Orientador: Cecilia A. C. Zavaglia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-08-07T02:27:14Z (GMT). No. of bitstreams: 1 Uzumaki_EmiliaTieko_D.pdf: 44142340 bytes, checksum: e8db433907d2fd2f88b260ed4cd8a791 (MD5) Previous issue date: 2006 / Resumo: O carbono tipo diamante ("diamond-like carbon" - DLC) tem demonstrado características, como alta dureza, baixo atrito, resistência ao desgaste e à corrosão, e biocompatibilidade, que podem melhorar as propriedades de implantes sólidos e articulados. O processo de imersão em plasma vem sendo usado para depositar DLC em substratos tridimensionais, pois, com esta técnica, se consegue melhor adesão do que com as técnicas convencionais. Neste trabalho, filmes de DLC foram depositados, pelo processo de imersão em plasma, em lamínulas de vidro, silício e liga de titânio Ti-13Nb-13Zr. A caracterização da microestrutura, morfologia, dureza e adesão dos filmes de DLC foi feita por espectroscopia Raman, microscopia eletrônica de varredura (MEV), microscopia de força atômica (MFA), nanoindentação e ensaio de puxamento. Como exemplos de peças tridimensionais, filmes de DLC foram depositados em implantes osteoarticulares de quadril e joelho, titânio poroso (esponja de célula-aberta, semelhante à estrutura do osso esponjoso, expandida a vácuo), facas industriais, bisturi cirúrgico, engrenagem de motor, tubos de quartzo e de alumínio, e outros objetos. O filme obtido apresentou boas propriedades mecânicas, aumentando em 2 vezes a dureza da liga de Ti, alta adesão (filme sem interface definida e sem delaminação), resistência ao desgaste, baixa rugosidade e uniformidade de deposição em superfícies tridimensionais. Os resultados de corrosão (polarização de Tafel e espectroscopia de impedância eletroquímica em fluido corporal simulado) mostraram que o revestimento de DLC melhora a resistência da liga de Ti à corrosão. No ensaio de biocompatibilidade in vitro, com células fibroblásticas, foram estudados a citotoxicidade, adesão e morfologia celular (estudo citoquímico, microscopia de contraste de fase, MEV e MFA). No ensaio de biocompatibilidade in vivo, a liga de Ti-13Nb-13Zr, revestida com DLC, foi investigada em tecido muscular e ósseo de ratos após 4 e 12 semanas do procedimento cirúrgico. A interface formada entre o DLC e o tecido foi investigada por histologia convencional, e os implantes retirados por MEV. A interface entre o implante e o tecido ósseo, não descalcificado, foi estudada por MEV na modalidade retroespalhamento. Os resultados mostraram a biocompatibilidade in vitro e in vivo do filme de DLC, e foi verificado também que os implantes revestidos com DLC possuem resposta biológica mais favorável do que os implantes não revestidos / Abstract: Diamond-like carbon (DLC) films are often considered a suitable coating material for orthopaedic applications. It has proven characteristics, such as hardness, wear resistance, low friction coefficient and biocompatibility that improve the properties of solid and articulated implants. Recently, the plasma immersion process was used to deposit DLC films with superior adhesion properties to those prepared with conventional techniques. DLC coatings were deposited on glass coverslips, silicon (Si) and Ti-13Nb-13Zr substrates using the plasma immersion process. The microstructure, morphology, roughness, hardness and adhesion of DLC films were characterized using Raman spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), nanoindentation and pull-test. As examples, DLC films produced by plasma immersion were deposited on industrial knives, surgical knives, knee implants, femoral heads (of hip prostheses), titanium foams, transmission gears of motorcycles, aluminium pipes, quartz pipes, and others objects. The corrosion susceptibility of DLC coatings produced by plasma immersion was studied in a simulated body fluid environment (Hanks' solution) using polarization test and electrochemical impedance spectroscopy (EIS). Electrochemical results showed that DLC coating produced by plasma immersion could improve corrosion resistance, and no significant damage has been observed. Vero cells (fibroblasts) were utilized for the in vitro biocompatibility studies, by cytotoxicity, adhesion and cell morphology (phase contrast microscopy, SEM, AFM, and cytochemical study). DLC-coated Ti-13Nb-13Zr was investigated in an animal model using the muscular tissue and femoral condyles of rats for intervals of 4 and 12 weeks postoperatively. The interface between the implants and tissue were analysed by light microscopy, and the removed implants by SEM. The SEM by backscattering was used to access the interface between the implants the bone tissue without decalcifying. Our results indicate that DLC coatings are biocompatible in vitro and in vivo / Doutorado / Materiais e Processos de Fabricação / Doutor em Engenharia Mecânica

Orientador: Lourenço Sbragia Neto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T14:56:26Z (GMT). No. of bitstreams: 1 Goncalves_FrancesLilianLanhellas_M.pdf: 3789177 bytes, checksum: e9b1048e4d4f077e540d297c2a998419 (MD5) Previous issue date: 2008 / Resumo: Gastrosquise é um defeito congênito da parede abdominal anterior no qual as alças intestinais ficam herniadas e em contato com o líquido amniótico (LA). Assim, a exposição ao LA resulta em várias disfunções intestinais pós-natal. Para reduzir o tempo de exposição ao LA em modelo animal, usou-se um hidrogel de N-isopropilacrilamida (NIPAAm) copolimerizado com ácido acrílico (Aac), que rapidamente intumesce na presença de LA. O hidrogel foi usado para cobrir as alças expostas até o fim da gestação. A gastrosquise foi induzida em fetos de ratas fêmeas da raça Sprague-Dawley através de um corte paramediano à direita do cordão umbilical para exposição parcial das alças com 18,5 dias de gestação. Os fetos foram separados em quatro grupos: controle (C), apenas gastrosquise (G), gastrosquise + cobertura das alças com adesivo de fibrina - Beriplast® (GA) e gastrosquise + cobertura das alças com adesivo de fibrina e aderido um pedaço de hidrogel seco (GAH). Os animais foram colhidos por cesárea com 21,5 dias de gravidez e o hidrogel foi cuidadosamente removido. Os fetos e as alças intestinais foram pesados e análise morfométrica foi realizada. Resultados mostraram que o hidrogel após intumescimento pesou 34X que seu peso seco; ele possui carga elétrica assim como a maioria das proteínas presentes no LA e sua retirada não provocou lesão à camada serosa do intestino exposto como visto na MEV. A comparação dos grupos C e GAH com os grupos G ou GA mostrou que o peso, o diâmetro, a espessura das camadas e da parede intestinais foi significativamente menor nos grupos C e GAH quando comparados aos grupos G e GA indicando processo inflamatório. Sendo assim, a aplicação do hidrogel aderido pelo adesivo de fibrina mostrou servir como uma efetiva proteção das alças herniadas, com uma redução significante da inflamação na gastrosquise. / Abstract: Gastroschisis is a congenital defect of the anterior abdominal wall which leads the fetal bowel to herniate into the amniotic cavity. There, exposition to amniotic fluid (AF), results in severe postnatal intestinal dysfunction. In order to reduce exposition time to AF in an animal model, has used a hydrogel of N-isopropylacrylamide (NIPAAm) copolymerized with acrylic acid (Aac), which undergoes rapid swelling in the amniotic fluid. The hydrogel was used to coat the bowel hernia until pregnance is completed. Gastroschisis was induced in the fetuses of female Sprague-Dawley rats by partial evisceration of the bowel through a right paramedian opening of the abdominal wall in day 18,5 of pregnancy. The fetuses were separated in four groups: control (C), gastroschisis alone (G), gastroschisis + coating of the bowel hernia with fibrin adhesive -Beriplast® (GA) and gastroschisis + coating of the bowel hernia with fibrin adhesive topped by a piece of adhered dry hydrogel (GAH). Animals were harvested by cesarean section at day 21.5 of pregnancy and the hydrogel was carefully removed. Fetuses and intestinal tract were weighed and morphometric analysis was performed. Results showed that the hydrogel weight was 34X heavier than its dry weight; its electric charge and also the AF charge were negative and there was no damage to serosa layer of the intestine exposed. Comparison of the C and GAH groups with G and GA showed that the bowel weight, diameter, the layers and wall thickness was significantly reduced in C and GAH compared to G and GA. Thus, application of the hydrogel bound onto the fibrin adhesive was shown to provide an effective protection of the herniated bowel, with a significant reduction of inflammation in gastroschisis. / Mestrado / Pesquisa Experimental / Mestre em Cirurgia

Made available in DSpace on 2014-07-29T15:07:31Z (GMT). No. of bitstreams: 1 Daniel Barbosa.pdf: 1143657 bytes, checksum: d56a1f0787d16e510afed007895c5973 (MD5) Previous issue date: 2010-10-24 / Abdominal wall defects may occur in fairly all animal species, and frequently demand corrective surgical treatment including implants of meshes, such as polypropylene. However, this biomaterial is not free from complications, what stimulates constant researches for new biomaterials that present certain advantages. Due to its well known biocompatibility, poliHEMA hydrogel was the chosen biomaterial for experimental abdominal wall implant associated to polypropylene mesh. This essay compared tissue responses to the employment of polypropylene mesh alone (group PP) and polyHEMA Hydrogel film associated to polypropylene mesh (group PH) on the correction of induced abdominal wall defects on bitches. Twelve mongrel adult female dogs, weighting from 10 to 20 kg, were divided in two equal groups. The animals from group PP received the polypropylene mesh implant whilst those from group PH received polyHEMA hydrogel coated polypropylene mesh to replace the abdominal transverse muscle. Six animals from each group were submitted to surgical procedure to remove the meshes at 30 and 60 days of the postoperative period. Tissue repairing phenomena such as: chronic inflammatory reaction, giant cell presence (foreign body chronic granulomatous inflammatory reaction) and connective tissue proliferation were microscopically evaluated. It may be concluded that polyHEMA hydrogel as a coating agent on polypropylene mesh implanted onto abdominal wall of female dogs triggers greater deposition of type I collagen, than polypropylene mesh alone. / Os defeitos na parede abdominal estão presentes em praticamente todas as espécies animais e não raro demandam tratamento cirúrgico corretivo com implantação de telas como a de polipropileno. Contudo esse biomaterial não é livre de complicações, o que impulsiona a constante pesquisa na busca de novos biomateriais que apresentem vantagens. O hidrogel de poliHEMA por ser reconhecidamente biocompatível foi o biomaterial de escolha para a implantação experimental em associação à tela de polipropileno na parede abdominal. Este estudo comparou as respostas teciduais do uso da tela de polipropileno isolada (grupo PP) e o filme de hidrogel de poliHEMA associado à tela de polipropileno (grupo PH) quando utilizados na correção de defeitos criados na parede abdominal de cadelas. Foram utilizadas 12 cadelas sem raça definida, adultas, pesando entre 10 e 20 kg, divididas em dois grupos de seis. Os animais do grupo PP receberam o implante da tela de polipropileno e os animais do grupo PH receberam a tela de polipropileno revestida de hidrogel de poliHEMA em substituição ao músculo transverso do abdome. Seis animais de cada grupo foram submetidos ao procedimento cirúrgico para a retirada das telas aos 30 e 60 dias do pós-operatório. Foram avaliados microscopicamente os fenômenos de reparo tecidual como: reação inflamatória crônica, presença de células gigantes (reação inflamatória crônica granulomatosa tipo corpo estranho), e proliferação conjuntiva. Conclui-se que o hidrogel de poliHEMA como agente de revestimento da tela de polipropileno quando implantado na parede abdominal de cadelas desencadeia maior deposição de colágeno tipo I quando comparado a tela de polipropileno isolada.

Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces.    Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H. Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood. In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering platelets, which highlights the importance of regulating both complement and platelets to lower inflammatory events. In addition, a strategy to enhance the biocompatibility of biomaterials and cells by simultaneously targeting ADP-dependent platelet activation and the alternative complement C3-convertase is proposed.

Une lésion au niveau d’un nerf périphérique peut provoquer la perte de fonction sensorielle et motrice, et dans le cas de neurotmésis, la régénération spontanée ne se produira pas. De plus, si l’espace entre les deux segments de nerf est trop important, une suture directe n’est pas possible et l’implantation d’une greffe est nécessaire afin de créer une liaison entre les deux segments de nerf. L’autogreffe de nerf est le « gold standard » pour des procédés de réparation nerveuse : une portion d’un nerf sein (qui est considéré comme un nerf moins important) est prise du même patient et implantée au site de la lésion. Cependant, il existe plusieurs désavantages avec ce procédé comme une deuxième chirurgie, la perte de fonction au site du don, la possibilité de développer un neurome sur ce même site, ainsi qu’un taux de réussite de 50% dans les cas où l’espace entre les deux segments de nerf est très important. Il reste donc, un besoin de trouver un procédé alternatif afin d’augmenter le taux de réussite et d’éliminer les désavantages de l’autogreffe. L’objectif de cette étude est d’avancer vers une solution alternative de l’autogreffon en utilisant des biomatériaux. Cette thèse se divise en trois parties. La première se focalise sur le développement d’un modèle de guide nerveux basé sur des nanofibres de fibroïne de soie. Ce matériau est composé d’une organisation complexe qui inclut deux surfaces de nanofibres alignées avec une couche de nanofibres aléatoires à l’intérieur afin d’améliorer des propriétés mécaniques du matériau sans la perte d’orientation des fibres pour la régénération nerveuse. Le matériau est ensuite manipulé pour fabriquer un tube, multi-canaux avec une « enveloppe » supplémentaire afin de faciliter le procédé d’implantation chirurgicale. Ce guide nerveux a été soumis pour l’obtention d’un brevet européen le 12 juillet 2017 et cela est le sujet d’un deuxième article qui a été soumis pour publication. La deuxième partie de cette étude explore des possibilités d’une fonctionnalisation du matériau afin d’améliorer son efficacité pour la régénération nerveuse. Cette étude explore la fonctionnalisation de la fibroïne de soie avec une deuxième protéine, plusieurs facteurs de croissance, et des nanoparticules. Chacune de ces fonctionnalisations donne une possibilité d’ajouter des propriétés favorables à la fibroïne de soie, un matériau naturel et biocompatible. La troisième partie de cette étude examine l’efficacité d’un guide nerveux composé de la fibroïne de soie fonctionnalisée avec des facteurs de croissance pour la régénération nerveuse périphérique en comparaison avec un guide nerveux composé de la fibroïne de soie sans aucune fonctionnalisation et une suture direct (qui simule une autogreffe). Trois techniques d’évaluation différentes de la régénération nerveuse ont été réalisées afin d’obtenir une analyse plus complète. Il y a de nombreux mécanismes impliqués dans la régénération nerveuse, il est donc nécessaire d’étudier différents paramètres pour analyser l’efficacité de régénération. Les résultats d’analyses histologiques, d’électromyographie, et de capture de mouvement, ont été considérées ensemble afin d’arriver à une conclusion sur la réussite d’une régénération nerveuse pendant cette étude. Pour conclure cette étude, les guides nerveux fonctionnalisés avec une combinaison de facteurs de croissance démontrent une meilleure régénération nerveuse et une récupération de fonction supérieure. / Injury to a peripheral nerve can cause loss of sensory and motor function, and if the injury is very severe where the nerve undergoes neurotmesis, unassisted nerve regeneration may not occur. In this case, where the gap between nerve segments is too large to carry out a direct end to end suture, a graft is sutured to bridge the gap between sectioned nerve segments. The autologous nerve graft, where a portion of a less important nerve from the same patient is removed and grafted between nerve segments, continues to be the gold standard procedure for nerve repair. However, there are several drawbacks of this technique including a second surgical procedure, loss of function at the donor site, possibility of developing a painful neuroma at the donor site, and the 50% success rate of autografts used in large gaps. There is therefore a need for a tissue engineered nerve graft that can replace the autograft, and this study aims to advance toward an effective autograft alternative. This PhD is presented as a three part study consisting first of the development of a novel nerve guidance conduit based on a tri-layered silk fibroin nanofiber material comprised of a complex organization including two aligned fiber surfaces and a randomly deposited fiber interior to improve the mechanical properties of the material while not compromising the guidance capabilities of aligned nanofibers for nerve regeneration. The material is then used to fabricate a multi-channeled tube with an additional “jacket layer” in order to facilitate surgical implantation. This NGC has been submitted to be patented on July 12, 2017 and is the subject of the second article submitted for review for publication. The second part of this study explores the different possibilities of the functionalization of the material in order to improve the effectiveness for nerve regeneration. This study explores functionalizing the silk fibroin with a second protein, several growth factors, and nanoparticles that all have potential to add favorable properties to the natural biocompatible silk fibroin material. The final part of this study tests the effectiveness of growth factor-embedded silk fibroin NGCs for peripheral nerve regeneration in comparison with non-functionalized silk fibroin devices and a direct suture to simulate results obtained with an autograft. Three different techniques for the evaluation of nerve regeneration were used in order to produce a more comprehensive analysis. As there are many mechanisms involved in nerve regeneration, only one or two analysis techniques cannot paint a complete picture of the success of nerve regeneration. Therefore, histological analyses, electromyography analyses, and motion capture analyses were carried out and considered together in order to make a conclusion on the level of nerve regeneration success during this study. The conclusions from this study were that a NGC functionalized with a combination of growth factors appeared to exhibit the most successful nerve regeneration and functional recovery.

The role of a protein-repelling coating is to limit the interaction between a device and its physiological environment. Plasma-polymerized-PEG (pp-PEG) surfaces are of great interest since they are known to avoid protein adsorption. and cell attachment. However, in all the studies previously published in the literature, the PEG coatings have been prepared using low pressure processes. <p>In this thesis, we synthesize biocompatible pp-PEG coatings using atmospheric pressure plasma. Two original methods are developed to obtain these pp-PEG films. 1. Atmospheric pressure plasma liquid deposition (APPLD) consists in the injection of the precursor, tetra(ethylene glycol)dimethylether (tetraglyme), by means of a liquid spray, directly in the post-discharge of an atmospheric argon plasma torch. 2. In atmospheric pressure plasma-enhanced chemical vapor deposition (APPECVD), tetraglyme vapors are brought in the post-discharge trough a heating sprinkler. The chemical composition, as well as the non-fouling properties of the APPLD and APPECVD films, are compared to those of PEG coatings synthesized by conventional low pressure plasma processes.<p>In the first part of the study, the effect of the power on the chemical composition of the films has been investigated by infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS) and secondary ions mass spectroscopy (SIMS). <p>The surface analysis reveals that for the APPECVD samples, the fragmentation of the precursor increases as the power of the treatment is increased. In other terms, the lower the plasma power is, the higher the “PEG character” of the resulting films is. Indeed, the C-O component (286.5 eV) of the XPS C 1s peak is decreasing while the hydrocarbon component (285 eV) is increasing as the power of the plasma is increased. The same conclusion can be drawn from the signature ToF-SIMS peaks (m/z = 45 (CH3&61485;O&61485;CH2+ and +CH2CH2&61485;OH), 59 (CH3&61485;O&61485;CH2&61485;CH2+), 103 (CH3&61485;(O&61485;CH2&61485;CH2)2+)) that are decreasing in the case of high power treatments. Accordingly, IRRAS measurements show that the C-O stretching band is decreasing for high power plasma deposition. This is in agreement with the observations made from the analysis of the LP PECVD coatings and from the literature.<p>The films deposited by the APPLD process do not show the same behavior. Indeed, whatever the power injected into the discharge is, we are able to achieve films with a relatively high PEG character (&61566;83 %).<p>The second part of this study is dedicated to the evaluation of the non-fouling properties of the coatings by exposing them to proteins (bovine serum albumin and human fibrinogen) and cells (mouse fibroblasts (L929 and MEF)) and controlling the adsorption with XPS (proteins) and SEM (cells).<p>For the APPECVD samples, a low plasma power (30 W) leads to an important reduction of protein adsorption and cell adhesion (over 85%). However, higher-powered treatments tend to reduce the non-fouling ability of the surfaces (around 50% of reduction for a 80 W deposition). <p>The same order of magnitude (over 90% reduction of the adsorption) is obtained for the APPLD surfaces, whatever is the power of the treatment. <p>Those results show an important difference between the two processes in terms of power of the plasma treatment, and a strong relationship between the surface chemistry and the adsorption behavior: the more the PEG character is preserved, the more protein-repellent and cell-repellent is the surface. / Le rôle d’une couche empêchant l’adsorption de protéines est de limiter les interactions entre un implant et le milieu physiologique auquel il est exposé. Les films de poly(éthylène glycol) polymérisés par plasma (pp-PEG) sont d’intérêt majeur car ils sont connus pour empêcher l’adsorption de protéines ainsi que l’attachement cellulaire. Cependant, dans toutes les études publiées précédemment, les couches de type PEG ont été réalisées sous vide.<p>Dans cette thèse de doctorat, nous synthétisons des couches de type pp-PEG biocompatibles par plasmas à pression atmosphérique. A cette fin, deux méthodes originales ont été développées. 1. La première méthode consiste en l’injection du précurseur, le tetra(éthylène glycol) diméthyl éther (tetraglyme), en phase liquide, en nébulisant ce dernier au moyen d’un spray, directement dans la post-décharge d’une torche à plasma atmosphérique fonctionnant à l’argon. En anglais, nous appelons ce procédé « Atmospheric pressure plasma liquid deposition (APPLD) ». 2. Dans la deuxième méthode, appelée en anglais « Atmospheric pressure plasma-enhanced chemical vapor deposition (APPECVD)», le tetraglyme est amené en phase vapeur dans la post-décharge, au moyen d’un diffuseur chauffant. La composition chimique des dépôts de type APPLD et APPECVD, ainsi que leurs propriétés d’anti-adsorption sont évaluées, et comparées aux dépôts pp-PEG obtenus par les méthodes à basse pression conventionnelles.<p>Dans la première partie de cette étude, nous nous focalisons sur la composition chimique des films déposés, et plus particulièrement sur l’influence de la puissance injectée dans le plasma sur cette composition chimique. A cette fin, nous avons fait appel à des techniques d’analyse telles que la spectroscopie de réflexion-absorption infrarouge (IRRAS), la spectroscopie des photoélectrons X (XPS) et la spectrométrie de masse des ions secondaires (SIMS). <p>Il en ressort que les films de type APPECVD perdent progressivement leur « caractère PEG » à mesure que la puissance de la décharge plasma est élevée. Cela serait dû à une plus grande fragmentation du précurseur dans la post-décharge d’un plasma plus énergétique. Cette tendance est cohérente avec ce que nous avons observé pour les dépôts à basse pression ainsi que dans la littérature.<p>Dans le cas des films de type APPLD, un tel comportement n’a pas été mis en évidence :quelle que soit la puissance dissipée dans le plasma, les films présentent un « caractère PEG » relativement élevé.<p>La deuxième partie de cette thèse est dédiée à l’évaluation des propriétés d’anti-adsorption des films synthétisés, en les exposant à des protéines (albumine de sérum bovin et fibrinogène humain) et des cellules (fibroblastes de souris, L929 et MEF). L’adsorption de protéines est contrôlée par XPS tandis que l’attachement cellulaire est contrôlé par imagerie SEM.<p>Pour les échantillons de type APPECVD, un dépôt à faible puissance (30 W) mène à une importante réduction de l’adsorption de protéines et de cellules (> 85%) tandis qu’à de plus hautes puissances (80 W), l’anti-adsorption est sensiblement diminuée (50% de réduction). Dans le cas des dépôts de type APPLD, quelle que soit la puissance du plasma, une forte diminution de l’adsorption de protéines et de cellules est observée (> 90 %).<p>Ces résultats montrent une différence majeure entre les deux procédés quant à l’influence de la puissance du plasma ainsi qu’une forte relation entre la composition chimique de la surface synthétisée et son pouvoir d’anti-adsorption :plus le « caractère PEG » du dépôt est conservé, plus la surface empêchera l’interaction avec les protéines et les cellules. <p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La première partie du travail présenté ici se concentre sur l’optimisation d’une cathode à oxygène. Tout d’abord, l’utilisation d’une nouvelle enzyme (la BOD de Magnaporthe oryzae) permet de multiplier le courant de réduction de l’oxygène en eau jusqu’à neuf fois. Ensuite la synthèse d’un polymère rédox adapté a permis d’améliorer le coefficient de diffusion des électrons dans l’hydrogel résultant en l’augmentation de la densité de courant générée. Enfin nous sommes passés d’uneélectrode de carbone en 2 dimensions à une fibre d’or poreuse tridimensionnelle. Après modification de cette fibre avec l’hydrogel rédox à base de BOD de M. oryzaenous avons évalué sa biocompatibilité : in vitro les tests ont montré l’absence totale de cytotoxicité et seule une très faible réponse inflammatoire ; in vivo aucune infection ne s’est déclarée pendant les 8 semaines d’implantation dans les souris etla formation d’une capsule fibrotique autour de l’électrode traduit sa bonne intégration dans les tissus de l’animal. La seconde partie concerne la biopile dans son intégralité, construite à partir de la cathode optimisée et d’une anode adaptée à base de GDH. Elle permet de générer jusqu’à 240 μW.cm-2 dans du tampon Pipes/CaCl2 à 5mM de glucose. La biopile a ensuite été testée dans du sang humain total. Un maximum de 129 μW.cm-2 a été obtenu dans un échantillon avec une glycémie de 8,2 mM sous air. De plus nous avons constaté que la densité de puissance délivrée augmente proportionnellement avec la glycémie des différents échantillons de sang testés, faisant de la biopile à la fois une source d’électricité et un biocapteur à glucose ce qui n’avait jamais été démontré auparavant. / The first part of the work presented here focuses on the optimization of an oxygen cathode. First, the use of a new enzyme (BOD from Magnaporthe oryzae) permit to increase the current of reduction of oxygen into water by a factor nine. Then the synthesis of a suitable redox polymer greatly improved the diffusion coefficient of electrons in the hydrogel, resulting in an increase of the current density. Finally we switched from a two-dimensional carbon electrode to a three-dimensional porous gold fiber. After modification of the fiber with the redox hydrogel based on BOD from M. oryzae, we assessed its biocompatibility: in vitro the tests showed the total absence of cytotoxicity and only a very low inflammatory response; in vivo noinfection appeared during the 8 weeks of implantation in mice and the formation of afibrotic capsule around the device reflects its successful integration into the animal tissues.The second part concerns the full biofuel cell, elaborated from the optimized cathode and an adapted GDH-based anode. It could generate up to 240 μW.cm-2 at 5mMglucose in Pipes/CaCl2 buffer. The biofuel cell was then tested in whole human blood. A maximum of 129 μW.cm-2 was obtained in a sample with 8,2 mM glycaemiaunder air. In addition we observed that the delivered power density increased proportionally with the glycaemia of the different blood samples tested, making the biofuel cell both a power source and a glucose biosensor at the same time which had never been shown before.

Actuellement, la presbytie peut être corrigée chirurgicalement à l’aide d’implants intraoculaires réfractifs ou diffractifs multifocaux (chirurgie endoculaire invasive et irréversible) ou en intracornéen avec une correction multifocale réfractive (correction laser irréversible, ou insertion d’un implant dans le stroma). L’objectif de ce travail est de développer un nouvel implant permettant de corriger la presbytie, qui allie l’innocuité et la réversibilité d’une correction intracornéenne, à l’efficacité du diffractif. Le design des profils optiques bifocaux a été permis grâce au développement d’outils de simulation optique. Les efficacités de diffraction sont calculées à partir de la propagation du champ électrique par spectre angulaire. La qualité optique est déterminée d’après les simulations de Fonction de Transfert de Modulation obtenues sous Zemax. Des simulations de rendu d’images permettent de visualiser les effets de différents profils envisagés. Les paramètres critiques du design optique sont déterminés. Le choix du matériau dépend des contraintes de biocompatibilité de l’implant et des techniques de fabrication. La solution retenue est un hydrogel à forte teneur en eau, couplé à une nouvelle architecture de l’implant. L’hydrogel est obtenu par polymérisation radicalaire de macromonomères difonctionnels de poly(éthylène glycol) de masses molaires de l’ordre de 8000 g.mol‐1 qui conduisent à des propriétés mécaniques et une perméabilité aux nutriments compatibles avec l’application. La réalisation, la stérilisation et la caractérisation optique de prototypes ont abouti à la preuve du concept d’un implant bifocal diffractif intracornéen / Presbyopia can be corrected with surgery by means of refractive or diffractive multifocal intraocular lenses (which imply an irreversible and invasive endocular surgery) or by intracorneal multifocal refractive correction (irreversible laser correction, or insertion of an intrastromal implant). This work aims at developing a new implant to correct presbyopia, which takes advantage of both the harmlessness and the reversibility of an intracorneal correction, and the efficiency of diffractive optics. The design of the bifocal optical profiles was based on the development of optical simulation tools. The diffractive efficiencies are calculated from the distribution of the electric field with the method of angular spectrum. The optical quality is determined according to the simulations of Modulation Transfer Function obtained with Zemax. Images simulations show the effects of the different profiles studied. The critical parameters of the optical design are also determined. The choice of the material depends on several constraints such as biocompatibility and techniques of manufacturing. The adopted solution relies on the used of an hydrogel with high water content and the design of a new implant architecture. The hydrogel is obtained by radical polymerization of difunctional macromonomers of poly(ethylene glycol) with molar masses around 8000 g.mol‐1, allowing mechanical properties and permeability to nutriments compatible with the application. The realization, the sterilization and the characterization of prototypes showed the proof of the concept of a diffractive bifocal intracorneal implant

La lésion de nerfs périphériques peut engendrer des déficits moteurs et/ou sensoriels permanents. En dépit des progrès techniques réalisés au cours de ces 25 dernières années, une récupération complète suite à ces lésions n’est pas encore possible aujourd'hui. L’autogreffe nerveuse, toujours considérée comme le standard clinique, est la seule technique capable d’offrir les meilleurs résultats en termes de récupération fonctionnelle. Cependant, la survenue de complications post-opératoires lors d’autogreffes d’un nerf et la quantité limitée de nerfs disponibles conduisent à mettre au point d’autres stratégies alternatives. Dans ce contexte, la mise au point de biomatériaux pour substituts nerveux devient une nécessité clinique. Malgré les efforts de la recherche, ces prothèses ne permettent toujours pas une régénération du nerf à la hauteur de l’autogreffe. Le biomatériau utilisé doit notamment présenter des propriétés physiques et chimiques proches de celui du nerf natif. La soie, aux propriétés mécaniques uniques, représente une bonne alternative pour mettre au point ce type de prothèses. En effet, la protéine de soie déjà utilisée dans le domaine biomédical est biocompatible. Les modifications chimiques de cette protéine améliore et favorise l’adhérence et la croissance cellulaires par l’incorporation de facteurs de croissance ou d’autres molécules d'intérêt. Ce travail de thèse propose de développer un nouveau type de biomatériau à base de soie fonctionnalisée par deux facteurs de croissance : le Nerve Growth Factor (NGF) et le Ciliary NeuroTrophic Factor (CNTF). Étant donné l’architecture complexe qui compose la structure nerveuse, une matrice supportant la repousse des tissus de façon orientée semble primordiale. Nous démontrons, dans un premier temps, le pouvoir de ces nanofibres alignées (produites par electrospinning) à orienter la régénération tissulaire de différents organes par culture d’explants. Les nanofibres de soie alignées, biocompatibles sont bio-activées par ajout de NGF spécifique de la régénération nerveuse. Cette matrice créée présente un gradient de concentration en NGF qui permet d’orienter la repousse axonale en stimulant la croissance axonale dans une seule direction. Afin d’optimiser la croissance de deux populations cellulaires, nous avons incorporé du CNTF pour produire des nanofibres bifonctionnalisées. Ces nanofibres bifonctionnalisées ont conduit à une longueur des neurites 3 fois plus grande à leurs contacts, stimulant la croissance des cellules gliales. Ainsi, nous avons produit des conduits nerveux à base de soie biofonctionnalisée pour implantation chez le rat. Les analyses physico-chimiques et les propriétés mécaniques démontrent le caractère biomimétique de nos tubes de guidage. Les premières études de la locomotion et l’observation de coupes du nerf sciatique de rat, suite à l’implantation de nos conduits donnent des résultats très prometteurs. L’ensemble de ces travaux démontre l’efficacité de nos guides nerveux à base de soie et les présente comme une alternative prometteuse à l’autogreffe nerveuse pratiquée en clinique. / Peripheral nerve injury causes sensory and/or motor functions deficits. Despite technological advances over the past 25 years, a complete recovery from these injuries remains unsatisfactory today. The autograft still considered the "gold standard" in clinical practice. This is the only technique able to offer complete functional recovery. However, the occurrence of postoperative complications in autologous nerve and the limited amount of available nerves lead to develop alternatives strategy.In this context, development of nerve graft substitutes becomes by far a clinical necessity. Despite research efforts, these artificial prostheses design based on biomaterial doesn’t allow nerve regeneration as found in autograft nerve procedures. The biomaterial used must have the physical and chemical properties similar to that of the native nerve. Silk, well known for its unique mechanical properties, proposes a good alternative to develop these prostheses. Indeed, the silk protein is commonly used in the biomedical field and regenerative medicine. This protein biocompatibility may be improved through chemical modifications to promote adhesion and cell growth by the incorporation of growth factors or other molecules of interest. Therefore, this thesis proposes to develop a new type of functionalized silk biomaterial based on two growth factors : Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF). Given the complex architecture that consists of nerve structure, a matrix which is able to support and manage the outgrowth of tissue becomes essential. We demonstrate the power of these aligned nanofibers (produced by electrospinning) to guide and manage tissue regeneration from different organ explants culture. Aligned silk nanofibers, were biocompatible and bio-activated by adding NGF involved for nerve regeneration. This matrix has been created with a concentration gradient of NGF to guide neuritis outgrowth in only one direction. The presence of this gradient demonstrated a better axonal growth in one direction versus the uniform concentration conditions. Nerve cells consist essentially of two cell populations which are neurons and Schwann cells. To optimize the culture and growth of these two populations, in addition to NGF, we incorporated CNTF to produce bifunctionalized nanofibers. These biofunctionalised nanofibers led to a length 3 times larger on contact with neurites. The glial cells growth, alignment and migration were stimulated by CNTF. Thus, we produced bi-functionalized nerve guidance conduits for rat implantation. The physico-chemical analyzes demonstrate the biomimetic of our guide tubes. Early studies of locomotion and observing histological sections of rat sciatic nerve, following the implementation of our conduits gave very promising results.These studies demonstrate the relevance of our nervous guides’ silk-based developed as an effective alternative to nerve autograft performed in the clinic.

Background: In addition to conventional chemotherapeutics, nucleic acid-based therapeutics like antisense oligodeoxynucleotides (AS-ODN) represent a novel approach for the treatment of bladder cancer (BCa). An efcient delivery of AS-ODN to the urothelium and then into cancer cells might be achieved by the local application of multiwalled carbon nanotubes (MWCNT). In the present study, pristine MWCNT and MWCNT functionalized with hydrophilic moieties were synthesized and then investigated regarding their physicochemical characteristics, dispersibility, biocompatibility, cellular uptake and mucoadhesive properties. Finally, their binding capacity for AS-ODN via hybridization to carrier strand oligodeoxynucleotides (CS-ODN), which were either non-covalently adsorbed or covalently bound to the diferent MWCNT types, was evaluated. Results: Pristine MWCNT were successfully functionalized with hydrophilic moieties (MWCNT-OH, -COOH, -NH2, -SH), which led to an improved dispersibility and an enhanced dispersion stability. A viability assay revealed that MWCNTOH, MWCNT-NH2 and MWCNT-SH were most biocompatible. All MWCNT were internalized by BCa cells, whereupon the highest uptake was observed for MWCNT-OH with 40% of the cells showing an engulfment. Furthermore, all types of MWCNT could adhere to the urothelium of explanted mouse bladders, but the amount of the covered urothelial area was with 2–7% rather low. As indicated by fuorescence measurements, it was possible to attach CS-ODN by adsorption and covalent binding to functionalized MWCNT. Adsorption of CS-ODN to pristine MWCNT, MWCNT-COOH and MWCNT-NH2 as well as covalent coupling to MWCNT-NH2 and MWCNT-SH resulted in the best binding capacity and stability. Subsequently, therapeutic AS-ODN could be hybridized to and reversibly released from the CS-ODN coupled via both strategies to the functionalized MWCNT. The release of AS-ODN at experimental conditions (80 °C, bufer) was most efective from CS-ODN adsorbed to MWCNT-OH and MWCNT-NH2 as well as from CS-ODN covalently attached to MWCNT-COOH, MWCNT-NH2 and MWCNT-SH. Furthermore, we could exemplarily demonstrate that AS-ODN could be released following hybridization to CS-ODN adsorbed to MWCNT-OH at physiological settings (37 °C, urine). Conclusions: In conclusion, functionalized MWCNT might be used as nanotransporters in antisense therapy for the local treatment of BCa.