L'intégrine β1 appartient à une large famille de récepteurs de première importance pour les interactions cellule/matrice extracellulaire. La délétion spécifique d'un régulateur négatif de l'intégrine β1, ICAP-1α, induit de sévères défauts osseux. Nous avons pu montrer que la perte d'ICAP-1α est accompagnée d'une augmentation de l'activité de l'intégrine β1, affectant le dépôt des matrices de fibronectine et de collagène de type I. De plus, nous avons pu montrer qu'ICAP-1α a une action antagoniste sur le recrutement de la kindline-2 au niveau du domaine cytoplasmique de l'intégrine β1 (Brunner et al. JCB 2011). Nous nous sommes ensuite intéressés au rôle de l'intégrine β1 elle-même dans l'ostéogenèse afin de comprendre comment les ostéoblastes intègrent les signaux du microenvironnement pour coordonner la formation et le remodelage osseux. Dans cette optique, nous avons généré un modèle de souris délétées pour l'intégrine β1 spécifiquement dans les ostéoblastes en cours de maturation. Ces souris présentent un sévère phénotype osseux caractérisé par des réductions importantes de la minéralisation et de la dynamique osseuse, ainsi que des déformations osseuses et des fractures rappelant le syndrome d'Ostéoporose Juvénile. L'analyse in vitro d'ostéoblastes n'exprimant pas l'intégrine β1 a révélé un défaut majeur de prolifération impliquant non pas la voie canonique MAPK/ERK mais plutôt un défaut d'activation du co-facteur de transcription YAP. De plus, nous avons pu montrer que les intégrines β1 régulaient le niveau d'AMP cyclique (AMPc) dans les ostéoblastes et que ceci était corrélé à l'inactivation de YAP. De même, nous avons pu relier l'inactivation de YAP à la dynamique d'endocytose des rafts. Finalement, des analyses in vivo et in vitro ont révélé un défaut fonctionnel des ostéoblastes dépourvus d'intégrine β1. Nous avons pu montrer que cette incapacité fonctionnelle était due à une réduction de la réponse au BMP-2, facteur de croissance ostéoblastique majeur, non pas au niveau de son récepteur mais probablement au niveau de l'activation des promoteurs BMP-dépendants. Nos résultats montrent ainsi que l'intégrine β1 est un régulateur clé de la prolifération ostéoblastique dépendante de YAP et de la signalisation BMP régulant la fonction ostéoblastique, la minéralisation et la formation osseuse. / Β1 integrins belong to a large family of receptors that have been shown to be of paramount importance for cell/extracellular matrix interactions. The ablation of the specific β1 integrin regulator ICAP-1α results in severe bone and mineralization defects. By combining mouse and cell biology we could demonstrate that loss of ICAP-1α was accompanied by an increase of β1 integrin activity that affects fibronectin and collagen deposition. Moreover, we could show that ICAP-1 is an important negative regulator of kindlin-2 recruitment on β1 integrin cytoplasmic domain (Brunner et al. JCB 2011). We then wanted to address the functional role of β1 integrin per se in osteogenesis and to understand how osteoblasts integrate environmental cues to coordinate bone formation and remodeling. For this we generated osteoblast specific β1 integrin deficient mice. These mice showed severe bone defects characterized by reduced bone mineralization and dynamic, as well as bending and fractures reminding human Juvenile Osteoporosis symptoms. In vitro analyses of β1 integrin deficient osteoblasts revealed proliferation defect which is not due to defective canonical MAPK/ERK pathway, but rather to defective activity of the co-transcription factor YAP. Then, we showed that β1 integrins are regulating cAMP level in osteoblasts and that the cAMP level correlates with YAP inactivation. We also linked YAP inactivation with raft endocytosis. Finally, in vivo and in vitro analyses revealed a functional incapacity of β1 integrin deficient osteoprecursors. We could show that the lazy phenotype of β1 integrin deficient osteoblasts is likely due to a reduced response to BMP signaling, a major osteoblast growth factor. Taken together, our findings demonstrate that β1 integrin is a key regulator of YAP-dependent osteoblast proliferation and BMP signaling allowing osteoblast functionality, mineralization and bone formation.

As coberturas de hidroxiapatita (HAp) são utilizadas para aumentar a osteointegração em implantes de titânio (Ti), devido à sua capacidade de promover a biomineralização para corrigir defeitos esqueléticos e craniofaciais. O objetivo desta pesquisa foi avaliar a influência dos revestimentos sobre culturas primárias de osteoblastos. Na primeira fase de estudos, desenvolvemos uma nova abordagem de revestimento baseada em filmes Langmuir-Blodgett (LB) de dihexadecilfosfato (DHP) e ácido octadecilfosfônico (OPA) depositados em discos Ti, e crescimento subsequente de cristais de HAp. Analisamos a viabilidade dos osteoblastos, a atividade da fosfatase alcalina (ALP) e a formação da matriz mineralizada por métodos colorimétricos e a morfologia das culturas por microscopia eletrônica de varredura e microscopia confocal. Os resultados revelaram que o revestimento DHP/HAp aumentou a viabilidade dos osteoblastos até 150% em comparação com o controle em todos os dias testados. O revestimento OPA/HAp promoveu a maior viabilidade ao 14 dias (190%). A atividade de ALP foi aumentada apenas pelo revestimento de DHP/HAp ao 14º dia em comparação com o controle e Ti limpo. A microscopia eletrônica de varredura e as microfotografias confocais revelaram diferenças morfológicas entre os osteoblastos cultivados em ambos os revestimentos, aumentando o seu número e o espalhamento. O revestimento de DHP/HAp aumentou a produção de nódulos biomineralizados. O ensaio de biomineralização pela técnica do Vermelho de Alizarina mostrou que o revestimento de OPA/HAp possuía uma concentração de cálcio (Ca2+) 1,88 vezes superior à cobertura de DHP/HAp. Uma vez que a literatura relata que o Ca2+ pode estimular ou inibir a atividade da ALP e, consequentemente, o processo de biomineralização, as diferenças no comportamento desses dois revestimentos podem estar relacionadas às diferenças de concentração de superfície de Ca2+. O bom desempenho do revestimento de DHP/HAp pode estar relacionado às características da composição química, adicionada à técnica de deposição LB. Na segunda fase da pesquisa, as monocamadas de Langmuir de DHP e dipalmitoil fosfatidilcolina (DPPC) foram testadas e utilizadas para incorporar o paratormônio 1-34 (PTH 1-34) (DHP/Ca+PTH e DPPC/Ca+PTH, respectivamente). Também foram testadas as ações dos revestimentos DHP/HAp com PTH em solução (DHP/HAp+PTH S) e gotejado (DHP/HAp+PTH G) em culturas de osteoblastos. Um potencial zeta negativo em pH 7,4 foi encontrado (-14,9 mV) para o PTH 1-34. A isoterma de DPPC mostrou um aumento da área mínima ocupada por molécula lipídica após a injeção de PTH na subfase de água (50 ?L de solução 0,5 mg/mL) em 10,97 Å2, o que pode ser devido à inserção de PTH neste filme. A área mínima de DHP foi alterada em 2,3 Å2, o que não é estatisticamente significativo. A análise de QCM mostrou um depósito de 72,5 ng de PTH em filme de DPPC e 29,3 ng de PTH em filme de DHP para cada 25 ?g de PTH injetado na cuba de Langmuir. A viabilidade celular e a formação da matriz mineralizada de culturas de osteoblastos crescidas em DHP/Ca+PTH e revestimentos DPPC/Ca+PTH diminuíram quando comparadas com Ti limpo. Os revestimentos DHP/HAp+PTH S e DHP/HAp+PTH G mostraram ser tão eficientes quanto o Ti DHP/HAp para estimular o processo de biomineralização. Mas a cobertura de DHP/HAp+PTH G aumentou a viabilidade dos osteoblastos e a formação de matriz mineralizada quando comparada com Ti DHP/HAp. Esta é uma cobertura inovadora que abre precedentes para o uso da técnica de gotejamento em HAp para outros hormônios e drogas que agem sobre o tecido ósseo. / Due to their ability to promote biomineralization, Hydroxyapatite (HAp) coatings are used to increase the osteointegration in titanium (Ti) implants, in order to correct skeletal and craniofacial defects. The objective of the research was to evaluate the influence of the coatings on osteoblasts primary cultures. In the first phase of the research we developed a new coating approach based on Langmuir-Blodgett (LB) films of dihexadecyl phosphate (DHP) and octadecylphosphonic acid (OPA) deposited on Ti discs and subsequent growth of HAp crystals. We analyzed the osteoblast viability, alkaline phosphatase (ALP) activity and mineralized matrix formation by colorimetric methods, and the morphology of the cultures by scanning electron microscopy and confocal micrographies. The results revealed that the DHP/HAp coating increased osteoblast viability up to 150% compared to the control at all days tested. The OPA/HAp coating promoted the highest viability on the 14th day (190%). The ALP activity was enhanced only by the DHP/HAp coating on the 14th day compared to control, and clean Ti. To explore the morphology of the cells, the scanning electron microscopy and confocal micrographies were obtained, and revealed morphological differences between osteoblasts grown on both coated Ti compared to clean Ti. Both coatings increased the number and spreading of osteoblasts, while the DHP/HAp coating enhanced the production of biomineralized nodules. The Alizarin Red assay showed that OPA/HAp coating has 1.88 times higher calcium (Ca2+) concentration than DHP/HAp. The same test confirmed the increase of mineralization only by DHP/HAp coating compared to clean Ti. Since literature reports that Ca2+ can stimulate or inhibit the ALP activity and consequently, the biomineralization process, the differences on the behavior of these two coatings could be related to the Ca2+ surface concentration differences. The good performance of the DHP/HAp coating can be explained due to the characteristics of the chemical composition, added to the LB deposition technique. In the second phase of the research, Langmuir monolayers of DHP and dipalmitoyl phosphatidylcholine (DPPC) was tested and used to incorporate 1-34 parathyroid hormone (PTH 1-34) (DHP/Ca+PTH, and DPPC/Ca+PTH, respectively). DHP/HAp coatings with PTH in solution (DHP/HAp+PTH S), and dropped (DHP/HAp+PTH G) also were tested on osteoblasts cultures. A negative zeta-potential at pH 7.4 was found (-14.9 mV) to PTH 1-34. The Langmuir isotherm of DPPC showed an increase of the minimum area occupied per lipid molecule after the PTH injection into the water subphase (50 ?L of 0.5 mg/mL solution) by 10.97 Å2, which could be due to the insertion of PTH in this film. The DHP minimum area changed by 2.3 Å2, which is not statistically significant. The QCM analysis showed the deposit of 72.5 ng of PTH on DPPC film, and 29.3 ng of PTH on DHP film for each 25 ?g of PTH injected into the Langmuir trough. The cell viability and matrix mineralization of osteoblasts cultures grown on DHP/Ca+PTH, and DPPC/Ca+PTH coatings decreased when compared to clean Ti. DHP/HAp+PTH S and DHP/HAp+PTH G coatings proved to be as efficient as Ti DHP/HAp to stimulate the biomineralization process. But DHP/HAp+PTH G increased the osteoblast viabilitiy and formation of mineralized matrix when compared to Ti DHP/HAp. This is an innovative coating that sets the precedent for the use of the drip technique on HAp for other hormones and drugs that act on bone tissue.

Materiais vítreos e vitrocerâmicos bioativos podem ser usados particulados ou como scaffolds em diferentes tratamentos de defeitos ósseos. Tratamentos térmicos que possibilitam o desenvolvimento de scaffolds a partir de composições de vidros bioativos introduzem fases cristalinas em sua estrutura amorfa com potencial impacto na bioatividade e biocompatibilidade do material. O objetivo do presente estudo foi avaliar, qualitativa e quantitativamente, o desenvolvimento do fenótipo osteogênico de culturas de células osteoblásticas sobre substratos vítreos e vitrocerâmicos bioativos. Células MC3T3-E1 foram cultivadas em condições osteogênicas por períodos de até 21 dias sobre superfícies de Bioglass® 45S5, de duas preparações de vitrocerâmica bioativa e altamente cristalina, Biosilicato® e Biosilicato® para scaffold, e de borosilicato (vidro bioinerte). Foram avaliados, nos períodos de 7, 12 e 21 dias, morfologia celular, formação de matriz mineralizada e expressão de genes relacionados à osteogênese. Os resultados mostraram confluência das culturas sobre as superfícies de vidros e vitrocerâmicas, com progressiva formação de multicamadas celulares. A quantificação de vermelho de Alizarina revelou aumento de mineralização para culturas sobre materiais bioativos, com os maiores valores para Biosilicato® para scaffold. Expressão diferencial de genes foi observada nos 3 períodos de culturas sobre os materiais vítreo e vitrocerâmicos bioativos em comparação ao vidro bioinerte e sobre as vitrocerâmicas em comparação ao vidro bioativo. Os resultados permitem concluir que modificações em aspectos químicos de materiais vítreos e vitrocerâmicos, com efeitos sobre sua bioatividade, resultam em alteração do potencial osteogênico e do perfil de expressão gênica de células osteoblásticas in vitro. A maior atividade osteogênica sobre o Biosilicato® para scaffold permite considerar esse material um potencial candidato para aplicações em defeitos ósseos. / Bioactive glasses and glass-ceramics have been used as bone substitutes in either particulate or scaffold forms. Various thermal treatments that allow the development of scaffolds from bioactive glasses may create varied proportions of new crystalline phases in the amorphous phase with a potential impact on the bioactivity and biocompatibility of the material. The aim of the present in vitro study was to qualitatively and quantitatively evaluate the development of the osteogenic phenotype in osteoblastic cell cultures grown on bioactive glass and glass-ceramic surfaces. MC3T3-E1 cells, subclone 14, were cultured under an osteogenic condition for periods of up to 21 days on the following disc surfaces: Bioglass® 45S5 (bioactive glass), Biosilicate® (bioactive glass-ceramic), Biosilicate® as the material for scaffold preparation (Bio-sc, bioactive glass-ceramic), and borosilicate (bioinert glass). At days 7, 12, and 21 post-plating, cell morphology, mineralized matrix formation and the expression profile of genes associated with osteogenesis were evaluated. Epifluorescence of actin cytoskeleton and DAPI DNA stain revealed confluent cell cultures at day 7 for all groups, with progressive cell multilayering formation. The quantitative analysis of Alizarin red-stained cultures at day 21 revealed significantly enhanced mineralization in cultures grown on bioactive materials compared with the ones on borosilicate and the highest absorbance intensities for the Bio-sc group. Differential gene expression profiles were detected at the three time points evaluated in cultures grown on the bioactive materials in comparison with borosilicate, and on the glass-ceramics in comparison with Bioglass® 45S5. From the results presented, it can be concluded that changes in chemical characteristics of glass and glass-ceramic that may have an impact on their bioactivity index can affect the osteogenic potential and the gene expression profile of osteoblastic cells in vitro. The highest osteogenic activity on Bio-sc renders this material a good candidate for bone defect applications.

O crescimento, desenvolvimento e manutenção do tecido ósseo são processos altamente regulados. Diversas proteínas como hormônios, fatores de crescimento e citocinas estão envolvidas nestes processos e exercem atividade direta sobre células osteoblástica e osteoclástica, atuando em sua diferenciação e ativação metabólica. O processo de regeneração óssea é iniciado por fatores estimuladores locais como as proteínas morfogenética óssea (BMP Bone Morphogenetic Proteins). As BMPs são um produto do metabolismo dos osteoblastos, odontoblastos e de várias células tumorais, sendo armazenadas na forma de concentrados no osso, dentina e em células neoplásicas do osteossarcoma e de certos tumores odontogênicos, tais como: fibroma cementificante, cementoblastoma benigno, dentinoma, fibroma odontogênico e odontoma. Esclarecer os mecanismos que controlam a remodelação óssea é uma questão bastante relevante. Nesse sentido, as células-tronco mesenquimais têm despertado grande interesse devido ao seu potencial envolvimento no processo de reparo tissular. A obtenção de osteoblastos funcionais a partir de células-tronco mesenquimais tem sido utilizada na engenharia de tecidos e terapia celular. Desse modo, no presente trabalho foi realizada uma análise proteômica das proteínas envolvidas nas diversas fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea de rato Wistar e humana, no sentido de obter maiores informações sobre a diferenciação celular e a biologia do tecido ósseo. Células-tronco mesenquimais obtidas de medula óssea foram cultivadas em meio osteogênico por diferentes períodos para obter células em diversas fases da diferenciação osteoblástica. Para análise proteômica foram utilizadas ferramentas como a estratégia de shotgun proteomics e quantificação relativa (iTRAQ - Isobaric Tag for Relative and Absolute Quantitation) para separação de proteínas e a espectrometria de massas para a identificação e quantificação relativa de proteínas e peptídeos. Neste contexto, os nossos resultados nos levam a concluir que: as CTMs de medula óssea de rato Wistar expressam genes que estão envolvidos na diferenciação osteogênica quando estimuladas in vitro formando matriz óssea no período de 14 dias, ou seja, o fator estimulante no microambiente é de fundamental importância; as CTMs de medula óssea humana apresentaram resultados semelhantes com as CTMs de ratos em nível genômico durante a diferenciação osteogênica, entretanto quando estimuladas in vitro formaram a matriz óssea no período de 21 dias; utilizando duas abordagens proteômicas, foi possível identificar proteínas importantes que estão envolvidas no processo de diferenciação. Mas cabe salientar que, embora tenham sido detectados genes que parecem envolvidos no processo de diferenciação, isso não teve reflexo no proteoma dessas células nos períodos de 7 e 14 dias da indução de diferenciação à osteogênese, o que indica que a maior parte da funcionalidade dessas células quanto aos outros processos biológicos estão preservados, como por exemplo a proliferação celular permaneceu sem grandes alterações. Isso indica que manipulações de isolamento, cultivo e indução da diferenciação dessas células não afetaram o proteoma, com aspectos positivos para a utilização de células-tronco mesenquimais em terapia celular. Do ponto de vista metodológico, esse trabalho abre perspectivas da utilização de estratégias proteômicas baseadas na marcação por isóbaros em combinação com separação de proteínas por eletroforese unidimensional SDS-PAGE para a análise de amostras biologicamente complexas e de quantidades limitadas de obtenção como células-tronco mesenquimais. O estudo da expressão de proteínas durante as fases de diferenciação osteoblástica de células-tronco mesenquimais de medula óssea deve refletir seu estado funcional e contribuir para o entendimento das diversas vias envolvidas no processo de diferenciação. / The growth, development and maintenance of bone tissue are highly regulated processes. Several proteins such as hormones, growth factors and cytokines are actively involved in these processes and exert direct activity on osteoblastic and osteoclastic cells, acting in their differentiation and metabolic activation. The process of bone regeneration is initiated by local stimulating factors as bone morphogenetic proteins (BMP). BMPs are a product of the metabolism of osteoblasts, odontoblasts and various tumor cells and is stored in the form of concentrates in bone, dentin and neoplastic cells of osteosarcoma and certain odontogenic tumors such as fibroma cementifying, cementoblastoma benign dentinoma, odontogenic fibroma and odontoma. Clarify the mechanisms that control bone remodeling is a very relevant issue. Accordingly, the mesenchymal stem cells have attracted great interest because of its potential involvement in the process of tissue repair. Obtaining functional osteoblasts from mesenchymal stem cells has been used in tissue engineering and cell therapy. Thus, this present work performed a proteomic analysis of proteins involved in various stages of osteoblast differentiation of mesenchymal stem cells from bone marrow of Wistar rat and human, in order to obtain more information on the biology of cell differentiation and bone tissue. Mesenchymal stem cells obtained from bone marrow were cultured in osteogenic medium for different periods to obtain cells at different stages of osteoblast differentiation. For proteomics analysis tools were used as the strategy of shotgun proteomics and relative quantification (iTRAQ - isobaric Tag for Relative and Absolute quantitation) for protein separation and mass spectrometry to identify proteins. In this context, our results take us to conclude that the MSCs of Wistar rat bone marrow express genes that are involved in osteogenic differentiation in vitro when stimulated to form bone matrix during the 14 days, ie stimulating factor in the microenvironment is of fundamental importance, the MSCs from human bone marrow showed similar results with rat MSCs at the genomic level during osteogenic differentiation, however, when stimulated in vitro formed bone matrix within 21 days, using two proteomic approaches, we could identify proteins important that are involved in the process of differentiation. But it should be noted that although it has been identified genes that seem involved in the process of differentiation, it was not reflected in the proteome of these cells at 7 and 14 days after induction of the osteogenic differentiation, indicating that most of the functionality of these cells and other biological processes are preserved, such as cell proliferation remained without major changes. This indicates that manipulations of isolation, culture and induction of differentiation of these cells did not affect the proteome, with positive aspects to the use of mesenchymal stem cells in cell therapy. From the methodological point of view, this work opens up the use of proteomic strategies based on the score for isobars in combination with protein separation by electrophoresis, one-dimensional SDS-PAGE for the analysis of complex biological samples and limited quantities of production as mesenchymal stem cells. The study of protein expression during stages of osteoblast differentiation of mesenchymal stem cells from bone marrow should reflect their functional status and contribute to the understanding of pathways involved in the process of differentiation.

Eventos como fosforilação e desfosforilação estão presentes nos processos de crescimento e diferenciação celular. As proteínas tirosina fosfatases estão envolvidas nestes processos. Estas enzimas são encontradas em animais, plantas e ocorrem em diversas formas, diferindo no peso molecular, substrato específico e sensibilidade a inibidores. As enzimas que possuem baixo peso molecular (entre 18-20 KDa), hidrolisam p-nitrofenilfosfato e são sensíveis ao p-hidroximercuribenzoato são chamadas como proteínas tirosina fosfatases de baixo peso molecular relativo (PTP-BMr) ou fosfatases ácidas. Vários dados sugerem que tipos de células de osso, como osteoblastos, podem expressar esta enzima ativa. Aqui, culturas de osteoblastos derivadas da medula removida do fêmur de rato foram investigadas para padronizar a metodologia de obtenção da PTP-BMr. A expressão e atividade catalítica desta enzima em diferentes estágios de crescimento de osteoblastos também foram verificadas. Foi observado que são necessários de 16-19 dias de cultura para obter maiores níveis de atividade da PTP-BMr em extrato citoplasmático. O nível de expressão do gene da PTP-BMr foi determinado por PCR em tempo real e uma maior quantificação de RNAm foi obtida em 16 dias de crescimento de osteoblasto. A caracterização bioquímica parcial confirma uma banda de atividade em gel de poliacrilamida com peso molecular de 17,6 KDa, e pH ótimo de 5,5. A hidrólise do p-nitrofenilfosfato demonstra uma pequena cooperatividade relativa (n=1,2) com K0,5= 0,12 e Vmax= 3,5U/mg. Esta atividade foi fortemente inibida (60 a 75%) por molibdato de amônio (10mM); fosfato de sódio (10mM); ortovanadato de sódio (10mM) e p-hidroximercuribenzoato de sódio (10mM). Estas propriedades são típicas desta classe. A interação entre osteoblastos e diferentes superfícies pode ativar ou desativar genes. Este presente projeto investigou se a interação com superfície de titânio pode modificar o perfil de atividade da PTP-BMr. Quando em presença de uma superfície de titânio há um maior aumento na atividade catalítica do que nos níveis de RNAm da PTP-BMr o que sugere uma estimulação da enzima a qual pode estar auxiliando no processo de formação óssea. / Events as phosphorilation and desphosphorilation are experienced in cell growth and differentiation processes. Tyrosine phosphatases proteins are involved in these processes. These enzymes are found in animals, plants and occur in multiple forms, differing in relative molecular weight, substrate specificity and sensitivity to inhibitors. The enzymes that have low molecular weight (between 18-20KDa), hydrolize p-nitrophenilphosphate and are sensible to p-hidroximercuribenzoate are known as low molecular weight relative tyrosine phosphatase proteins (PTP-LMWr) or acid phosphatase. Several data suggest that bone cell types, such as osteoblasts, may express this enzyme activity. Here, osteosblast cultures derived from medulla removed from rat femur were investigated to standardize the methodology of PTP-LMW obtainment. The expression and the catalytic activity of this protein in different stages of osteoblast growth were checked as well. It was observed that 16-19 culture days are necessary to obtain greater levels of activity of PTP-LMW in the cytoplasmatic extracts. The gene expression level of PTP-LMW was determined by quantitative real-time PCR, and a greater quantity of mRNA was observed within sixteen days of osteoblast growth. The partial biochemistry characterization confirms a single active band in poliacrilamide gel with molecular weight of about 17.6KDa, and a pH-optimum around 5.5. p-nitrophenilphosphate hydrolysis shows a small cooperative behavior (n=1.2) with K0.5=0.12mM and Vmax=3.5U/mg. This activity was strongly inhibited (about 60-95%) by ammonium molybdate (10mM); sodium phosphate (10mM); sodium orthovanadate (10mM) and sodium p-hydroximercuribenzoate (10mM). These properties are typical for this enzyme class.

Abstract Bone remodelling occurs in humans throughout life, therefore bone is continuously renewed to better respond to changes in weightbearing circumstances. Bone remodelling is extremely vulnerable during fracture healing and integration of prostheses into the surrounding bone. Bone remodelling is a complex system in which many growth factors, cytokines and enzymes, which are essential for the differentiation of osteoblasts and osteoclasts, are involved. Some widely used drugs can affect this sensitive system of remodellation in unexpected manner. Painkillers such as cyclooxygenase (COX) inhibitors have been demonstrated in animal studies to interfere with fracture healing and a few retrospective clinical studies confirm these observations. Bisphosphonates (BP), main target of which is the bone resorbing osteoclast, have been suggested to be the drug of choice to improve periprosthetic bone density and thus prevent aseptic loosening of implants. The exact mechanism of action of clodronate (CLO), a non-amino-BP, which was selected for the study, has not been clarified thus far. In order to gain a deeper understanding of the role of the COX enzyme in the differentiation of osteoblasts we studied human mesenchymal stem cell (hMSC) cultures in the presence of different COX-inhibitors; indomethacine, parecoxib and NS398, a specific COX-2 inhibitor. We used the liposome encapsulated CLO metabolite (AppCCl2p) to study in detail the mechanism of BP induced apoptosis in osteoclast. The effects of different BPs CLO, pamidronate (PAM) and zoledronic acid (ZOL), on the differentiation of osteoblasts and osteoclasts were tested in vitro. The optimal concentration for in situ CLO rinsing in clinical study was found. Finally, the effects of in situ and per oral CLO on the periimplant bone density and integration of prostheses were studied in vivo. All tested COX-inhibitors significantly inhibited osteoblast differentiation from hMSCs and stimulated the differentiation of adipocytes. It was also demonstrated that AppCCl2p inhibits mitochondrial function by a mechanism that involves competitive inhibition of ADP/ATP translocase. In the comparison of BPs, ZOL seemed to posses the properties of both non-amino- and amino-BPs and it thus belongs to a new class of BPs. Peroral and in situ CLO seemed to have different mechanisms of action. Peroral CLO delayed the integration of prosthesis to the bone and increased peri-implant osteolysis while is situ CLO accelerated integration. In conclusion, we can alter normal bone remodellation during fracture healing and prosthesis integration. On the other hand, we can also improve the circumstances for the integration of implant to the surrounding bone by in situ BP rinsing, thus creating a better environment for bone ingrowth. / Tiivistelmä Läpi elämän luustossa tapahtuu uudelleenmuotoutumista, remodelaatiota, jonka seurauksena luu pystyy paremmin vastaamaan muuttuneisiin kuormitusolosuhteisiin. Remodelaatioprosessi on hyvin haavoittuvainen murtuman luutumisen aikana sekä proteesin kiinnittyessä ympäröivään luuhun. Luun remodelaatioon osallistuvat kasvutekijät, sytokiinit ja entsyymit, jotka puolestaan ovat välttämättömiä osteoblastien ja osteoklastien erilaistumiselle. Monet lääkeaineet voivat yllättävällä tavalla vahingoittaa tätä herkkää remodelaatiosysteemiä. Kipulääkkeet, kuten syklo-oksygenaasi (COX) estäjät, voivat häiritä murtuman luutumista aikaisempien eläintöiden ja muutamien retrospektiivisten potilastutkimusten mukaan. Lisäksi bisfosfonaatit, joiden päävaikutuskohde on luuta hajoittava osteoklasti, voisivat olla lupaavia lääkkeitä myös parantamaan proteesia ympäröivän luun laatua ja siten estämään aseptista implantin irtoamista. Tutkimuksen yhtenä tarkoituksena oli selvittää klodronaatin, ensimmäisen polven typpi-ryhmää sisältämättömän bisfosfonaatin tarkka vaikutusmekanismi. Viljelemällä ihmisen luuytimen kantasoluja indometasiinia, parekoksibia tai spesifistä COX-2 estäjää NS 398:a, sisältävässä kasvatusliuoksessa selvitettiin COX-entsyymin merkitys osteoblastien erilaistumiselle. Liposomien sisälle pakattua klodronaatin metaboliittia (AppCCl2p) käytettiin tutkittaessa millä vaikutusmekanismilla klodronaatti aiheuttaa osteoklastien apoptoosin. Bisfosfonaattien; klodronaatin, pamidronaatin ja tsoledronaatin vaikutusta osteoklastien ja osteoblastien erilaistumiseen tutkittiin soluviljelmämallissa ja määritettiin kliinisessä potilastyössä paikallisesti käytettävän klodronaattiliuoksen pitoisuus. Lopuksi potilastyössä selvitettiin paikallisen klodronaattihuuhtelun ja suun kautta annostellun klodronaatin vaikutus proteesia ympäröivän luun tiheyteen ja proteesin kiinnittymiseen ympäristöönsä. Tutkimukseen valitut COX-estäjät vähensivät ihmisen kantasolujen erilaistumista osteoblasteiksi ja lisäsivät erilaistumista rasvasoluiksi. Lisäksi todettiin, että AppCCl2p estää mitokondrioissa tapahtuvaa hengitystä estämällä ADP/ATP-vaihtajan toiminnan, saaden aikaan solukuoleman. Vertailtaessa bisfosfonaatteja, tsoledronaatilla vaikutti olevan sekä ensimmäisen, että kolmannen polven (sisältää typpi-ryhmän) bispfosfonaattien vaikutuksia, joten tsoledronaatti kuuluu aivan uuteen bisfosfonaattiryhmään. Potilastutkimuksessa suun kautta ja paikallisesti reisiluun ytimeen annostellulla klodronaatilla oli täysin erilainen vaikutus. Suun kautta syötynä klodronaatti hidasti proteesin kiinnittymistä ja aiheutti osteolyysiä. Sen sijaan paikallinen klodronaatti nopeutti merkittävästi proteesin kiinnittymistä ympäröivään luuhun. Näiden tutkimustulosten perusteella voidaan olettaa, että COX-estäjät, samoin kuin peroraalinen bisfosfonaatti, voivat tahattomasti häiritä luun remodelaatiota.

Les infections ostéo-articulaires à Staphylococcus aureus sont des pathologies fréquentes dont les conséquences peuvent aller de la simple altération cellulaire à un retard de la réparation osseuse ou une réponse inflammatoire excessive. Afin d’étudier ce phénomène, nous avons, dans un premier temps, développé deux modèles d’infections in vitro faisant interagir Staphylococcus aureus et des cellules osseuses primaires issues d’explants chirurgicaux humains. Ces cellules ont été préalablement cultivées dans un milieu standard ou un milieu ostéogénique afin d’obtenir 2 populations à des stades de maturation différents. L’étude de l’internalisation de Staphylococcus aureus, de la mortalité cellulaire et de la production de médiateurs inflammatoires pour ces 2 populations a permis d’établir si l’impact de Staphylococcus aureus variait en fonction de la maturation cellulaire. Dans un second temps, nous avons étudié l’impact de Staphylococcus aureus sur des cellules souches mésenchymateuses dérivées du cordon ombilical. En effet, dans le cadre d’une régénération osseuse en site infecté, les cellules souches mésenchymateuses pourraient être amenées à interagir avec Staphylococcus aureus. Nous avons donc caractérisé la capacité de ces cellules à internaliser Staphylococcus aureus, à survivre face à l’infection et à produire des médiateurs inflammatoires dans notre modèle in vitro d’infection aiguë. Ce projet nous a permis de valider nos modèles d’infection in vitro et de caractériser l’impact de Staphylococcus aureus sur différentes cellules du microenvironnement osseux, donnant ainsi de nouvelles pistes pour le développement de stratégies pour la lutte antibactérienne et l’ingénierie tissulaire osseuse. / Staphylococcus aureus-related bone and joint infections are common diseases whose consequences can range from simple cell damage to delayed bone repair or excessive inflammatory response. To study this phenomenon, we have developed two models of in vitro infection with Staphylococcus aureus and primary bone-forming cells derived from human surgical explants. These cells have been previously cultured in a standard medium or osteogenic medium to obtain two populations at different stages of maturation. The study of Staphylococcus aureus internalization, cell death and production of inflammatory mediators in these 2 populations allowed us to establish whether the impact of Staphylococcus aureus varied depending on cell maturation. We also studied the impact of Staphylococcus aureus on mesenchymal stem cells derived from umbilical cord. In case of bone regeneration in infected site, mesenchymal stem cells may have to interact with Staphylococcus aureus. Therefore, we characterized the ability of these cells to internalize Staphylococcus aureus, to survive against the infection and to produce inflammatory mediators in our in vitro model of acute infection. This project allowed us to validate our in vitro infection models and to characterize the impact of Staphylococcus aureus on different cells in the bone microenvironment, providing new approaches for the development of antibacterial strategies and bone tissue engineering.

Des résultats d’expériences cliniques de reconstruction de l’os maxillaire faites à partir de la greffe d’une "pâte osseuse" gélifiée par l’ajout de facteur tissulaire ont été le primum movens de ce travail. Cette "pâte osseuse", faite d’os en poudre et de plasma enrichi en plaquette (PRP) à laquelle on ajoute du facteur tissulaire, est un modèle à la fois de la coagulation et de la régénération osseuse.<p>Pour analyser des effets de la coagulation, nous avons utilisé un modèle connu :la culture primaire de cellules endothéliales (HUVEC). Les effets in vitro des facteurs de coagulation, dénommés protéases de la coagulation, pris séparément, ont été bien étudiés dans ces cellules, néanmoins aucune information sur l’effet combiné de ces protéases ou du plasma en coagulation n’était connue. Nous avons mesuré la "signalisation calcium" comme réponse cellulaire aux différents agents et ces mesures de la signalisation calcium ont été complétées par la mesure d’une autre réponse biologique, à savoir la sécrétion de cytokines pro-inflammatoires (IL-6 et IL-8). Pour l’étude de la régénération osseuse, la signalisation calcium a été mesurée sur une lignée d’ostéosarcomes humains (SaOS-2), stimulée par des protéases de la voie extrinsèque de la coagulation (facteur VIIa, facteur Xa et thrombine). Comme réponse biologique complémentaire, nous avons évalué l’effet des protéases d’intérêt sur l’apoptose induite par l’absence de sérum dans le milieu de culture.<p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

L'interleukine-17A (IL-17A) et le facteur de nécrose tumorale alpha (TNF-α) sont des cytokines pro-inflammatoires impliquées dans la pathogénèse de plusieurs maladies articulaires. Au cours de la polyarthrite rhumatoïde (PR), une augmentation de la destruction osseuse ainsi qu'un defaut de réparation sont responsables des dommages articulaires. Cependant au cours de la spondylarthrite ankylosante (AS), une importante ossification ectopique est observée, conduisant à la formation de syndesmophytes, associé à une perte de la masse osseuse systémique. Récemment, l'étude de ces cytokines a conduit à la publication de résultats contradictoires. Notre objectif a donc été d'étudier l'effet de ces deux cytokines sur la différenciation ostéogénique de cellules souches mésenchymateuses humaines isolées (hMSCs) et de fibroblastes de la membrane synoviale (FLS). Tous les modèles de cellules utilisés, ont démontré que l'IL-17A et le TNF-α augmentent de manière synergique l'ostéogénèse. Ceci semble se rapprocher du modèle de l'AS où une formation d'os ectopique est observée dans laquelle l'IL-17A et le TNF-α jouent un rôle majeur. En parallèle, ces deux cytokines stimulent localement les ostéoclastes, entraînant une perte de masse osseuse observée à la fois dans la PR et dans l'ostéoporose. Cibler simultanément l'IL-17A et le TNF-α pourrait conduire à une diminution de l'infiltration de cellules et de la destruction articulaire observée dans la PR et pourrait ainsi réduire les effets des FLS PR sur l'activation de l'ostéoclastogénèse / Interleukin-17A (IL-17A) and tumor necrosis factor alpha (TNF-α) are pro-inflammatory cytokines involved in the pathogenesis of several arthritic diseases. In rheumatoid arthritis (RA), joint damage is a result of an increase in bone destruction and a decrease in bone repair. In contrast, in ankylosing spondylitis (AS), a bone mass loss accompanied by a significant ectopic ossification is observed leading to the formation of syndesmophytes. Recent studies led to contradictory findings regarding the role of IL-17A and TNF-α in arthritic disease. Therefore, our objective was to study the effect of these two cytokines on the osteogenic differentiation of isolated human mesenchymal stem cells (hMSCs) and fibroblasts of the synovial membrane (FLS). In all the cell models used, we demonstrated that Il-17A and TNF α synergistically increase osteogenesis. This seems to approach the model of AS where ectopic bone formation is observed and in which IL-17A and TNF-α both are involved. These cytokines stimulate osteoclasts locally resulting in loss of bone mass observed in both RA and osteoporosis. Thus, targeting IL-17A and TNF-α could lead to a decrease in cell infiltration and joint destruction which is observed in RA and may reduce the effects of RA FLS on the activation of osteoclastogenesis

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-&#946; (Transforming Growth Factor &#946; ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto \"in vitro\" como \"in vivo,\" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação \"in vitro\" e formação de osso \"in vivo\", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil. / The BMPs (Bone Morphogenetic Proteins) are members of the TGF-&#946; (Transforming Growth Factor &#946;) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction \"in vitro\" and \"in vivo\" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation \"in vitro\" and bone formation \"in vivo\" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil.