200 nm, exhibiting standard deviations below 15% across full wafers were realized using self-assembly of block copolymer colloids. To investigate if modifications of surface chemistry could further improve the modulation of hMSC differentiation, mimetic peptides were grafted on the fabricated nanoarrays. A peptide known for its ability to ameliorate cell adhesion (RGD peptide), a synthetic peptide able to enhance osteogenesis (BMP-2 mimetic peptide), and a combination or both molecules were covalently grafted on the nanostructures.Immunofluorescence and quantitative polymerase chain reaction (RT-qPCR) measurements reveal clear dependence of osteogenic differentiation of hMSCs on the diameter and periodicity of the arrays. Moreover, the differentiation of hMSCs was found to be dependent on the age of the donor. Surface functionalization allowed additional enhancement of the expression of osteogenic markers, in particular when RGD peptide and BMP-2 mimetic peptide were co-immobilized. These findings can contribute for the development of personalized treatments of bone diseases, namely novel implant nanostructuring depending on patient age.

Poly (glycerol-sebacate) (PGS) is a synthetic bioelastomer with a covalently crosslinked, three-dimensional network of random coils with hydroxyl groups attached to its backbone. This biodegradable polymer is biocompatible (in vitro and in vivo), tough, elastic, inexpensive, and flexible, and finds potential applications in tissue engineering and regenerative medicine. Due to the slow rate of step-growth polymerisation, the synthesis of PGS prepolymer requires 24-48 h. A batch and a continuous process, if developed, could address the inherent deficiencies (eg. long residence time, venting) associated with the large-scale synthesis of such bioelastomers. However, in order to assess whether this particular system may be adapted to continuous processes, such as reactive extrusion, studies on kinetics of controlled condensation reactions are of vital importance. FT-Raman spectroscopy was used to study the kinetics of the step-growth reactions between glycerol (G) and sebacic acid (SA) at three molar ratios (G:SA= 0.6,0.8,1.0) and three temperatures (120, 130, 140 ˚C). The rate curves followed first-order kinetics with respect to sebacic acid concentration in the kinetics regime. An increase in the molar ratio (G : SA) of the reactants decreased the average functionality of the system and the crosslinking density, resulting in the lowering of the activation energy and pre-exponential factor. The average functionality of the system had a profound effect on the crosslinking density, mechanical properties, and the reaction kinetics of the system. Three different PGS oligomers and films (PGS 0.6, PGS 0.8, PGS 1.0) were thoroughly characterised using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), wide angle X-ray scattering (WAXS), differential scanning calorimetry (DSC), and contact angle measurements. FTIR spectra of PGS oligomers confirmed the formation of ester bonds (1740 cm -1). Quantification of various functional groups in PGS films using XPS was in agreement with the theoretical values of the proposed structure. WAXS results indicated that PGS system with a higher average functionality possesses a higher degree of crystallinity. Crystallisation exotherms and melting endotherms of PGS systems revealed that the average functionality influences the density of crosslinking, degree of crystallinity, and the network structure of bioelastomers. Contact angle studies confirmed that an increase in the average functionality of PGS system increases hydrophilicity, and the surface treatment through aminolysis further increases the hydrophilicity of the films. Batch studies were performed on a Brabender Plasticorder®. The samples collected over a reaction period of 5 h were characterised using Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The number-average molecular weight (Mn) and the weight-average molecular weight (Mw) of the oligoesters were determined using matrix-assisted laser desroption/ionization time-of-flight spectroscopy (MALDI-TOF) and compared with the corresponding values from the benchtop synthesis. It was found that due to higher shear-mixing and better orientation of functional groups, the degree of polymerisation at any stage of the reaction was higher in the Brabender than in the benchtop process. The gel-point of the reaction was determined from the crossover point of storage and loss moduli, and the reaction rate constant was calculated using the torque vs time data of the rheometer. The kinetics rate constant and the extent of the reaction in the Brabender were found to be higher than the corresponding values obtained from the conventional benchtop process by a factor of 2. PGS was found to be thermo-mouldable and adaptable to high-shear mixing, and hence is a better candidate for making thermoplastic elastomers using reactive extrusion. The challenges and possibilities in scaling up a batch process to a continuous process were investigated. The use of a wiped film reactor or a disk reactor along with reactive extrusion and batch-mixing (as a post-extrusion operation) is a commercially viable method to synthesise PGS oligomers. Such a continuous process will boost the production of bioelastomers for tissue engineering application by addressing the constraints in step-growth polymerisation. Finally, the effect of PGS substrate stiffness and surface treatment (aminolysis, hydrolysis, layer-by-layer deposition) on the morphology and lineage of mesenchymal stem cells – which have a capacity to differentiate themselves into cartilage, adipose, tendon, and muscle tissues – was analysed using fluorescence microscopy and DNA and protein assays. Stiffness of the PGS surface and the method of treatment influenced the cell attachment and spreading on different surfaces. However, cells did not differentiate into definite phenotypes at the end of 14 d time-point, indicating that higher time-points are needed to be considered to study the effect of matrix stiffness and surface treatment on cell attachment and phenotype differentiation.