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[EN] Articular cartilage is a tissue that consists of chondrocytes surrounded by a dense extracellular matrix (ECM). The ECM is mainly composed of type II collagen and proteoglycans. The main function of articular cartilage is to provide a lubricated surface for articulation. Articular cartilage damage is common and may lead to osteoarthritis. Articular cartilage does not have blood vessels, nerves or lymphatic vessels and therefore has limited capacity for intrinsic healing and repair. Tissue engineering (TE) is a powerful approach for healing degenerated cartilage. TE uses three-dimensional (3D) scaffolds as cellular culture supports. The scaffold provides a structure that facilitates chondrocyte adhesion and expansion while maintaining a chondrocytic phenotype and limiting dedifferentiation, which is a problem in two-dimensional (2D) systems. Cell attachment to the scaffolds depends on the physical and chemical characteristics of their surface (morphology, rigidity, equilibrium water content, surface tension, hydrophilicity, presence of electric charges). The primary aim of this thesis was to study the influence of different kinds of biomaterials on the response of chondrocytes to in vitro culture. 3D scaffold constructs must have an interconnected porous structure in order to allow cell development through the network, to maintain their differentiated function, as well as to allow the entry and exit of nutrients and metabolic waste removal. Therefore, the effect of the hydrophilicity and pore architecture of the scaffolds was studied. A series of polymer and copolymer networks with varying hydrophilicity was synthesised and biologically tested in monolayer culture. Cell viability, proliferation and aggrecan expression were quantified. When human chondrocytes were cultured on polymer substrates in which the hydrophilic groups were homogeneously distributed, adhesion, proliferation and viability decreased with the content of hydrophilic groups. Nevertheless, copolymers in which hydrophilic and hydrophobic domains alternate showed better results than the corresponding homopolymers. Biostable and biodegradable scaffolds with different hydrophilicity and porosity were synthesised using a template of sintered microspheres of controlled size. This technique allows the interconnectivity between pores and their size to be controlled. Periodic and regular pore architectures and reproducible structures were obtained. The mechanical behaviour of the porous samples was significantly different from that of the bulk material of the same composition. Cells fully colonised the scaffolds when the pores' size and their interconnection were sufficiently large. Another objective was to assess the chondrogenic redifferentiation in a biodegradable 3D scaffold of polycaprolactone (PCL) of human autologous chondrocytes previously expanded in monolayer. This study demonstrated that chondrocytes cultured in PCL scaffolds without fetal bovine serum (FBS) efficiently redifferentiated, expressing a chondrocytic phenotype characterised by their ability to synthesise cartilage-specific ECM proteins. The influence that pore connectivity and hydrophilicity of caprolactone-based scaffolds has on the chondrocyte adhesion to the pore walls, proliferation and composition of the ECM produced was studied. The number of cells inside polycaprolactone scaffolds increased as porosity was increased. A minimum of around 70% porosity was necessary for this scaffold architecture to allow seeding and viability of the cells within. The results suggested that some of the cells inside the scaffold adhered to the pore walls and kept the dedifferentiated phenotype, while others redifferentiated. In conclusion, the findings of this thesis provide valuable insight into the field of cartilage regeneration using TE techniques. The studies carried out shed light on the right composition, porosity and hydrophilicity of the scaffolds to be used for optimal cartilage production. / [ES] El cartílago articular es un tejido compuesto por condrocitos rodeados por una densa matriz extracelular (MEC). La MEC se compone principalmente de colágeno tipo II y de proteoglicanos. La función principal del cartílago articular es proporcionar una superficie lubricada para las articulaciones. Las lesiones en el cartílago articular son comunes y pueden derivar a osteoartritis. El cartílago articular no tiene vasos sanguíneos, nervios o vasos linfáticos y, por tanto, tiene una capacidad limitada de auto-reparación. La ingeniería tisular (IT) es un área prometedora en la regeneración de cartílago. En la IT se utilizan "andamiajes" (scaffolds) tridimensionales (3D) como soportes para el cultivo celular y tisular. Los scaffolds proporcionan una estructura que facilita la adhesión y la expansión de los condrocitos, manteniendo un fenotipo condrocítico limitando su desdiferenciación; que es el mayor problema en los sistemas bidimensionales (2D). La adhesión celular a los scaffolds depende de las características físicas y químicas de su superficie (morfología, rigidez, contenido de agua en equilibrio, tensión superficial, hidrofilicidad, presencia de cargas eléctricas). El objetivo general de esta tesis fue estudiar la influencia de diferentes tipos de biomateriales en la respuesta de los condrocitos en cultivo in vitro. Los scaffolds deben tener una estructura porosa interconectada para permitir el desarrollo celular a través de toda la estructura 3D, potenciando que los condrocitos mantengan su fenotipo, así como permitiendo entrada de nutrientes y eliminación de desechos metabólicos. Se estudió el efecto de la hidrofilicidad y de la arquitectura de poro. Se cuantificó la viabilidad celular, la proliferación y la expresión de agrecano. Cuando los condrocitos humanos se cultivaron en sustratos poliméricos donde los grupos hidrófilos se distribuyeron de manera homogénea, la adhesión, la proliferación y la viabilidad disminuyó con el contenido de grupos hidrófilo. Sin embargo, los copolímeros en los que los dominios hidrófilos e hidrófobos se alternaban mostraron mejores resultados que los homopolímeros correspondientes. Se sintetizaron series de scaffolds bioestables y series biodegradables con diferente hidrofilicidad y porosidad utilizando plantillas de microesferas sinterizadas. Se obtuvieron arquitecturas de poros regulares y reproducibles. Las células colonizaron el scaffold en su totalidad cuando los poros y la interconexión entre ellos era lo suficientemente grande. Se evaluó la rediferenciación condrogénica de condrocitos autólogos humanos, previamente expandidos en monocapa, sembrados en un scaffold biodegradable de policaprolactona (PCL). Se demostró que los condrocitos cultivados en scaffolds de PCL con medio sin suero bovino fetal (FBS), se rediferenciaban de manera eficiente; expresando un fenotipo condrocítico, caracterizado por su capacidad de sintetizar proteínas de la MEC específicas de cartílago hialino. Se estudió la influencia de la hidrofilicidad y la conectividad de los poros de los scaffolds de caprolactona sobre la adhesión de los condrocitos a las paredes de los poros, su capacidad proliferativa y la composición de MEC sintetizada. Se observó que un mínimo de 70% de porosidad era necesario para permitir la siembra de los condrocitos en el scaffold y su posterior viabilidad. El número de células aumentaba a medida que aumentaba la porosidad del scaffold. Los resultados sugieren que parte de las células que se adherían a las paredes internas de los poros mantenían el fenotipo desdiferenciado de condrocitos cultivados en monocapa, mientras que otros se rediferenciaban. En conclusión, los resultados de esta tesis aportan un avance en el campo de la regeneración de cartílago articular utilizando técnicas de IT. Los estudios realizados proporcionan directrices sobre la composición, la porosidad y la hidrofilicidad más adecuada para l / [CA] El cartílag articular és un teixit format per condròcits envoltats per una densa matriu extracel·lular (MEC). La MEC es compon principalment de col·lagen tipus II i de proteoglicans. La funció principal del cartílag articular és proporcionar una superfície lubricada a les articulacions. Les lesions en el cartílag articular són comuns i poden derivar en osteoartritis. El cartílag articular no té vasos sanguinis, nervis ni vasos limfàtics i, per tant, té una capacitat limitada d'auto-reparació. L'enginyeria tissular (IT) és una àrea prometedora en la regeneració del cartílag. A la IT s'utilitzen "bastiments" (scaffolds) tridimensionals (3D) com a suports per al cultiu cel·lular i tissular. Els scaffolds proporcionen una estructura que facilita l'adhesió i l'expansió dels condròcits, mantenint un fenotip condrocític limitant la seua desdiferenciació; que és el major problema en els sistemes bidimensionals (2D). L'adhesió cel·lular als scaffolds depèn de les característiques físiques i químiques de la superfície (morfologia, rigidesa, contingut d'aigua en equilibri, tensió superficial, hidrofilicitat i presència de càrregues elèctriques). L'objectiu general d'aquesta tesi va ser estudiar la influència de diferents tipus de biomaterials en la resposta dels condròcits en cultiu in vitro. Els scaffolds han de tindre una estructura porosa interconnectada per a permetre el desenvolupament cel·lular a través de tota l'estructura 3D, potenciant que els condròcits mantinguen el seu fenotip així com permetent l'entrada de nutrients i l'eliminació de productes metabòlics. S'ha estudiat l'efecte de la hidrofilicitat i de l'arquitectura de porus dels scaffolds. Es va quantificar la viabilitat cel·lular, la proliferació i l'expressió de agrecà. Quan els condròcits humans es van cultivar en substrats polimèrics en els quals els grups hidròfils es van distribuir de manera homogènia, l'adhesió, la proliferació i la viabilitat van disminuir amb el contingut de grups hidròfils. No obstant això, els copolímers en els quals els dominis hidròfils i hidròfobs s'alternaven van mostrar millors resultats que els homopolímers corresponents. Es van sintetitzar sèries de scaffolds bioestables i sèries biodegradables amb diferent hidrofilicitat i porositat utilitzant plantilles de microesferes sinteritzades. Es van obtindre arquitectures de porus regulars i reproduïbles. Les cèl·lules van colonitzar el scaffold en la seua totalitat quan els porus i la interconnexió entre ells era suficientment gran. Es van avaluar la rediferenciació condrogènica de condròcits autòlegs humans, prèviament expandits en monocapa, en un scaffold biodegradable de policaprolactona (PCL). Es va demostrar que els condròcits cultivats en scaffolds de PCL sense sèrum boví fetal (FBS) es rediferenciaven de manera eficient, expressant un fenotip condrocític caracteritzat per la seua capacitat de sintetitzar proteïnes de la MEC específiques de cartílag hialí. També es va estudiar la influència de la hidrofilicitat i la connectivitat dels porus dels scaffolds de caprolactona sobre l'adhesió dels condròcits a les parets dels porus, la seua capacitat proliferativa i la composició de MEC sintetitzada. Es va observar que un mínim del 70% de porositat sembla ser necessari per permetre la sembra dels condròcits i la seua posterior viabilitat en el scaffold. El nombre de cèl·lules augmentava a mesura que augmentava la porositat del scaffold. Els resultats suggereixen que part de les cèl·lules que s'adherien a les parets internes dels porus mantenien el fenotip desdiferenciat de condròcits cultivats en monocapa, mentre que altres es rediferenciaven. En conclusió, els resultats d'aquesta tesi proporcionen informació valuosa en el camp de la regeneració de cartílag utilitzant tècniques d'IT. Els estudis realitzats proporcionen directrius sobre la composició, la porositat i la hidrofilicitat m / Pérez Olmedilla, M. (2015). Tissue engineering techniques to regenerate articular cartilage using polymeric scaffolds [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58987

Notre équipe a développé une stratégie innovante de fonctionnalisation d’implants nanofibreux synthétiques à base de nanoréservoirs actifs pour la médecine régénérative osseuse. Notre objectif essentiel est de proposer un implant synthétique, biodégradable, et nanostructuré permettant d’accélérer la réparation du tissu osseux. Ces nouveaux implants synthétiques représentent un choix alternatif aux membranes de collagène d’origine animale. Notre stratégie consiste à construire des nanoréservoirs de chitosane, contenant des facteurs ostéoinducteurs tels que la BMP-2 afin d’enrober les nanofibres de nos implants. L’implant synthétique et biomimétique a été conçu à partir du le poly(ε-caprolactone) (PCL),polymère biocompatible et biodégradable approuvé par la FDA, et élaboré grâce à la technique de l’electrospinning afin de mimer la matrice extracellulaire. L’optimisation de ce procédé a permis la mise en oeuvre d’implants d’épaisseurs différentes (jusqu’à 10mm). La double fonctionnalisation de l’implant a permis de le rendre bioactif et vivant en utilisant la combinaison de facteur de croissance et de cellules souches mésenchymateuses. L’efficacité de la double fonctionnalisation des implants de PCL a ainsi été mise en évidence par l’accélération de la régénération osseuse in vivo.L’activité de ces implants fonctionnalisés de nanoréservoirs bioactifs est en cours d’analyse dans le cadre de tests précliniques pour une application maxillo-faciale, parodontale et orthopédique en vu d’obtenir un marquage CE. De plus, une start-up (ARTiOS NanoMed) basée sur cette nanotechnologie a été crée. En conclusion, nous pensons que la technologie développée par notre laboratoire a permis une avancée dans le domaine de la régénération osseuse et que cette technologie présente un fort potentiel d’application en clinique. / Our team has developped a novel and unique strategy to functionnalize nanofibrous and synthetic implants based on active nanoreservoirs for bone regeneration. We propose a new synthetic biodegradable and nanostructured implant to accelarate restoration of bone tissue. These new implants could replace collagen membranes from animal origin. The nanoreservoirs are based on chitosan containing osteoinductive growth factors such as BMP-2. Poly(ε-caprolactone) (PCL) is a biodegradable and biocompatible polymer approved by FDA and has been used to produce the synthetic and biomimetic implants by electrospinning in order to mimic the bone extracellular matrix. Optimization of this process has allowed the elaboration of nanofibrous implants with different thicknesses reaching 10 mm. Using the combination of growth factors and mesenchymal stem cells in a double functionalization created a bioactive and living implant. This strategy has been validated in vitro and in vivo thanks to bone site implantation in murin model. Acceleration of bone regeneration in vivo has brought to light the efficiency of the double functionalization onto the PCL implants.The functionalized implants bioactivity is still currently in study for pre-clinical trials in order to obtain authorization for applications in maxillo-facial, parodontal, and orthopaedic fields. Moerover, astat-up (ARTiOS NanoMed) based on this nanotechnology has been founded.To conclude, we believe that our nanotechnology could lead to a new generation of engineered bone implants which has a great potential to be used in the clinic.

La cicatrisation parodontale est un processus complexe, composé de quatre phases hautement intégrées (hémostase, inflammation, prolifération, remodelage), qui nécessite une interaction complexe entre les différents types tissulaires (épithélium, conjonctif, os) ainsi que la synthèse de médiateurs, tels que les hormones et les facteurs de croissance. La difficulté à pouvoir obtenir une régénération des tissus parodontaux est en partie due à la réponse inflammatoire qui interfère avec le processus de cicatrisation, via la surexpression des cytokines pro-inflammatoires, ainsi qu’à la croissance rapide des cellules épithéliales le long de la surface de la racine qui porte atteinte à la vraie organisation des tissus, essentielle à la régénération parodontale. Notre objectif a été de mettre au point des membranes nanofibreuses implantables à base de polycaprolactone (PCL) fonctionnalisés par plusieurs molécules actives (Alpha-Melanocyte Stimulating Hormone (α-MSH)), ibuprofène, atorvastatine) et implantables, permettant à la fois un contrôle physique et biochimique de la cicatrisation parodontale. En d’autres termes, nous avons cherché à ralentir la colonisation de la surface radiculaire par les cellules épithéliales et à moduler l’inflammation de la phase post-chirurgicale afin de promouvoir la cicatrisation parodontale. Pour cela, nous avons mis au point un modèle d’inflammation in vitro mimant le tissu superficiel du parodonte en utilisant des cellules parodontales, à savoir des kératinocytes et fibroblastes gingivaux humains, stimulées par du lipopolysaccharide de Porphyromonas gingivalis (LPS-Pg). Les résultats obtenus ont montré une bonne biocompatibilité des systèmes (α-MSH, ibuprofène) ainsi qu’une diminution de la prolifération, migration des kératinocytes, fibroblastes gingivaux humains et une diminution significative de l’expression des marqueurs pro- ou anti-inflammatoires (TNF-α, TGF-β, IL-6, IL-8), des marqueurs d’adhérence, de prolifération (Intégrine, Laminine, Fibronectine) et de remodelage (COL-IV). En conclusion, les stratégies développées (α-MSH, ibuprofène) au sein de notre laboratoire ont permis de mettre en évidence l’intérêt de délivrer une molécule anti-inflammatoire à partir d’un biomatériau et représentent un fort potentiel d’application clinique pour la parodontologie mais aussi pour la médecine de demain. / Periodontal wound healing is a process involving hemostasis, inflammatory phase, proliferation and maturation/matrix remodeling. These phases require cell-to-cell interaction of different cell types (epithelial cells, fibroblasts, osteoblasts, and cementoblasts) orchestrated by growth factors, cytokines and extracellular matrix components. After conventional periodontal therapy, wound healing corresponds more to tissue reparation than regeneration. This absence of true regeneration is considered to be mainly due to the competition between the different periodontal tissues (gingiva, cementum, alveolar bone) and the differential rate of proliferation, migration and differentiation of periodontal cells during wound healing. Therefore, the inflammatory response could interfere with the healing process depending on the secretion/activity level of matrix metalloproteinase (MMPs), cytokines, chemokines and also the imbalance with their antagonists/inhibitors, which leads to fibrosis and excessive scarring. Our aim was to develop implantable nano-fibrous membranes based on polycaprolactone (PCL) and functionalized by several active molecules (Alpha-melanocyte stimulating hormone (α-MSH)), ibuprofen, atorvastatin) allowing both physical control and biochemical periodontal healing features. Furthermore, we developed an in vitro inflammatory model mimicking the periodontal tissue surface, using periodontal cells ; keratinocytes and human gingival fibroblasts stimulated with lipopolysaccharide of Porphyromonas gingivalis (Pg-LPS). The results obtained showed good biocompatibility systems (α-MSH, ibuprofen) and a decrease in the proliferation and migration of keratinocytes, human gingival fibroblasts. Moreover, a significant decrease of pro- or anti-inflammatory markers expression (TNF-α, TGF-β, IL-6, IL-8), adhesion markers of proliferation (Integrin, laminin, fibronectin) and remodeling (COL-IV) could be achieved. In conclusion, the strategies developed in our laboratory (α-MSH, ibuprofen), have helped to highlight the interest of the release of an anti-inflammatory molecule from a biomaterial, and represented a strong potential for clinical application not only in periodontics but also in general medicine.

A doença de Chagas é causada pelo Trypanosoma cruzi e acomete milhões de pessoas, principalmente as de baixa renda em países subdesenvolvidos. É considerada uma doença negligenciada, não existindo uma terapia eficaz contra os parasitas na fase crônica da doença. Estudos preliminares demonstraram que o ácido ursólico apresenta atividade tripanocida, entretanto, este fármaco possui baixa solubilidade em água, o que prejudica a sua biodisponibilidade. Com o intuito de viabilizar a terapia com ácido ursólico, as nanopartículas poliméricas são sistemas de liberação promissores, devido a sua capacidade de liberação modificada. Além disso, os sistemas nanoencapsulados destacam-se pela alta eficiência de encapsulação do fármaco, proteção contra degradação, e menor possibilidade de causar toxicidade. O objetivo deste trabalho foi o desenvolvimento e a caracterização de nanopartículas de policaprolactona para a veiculação de ácido ursólico, visando à otimização da terapia da Doença de Chagas. O estudo teve início com o desenvolvimento das nanopartículas poliméricas contendo ácido ursólico. A formulação obtida neste estudo pela técnica da nanoprecipitação apresentou menor valor de tamanho de partícula (173,17±4,20) e índice de polidispersividade (0,09±24,77), com perfil monomodal, potencial zeta de -36 mV e eficiência de encapsulação de 94,1±1,31%. O tamanho das partículas observado na microscopia eletrônica de varredura demonstrou ser compatível com os valores observados nas análises de espalhamento dinâmico de luz, embora tenha apresentado uma característica agregada e ligeiramente esférica. Através da determinação do coeficiente de partição do ácido ursólico foi possível avaliar a alta lipofilicidade do ácido ursólico. Na determinação do coeficiente de solubilidade do ácido ursólico, o lauril sulfato de sódio foi o tensoativo de escolha para o estudo in vitro do perfil de liberação, solubilizando aproximadamente 300 ?g.mL-1 de fármaco, mas não foi possível a realização do estudo in vitro do perfil de liberação, devido a sua característica altamente lipofílica. O estudo da citotoxicidade por ensaio de resazurina mostrou que a formulação escolhida não alterou a viabilidade celular de células LLCMK2, portanto sem toxicidade para o meio biológico, bem como evidenciou a capacidade das nanopartículas poliméricas contendo ácido ursólico de reduzir a viabilidade dos parasitas em aproximadamente 50%. A avaliação da atividade biológica do ácido ursólico em camundongo C57BL/6 infectados com a cepa Y do Trypanosoma cruzi apresentou uma redução acentuada (p<0,001) dos tripomastigotas quando comparados ao grupo I, sugerindo uma liberação sustentada do ácido ursólico nesse modelo de nanopartículas poliméricas. Como conclusão, as nanopartículas poliméricas contendo ácido ursólico podem ser propostas como uma abordagem quimioprofilática da doença de Chagas, considerando a necessidade dessa medida de segurança para pacientes que recebem transfusão sanguínea no Sistema Único de Saúde no Brasil. / Chagas disease is caused by parasite Trypanosoma cruzi and affects millions of lowincome in developing countries and because of that it is neglected by the pharmaceutical industry and there is no effective therapy against parasites in the chronic phase of the disease. Preliminary studies showed that ursolic acid presents tripanocidal activity, however, it has low water solubility, which reduces its bioavailability. Among the existing drug delivery systems, polymeric nanoparticles play a central role, due to their ability to sustain or control the release of drugs. Moreover, nanocoated systems are distinguished by high drug encapsulation efficiency, protection from degradation, and less likely to cause irritation. The aim of this work is development and characterization of the polymeric nanoparticles containing ursolic acid, aiming to optimize the treatment of Chagas disease. The study began with the development of polymeric nanoparticles containing ursolic acid. The formulation obtained in this study by the nanoprecipitation technique showed the lowest particle size (173.17 ± 4.20) and polydispersity index (0.09 ± 24.77), with monomodal profile, zeta potential of -36 mV and encapsulation efficiency was 94.1% ± 1.31. The size of the particles observed by scanning electron microscopy showed to be compatible with the values observed in the analysis of dynamic light scattering, although it had an aggregate and slightly spherical characteristic. By determining the coefficient of ursolic acid partition was possible to evaluate the high lipophilicity of ursolic acid. In determining the solubility coefficient of ursolic acid, sodium lauryl sulfate was the surfactant of choice for studying in vitro release profile, solubilizing approximately 300 ?g.mL-1 of the drug, but it has not been possible to conduct the study in vitro release profile, due to its highly lipophilic character. The study of resazurin cytotoxicity assay showed that the formulation did not alter the cell viability of LLCMK2 cells, and therefore, without toxicity to the biological environment and demonstrated the ability of the polymeric nanoparticles containing ursolic acid to reduce the viability of parasites in approximately 50 %. The evaluation of biological activity of ursolic acid in mice C57BL/6 mice infected with the Y strain of Trypanosoma cruzi showed a marked reduction (p<0.001) of trypomastigotes when compared to group I, suggesting a sustained release of ursolic acid in polymeric nanoparticles model. In conclusion, polymeric nanoparticles containing ursolic acid may be proposed as a chemoprophylactic approach of Chagas disease, considering the need for this safety measure for patients receiving blood transfusion Health System in Brazil.

Uma potencial alternativa para o transplante de tecidos é a engenharia de tecidos. Células-tronco mesenquimais e scaffolds eletrofiados são comumente utilizados nesta área devido à capacidade multipotente de diferenciação destas células e à rede de poros interconectados destas estruturas fibrosas. Além disso, bioreatores de perfusão podem ser utilizados para melhorar o transporte de nutrientes e reduzir o acúmulo de metabolitos tóxicos. Neste contexto, uma maneira de estudar e otimizar o sistema de cultivo é utilizar técnicas de modelagem para descrever interações ou processos individuais envolvidos no crescimento celular. Deste modo, o objetivo geral deste estudo é realizar o cultivo de células-tronco mesenquimais da polpa de dente decíduo (DPSCs) utilizando scaffolds tridimensionais eletrofiados de policaprolactona (PCL), biorreatores e técnicas de modelagem. Inicialmente foram testadas diferentes misturas de solventes (clorofórmio e metanol), a fim de produzir scaffolds com poros adequados ao cultivo tridimensional. Os diâmetros de fibra e de poro foram determinados por microscopia eletrônica de varredura (MEV). O crescimento e o metabolismo das células foram avaliados através da determinação da atividade metabólica e das concentrações de glicose e lactato do meio de cultivo, e a infiltração celular foi observada com a marcação do núcleo celular. Depois de estabelecidos os parâmetros de eletrofiação, o efeito da perfusão direta no desprendimento de DPSCs de scaffolds eletrofiados de PCL foi estudado. A atividade metabólica das células foi determinada para diferentes tempos de adesão, vazões e densidades de semeadura, e a tensão de cisalhamento na parede do poro foi calculada para cada vazão. A morfologia das células foi avaliada através de imagens de microscopia confocal e MEV. Paralelamente, foram realizadas simulações utilizando o software OpenFOAM para estudar como os parâmetros e variáveis de entrada (concentração inicial de glicose, porosidade e espessura do scaffold) afetam as saídas (fração volumétrica de células e concentração de substrato) de um modelo de proliferação celular que considera a difusão e o consumo de glicose. As contribuições do teor de oxigêno na cinética de crescimento de Contois e da variação da porosidade com o tempo devido à degradação do polímero também foram avaliadas. Inicialmente, foi observado que apenas um tamanho de poro maior que o diâmetro da célula permitiu a infiltração das células no scaffold. Então, observou-se que o aumento do tempo de adesão acarretou em maior espalhamento das células e, assim como a diminuição da densidade de semeadura e da tensão de cisalhamento, resultou em uma redução do desprendimento das células sob perfusão. Quanto ao modelo fenomenológico, observou-se maior sensibilidade à concentração inicial de glicose e à porosidade do scaffold, e aos parâmetros adimensionais relacionados à proliferação e morte celular e ao consumo de nutrientes. Além disso, o número inicial de células apresentou maior impacto no transporte de massa do que no crescimento celular. Neste estudo, foi possível obter scaffolds eletrofiados e conduções de cultivo dinâmico adequadas ao cultivo tridimensional de DPSCs, e elucidar os efeitos da limitação do transporte de massa e do oxigênio no crescimento celular, e da degradação do polímero no transporte de massa. / A potential alternative to tissue transplant is tissue engineering. Mesenchymal stem cells and electrospun scaffolds are commonly used in this field due to the multipotent differentiation capacity of these cells and the interconnected pore network of these fibrous structures. In addition, perfusion bioreactors can be used to enhance nutrient transport and reduce the accumulation of toxic metabolites. In this context, one way to study and optimize the culture system is to use modeling techniques to describe interactions or individual processes involved in cell growth. Thus, the objective of this study is to perform the three-dimensional culture of mesenchymal stem cells of dental pulp (DPSCs) using electrospun polycaprolactone (PCL) scaffolds, bioreactors and modeling techniques. Initially, different solvent mixtures (chloroform and methanol) were tested to produce scaffolds with pores suitable to three-dimensional culture. Fiber and pore diameter was determined using a scanning electron microscope. Cell growth and metabolism were evaluated through the metabolic activity and the culture medium concentration of glucose and lactate, and the cell infiltration was observed with cell nuclei staining. After the establishment of the elesctrospinning parameters, the effect of direct perfusion on DPSCs detachment from PCL electrospun scaffolds was investigated. The metabolic activity of the cells was determined for different adhesion times, flow rates and seeding densities and the pore wall shear stress was calculated for each flow rate. The cell morphology was evaluated through scanning electron and confocal microscopy imaging. In parallel, simulations with the software OpenFOAM were performed to study how parameters and inputs (initial glucose concentration, porosity and thickness of the scaffold) affect the outputs (cell volume fraction and substrate concentration) of a model of cell proliferation and glucose diffusion and consumption. The contribution of the oxygen in the Contois growth kinetics and the porosity variation with time due to polymer degradation was also evaluated. Initially, it was observed that only a pore size higher than the cell diameter allowed the infiltration of the cells through the scaffold. Then, it was observed that a higher adhesion time leaded to higher cell spreading in static conditions and, similar to smaller seeding densities and shear stresses, reduced cell detachment under perfusion. Regarding the phenomenological model, it was observed that the model is more responsive to the initial glucose concentration and scaffold porosity, and to the dimensionless parameters related to cell proliferation, death and nutrient uptake. Furthermore, the initial cell number had a more significant impact on mass transport than on cell growth. In this study, it was possible to obtain an electrospun scaffold and dynamic culture conditions suitable for the three-dimensional culture of DPSCs, and to elucidate the effects of transport limitations and of oxygen on cell growth, and of polymer degradation on mass transport were elucidated.

L'ostéoporose est la maladie métabolique la plus fréquente qui touche l'os. Plusieurs substances actives sont utilisées pour le traitement pharmacologique de cette maladie. Cependant, ce sont les bisphosphonates et surtout l'alendronate de sodium, qui sont prescrits en première intention. L'alendronate de sodium est, en effet, très efficace mais présente une faible absorption quand il est administré par la voie orale. Sa solubilité dans l'eau est de 20 mg/ml. Il présente en outre une faible biodisponibilité (de 0,6 à 0,7%). Cette substance active est aussi à l'origine d'effets indésirables d'irritation au niveau de l'oesophage, l'estomac et l'intestin. Ces effets sont dus à un contact local des cristaux de la substance active avec la muqueuse. L'approche d'encapsulation des substances actives dans des particules polymériques a permis d'obtenir plusieurs bénéfices thérapeutiques comme l'amélioration de la biodisponibilité et la diminution des effets indésirables. Dans la première partie de notre étude, on a réalisé l'encapsulation de l'alendronate dans des nanoparticules à base de poly-epsilon-caprolactone en utilisant la nanoprécipitation et l'émulsion double. Les nanoparticules obtenues ont une forme sphérique et une taille comprise entre 200 et 450 nm. Le meilleur pourcentage d'encapsulation a été de 34% et il a été obtenu avec la technique d'émulsion double. Ceci confirme que cette méthode est plus adaptée à l'encapsulation des molécules hydrophiles. Le profil de libération in vitro a montré deux phases : une première phase de libération relativement rapide et une deuxième phase beaucoup plus lente. L'analyse par modélisation mathématique a montré que la libération in vitro de l'alendronate se fait par diffusion et relâchement des chaines polymériques / Osteoporosis is the most frequent metabolic disease that affects bone. Many actives have been used as pharmacological treatment of this disease. However, bisphosphonates, especially, alendronate sodium, are indicated as first line regimen. Alendronate is highly efficient but presents low absorption after oral administration. Its solubility in water is 20 mg/ml. It has also poor bioavailability (0.6-0.7%). In addition, this active could lead to many side effects, which are mainly related to the esophagus, the stomach and the intestine. Such effects are linked to a local contact of drug crystals with the mucosa. Encapsulation of active molecules allowed the obtaining of many advantages over conventional pharmaceutical forms such as, bioavailability and tolerance enhancement. In the first part of our study, we managed to encapsulate alendronate sodium in poly-epsilon-caprolactone nanoparticles via two techniques: nanoprecipitation and double emulsion. Obtained nanoparticles presented a spherical form. Their size ranged between 200 and 450 nm. The highest encapsulation efficiency value was 34% and was obtained via double emulsion technique. This confirms that double emulsion is more suitable for hydrophilic drugs encapsulation. In vitro release profile showed two phases: first phase of burst release and a second more prolonged phase. Mathematical modeling showed that alendronate in vitro release occurs by drug diffusion and polymer chain relaxation. In the second experimental part, we managed to find a more interesting alternative. In fact, we opted for the use of chitosan which is a natural hydrophilic polymer. One of the obtained advantages is the avoidance of organic solvents use. In addition, this approach allowed the enhancement of encapsulation efficiency as this value increased to 70%. The used technique is ionic gelation. It is a simple encapsulation technique that is based on the transformation of a dissolved polymer to a gel-like state

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.