Un substitut osseux hybride, composé d’un biomatériau support (scaffold) et de cellules vivantes, a été étudié, développé par la méthode d’ingénierie tissulaire et caractérisé. Il devait répondre aux attentes spécifiques de la chirurgie maxillofaciale : un protocole standard pouvant s’adapter aux géométries complexes des défauts osseux de chaque patient, une forme souple et manipulable, une pré-vascularisation et une cohésion mécanique suffisante. Une forme de feuillet fin et plat a ainsi été définie et développée au sein d’une chambre de culture parallélépipédique spécifique, en utilisant une monocouche de granules de phosphate de calcium comme support. Après une caractérisation biologique et mécanique complète à partir d’une lignée cellulaire, le procédé a été validé puis transposé à une coculture de cellules primaires humaines (cellules souches et endothéliales). La bonne différenciation et la pré-vascularisation ont été constatées mais le maintien mécanique pouvait être considéré comme insuffisant pour assurer une manipulation en cours d’opération chirurgicale. La dernière partie de ce travail de thèse a donc consisté dans la mise en place d’un montage de production de fibres électrospinnées et leur utilisation comme nouveau support de culture. La formation de ces matériaux a été rendue opérationnelle de façon optimale pour différents polymères. Leur potentiel en tant que scaffold favorisant la différenciation en os ou en tendon a été vérifié et comparé à d’autres matériaux fibreux obtenus dans le cadre de collaborations nationales et internationales. La faisabilité de l’application de sollicitations mécaniques aux substituts en cours de culture a également été étudiée. / An hybrid bone substitute, based on a specific biomaterial (scaffold) and living cells, was studied, developed with a tissue engineered method and characterized. It should meet the expectations of the maxillofacial surgery : a standard process which could fit with the complex geometries of each patient’s bone mass loss, a flexible shape with an easy handling, a prevascularization and a sufficient mechanical cohesion. A sheet-like shape was thus designed and developed in a specific flat cell culture chamber, with a monolayer of calcium phosphate granules as a scaffold. After both biological and mechanical full characterizations with a cell line, the process was adapted to a coculture of human primary cells (stem and endothelial cells). Relevant differentiation and prevascularization were highlighted but the mechanical cohesion could be noticed as too low to ensure an easy handling during the surgery. The last part of this thesis project was thus the set-up of a device for electrospun polymer fibers in order to use them as a new scaffold. The production of these materials was efficiently performed for several polymers. The differentiation potential for bone and tendon lineages was studied and compared to other scaffolds from national and international collaborations. The application of mechanical solicitations to the substitutes during cellculture was also studied.

Ce travail porte sur la recherche et le développement d'un matériau de substitution osseuse permettant une implantation par chirurgie mini invasive, limitant les infections post-opératoires et dont la résorbabilité serait adaptée à la cinétique de régénération osseuse. Nous nous sommes intéressés à un ciment à base de carbonate et de phosphate de calcium (CaCO3 – CaP) dont la réaction de prise conduit à la formation d'une apatite nanocristalline analogue au minéral osseux. Dans une première partie la cinétique de prise et le produit de réaction ont été caractérisés par différentes techniques, notamment la diffraction des RX et les spectroscopies FTIR et RMN du solide. Un sel d'argent – Ag3PO4 ou AgNO3, choisis pour leurs propriétés antibactériennes – a été ensuite introduit dans la formulation. Son effet sur la cinétique de la réaction chimique de prise a été mis en évidence par traitement des spectres FTIR et RMN et un mécanisme réactionnel original impliquant les ions argent et nitrate dans la formation de l'apatite a été proposé. L'ajout d'un polysaccharide, la carboxyméthylcellulose (CMC), dans la phase solide du ciment a montré une très nette amélioration de l'injectabilité de la pâte, avec la disparition du phénomène de séparation des phases qui limite généralement l'injectabilité des ciments minéraux. La résistance à la compression et le module élastique des ciments composites ont été par ailleurs augmentés, parallèlement à une diminution de leur porosité. Différentes études in vitro en présence de cellules ou de bactéries ont enfin été réalisées et ont mis en évidence respectivement la cytocompatibilité des différentes compositions de ciments étudiées et le caractère antibactérien de ces matériaux à partir d'une certaine concentration en argent. L'implantation in vivo de compositions choisies a présenté des résultats très prometteurs quant à la résorbabilité d'un ciment composite CaCO3 - CaP/CMC/Ag et à la néoformation osseuse. / The present work concerns research and development of a material for bone substitution, enabling implantation through a mini-invasive surgery, limiting post-operative infections and whose resorbability is adapted to bone regeneration kinetics. This study focused on a calcium carbonate and phosphate based cement, whose setting reaction leads to the formation of a nanocrystalline apatite, similar to bone mineral. First, the setting kinetics and the reaction products were characterised using different techniques, especially X-ray diffraction and FTIR and solid-state NMR spectroscopies. A silver salt – Ag3PO4 or AgNO3, chosen for their antibacterial properties – was then introduced in the formulation. Its effect on the setting reaction kinetics was revealed by data processing of FTIR and NMR spectra and an original reaction mechanism which involves silver and nitrates in the formation of apatite was proposed. The addition of a polysaccharide, carboxymethylcellulose (CMC), in the solid phase of the cement showed a clear improvement of the injectability of the paste, preventing the occurrence of filter-pressing phenomenon, often limiting the injectability of mineral cements. The resistance to compressive strength and elastic modulus of the composite cement were also improved together with a decrease in their porosity. Different in vitro studies were carried out in the presence of cells or bacteria and demonstrated the cytocompatibility of different cement compositions and their antibacterial properties starting at a certain silver concentration, respectively. In vivo implantation of selected compositions showed promising results concerning resorbability of a composite CaCO3 - CaP/CMC/Ag cement and the associated bone neoformation.

Nanocompósitos de Al2O3/ZrO2 apresentam-se promissores para serem utilizados como biomateriais, por promoverem melhorias significativas quanto à homogeneidade microestrutural, propriedades mecânicas e serem biologicamente inerte. Dentre as técnicas capazes de tornar seu desempenho biológico mais adequado, o recobrimento biomimético tem se destacado, sendo que sua eficiência pode ser melhorada a partir de prévios tratamentos superficiais. Nesse sentido, o objetivo desse trabalho foi estudar a adesão e a uniformidade do revestimento de fosfato de cálcio em nanocompósitos de Al2O3 contendo 5% em volume de inclusões nanométricas de ZrO2. Para isso, nanocompósitos foram conformados, sinterizados, tratados quimicamente com soluções de H3PO4, HNO3 e NaOH e por plasma em diferentes condições de processo: 20%N2-80%H2, 40%N2-60%H2, 40%N2-40%H2-20%O2, 30%N2-50%H2-20%O2, 100%N2 e 100%O2 e recobertos biomimeticamente com soluções que simulam o plasma sanguíneo (SBF 1,5x e 5,0x) nos tempos de 7, 14, 21 e 28 dias de incubação. A partir dos resultados obtidos pode-se observar que os prévios tratamentos superficiais promoveram variações na rugosidade média superficial (Ra) entre 0,045 e 0,079 µm. Além disso, independentemente do tratamento superficial, observou-se a formação de apenas três fases de fosfatos de cálcio: hidroxiapatita (HA), α-fosfato tricálcico (α-TCP) e β-fosfato tricálcico (β-TCP). Observou-se ainda, que o percentual das fases formadas variou de acordo com o tratamento, sugerindo a possibilidade de controlar a razão TCP:HA. Nos resultados obtidos pelo teste de riscamento observou-se que tratamentos realizados com plasma, H3PO4 e HNO3 aumentaram a carga crítica (Lc) entre 2,0 e 2,9N, melhorando assim a aderência da camada de fosfato de cálcio formada. Os testes de proliferação celular utilizando linhagem de células de hamster chinês (CHO) demonstraram que os nanocompósitos tratados com H3PO4, HNO3 e NaOH e recobertos biomimeticamente com SBF 5,0x são biocompatíveis. / Nanocomposite Al2O3/ZrO2 have to be promising to be used as biomaterials for promoting significant improvements in the microstructural homogeneity, mechanical properties and be biologically inert. Among the techniques to make it more useful biological performance, the biomimetic coating has been highlighted, and its efficiency can be improved from previous surface treatments. In this sense, the objective of this work was to study the adhesion and uniformity of calcium phosphate nanocomposite coating of Al2O3 containing 5% by volume of nanometric inclusions of ZrO2. For this nanocomposites were shaped, sintered, chemically treated with solutions of H3PO4, HNO3 and NaOH, and plasma in different process conditions: 20%N2-80%H2, 40%N2-60%H2, 40%N2-40%H2-20%O2, 30%N2-50%H2-20% O2, 100% N2 and 100%O2 and covered with solutions biomimetic that simulate the blood plasma (SBF 1.5x and 5.0x) in the times of 7, 14, 21 and 28 days of incubation. From the results obtained it can be seen that prior surface treatments promoted variations in average surface roughness (Ra) from 0.045 to 0.079 micrometers. Furthermore, regardless of surface treatment, formation of the observed only three-phase calcium phosphate: hydroxyapatite (HA), tricalcium α-phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). It was also observed that the percentage of phases formed varied according to the treatment, suggesting the possibility of controlling the ratio TCP:HA. In the results obtained by scratching test was observed that plasma treatments performed, HNO3 and H3PO4 increased the critical load (Lc) between 2.0 and 2,9N, thereby improving the adhesion of the calcium phosphate layer is formed. Cell proliferation assays The strain using chinese hamster cells (CHO) have demonstrated that nanocomposites treated with H3PO4, HNO3 and NaOH, and coated with solution biomimetic SBF 5.0x are biocompatible.

Os objetivos deste trabalho foram : 1) sintetizar e caracterizar partículas de fosfato di-cálcico di-hidratado (DCPD) funcionalizadas com dimetacrilato de trietileno glicol (TEGDMA), com o propósito de reduzir a aglomeração e melhorar a interação entre as partículas e a matriz orgânica dos materiais resinosos e 2) avaliar o efeito da adição de partículas funcionalizadas em uma matriz resinosa em relação a liberação de íons, propriedades ópticas, grau de conversão, propriedades mecânicas e microestrutura do material. Na primeira etapa do trabalho foram sintetizadas pelo método sol-gel dois tipos partículas de DCPD: funcionalizadas e puras. Além dessas, caracterizou-se nesta etapa partículas comerciais do mesmo composto. As partículas foram caracterizadas por difração de raio X (DRX), espectroscopia no infravermelho com transformada de Fourier (FTIR), análise elementar, análise termogravimétrica (TGA), isortermas de adsorção de nitrogênio e método BET, microscopia eletrônica de transmissão e espalhamento de luz dinâmico (DLS). Na segunda etapa do trabalho, um novo lote de partículas funcionalizadas foi sintetizado, e como controle, sintetizou-se partículas não funcionalizadas. Quatro materiais resinosos foram formulados, um deles contendo apenas BisGMA e TEGDMA (1:1 em mols) e os demais contendo, além da matriz resinosa, 20% (em volume) de partículas funcionalizadas, partículas não funcionalizadas ou sílica coloidal silanizada. Os materiais foram avaliados quanto à liberação de íons, propriedades ópticas (transmitância total e E em relação à resina sem carga), grau de conversão (GC), resistência à flexão biaxial (RFB), módulo de elasticidade e análise microestrutural. Os dados foram submetidos à análise de variância (ANOVA), complementada pelo teste de Tukey, com nível de significância de 5 %. A composição das partículas foi confirmada por DRX e a funcionalização das mesmas foi confirmada pelas análises de FTIR, análise elementar e TGA. As partículas funcionalizadas apresentaram área superficial até 3 vezes maior quando comparadas às partículas puras e comerciais. Esses dados foram confirmados pela análise de DLS, que encontrou aglomerados maiores para as partículas dos compostos puro e comercial. Na segunda etapa do estudo, observou-se que a funcionalização das partículas não afetou a liberação de íons Ca2+ dos materiais bioativos; entretanto, a liberação de íons HPO42- foi 51% menor para o material contendo partículas funcionalizadas. Todos os materiais apresentaram GC semelhantes. O compósito com sílica apresentou valores de transmitância significantemente menores e E superior ao obtido para os demais materiais. A funcionalização das partículas aumentou a RFB em 32%, porém ainda inferior à do compósito com sílica. A funcionalização não afetou o módulo de elasticidade dos materiais. A síntese de partículas de DCPD funcionalizadas com TEGDMA foi realizada com êxito. O monômero funcionalizante contribuiu para a diminuição do tamanho dos aglomerados. Além disso, a funcionalização favoreceu a interação entre as partículas de DCPD e a matriz orgânica dos materiais, evidenciado pelo aumento no valor de resistência à flexão, sem redução substancial na liberação de íons cálcio do material. / The aims of this study were: 1) to synthesize and characterize dicalcium phosphate dihidrate (DCPD) particles functionalized with triethylene glycol dimethacrylate (TEGDMA), with the purpose of reducing agglomeration and improve the interaction between the particles and the organic matrix of resin-based materials and 2) evaluate the effect of the addition of functionalized particles in a resin matrix regarding ion release, optical properties, degree of conversion, mechanical properties and microstructure. In the first part of the study, two types of DCPD particles were synthesized by a sol-gel method: functionalized and bare. Additionally, proprietary DCPD particles were also characterized. Particles were characterized by X-ray diffraction (DRX), Fourier-transformed infrared spectroscopy (FTIR), elemental analysis, thermogravimetric analysis (TGA), Nitrogen adsorption isotherms and BET method, transmission electron microscopy and dynamic light scattering (DLS). In the second part, a new batch was synthesized and, as a control, non-functionalized nanoparticles were synthesized. Four resin-based materials were prepared, one containing only BisGMA and TEGDMA (1:1 in mols) and the other containing besides the resin, 20 % (in volume) of functionalized, non-functionalized or silanized colloidal silica particles. Materials were evaluated for ion release, optical properties (total transmittance and E, in relation to the unfilled resin), degree of conversion (DC), biaxial flexural strength (BFS), elastic modulus and microstructural analysis. Data were subjected to analysis of variance (ANOVA), complemented by Tukey test, with a global significance level of 5%. Particles composition was confirmed by DRX, while functionalization was confirmed by FTIR , elemental analysis and TGA. Functionalized particles presented surface area up to three times higher compared to bare and proprietary particles. These findings were confirmed by DLS, which found larger agglomerates for the bare and proprietary particles. In the second part of the study, it was observed that particle functionalization did not affect Ca2+ release from the bioactive materials; however, HPO42- release was 51% lower from the material containing functionalized particles. All materials presented similar DC. The silica composite presented transmittance values significantly lower and higher E than the other materials. Functionalization increased BFS in 32%, however, it remained lower than the silica-containing composite. Functionalization did not affect elastic modulus. The synthesis of functionalized particles was accomplished with success. The functionalized monomer contributed for reducing agglomerate size. Also, functionalization favored the interaction between DCPD particles and the resin matrix of the materials, evidenced by the increase in flexural strength, without substantially reducing calcium release from the material.

De nombreux traitements de surface (sablage, attaques acides…) ont été mis au point sur les implants dentaires afin de favoriser leur ostéointégration. Par ailleurs, depuis plusieurs années des revêtements à base de phosphates de calcium sont également développés dans le même but.L’objectif principal de la thèse est de mettre au point un procédé de revêtement à basse température afin de déposer à la surface de l’implant en titane une couche mince de phosphate de calcium, de structure et de composition analogue au minéral osseux en vue de favoriser l’ostéointégration. Il est aussi souhaité que ce revêtement présente des propriétés antibactériennes afin de lutter contre les infections post-opératoires. Dans un premier temps, un traitement de surface de l’implant en titane composé d’une étape de sablage et d’une attaque acide a été développé. Il permet d’obtenir une rugosité moyenne de surface comprise entre 1,4 et1,8 µm ainsi qu’une texture microporeuse et une mouillabilité de surface améliorée. Puis, les procédés de revêtement d’électrodéposition et d’immersions successives ont été étudiés. L’étapede centrifugation implémentée dans le procédé d’immersions successives s’est révélée cruciale et un revêtement d’environ 2 µm d’épaisseur composé d’apatite biomimétique a été obtenu. La composition et l’épaisseur du revêtement élaboré par électrodéposition est fortement influencée par la durée du dépôt. Ainsi une durée d’électrodéposition de 1 mn menée à un potentiel de -1,6V/ECS permet d’obtenir un revêtement d’environ 1,5 µm d’épaisseur composé d’une couche de phosphate octocalcique et de cristaux de brushite. Un test de vissage/dévissage dans une mâchoire artificielle a démontré la tenue mécanique des revêtements obtenus selon les deux procédés. Enfin, des ions antibactériens tels que l’argent, le cuivre ou le zinc ont été incorporésaux revêtements. Il a été démontré que des taux importants d’incorporation allant jusqu’à 40 %molaire par rapport au calcium peuvent être atteints pour le cuivre et le zinc. Des tests biologiques permettant d’évaluer l’effet de ces revêtements sur l’activité biologique de cellules mésenchymateuses humaines ainsi que sur la formation d’un biofilm (modèle de péri-implantite)en vue de la prévention des infections post-opératoires ont conduit à des résultats prometteurs pour le développement de tels revêtements bioactifs. Ces travaux de thèse s’inscrivent dans le cadre du projet BIOACTISURF, soutenu par la Région Midi-Pyrénées, et réalisé en collaboration avec le Laboratoire de Génie Chimique (LGC) ainsi qu’un partenaire industriel / Numerous surface treatments have been developed in order to improve osseointegration of dentalimplants (sandblasting, acid etching…). Moreover, various strategies involving calcium phosphate coatings have emerged for the same target for a few decades. The main purpose of this work is todevelop a low temperature process allowing the deposition of a thin calcium phosphate layer at the titanium implant surface. The composition and structure of this calcium phosphate coating have to be close to the bone mineral to enable osseointegration improvement. Moreover, thecoating should have antibacterial properties in order to prevent post-operative infections. A surfacetreatment protocol composed of sandblasting and acid etching was firstly developed creating anaverage roughness of 1.4 – 1.8 µm with micropits and improved wettability. Secondly, two processes were studied to produce the calcium phosphate coating: the alternate soaking processand the electrodeposition. It was demonstrated that the centrifugation step implemented in thealternate soaking process is crucial and a coating of about 2 µm thick composed of biomimeticapatite was obtained. Among all the operating parameters of the electrodeposition process, time ofelectrodeposition has the major impact on the composition and the thickness of the coating. An electrodeposition of 1 mn at -1.6 V/SCE leads to a 1.5 µm thick coating composed of a layer ofoctacalcium phosphate and dicalcium phosphate dihydrate crystals. A screw/unscrew test demonstrated the mechanical stability of the coatings obtained by both processes. Finally,antibacterial ions such as silver, copper and zinc were incorporated in the coatings. Highincorporation rate up to 40 mol.% compared to calcium were determined for copper and zinc.Biological tests were conducted to qualify the effect of these coatings on the biological activity ofhuman mesenchymal cells and on the formation of a biofilm (peri-implantitis model) to preventpost-operative infections. They led to promising results for the development of such bioactivecoatings. This work is part of the BIOACTISURF project, supported by the Midi-Pyrénées Region,and carried out in collaboration with the Laboratory of Chemical Engineering (LGC) and an industrial partner

La presente Tesis Doctoral se titula "Obtención y caracterización de nuevos cementos óseos de fosfatos de calcio en el sistema DCP&#8213;alfa-TCP". Los cementos de fosfatos de calcio (CFC) son materiales biocompatibles que tienen la propiedad de endurecer en condiciones fisiológicas formando una estructura porosa de cristales interconectados de hidroxiapatita, i.e. la fase mineral de los tejidos óseos. Estos materiales pueden ser diseñados para que sean inyectables y tengan distintos porcentajes de porosidad. Este hecho permite obtener cementos con distintas tasas de reabsorción ósea que pueden aplicarse con técnicas de mínima invasión. En general, estas propiedades otorgan a estos biomateriales un excelente interés en campos de aplicación de prevención, reconstrucción o estabilización ósea (p.e.: osteoporosis, cirugía craneofacial, vertebroplastia, cifoplastia).Esta Tesis Doctoral consta de 6 capítulos. El Capítulo 1 (Cementos óseos de fosfatos de calcio) recoge el estado del arte de los CFC. Se realiza una revisión bibliográfica crítica de toda la información de interés. Se concluye la necesidad de realizar estudios cinéticos que expliquen adecuadamente las propiedades de fraguado de estos cementos y permitan diseñar cementos con mejores propiedades. El Capítulo 2 (Materiales y métodos utilizados en la fabricación y caracterización de cementos óseos de fosfatos de calcio) describe el protocolo de trabajo seguido para caracterizar los CFC física y químicamente. Este protocolo permite establecer correlaciones entre la microestructura y las propiedades macroscópicas. El Capítulo 3 (Estudio de la cinética de la reacción de fraguado de un cemento óseo de fosfato de calcio) investiga la cinética de fraguado de un cemento octocálcico en el sistema DCP&#8213;alfa-TCP. Los resultados mostraron que: (a) sólo el alfa-TCP reaccionó para dar una hidroxiapatita deficiente en calcio (CDHA); (b) el porcentaje de reacción y la resistencia mecánica a compresión estaban correlacionadas; (c) el cemento presentó una estructura de cristales interconectados con distinto grado de compacidad. El Capítulo 4 (Estudio de nuevas formulaciones de cementos de fosfatos de calcio en el sistema DCP&#8213;alfa-TCP) investiga las propiedades de fraguado y de resistencia mecánica de cementos del sistema DCP&#8213;alfa-TCP en el intervalo de relaciones Calcio/Fósforo (Ca/P) 1.26<Ca/P<1.50. Se estudia cómo varían la trabajabilidad, los tiempos de fraguado y la resistencia a la compresión. También se estudia el efecto de la adición de carbonato cálcico (CC) sobre las propiedades de fraguado ya que se espera obtener como producto final de la reacción de fraguado una hidroxiapatita deficiente en calcio carbonatada y, por lo tanto, más semejante a la apatita de la fase mineral ósea. Los resultados mostraron que la adición de un 10 % de CC produjo un efecto fluidificante sobre los cementos con un incremento en los valores de los tiempos de fraguado. El análisis de la evolución de la resistencia a la compresión con el tiempo demostró que el CC actúa como un retardador. Sin embargo, la resistencia final alcanzada representa en muchos casos un incremento de más del 40 %. El Capítulo 5 (Discusión general) discute los resultados más relevantes analizados a lo largo de los Capítulos 3 y 4 para comprender las posibilidades clínicas que este tipo de materiales nos ofrece. El Capítulo 6 (Conclusiones) presenta una serie de conclusiones obtenidas a partir de los resultados de los Capítulos 3 y 4. Son conclusiones extraídas a partir del análisis de los resultados de determinadas poblaciones de muestras. Como los intervalos de variabilidad de los parámetros de cada población son diferentes, muchas de las conclusiones no son generalizables a todo el sistema DCP&#8213;alfa-TCP, sino que se han de leer dentro de este sistema y en las condiciones experimentales estudiadas. Finalmente, la Tesis Doctoral lista el conjunto de referencias bibliográficas utilizadas en este trabajo de investigación.

Los estímulos mecánicos son uno de los factores que afectan a la diferenciación celular en el proceso de regeneración del tejido óseo, por lo tanto, en el desarrollo de andamios para ingeniería de tejidos, se pueden aplicar las cargas mecánicas con el fin de inducir la actividad de las células. Cuando se aplican cargas mecánicas, los estímulos mecánicos específicos transmitidos a las células a nivel microscópico pueden estudiarse mediante técnicas numéricas. El objetivo de esta tesis fue estudiar la mecanoregulación de la diferenciación de tejido en andamios de fosfato de calcio utilizando modelos de elementos finitos basados en micro tomografía axial computarizada.Dos muestras de materiales porosos basados en fosfato de calcio fueron utilizadas. Se desarrollaron mallas de elementos finitos congruentes, discretizando la fase sólida y los macro poros interconectados, con el fin de tener en cuenta la morfología irregular de los andamios.En primer lugar, se estudió la distribución de los estímulos mecánicos. La fase sólida y el fluido intersticial se simularon como material elástico lineal y como fluido Newtoniano, respectivamente. Se simuló una compresión del 0.5% en el sólido y un fluido con velocidades de entrada de 1, 10 y 100 µm/s en los poros. Se encontraron distribuciones de deformación similares en las paredes ambos materiales, con valores máximos de 1.6% en compresión y de 0.6% en tracción. En algunos poros, la velocidad del fluido aumentó a 100 y 1000 veces la velocidad de entrada. Este estudio mostró como estímulos mecánicos macroscópicos pueden causar distintos niveles de estímulos mecánicos microscópicos dentro los andamios, debido a la morfología.A continuación se realizó un estudio en el tiempo de la diferenciación de tejido en un andamio sometido a condiciones in vitro. La compresión y la perfusión se modelaron como en el estudio anterior. Se simularon una compresión del 0.5% y una velocidad de entrada de fluido constante de 10 µm/s o una presión de entrada de fluido constante de 3 Pa. La deformación cortante octaédrica y el esfuerzo cortante del fluido se utilizaron como estímulos mecano-regulatorios basándose en la teoría de Prendergast et al. (1997). Al aplicar velocidad constante, se predijeron fluctuaciones entre los estímulos equivalentes a la formación de tejido y a la muerte celular, debido al aumento en el esfuerzo cortante del fluido cuando el tejido comienza a llenar los poros. Sin embargo, al aplicar presión constante, se predijo estímulo equivalente a la diferenciación de tejido óseo en la mitad del volumen de los poros. Estos resultados sugieren que para permitir la diferenciación de tejido, la velocidad del fluido debe disminuirse cuando el tejido empieza a mineralizarse.Finalmente, se llevó acabo un estudio en el tiempo de la angiogénesis y de la diferenciación de tejido en un andamio bajo condiciones in vivo. La deformación cortante octaédrica y la velocidad relativa del fluido se utilizaron como estímulos mecano-regulatorios. Las fases sólida y porosa fueron tratadas como materiales poroelásticos. Se simuló la actividad individual de las células. Compresiones de 0.5 y 1% fueron simuladas. La mayoría de los vasos crecieron en los poros de la periferia del andamio y se bloquearon por las paredes. Se formaron redes capilares similares independientemente de la magnitud de deformación utilizada. Al aplicar 0.5% de compresión, estímulos correspondientes a la formación de hueso se predijeron en el 70% del volumen de los poros, sin embargo, sólo el 40% del volumen se llenó de osteoblastos debido a la falta de oxigeno. Este estudio mostró el efecto de la falta de vascularización en el centro del andamio en la diferenciación de tejido.Ese tipo de estudios, combinados con estudios in vitro, deberían contribuir a la comprensión del proceso de diferenciación de los tejidos dentro de los andamios y por lo tanto a la mejora de los métodos de diseño de andamios. / Mechanical stimuli are one of the factors that affect cell differentiation in the process of bone tissue regeneration; therefore, in the development of scaffolds for tissue engineering, mechanical loads can be applied in order to induce cell activity. The specific mechanical stimuli transmitted to cells at a microscopic level when mechanical loads are applied can be studied using numerical techniques. The objective of this thesis was to study the mechanoregulation of tissue differentiation within calcium phosphate scaffolds using micro computed tomographed based finite element models.Two samples of porous calcium phosphate based materials were used. Congruent finite element meshes, with the solid phase and the interconnected pores discretized, were developed in order to account for the scaffold irregular morphology.First, a study of the distribution of mechanical stimuli was performed. The solid phase and the fluid flow within the pores were modeled as linear elastic solid material and Newtonian fluid respectively. Compressive strains of 0.5% of total deformation applied to the solid and interstitial fluid flows with inlet velocities of 1, 10 and 100 µm/s applied to the pores were simulated. Similar strain distributions for both materials were found, with compressive and tensile strain maximal values of 1.6% and 0.6% respectively. For the fluid flow models, the fluid velocity in some of the scaffold pores increased to 100 and 1000 times the inlet velocity. This study showed how mechanical loads and fluid flow applied to the scaffolds caused different levels of mechanical stimuli within the samples according to the morphology of the materials.Next, a study of the mechanoregulation of tissue differentiation over time in a scaffold subjected to in vitro loads was performed. The solid phase and the fluid flow were modeled as in the study described above. Compressive strain of 0.5% and fluid flow with constant inlet velocity of 10 µm/s or constant inlet pressure of 3 Pa were applied. Octahedral shear strain and fluid shear stress were used as mechano-regulatory stimuli based on the theory of Prendergast et al. (1997). When a constant velocity was simulated, fluctuations between stimuli equivalent to tissue formation and cell death were predicted due to the increase in the fluid shear stress when tissue started to fill the pores. However, when constant pressure was applied, stimuli equivalent to bone formation were predicted in about half of the pore volume. These results suggest that in order to allow tissue differentiation within a scaffold, the fluid velocity should be decreased when tissue starts mineralizing.Finally, a study of the angiogenesis and the mechanoregulation of tissue differentiation over time in a scaffold subjected to in vivo conditions was performed. Octahedral shear strain and relative fluid velocity were used as mechano-regulatory stimuli. The solid and pore phases were treated as poroelastic materials. Individual cell activity was simulated within the pore domain. Compressive strains of 0.5 and 1% of total deformation were simulated. Most vessels grew in the pores at the periphery of the scaffolds and were blocked by the scaffold walls. Similar capillary networks were formed independently of the magnitude of the mechanical strain applied. When 0.5% of strain was applied, 70% of the pore volume was affected by mechano-regulatory stimuli corresponding to bone formation; however, because of the lack of oxygen, only 40% of the volume was filled with osteoblasts. This study showed the effect of the lack of vascularization in the center of the scaffold on the tissue differentiation.Such kind of studies, combined with in vitro studies, should contribute to the understanding of the process of tissue differentiation within the constructs and therefore to the improvement of scaffold design methods.

COLLAGEN IN SITU GROWN CALCIUM PHOSPHATE SCAFFOLDS FOR OSTEOGENIC DIFFERENTIATION OF WHARTON&rsquo / S JELLY AND MENSTRUAL BLOOD STEM CELLS Karadas, &Ouml / zge M.Sc., Department of Biotechnology Supervisor : Prof. Dr. Vasif Hasirci Co-Supervisor: Assoc. Prof. Dr. Gamze Torun K&ouml / se February 2011, 91 pages The importance of developing new techniques for the treatment of bone and joint diseases is increasing continuosly together with the increase of human population and the average life span. Especially bone fractures as a result of osteoporosis are often seen in humans older than 50 years old. The expenses of bone and joint disease operations are very high and the duration of recovery is long. Because of these reasons World Health Organization, The United Nations and 37 countries announced that the years 2000-2010 is the Bone and Joint Decade. Tissue engineering is an alternative approach to clinically applied methods. In this study collagen scaffolds crosslinked with genipin, to improve the stability of foams in culture media, were prepared by lyophilization. To mimic the natural bone structure calcium phosphate mineral phase in the foam was formed by wet chemical precipitation. Collagen concentration (0.75% and 1%, w/v), freezing temperature (-20 oC and -80 oC) of the collagen solution before lyophilization and immersion duration (2x4 h and 2x48 h) of the foams in calcium and phosphate solutions for wet chemical precipitation were changed as process v parameters of foam production. Pore size distribution and porosity analysis as well as compression test were performed for characterization of the scaffolds. The foam with 1% w/v collagen concentration, frozen at -20 oC before lyophilization and immersed for 2x4 h in calcium and phosphate solution was chosen for in vitro cell culture studies. The defined foam had 70% porosity and pore sizes varying between 50 and 200 &mu / m. The elastic modulus and compressive strength of the foam was calculated as 127.1 kPa and 234.5 kPa, respectively. Stem cells isolated from Wharton&rsquo / s jelly (WJ) and menstrual blood (MB) were seeded to foams to compare their osteogenic differentiation. Both cells are isolated from discarded tissues and used in this study as an alternative to the commonly used cells which are isolated by invasive techniques such as bone marrow stem cells. Cells were seeded to collagen foams with and without calcium phosphate (CaP). It was observed that WJ cells proliferated during 21 days on collagen foams without CaP, but MB cell number decreased after day 14. Collagen foams with CaP supported the alkaline phosphate (ALP) activity compared to tissue culture polystyrene (TCPS) and foams without CaP. Contrarily lower cell numbers achieved on CaP containing collagen foams, possibly because of the calcium and phosphate concentration changes in the medium and as the result of osteogenic differentiation. ALP activity of both cell types increased almost 10 times and specific ALP activity (activity per cell) increased 40 times and 150 times for WJ and MB cells, respectively on the CaP containing foams compared to TCPS. Therefore, in this study it was shown that in situ CaP formed collagen foams induce osteogenic differentiation of WJ and MB cells, and these cells isolated from discarded tissues can be used as alternative cell sources in bone tissue engineering applications.

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Ziel der vorliegenden Arbeit war die biologische Charakterisierung neuartiger nanostrukturierter und für die Knochenregeneration geeigneter Calciumphosphatzemente (CPC). Hierzu wurde ein aus α-Tricalciumphosphat, Calciumhydrogenphosphat, gefälltem Hydroxylapatit und Calciumcarbonat bestehender CPC verwendet, der mit den Biomolekülen Cocarboxylase, Glucuronsäure, Weinsäure, Glucose-1-phosphat, Arginin, Lysin und Asparaginsäure-Natriumsalz modifiziert wurde. Ermittelt wurde dabei der Einfluss der Modifikationen auf die Proteinadsorption und die Biokompatibilität. In Vorversuchen wurden die Zementmodifikationen hinsichtlich ihrer Bindungskapazität für humane Serumproteine und für das knochenspezifische Protein Osteocalcin (OC) sowie hinsichtlich ihrer Eignung für die Adhäsion, Proliferation und osteogene Differenzierung von humanen fötalen Osteoblasten (hFOB 1.19) und humanen mesenchymalen Stammzellen (hMSC) untersucht. Dabei erwiesen sich die Modifikationen mit Cocarboxylase, Arginin und Asparaginsäure-Natriumsalz als besonders günstig. Mit diesen „Favoriten“ erfolgte eine detailliertere Analyse der Adsorption humaner und boviner Serumproteine sowie der knochen-spezifischen Proteine Osteocalcin, BMP-2 und VEGF. Dabei führte sowohl der Zusatz von Cocarboxylase, als auch der von Arginin und Asparaginsäure-Natriumsalz zu einer erhöhten Adsorption von Serumproteinen. Die Bindungsaffinität des Basiszements gegenüber Osteocalcin, BMP-2 und VEGF konnte durch Funktionalisierung mit Arginin gesteigert werden. Während die Modifizierung mit Cocarboxylase nur die VEGF-Adsorption förderte, bewirkte der Zusatz von Asparaginsäure-Natriumsalz eine Erhöhung der Osteocalcin- und BMP-2-Adsorption. Bedingt durch die größere spezifische Oberfläche der noch nicht abgebundenen Zemente, war die Menge adsorbierter Proteine auf frisch hergestellten Zementproben im Vergleich zu abgebundenen und ausgehärteten Zementen signifikant höher. Die Eignung der ausgewählten Zementvarianten als Knochenersatzmaterialien wurde mithilfe humaner mesenchymaler Stammzellen zweier verschiedener Spender getestet. Bei Verwendung abgebundener und ausgehärteter Zemente waren die hMSC in der Lage, auf allen Modifikationen zu adhärieren, zu proliferieren und in die osteogene Richtung zu differenzieren. Eine vorherige Inkubation der Zementproben mit humanem Serum förderte dabei vor allem die Zelladhäsion. Weiterhin konnte gezeigt werden, dass hMSC im Gegensatz zu anderen Studien auch auf frisch hergestellten Zementproben adhärieren, proliferieren und differenzieren können. Die Modifizierung des Basiszements mit Cocarboxylase führte hierbei zu einer gegenüber den anderen Modifikationen signifikant erhöhten Zelladhäsion und -vitalität. Neben den verschieden modifizierten Pulver/Flüssigkeitszementen wurden im Rahmen dieser Arbeit neuartige ready-to-use Zementpasten untersucht. Diese zeigten allerdings im Vergleich zu den herkömmlichen Zementen eine geringere Proteinbindungsaffinität. HMSC, die auf den Pastenzementen kultiviert wurden, war es wiederum möglich zu adhärieren, zu proliferieren und den osteoblastenspezifischen Marker Alkalische Phosphatase zu exprimieren. Hinsichtlich ihrer Biokompatibilität sind sie damit vergleichbar zu den herkömmlichen Pulver/Flüssigkeitszementen.