Mesenchymal stem cells (MSC) possess a multilineage differentiation potential and have the ability to regulate reactivity of the immune system. They are usually isolated and expanded from the bone marrow, adipose tissue or umbilical cord. MSC represent promising cell population for the treatment of some severe diseases, such as amyotrofic lateral sclerosis (ALS), due to the combination of regenerative and immunomodulatory properties. The aim of this study is to compare MSC from ALS patients and healthy donors in their phenotype, proliferative activity and mainly their immunomodulatory properties. The assessment of impact of the disease on the properties of MSC is important for their autologous use in clinical trials. In this study we used MSC isolated from bone marrow of 14 ALS patients and 15 patients undergoing mostly orthopedic surgery as control group. We also used MSC stimulated for 24 hours by poinflammatory cytokines. Cells were compared in terms of immunophenotype, differentiation in adipocytes and osteoblasts, metabolic activity, expression of selected genes for immunomodulatory molecules and for inhibition of lymphocyte proliferation. Further experiments were focused on evaluation of immunomodulatory properties of MSC. The effect of MSC on peripheral blood mononuclear cells stimulated...

Origin of mammalian somatic cells in the developing testes remains unclear. This origin could be explained by established cell culture derived from testes of Xenopus tropicalis juvenile male. The expression profile of the cell culture showed transcription of some pluripotency genes, somatic Sertoli and peritubular myoid cell markers and last but not least, the mesenchymal stem cell markers. Conversely, germ cell genes were downregulated. Immunocytochemical analysis revealed expression of Vimentin, Sox9 and α-smooth muscle actin, indicating that the testicular cell culture is a common mesenchymal progenitor of the Sertoli and peritubular myoid cells and that the cell culture did not arise from spermatogonial stem cells undergoing incomplete reprogramming in vitro. Testing of X. tropicalis cell culture during induction of differentiation in vitro revealed that these cells are probably multipotent with the ability to differentiate into adipocytes, chondroblasts and osteoblasts. The ability to derive multipotent stem cells from the juvenile testes opens new possibilities of using these cells for biotechnology and medicine. Keywords: Testicular somatic cells, Xenopus tropicalis, progenitor, mesenchymal stem cells, induction of differentiation, multipotency

L'ingénierie tissulaire est une discipline récente aux enjeux ambitieux et prometteurs : la régénération de tissus ou d'organes lésés voire détruits en mettant à profit des connaissances et compétences dans différents domaines à l'interface de la chimie et de la biologie. Pour répondre à la demande d'alternatives aux techniques chirurgicales actuelles de réparation du ligament antérieur croisé, nous avons décidé d'appliquer l'ingénierie tissulaire à ce tissu en associant matrices en polymères dégradables et cellules souches mésenchymateuses (CSM). Dans un premier temps, nous avons donc travaillé à la synthèse de polymères adaptés à l'application en cherchant à mettre l'accent sur l'obtention de propriétés élastiques. De nouveaux élastomères dégradables obtenus par des approches originales de photoréticulation chimique de poly(lactide) (PLA) et de poly(ε-caprolactone) (PCL) par voie nitrène ou thiol-yne ont notamment été développés avec des résultats prometteurs. En parallèle, des copolymères thermoplastiques multiblocs à base de PLA et poloxamine ou poloxamère nous ont permis de mener une étude plus appliquée. Ces copolymères ont en effet montré, en particulier au cours d'une étude de dégradation in vitro de 7 semaines, des propriétés, notamment thermiques et mécaniques, qui en font d'eux des candidats intéressants pour le conception d'une matrice ligamentaire. C'est pourquoi ils ont été utilisés pour la conception de prototypes de matrices de régénération textiles dont les propriétés mécaniques se sont révélées être très proches de celles du ligament. Après avoir démontré l'excellente cytocompatibilité de ces matrices avec des CSM, nous avons finalement mené des expériences de différenciation in vitro de ces CSM et sommes parvenus à favoriser leur orientation vers un phénotype ligamentocytaire, notamment grâce à un procédé de stimulation mécanique cyclique des cellules ensemencées sur les matrices textiles. / Tissue engineering is a recent discipline with ambitious and promising stakes: the regeneration of wounded or destroyed tissues or organs by taking advantage of knowledge and skills in various fields at the interface of chemistry and biology. In order to meet the need for alternatives to current surgical techniques of anterior cruciate ligament repair, we decided to apply the tissue engineering approach to this tissue by associating degradable polymer scaffolds and mesenchymal stem cells (MSCs). At first we worked on the synthesis of biodegradable polymers suitable for the application and focused on getting elastic properties. New degradable elastomers obtained by chemical photocrosslinking of poly(lactide) (PLA) and poly(ε-caprolactone) (PCL) were developed by following nitrene or thiol-yne strategies and yielded promising results. In parallel, a more in depth and practical study was performed with PLA based thermoplastic multiblock copolymers embedding poloxamer or poloxamine. These copolymers exhibited properties that make them attractive candidates for the design of ligament regeneration scaffolds, and especially their thermal and mechanical properties during a 7 week in vitro degradation test. That is why they were used to design prototypes of textile scaffolds whose mechanical properties were found to be very close to the ligament's ones. After demonstrating the excellent cytocompatibility of these scaffolds with MSCs, we finally carried out in vitro differentiation experiments on these MSCs and managed to induce their orientation towards a ligamentocyte phenotype, particularly through a process of cyclic mechanical stimulation of cells seeded on the textile scaffolds.

Le lumicanne est un petit protéoglycanne riche en leucine de la matrice extracellulaire impliqué, entre autre, dans le contrôle de l'angiogenèse, en particulier l'angiogenèse tumorale. Nous avons déjà démontré que le lumicanne inhibe la progression du mélanome in vivo. Le mécanisme d'action anti-tumoral du lumicanne a été partiellement décrit dans notre laboratoire. L'intégrine alpha2beta1 a été caractérisée comme un récepteur direct du lumicanne. Dans l'étude que nous présentons, nous avons décrit le rôle du lumicanne dans le contrôle de la transition des cellules souche mésenchymateuse (CSM) en cellules progénitrices endothéliales, pouvant contribuer à l'angiogenèse tumorale. Nous avons montré que le lumicanne inhibe spécifiquement la migration et l'invasion des CSM par une diminution de l'expression et de l'activité de la MMP-14. De plus, nous avons démontré que le lumicanne est un inhibiteur compétitif de la MMP-14. Il se lie directement au domaine catalytique de l'enzyme avec une affinité modérée (KD=275,4±16.12nM). De plus, nous avons démontré que le lumicanne diminue la phosphorylation de récepteurs (EGFR, Mer, EphB2, EphB6, ROR) et de protéines kinases (AKT, GSK3 beta et p130CAS). Une diminution de l'expression de la beta-caténine a également été détectée.Notre équipe a précédemment identifié une séquence de 17aa dans la protéine de cœur du lumicanne, la lumcorine, qui est capable de reproduire l'effet anti-migratoire du lumicanne. Nous proposons ici deux mécanismes d'action de la lumcorine: une inhibition de la phosphorylation de FAK et une diminution de l'activité de la MMP-14. De plus, nous avons identifié, au sein de la séquence de la lumcorine, un peptide court de 10aa (L9M) qui est capable de reproduire l'effet anti-tumoral de la lumcorine. Nous montrons que le peptide cyclique L9M diminue la croissance tumorale in vivo.Nos travaux permettent donc de mieux comprendre les mécanismes impliqués dans l'effet anti-tumoral du lumicanne et mettent en évidence de nouveaux peptides prometteurs pour des applications anticancéreuses. / Lumican is a small leucine-rich proteoglycan of the extracellular matrix involved in the control of angiogenesis, particularly tumor angiogenesis. We have previously demonstrated that lumican inhibits melanoma progression in vivo. The anti-tumor mechanism of action of lumican was partially described in our laboratory. The alpha2beta1 integrin was characterized as a direct receptor of lumican. In the present studies, we first described the role of lumican in the control of Mesenchymal Stem Cells (MSC) transition to functional Endothelial Progenitor Cells (EPC), which can contribute to tumor angiogenesis. We showed that lumican inhibits specifically the migration and invasion of MSC by decreasing the expression and activity of MMP-14. Moreover, we demonstrated that lumican reduces the activity of MMP-14 in melanoma cells. We next showed that lumican directly inhibits MMP-14 activity as a competitive inhibitor which binds to the catalytic domain of the enzyme with moderate affinity (KD=275.4±16.12nM). Moreover, we demonstrated that lumican decreases the phosphorylation of some cell surface receptors (EGFR, Mer, EphB2, EphB6, ROR), some kinases (AKT, GSK3 beta and p130CAS) and alters the expression of beta-catenin.Previous works from our laboratory identified a sequence of 17aa within the leucine-rich repeat 9, named lumcorin, which was able to reproduce the anti-migratory effect of lumican. Here, we propose two mechanisms of action of lumcorin: inhibition of phosphorylation of FAK and a decrease of the MMP-14 activity. We also identified in the sequence of lumcorin a short 10aa peptide (L9M) which was capable to reproduce the anti-tumor effect of lumcorin. In addition, the cyclic peptide L9M was demonstrated to reduce tumor growth in vivo.Altogether, our results help to better understand the mechanisms involved in the anti-tumor effect of lumican and identify lumican-derived peptides which could have potential anti-cancer applications.

Le silicium poreux est un biomatériau prometteur pour l'ingénierie tissulaire car il est non toxique et biorésorbable. Des modifications de surface permettent de contrôler sa vitesse de dégradation et peuvent favoriser l'adhésion cellulaire. Les cellules souches de la pulpe dentaire (DPSC) sont des cellules souches mésenchymateuses retrouvées dans la pulpe dentaire, à l'intérieur des dents, et constituent une source accessible de cellules souches. Regrouper les capacités de prolifération et différenciation des DPSC avec les propriétés morphologiques et biochimiques du pSi représente une approche intéressante pour des applications thérapeutiques de médecine régénératrice. Dans cette thèse, nous avons étudié le comportement de DPSC humaines sur des supports de pSi, avec des pores variant de quelques nanomètres à plusieurs centaines de nanomètres. Nous avons travaillé sur différentes fonctionnalisations chimiques afin d'optimiser l'adhésion cellulaire et de stabiliser le matériau : oxydation thermique, silanisation et hydrosilylation. L'adhésion, la prolifération et la différenciation osseuse ont été évaluées par microscopie à fluorescence, microscopie électronique à balayage, activité enzymatique, tests de prolifération (activité mitotique), immunofluorescence et spectroscopie FTIR. Le pSi avec des pores de 30 à 40 nm de diamètre s'est révélé être le plus approprié pour l'adhésion, la prolifération cellulaire et la différenciation ostéoblastique. De plus, la structure nanométrique et le relargage d'acide silicique par le pSi a démontré un effet positif sur l'induction osseuse et la formation d'une matrice minéralisée. Le pSi est donc apparu comme un matériau prometteur pour l'adhésion de cellules souches mésenchymateuses, que ce soit pour une transplantation immédiate in vivo ou pour expansion et différenciation in vitro. / Porous silicon (pSi) is a promising biomaterial for tissue engineering as it is both non-toxic and bioresorbable. Moreover, surface modification can offer control over the degradation rate of pSi and can also promote cell adhesion. Dental pulp stem cells (DPSC) are mesenchymal stem cells found within the teeth and constitute a readily source of stem cells. Coupling the good proliferation and differentiation capacities of DPSC with the textural and chemical properties of the pSi substrates provides an interesting approach for therapeutic use. In this thesis, the behavior of human DPSC is analyzed on pSi substrates presenting pore of various sizes, from few to hundreds nanometers. We investigated different chemical surface treatments, in order to enhance cell adhesion and stabilize the material: thermal oxidation, silanization and hydrosilylation. DPSC adhesion, proliferation and further osteodifferentiation were followed for up to 3 weeks by fluorescence microscopy, scanning electron microscopy (SEM), enzymatic activity assay, BrdU assay for mitotic activity, immunostaining and FTIR spectroscopy. Porous Silicon with pore size ranging from 30 to 40 nm was found to offer the best adhesion, the fastest growth rate for DPSC and the highest osteoinductive effect. Moreover, the pSi nanostructure and the release of silicic acid had a positive effect on precursor cells osteodifferentiation and mineralized matrix formation. Porous silicon appeared to be an appropriate biomaterial for mesenchymal stem cells adhesion and immediate in vivo transplantation, or for long term in vitro culture, for stem cells proliferation and differentiation.

Résumé français manquant / Scientific background: GAGs mimetics properties on regenerative process.Glycosaminoglycans (GAGs) are sulfated polysaccharides actually considered as major structural components of the extracellular matrix as well as regulators of cells functions during homeostatic and pathological processes. These GAGs activities are based on their ability to interact with heparin binding growth-factors (HBGF), chemokines and enzymes, to protect them from proteolytic degradation and to potentialyze their interaction with cell surface specific receptors and/or other components of the ECM. GAGs are characterized by their extensive structural diversity, based on the number and location of sulfate or acetylate groups, that would determine specific biological interactions.As comparative tool to study the relationship between the complexity of GAGs chemical structures and their biological functions, we used synthetic GAGs mimetics, derivate from a polymer of dextran and functionalized with carboxylate, sulfate and/or acetate groups. They are structurally and functionally related to natural heparan sulfates. These compounds improved both the rate and quality of regenerative process in numerous animal models of injury after topical treatment.Our hypothesize is that specific HS cooperative interactions with HBGF and ECM compounds could influence both therapeutic progenitors and stem cells properties by compartmentalizing them to specific microenvironment niches, and protecting them against deleterious signals. Such abilities to modulate stem cell biology could be a new way to explain and to take advantage of regenerative properties of these compounds. The principal aim of this work was to demonstrate the effects of GAGs mimetics on Mesenchymal Stem Cells (MSC) properties for application in bone repair. GAGs mimetics as new potentializing agents of mesenchymal stem cells propertiesDuring osteogenesis, a controlled expression of functional HS is required to interact and regulate the activity of growth promoting and osteogenic differentiation factors. However effects of GAGs on MSC properties remain to be analyzed. We focus on two GAGs mimetics leader molecules [OTR4131] and [OTR4120], with distinct chemical characteristics, since sulfated mimetic [OTR4120] was previously shown to stimulate bone repair in vivo. We demonstrate that its acetylated and sulfated counterpart [OTR4131] enhances proliferation, whereas [OTR4120] clearly stimulates migration and osteogenic differentiation properties of rat MSC in vitro, that could explain its bone regenerative effect in vivo. This indicates that GAGs mimetics would be of great interest for potential application in therapy, since according to their structural signature they could modulate specific activities of progenitors and stem cells, and represent an alternative to exogenous growth factor treatments. New matricial strategy for bone repair associating GAGs mimetics to biomaterials and human MSCCell based therapy associated to biomaterials for repair of bone defects are promising but not enough efficient. We proposed to develop matricial strategy, associating efficient micro-environment molecules such as GAGs mimetics, to optimize cell therapeutic approaches. First we validated that GAGs mimetics are effective on human MSC proliferation, migration and differentiation properties in vitro. We demonstrated that colonization efficiency of hydroxyapatite/β-tricalcium phosphate biomaterial scaffolds by human MSC was improved when scaffolds are functionalized with GAGs mimetics in vitro. Finally osteoformation in vivo was evaluated after ectopic transplantation of functionalized and/or cellularized biomaterials in nude mice: few effects were observed on bone formation, whereas osteoclastogenesis and vascularization were clearly modulated by GAGs mimetics immobilized. GAGs mimetics as new mobilizing agents of stem cells...

Background: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. Results: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O-2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Conclusions: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.

Ce travail de thèse avait pour objectif le développement de systèmes de culture cellulaire, en 2D et en 3D, en mettant à profit les propriétés d’un transporteur d’oxygène marin, HEMOXCell®. Notre approche générale était articulée selon deux grands axes : un premier concernant l’évaluation de l’utilisation d’HEMOXCell® dans la culture de deux modèles cellulaires, et un second, utilisant les résultats obtenus à des fins d’ingénierie tissulaire. Dans le premier axe, l’évaluation de l’effet dose-réponse d’HEMOXCell® dans la culture des cellules CHO-S et des cellules souches mésenchymateuses (CSM), a permis de déterminer des concentrations de travail optimales, favorisant la viabilité et la prolifération cellulaire. Le modèle cellulaire CHO-S a contribué à la mise en place d’un test de performance de la molécule, et encouragé son utilisation dans des systèmes de bioproduction. Les essais menés sur les CSM ont quant à eux permis de valider l’innocuité de la molécule à de faibles doses et le maintien de l’état « souche ». L’idée d’associer les CSM à des supports poreux est prometteuse pour des applications d’ingénierie tissulaire, mais est soumise aux problèmes liés à l’oxygénation en profondeur des supports. Dans le second axe de ce projet, nous avons oeuvré à améliorer la colonisation de substituts osseux et méniscaux, en culture statique et dynamique, en présence d’HEMOXCell®. Parallèlement, une étude a été menée pour tenter de caractériser les cellules méniscales. Les analyses de la colonisation des biomatériaux suggèrent un effet bénéfique d’HEMOXCell® lorsqu’il est utilisé en complément des milieux de différenciation cellulaire. Ce travail a contribué à améliorer la compréhension de ce transporteur d’oxygène et à l’élargissement de ses potentiels champs d’utilisation notamment dans un cadre thérapeutique. / This work aimed to develop cell culture systems, in 2D and 3D, based on the properties of HEMOXCell®, a marine oxygen carrier. Our approach was articulated in two main parts: the first one dealing with the assessment of the use of HEMOXCell® in the culture of two cellular models, and the second one, exploiting the results obtained for tissue engineering purposes. In this first axis, the dose-response effect of HEMOXCell® in the CHO-S cells and mesenchymal stem cells (MSC) in vitro culture, allowed the identification of optimal working concentrations, which can promote cell viability and proliferation. The CHO-S model has contributed to the establishment of a performance test of the molecule, and encouraged its use for bioproduction stimulation. The tests performed on MSCs were used to validate the harmlessness of the molecule at low doses and the maintenance of "stemness". The idea to associate MSCs with porous scaffolds is a promising approach for tissue engineering applications, but it is confronted to the lack of oxygen in the depth of the substitutes. In the second part of this project, we worked at improving the cellularization of bone and meniscal substitutes, under static and dynamic culture systems, w/ and w/o HEMOXCell®. In parallel, a study was conducted to attempt to characterize the meniscal cells. Analyses of cellularized biomaterials suggest a beneficial effect of HEMOXCell® when used as a differentiation media supplement. This work contributed to improve this oxygen carrier understanding and to extend the field of its potential uses particularly for therapeutic applications.

A osteogênese imperfeita (OI) é caracterizada como uma desordem genética na qual uma osteopenia generalizada leva a baixa estatura, fragilidade óssea excessiva e deformidades ósseas graves. As células mesenquimais estromais multipotentes (CTMs) são precursores presentes na medula óssea adulta capazes de se diferenciar em osteoblastos, adipócitos e mioblastos que passaram a ter grande importância como fonte terapia celular. O objetivo do presente estudo foi analisar o perfil de expressão gênica durante a diferenciação osteogênica a partir de células mesenquimais estromais multipotentes da medula óssea obtidas de pacientes diagnosticados com Osteogênese Imperfeita e de indivíduos controle. Foram coletadas amostras de três indivíduos normais e cinco amostras de pacientes portadores de Osteogênese Imperfeita. As células mononucleares (CMN) foram isoladas para a obtenção de células mesenquimais que foram expandidas até a terceira passagem quando iniciou-se o estímulo para diferenciação osteogênica. Também foram realizadas análises para contagem de CFU-F e para quatro das cinco amostras de pacientes portadores de OI, o número de CFU-F observado foi inferior ao geralmente encontrado para amostras de doadores normais. Foram coletadas células para análises de imunofenotipagem celular por citometria de fluxo e o RNA foi extraído originando a amostra denominada T0. As garrafas restantes tiveram suas células estimuladas para diferenciação osteogênica. Após um dia em cultura com estímulo, mais uma garrafa teve o RNA de suas células extraído (T1), e o mesmo procedimento foi realizado nos dias 2 (T2), 7 (T7), 12 (T12), 17 (T17) e 21 (T21). Todas as amostras demonstraram possuir potencial de diferenciação in vitro em osteoblastos e adipócitos. A imunofenotipagem de células mesenquimais foi realizada e as amostras de todos os pacientes apresentaram perfil imunofenotípico compatível com trabalhos anteriores. Foram identificadas mutações nos genes COL1A1 e/ou COL1A2 responsáveis pelo desenvolvimento da doença para quatro dos cinco pacientes avaliados. Para o paciente portador de Osteogênese Imperfeita e Síndrome de Bruck a região codificadora do gene PLOD2 também foi seqüenciada, porém não foram encontradas mutações. A análise da expressão gênica foi realizada pela técnica de microarranjos e foram identificados vários genes com expressão diferencial. Alguns genes com importância fundamental na diferenciação osteoblástica apresentaram menor expressão nas amostras dos pacientes portadores de OI, sugerindo um menor comprometimento das CTMs desses pacientes com a linhagem osteogênica. Outros genes também tiveram sua expressão diferencial confirmada por PCR em Tempo Real. Foi observado um aumento na expressão de genes relacionados a adipócitos, sugerindo um aumento da diferenciação adipogênica em detrimento à diferenciação osteogênica. A expressão das variantes do gene PLOD2 mostrou-se diferencial entre amostras normais, de OI e do paciente portador de Síndrome de Bruck. Também foi evidenciada uma expressão diferencial do microRNA 29b, um microRNA com papel estabelecido durante a diferenciação osteogênica, sugerindo um mecanismo de regulação dependente da quantidade de RNAm do seu gene alvo, o COL1A1. / Osteogenesis imperfecta (OI) is characterized as a genetic disorder in which a generalized osteopenia leads to short stature, bone fragility and serious skeletal deformities. Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that can differentiate into osteoblasts, adipocytes and myoblasts that have been given great importance as a source cell therapy. The aim of this study was to analyze the gene expression profile during osteogenic differentiation from mesenchymal stem cells from bone marrow taken from patients diagnosed with Osteogenesis Imperfecta and control subjects. Samples were collected from three normal individuals and five samples from patients with Osteogenesis Imperfecta. Mononuclear cells (MON) were isolated to obtain mesenchymal cells that were expanded until third passage when the stimulus for osteogenic differentiation was induced. Analyses were also conducted to count the CFU-F and for four of the five samples from patients with OI, the number of CFU-F observed was lower than generally found for normal samples. Cells were collected for analysis of cell immunophenotyping by flow cytometry and RNA was extracted from the resulting sample called T0. Remaining cells were stimulated for osteogenic differentiation. After a day in culture with stimulation, cells from another bottle had their RNA extracted (T1), and the same procedure was performed on days 2 (T2), 7 (T7), 12 (T12), 17 (T17) and 21 (T21). All samples have shown potential of in vitro differentiation into osteoblasts and adipocytes. Immunophenotyping of mesenchymal cells was performed and samples of all patients had immunophenotypic profile consistent with previous works. We identified mutations in COL1A1 and / or COL1A2 responsible for developing the disease for four of five patients. For the patient with Osteogenesis Imperfecta and Bruck Syndrome, coding region of the gene PLOD2 was also sequenced, but no mutations were found. The gene expression analysis was performed by microarray and identified several genes with differential expression. Some genes of fundamental importance in osteoblast differentiation showed lower expression in samples from patients with OI, suggesting a minor involvement of MSCs of patients with osteogenic lineage. Other genes also confirmed their differential expression by Real Time PCR. We observed an increased expression of genes related to adipocytes, suggesting an increased adipogenic differentiation at the expense of osteogenic differentiation. The expression of PLOD2 gene variants proved to be different between normal samples, OI and the patient with Bruck Syndrome. There was also evidence of differential expression of 29b microRNA, with established role during osteogenic differentiation, suggesting a mechanism dependent regulation of mRNA abundance of its gene target, COL1A1.

A menopausa é provocada pela falência da produção de hormônios ovarianos e tem como consequências alterações desfavoráveis no metabolismo e perda de massa óssea. O declínio da produção de estrógeno é considerado um grande fator de risco para o desenvolvimento da osteoporose em mulheres e como tratamento faz-se o uso da Terapia de Reposição Hormonal. No entanto, esta terapia tem trazido riscos á saúde de alguns grupos de mulheres. Como alternativa ao tratamento tradicional, tem-se os fitoestrógenos, e com eles as isoflavonas, encontradas principalmente na soja, Trifolium pratense e Cimicifuga racemosa. Este estudo teve como objetivo comparar a capacidade de estimular a osteogênese in vitro, a partir de cultura de osteoblastos derivados de células tronco mesenquimais, em duas preparações de fitoestrógenos: O extrato de soja biotransformado pelo fungo Aspergillus awamori (ESBF), e o Menoflavon® 40mg (Melbrosin International) composto pela isoflavona Trifolium pratense. Para este objetivo foram realizadas: a) Avaliação do crescimento e proliferação celular b) Viabilidade e crescimento das culturas de osteoblastos. c) Dosagem de proteína total das culturas de osteoblastos. d) Determinação da atividade específica da enzima fosfatase alcalina. e) Formação da matriz mineralizada. O Menoflavon® foi testado nas concentrações de 28,75 nM de daidzeína (D) + 7,5 nM de genisteína (G) (0,5 ?g/mL de Menoflavon®); 57,5 nM de D + 15 nM de G (1 ?g/mL de Menoflavon®); e 230 nM de D + 60 nM de G (4 ?g/mL de Menoflavon®); controle padrão de 57,5 nM de D + 15 nM de G comercial e controle de dimetilsulfóxido (DMSO). O ESBF foi testado nas concentrações de 1,181 nM de D + 0,922 nM de G (0,5 ?g/mL de ESBF); 2,361 nM de D + 1,845 nM de G (1 ?g/mL de ESBF); 9,445 nM de D + 7,379 nM de G (4 ?g/mL de ESBF); controle padrão de 2,361 nM de D + 1,845 nM de G comercial e controle de DMSO. Com a metodologia do MTT (3[4,5-dimetiltiazol-2-il]-2,5-brometo difenil tetrazolium) e da Resazurina comprovamos que não houve morte celular nas concentrações testadas com as duas formulações. A dosagem de proteínas totais manteve-se constante com as duas formulações e a formação de matriz mineralizada também manteve-se constante em relação ao controle para ambos. A atividade específica da fosfatase alcalina teve um decréscimo significativo ao 14º dia com todas as concentrações testadas e ao 21º dia com algumas concentrações de Menoflavon e com o ESBF decresceu em alguns dias, no entanto manteve-se estável no restante do teste. O ESBF mostrou ser melhor que o Menoflavon®, já que obtivemos resultados semelhantes e sua concentração é 24 vezes menor. No entanto, o estudo realizado mostrou que tanto o ESBF quanto o Menoflavon® não são capazes de estimular a osteogênese in vitro, a partir de cultura de osteoblastos derivados de células tronco mesenquimais. / Menopause is caused by failure in the production of ovarian hormones and its consequences are unfavorable changes in metabolism and bone loss. The decline in estrogen production is considered a major risk factor for the development of osteoporosis in women, and the Hormone Replacement Therapy is used as treatment. However, this therapy has brought some risks to the health of some groups of women. As an alternative to the traditional treatment, phytoestrogens as isoflavones, found mainly in soy, Trifolium pratense, Cimicifuga racemosa and rye can be used. This study aimed to compare the ability of two preparations of phytoestrogens to stimulate osteogenesis in vitro (from cultures of osteoblasts derived from mesenchymal stem cells): soy extract biotransformed by the fungus Aspergillus awamori (ESBF), and Menoflavon® 40mg (Melbrosin International) composed by the isoflavone Trifolium pratense. With this objective, were used: a) Evaluation of cell growth and proliferation; b) Viability of the cultures of osteoblasts; c) Determination of total protein from the cultures of osteoblasts; d) Determination of the specific activity of the enzyme alkaline phosphatase; e) Formation of mineralized matrix. Menoflavon® was tested at the concentrations of 28.75 nM of daidzein (D) + 7.5 nM of genistein (G) (Menoflavon® 0.5 ?g/mL); 57.5 nM D + 15 nM G (Menoflavon® 1 ?g/mL); and 230 nM D + 60 nM G (Menoflavon® 4 ?g/mL); standard control of commercial 57.5 nM D + 15 nM G and DMSO control. ESBF was tested at the concentrations of 1.181 nM D + 0.922 nM G (ESBF 0.5 ?g/mL); 2.361 nM D + 1.845 nM G (ESBF 1 ?g/mL); 9.445 nM D + 7.379 nM G (ESBF 4 ?g/mL); standard control of commercial 2.361 nM D + 1.845 nM G and dimetilsulfoxide (DMSO) control. With the MTT and Resazurin methods we verified that there was no cell death for all concentrations tested with the two formulations. The amount of total protein remained constant with the two formulations, and the formation of mineralized matrix also were the same as the control. The specific activity of alkaline phosphatase decreased significantly on day 14 for all concentrations tested, and at day 21 for some concentrations of Menoflavon®, with the ESBF decreased some days, however remained constant in the other tests. Therefore, ESBF proved better than Menoflavon®, since we obtained similar results for both, but the concentration of ESBF is 24 times smaller than the concentration of Menoflavon®. However, both the ESBF and the Menoflavon® were not capable of stimulating osteogenesis in vitro from cultures of osteoblasts derived from mesenchymal stem cells.