The overall objective of this work was to assess the suitability of poly(3-octylthiophene) (P3OT) to sustain MC3T3-E1 osteoblast attachment and growth. The central hypothesis was that specific P3OT film properties (i.e., thickness, film preparation conditions, and level of doping) are able to regulate osteoblast functions (i.e., attachment and proliferation). Discrete and combinatorial techniques were utilized to prepare and characterize thin films of P3OT, a semiconductor in its undoped state, and to study its interaction with MC3T3-E1 osteoblasts. In this work we demonstrate that P3OT is a suitable surface to sustain MC3T3-E1 attachment and proliferation with no observed cytotoxicity. We show that P3OT has an effect on MC3T3-E1 attachment and proliferation as area, circularity, and proliferation ratio are significantly different for P3OT compared to control surfaces. We also demonstrate that P3OT doping and film preparation conditions have an effect on osteoblast attachment and proliferation but that thickness over a low and high range does not affect osteoblast functions. This work is significant because it contributes to the growing area of conducting polymers in biomedical applications and establishes P3OT as a potential cell substrate that sustains MC3T3-E1 attachment and promotes high levels of cell proliferation.

Le syndrome d’Andersen est une maladie rare et associée à la perte de fonction du canal potassique Kir2.1. Afin d’étudier sa physiopathologie, nous avons généré et caractérisé des cellules souches pluripotentes induites (iPS) contrôle et Andersen. Nous avons ensuite différencié ces cellules iPS en cellules souches mésenchymateuse (MSC). Les cellules MSC de patients présentent une capacité de différenciation en ostéoblastes et en chondrocytes diminuée par rapport aux cellules contrôle. En effet, la production de matrice extracellulaire et l'expression des master gènes des différenciations osseuses et cartilagineuses, est réduite chez les patients. Ces travaux de thèse montrent que le canal Kir2.1 est essentiel au développement osseux. Les défauts de différentiation observés pourraient expliquer les dysmorphies associées avec le syndrome d’Andersen. / Andersen's syndrome is a rare disorder associated with a Kir2.1 potassium channel loss of fuction. To study the pathophysiology, we have generated and characterized induced Pluripotent Stem cells (iPS) from control and patient cells. We have then differentiated those iPS cells into mesenchymal stem cells (MSC). Patient's MSc have a lower osteoblastic and chondrogenic differnciation ability compared to control cells. Indeed, extracellular matrix production and master gene expression of osteoblastic and chondrogenic differenciation are reduced in patient’s cells. Alltogether, these results shown that Kir2.1 channel is required for bone developement. The differenciation defects saw in patient cells could explain the Andersen's syndrome associated dysmorphies.

Made available in DSpace on 2014-06-11T19:31:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-04Bitstream added on 2014-06-13T20:02:12Z : No. of bitstreams: 1 paulo_ao_dr_arafo.pdf: 1640675 bytes, checksum: 92907baf8888322bc6931c68024509f2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi avaliar a associação de células derivadas de periósteo autógeno (CDPA) e xenoenxerto de hidroxiapatita e colágeno I (HA/Col) no reparo de defeito crítico de crânio de ratos. CDPA de 10 ratos Wistar foram semeadas na densidade de 1,0x106 células sobre discos de HA/Col (8x2mm) em DMEM:HAMF12 com 10% soro fetal bovino e dexametazona, ácido ascórbico e glicerofosfato por 6 dias. Ensaio funcional comparou efeito de coágulo sanguíneo (G1), osso autógeno (G2), HA/Col (G3) e HA/Col+CDPA (G4) preenchendo defeito de 8 mm de crânio de ratos (n=40) em 1 e 3 meses. A análise radiográfica não exibiu variação temporal, tendo G1 e G2 discreto novo osso marginal; radiopacidade dos materiais em G2, G3 e G4 impediu confirmar osteogênese central. A análise histológica dos grupos mostrou em G1 tecido conjuntivo denso e ilhas de ossificação (1-3m), em G2 coalescência do material e osteogênese (1-3m), em G3 ossificação intramembranosa (1m) e novo osso homogêneo ao redor e substituindo HA (3m) e em G4 abundante tecido conjuntivo frouxo permeando material (1m) e novo osso heterogêneo (3m); não houve necrose, inflamação crônica ou exuberância de células gigantes tipo corpo estranho. As análises histomorfométrica e estatística mostraram diferença signif icativa (p<0,05) para biomaterial (1m) de G2 (23,3%) com G3 (44,6%) e G4 (47,5%) e para ganho ósseo (3m) de G2 (10,9%) com G1 (4,6%) e G4 (5,1%), tendo G3 (8,5%) neoformação óssea próxima a G2. Conclui-se que CDPA não aumentam formação óssea se associadas a HA/Col. / The aim of this study was evaluate the association of autogenous periosteum derived cells (APDC) and xenograf t made up hydroxyapatite and collagen I (HA/Col) in repair of critical size defect in rat calvaria. APDC of 10 Wistar rats were seeded with density of 1,0x106 cells above discs of HA/Col (8x2mm) in DMEM:HAMF12 with 10% fetal bovine serum and dexamethasone, ascorbic acid and glicerophosphate until 6 days. Functional assay compared ef fect of blood clot (G1), autogenous bone (G2), HA/Col (G3) and HA/Col+CDPA (G4) f illing the 8mm-defect in rat skull (n=40) at 1 and 3 months. Radiographic analysis did´t have time variation, with G1 and G2 showing mild peripheral new bone; radiopacity of materials in G2, G3 and G4 didn’t enable to conf irm central osteogenesis. Histologic analysis of groups showed in G1 dense connective tissue and bone islets (1-3m), in G2 fusion of material and osteogenesis (1-3m), in G3 intramembranous ossification (1m) and homogeneous new bone around and substitute HA (3m) and in G4 abundant loose connective tissue permeating the material (1m) and heterogeneous new bone (3m); there is not necrosis, chronic inf lammation or exuberance of giant cell like foreign body reaction. Histomorphometric and statistic analysis showed signif icative dif ferences (p<0.05) for biomaterial (1m) f rom G2 (23,3%) to G3 (44,6%) and G4 (47,5%) and for bone gain (3m) f rom G2 (10,9%) to G1 (4,6%) and G4 (5,1%), with G3 (8,5%) showing new bone formation closer to G2. It’s possible to conclude that APDC don’t increase bone formation if associate to HA/Col.

Made available in DSpace on 2014-06-11T19:23:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-01Bitstream added on 2014-06-13T18:19:48Z : No. of bitstreams: 1 pereira_ccs_me_araca.pdf: 1524857 bytes, checksum: 29dbf40d228058d4769e3d3ef3821153 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O propósito desse trabalho foi avaliar o efeito da osteotomia para implantes sobre a viabilidade celular óssea imediata, comparando a utilização de fresas do sistema convencional com a piezocirurgia em tíbias de coelhos. Foram utilizados 6 coelhos machos, divididos em 2 etapas: (1) Fresas – 5 grupos (G1 a G5) correspondentes às osteotomias 10, 20, 30, 40 e 50 vezes, respectivamente. Cada leito ósseo recebeu a sequência de fresas: lança e helicoidais de 2,0 mm, 2,8 mm, 3,0 mm e 3,15 mm; (2) Piezocirurgia – 5 grupos (P1 a P5) correspondentes às osteotomias 10, 20, 30, 40 e 50 vezes, respectivamente, efetuadas pela sequência de pontas de 2,0 e 3,0 mm, mediante vibração ultrassônica piezoelétrica. As áreas osteotomizadas foram coletadas e processadas laboratorialmente. A análise imunoistoquímica demonstrou equilíbrio das expressões de OPG e RANKL, ou seja, formação e reabsorção óssea, tanto nas osteotomias com fresas, quanto na piezocirurgia, embora mais intensas nesta última. A expressão de OC apresentou-se bastante intensa nos grupos de piezocirurgia, mas com redução da imunomarcação a partir da 30ª osteotomia, enquanto manteve-se constante nos grupos fresados. A CAS3 evidenciou a viabilidade osteoblástica a partir da 20ª osteotomia com piezocirurgia e manteve-se constante até a 50ª. No grupo de fresas, notou-se aumento gradativo da expressão dessa proteína, conforme o aumento do número de osteotomias. De acordo com a metodologia aplicada, foi possível concluir que a piezocirurgia propicia maior viabilidade celular osteoblástica do que o sistema de fresas convencional / The purpose of this study was to evaluate the effect of the osteotomy implant on bone cell viability immediately, comparing the use of conventional drilling system with piezosurgery® in tibiae of rabbits. We used 6 male rabbits were divided into 2 stages: (1) Drilling - 5 groups (G1 to G5) corresponding to the 10 osteotomies, 20, 30, 40 and 50 times respectively. Each received a bone bed following drills: Spear drill and 2.0 mm, 2.8 mm, 3.0 mm and 3.15 mm helicoidal drills, (2) Piezosurgery® - 5 groups (P1 to P5) corresponding to the 10 osteotomies, 20, 30, 40 and 50 times, respectively, analyzed by the sequence of inserts 2 and 3 mm by piezoelectric ultrasonic vibration. The receptor-beds were collected and processed in a laboratory. The immunohistochemical analysis showed balance expressions of OPG and RANKL, ie bone formation and resorption in both the osteotomies with drills and piezosurgery®, although more intense in the latter. The expression of OC had become quite intense in piezosurgery® groups, but with reduced immunostaining from the 30th osteotomy, while remained unchanged in groups drilled. The CAS3 showed the viability of the osteoblast from the 20th osteotomy with piezosurgery® and remained constant until the 50th. In the group of drills noticed a gradual increase in the expression of this protein, as the increase in the number of osteotomies. According to the methodology, it was concluded that the piezosurgery® provides greater osteoblastic cell viability than the system of conventional drilling

Resumo: O objetivo deste estudo foi avaliar a associação de células derivadas de periósteo autógeno (CDPA) e xenoenxerto de hidroxiapatita e colágeno I (HA/Col) no reparo de defeito crítico de crânio de ratos. CDPA de 10 ratos Wistar foram semeadas na densidade de 1,0x106 células sobre discos de HA/Col (8x2mm) em DMEM:HAMF12 com 10% soro fetal bovino e dexametazona, ácido ascórbico e glicerofosfato por 6 dias. Ensaio funcional comparou efeito de coágulo sanguíneo (G1), osso autógeno (G2), HA/Col (G3) e HA/Col+CDPA (G4) preenchendo defeito de 8 mm de crânio de ratos (n=40) em 1 e 3 meses. A análise radiográfica não exibiu variação temporal, tendo G1 e G2 discreto novo osso marginal; radiopacidade dos materiais em G2, G3 e G4 impediu confirmar osteogênese central. A análise histológica dos grupos mostrou em G1 tecido conjuntivo denso e ilhas de ossificação (1-3m), em G2 coalescência do material e osteogênese (1-3m), em G3 ossificação intramembranosa (1m) e novo osso homogêneo ao redor e substituindo HA (3m) e em G4 abundante tecido conjuntivo frouxo permeando material (1m) e novo osso heterogêneo (3m); não houve necrose, inflamação crônica ou exuberância de células gigantes tipo corpo estranho. As análises histomorfométrica e estatística mostraram diferença signif icativa (p<0,05) para biomaterial (1m) de G2 (23,3%) com G3 (44,6%) e G4 (47,5%) e para ganho ósseo (3m) de G2 (10,9%) com G1 (4,6%) e G4 (5,1%), tendo G3 (8,5%) neoformação óssea próxima a G2. Conclui-se que CDPA não aumentam formação óssea se associadas a HA/Col. / Abstract: The aim of this study was evaluate the association of autogenous periosteum derived cells (APDC) and xenograf t made up hydroxyapatite and collagen I (HA/Col) in repair of critical size defect in rat calvaria. APDC of 10 Wistar rats were seeded with density of 1,0x106 cells above discs of HA/Col (8x2mm) in DMEM:HAMF12 with 10% fetal bovine serum and dexamethasone, ascorbic acid and glicerophosphate until 6 days. Functional assay compared ef fect of blood clot (G1), autogenous bone (G2), HA/Col (G3) and HA/Col+CDPA (G4) f illing the 8mm-defect in rat skull (n=40) at 1 and 3 months. Radiographic analysis did't have time variation, with G1 and G2 showing mild peripheral new bone; radiopacity of materials in G2, G3 and G4 didn't enable to conf irm central osteogenesis. Histologic analysis of groups showed in G1 dense connective tissue and bone islets (1-3m), in G2 fusion of material and osteogenesis (1-3m), in G3 intramembranous ossification (1m) and homogeneous new bone around and substitute HA (3m) and in G4 abundant loose connective tissue permeating the material (1m) and heterogeneous new bone (3m); there is not necrosis, chronic inf lammation or exuberance of giant cell like foreign body reaction. Histomorphometric and statistic analysis showed signif icative dif ferences (p<0.05) for biomaterial (1m) f rom G2 (23,3%) to G3 (44,6%) and G4 (47,5%) and for bone gain (3m) f rom G2 (10,9%) to G1 (4,6%) and G4 (5,1%), with G3 (8,5%) showing new bone formation closer to G2. It's possible to conclude that APDC don't increase bone formation if associate to HA/Col. / Orientador: Idomeo Bonetti Filho / Coorientador: José Mauro Granjeiro / Banca: Mário Tanomaru Filho / Banca: Fábio Luiz Camargo Villela Berbert / Banca: Manoel Eduardo de Lima Machado / Banca: Arlindo Di Spagna Souza / Doutor

Os eventos celulares e extracelulares que ocorrem durante o processo de osseointegração do titânio (Ti) são bastante influenciados por suas propriedades de superfície, como morfologia, topografia e composição química. A modificação bioquímica da superfície do Ti consiste em imobilizar proteínas ou peptídeos nessa superfície com a finalidade de induzir respostas celulares e teciduais específicas na interface osso-implante que acelerem ou aumentem a osseointegração. O objetivo deste estudo foi avaliar o desenvolvimento do fenótipo osteoblástico em culturas de células crescidas sobre Ti revestido com colágeno tipo I. Para tanto, células osteoblásticas derivadas de fragmentos ósseos do processo alveolar de humanos foram cultivadas sobre discos de Ti usinados revestidos (Ti-col) ou não (Ti-usinado) com colágeno tipo I e foram avaliados os seguintes parâmetros: adesão, morfologia e proliferação celulares, síntese de proteína total, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, e expressão de genes marcadores do fenótipo osteoblástico por reação em cadeia da polimerase em tempo real (PCR em tempo real). O Ti-col alterou o crescimento e a expressão gênica das culturas e não teve efeito na adesão e morfologia celulares, síntese de proteína total, atividade de ALP e formação de matriz mineralizada comparado ao Ti-usinado. Esses resultados indicam que a superfície Ti-col pode favorecer um maior crescimento da cultura durante a fase proliferativa e um aumento e/ou aceleração da diferenciação, como indicado por alterações na expressão gênica de marcadores do fenótipo osteoblástico. Portanto, essa modificação de superfície pode ter um impacto nos processos de reparo e remodelação do tecido ósseo adjacente a implantes, favorecendo a ocorrência de maior formação óssea. / Cellular and extracellular events that occur during titanium (Ti) osseointegration process are highly influenced by its surface properties, such as morphology, topography and chemical composition. The objective of biochemical modification of Ti is to immobilize proteins or peptides on its surface in order to induce specific cellular and tissue responses at the boneimplant interface in order to accelerate or enhance osseointegration. The aim of this study was to evaluate the osteoblastic phenotype development in cells grown on collagen type I-coated Ti surface. Osteoblastic cells from human alveolar bone fragments were cultured on turned Ti either coated with collagen type I (col-Ti) or not (turned-Ti) and the following parameters were assessed: cell adhesion, morphology, and proliferation, total protein content, alkaline phosphatase (ALP) activity, bone-like formation and gene expression of osteoblastic markers by real-time polymerase chain reaction (real-time PCR). Col-Ti altered culture growth and gene expression of osteoblastic markers without affecting cell adhesion, morphology, protein synthesis, ALP activity, and matrix mineralization. These results demonstrated that col-Ti favours cell growth during the proliferative phase and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase, suggesting that this Ti surface modification may affect the processes of bone healing and remodelling.

Apesar da ampla aplicação clínica de plasma rico em plaquetas (PRP), a sua eficácia no reparo de defeitos ósseos e na osseointegração de implantes metálicos continua sendo questionada. Em vista disso, objetivo do presente estudo foi avaliar os efeitos de um coquetel contendo os principais fatores de crescimento (GFs) e proteínas de PRP no desenvolvimento do fenótipo osteogênico in vitro sobre titânio (Ti). O coquetel referido continha PDGF-BB, TGF-&beta;1, TGF- &beta;2, albumina, fibronectina e trombospondina. Células da linhagem osteoblástica foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sob condições osteogênicas convencionais até a subconfluência, sendo, em seguida, subcultivadas sobre superfície de Ti. As subculturas foram expostas durante os 7 primeiros dias a meio osteogênico, suplementado com GFs e proteínas, e apenas ao meio osteogênico nos 7 dias subseqüentes. Os grupos controles foram expostos apenas ao meio osteogênico. Nos experimentos dose-resposta foram utilizadas culturas primárias de calvária de ratos, as quais foram expostas ao coquetel de GFs e proteínas e às suas diluições de 1:10 e 1:100. Culturas derivadas de osso alveolar humano expostas ao coquetel de GFs e proteínas apresentaram: aumento significativo do número de células a partir do dia 4 e da proliferação celular em 1 e 4 dias; redução significativa nos níveis de atividade de fosfatase alcalina (ALP) em 4, 7 e 10 dias e ausência de marcação com vermelho de Alizarina em 14 dias. Apesar de as diluições 1:10 e 1:100 restaurarem a atividade proliferativa das culturas aos níveis controles, formações de matriz calcificada foram observadas apenas na diluição 1:100. Os resultados do presente trabalho mostram que o coquetel de GFs e proteínas inibe o desenvolvimento do fenótipo osteogênico de culturas de células osteoblásticas humanas e de ratos crescidas sobre Ti. / Background: Despite wide clinical application, the efficacy of platelet-rich plasma (PRP) for repairing bone defects and enhancing osseointegration of metal implants is still subject of debate. The objective of the present study was to evaluate the effects of a well-defined mixture of growth factors (GFs) and proteins (GFs+proteins) on the development of the osteogenic phenotype on titanium (Ti) in vitro. The composition of the mixture was based on the major components found in PRP preparations. Methods: The PRP-like mixture contained PDGF-BB, TGF-&beta;1, TGF-&beta;2, albumin, fibronectin, and thrombospondin. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic condition until subconfluence. They were then subcultured on Ti discs up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with GFs+proteins and to osteogenic medium alone thereafter. Control cultures were exposed to only osteogenic medium throughout the culture interval. Dose-response experiments were carried out using rat primary calvarial cells exposed to GFs+proteins and 1:10 or 1:100 dilutions of the mixture. Results: Treated human-derived cell cultures exhibited a significantly higher number of cells from day 4 on and of cycling cells at days 1 and 4, significantly reduced levels for alkaline phosphatase (ALP) activity, and no Alizarin red stained areas at day 14. Although the 1:10 and 1:100 dilutions restored the proliferative activity of rat calvaria-derived osteogenic cells to control levels, mineralized bone-like nodule formation was only observed with the 1:100 dilution. Conclusions: The present results demonstrated that a PRP-like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblastic cell cultures grown on Ti.

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.

La maladie osseuse est une complication sévère pour les patients atteints de mucoviscidose (Cystic Fibrosis, CF). Les fractures vertébrales et costales impactent les capacités pulmonaires et la clairance du mucus bronchique. La meilleure prise en charge des symptômes des patients CF a permis l’amélioration de leurs qualité et espérance de vie. Cependant malgré l’optimisation de facteurs impactant le métabolisme osseux aucune amélioration notable n’a été observée dans la fragilité osseuse. Plus de 80% des patients CF sont porteurs de la mutation F508del du gène CFTR (Cystic Fibrosis Transmembrane conductance Regulator) sur au moins un allèle. L’implication directe de la mutation F508del dans la maladie osseuse a été montrée, bien que son rôle dans le dysfonctionnement du métabolisme osseux reste encore à élucider. Notre travail a permis de mettre en évidence que la mutation F508del portée par les ostéoblastes humains est responsable d’une dérégulation importante de la voie de signalisation RANK/RANKL/OPG aboutissant à un ratio RANKL/OPG élevé, potentiateur de l’ostéoclastogénèse et de la résorption osseuse. Nous avons également mis en évidence que le contexte inflammatoire chronique de la pathologie pourrait exacerber la perte osseuse, les cellules CF étant plus sensibles à ces stimulations. Par ailleurs, nous avons montré que l’utilisation de molécules pharmaceutiques comme des correcteurs et potentiateurs de CFTR, actuellement utilisés en essais cliniques, permettent une normalisation au moins partielle des dérégulations observées des ostéoblastes CF, et apparaissent comme des nouvelles stratégies thérapeutiques. / Bone disease is a serious complication for patients with Cystic Fibrosis (CF). Rib and vertebral fractures worsen lung function and mucus clearance. Better care of CF patient’s symptoms enable an improvement in life quality and expectancy. Despite optimization of factors impacting bone metabolism no improvement was observed in bone loss of patients with CF. More than 80% CF patients carried the F508del mutation on the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene on at least one allele. The role of the F508del mutation in the dysfunction of bone metabolism is yet unclear, but its involvement has been already shown in clinical studies and mouse models.Our work shown that F508del mutation on human osteoblasts causes a dysregulation in the RANK/RANK-L/OPG signalling leading to a high RANK-L to OPG ratio that may improve osteoclastogenesis, and thus the bone résorption. We also show that chronic inflammatory status of CF patients could exacerbate bone loss because of a high sensitivity of osteoblasts with the F508del-CFTR mutation. In addition, we demonstrate that the use of drugs as CFTR correctors and potentiators cause an improvement of the dysregulation observed and seems to be a promising therapeutic strategy.

L’exposition à l’uranium, qu’elle soit naturelle ou accidentelle, est un sujet de préoccupation de santé publique. Cet actinide est utilisé dans de nombreuses applications civiles ou de défenses. Il est également présent de façon naturelle dans l’eau de boisson et dans l’alimentation. Sa toxicité chimique cible en majorité le rein, organe de stockage à court terme, et les tissus squelettiques, où l’uranium est retenu pendant des années. L’uranium induisant une perte osseuse à long terme, il est susceptible d’affecter les cellules impliquées dans le processus de remodelage osseux : les ostéoclastes (OCs) qui résorbent la matrice osseuse, les ostéoblastes (OBs), qui la reconstitue via la minéralisation, et les ostéocytes (OSTs), les capteurs mécano-sensibles, orchestrant le cycle de remodelage. Par ailleurs, aucune thérapie de chélation (comme il en existe pour d’autres métaux lourds) n’est véritablement efficace pour diminuer l’exposition interne et éliminer cet actinide en cas de contamination à l’uranium. Dans l’optique de comprendre les mécanismes de toxicité mis en jeu sur les cellules osseuses, la première partie de cette thèse est ainsi consacrée à l’étude des effets moléculaires et fonctionnels d’une exposition aiguë et chronique à l’uranium sur une lignée ostéocytaire, les cellules MLO-A5. Nous avons d’abord montré que ces cellules présentent une IC50 plus élevée que les OCs et les OBs et qu'une exposition aiguë stimule le processus autophagique. Dans un second temps, nous avons analysé la réponse de ces cellules lors d’une exposition chronique à des concentrations sub-toxiques d’uranium, et observé une inhibition drastique de leur capacité de minéralisation, sans toutefois affecter leur viabilité.Dans une seconde partie, nous nous sommes intéressés aux effets d’un décorporant potentiel de l’uranium, le 3,4,3-LI(1,2-HOPO), sur les OBs et les OCs, dans le but de mettre au point une méthode de criblage de nouveaux décorporants. Les résultats ont montré que le 3,4,3-LI(1,2-HOPO) permet une restauration partielle des capacités de différenciation et de résorption des OCs exposés à l'uranium. Ces résultats permettront d’adapter cette méthode pour le criblage de futurs décorporants dans une démarche de valorisation. Ainsi, ces travaux ont permis d’accroître les connaissances de la toxicité chimique de l’uranium sur les cellules osseuses, et ont apporté de nouvelles données toxicologiques in vitro concernant les effets d’un agent décorporant, le 3,4,3-LI(1,2-HOPO), dans ces modèles cellulaires. / Natural or accidental uranium exposure is a health care concern. Uranium is a natural metal found in the environment and is both used for civil or military applications. It is also naturally present in food and water. It exhibits both a radiological and a chemical toxicity, the latter being considered largely predominant for natural uranium. Kidneys and bones are the main targeted organs of its toxicity. The skeleton is the storage organ of uranium and can be retained there for years, causing a long-term bone loss. Bone cells, osteoclasts cells in charge of bone resorption, osteoblasts involved in matrix production and mineralization, and osteocytes, considered the major orchestrators of bone remodeling, are therefore likely to exhibit impaired functions. Furthermore, current chelation therapy treatments failed to demonstrate a true decorporating efficiency after internal uranium contaminations.The first part of this study describes molecular and cellular effects of acute and chronic uranium exposure on a murine osteocytic cell line MLO-A5. Acute exposure enhanced their autophagic process and CI50 determination shows less toxicity on osteocytes than on osteoclasts and osteoblasts. Moreover, mineralization capacity of these cells was strongly inhibited after a chronic exposure without affecting cell viability. In a second part, we determined the in vitro effects of 3,4,3-LI(1,2-HOPO), a potential decorporating agent, on osteoclasts and osteoblasts with the intend to develop methods for decorporating agent screening. This molecule shows an ability to partly restore differentiation and resorption function of exposed osteoclasts to uranium. These results constitute a step forward in the development of screening methods to evaluate the potential of new decorporating agent.Taken together, these results enhanced our knowledge of uranium toxicity mechanisms on bone cells and brought new toxicological data regarding the use of 3,4,3-LI(1,2-HOPO) in our cellular models.