O objetivo deste estudo foi avaliar a imunolocalização do fator de crescimento BMP- 2 em enxerto ósseo autógeno em bloco recoberto ou não por membrana de PTFE-e na fase inicial da reparação óssea. MATERIAL E MÉTODOS: Quarenta e oito ratos \"wistar\" foram divididos em dois grupos de estudo, um que recebeu somente o enxerto ósseo autógeno (E) e o outro que recebeu o enxerto ósseo autógeno recoberto por membrana (ME). Os períodos de avaliação foram: 0 hora, 3, 7, 14, 21 e 45 dias. Os cortes foram submetidos à reação imunoistoquímica. Foram realizadas as análises, qualitativa e quantitativa: contagem de células com marcação intracitoplasmática nas estruturas bordo, enxerto, tecido conjuntivo superior ao enxerto, interface e leito. RESULTADOS: No leito o maior número de células marcadas foi observado em 3 dias nos grupos (E) e (ME). A região de bordo apresentou maior número de células marcadas em 7 dias no grupo (E) e em 14 dias no grupo (ME), onde a marcação foi predominante em osteoblastos e células osteoprogenitoras em áreas de remodelação óssea. No tecido conjuntivo superior ao enxerto a maior proporção de marcação no grupo (E) ocorreu em 7 dias e no grupo (ME) em 21 dias, no entanto, neste último, ainda havia marcação em células osteoprogenitoras ao final dos 45 dias. Na interface, o maior número de células marcadas ocorreu entre 7 e 21 dias, considerando os grupos (E) e (ME), quando células osteoprogenitoras apresentaram-se positivas nas áreas de pontes de osso imaturo entre o leito e o enxerto. A maior proporção de marcação no enxerto foi variável entre os períodos de 3 dias no grupo (E) e 21 dias no grupo (ME), mas esta foi considerada de menor proporção quando comparada às marcações observadas nas outras estruturas. CONCLUSÕES: Os dados observados mostram que as estruturas de maiores proporções de marcação são aquelas relacionadas à intensa revascularização e osteogênese. Células osteoprogenitoras, osteoblastos e osteócitos apresentaram marcação intracitoplasmática, independentemente do período ou da estrutura analisada. À exceção da interface, o grupo (ME) apresentou marcação mais tardia quando comparado ao grupo (E) para as estruturas bordo e tecido conjuntivo superior ao enxerto. / The aim of this study was to evaluate the immunolocalization of the BMP-2 growth factor in an autogenous block graft covered or not with an e-PTFE membrane on the early phases of bone repair. MATERIAL AND METHODS: Forty-eigth wistar male rats had their mandibles augmented by either an autogenous bone block graft (B) or an autogenous bone block graft covered with an e-PTFE membrane (MB). The specimens were evaluated at baseline, 3, 7, 14, 21 and 45 days after surgery by immunohistochemistry. Qualitative and quantitative analysis were performed. RESULTS: On the receptor bed the greatest number of staining cells was observed within three days on groups (B) and (MB). The border region has shown a largest number of staining cells in seven days on (B) group and in fourteen days on (MB) group, where the staining was prominent in osteoblasts and osteoprogenitor cells in bone remodeling areas. On the connective tissue above the graft, the greatest staining proportion occurred on (B) group within seven days and on (MB) group in twenty one days. However, there was still staining in osteoprogenitor cells in the end of the experiment, (at fourty-five day period). On the interface, the greatest number of staining cells occurred between seven and twenty one days for groups (B) and (MB) respectively, when osteoprogenitor cells were positive in woven bone edges between the receptor bed and the graft. The greatest proportion of staining on the graft was variable among tree days on group (B) and twenty one days on group (MB), but this was considered a smaller proportion when compared to other structures staining. CONCLUSIONS: The data suggest that the greatest staining proportions structures are related to intense revascularization and osteogenesis. Osteoprogenitor cells, osteoblasts and osteocytes showed intracitoplasmatic staining, independently of the period or structure analyzed. Except for the interface, the (MB) group has shown later staining compared to group (MB) for border and connective tissue above the graft.

Os tecidos ósseos alógenos são utilizados nas cirurgias de reconstrução ortopédicas e odontológicas. O número crescente de transplantes de ossos na última década beneficia diversos pacientes. Os profissionais dos Bancos de Tecidos utilizam protocolos que reduzem os riscos de transmissão de doenças infectocontagiosas, entretanto, não eliminam esta possibilidade. Portanto, é importante que tecidos sejam esterilizados por um método eficaz, sendo usualmente aplicada a radiação ionizante. Porém, a radiação ionizante pode acarretar alterações estruturais e biológicas nos ossos em relação à dose. O objetivo deste trabalho foi estudar os efeitos da aplicação da radiação gama e de feixe de elétrons, nas doses de 15 kGy, 25 kGy e 50 kGy nos tecidos ósseos desmineralizados preservados liofilizados ou congelados e avaliar as possíveis alterações no colágeno, na concentração de proteínas totais, BMP-2 e BMP-7. Para tanto foram utilizadas as técnicas de espectroscopia Raman, Bradford e ELISA. Foram utilizadas 5 diáfises de femur humano e parte das amostras foram desmineralizadas. As proteínas ósseas foram extraídas e quantificadas. Com os resultados da espectroscopia Raman, observamos que a eficiência da desmineralização foi de 85 a 90% porém com alterações na estrutura do colágeno. Alterações também foram observadas nos ossos congelados e irradiados nas doses de 25 kGy e 50 kGy. Nos resultados da quantificação de proteínas totais e específicas, ocorreram diminuições gradativas das concentrações médias das proteínas em relação à dose de radiação nos grupos estudados. Nas doses de radiação usualmente aplicadas aos tecidos ósseos, 15 kGy ou 25 kGy, as reduções nas concentrações das BMP-2 e BMP-7, foram menores que 20%. As reduções na dose de 50 kGy foram entre 27% a 53%, sendo influenciadas pelo tipo de radiação e pelo tipo de preservação dos ossos. / Allogenic bone tissues are used in orthopedic and dental reconstructive surgery. The growing number of bone transplants in the last decade benefits many patients. The professionals of Tissue Banks use protocols that reduce the risk of transmission of infectious diseases, however do not eliminate this possibility. Therefore, it is important that tissues are sterilized by an effective method, usually being applied ionizing radiation. However, ionizing radiation can cause structural and biological changes in the bones relative to the dose. The objective of this work was to study the effects of the application of gamma radiation and electron beam at doses of 15 kGy, 25 kGy and 50 kGy in the bone tissues demineralized preserved freeze-dried or frozen and to evaluate possible changes in collagen, the protein concentration total, BMP-2 and BMP-7. For both Raman spectroscopy techniques, Bradford and ELISA were used. They used 5 diaphyses human femur and part of the samples was demineralized. Bone proteins were extracted and quantified. With the results of Raman spectroscopy, we found that the efficiency of demineralization was 85-90% but with changes in the structure of collagen. Changes were also observed in frozen and irradiated bone at doses of 25 kGy and 50 kGy. The results of quantification of total and specific proteins, there were gradual decreases in average concentrations of proteins in relation to radiation dose in both groups. In the radiation doses usually applied to the bone tissue (15 kGy and 25 kGy) reductions in concentrations of BMP- 2 and BMP-7, were lower than 20%. The reductions at a dose of 50 kGy were between 27% to 53%, being influenced by the type of radiation and the kind of preservation of the bones.

Les amphibiens urodèles (e.g. les axolotls) possèdent la remarquable capacité de régénérer plusieurs parties de leur corps. Ils peuvent, entre autres, régénérer parfaitement un membre amputé par épimorphose, un processus biphasique comprenant une phase de préparation, spécifique à la régénération, et une phase de redéveloppement, commune à l’épimorphose et au développement embryonnaire. Durant la phase de préparation, les cellules du moignon se dédifférencient en cellules pseudo-embryonnaires, prolifèrent et migrent distalement au plan d’amputation pour former un blastème de régénération. Parmi les vertébrés, la dédifférenciation est unique aux urodèles. Afin de mieux comprendre le contrôle moléculaire de la régénération chez les urodèles, nous avons choisi d’étudier BMP-2, un facteur de croissance, en raison de son implication dans la régénération des phalanges distales chez les mammifères. Le facteur de transcription MSX-1 a également été sélectionné en raison de sa capacité à induire la dédifférenciation cellulaire in vitro et de son interaction potentielle avec la signalisation des BMPs. Les résultats présentés dans cette thèse démontrent que BMP-2 et MSX-1 sont exprimés lors des phases de préparation et de redéveloppement de l’épimorphose, et que leur profil d'expression spatio-temporel est très semblable, ce qui suggère une interaction de leurs signaux. En outre, chez les tétrapodes amniotes, l’expression de Shh est restreinte au mésenchyme postérieur des membres en développement et chevauche l’expression de BMP-2. Toutefois, l’expression de BMP-2 n’est pas restreinte à la région postérieure mais forme un gradient postéro-antérieur. Shh est le principal régulateur de la formation du patron de développement antéro-postérieur du ii membre. Étant donné les domaines d’expression chevauchants de BMP-2 et Shh et la restriction postérieure d’expression de Shh, on croit que Shh régule la formation du patron de développement de postérieur à antérieur par l’activation de l’expression de BMP-2. Fait intéressant, l’axolotl exprime également Shh dans la région postérieure, mais le développement des pattes se fait de la région antérieure à la région postérieure au lieu de postérieur à antérieur comme chez les autres tétrapodes, et ceci durant le développement et la régénération. Nous avons utilisé cette caractéristique de l’axolotl pour démontrer que la signalisation Shh ne structure pas l’autopode via BMP-2. En effet, l’expression de BMP-2 n'est pas régulée par l'inhibition de la signalisation Shh, et son expression est du côté opposé à celle de Shh durant le développement et la régénération des pattes de l’axolotl. Il a été observé durant le développement du membre chez la souris que MSX-1 est régulé par la signalisation Shh. Nos résultats ont démontrés que chez l’axolotl, MSX-1 ne semble pas régulé par l'inhibition de la signalisation Shh au cours de la régénération du membre. De plus, nous avons démontré que contrairement à l’expression de Shh, l’expression de BMP-2 est corrélée avec l’ordre de formation des phalanges, est impliquée dans la condensation cellulaire et dans l'apoptose précédant la chondrogenèse. L’ensemble de ces résultats suggère un rôle de BMP-2 dans l’initiation de l’ossification endochondrale. Enfin, nous avons démontré que la signalisation BMP est indispensable pour l’épimorphose du membre durant la phase de redéveloppement. / Urodele amphibians (e.g. the axolotls) have a remarkable ability to regenerate parts of their body. They will, among other things, fully regenerate an amputated limb by epimorphosis, a biphasic process comprising a preparation phase, specific to the regeneration, and a redevelopment phase, common to epimorphosis and embryonic development. During the preparation phase, the cells of the stump dedifferentiate into embryonic-like cells, proliferate and migrate distally from the level of amputation to form a regeneration blastema. Among vertebrates, the process of dedifferentiation is unique to urodeles. To better understand the molecular control of regeneration in urodeles, we chose to study BMP-2, a growth factor, because of its involvement in mammalian digit tip regeneration. The transcription factor MSX-1 has also been selected because of its ability to induce cellular dedifferentiation in vitro and its potential interaction with BMPs signaling. The results presented in this thesis show that BMP-2 and MSX-1 are expressed during phases of preparation and redevelopment of epimorphosis, and their spatio-temporal expression profiles are very similar at each stage of epimorphosis, suggesting an interaction of their signals during regeneration. In addition, in tetrapod amniotes, the expression of Shh is restricted to the posterior mesenchyme of developing limbs and overlaps with the expression of BMP-2. However, the expression of BMP-2 is not restricted to the posterior region but forms a posterior-anterior gradient. Shh is the main regulator of the anterior-posterior pattern formation of developing limbs. Given the overlapping expression domains of Shh and BMP-2, and the expression restriction of Shh in posterior, Shh is believed to iv regulate the pattern formation of developing limbs by the activation of BMP-2 expression. Interestingly, the axolotl also expresses Shh in the posterior region, but the limb develops from anterior to posterior rather than posterior to anterior as in other tetrapods, and this, during development and epimorphosis. We used this feature of the axolotl to demonstrate that Shh signaling does not regulate pattern formation through BMP-2. Indeed, the expression of BMP-2 is not regulated by the inhibition of hh signaling, and its expression is opposite to that of Shh during development and regeneration of the axolotl limb. It was observed, during limb development in mice that MSX-1 is regulated by Shh signaling. Our results suggest that in the axolotl, MSX-1 is not regulated by the inhibition of Shh signaling during limb regeneration. Furthermore, we demonstrated that unlike the expression of Shh, the expression of BMP-2 is correlated with the order of formation of the phalanges, is involved in cell condensation and apoptosis preceding chondrogenesis. Taken together, these results suggest a role for BMP-2 in the initiation of endochondral ossification. Finally, we demonstrated that BMP signaling is essential for the redevelopment phase of limb epimorphosis.

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.

Os tecidos ósseos alógenos são utilizados nas cirurgias de reconstrução ortopédicas e odontológicas. O número crescente de transplantes de ossos na última década beneficia diversos pacientes. Os profissionais dos Bancos de Tecidos utilizam protocolos que reduzem os riscos de transmissão de doenças infectocontagiosas, entretanto, não eliminam esta possibilidade. Portanto, é importante que tecidos sejam esterilizados por um método eficaz, sendo usualmente aplicada a radiação ionizante. Porém, a radiação ionizante pode acarretar alterações estruturais e biológicas nos ossos em relação à dose. O objetivo deste trabalho foi estudar os efeitos da aplicação da radiação gama e de feixe de elétrons, nas doses de 15 kGy, 25 kGy e 50 kGy nos tecidos ósseos desmineralizados preservados liofilizados ou congelados e avaliar as possíveis alterações no colágeno, na concentração de proteínas totais, BMP-2 e BMP-7. Para tanto foram utilizadas as técnicas de espectroscopia Raman, Bradford e ELISA. Foram utilizadas 5 diáfises de femur humano e parte das amostras foram desmineralizadas. As proteínas ósseas foram extraídas e quantificadas. Com os resultados da espectroscopia Raman, observamos que a eficiência da desmineralização foi de 85 a 90% porém com alterações na estrutura do colágeno. Alterações também foram observadas nos ossos congelados e irradiados nas doses de 25 kGy e 50 kGy. Nos resultados da quantificação de proteínas totais e específicas, ocorreram diminuições gradativas das concentrações médias das proteínas em relação à dose de radiação nos grupos estudados. Nas doses de radiação usualmente aplicadas aos tecidos ósseos, 15 kGy ou 25 kGy, as reduções nas concentrações das BMP-2 e BMP-7, foram menores que 20%. As reduções na dose de 50 kGy foram entre 27% a 53%, sendo influenciadas pelo tipo de radiação e pelo tipo de preservação dos ossos. / Allogenic bone tissues are used in orthopedic and dental reconstructive surgery. The growing number of bone transplants in the last decade benefits many patients. The professionals of Tissue Banks use protocols that reduce the risk of transmission of infectious diseases, however do not eliminate this possibility. Therefore, it is important that tissues are sterilized by an effective method, usually being applied ionizing radiation. However, ionizing radiation can cause structural and biological changes in the bones relative to the dose. The objective of this work was to study the effects of the application of gamma radiation and electron beam at doses of 15 kGy, 25 kGy and 50 kGy in the bone tissues demineralized preserved freeze-dried or frozen and to evaluate possible changes in collagen, the protein concentration total, BMP-2 and BMP-7. For both Raman spectroscopy techniques, Bradford and ELISA were used. They used 5 diaphyses human femur and part of the samples was demineralized. Bone proteins were extracted and quantified. With the results of Raman spectroscopy, we found that the efficiency of demineralization was 85-90% but with changes in the structure of collagen. Changes were also observed in frozen and irradiated bone at doses of 25 kGy and 50 kGy. The results of quantification of total and specific proteins, there were gradual decreases in average concentrations of proteins in relation to radiation dose in both groups. In the radiation doses usually applied to the bone tissue (15 kGy and 25 kGy) reductions in concentrations of BMP- 2 and BMP-7, were lower than 20%. The reductions at a dose of 50 kGy were between 27% to 53%, being influenced by the type of radiation and the kind of preservation of the bones.

O objetivo deste estudo foi avaliar a imunolocalização do fator de crescimento BMP- 2 em enxerto ósseo autógeno em bloco recoberto ou não por membrana de PTFE-e na fase inicial da reparação óssea. MATERIAL E MÉTODOS: Quarenta e oito ratos \"wistar\" foram divididos em dois grupos de estudo, um que recebeu somente o enxerto ósseo autógeno (E) e o outro que recebeu o enxerto ósseo autógeno recoberto por membrana (ME). Os períodos de avaliação foram: 0 hora, 3, 7, 14, 21 e 45 dias. Os cortes foram submetidos à reação imunoistoquímica. Foram realizadas as análises, qualitativa e quantitativa: contagem de células com marcação intracitoplasmática nas estruturas bordo, enxerto, tecido conjuntivo superior ao enxerto, interface e leito. RESULTADOS: No leito o maior número de células marcadas foi observado em 3 dias nos grupos (E) e (ME). A região de bordo apresentou maior número de células marcadas em 7 dias no grupo (E) e em 14 dias no grupo (ME), onde a marcação foi predominante em osteoblastos e células osteoprogenitoras em áreas de remodelação óssea. No tecido conjuntivo superior ao enxerto a maior proporção de marcação no grupo (E) ocorreu em 7 dias e no grupo (ME) em 21 dias, no entanto, neste último, ainda havia marcação em células osteoprogenitoras ao final dos 45 dias. Na interface, o maior número de células marcadas ocorreu entre 7 e 21 dias, considerando os grupos (E) e (ME), quando células osteoprogenitoras apresentaram-se positivas nas áreas de pontes de osso imaturo entre o leito e o enxerto. A maior proporção de marcação no enxerto foi variável entre os períodos de 3 dias no grupo (E) e 21 dias no grupo (ME), mas esta foi considerada de menor proporção quando comparada às marcações observadas nas outras estruturas. CONCLUSÕES: Os dados observados mostram que as estruturas de maiores proporções de marcação são aquelas relacionadas à intensa revascularização e osteogênese. Células osteoprogenitoras, osteoblastos e osteócitos apresentaram marcação intracitoplasmática, independentemente do período ou da estrutura analisada. À exceção da interface, o grupo (ME) apresentou marcação mais tardia quando comparado ao grupo (E) para as estruturas bordo e tecido conjuntivo superior ao enxerto. / The aim of this study was to evaluate the immunolocalization of the BMP-2 growth factor in an autogenous block graft covered or not with an e-PTFE membrane on the early phases of bone repair. MATERIAL AND METHODS: Forty-eigth wistar male rats had their mandibles augmented by either an autogenous bone block graft (B) or an autogenous bone block graft covered with an e-PTFE membrane (MB). The specimens were evaluated at baseline, 3, 7, 14, 21 and 45 days after surgery by immunohistochemistry. Qualitative and quantitative analysis were performed. RESULTS: On the receptor bed the greatest number of staining cells was observed within three days on groups (B) and (MB). The border region has shown a largest number of staining cells in seven days on (B) group and in fourteen days on (MB) group, where the staining was prominent in osteoblasts and osteoprogenitor cells in bone remodeling areas. On the connective tissue above the graft, the greatest staining proportion occurred on (B) group within seven days and on (MB) group in twenty one days. However, there was still staining in osteoprogenitor cells in the end of the experiment, (at fourty-five day period). On the interface, the greatest number of staining cells occurred between seven and twenty one days for groups (B) and (MB) respectively, when osteoprogenitor cells were positive in woven bone edges between the receptor bed and the graft. The greatest proportion of staining on the graft was variable among tree days on group (B) and twenty one days on group (MB), but this was considered a smaller proportion when compared to other structures staining. CONCLUSIONS: The data suggest that the greatest staining proportions structures are related to intense revascularization and osteogenesis. Osteoprogenitor cells, osteoblasts and osteocytes showed intracitoplasmatic staining, independently of the period or structure analyzed. Except for the interface, the (MB) group has shown later staining compared to group (MB) for border and connective tissue above the graft.

Les films multicouches de polyélectrolytes sont des auto-assemblages de polymères chargés formant des films dont l'épaisseur peut être variée de quelques nm à quelques µm. Un nombre croissant de travaux concerne la compréhension de leur mécanisme d'auto-assemblage et leur utilité pour modifier les propriétés physico-chimiques, topographiques ou mécaniques de surface de (bio)matériaux. Dans cette thèse, nous avons étudié les propriétés de films à base de poly(L-lysine) et de polysaccharides connus pour leur rôle physiologique, notamment le hyaluronane, la chondroïtine sulfate et l'héparine. Les compositions internes de ces films mono-constituants ou à base de mélanges de polyélectrolytes ont été sondées. L'influence de la chimie des polyélectrolytes sur la formation des films, en particulier l'importance des groupements sulfates, a été mise en évidence. Leur potentiel comme vecteur de délivrance d'un facteur de croissance, la BMP-2, a été évalué. De fortes quantités de BMP-2 ont pu être chargées dans les films à base de hyaluronane. Nous avons pu contrôler les quantités insérées en faisant varier la composition chimique des films, leur épaisseur ou la concentration en BMP-2 de la solution de chargement. Puis nous avons mis en évidence une différenciation contrôlée de façon dose-dépendante de cellules C2C12 pluripotentes sur les films bioactifs : différenciation myogénique (en absence de facteur) ou ostéoblastique. De plus, nous montrons qu'un contact des cellules avec le film bioactif est nécessaire pour induire leur différenciation. La protéine est donc présentée par « la phase solide », ce qui constitue un mode de présentation du facteur proche des conditions physiologiques. Des résultats préliminaires obtenus en recouvrant des biomatériaux orthopédiques par les films bioactifs laissent penser que ces films offrent des perspectives intéressantes dans le domaine de la régénération osseuse in vivo. / Polyelectrolyte multilayer films are self-assembled architectures forming nm to µm thick films. During the last decade, they have emerged as an efficient way of modifying materials surface properties such as chemistry, physico-chemical properties, topography as well as mechanical properties. Thanks to the technology's versatility and ease of use, polyelectrolyte multilayer films are now recognized as a new tool for modifying biomaterial surfaces and mediating cell behaviours and implant bio-integration. In this thesis, we studied the properties of poly(L-lysine) and polysaccharide-based multilayer films and focused on their physical-chemical properties as well as on their internal composition. In particular, we studies the influence of their chemistry (presence of carboxylic or sulfate groups) on film formation and characteristics. Three polysaccharides with increasing sulfate group content were chosen for this purpose: hyaluronan, chondroitin sulfate and heparin. The capacity of these films to act as a drug delivery vehicle for BMP-2 (a growth factor able to induce osteo-differentiation) was then assessed. High BMP-2 amounts were successfully loaded and retained in the films in a controlled manner. The loaded amounts could be modulated by varying the film's chemistry, film thickness or BMP-2 concentration in the loading solution. We showed that it is possible to control the extent of C2C12 cell differentiation in osteoblasts when cultured on the bioactive films. Importantly, when no BMP-2 is loaded in the films, the cells differentiated in to myotubes, their most common differentiation pathway. Cells needed a direct contact with the bioactive films to respond to BMP-2, suggesting that BMP-2 is mainly presented to the cells from the solid phase. Preliminary in vivo tests on film-coated orthopaedic biomaterials are encouraging. They showed that these films are interesting candidates for surface modification of orthopaedic biomaterials and may foster bone regeneration.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.