Für das Tissue Engineering von Knochen werden poröse dreidimensionale Substrate (Scaffolds) als Zellträger benötigt, die in der vorliegenden Arbeit über keramische Technologie hergestellt wurden. Neben dem strukturierten und getrockneten Verbundwerkstoff (Grünkörper) und der Sinterkeramik wurde auch der Zwischenzustand nach Ausheizen der organischen Phase (Braunkörper) evaluiert. Bei der Herstellung blieb die Architektur der parallel orientierten Kanalporen, die über den Sol-Gel-Prozess der gerichteten ionotropen Gelbildung des Alginates erzeugt wurde, in allen Materialzuständen erhalten. Die Herstellungstechnologie wurde derart optimiert, dass die neuartigen anisotropen Scaffolds allen prinzipiell gestellten Forderungen für das Tissue Engineering entsprachen – sie waren porös mit weithin einstellbarer Porengröße, sterilisierbar, gut handhabbar unter Zellkulturbedingungen, biokompatibel und degradabel. Der unerwartete Favorit der Biomaterialentwicklung, der Braunkörper – eine nanokristalline, poröse Hydroxylapatit-Biokeramik – lag in einer ersten in vivo-Studie nach 4 Wochen integriert im Knochen vor. Die beobachtete Knochenneubildung deutete auf eine osteokonduktive Wirkung des Materials hin. Die in der vorliegenden Arbeit untersuchten Technologien und Biomaterialien bieten eine Basis für weitere Forschung und motivieren zur Weiterentwicklung und Nutzung als Scaffold für das Tissue Engineering oder Knochenersatzmaterial unter Verwendung der interessanten Architektur.

Recombinant human bone morphogenic proteins (rhBMPs) are extensively studied and employed clinically for treatment of various bone defects. Current clinical delivery vehicles suffer wasteful burst releases that mandate supra-physiological dosing driving concerns over safety and cost. It was therefore investigated whether a unique drug delivery vehicle sequestered within a composite scaffold could lower the burst release of rhBMP-2. PLGA-calcium phosphate tri-phasic composite scaffolds delivered model protein BSA with burst release of ~13% and sustained kinetics of 0.5-1.5% BSA/day up to 45 days. rhBMP-2 was delivered with zero burst release however at much lower levels, totaling 0.09% to 0.9 % release over 10 days, but had up to 6.3-fold greater bioactivity than fresh rhBMP-2 (p<0.05). In conclusion, the three-phase composite scaffold can deliver bioactive proteins with a reduced burst release and sustained secondary kinetics.

Recombinant human bone morphogenic proteins (rhBMPs) are extensively studied and employed clinically for treatment of various bone defects. Current clinical delivery vehicles suffer wasteful burst releases that mandate supra-physiological dosing driving concerns over safety and cost. It was therefore investigated whether a unique drug delivery vehicle sequestered within a composite scaffold could lower the burst release of rhBMP-2. PLGA-calcium phosphate tri-phasic composite scaffolds delivered model protein BSA with burst release of ~13% and sustained kinetics of 0.5-1.5% BSA/day up to 45 days. rhBMP-2 was delivered with zero burst release however at much lower levels, totaling 0.09% to 0.9 % release over 10 days, but had up to 6.3-fold greater bioactivity than fresh rhBMP-2 (p<0.05). In conclusion, the three-phase composite scaffold can deliver bioactive proteins with a reduced burst release and sustained secondary kinetics.

Since current prosthetic heart valve replacements are costly, cause medical complications, and lack the ability to regenerate, tissue-engineered heart valves are an attractive alternative. These could provide an unlimited supply of immunological-tolerated biological substitutes, which respond to patients' physiological condition and grow with them. Since collagen is a major extra cellular matrix component of the heart valve, it is ideal material for constructing scaffolds. Collagen sources have been shown to influence the manufacturing of collagen scaffolds, and two commercial sources of collagen were obtained from Sigma Aldrich and Devro PLC for comparison. Consistencies between the collagens were shown in the primary and secondary structures of the collagen, while inconsistencies were shown at the tertiary level, when a higher level of natural crosslinking in the Sigma collagen and longer polymer chains in the Devro collagen were observed. These variations were reduced and the consistency increased by introducing crosslinking via dehydrothermal treatment (DHT). Collagen scaffolds produced via freeze-drying (FD) and critical point-drying with cross-linking via DHT or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide /N-hydroxysuccinimide (EDC/NHS) were investigated. All the scaffolds were compatible with mesenchymal stem cells (MSCs) according to the proliferation of the cells and their ability to produce ECM, without differentiating between osteogenic, chondrogenic or endothelial lineages. The FD EDC/NHS scaffold demonstrated the most suitable physical property of all. This result illustrates that FD EDC/NHS crosslinking is the most suitable scaffold investigated as a start for heart valve tissue engineering. To prepare a scaffold with a controlled local, spatial and temporal delivery of growth factor, a composite scaffold comprising poly (lactic-co-glycolic acid) (PLGA) microspheres was developed. This composite scaffold demonstrated the same compatibility to the MSCs as untreated scaffold. However, the PLGA microspheres showed an increase in the deterioration rate of Young's modulus because of the detachment of the microspheres from the scaffold via cellular degradation.

The damage of ocular surface represents one of the most common causes of decreased quality of vision or even blindness. If the injury is extensive and includes the region of limbus, niche of limbal stem cells (LSC), LSC deficiency occurs and the natural corneal regeneration is stopped. Conjunctival epithelium migrates into the injured area. Neovascularization, local inflammation and corneal opacity occur. Corneal transplantation is an insufficient treatment in such case. If the injury is bilateral, the allogenic limbal graft or LSC transplantation is required. In such cases systemic immunosuppressive drugs with many negative side-effects must be administered. The search for an adequate autologous substitution is important for avoid immunosuppressive medication. Mesenchymal stem cells (MSC) represent a perspective substitution for the reason of their immunomodulatory properties and the capability to differentiate in many cell types. There is possibility to isolate sufficient number of these cells from adipose tissue or bone marrow which are relatively easily accessible. Our goal was to observe local production of cytokines and other molecules which are present in inflammatory reaction after the chemical burn of the murine cornea and after the treatment with stem cells growing on nanofiber scaffold....

Les phosphates de calcium tels que le β-TCP sont utilisés depuis des décennies comme substitut osseux synthétique. Leurs bonnes propriétés chimiques et leur comportement analogue au tissu osseux in vivo et in vitro peuvent être améliorés par la technique de mise en forme employée. Il est aujourd'hui largement admis qu'une architecture poreuse optimisée aura un impact positif sur la bioactivité du matériau. Cette étude vise à étudier les liens existant entre une structure macroporeuse en β-TCP et la prolifération et différenciation cellulaire. Le β-TCP est fabriqué par précipitation aqueuse. Les paramètres de synthèse sont optimisés afin d'avoir un produit répondant aux normes ISO 13175 et 13779. Trois méthodes de mise en forme ont été choisies pour leur aptitude à générer une macroporosité originale. L'imprégnation d'une structure polymérique par une suspension génère un réseau de pores sphériques (PS), la stéréolithographie génère des pores cubiques interconnectés (3D) et la congélation orientée produit un réseau de pores tubulaires ellipsoïdaux parallèles au sens de la congélation (CO). Deux tendances émergent des cultures de cellules souches mésenchymateuses humaines: PS et 3D favorisent la prolifération alors que CO favorise la pénétration cellulaire et l'activité de la phosphatase alcaline. Cette dernière est favorisée par le β-TCP et cette aptitude est améliorée par la congélation orientée. Cela pourrait s'expliquer par l'état d'avancement de la différenciation cellulaire: les cellules sur les échantillons CO semblent être à un stade de différenciation plus avancé. Des essais complémentaires sur l'expression de gènes clés sont en cours pour vérifier cette hypothèse. / Calcium phosphates such as β-TCP have been used for decades as synthetic bone substitutes. Its good chemical properties and its similar behavior to that of the bone in vivo and in vitro can be enhanced by the chosen shaping method. It is nowadays largely accepted that an optimized porous architecture will have a positive impact on the material's bioactivity. This study aims at studying the links between a porous architecture and cell proliferation and differentiation. β-TCP was manufactured by aqueous precipitation. Synthesis parameters were optimized in order to get a product complying with ISO 13779 and 13175 requirements. Three shaping methods were chosen for their ability to generate original structures. The impregnation of a polymeric scaffold yields a network of interconnected spherical pores (PS), stereolithography yields a network of interconnected cubical pores (3D) and ice templating yields a network of parallel ellipsoidal channel-like structure (CO). Two different trends emerged from the human mesenchymal stem cell culture: PS and 3D favored cell proliferation whereas CO promoted cell penetration and alkaline phosphatase activity. The latter is stimulated by β-TCP and this ability is enhanced by freeze casting. This could be explained by the state of cell differentiation: cells on CO samples seem to be far more differentiated than the other ones. However the study of key genes expression is needed to confirm this hypothesis.

Escafoldes em alumina foram fabricados e em suas superfícies impregnou-se biovidro e hidroxiapatita; realizou-se análise das propriedades mecânica e de interação célula-escafolde in vitro. Estruturas porosas denominadas escafoldes são utilizadas como suportes para crescimento de tecidos, devem apresentar poros abertos interconectados, com morfologia, distribuição e quantidade de poros que confiram resistência mecânica e induzam o crescimento ósseo. Os escafoldes simulam a matriz extracelular e são a chave para a engenharia de tecidos que está conceituada na cultura prévia de células com proteínas morfogenéticas, oferecendo suporte para o crescimento celular na formação do tecido maduro. Neste trabalho desenvolveu-se técnica de manufatura onde foram conformados escafoldes como corpos-de-prova em alumina, em hidroxiapatita e em alumina infiltrada com biovidro e hidroxiapatita. Os escafoldes foram submetidos a ensaios mecânicos de compressão e sofreram análise de interação com células in vitro. A morfologia e a concentração da porosidade dos escafoldes foram analisadas por microscopia de varredura eletrônica e apresentaram porosidade volumétrica de aproximadamente 70% e diâmetro médio de poros em torno de 190 µm. Observou-se interação das células mais vigorosas e com pronunciada mitose nos escafoldes infiltrados relativamente aos escafoldes de alumina e hidroxiapatita. Os resultados indicaram resistência mecânica para os corpos infiltrados de 43,27 MPa, valor inferior ao observado nos escafolde de alumina 52,27 MPa e muito superior aos de hidroxiapatita 0,28 MPa. Conclui-se que os escafoldes de alumina infiltrados com biovidro e hidroxiapatita apresentaram uma combinação promissora nas características mecânicas e biológicas in vitro com viabilidade econômica. / Alumina scaffolds were manufactured and surface impregnated with bio-glass and hydroxyapatite; the mechanical properties and the in vitro bone-cell and scaffold interaction were analyzed. Porous matrices are usually denominated as scaffolds in tissue engineering and they are used as supports for the tissue growing; they may have open and interconnected pores, with known porous geometry and distribution and with good mechanical strength and be able to induce the tissue cells growing. Scaffolds can work as extra cell matrices, mimic the desired tissue and are considered as the key for the tissue engineering, offering support for the cellular growing in the formation of mature tissue. In this work, manufacture techniques were developed where scaffolds were conformed in alumina, in hydroxyapatite and in alumina infiltrated with bio-glass and hydroxyapatite, as test bodies. The scaffolds were submitted to mechanical compression tests and to the interaction with bone cells in vitro. The morphology and the concentration of the scaffold porosity were analyzed by scanning electronic microscopy (SEM) and they presented porosity concentration near 70,0 vol% and medium diameter of pores around 190,0 µm. The cells interaction strongest and more vigorous bone cell interaction with pronounced mitosis was observed in the alumina scaffolds infiltrated with bio-glass and hydroxyapatite when compared with the alumina scaffolds and hydroxyapatite scaffolds. The results obtained shown lower values of the mechanical strength for the infiltrated scaffolds (43,27 MPa), higher values for non infiltrated alumina scaffold (52,27 MPa) and very low values for the hydroxyapatite scaffolds (0,28 MPa). As observed, final results shown that alumina scaffolds infiltrated with bio-glass and hydroxyapatite presented a promising combination in the mechanical and biological in vitro characteristics with economic viability.

This thesis concerns the development of a new series of Sr-doped phosphate-based glasses for biomedical applications. Such glasses in powder form are envisaged to have applications in novel composite restorations where the following is achievable: dentin cell-mediated bioremineralization, dental pulp regeneration and as carrier for therapeutics or antibacterial ions.The initial aim was to produce soluble porous phosphate glasses using the sol-gel method (phosphate-alkoxide based sol-gel process). Knowing the effect that the variation of Ca content has on the dissolution properties of the glass, a series of glasses where Ca was progressively increased at the expense of Na was produced. The structure of the prepared samples was probed by XRD, XRF and FTIR to confirm the successful synthesis of the target phosphate-based glass compositions. After that a promising methodology was established, attempts were made to replace Ca with Sr. Different Sr sources were used without success due to the difficulty to fully dissolve those precursors in the sol-gel mixture. Subsequently, the issue of the toxicity of some precursors and solvents used in the sol-gel procedure was recognised. To overcome this obstacle, efforts were made to replace the toxic precursor chemicals with safer ones. Nevertheless, due to the low solubility of some new precursors and the low reactivity of others, the sol-gel process did not proceed in a predictable and reproducible fashion. At this stage, the sol-gel route was put aside, and two alternative soft and water-based chemical approaches were experimented: the precipitation method and the coacervation process. The first one was found to be unsuitable for our needs for two main reasons: 1) the presence of Na in the composition generated a crystalline material (instead of a glassy amorphous one); 2) the Ca/P ratio of our composition fell in the range of crystalline phase by using this method. In addition, the yield was really low. The second method (coacervation process) was a complete success. The glassy nature of the materials obtained was proved by XRD and XRF and the surface features were tested by BET and SEM. The process was retained for a while as the preferred synthesis route and both the scale-up effect and the possibility to add Sr were analysed. The production scale of the material was increased by 5 times and different Sr sources were tested to find the best one. XRD and XRF analysis proved both the success of the scale-up and the incorporation of the Sr in glass composition / This thesis concerns the development of a new series of Sr-doped phosphate-based glasses for biomedical applications. Such glasses in powder form are envisaged to have applications in novel composite restorations where the following is achievable: dentin cell-mediated bioremineralization, dental pulp regeneration and as carrier for therapeutics or antibacterial ions.The initial aim was to produce soluble porous phosphate glasses using the sol-gel method (phosphate-alkoxide based sol-gel process). Knowing the effect that the variation of Ca content has on the dissolution properties of the glass, a series of glasses where Ca was progressively increased at the expense of Na was produced. The structure of the prepared samples was probed by XRD, XRF and FTIR to confirm the successful synthesis of the target phosphate-based glass compositions. After that a promising methodology was established, attempts were made to replace Ca with Sr. Different Sr sources were used without success due to the difficulty to fully dissolve those precursors in the sol-gel mixture. Subsequently, the issue of the toxicity of some precursors and solvents used in the sol-gel procedure was recognised. To overcome this obstacle, efforts were made to replace the toxic precursor chemicals with safer ones. Nevertheless, due to the low solubility of some new precursors and the low reactivity of others, the sol-gel process did not proceed in a predictable and reproducible fashion. At this stage, the sol-gel route was put aside, and two alternative soft and water-based chemical approaches were experimented: the precipitation method and the coacervation process. The first one was found to be unsuitable for our needs for two main reasons: 1) the presence of Na in the composition generated a crystalline material (instead of a glassy amorphous one); 2) the Ca/P ratio of our composition fell in the range of crystalline phase by using this method. In addition, the yield was really low. The second method (coacervation process) was a complete success. The glassy nature of the materials obtained was proved by XRD and XRF and the surface features were tested by BET and SEM. The process was retained for a while as the preferred synthesis route and both the scale-up effect and the possibility to add Sr were analysed. The production scale of the material was increased by 5 times and different Sr sources were tested to find the best one. XRD and XRF analysis proved both the success of the scale-up and the incorporation of the Sr in glass composition

L'ingénierie tissulaire, qui consiste à remplacer un tissu lésé par un biosubstitut constitué de cellules réparatrices ensemencées dans une matrice de support biodégradable, possède un potentiel prometteur pour la réparation du Ligament Croisé Antérieur (LCA). Or, aucune solution opérationnelle n'a encore été proposée à ce jour, notamment au vu du nombre de domaines scientifiques impliqués. Dans ce travail, nous avons dressé un cahier des charges pour la définition de cette matrice en nous appuyant sur l'état de l'art. Une matrice de support tressée multicouche constituée de fibres de P(LL85/CL15) a été imaginée, puis les outils nécessaires à sa fabrication à l'échelle du laboratoire ont été mis en place. Nous avons ensuite développé des outils numériques spécifiques permettant la modélisation de sa géométrie et de son comportement biomécanique multi-échelles, qui ont été mis à profit afin d?optimiser les caractéristiques de la matrice compte tenu du cahier des charges établi. De plus, des caractérisations biologiques ont montré que la matrice était compatible avec la culture de cellules souches, et était susceptible d?accueillir la formation d'un néo-tissu. Par ailleurs, nous avons mis en place un bioréacteur spécifique permettant d'imposer à la matrice de support des cycles de traction-torsion sous environnement contrôlé. L'utilisation des informations locales issues de la modélisation biomécanique, afin d'interpréter ou d'optimiser les résultats de culture cellulaire sous sollicitations cycliques, constitue une perspective majeure du présent travail. Notre investigation permet en outre de penser qu'un nouveau biosubstitut pour le LCA pourrait prochainement être proposé / Tissue engineering, which consists in replacing an injured tissue with a biodegradable scaffold seeded with cells, has the potential to overcome the limitations associated with current reconstructions strategies of the Anterior Cruciate Ligament (ACL). However, no relevant solution has been proposed yet, especially due to the variety of scientific fields involved in this approach. In the current study, the key requirements for the design of a new scaffold have been listed from the current state of art. A scaffold based on P(LL85/CL15) fibers arranged into a multilayer braided structure has been proposed, and the tools needed to process this scaffold have been developed. Dedicated numerical tools have been proposed in order to predict the morphological and multiscale biomechanical behavior of the scaffold. These simulation tools have enabled to optimize the scaffold geometry in order to match the selected key requirements for ACL tissue engineering. Moreover, preliminary biological assessments have shown that the scaffold was suited for the culture of stem cells and for tissue formation. In addition, a dedicated bioreactor has been developed in order to prescribe tension-torsion cycles within a controlled environment. The use of local information issued from the biomechanical simulations open large perspectives as far as the optimization of culture conditions and the understanding of mechanisms that govern the formation of a ligamentous tissue are concerned. As a conclusion, the present study is likely to enable a new solution for ACL tissue engineering to emerge in the next years

Depuis plus d’une cinquantaine d’années, de formidables avancées ont été initiées dans le domaine de l’ingénierie tissulaire cutanée menant à la reconstruction in vitro de substituts de peau. La plupart sont des substituts dermiques destinés à être utilisés comme aide à la cicatrisation des plaies aigües et chroniques en complément des traitements de greffes conventionnels ainsi que pour l’augmentation des tissus mous. Bien qu’un nombre croissant de patients aient pu bénéficier de ces matrices dermiques, leur application clinique reste encore restreinte, en raison de leur coût élevé mais également à cause de résultats cicatriciels parfois peu satisfaisants. Par conséquent, il reste un défi de taille, celui de développer des substituts dermiques stimulant activement la cicatrisation, présentant un faible coût de production, sans propriétés antigéniques et possédant des propriétés mécaniques adaptées. Dans ce cadre, les hydrogels à base de fibrine constituent des candidats prometteurs, en particulier en raison du rôle central de cette protéine dans la cicatrisation. Le principal inconvénient est qu’à concentration physiologique, ces hydrogels sont faibles mécaniquement, ce qui les rend difficilement manipulables. L’objectif de cette thèse a été la mise au point ainsi que la caractérisation de différents hydrogels destinés à être utilisés comme substituts dermiques. Ces derniers présentent l’avantage d’associer les propriétés biologiques de la fibrine avec les propriétés mécaniques d’un polymère synthétique, le polyéthylène glycol dans une architecture de réseaux interpénétrés de polymères (RIP). Les résultats obtenus ont permis : - de confirmer les propriétés physico-chimiques des RIP développés initialement par nos collaborateurs de l’université de Cergy-Pontoise, - de valider en trois étapes (in vitro, ex vivo puis in vivo) la biocompatibilité de ces nouvelles matrices, destinées à être utilisées comme supports de culture 2D et pour l’augmentation des tissus mous, - d’élaborer et de caractériser des matrices macroporeuses, optimisées pour la culture 3D de fibroblastes de dermes humains. / Over the past five decades, we assisted in extraordinary advances in the field of skin tissue engineering which led to the in vitro reconstruction of a wide range of skin substitutes. Most of them are dermal substitutes: Their clinical application ranges from treating acute and chronic wounds to soft tissue augmentation. Although increasing numbers of patients have been treated with dermal substitutes, their clinical application has been limited by their substantial cost and some poor healing outcomes. Hence, there is still a challenge to produce a dermal substitute which enhance sufficiently wound healing. To this end, the substitute should exhibit suitable properties for enabling the repair process. Other requirements such as excellent biocompatibility, minimal antigenicity, ease to handle and cost-effective production are also essential. In this context, fibrin hydrogels constitute promising candidates for skin tissue engineering since fibrin fibers form a physiological and provisional backbone during wound healing. However, the poor mechanical properties of fibrin-based hydrogels at physiological concentration are an obstacle to their use. In this study, our aim was to design and characterize mechanically reinforced fibrin-based hydrogels by combining the intrinsic properties of a fibrin network with the mechanical features of a polyethylene glycol network using an interpenetrating polymer network (IPN) architecture. They are intended to be used as dermal scaffolds. The results obtained in this thesis: - Confirmed the suitable physico-chemical properties of IPN, first developed by our partner of the University of Cergy-Pontoise. - Validated their biocompatibility using a three-step approach (in vitro, ex vivo and in vivo assays). - Led to the synthesis and characterization of a new type of fibrin-based macroporous matrices, optimized for 3D dermal fibroblast culture.