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

Orientador: Cecília Amélia de Carvalho Zavaglia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-20T20:36:31Z (GMT). No. of bitstreams: 1 Rodrigues_LeonardoRibeiro_D.pdf: 27919489 bytes, checksum: c5885ca5237fb42cea7f7189a4070b0e (MD5) Previous issue date: 2012 / Resumo: A engenharia tecidual associada à nanotecnologia ganhou destaque no meio científico devido a sua multidisciplinaridade e capacidade de atingir várias áreas de estudo, inclusive no campo da medicina regenerativa. Para que o processo de regeneração tecidual óssea ocorra de forma esperada é possível utilizar scaffolds, que com sua estrutura porosa e com poros interconectados estruturam e organizam a região injuriada durante a recuperação do tecido. A organização da estrutura 3D da região afetada deve ser estudada para que seja possível criar scaffolds apropriados ao tamanho e forma do defeito existente. Com o scaffold já implantado deve ser avaliada a interação dos materiais com as células da região danificada. A partir desse princípio, o objetivo deste trabalho foi sintetizar nanopartículas de hidroxiapatita (HA) e betafosfato tricálcico ('beta'-TCP) utilizando a nova rota química baseada na utilização da sacarose como agente formador do gel (sucrose-based route) para síntese das nanopartículas, que são utilizadas na produção dos scaffolds, formando compósitos HA/TCP que serão utilizados como suporte para células tronco mesenquimais (MSCs) nos testes de substituição de tecidos ósseos. As nanopartículas foram caracterizadas por DRX, FRX, equação de Scherrer, MEV, MET, EELS, ESEM, NTA, FTIR e potencial Zeta. A partir dos dois fosfatos de cálcio (HA e 'beta'-TCP), foram feitos dois tipos de compósitos (esponja cerâmica e pastilha porosa) que foram caracterizados por ensaio mecânico de compressão axial, DRX, picnometria, microtomografia de raios X (Micro- CT), ESEM, FTIR e ensaio de degradação. Após a caracterização dos compósitos, os scaffolds foram submetidos a testes in vitro e in vivo e caracterizados por lupa de fluorescência, microscopia confocal, MEV, EDS, radiografia, MTT e histologia. Os resultados sugerem que os compósitos obtidos possam ser utilizados na potencialização da diferenciação osteogênica de MSCs provendo o desenvolvimento de novos modelos de bioengenharia tecidual na reconstrução óssea / Abstract: Tissue engineering associated with nanotechnology stood out because of its multidisciplinarity and results, in addition to targeting many areas of study in the field of regenerative medicine. For the process of bone regeneration to occur in appropriate form, the injured area must be organized. The reorganization of the 3D structure in the affected area should be studied in order to create scaffolds of appropriate size and shape similar to the existing defect. The implanted scaffold should be evaluated regarding the interaction of the material with the cells on the damaged region. From this principle, the objective of this work was to synthesize nanoparticles of hydroxyapatite (HA) and tricalcium phosphate ('beta'-TCP) using the new sucrosebased route which is based on the use of sucrose as a chelating agent for synthesis of nanoparticles, which are used in the production of scaffolds, forming composite HA / TCP that will be used as support for mesenchymal stem cells (MSC) in the replacement tests of bone tissue. The nanoparticles were characterized by XRD, XRF, Scherrer equation, SEM, TEM, EELS, ESEM, NTA, FTIR and Zeta potential. With two calcium phosphates (HA and TCP) it was prepared two types of composites (ceramic sponge and porous ceramic cylinder), which were characterized by mechanical test, XRD, pycnometry, X-ray microtomography (Micro-CT) ESEM, FTIR and degradation test. Scaffolds were tested in vivo and in vitro and they were characterized by magnifying fluorescence, confocal microscopy, SEM, EDS, radiography, MTT and histology. The results suggest that the scaffolds obtained can be used to improve the osteogenic differentiation of the MSC providing the development of new types of bone tissue engineerinG / Doutorado / Materiais e Processos de Fabricação / Doutor em Engenharia Mecânica

O tecido ósseo tem sido amplamente estudado devido às suas inúmeras funções e capacidade de auto-regeneração. Entretanto, muitas vezes a reparação óssea completa pode ser prejudicada quando as fraturas ósseas são graves. Muitas pesquisas visando a regeneração do tecido ósseo estão sendo realizadas tanto nas abordagens que envolvem a utilização de enxertos, quanto na aplicação de terapia celular. O objetivo deste trabalho foi obter e caracterizar uma linhagem celular proveniente do tecido ósseo de fetos caninos. Para isso, foi realizado método de dissociação enzimática do osso fetal canino para obtenção da cultura celular, estabelecendo assim o cultivo das células OSTBN6. Além disso, foram realizados técnicas do teste de viabilidade e proliferação celular por MTT, de imunofenotipagem das células, expressão gênica, diferenciação celular em linhagens adipogênica, osteogênica e condrogênica, e análise do potencial tumorigênico das células derivadas de tecido ósseo fetal canino. Sendo assim, a população isolada de células derivadas de tecido ósseo fetal canino isolada apresentou duas morfologias distintas: formato fibroblastóide e formato triangular. Essas células são viáveis e possuem ótima taxa de proliferação, como indicou o ensaio de MTT. As células foram positivas para pluripotência e para células de origem mesenquimal, e foram negativas para marcadores de células de origem hematopoiéticas. As OSTBN6 foram capazes de se diferenciar em linhagem adipogênica, osteogênica e condrogênica, característica de células mesenquimais. Por fim, foi realizado o teste do potencial tumorigênico em camundongos nude, não havendo formação de tumores. Desse modo, concluimos que a célula proveniente do tecido ósseo fetal canino constitui uma fonte segura para utilização na medicina regenerativa e terapia celular / Bone tissue has been widely studied due to its numerous functions and capacity for self-regeneration. However, often the complete bone repair may be impaired when bone fractures are severe. Many research aiming to regenerate the bone tissue are being conducted in both approaches involving the use of grafts, as the application of stem cell therapy. The objective of this study was to obtain and characterize a cell line derived from the canine fetuses bone. To do so, it was carried out an enzymatic dissociation method for obtaining canine fetal bone cell culture, thus establishing OSTBN6 cells culture. In addition, there were carried out technical viability and cell proliferation by MTT test, immunophenotyping of cells, gene expression, cellular differentiation in adipogenic, osteogenic and chondrogenic lineages, and analysis of tumorigenic potential of cells derived from canine fetal bone. Thus, the population of cells derived from isolated canine fetal bone tissue showed two distinct morphologies: fibroblastoid shape and triangular shape. These cells are viable and have optimal rate of proliferation, as indicated by the MTT assay. Cells were positive for pluripotency and as mesenchymal cells and were negative for hematopoietic origin cell markers. The OSTBN6 were able to differentiate into adipogenic, osteogenic and chondrogenic lineage, characteristic of mesenchymal cells. Finally, it was performed the tumorigenic potential test in nude mice, with no tumor formation. Thus, it was concluded that the cell derived from canine fetal bone is a reliable source for use in regenerative medicine and cell therapy

As doenças genéticas degenerativas atingem milhões de crianças em todo o mundo. Dentre essas doenças, a distrofia muscular, caracterizada como uma doença monogênica poderia ser tratada na sua origem através da terapia gênica. Assim, este estudo propõe à correção da mutação no gene da distrofina, causador da distrofia muscular através de modificações genéticas específicas. A criação de novas classes de terapêuticos que podem desencadear rearranjos no DNA genômico de maneira específica representa uma nova promessa para experimentos em terapia gênica. A tecnologia usada foi o RNA interferente (RNAi) que é utilizada para regulação da expressão gênica pós-transcricional. A Ku 70 é uma das proteínas específicas para a recombinação não homóloga, o RNAi foi usado na tentativa de atenuar a Ku70, prevalecendo então a expressão da recombinação homóloga, com intuito de corrigir a mutação gênica causadora da distrofia muscular em cães. Para tal avaliação, utilizamos linhagens de células tronco (CT) mesenquimais recentemente isoladas, oriundas de populações mononucleares da médula óssea de cães jovens afetados pela distrofia muscular, apresentando bons resultados em cultivo e caracterização. Este trabalho proporciona além da criação de uma nova terapêutica específica para a correção da distrofia muscular, o aumento do conhecimento e entendimento na indução de modificações genômicas em células, no desenvolvimento de novas classes de agentes terapêuticos moleculares que representam um grande potencial em estudos e no tratamento de várias doenças genéticas e infecciosas, degenerativas ou adquiridas. O presente trabalho apresenta métodos de isolamento e caracterização de células tronco-mesenquimais bem como a utilização de RNAi visando promover a recombinação homóloga entre o DNA transfectado e o alvo no DNA genômico. / The degenerative genetic diseases affect millions of children around the world. Among these diseases, muscular dystrophy, characterized as a monogenic disease can be treated at its source through gene therapy. Thus, this study proposes the correction of the gene that causes muscular dystrophy through genetic modification specific. The creation of new classes of therapeutics that can trigger rearrangements in the genomic DNA in a specific manner represents a new promise for gene therapy experiments. The technology will be used by RNA interference (RNAi) and that used to regulate gene expression post-transcriptional. The 70 Ku is a protein specific to the non-homologous recombination, RNAi was used in an attempt to mitigate the Ku70, then the prevailing expression of homologous recombination, aiming to correct the mutation that causes muscular dystrophy in dogs. For this evaluation, we use mesenchymal stem cell lines recently isolated populations derived from bone marrow mononuclear cells of young dogs affected by muscular dystrophy, presenting good results in characterization and culture. This work also provides the creation of a new specific therapy for the correction of muscular dystrophy, increased knowledge and understanding in the induction of genomic changes in cells in the development of new classes of molecular therapeutic agents have great potential in studies and treatment of various genetic and infectious diseases, degenerative or acquired. This paper presents methods for isolation and characterization of mesenchymal stem cells and the use of RNAi to promote homologous recombination between transfected DNA and genomic target DNA.

Abstract The stem cell is a primitive cell that is capable of dividing to reproduce itself and can give rise to a selection of differentiated progeny. Stem cells are thought to be involved in or even main factors in many diseases. In postnatal humans, mesenchymal tissues have the capacity to regenerate from stem cells called mesenchymal stem cells (MSC). It is currently thought that these cells will become the basis of therapy for many diseases. In the present study, a novel in vitro method was developed to examine human bone marrow derived MSC differentiation into osteoblast lineage, and to study the role of MSC in a variety of mesenchymal diseases. The ability of MSCs to differentiate into osteoblasts was investigated during aging. In addition, the interindividual variability in the osteogenesis of MSCs and in the osteoblastic response of MSC to estrogen and testosterone was studied. Furthermore, an ex vivo model using a human aortic valve microenvironment was developed to explore whether the extracellular matrix influences the osteoblastic differentiation of the MSC. Finally, the role of MSC in neurofibromatosis type 1 (NF1) related congenital pseudarthrosis of the tibia (CPT) was studied. It was found that after menopause the osteogenic potential of MSCs does not decrease. It was also found that estrogen receptor (ER) alpha genotype confers interindividual variability of response to estrogen and testosterone in MSC derived osteoblasts. In addition, it was found that the non-calcified valves with living valve cells inhibited osteogenesis of co-cultured MSCs, whereas the calcified and devitalised valves promoted differentiation towards an osteoblastic lineage. Finally, MSCs from NF1-related pseudarthrosis showed altered NF1 gene expression, poor osteoblastic differentiation and bone formation. In conclusion, MSC can be easily isolated from the bone marrow and MSC has the capacity to regenerate tissue even at later stages of life. These results could help explain the contradictory effects of 17β-estradiol (E2) on osteoblasts in vitro and might also provide new insights into understanding the differences in responses to hormone replacement therapy. It seems that adult stem cells from bone marrow undergo milieu-dependent differentiation to express phenotypes that are similar to cells in the local microenvironment. Finally, the NF1 gene was shown to have a role in bone development and remodelling.

In the first part of these studies, we have performed standard microsurgical procedures provide a solution for long standing bone and soft tissue defects, even in cases of longstanding osteomyelitis of long bones. When long bony segments are missing, the microvascular bone transfer provides a reliable method. In smaller soft tissue and bone defects, the application of a descending genicular osteomyocutaneous flap provides an option with low donor site morbidity. In the second part, we have focussed on reducing the donor site morbidity and expanded on the application of tissue engineering methods. MSCs derived from bone marrow can be injected percutaneous or be combined with an autologous bony scaffold for treatment of delayed union and nonunion. The outcome of our studies, however, limited in number of patients, clearly showed the possibilities and advantages of this new approach. A multimodality approach is essential, but it can provide promising solutions. Well-established microvascular and modern biotechnology methods will improve patient satisfaction and functional recovery in severe limb trauma, often the result of high-energy motorcycle accidents. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Un patient peut souffrir d’une perte de substance osseuse de taille critique suite à des accidents ou des pathologies. Aujourd’hui, le traitement le plus fréquent consiste en la greffe du tissu osseux (autogreffe ou allogreffe). Compte tenu des complication rencontrées (réponse immunologique, morbidité du site donneur), la recherche actuelle s’inscrit dans la recherche de synthèse d’un biomatériau bioactif favorisant la régénération osseuse. Ces matériaux devraient imiter les qualités de la matrice extracellulaire osseuse pour stimuler la formation osseuse.Les cellules souches mésenchymateuses jouent un rôle important du fait de leur capacité de prolifération et différentiation en ostéoblastes. Pour profiter de ce potentiel des cellules souches, il est nécessaire de comprendre comment contrôler leur comportement et mesurer l’impact du microenvironnement cellulaire sur la différenciation ostéogénique de ces cellules. Comme les cellules souches mésenchymateuses sont capables de différencier en plusieurs phénotypes différents, il est indispensable de les diriger dans la direction désirée. Plusieurs facteurs qui influencent le devenir cellulaire ont été identifiés, comme certains peptides bioactifs, des facteurs mécaniques comme la rigidité, ou la topographie de surface de matériaux.Dans la matrice extracellulaire naturelle du tissu osseux, les cellules souches mésenchymateuses sont entourées d’une variété de principes actifs, dont le plus abondant est le collagène I. Cette protéine s’assemble pour former des nanofibres qui présentent une nanomorphologie périodique avec une périodicité bien définie. La question posée au début de ce travail était: Cette structure a-t-elle un impact sur la différenciation des cellules souches?Pour étudier l’impact de la périodicité nanofibrillaire, nous proposons dans ce travail de recherche l’utilisation d’hélices modèles qui miment en partie la morphologie du collagène. Les hélices nanométriques auto-assemblées des surfactants gemini peuvent avoir un pas d’hélice et un diamètre similaires à ceux du collagène. La modulabilité de ces paramètres et la possibilité de modifier ces structures par des molécules bioactives permettent de moduler les caractéristiques des nanoobjets et d’étudier l’impact de ces nanomatériaux sur les cellules souches mésenchymateuses. / Tissue engineering is a field related to regenerative medicine which aims at replacing or regenerating a patient’s tissue, usually using a combination of cells and bioactive material designed to influence cell behaviour. In approaches for bone regeneration, human mesenchymal stem cells (hMSCs) are a common choice because of their ability to proliferate and differentiate into osteoblasts. Harnessing this potential requires biomaterials which promote osteoblastic differentiation, for example by mimicking the conditions in natural bone. Collagen I is a common protein in human bone; it forms fibrils with a characteristic periodic structure, which raises the question whether this morphology has in impact on stem cell fate. Collagen-mimicking nanomaterials can help investigate this question: Gemini surfactants with chiral counterions form twisted bilayers the morphology of which can be tuned by variation of enantiomeric excess, time and temperature. The self-assembled helical nanoribbons which are obtained by this process can be transformed by a sol-gel condensation to form silica nanohelices the size and twist pitch of which resembles that of collagen fibres. The objective of this study is to prepare 2D culture environments featuring these nanomaterals (with and without bioactive peptide functionalisation) to explore the effect of these materials on hMSC differentiation.Silica helices are fabricated by synthesis of surfactants with tartrate as counterion, and organic-inorganic transcription using a silica precursor compound. They can be modified by reaction with APTES and an N-hydroxysuccinimide ester and covalent immobilisation of a peptide. Two peptides were used in this study, one adhesion-promoting peptide and the active domain of the osteogenesis-inducing peptide BMP2. Helices with or without this bioactive functionalisation were covalently grafted to glass substrates using APTES and EDC/NHS-coupling. The presence of peptides on helices was shown by the absorption of helix-grafted peptides bearing the FITC-fluorophore. Successful peptide grafting onto glass surfaces was verified by XPS and fluorescence microscopy. The morphology of helices was monitored with TEM and SEM. SEM images were used to determine the amount of helices on surfaces. HMSCs were cultivated for four weeks on surfaces modified with APTES, peptide(s) or left- or righthanded nanohelices, functionalised or not with bioactive peptide(s). After fixation, the quantities of the osteogenic markers Runx2 and Osteocalcin (OCN) in the cells were evaluated. The results show that BMP2-functionalised surfaces exhibited an elevated level of Runx2 and OCN expression. Some helix-grafted materials exhibited a significantly higher Runx2 and/or OCN expression than the corresponding homogeneous materials, but these differences were not consistent across samples of the same chiral orientation or bioactive functionalisation. Therefore, conclusive general statements about differences in osteogenic effect between helix functionalisations and handednesses aredifficult to make. A potential reason for this is the variability of surface coverage of helix-grafted materials: As the quantity of helices that are immobilised onto the surfaces is lower than expected and varies greatly between the samples, the number of cells that are not in contact with the helices might change as well, which can lead to false negatives.The results of a proteomic experiment have shown which proteins are differentially expressed in cells cultured on helices with or without BMP-functionalisation, compared to bare glass. Comparison with other proteomic studies shows that proteins which are known to be upregulated during osteogenic differentiation are overexpressed most frequently in cells cultured on BMPmodified helices. The proteins that were identified with this method might serve as starting point for future investigations.

L’arthrose et la polyarthrite rhumatoïde sont deux atteintes ostéo-articulaires qui affectent les tissus cartilagineux et osseux. Elles représentent un réel problème de santé publique de par leur prévalence et le manque de traitements curatifs. Des approches innovantes de thérapie cellulaire sont en cours d’évaluation dans ces maladies. Elles sont basées sur l’utilisation des cellules souches mésenchymateuses (CSM), qui possèdent des propriétés immunosuppressives et régénératrices, et pourraient apporter de nouveaux espoirs aux patients. Ces cellules sécrètent des vésicules extracellulaires, moyen de communication intercellulaire puissant, qui sont classées en 3 populations : exosomes, microparticules (MPs) et corps apoptotiques. Dans cette étude, nous proposons d’étudier et de comparer les effets in vitro et in vivo des exosomes et des MPs sécrétés par les CSMs. Nous montrons pour la première fois que les exosomes et les MPs de CSMs exercent in vitro des effets anti-inflammatoires, anti-apoptotiques et chondroprotecteurs similaires. In vivo, seuls les exosomes exercent un effet thérapeutique dans un modèle expérimental de polyarthrite rhumatoïde. En revanche dans un modèle expérimental d’arthrose, les exosomes et les MPs protègent le cartilage et l’os de la dégradation et ce, de manière équivalente. Cette étude est la première preuve de concept que des exosomes et/ou des MPs isolés de CSMs ont un effet thérapeutique dans des maladies rhumatismales. / Osteoarthritis and rheumatoid arthritis are osteoarticular diseases affecting primarily cartilage and bone. They are a high public health problem because of their prevalence and absence of curative treatment. Innovating cell therapy approaches are being evaluated in the clinic for treating these diseases. They rely on the use of mesenchymal stem cells (MSCs), which display immunosuppressive and regenerative functions and could provide new hope for patients. These cells release extracellular vesicles, which are very powerful tools for intercellular communication and are classified into 3 populations: exosomes, microparticles (MPs) and apoptotic bodies. In the present study, we characterize and compare the in vitro and in vivo effects of MSC-derived exosomes and MPs. We show for the first time that exosomes and MPs exert in vitro anti-inflammatory, anti-apoptotic and chondroprotective functions. In vivo, only exosomes exert a therapeutic effect in an experimental model of inflammatory arthritis. However in a model of osteoarthritis, both exosomes and MPs protect cartilage and bone from degradation, in a similar fashion. This study provides the proof-of-concept that MSC-derived exosomes and/or MPs exert a therapeutic effect in rheumatic diseases.

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Malgré l’augmentation des cas de cancers, l’amélioration des thérapies anti-cancéreuses a permis d’accroitre la survie des patients. Bien qu’efficace, la radiothérapie peut induire des complications sévères. La sphère abdomino-pelvienne concentre de nombreux cancers (prostate, vessie…), et des organes à risque lors de la radiothérapie. Le côlon-rectum développe des séquelles sévères chez 20% des patients 20 ans après traitement. La fibrose colorectale est la principale de ces complications. Les traitements actuels de ces lésions sont palliatifs. Les Cellules Souches Mésenchymateuses (CSM) favorisent la régénération tissulaire dans de nombreuses pathologies, y compris fibrosantes, et semblent donc adaptées au traitement des atteintes radio-induites. Néanmoins, les effets des CSM sur la tumeur et sur les complications des radiothérapies sont méconnus. Nos travaux évaluent la sureté et l’efficacité de la transplantation de CSM avant et après radiothérapie colorectale chez le rat. Sur un modèle de tumorigénèse colorectale suivie de radiothérapie, l’injection de CSM a inhibé la croissance tumorale en modifiant le profil des lymphocytes T et macrophages du microenvironnement tumoral. Dans un second modèle, les CSM ont induit une suppression durable de la fibrose colorectale radio-induite. Les protéines HGF et TSG-6 sécrétées par les CSM bloquent l’acquisition du phénotype pro-fibrosant par les cellules sécrétrices de matrice extracellulaire dans le côlon-rectum. Les CSM ont amélioré la survie des animaux dans ces deux modèles. Dans l’ensemble, nos résultats supportent l’utilisation des CSM dans le contexte des complications des radiothérapies abdomino-pelviennes. / Despite the growing number of cancer cases, current anti-cancer treatments greatly improve patients’ survival. Although it is efficient, radiotherapy can induce severe complications. The abdomino-pelvic area regroups cancers with high prevalence (prostate, bladder…) and organs at risk during radiotherapy. Colon and rectum display severe side effects in 20% of patients 20 years after treatment. Colorectal fibrosis is the most frequent of these complications. Existing treatments are only palliative. Mesenchymal Stem Cells (MSCs) promote tissue regeneration in a wide variety of pathologies, fibrosis included, and thus seem fitted for the treatment of radiation-induced disorders. However, the effects of MSCs on tumor growth and radiotherapy induced damages are still unclear. Our work evaluates the safety and efficacy of MSC transplantation before and after colorectal radiotherapy in rats. In a model of chemically-induced colorectal carcinogenesis, followed by radiotherapy, MSC injection suppressed tumor growth by modifying the phenotype of T lymphocytes and macrophages of the tumor microenvironment. In a second model, transplanted MSCs suppressed radiation-induced fibrosis. Two proteins secreted by MSCs, HGF and TSG-6, are responsible for inhibiting extracellular matrix-producing cells, which are the major contributors to fibrosis. MSC injection was associated with increased survival in both studies. Overall, our results support the use of MSCs to treat the side effects of abdomino-pelvic radiotherapy.