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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

Tesis por compendio / [ES] El uso masivo de plásticos sintéticos y su impacto medioambiental obliga a buscar alternativas biodegradables para el envasado de los alimentos,etapa necesaria para su adecuada conservación.Así mismo,la necesidad de incrementar la vida útil de los alimentos ha despertado gran interés en el desarrollo de materiales activos(antimicrobianos y antioxidantes)que mantengan su calidad y seguridad por más tiempo,mediante el uso de compuestos de origen natural,seguros para el consumidor.En este sentido,el desarrollo de materiales biodegradables activos para el envasado de alimentos constituye hoy en día un reto importante para la industria alimentaria.En la presente Tesis Doctoral,se ha estudiado la encapsulación de carvacrol mediante el electroestirado o extensión y secado de diferentes disoluciones poliméricas con carvacrol.Se han utilizado polímeros biodegradables portadores de diferente polaridad(almidón termoplástico:TPS,polivinil-alcohol:PVA, policaprolactona:PCL o ácido poliláctico:PLA)disueltos en el solvente adecuado,con el fin de obtener capas activas.Estas capas se han combinado con otras de polímeros con propiedades complementarias,para obtener laminados activos adecuados para el envasado de alimentos.Los laminados combinaron polímeros polares(TPS o PVA)y poliésteres no polares(PCL o PLA)incorporando el carvacrol en una de las capas.Se evaluó la cinética de liberación del activo,así como la acción antimicrobiana de los materiales obtenidos.Los laminados se caracterizaron en su funcionalidad como material de envase(prop. de barrera,mecánicas u ópticas)así como en su estructura y comportamiento térmico.Los estudios de encapsulación revelaron un mayor potencial encapsulante del carvacrol para los polímeros no polares(PCL;PLA),aunque el PVA mostró también una buena afinidad con el compuesto activo.La matriz de PVA mostró una mayor retención de carvacrol mediante electroestirado de sus disoluciones acuosas que por extensión y secado,sin necesidad de adición de tensoactivos como el Tween85.Para la encapsulación en PLA,se usaron mezclas binarias de solventes aptos para contacto con los alimentos(acetato de etilo y DMSO).En este caso,se obtuvo una mayor eficiencia encapsulante del PLA en los materiales obtenidos por extensión y secado que en los electroestirados.La cinética de liberación del carvacrol de las fibras de PCL explicó el mayor efecto antibacteriano contra E.coli,y el escaso efecto antilisteria.La velocidad de liberación del activo aumentó cuando disminuyó la polaridad de los simulantes alimentarios, mostrando una liberación completa en los sistemas apolares,pero solo hasta 75% en los sistemas acuosos,que requerirían una mayor proporción del activo en el envase para potenciar su efectividad.La combinación de láminas de TPS con fibras de PCL cargadas con carvacrol dio lugar a materiales con una permeabilidad al vapor de agua mejorada,en comparación con los films de almidón,sin efectos relevantes sobre las otras propiedades funcionales estudiadas.Cuando los laminados se probaron in vitro contra cepas G(+) y G(-) mostraron un efecto antibacteriano similar al de las fibras de PCL con carvacrol,pero retrasado en el tiempo.Los estudios de desintegración-biodegradación de los laminados almidón-PCL revelaron que las películas con carvacrol afectaron la actividad del inóculo del compost,disminuyendo ligeramente la biodegradabilidad de las películas,pero alcanzando valores de desintegración similares(75-80%)a las muestras libres de carvacrol.Se obtuvieron también laminados de PLA y PVA mediante la extensión y secado de disoluciones acuosas de PVA con carvacrol.La superficie del PLA fue sometida a aminolización a fin de mejorar la extensibilidad de las disoluciones acuosas.A pesar del incremento de la componente polar de la energía superficial del PLA y su mejorada humectabilidad con las soluciones de PVA,estas bicapas no mostraron una mejora significativa en las propied / [CA] L'ús massiu de plàstics sintètics i el seu impacte mediambiental obliga a buscar alternatives biodegradables per a l'envasament dels aliments necessari per a la seua conservació.Així mateix,la necessitat d'incrementar la vida útil dels aliments ha despertat gran interés en el desenvolupament de materials actius(antimicrobians i antioxidants)que mantinguen la seua qualitat i seguretat per més temps,per mitjà de l'ús de compostos d'origen natural,segurs per al consumidor.En este sentit,el desenvolupament de materials biodegradables actius per a l'envasament d'aliments constituïx un repte important per a la indústria alimentària.En la present Tesi Doctoral,s'ha estudiat l'encapsulació de carvacrol per mitjà de l'electroestirat o extensió i assecat de diferents dissolucions polimèriques amb carvacrol.S'han utilitzat polímers biodegradables portadors de diferent polaritat(midó termoplàstic:TPS, polivinil-alcohol:PVA, policaprolactona:PCL o àcid poliláctic:PLA)dissolts en el solvent adequat,a fi d'obtindre capes actives.Estes s'han combinat amb altres de polímers amb propietats complementàries,per a obtindre laminats actius adequats per a l'envasament d'aliments.Els laminats van combinar polímers polars(TPS o PVA)i poliésters no polars(PCL o PLA)incorporant el carvacrol en una de les capes.Es va avaluar la cinètica d'alliberament de l'actiu,així com l'acció antimicrobiana dels materials obtinguts.Els laminats es van caracteritzar en la seua funcionalitat com a material d'envàs(propietats de barrera, mecàniques o òptiques),així com en la seua estructura i comportament tèrmic.Els estudis d'encapsulació van revelar un major potencial encapsulant del carvacrol per als polímers no polars(PCL i PLA),encara que el PVA va mostrar també una bona afinitat amb el compost actiu.La matriu de PVA va mostrar una major retenció de carvacrol per mitjà d'electroestirat de les seues dissolucions aquoses que per extensió i assecat,sense necessitat d'addició de tensioactius com el Tween 85.Per a l'encapsulació en PLA,es van usar mescles binàries de solvents aptes per a contacte amb els aliments(acetat d'etil i DMSO).Es va obtindre una major eficiència encapsulant del PLA en els materials obtinguts per extensió i assecat que en els electroestirats.La cinètica d'alliberament del carvacrol de les fibres de PCL va explicar el major efecte antibacterià contra Escherichia coli,i l'escàs efecte antilisteria.La velocitat d'alliberament de l'actiu va augmentar quan va disminuir la polaritat dels simulants alimentaris,mostrant un alliberament complet en els sistemes no polars, però només fins a un 75% en els sistemes aquosos,que requeririen una major proporció de l'actiu en l'envàs per a potenciar la seua efectivitat.La combinació de làmines de TPS amb fibres de PCL carregades amb carvacrol va donar lloc a materials amb una permeabilitat al vapor d'aigua millorada,en comparació amb els films de midó, sense efectes rellevants sobre les altres propietats funcionals.Quan els laminats es van provar in vitro contra ceps Gram(+) i Gram(-) van mostrar un efecte antibacterià semblant al de les fibres de PCL amb carvacrol,però retardat en el temps.Els estudis de desintegració-biodegradació dels laminats midó-PCL van revelar que les pel·lícules amb carvacrol van afectar l'activitat de l'inocule del compost,disminuint lleugerament la biodegradabilitat,però aconseguint valors de desintegració semblants(75-80%)a les mostres lliures de carvacrol.Es van obtindre també laminats de PLA i PVA per mitjà de l'extensió i assecat de dissolucions aquoses de PVA amb carvacrol.La superfície del PLA va ser sotmesa a aminolizatció a fi de millorar l'extensibilitat de les dissolucions aquoses.A pesar de l'increment de la component polar de l'energia superficial del PLA i la seua millorada mullabilitat amb les solucions de PVA,estes bicapes no van mostrar una millora significativa en les propietats mecàniques i de barrera / [EN] The massive use of synthetic plastics and their environmental impact makes necessary the search for biodegradable alternatives for food packaging. Likewise, the need to increase the shelf life of food has aroused great interest in the development of active materials (antimicrobial and antioxidant) that maintain food quality and safety for longer periods of time through the use of compounds of natural origin, safe for the consumer. In this sense, the development of active biodegradable materials for food packaging is both a major imperative and challenge for the food industry today. In the present Doctoral Thesis, the encapsulation of carvacrol has been studied by means of the electrospinning or casting of different polymeric solutions with carvacrol. Biodegradable polymers with different polarities (thermoplastic starch: TPS, poly(vinyl-alcohol): PVA, poly-(¿-caprolactone): PCL or poly(lactic acid): PLA) dissolved in the appropriate solvent have been used to obtain active layers. These have been combined with other polymers with complementary properties, to obtain active laminates suitable for food packaging. The laminates combined polar polymers (TPS or PVA) and non-polar polyesters (PCL or PLA) incorporating carvacrol in one of the layers. The release kinetics of the active ingredient was evaluated, as well as the antimicrobial action of the materials obtained. The laminates were characterized in their functionality as a packaging material (barrier, mechanical or optical properties), as well as in their structure and thermal behaviour. Encapsulation studies revealed a higher encapsulating potential of carvacrol for non-polar polymers (PCL and PLA), although PVA also showed a good affinity with the active compound. The PVA matrix showed a higher retention of carvacrol by electrospinning of its aqueous solutions than by casting, without the need for addition of surfactants such as Tween 85. For the encapsulation in PLA, binary mixtures of solvents suitable for food contact (ethyl acetate and DMSO) were used. A higher encapsulation efficiency of PLA was obtained in the materials produced by casting than by electrospinning. The carvacrol release kinetics of PCL fibres explained the higher antibacterial effect against Escherichia coli and the lower antilisterial effect. The release ratio of the active ingredient increased when the polarity of the food simulants decreased, showing a complete release in non-polar systems and only up to 75% in aqueous systems that would require a higher proportion of the active ingredient in the packaging material to enhance its effectiveness. The combination of TPS films with carvacrol loaded PCL fibres resulted in materials with improved water vapour permeabilities, compared to starch films, with no relevant effects on the other functional properties. When the laminates were tested in vitro against Gram (+) and Gram (-) strains, they showed a similar antibacterial effect to that of PCL fibres with carvacrol, but delayed in time. Disintegration-biodegradation studies of PCL-starch laminates revealed that carvacrol films affected the activity of the compost inoculum, slightly decreasing the biodegradability of the laminates, but reaching similar disintegration values (75-80%) to the carvacrol-free samples. PLA and PVA laminates were also obtained by casting aqueous PVA solutions with carvacrol. The surface of PLA was submitted to aminolization in order to improve the extensibility of the aqueous solutions. Despite the increase in the polar component of the PLA surface energy and its improved wettability with PVA solutions, these bilayers did not show significant improvement in mechanical and barrier properties over the PLA monolayers. / The authors would like to thank the Ministerio de Economia y Competitividad of Spain, for funding this study as part of projects AGL2013-42989-R and AGL2016-76699-R and predoctoral research grant # BES-2014-068100. / Tampau, A. (2020). Carvacrol encapsulation by electrospinning or solvent casting to obtain biodegradable multilayer active films for food packaging applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/140313 / Compendio

Tesis por compendio / [ES] La presente Tesis Doctoral se ha centrado en el aislamiento y caracterización de materiales celulósicos y extractos activos, procedentes de las cascarillas de arroz y café, y su incorporación a películas de almidón y mezclas compatibilizadas de almidón-PLA, para mejorar sus propiedades funcionales como materiales para el envasado de alimentos. Las fibras de celulosa (CF) se obtuvieron mediante tratamiento alcalino y de blanqueo, con un rendimiento de 41 y 53 g fibras/100 g cascarilla, respectivamente para cascarilla de arroz y café. Los nanocristales de celulosa (CNC) se aislaron de las fibras mediante hidrólisis ácida, con un rendimiento del 5% respecto a las fibras y con alta cristalinidad (90-92%), resistencia térmica y relación de aspecto (L/d: 20-40). Los compuestos activos se obtuvieron mediante extracción hidrotérmica (180 ºC; 9,5 bares), con un rendimiento de 17-18 g/ 100 g de cascarilla. Dichos extractos exhibieron capacidad antioxidante (EC50: 5,37-5,29 mg sólidos extraídos/ mg DPPH) y antimicrobiana (cuantificada en términos de concentración mínima inhibitoria: MIC) frente a L. innocua (MIC: 48-52 mg polvo/mL) y E. coli (MIC: 50-66 mg polvo/mL). Los materiales celulósicos procedentes de cascarilla de arroz y café se incorporaron a películas de almidón termoplástico (TPS), obtenidas mediante mezclado en fundido y moldeo por compresión. El módulo elástico aumentó un 186 y 121% cuando se incorporó a la matriz un 1% (p/p) de CNC de cascarilla de arroz y café, respectivamente. Del mismo modo, las CF se añadieron a las películas de TPS al 1, 5 y 10 pt%. Ambas CF aumentaron la rigidez y redujeron la extensibilidad de los films, aunque las CF de cascarilla de café mantuvieron mejor la ductilidad al 1 y 5% (p/p). La permeabilidad al vapor de agua de las películas de TPS no se redujo en los materiales compuestos, aunque la permeabilidad al oxígeno se redujo en aproximadamente un 17%. Al incorporar extractos activos a los films de almidón, mejoraron sus propiedades de tracción; el módulo elástico aumentó un 350%, a la vez que se hicieron menos extensibles. Las fibras de celulosa de ambos residuos fueron más efectivas como agentes de refuerzo en los films con extractos sólidos que en los de almidón solo. Se estudiaron también mezclas de almidón-PLA utilizando como compatibilizador policaprolactona funcionalizada con anhídrido maléico y/o glicidil metacrilato (PCLMG o PCLG). Se analizó el efecto de la proporción de PLA en la mezcla (20 y 40% respecto al almidón), y la de ambos compatibilizadores (2,5 y 5%), en las propiedades de los films. Los análisis de la microestructura, el comportamiento térmico y las propiedades funcionales (mecánicas, ópticas y de barrera) de los films, demostraron que sustituir el 20% del almidón por PLA e incorporar el 5% de PCLG podría ser una buena estrategia para obtener materiales adecuados para envasado de alimentos. Además, se estudió el efecto de la adición de rellenos celulósicos (CF y CNC) y del extracto antioxidante de cascarilla de café en la mezcla de almidón-PLA compatibilizada seleccionada. Las propiedades antioxidantes de los films se probaron a través de su eficacia para preservar al aceite de girasol de la oxidación. Se observaron diferencias significativas en las propiedades funcionales de los films cuando los CNC se incorporaron mediante dos métodos diferentes. El efecto de refuerzo de los materiales celulósicos en mezclas de S-PLA fue menos notable que en las películas de almidón, probablemente debido a la superposición del efecto de refuerzo de PLA. El extracto antioxidante no mejoró el comportamiento mecánico en la mezcla, pero le confirió capacidad antioxidante, adecuada para aplicaciones en el envasado de alimentos. / [CA] La present Tesi Doctoral s'ha centrat en l'aïllament i caracteritzaciò de materials cel.lulòsics i extractes actius, procedents de pellorfa d'arròs i café, i la seua incorporació a pel·lícules de midó i mescles compatibilitzades de midò-PLA, per a millorar les seues propietats funcionals com materials per al envasat d'aliments. Les fibres de cel.lulosa (CF) s'obtingueren mitjançant tractament alcalí i de blanqueig, amb un rendiment de 41 i 53 g fibres/100g pellorfa, respectivament per a pellorfa d'arròs i cafè. Els nanocristalls de cel·lulosa (CNC) es van aïllar de les fibres de cel·lulosa per mig d'hidròlosi àcida, amb un rendiment del 5% respecte a les fibres; en tots dos casos, amb alta cristal·línitat (90-92%), resistència tèrmica i relaciò d'aspecte (L/d: 20-40). Els composts actius s'obtingueren mitjançant l'extracció hidrotèrmica (180 ºC; 9,5 bars), amb un rendiment del 17-18 g/100 g de pellorfa. Aquests composts exhibiren capacitat antioxidant (EC50: 5,37-5,29 mg extracte solit/ mg DPPH) i antimicrobiana, (quantificada en termes de concentració mínima inhibitòria: CMC) enfront a L. innocua (MIC: 48-52 mg pols/mL) i E. coli (MIC: 50-66 mg pols/ mL). Els materials cel·lulòsics procedents de pellorfa d'arròs i cafè es van incorporar a pel·lícules de midó termoplàstic (TPS), obtingudes mitjançant mesclat en fos i modelatge per compressió. El mòdul elàstic va augmentar un 186 i 121% quan es va incorporar a la matriu un 1 pt% CNC de pellorfa d'arròs i café, respectivament. De la mateixa manera, les CF es van afegir a les pel·lícules de TPS al 1, 5 i 10 pt%. Ambdues CF va augmentar la rigidesa de les pel·lícules i es va reduir la seua capacitat d'estirament. No obstant, les CF de pellorfa de cafè mantingueren millor la ductilitat al 1 i 5%. La permeabilitat al vapor d'aigua de les pel·lícules de TPS no es va reduir en els materials compostos, encara que la permeabilitat a l'oxigen es va reduir en aproximadament un 17%. A l'incorporar extractes actius a les pel·lícules de midó, milloraren les propietats de tracció de les pel·lícules ; el mòdul elàstic va augmentar un 350%, mentre que les pel·lícules es feren menys extensibles. Les CF dels dos residus foren més efectives com agents de reforç en pel·lícules que contenien extractes actius, que en pel·lícules de midó pur. També es van estudiar mescles de midò-PLA utilitzant com a compatibilitzador policaprolactona funcionalitzada amb anhídrid maleic i/o glicidil metacrilat (PCLMG o PCLG). Es va analitzar l'efecte de la proporció de PLA en la mescla (20 i 40% respecte al midó), i de la tots dues compatibilitzadors (2,5 i 5%), en les propietats de les pel·lícules. Els anàlisis de la microestructura, el comportament tèrmic i les propietats funcionals (mecàniques, óptiques i de barrera) de les pel·lícules, demostraren que substituir el 20% del midó per PLA i incorporar el 5% de PCLG podria ser una bona estratègia per a obtindré pel·lícules adequades per a l'envasat d'aliments. A demés, es va estudiar l'efecte de l'addició de reforçaments cel·lulòsics (CF i CNC) i extracte antioxidant de pellorfa de cafè, en mescles de midó-PLA compatibilitzades. Les propietats antioxidants de les pel·lícules s'analitzaren a través de la seua eficàcia per a preservar de l'oxidació l'oli de gira-sol. S'observaren diferències significatives en les propietats funcionals de les pel·lícules quan els CNC s'incorporaren mitjançant dos mètodes diferents. L'efecte de reforç dels materials cel·lulòsics en mescles de S-PLA va ser menys notable que en les pel·lícules de midó, provablement degut a la superposició de l'efecte de reforç del PLA. L'extracte antioxidant no va millorar el comportament mecànic en les mescles, però li va conferir la capacitat antioxidant adequada per a aplicacions a l'envasat d'aliments. / [EN] This Doctoral Thesis has focused on the isolation and characterisation of cellulosic materials and active extracts from coffee and rice husks, and their incorporation into starch films and starch-PLA compatibilised blend films in order to improve their functional properties as food packaging materials. Cellulose fibres were obtained through alkali and bleaching treatment with a final yield of 41 and 53 g fibres/100 g husk, respectively for rice and coffee husks. Cellulose nanocrystals were isolated from the bleached fibres by acid hydrolysis, with a yield of 5% with respect to bleached fibres, in both cases, with high crystallinity (90-92%), thermal resistance and aspect ratio (L/d: 20-40). The active compounds were obtained by hydrothermal extraction (180 ºC, 9.5 bar) with yields of 17 -18 g/100 g husks. They exhibited antioxidant properties (EC50: 5.37-5.29 mg extract solids/mg DPPH) and antibacterial activity against L. innocua (MIC: 48-52 mg powder/mL) and E. coli (MIC: 50-66 mg powder/mL), which were quantified in terms of the minimal inhibitory concentration. Cellulosic material from rice and coffee husks were incorporated into thermoplastic starch films (TPS) by melt blending and compression moulding. The elastic modulus increased by 186 and 121% when 1 wt% of cellulose nanocrystals (CNC) from rice and coffee husks, respectively, was incorporated into the matrix. Likewise, cellulose fibres (CF) were incorporated into TPS films at 1, 5 and 10 wt%. Both CF increased the film stiffness while reducing its stretchability. However, CF from coffee husk better maintained the film ductility at 1 and 5 wt%. The water vapour permeability of TPS films was not reduced in composites, although oxygen permeability was lowered by about 17%. When active extracts were incorporated into starch films, they improved the tensile properties; the elastic modulus increased by about 350%, while films became less stretchable. The cellulosic fibres from both residues were more effective as reinforcing agents in films containing extract solids than in net starch films. Starch-PLA blend films were also studied using grafted polycaprolactone with maleic anhydride and/or glycidyl methacrylate (PCLMG or PCLG) as compatibilisers. The effect of both the PLA ratio in the blend (20 and 40% with respect to starch) and the amount of both compatibilisers (2.5 and 5%) on the film properties was analysed. The analyses of microstructure, thermal behaviour and functional properties (mechanical, optical and barrier) of the films led to the conclusion that substituting 20% of the starch by PLA, and incorporating 5% of PCLG would be a good strategy to obtain films suitable for food packaging. The effect of the addition of cellulosic fillers (CF and CNC) and antioxidant aqueous extract from coffee husk to compatibilised starch-PLA blends was also studied. The antioxidant properties of the films were tested through their efficacy at preserving sunflower oil from oxidation. Significant differences were observed in the functional properties of the films when CNC was incorporated by two different methods. The reinforcing effect of cellulosic materials in S-PLA blends was less noticeable than in starch films, probably due to the overlapping of the PLA reinforcing effect. The antioxidant extract did not improve the mechanical performance in the blends, but conferred antioxidant capacity suitable for food packaging applications. / Collazo Bigliardi, S. (2019). Lignocellulosic fractions from rice and coffee husks to improve functionality of biodegradable films based on starch and poly-lactic acid [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/123055 / Compendio

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.