Made available in DSpace on 2016-06-02T19:22:50Z (GMT). No. of bitstreams: 1 2036.pdf: 748244 bytes, checksum: f28f7da3d00f1b29890db0e264cd0b46 (MD5) Previous issue date: 2008-08-29 / Universidade Federal de Sao Carlos / The mercury is used in the industry, medicine, agriculture and other fields. However, little is known about its involvement with the bone metabolism. The aim of this study was to evaluate the physical, biochemical and biomechanical bone parameters in adult rats contaminated with mercuric chloride during the development phase. The animals were separated as control group: 10 male rats treated with saline 0.9% (0,1 ml/100g BW) and contaminated group: 8 male rats treated with mercuric chloride (2.5 mg/Kg BW). The animals were treated during 60 days, 5 days/week, by stomacal gavage. The body weight, femoral length and diaphysis thickness were measured. The mechanical properties (maximum and failure forces, stiffness and yield) of femurs were evaluated by the three-point bending test using the universal machine Instron model 4444. The bone volume was estimated by the Archimedes Principle. The wet and ash weight were also determined. The body weight, length and minor diameter of the femurs were not different between the groups; however the major diameter increased in contaminated group (p=0.0002). It was observed an increase (p=0.0044) of bone volume and a decrease of the bone mineral density (p=0.0009) and of the percentage of mineral material (p=0.0001) of the femurs of the contaminated group. The bone response to biomechanical test was similar in both groups. The rat contamination with mercuric chloride in the dose of 2.5 mg/Kg BW caused small alteration in the development of bones, as observed by the biometrical and biophysical parameters analyzed. However these alterations were not enough to induce modification of the biomechanical and biochemical parameters. / O mercúrio é utilizado na indústria, medicina, agricultura e outros campos. Pouco se sabe sobre seu envolvimento com o metabolismo ósseo. O objetivo desse estudo foi avaliar possíveis alterações físicas, bioquímicas e biomecânicas ósseas em ratos adultos intoxicados com cloreto mercúrico durante seu desenvolvimento. Os animais foram divididos em grupo contaminado e controle, sendo 10 ratos machos tratados com salina 0,9% (0,1 ml/100g PC) e 8 ratos machos tratados com cloreto mercúrico (2,5 mg/Kg PC), durante 5 dias/semana, por 60 dias pelo método de gavagem gástrica. O peso corporal, comprimento femoral e espessura das diáfises femorais foram mensurados. As propriedades (força máxima e de ruptura, rigidez e resiliência) dos fêmures foram mensuradas através do teste de flexão à três pontos em uma máquina universal modelo Instron 4444. Através do Princípio de Arquimedes, calculou-se o volume ósseo, e a partir deste parâmetro as densidades óssea e mineral foram determinadas utilizando as propriedades físicas: peso seco, peso imerso, peso úmido e das cinzas. O peso corporal, comprimento e o diâmetro menor do fêmur não foram diferentes entre os grupos, porém o diâmetro maior aumentou no grupo contaminado (p=0,0002). Houve aumento do volume ósseo (p=0,0044) e diminuição da densidade mineral (p=0,0009) e percentual de material mineral (p=0,0001) dos fêmures do grupo contaminado, porém essas alterações não foram suficientes para provocar alteração dos parâmetros biomecânicos. A contaminação com cloreto mercúrico na dose de 2,5 mg/Kg PC causou pequena alteração do desenvolvimento ósseo dos ratos, como pode ser observado pela alteração dos parâmetros biométricos e biofísicos, mas não suficiente para alterar os parâmetros bioquímicos e biomecânicos do fêmur.

Made available in DSpace on 2016-06-02T19:22:51Z (GMT). No. of bitstreams: 1 2339.pdf: 646629 bytes, checksum: a664e5d6f2b2d647db3059c99759df5b (MD5) Previous issue date: 2009-04-09 / Universidade Federal de Sao Carlos / Lead is a ubiquitous pollutant in the environmental ecosystem which distribution is mainly anthropogenic, come from burning fossil fuels, mining and various industrial products. There is little knowledge of the involvement of this element on the bone metabolism. This study aimed to determine possible changes in adult rat bone, contaminated with lead acetate during the development phase, through the incorporation of lead in the tissue. Biometric, biophysical, and biomechanical bone parameters were analyzed. The animals were divided into 3 groups: control, 9 rats treated with saline 0.9% (0.1 ml/100g BM) and contaminated groups C-100 and C-200 with 10 animals each, poisoned with lead acetate (100 and 200 mg / kg BM, respectively), once per week for 8 weeks by gastric gavage method. The incorporation of lead was examined with scanning electron microscopy and the concentration of this element in bone tissue with atomic absorption spectrometry with flame. We calculated the bone volume, and from this parameter the bone density was determined. The biomechanical properties of femurs were obtained by three points bending test. Contamination with lead acetate promoted incorporation and increased the concentration of lead in bone for the two doses tested, however were not sufficient to cause changes in the somatic and bone development of these animals. The dose of 100 mg / kg BM was not enough to cause bone changes and the dose of 200 mg / kg BM caused reduced bone density and strength. Therefore, the results indicate the ability of this element in damage the quality of the bone and maybe, serve as a source for endogenous subsequent contamination. / Chumbo é um poluente ubíquo no ecossistema cuja distribuição ambiental é principalmente antropogênica, oriunda da queima de combustíveis fósseis, mineração e fabricações industriais diversas. Há pouco conhecimento do envolvimento deste elemento sobre o metabolismo ósseo. O objetivo deste estudo foi estabelecer possíveis alterações ósseas em ratos adultos, contaminados com acetato de chumbo durante a fase de desenvolvimento, por meio da incorporação de chumbo neste tecido. Foram analisados parâmetros biométricos, biofísicos, e biomecânicos ósseos. Os animais foram divididos em 3 grupos: controle, com 9 ratos tratados com salina 0,9% (0,1 ml/100g MC) e contaminados C-100 e C-200 com 10 animais cada, intoxicados com acetato de chumbo (100 e 200 mg/kg MC, respectivamente), 1 vez por semana, por 8 semanas pelo método de gavagem gástrica. A incorporação de chumbo foi analisada com microscopia eletrônica de varredura e as concentrações deste elemento no tecido ósseo com espectrometria de absorção atômica com chama. Calculou-se o volume ósseo, e a partir deste parâmetro a densidade óssea foi determinada. As propriedades biomecânicas dos fêmures foram obtidas pelo teste de flexão a três pontos. A contaminação com acetato de chumbo promoveu incorporação e aumento da concentração de chumbo no tecido ósseo para as duas doses testadas, entretanto não foram suficientes para provocar alterações no desenvolvimento somático e ósseo destes animais. A dose de 100 mg/kg MC não foi suficiente para provocar alterações ósseas e a dose de 200 mg/kg MC provocou redução da densidade e resistência óssea. Portanto, os resultados apontam a capacidade deste elemento em prejudicar a qualidade do tecido ósseo e talvez servir como fonte endógena para contaminações subseqüentes.

Made available in DSpace on 2016-06-02T19:23:00Z (GMT). No. of bitstreams: 1 5830.pdf: 3124907 bytes, checksum: 55c12c9ea3515d048b2237215c49333a (MD5) Previous issue date: 2014-05-06 / Financiadora de Estudos e Projetos / Resveratrol (trans 3,5,4 'trihydroxystilbene) is a polyphenol found in some plants and fruits. There are indications that it may be associated with the prevention of osteoporosis due to its action similar to a SERM (Selective Estrogen Receptor Modulators), a substance used to treat osteoporosis (among other diseases) in women whose use of hormone replacement therapy does not is indicated. However, more specific information about their effect in various quotas of bone tissue is still scarce. The aim of this study was to evaluate the ability of resveratrol to protect against bone changes characteristics of estrogen deficit in young adult ovariectomized rats. Were utilized 30 animals (Wistar rats) distributed into 3 groups: intact group (INT) with 10 intact animals, ovariectomized group (OVX) with 10 ovariectomized animals treated with a saline and dimethylsulfoxide (DMSO) solution, ovariectomized + resveratrol group (OVX + RES) with 10 ovariectomized animals treated with a resveratrol, saline and DMSO solution [0.7 mg/kg BW (body weight)], 7 times a week, for 12 weeks. Biometric, biophysical, biomechanical, microtomographical and radiographic density parameters were analyzed from bones with higher cortical (femur) and trabecular quota (vertebrae). The biomechanical properties of femurs were obtained by the three-point bending test and the lumbar vertebrae obtained by bone compression test. Lumbar vertebrae were used to perform the microtomographical analysis and radiographic density analysis. The ovariectomized rats gained more body weight and had lower bone density and radiographic density, and also showed reduction of microtomographical parameters when compared to the intact group. The biomechanical parameters of femurs did not change in neither group. The animals treated with resveratrol did not shown decrease in microtomographical parameters and bone density of the vertebrae as the untreated group. Therefore, the results suggest resveratrol as a potential protector of ovariectomy induced spoliation of bone tissues with greater trabecular quota. / Resveratrol (trans 3,5,4 -trihidroxiestilbeno) é um polifenol encontrado em algumas plantas e frutos. Há indícios de que ele possa ser associado à prevenção da osteoporose devido a sua ação similar a de um SERM (Selective Estrogen Receptor Modulators), substância utilizada para tratamento da osteoporose (entre outras doenças) em mulheres cujo o uso da Terapia de Reposição Hormonal não é indicado. Entretanto, informações mais específicas sobre o seu efeito em diferentes contingentes do tecido ósseo ainda são escassas. O objetivo deste estudo foi avaliar a capacidade do resveratrol de proteger contra as alterações ósseas características da deficiência de estrógeno em ratas adultas jovens ovariectomizadas. Foram utilizados 30 animais (ratas Wistar) distribuidos em 3 grupos: grupo intacto (INT) com 10 animais intactos, grupo controle ovariectomizadas (OVX) com 10 animais ovariectomizados tratados com uma solução de salina e Dimetilsulfóxido (DMSO), grupo ovariectomizadas + resveratrol (OVX + RES) com 10 animais ovariectomizados tratados com uma solução de resveratrol, salina e DMSO intraperitonealmente [0,7 mg/kg MC (massa corpórea)], 7 vezes por semana, por 12 semanas. Foram analisados os parâmetros biométricos, biofísicos, biomecânicos ósseos, microtomográficos e de densidade radiográfica de ossos com maior contingente cortical (fêmures) e trabecular (vértebras). As propriedades biomecânicas dos fêmures foram obtidas pelo teste de flexão a três pontos e das vértebras lombares obtidas pelo teste de compressão óssea. Foram utilizadas vértebras lombares para a realização da análise microtomográfica e de densidade radiográfica. As ratas ovariectomizadas apresentaram maior ganho de massa corporal e diminuição da densidade óssea e densidade radiográfica, além de redução dos parâmetros microtomográficos quando comparadas ao grupo intacto. Os parâmetros biomecânicos dos fêmures não sofreram alteração em nenhum dos grupos. Os animais tratados com resveratrol não apresentaram a diminuição dos parâmetros microtomográficos e de densidade óssea das vertebras como os não tratados. Portanto, os resultados apontam o resveratrol como potencial protetor do tecido ósseo com maior contingente trabecular da espoliação induzida pela ovariectomia.

O osso requer que alguns de seus elementos detectem estímulos mecânicos e os transformem em sinais celulares adequados para que o tecido ósseo possa manter a eutrofia e adaptar-se às solicitações mecânicas a que é submetido. Assim, a estrutura óssea pode torna-se mais resistente quando a demanda mecânica aumenta e, da mesma forma, menos resistente em condições de hipoatividade como imobilizações, sequelas motoras, repouso prolongado ou em ambientes de microgravidade como as experimentadas por astronautas. Dessa interação podem resultar ossos com massa diminuída, o que caracteriza situações de osteopenia ou osteoporose. Uma das indicações do exercício de natação é estimular e manter o trofismo do tecido ósseo. No entanto, a relação entre natação e qualidade óssea em pessoas com osteopenia, não foi suficientemente estudada de forma a embasar recomendações clínicas. Assim, o objetivo desta pesquisa foi investigar a eficácia da natação na recuperação da qualidade óssea avaliada em tíbias de ratas com osteopenia induzida por suspensão dos membros posteriores. Foram utilizadas 50 ratas da raça Wistar divididas em cinco grupos experimentais (n=10/grupo); dois controles e três experimentais. Houve um grupo suspenso pela cauda durante 21 dias consecutivos (S) e seu controle que permaneceu em gaiolas comuns pelo mesmo período (CI). No grupo suspenso e treinado os animais permaneceram em suspensão durante 21 dias, e depois foram submetidos a 20 sessões de natação (ST). No grupo suspenso não treinado os animais foram suspensos por 21 dias e soltos em gaiolas comuns por 30 dias (SNT). No grupo controle II as ratas foram observadas em gaiolas comuns por 51 dias, sem intervenção (CII). A avaliação da qualidade óssea foi feita pela densitometria (DXA) e ensaio mecânico realizados na tíbia direita e histomorfometria da região metafisária proximal da tíbia esquerda. Para análise estatística todos os valores foram considerados diferentes significativamente em p<0,05. Os ensaios mecânicos mostraram que, para o grupo S os valores de força máxima (-14,03%, p=0,0003), rigidez (-21,68%, p=0,0055), DMO (-17,62%, p=0,019) e percentual de osso trabecular (-57,2%, p=0,0001), diminuíram significativamente em comparação com o grupo CI. A comparação das propriedades mecânicas simultâneas entre os grupos avaliados após 51 dias (STxCIIxSNT) mostrou diferença estatística significante entre os grupos para força máxima de (p=0,0014), rigidez (p=0,0010), DMO (p= 0,0095) e percentual de osso trabecular (p<0,0001). O grupo ST apresentou aumento significativo na força máxima (+10,23%, p<0,05), rigidez (+21,91%, p<0,001), DMO (+9,46%, p<0,05) e percentual de osso trabecular (+48,82%, p<0,001) em comparação com o grupo SNT. Na comparação dos grupos SNT e CII também houve diminuição significativa (-14,4%, p<0,05) para força máxima, rigidez (-25,21%, p<0,005), DMO (-13,34%, p<0,05) e percentual de osso trabecular (-52,06%, p<0,001). Finalmente, a comparação entre os grupos ST e CII não apresentou diferença estatística significante (p>0,05) para os valores de força máxima, rigidez, DMO e percentual de osso trabecular. Em relação à análise histológica qualitativa o grupo S apresentou ossos com características histológicas condizentes com osteopenia, com trabéculas ósseas bem mais delgadas em quantidade muito menor tanto na epífise quanto na metáfise, quando comparado ao grupo CI. O grupo suspenso apresentou ainda, diminuição na espessura do tecido ósseo compacto e redução significativa na quantidade de tecido esponjoso em sua porção mais interna. A espessura do tecido ósseo compacto se mostrou similar nos diferentes grupos (ST, SNT e CII). Todavia, a espessura e a quantidade de trabéculas do osso esponjoso diferiram expressivamente nos diferentes grupos. O grupo ST apresentou uma aparente maior quantidade de trabéculas ósseas, bastante delgadas, ao passo que o grupo CII apresentou, comparativamente, uma menor quantidade de trabéculas bem mais espessas. Comparado a estes últimos, o grupo SNT apresentou uma menor quantidade de trabéculas com delgada espessura. Com a suspensão por três semanas os parâmetros mostraram que houve deterioração da qualidade óssea expressa pela diminuição da densidade mineral óssea, enfraquecimento mecânico do osso e perda de massa óssea trabecular. A quantidade de osso trabecular na região metafisária foi o parâmetro mais afetado pela hipoatividade, mas também o elemento que apresentou a resposta mais rápida para a atividade motora. A conclusão foi que a natação reverteu mais rapidamente a deterioração osteopênica causada pela hipoatividade, com recuperação completa da qualidade óssea. / The bone is able to detect mechanical stimuli and to transform them into cell response so that the bone tissue can maintain its health and to adapt to different mechanical demands (mechanotransduction). Thus, the bone structure become more resistant when the mechanical solicitations are increased and, conversely, less resistant when in hypoactivity conditions as occurring during immobilizations, in locomotor sequela, prolonged bed rest or in a microgravity environment as experienced by astronauts. In general, hypoactivity leads the bone to develop a decreased and deteriorated mass, thus characterizing conditions of osteopenia or osteoporosis. Thus, physical activity such as swimming is indicated as one of the factors that contribute to the bone health. However, the relation between swimming and bone quality in individuals with osteopenia has not been sufficiently studied to fully support clinical recommendations. So, the aim of this study was to investigate the effectiveness of swimming in recovering bone quality evaluated in osteopenic tibias of rats that spent three weeks in hindlimb suspension. Fifty Wistar rats were used and divided into five groups (n = 10/group); two control and three experimental groups. There was a group suspended by the tail for 21 consecutive days (S) and its control kept in regular cages for the same period (CI). In the suspended and trained group, animals were suspended for 21 consecutive days and then underwent a 20 swimming sessions (ST). In the suspended, not trained group, animals were suspended for 21 days and released in regular cages for 30 days (SNT). In the control group II rats were kept in regular cages for 51 days without intervention (CII). The bone quality was assessed by densitometry (BMD), bending mechanical testing and histomorphometry. Statistical significance was set at p<0.05. Mechanical tests showed that for the suspended group there was a decrease of the maximum force (-14.03%, p = 0.0003), stiffness (-21.68%, p = 0.0055), BMD (-17.62 %, p = 0.019) and the percentage of trabecular bone (-57.2%, p = 0.0001) in comparison with the control group. Comparison among the groups after 51 days (STxCIIxSNT) showed statistically significant differences between groups for maximum force (p = 0.0014), stiffness (p = 0.0010), BMD (p = 0. 0095) and the percentage of trabecular bone (p<0.0001). The suspended and trained group showed a significant increase in maximum force (+10.23%, p <0.05), stiffness (+21.91%, p<0.001), BMD (+9.46%, p<0.05) and the percentage of trabecular bone (+48.82%, p <0.001) compared to the suspended and not trained group. When the suspended but not trained group was compared to control CII, there was also a significant decrease (- 14.4%, p<0.05) for maximum force, stiffness (-25.21%, p<0.005), BMD (-13.34%, p<0.05) and the percentage of trabecular bone (-52.06%, p<0.001). Finally, the comparison between group suspended and trained ad its control and CII showed no statistically significant difference (p>0.05) for all the parameters. Considering the qualitative histological analysis, suspended group presented bones with histological characteristics consistent with osteopenia, with thinner trabecular bone and in much less quantity both in the epiphysis and in the metaphysis. The suspended and trained group had a higher quantity of trabecular bone, with thinner trabeculae, while the CII group showed a comparatively smaller amount of much thicker trabeculae. Compared to the latter, the suspended and not trained group had an apparent smaller amount of trabeculae with a thinner thickness. In conclusion, the three week suspension caused a marked deterioration of the tibia bone tissue that was completely reverted by swimming and partially reverted with spontaneous cage activities taking in consideration all the parameters analyzed.

A regeneração de defeitos ósseos continua sendo um grande desafio na área de Odontologia e Medicina. É bem estabelecido que células-tronco mesenquimais (CTMs) e osteoblastos (OBs) desempenham um papel crítico na osteogênese, tornando-se candidatos a utilização em procedimentos de terapia celular que visam otimizar o processo de reparação óssea. Porém, pouco se sabe sobre a interação entre CTMs e OBs, e a maioria dos estudos enfatiza o efeito dos OBs sobre CTMs, fazendo com que a influência das CTMs na atividade osteogênica dos OBs continue sendo uma questão desafiadora. Baseados em nossos estudos anteriores, formulamos a hipótese de que a terapia celular que fizesse uso de uma associação de CTMs e OBs poderia ser mais eficaz para o reparo do tecido ósseo do que essas células isoladamente, principalmente como resultado da estimulação de OBs por CTMs. Para tal, foi realizado estudo in vitro para avaliar os efeitos das CTMs sobre os OBs e in vivo para avaliar os efeitos dessas células, isoladamente e combinadas, sobre a reparação óssea. CTMs da medula óssea de rato foram cultivadas em meio de crescimento para manterem-se como CTMs ou em meio osteogênico para diferenciarem-se em OBs. Após alcançar a subconfluência, as células foram cultivadas in vitro em três diferentes condições: (1) co-cultura direta de CTMs e OBs usando três proporções celulares (1:1, 1:2 e 2:1), (2) co-cultura indireta de CTMs e OBs usando insertos e (3) OBs cultivados em meio condicionado por CTMs. Para avaliação das respostas celulares foram realizados ensaios de proliferação celular, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteína óssea (BSP) e osteopontina (OPN) e migração celular. Para os experimentos in vivo, as células foram carreadas em esponja de colágeno através de vários ciclos de centrifugação. Após, defeitos ósseos em calvária de rato foram preenchidos com (1) esponja de colágeno sem células, (2) esponja de colágeno com CTMs, (3) esponja de colágeno com OBs e (4) esponja de colágeno com associação de CTMs e OBs. Para avaliação da reparação óssea in vivo após 4 semanas, foram realizadas análises histomorfométricas através de cortes histológicos e microtomografia computadorizada. Os dados foram comparados pelo teste de Kruskal-Wallis e, se necessário pelo teste de Mann-Whitney (p&le;0,05). Foi observado que CTMs têm efeito repressivo sobre a proliferação e as expressões fenotípicas e genotípicas de OBs (P&le;0,05). Em relação ao reparo dos defeitos ósseos, somente naqueles tratados com células observou-se formação óssea predominantemente como ilhotas isoladas e diferenças, principalmente qualitativas, entre os tipos celulares utilizados, com tendência de maior formação óssea em defeitos tratados com OBs em comparação ao uso de CTMs. Com base nos resultados obtidos, pôde-se concluir que as CTMs apresentam efeito inibitório sobre OBs e que a terapia celular com OBs parece ser mais eficaz no reparo do tecido ósseo. / The regeneration of bone defects remains a major challenge in the field of Dentistry and Medicine. It is well known that mesenchymal stem cells (MSCs) and osteoblasts (OBs) play critical roles in osteogenesis, making them promising alternatives to be employed in cell therapy procedures to enhance the process of bone regeneration. Studies about the crosstalk between MSCs and OBs are mainly focused on the effect of OBs on MSCs, thus how MSCs may affect OBs phenotype expression remains a challenging question. Based on our previous studies, we have hypothesized that cell therapy using a combination of MSCs and OBs could be more effective for the bone repair than these cells separately, mainly due to the stimulation of OBs by MSCs. For this, we carried out in vitro experiments to evaluate the effects of MSCs on the OBs and in vivo experiments to assess the effects of these cells either isolated or combined on bone repair. Rat bone marrow MSCs were cultured either in growth medium to keep MSCs features or in osteogenic medium to differentiate into OBs. After reaching subconfluence, cells were grown in vitro in three different conditions: (1) direct coculture of MSCs and OBs using three cell proportions (1:1, 1:2 and 2:1), (2) indirect coculture of MSCs and OBs using transwell porous filters and (3) OBs cultured in MSCs conditioned medium. Cell responses were evaluated by assaying cell proliferation, alkaline phosphatase activity (ALP), mineralized matrix formation, gene expression of osteoblast markers, immunolocalization of bone sialoprotein (BSP) and osteopontin (OPN), and cell migration. For in vivo experiments, cells were seeded into collagen sponge by a centrifugation method. After, calvarial defects were implanted with (1) collagen sponge without cells, (2) collagen sponge with MSCs, (3) collagen sponge with OBs, and (4) collagen sponge and association of MSCs with OBs. To evaluate bone repair at the end of 4 weeks, histomorphometric analyzes were carried out using histological slides and micro-computed tomography. Data were compared by Kruskal-Wallis test and, if appropriated, by Mann-Whtiney test (p&le;0.05). It was observed that MSCs repressed proliferation, phenotypic and genotypic expressions of OBs (P&le;0.05). Bone formation was observed only in cell treated defects as isolated islets and qualitative differences were noticed among cell types, with a tendency of more bone formation in OBs treated defects compared with MSCs ones. Based on these results, we can conclude that MSCs exhibited inhibitory effect on OBs and that cell therapy with OBs seemed to be more effective for bone repair.

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.

L’ingénierie tissulaire osseuse a récemment connu de nouveaux développements grâce à la prise en compte du phénomène de mécanotransduction dans la conception des bioréacteurs. Toutefois, des progrès restent à faire sur nos connaissances sur les mécanismes de la réponse des cellules souches mésenchymateuses (CSM) aux contraintes mécaniques en vue d’optimiser l’environnement mécanique tridimensionnel des cellules dans les bioréacteurs. L’objectif de cette thèse est double : déterminer le meilleur microenvironnement mécanique pour des CSM humaines et appliquer cet environnement au sein d’un bioréacteur. Pour cela, des CSM humaines ont été cultivées dans différentes conditions et soumises à des contraintes mécaniques. Leur réponse a été analysée via des marqueurs précoces de l’ostéogénèse. En parallèle, un modèle numérique a été développé pour simuler l’écoulement dans un bioréacteur à scaffold granulaire et déterminer le niveau et la répartition des contraintes ressentis par les cellules. Il a été mis en évidence que la réponse des cellules à une stimulation mécanique dépend très fortement de son environnement tridimensionnel. Ce travail à la fois mécanique et biologique permet de dégager des pistes d’amélioration des bioréacteurs et des scaffolds en vue de l’optimisation de l’environnement mécanique tridimensionnel des cellules en ingénierie tissulaire osseuse. / Bone tissue engineering is currently in full development and a growing field of research. The consideration of the mechanotransduction process is a key factor in the optimization of bioreactors. Mesenchymal stem cells (MSC) used in bone tissue engineering are known to be mechanosensitive but our knowledge of the mechanisms of cell response to mechanical stress needs to be improved. This thesis has a double goal: determining the best possible mechanical microenvironment for human MSC, and apply this environment in a bioreactor. To that aim, human MSC were grown in different conditions and subjected to mechanical stresses. Their response was analyzed through osteogenesis markers. A numerical model was also implemented to simulate the flow in bioreactor with a granular scaffold and evaluate levels and distributions of stresses felt by cells. It was shown that cell response to mechanical stress is strongly dependent on the tridimensional environment. This biological and mechanical study highlights tracks of improvement for bioreactors and scaffolds to optimize the mechanical tridimensional environment of cells in bone tissue engineering.

Abstract Bone tissue engineering has become a rapidly expanding research area because it offers a promising new approach for bone repair and regeneration. Compared to traditional autograft and allograft procedures, bone tissue engineering techniques based on the use of scaffolding materials in combination with autogenous stem cells can eliminate problems of donor site morbidity associated with the harvest of bone tissue, and its short supply. Clearly, the choices of material as well as a scaffold design that enhance bone regeneration are major challenges in the tissue engineering approach. Fibers in the micro-range in combination with textile-based technologies are consider as potential routes for the production of complex scaffolds since they can be used to generate a wide range of morphological structures and geometrically varied structures with high precision. Therefore in this thesis the specific objects were to: (i) develop a biocompatible composite fiber from poly(lactic acid) (PLA) and hydroxyapatite (HA) by melt spinning, (ii) design a 3D textile scaffold utilizing weaving and (iii) evaluate the scaffolds’ performance as a bone substitute material in vitro. In the present study PLA/HA composite fibers were successfully produced, and found to possess sufficient mechanical strength even at high loading concentrations (i.e. 20wt %), to be useful in a textile process. In addition, the material was shown to be biocompatible and the presence of HA in the PLA composite significantly enhanced the initial cell attachment. In a 3D woven scaffold, bone marrow derived human mesenchymal stem cells (hMSCs) differentiated into osteoblasts and mineralized bone formation in vitro was observed through-the-thickness of the scaffold. Taken together, these results indicate the potential feasibility of PLA/HA composite fiber in a 3D woven scaffold for use as a bone substitute material in tissue engineering applications. / Tiivistelmä Luupuutosten korvaaminen kudosteknologisesti on kehittynyt nopeasti ja tutkimustulokset tarjoavat lupaavia mahdollisuuksia tuottaa uutta luuta luupuutosalueelle. Perinteisiin potilaan omasta luusta tehtyihin luusiirteisiin ja pankkiluusiirteisiin verrattuna potilaan omat kantasolut voivat vähentää ongelmia, joita ovat siirremateriaalin rajallinen saatavuus ja vieraan kudoksen aiheuttamat reaktiot. On tärkeä etsiä hyviä materiaaleja, joista voidaan valmistaa sellaisia kolmiulotteisia (3D) rakenteita, joilla tehostetaan luun paranemista ja uuden luun muodostumista. Kutomalla tuotetut tukirakenteet mahdollistavat kantasolusiirteille kolmiulotteisuuden, jota voidaan säädellä monipuolisesti ja tarkasti. Tämän väitöstutkimuksen tarkoituksena oli: (i) kehittää bioyhteensopiva kuitu maitohappopolymeeristä poly lactic acid (PLA) ja hydroksiapatiitista (HA) kuituekstruusiolla, (ii) suunnitella ja kutoa tästä kuidusta 3D tekstiilirakenne, ja (iii) tutkia sen toimivuus ja ominaisuudet luunmuodostusta tukevana materiaalina soluviljelyolosuhteissa. Tämä tutkimus osoittaa, että PLA kuitua voidaan seostaa hydroksiapatiitilla, ja PLA/HA kuidut ovat mekaanisesti kestäviä sisältäessään jopa 20 painoprosenttia hydroksiapatiittia. Siten kuidut ovat tekstiilin valmistuksessa käyttökelpoisia. Lisäksi materiaali osoittautui bioyhteensopivaksi, ja hydroksiapatiitti paransi solujen tarttumista PLA kuituun viljelyn alkuvaiheessa. Ihmisen luuytimestä peräisin olevat sidekudoksen kantasolut (hMSCs) erilaistuivat soluviljelmässä luuta muodostaviksi soluiksi eli osteoblasteiksi, ja tuottivat mineralisoitunutta luun väliainetta kautta koko kudotun tukirakenteen. Johtopäätöksenä on, että PLA/HA yhdistelmäkuitua voidaan kutoa kolmiulotteiseksi tukirakenteeksi, ja sitä on mahdollista käyttää apuna korvattaessa luupuutoksia kudosteknologian keinoin.

L’ingénierie du tissu osseux est un domaine qui représente un enjeu majeur dans le cadre de la médecine régénératrice. Trois composants sont généralement décrits dans le cadre de l’ingénierie tissulaire : un biomatériau pour pallier le volume de tissu défectueux, une source de cellules progénitrices qui seront responsables de la synthèse des composants tissulaires ainsi que des facteurs de croissance ou des signaux issus des propriétés physico-chimiques du biomatériau afin de guider la prolifération et la différenciation cellulaire. Le but de cette étude a été de synthétiser des substrats phospho-calciques à l’aide de la technique de pulvérisation simultanée d’espèces réactives et de caractériser les différentes propriétés physico-chimiques des substrats obtenus. Nous avons pu démontrer la possibilité d’inclure des molécules organiques (chitosane et acide hyaluronique) à la phase minérale avec cette technique. Nous avons aussi montré la possibilité de faire varier des propriétés telles la rugosité (entre 300 et 700 nm), l’élasticité (entre 2 à 6 GPa), la composition chimique (phosphate octacalcique ou phosphate dicalcique dihydraté) et la bioactivité (précipitation des phosphates de calcium à la surface des substrats) avec la technique de pulvérisation. Par ailleurs, des cellules souches issues de la gelée de Wharton de cordons ombilicaux humains ont été isolées, puis caractérisées sur le plan génique et protéique. Ces cellules étant candidates pour l’utilisation en ingénierie tissulaire osseuse, nous nous sommes intéressés à plusieurs types de marqueurs dont les marqueurs mésenchymateux et les cytokines immuno-modulatrices.La dernière partie de cette thèse a concerné l’association des cellules souches isolées à partir de la gelée de Wharton aux substrats phospho-calciques obtenus à l’aide de la technique de pulvérisation. Nous avons pu démontrer que les cellules adhéraient sur ces substrats et s’organisaient en structures nodulaires au sein desquelles a été observée une couche de cellules sécrétrices entourant des fibres de collagène, des formations cristallines faîtes de phosphates de calcium et des cellules dont la morphologie rappelait celle des ostéocytes. Des variations dans l’expression de marqueurs ostéoblastiques ont aussi été observées, et ce en l’absence de facteurs solubles ostéogéniques dans le milieu de culture. En conclusion, les substrats phospho-calciques obtenus avec la technique de pulvérisation sont capables d’induire la différenciation de cellules souches issues du cordon ombilical en ostéoblastes. Ce modèle se révèle être prometteur pour la mise en place de thérapies en vue de la régénération du tissu osseux. / Bone tissue engineering is a major issue within regenerative medicine. There are three main components in the field of tissue engineering: a scaffold providing a structure for tissue development, a source of stem cells for tissue formation and growth factors or physical stimuli from the biomaterial to direct growth and differentiation of cells. The purpose of this study was to synthesize calcium phosphate substrates by simultaneous spraying of interacting species and to carry out the physico-chemical characterization of the built substrates. We showed that the spraying technique allows the inclusion of organic molecules such as chitosan and hyaluronic acid. The spraying technique allows several physio-chemical characteristics to be varied, rugosity (300 – 700 nm), elasticity (2 – 6 GPa), chemical composition (octacalcium phosphate or dicalcium phosphate dehydrate), but also studied the bioactivity of the substrates (calcium phosphate from the culture medium precipitates at thesurface of the substrates). In another hand, our aim was to isolate stem cells from human umbilical cords’ Wharton’s Jelly and to carry out their genic and proteic characterization by focusing on mesenchymal markers and immunomodulating cytokines, knowing that these cells are candidates for a use in bone regeneration therapy.The last purpose of our study was to evaluate the potential of Wharton’s jelly stem cells to adhere and proliferate onto the sprayed substrates, and also the formation of nodules. The ultrastructural analysis of nodules formed by Wharton’s jelly stem cells showed a layer of secretory cells surrounding collagen fibers, calcium phosphate crystals and cells with a similar morphology to that of osteocytes. Osteoblastic markers appeared to be regulated in cells cultured without osteogenic supplements. To conclude, sprayed calcium phosphate substrates seem to induce osteoblastic differentiation of Wharton’s jelly stem cells through the substrate’s physico-chemical properties. Our model appears as promising for further bone regenerative therapies.

La culture cellulaire traditionnelle en deux dimensions (2D) ne peut pas reproduire les propriétés des tissus observées dans des organes en trois dimensions (3D). Ces tissus, en particulier les tissus de soutien comme l'os, sont soumis à des contraintes mécaniques, facteurs majeurs de régulation des interactions cellulaires et cellules/matrices. Il y a donc un intérêt croissant pour les modèles 3D, afin de mieux comprendre les différents aspects du fonctionnement cellulaire et du remodelage osseux, dans des systèmes de moindre complexité que les modèles in vivo. Nous nous sommes intéressés ici aux cellules ostéoblastiques et à une de leur protéine matricielle, la sialoprotéine osseuse (BSP). La BSP appartient à la famille des "small integrin-binding ligand N-linked glycoproteins" (SIBLING), impliquées dans le développement, le remodelage et la minéralisation de l’os, et répondant rapidement à la contrainte mécanique. Notre objectif était d’analyser l’impact de cette protéine sur la différenciation ostéoblastique et l’ostéogénèse in vitro à partir de cellules de calvaria de souris présentant une extinction génique de la BSP (BSP-/-) cultivées en 2D et en 3D. Nous avons montré que les cellules BSP-/- présentaient un défaut de formation osseuse et de minéralisation qui est dépendant de la densité cellulaire. Puis nous avons développé et validé un bioréacteur perfusé et stimulable mécaniquement via le système ZetOsTM. Les premiers résultats obtenus avec cet outil contrôlé montrent que l’environnement 3D améliore la différenciation des cellules BSP-/-. Ces travaux restent à développer, notamment pour analyser l’effet des contraintes mécaniques sur ces cellules / Traditional (2D) cell culture cannot reproduce the tissue properties observed in 3 dimensional organs (3D). Weight-bearing organs such as bone are subjected to mechanical stresses, which are major regulating factors for cell and cell/matrix interactions. There is thus a growing interest in 3D culture models, in order to better understand the different aspects of cell function and bone remodeling in systems less complex than in vivo models. We are interested here in the osteoblastic cells and in one of their matrix proteins, the bone sialoprotein (BSP). BSP belongs to the family of the "small integrin -binding ligand N -linked glycoproteins" (SIBLING), involved in the development, remodeling and mineralization of bone, and responding quickly to mechanical stress. Our goal in this work was to analyze the impact of the absence of this protein on osteoblast differentiation and bone formation in vitro from mouse calvarial cells (MCC) with a gene knockout of BSP (BSP-/-), grown in 2D and 3D. We have shown that BSP-/- cells have a defect in bone formation and mineralization which is cell density dependent. We have developed and validated a perfused bioreactor able to apply mechanical stress to culture scaffolds via the ZetOsTM system. Our first results with this powerful tool show that a 3D environment improves BSP-/- cells differentiation. This work remains to be developed, in particular to analyze the effects of mechanical stress on these cells