Les champs électriques pulsés (CEP) offrent un réel intérêt dans le cadre de la traite des microalgues. En effet, ils permettent l'extraction sélective des composés hydrosolubles ou l'utilisation de solvants biocompatibles pour récolter les molécules hydrophobes. La viabilité des microalgues peut ainsi être conservée. L'objectif de ce travail de thèse est de définir des conditions de traitement permettant à la fois l'extraction des composés d'intérêt et le maintien de la viabilité des microalgues. Le modèle d'étude est la microalgue verte Haematococcus pluvialis. Au stade végétatif, celle-ci contient près d'un tiers de son poids de matière sèche en protéines et en conditions stressantes, elle accumule de l'astaxanthine, un caroténoïde à haute valeur ajoutée.L'application de CEP de 1 kV.cm-1 permet de collecter 50 % des protéines extractibles par broyage avec des microbilles. La mesure de cinq paramètres biologiques suite à ce traitement a mis en évidence que les cellules retrouvaient un état physiologique comparable à celui de microalgues non traitées au bout de 72 h. Cette condition de traitement constitue donc un bon compromis entre l'extraction des protéines et la survie des microalgues, ce qui renforce la faisabilité d'une traite de microalgues par CEP.Dans le cadre de l'extraction de l'astaxanthine, la paroi très résistante des kystes constitue le principal verrou à lever. Une optimisation des conditions de traitement (en particulier de la force des impulsions) et du stade cellulaire traité représentent les deux principales perspectives à étudier pour que l'utilisation des CEP dans le cadre de l'extraction de l'astaxanthine soit pertinente. / Pulsed electric fields (PEF) offer a real interest for microalgae milking. Indeed, they allow the selective extraction of water-soluble compounds or the use of biocompatible solvents to harvest the hydrophobic molecules. The viability of microalgae can thus be maintained. The aim of this PhD thesis work is to define the treatment conditions allowing both the extraction of compounds of interest and the maintenance of the microalgae viability. The biological model is the green microalga Haematococcus pluvialis. At the vegetative stage, it contains nearly one third of its dry matter weight in proteins and under stressful conditions, it accumulates astaxanthin, a high added value carotenoid.The application of PEF of 1 kV.cm-1 allows to collect 50% of the proteins extractable by bead milling. The measurement of five biological parameters highlights that treated cells recover a physiological state comparable to that of untreated microalgae after 72h. This treatment condition constitutes therefore a good compromise between the protein extraction and the survival of the microalgae, which reinforces the feasibility of microalgae milking by PEF.Within the context of astaxanthin extraction, the high resistance of the cell wall of the cysts constitutes the main limitation. Optimization of the treatment conditions (particularly pulse strength) and the cellular stage to treat represent the two main perspectives to study for the use of PEF for astaxanthin extraction to be relevant.

Des systèmes injectables de formation in situ ont été utilisés dans les dernières années pour l'obtention de formulations de préparation facile et permettant la libération prolongée de principes actifs. Ces systèmes utilisant des solvants biocompatibles et des polymères biodégradables sont des liquides (solutions ou émulsions) qui une fois injectés dans l'organisme donnent lieu à des implants (ISI) ou à des microparticules (ISM) solides. La formation de ces systèmes est induite par la précipitation du polymère à partir des solutions polymériques qu'ils contiennent lors du contact avec les fluides corporaux aqueux. Dans ce travail, des ISI et des ISM, réalisés à partir des polymères de l'acide lactique et/ou glycolique (PLA et PLGA) et des différents solvants biocompatibles, pour la libération prolongée d?ivermectine (IVM), un principe actif antiparasitaire faiblement biodisponible par la voie orale, ont été développés. Les profils de libération du principe actif in vitro et in vivo à partir de ces systèmes, ont été comparés avec ceux obtenus à partir de microparticules réalisées par la méthode classique dite d'émulsion simple - évaporation de solvant ; il s'agit d'une technique aux multiples étapes, à coût élevé et dont l'utilisation de solvants toxiques la font difficilement industrialisable. La libération du principe actif à partir des microparticules obtenues par émulsion simple/évaporation du solvant a été influencée par la forte interaction du principe actif avec les polymères mais aussi par la porosité. Dans le cas des systèmes in situ, la vitesse de libération d'IVM a été conditionnée par la solubilité dans l'eau du solvant biocompatible sélectionné et par les interactions solvant/polymère. Pour les ISM, des paramètres tels que la nature de la phase externe, aqueuse (ISM-O/W) ou huileuse (ISM-O/O), la solubilité dans l'eau du solvant de la phase interne, l'affinité entre les phases et l'affinité de l'IVM pour chacune des phases, ont déterminé la vitesse de libération du principe actif. La bonne stabilité ainsi que les profils de libération plus prolongés et présentant une faible libération initiale du principe actif in vivo et in vitro, ont montré que les ISI et les ISM réalisés à partir de solvants biocompatibles de faible solubilité dans l'eau tels que la triacetine sont les plus indiqués pour l'encapsulation d'IVM par rapport à ceux plus solubles dans l'eau comme la N-methyl-2-pyrrolidone et la 2-pyrrolidone. Ces systèmes représentent donc une alternative intéressante par rapport aux formulations conventionnelles d'IVM / In situ forming injectable systems have been used in the past years to obtain sustained drug release formulations which are easy to prepare. These systems using biocompatible solvents and biodegradable polymers are liquids (solutions or emulsions) that upon injection on the body lead to solid implants (ISI) or microparticles (ISM). These systems are formed in contact with water body fluids by polymer precipitation from the polymeric solution. In this work, ISI and ISM made from lactide and/or glycolide polymers (PLA and PLGA) and different biocompatible solvents were performed to obtain sustained release of ivermectin (IVM), an antiparasitic drug with a low oral bioavailability. In vitro and in vivo drug release profiles from these systems were compared with those from microparticles obtained by the classical simple emulsion/solvent evaporation method, which is difficult to propose in industry because of its multiple steps, high cost and the solvent toxicity. Drug release from simple emulsion/solvent evaporation microparticles was affected by the strong polymer/drug interactions and porosity. Concerning to in situ forming systems, the rate of IVM release was dependent on solvent water solubility and solvent/polymer interactions. The nature of the external phase, water (ISM-O/W) or oil (ISM-O/O), the water solubility of the solvent in the internal phase, phase affinity and IVM/phase affinity determined drug release from ISM. The good stability, the in vitro and in vivo sustained release and the low burst effect of IVM, indicated that ISI and ISM formulated from low hydrosoluble biocompatible solvents such as triacetin are more appropriated to IVM formulation instead of those based on more hydrophilic solvent (N-methyl-2-pyrrolidone and 2-pyrrolidone). These systems are an interesting alternative to conventional IVM formulations

L'émergence d’un nouveau système nanoparticulé dans le domaine biomédical, les matériaux hybride poreux du type MOF (pour Metal Organic Framework), a récemment attiré beaucoup d'attention en raison de leur grande versatilité structurale et chimique. En particulaire, le trimésate de fer(III) mésoporeux (MIL-100; MIL pour Matériau de l'Institut Lavoisier) a démontré des capacités remarquables de stockage de médicaments avec leur libération contrôlée dans des conditions physiologiques, ainsi que des propriétés en imagerie très intéressantes.Néanmoins, avant toute bioapplication, il estnécessaire d’étudier leur toxicité et leur profile de biodistribution, lesquels sont fortement affectés par plusieurs facteurs (composition, dégradabilité, chimie de surface, etc.). Ainsi, l’objectif principal de ce travail de thèse porte sur l'évaluation de la biocompatibilité de MOFs nanométriques et leur passage de barrières physiologiquespar différentes voiesd'administration (en particulier, par voie intraveineuse, orale et cutanée) en fonction de leurs propriétés physico-chimiques. / The recent emergence of nanometric porous metal-organic frameworks (nanoMOFs) in the biomedical field has recently attracted a great deal of attention owing to their large porosity and versatile composition. Particularly attractive is the mesoporous iron(III) trimesate (MIL-100; MIL stands for Material of Institute Lavoisier), which has shown exceptional loading of challenging drugs, together with their controlled release under physiological conditions and interesting imaging properties. Nevertheless, prior to any bioapplication, it is crucial investigate its toxicity and biodistribution profile, which are strongly affected by multiple factors (e.g. composition, degradability, surface engineering, etc.). Thus, the aim of this PhD work focuses on the evaluation of the nanoMOF biocompatibility and their physiological barrier crossing from different administration routes (specifically intravenous, oral and cutaneous) as a function of their physicochemical properties.

Ce doctorat portait sur le développement de nanoparticules (NPs) photosensibles constituées d’un cœur photolysablepoly(acrylate d'o-nitrobenzyle) (polymère hydrophobe biocompatible - PANB) et d’une couronne basée sur un dérivé du dextrane (polysaccharide bactérien, hydrophile et biodégradable). Dans un premier temps, le PANB-N3 a été synthétisé par i) polymérisation radicalaire contrôlée (SET-LRP) de l’acrylate d’o-nitrobenzyle puis ii) modification chimique de l’extrémité de chaîne par une fonction azoture. En parallèle, le dextrane a été hydrophobisé par quelques chaînes grasses dotées d’un groupe alcyne (obtention du DexAlcyne-15). Ces polymères précurseurs peuvent alors réagir par chimie click CuAAC (Cycloaddition azide-alcyne catalysée par Cu(I)) pour engendrer divers glycopolymères greffés Dex-g-PANB. Dans un deuxième temps, les NPs ont été formulées par deux procédés puis caractérisées en termes de taille, recouvrement en dextrane (quantité par gramme de PANB, épaisseur de la couche surfacique) et stabilité colloïdale en milieu salin, en présence de tensioactif compétitif ou dans un milieu de culture (DMEM). Le procédé de nanoprécipitation a été appliqué aux Dex-g-PANB présentant de fortes fractions massiques en PANB (>40%) alors que le procédé d’émulsion-évaporation de solvant organique a été mis en œuvre en utilisant le DexAlcyne-15 comme tensioactif hydrosoluble et le PANB-N3 comme matériau hydrophobe. Grâce à leurs fonctionnalités complémentaires, une réaction CuAAC peut (ou non) avoir lieu à l’interface liquide/liquide pendant l’élaboration des NPs et conduire à l’obtention de NPs « non clickées » ou « clickées ». Enfin, le caractère photosensible des NPs a été validé par irradiation UV en observant une disparition progressive des NPs résultant de la photolyse des PANB. Afin d’utiliser ces NPs comme systèmes stimulables de délivrance de médicaments, un anticancéreux (Doxorubicine - Dox) a été encapsulé au sein des NPs, pendant leur élaboration. Cette encapsulation a été optimisée et les NPs chargées de DOX ont été caractérisées en termes de taille et d’efficacité d’encapsulation. La libération de la DOX hors des NPs a ensuite été suivie par simple diffusion, ou provoquéepar irradiation UV. Enfin, le potentiel biologique de ces NPs a été évalué vis-à-vis d’une lignée cellulaire tumorale humaine d'origine intestinale isolée d'un adénocarcinome colique (Caco-2). Après vérification de leur biocompatibilité et de la résistance des Caco-2 aux irradiations UV, nous avons pu montrer que les NPs chargées pouvaient libérer suffisamment de DOX en seulement 30 secondes d’irradiation (puissane: 54mW/cm2) pour éradiquer plus de 50% de ces cellules cancéreuses. / This work was focused on the development of light-sensitive nanoparticles (NPs) based on a photodegradable poly(o-nitrobenzyl acrylate) core(PNBA, hydrophobic and biocompatible polymer) and a dextran derivative shell (dextran is a biodegradable and hydrophilic bacterial polysaccharide). Firstly, PNBA-N3 was synthesized by i) Single-Electron Transfer Living Radical Polymerization (SET-LRP) of o-nitrobenzyl acrylate then ii) introduction of one azide end-function. In the same time, DexAlkyne-15 carrying several alkyne groups was produced by hydrophobization of dextran. Such DexAlkyne-15 and PNBA-N3 can react by CuAAC (Cu(I)-catalyzed azide-alkyne cycloaddition) click chemistry leading to Dex-g-PNBA glycopolymers with various macromolecular parameters. Secondly, NPs were produced by comparing two processes then characterized in terms of size, dextran amount, shell thickness and colloidal stability in NaCl or cell culture media, or in presence of one strong surfactant. On one hand, NPs were made by nanoprecipitation of Dex-g-PNBA exhibiting high PNBA weight fractions (>40 %). On the other hand, NPs were produced by emulsion-evaporation of the organic solvent using DexAlkyne-15 as water-soluble surfactant and PNBA-N3 as hydrophobic materials. In this case, in situ CuAAC occurred (or not) at the liquid/liquid interface during the NPs formulation, leading to “clicked” and “not-clicked” NPs. Finally, NPs disruption was studied by UV irradiation according the PNBA chains photolysis. To use such NPs as smart drug delivery systems, Doxorubicin (DOX - an anticancer agent), was loaded inside the NPs during their elaboration. The experimental conditions were optimized to enhance the DOX encapsulation. The kinetics release of encapsulated DOX were studied by diffusion or under UV irradiation. Finally, the biological potential of these NPs was estimated towards Caco-2 (continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells). After checking the NPs biocompatibility and theCaco-2 strength under UV irradiation, we proved that such loaded NPs can release enough DOX under 30 second irradiation (power: 54mW/cm2) to decrease the Caco-2 viability about more than 50%.

O desenvolvimento atual de materiais ósseo-substitutos com potencialidade de promover o fechamento completo de um defeito ósseo crítico tem levantado questões quanto à sua atuação biológica. Uma opção de material ósseo utilizado são os enxertos ósseos de origem animal, pois possuem propriedades físicoquímicas similares ao osso humano. O osso bovino misto que preserva a estrutura colagênica e o mineral ósseo tem sido proposto e utilizado como material ósseosubstituto. O objetivo desse estudo foi avaliar a biocompatibilidade e o potencial osteogênico de um enxerto bovino ósseo subistituto, OrthoGen® (Baumer S.A.), na forma de partículas (OGp) e blocos (OGb). Para a avaliação da biocompatibilidade, 100 mg de OrthoGen® nas formas de partícula e bloco, foram implantados no subcutâneo de ratos (n=25) e o tecido reacional foi avaliado aos 7, 14, 21, 30 e 60 dias (n=5 animais/período) após a implantação. Para a análise do potencial osteogênico foram implantados 100 mg de Orthogen® nas formas de partícula e bloco em defeito critico na calvária de ratos (n=30), e a formação óssea foi mensurada aos 1, 3 e 6 meses (n=10/período) após a implantação. A análise radiográfica e histomorfométrica revelaram que no tecido subcutâneo o OGb foi melhor aceito pelo organismo quando comparado ao OGp com uma frequência menor de células gigante multinucleadas entre os períodos avaliados (OGb 0,23% vs OGp 2,19%) e consequentemente uma média de reabsorção também menor (OGb 13% vs OGp 38%). Em ambos os implantes não foi encontrado focos de infiltrado inflamatório composto por leucócitos polimorfonucleares, linfócitos e plasmócitos. Na calvária o OGb mostrou níveis de reabsorção inferiores e uma maior taxa de formação óssea quando comparado ao OGp após 6 meses (OGb 70 mm³ vs OGp 17 mm³). Baseado no modelo experimental utilizado neste estudo, concluímos que ambas as formas do Orthogen® são biocompatíveis em tecido subcutâneo, no entanto, sua forma em bloco promove uma maior formação óssea, possuindo uma capacidade osteogênica superior à forma em partícula, no modelo experimental avaliado. / The current development of bone graft materials with the potential to promote the complete closure of a critical size bone defect has raised questions as to its biological activity. An option of bone material used, are animal bone grafts since the human bone have similar physicochemical properties. Among the materials is the integral bone substitute of bovine origin, which preserves the organic and inorganic compound of the bone tissue, has been proposed and used as bone graft. The aim of this study was to evaluate the biocompatibility and osteogenic potential of a new integral bone substitute OrthogenTM (Baumer S.A.) in the form of particles (OGp) and block (OGb). For biocompatibility evaluation, 100mg OrthogenTM was implanted into dorsal subcutaneous pocket of rat (n= 25) and the reactional tissue was analyzed at 7, 14, 21, 30 and 60 days (n=5animals/period) after implantation. For osteogenic potential evaluation, 100mg OrthogenTM was implanted into critical-size defect in parietal bones of rat (n=30) and the bone formation, biomaterial reabsorption, connective tissue formation and osteoclast activity was evaluated at 1, 3 and 6 months (n=10/period) after implantation. Radiographic and histomorphometrical analysis showed that, in the subcutaneous tissue the OGb was more accepted by the host compared to OGp, with lower density of the multinucleated giant cells (OGb 0.23% vs. OGp 2.19%) and consequently a lower rate of matrix resorption (OGb 13%, vs. OGp 38%). In both implants was not found focus of inflammatory infiltrated composed by polymorphonuclear leucocytes, lymphocytes and plasmocytes. In rat calvaria the OGb showed lower rate of reabsorption and more volume of bone formation compared to OGp after 6 months (OGb 70 mm3 vs OGp 17 mm3). Based on experimental models used in this study we concluded that both forms of the OrthogenTM was biocompatible in subcutaneous tissue, however, its form of porous block promoted greater bone formation and has a higher osteogenic capacity than the particle shape, in the evaluated experimental model.

One strategy for bone regenerative engineering is to use matrices associated to osteogenic and angiogenic molecules to increase bone formation. The aim of the present study was to evaluate the efficacy of treatment of extensive cranial bone defects with the F1 protein obtained from latex adsorbed at different concentrations (0,01%, 0,025%, 0,05% e 1%) to two different bonesubstitutes biomaterials, deproteinized bovine bone (DBB) and biphasic calcium phosphate ceramics (pBCP) using a preclinical model in rats. Defects of 8-mm diameter were created in parietal bones of 72 rats filled with the pure biomaterial or carried with the different concentrations of F1 protein, in the microtomographic images a visual analysis of the microtomographic reconstructions of the skull through transverse, coronal and sagittal sections. Subsequently, the segmentation of the defect in the reconstructions will be done through an image processing algorithm to quantify the parameters. Analyzing, in the BP-G group, the total volume of bone (TV), in the CSBD-CG group, the total volume of new bone (TV-NB), and in the treated groups, the total volume of the grafted region (TV/GR), the total volume of new bone (TV-NB) and biomaterials (TV/DBB and TV/pBCP). In tissue sections stained with Hematoxylin and Eosin a descriptive histological analysis was performed to verify the tissue response to treatment with F1 protein and its association with osteoconductive biomaterial and correlate it with the histomorphometric determination to obtain percent values and volume of neoformed bone tissue, biomaterial, bone marrow and soft tissue. In the characterization of DBB and pBCP biomaterials, it was performed through the combined analytical methodology by SEM and SDD-EDS, to analyze external morphology and elemental chemical composition. All the results were compared between the groups by the ANOVA variance analysis and the Tukey tests at the 5% level of significance (Statistica v.5.1, StatSoft). After 12 weeks, defects treated with biomaterials without F1 presented greater bone formation in relation to the control group. The association of 0.025% and 0.05% of F1 plus DBB showed higher bone formation (32.6% and 25.1%, respectively) when compared to pBCP, being 19.3% and 15.1%, respectively. We conclude that the stimulation of angiogenesis and osteogenesis depends on its concentration of F1e and the physicochemical properties of the carrier material. / Uma estratégia da engenharia regenerativa óssea é usar matrizes associadas a moléculas osteogênicas e angiogênicas para aumentar a formação óssea. O objetivo do presente estudo foi avaliar a eficácia do tratamento de defeitos ósseos cranianos extensos com a proteína F1 obtida do látex adsorvido em diferentes concentrações (0,01%, 0,025%, 0,05% e 1%) a dois diferentes biomateriais ósseo-substitutos, osso bovino desproteinizado (DBB) cerâmica de fosfato de cálcio bifásica (pBCP) utilizando um modelo pré-clínico em ratos. Defeitos de 8 mm de diâmetro foram criados nos ossos parietais de 72 ratos preenchidos com o biomaterial puro ou carregados com as diferentes concentrações da proteína F1, nas imagens microtomográficas uma análise visual das reconstruções microtomográficas do crânio através de cortes transversais, coronais e sagitais. Subsequentemente, foi feito segmentação do defeito nas reconstruções através de algoritmo de processamento de imagem para quantificação dos parâmetros. Analisando, no grupo BP-G, o volume total de osso (TV), no grupo CSBD-CG o volume total de osso novo (TV-NB), e nos grupos tratados, o volume total da região enxertada (TV-GR), volume total de osso novo (TV-NB) e biomaterial (TV-DBB e TV-pBCP). Nos cortes teciduais corados pela Hematoxilina e Eosina foi realizado uma análise histológica descritiva para verificar a resposta tecidual frente ao tratamento com a proteína F1 e a sua associação com biomaterial osteocondutor e correlaciona-la com a determinação histomorfométrica para a obtenção dos valores percentuais e de volume de tecido ósseo neoformado, biomaterial, medula óssea e tecido conjuntivo. Na caracterização dos biomaterias DBB e pBCP, foi realizado através da metodologia analítica combinada por SEM e SDD-EDS, para analisar morfologia externa e composição química elementar. Todos os resultados foram comparados entre os grupos pela análise de variância ANOVA e o tests de Tukey ao nível de significância de 5% (Statistica v.5.1, StatSoft). Após 12 semanas, defeitos tratados com biomateriais sem F1 apresentaram maior formação óssea em relação ao grupo controle. A associação de 0,025% e 0,05% de F1 mais DBB mostraram maior formação óssea (32,6% e 25,1%, respectivamente) quando comparados com pBCP, sendo 19,3% e 15,1%, respectivamente.Nós concluímos que, a estimulação da angiogênese e osteogênese depende de sua concentração de F1e das propriedades físico-químicas do material carreador.

Este estudo avaliou o processo de reparo ósseo em cavidades cirúrgicas de tamanho não crítico, quando preenchidas por diferentes biomateriais. Um defeito ósseo de 5 mm de diâmetro por 8mm profundidade foi criado em cada metáfise tibial de 27 coelhos machos. Foram estabelecidos 4 grupos com 12 cavidades por material. As cavidades foram preenchidas com matriz óssea bovina inorgânica, GenOx Inorg® (grupo 1), matriz óssea bovina orgânica, GenOx Org® (grupo 2), matriz óssea bovina composta, GenMix® (grupo 3) e sulfato de cálcio di-hidratado (Grupo 4). Adicionalmente, seis cavidades preenchidas com coágulo sanguíneo foram utilizadas como controle (grupo 5). Os animais foram mortos após 30, 60 e 90 dias e as amostras coletadas foram submetidas ao processamento histotécnico para obter cortes de 5 m de espessura, corados com hematoxilina e eosina. Foram realizadas análises histomorfológica descritiva e histomofométrica, que consistiu na mensuração da área de tecido ósseo neoformado. Os dados obtidos foram submetidos ao teste estatístico de Kruskal-Wallis e Dunn. Aos 30 e 60 dias a área de tecido ósseo neoformado ocorreu de forma semelhante entre todos os grupos (P > 0,05). No período de 90 dias houve diferença significativa entre os grupos 2 e 4, bem como entre os grupos 4 e 5. Ao realizar a análise estatística de cada grupo em função dos 3 períodos experimentais, constatou-se que nos grupos 1, 2 e 3 a área de tecido ósseo neoformado aos 30 dias foi menor (P < 0,05) em relação a 60 e 90 dias. No grupo 4, ocorreu diferença significativa entre os períodos de 30 e 90 dias, já no grupo 5 houve diferença significante ao comparar 30 e 60 dias. Com base na microscopia observamos que os biomateriais estudados não inibiram o processo de reparo; o GenOx Inorg e a matriz inorgânica presente no GenMix exibiram características morfológicas de propriedade osteocondutora, mais intensa no primeiro grupo. O GenOx Org e o sulfato de cálcio di-hidratado apresentaram-se completamente reabsorvidos aos 30 dias. Foi possível concluir que os valores neoformativos proporcionados pelo GenMix não foram significativamente superiores, àqueles obtidos pelo GenOx Inorg, GenOx Org e sulfato de cálcio di-hidratado. Os diferentes biomateriais utilizados não apresentaram resultados superiores ao coágulo sanguíneo, exceto o sulfato de cálcio di-hidratado aos 90 dias. / This study evaluated the bone healing process in non-critical surgical cavity size, when grafted with different biomaterials. A 5mm wide and 8mm long defect were created in each tibial metaphysis of 27 male rabbits. Four groups of 12 cavities per material were established. The cavities were filled with deproteinized bovine bone, GenOx Inorg ® (group 1), demineralized bovine bone, GenOx Org® (group 2), composite bovine bone, GenMix® (group 3) and di-hydrate calcium sulfate (group 4). Additionally 6 cavities were filled with blood clot for control purposes (group 5). At 30, 60 and 90 days after surgery the animals were killed and the grafted areas were submitted to histologically process to obtain a 5 m thickness sections and stained with hematoxylin and eosin. The specimens were proceeded to descriptive and quantitative microscopic analysis, by measuring the area of new formed bone. The results were statistically analyzed by the Kruskal-Wallis and Dunn tests. At 30 and 60 days the area of new bone formation was similar among all groups (P > 0,05). In 90 days period there was a significant difference between groups 2 and 4, as well as groups 4 and 5. Performing the statistical analysis of each group in relation to the 3 experimental periods, it was found that in groups 1, 2 and 3 the area of new bone formed at 30 days was lower (P < 0,05) than 60 and 90 days. There was a significant difference in group 4 comparing the periods 30 and 90 days and in group 5 between 30 and 60 days. Based on microscopy findings we observed that the biomaterials studied did not inhibit the repair process; the GenOx Inorg and inorganic matrix present in GenMix exhibited morphological characteristics of osteoconductive property, more intense in the first one. The GenOx Org and di-hydrate calcium sulfate were completely reabsorbed within 30 days. It was concluded that the values of new bone formed area provided by GenMix were not significantly higher than obtained by GenOx Inorg, GenOx Org and di-hydrate calcium sulfate. The different biomaterials used did not show better results than blood clot, except di-hydrate calcium sulfate at 90 days.

Este trabalho está baseado no desenvolvimento do compósito poli(metacrilato de metila) (PMMA) microfibra de biovidro 45S5® (BV) com porosidade para aplicação direcionada a implantes ósseos. O PMMA é um material do tipo bioinerte amplamente utilizado na fabricação de implantes. O biovidro 45S5® é um biomaterial bioativo classe-A, que significa ser capaz de integração com tecidos moles e ósseos. O presente trabalho objetiva a conjugação das propriedades destes dois materiais em um conceito que introduz microfibra de biovidro 45S5® e porosidade em uma matriz de PMMA na busca de um material com elevada osteointegração e que permita a manufatura de implantes com adequada resistência a carregamentos mecânicos. Foram manufaturados e avaliados fisicamente e in vivo corpos de prova de PMMA com introdução de 20% de microfibra de biovidro (densos e porosos a 80%), corpos de PMMA com introdução de hidroxiapatita (densos e porosos a 80%) e de PMMA (densos e porosos a 80%) para controle. Foram realizados testes mecânicos de compressão e de flexão a três pontos para medidas de resistência e rigidez. Também foram realizadas medidas da microdureza Vickers e da densidade das amostras. Os resultados médios obtidos da tensão de escoamento e módulo elástico para os corpos de prova com introdução de 20% de microfibra de biovidro nos ensaios de compressão foram de 44 MPa e 1403 MPa respectivamente para as amostras densas e de 8 MPa e 13 MPa para as amostras porosas e nos ensaios de flexão foram de 48 MPa e 8129 MPa para os corpos densos e de 5 MPa e 97 MPa para os corpos porosos. A modelagem matemática a partir dos resultados permite a determinação antecipada da formulação para o atendimento específico de cada proposta de implante. Os implantes ensaiados in vivo com adição de microfibra de Biovidro 45S5® foram os que apresentaram os melhores resultados em integração óssea e controle da inflamação local. / This work is based on the development of poly(methyl methacrylate) (PMMA) - bioglass 45S5® microfibers composite with porosity for bone implants application. PMMA is a bioinert material widely used in the manufacture of biological implants. The 45S5® bioglass is a bioactive class-A biomaterial which indicates ability to integrate with bone and soft tissues. The present study aims to combine the properties of these two materials on a concept that introduces bioglass 45S5® microfibers and porosity in a matrix of PMMA in order to find a material with high bone integration and capable of manufacture implants with adequate resistance to mechanical loads. Samples of PMMA with the introduction of 20% of bioglass microfiber (dense and 80% of porous), PMMA with introduction of hydroxyapatite (dense and 80% of porous), and PMMA (dense and 80% of porous) for control were fabricated and evaluated both physically and in vivo. Mechanical tests such as compression and three points bending have been performed for measurements of strain and stiffness. Measurements of Vickers microhardness and density of the samples also were performed. The average results of yield stress and elastic modulus for the samples with the introduction of 20% microfiber bioglass in compression tests were 44 MPa and 1403 MPa respectively for dense samples and 8 MPa and 13 MPa for porous samples and in bending tests were 48 MPa and 8129 MPa for dense bodies and 5 MPa and 97 MPa for the porous bodies. The mathematical modeling based on the results allows the early determination of the formulation to meet the specific needs of each implant proposal. The implants tested in vivo with addition of 45S5® bioglass microfiber presented the best results in bone integration and control of local inflammation when compared with all the other groups with the scaffold presence.

Na clínica ortopédica e traumatológica existem diversos desafios clínicos envolvendo as extensas perdas de tecido ósseo, relacionados primordialmente com causas traumáticas, tumorais, congênitas e infecciosas. No tratamento, podem ser utilizados os enxertos autólogos, homólogos, xenólogos e os enxertos periostais. Os substitutos sintéticos, conhecidos como biomateriais, também são uma boa alternativa. O objetivo deste trabalho foi avaliar o processo de neoformação óssea durante o reparo de defeitos ósseos, provocados experimentalmente no crânio de animais enxertados com blenda polimérica, constituída de colágeno de tendão bovino e quitosana associada à hidroxiapatita. Foram utilizados 30 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para a criação de defeito ósseo no osso parietal esquerdo da calota craniana para o preenchimento com os biomateriais pesquisados. Foram divididos em 3 grupos com 10 animais cada, sendo um grupo controle sem a implantação de biomaterial, outro com implantação de blenda de colágeno e quitosana e o último com a utilização desta blenda em associação com hidroxiapatita. Cada grupo foi subdividido em dois subgrupos com 5 animais de acordo com o tempo do sacrifício pós-operatório, sendo com 3 semanas nos grupos de G1 a G3 e com 8 semanas de G4 a G6. Após o sacrifício, as calotas cranianas foram retiradas para foto documentação macroscópica e exames radiográficos. Em seguida, as amostras foram submetidas aos procedimentos histotécnicos de confecção das lâminas para avaliação histológica da neoformação óssea no local cirúrgico. A pesquisa foi aprovada pelo comitê de ética da Faculdade de Medicina de Jundiaí, protocolo nº 301/12. As análises macroscópicas e radiográficas demonstraram a biocompatibilidade das blendas utilizadas. Histologicamente, houve discreta neoformação óssea em continuidade com as margens da lesão óssea, porém predominou a presença de tecido conectivo na área cirúrgica. As blendas poliméricas apresentaram biocompatibilidade com o tecido receptor, porém com baixa capacidade osteogênica devido à permanência do defeito ósseo. Não houve a sua osteointegração em virtude da presença de tecido conectivo em grande quantidade e adjacente ao implante. / In orthopedics and traumatology there are several clinical challenges involving extensive bone loss, primarily related to trauma, tumors, congenital and infectious diseases. These conditions can be usually treated by autologous, periosteal, homologous and xenologous grafts. A good alternative is to use synthetic biomaterials as substitute. The aim of this study was to evaluate the process of bone formation during repair of bone defects experimentally induced in animals skull, grafted by a polymer blend consisting of bovine tendon collagen and chitosan associated with hydroxyapatite. A total of 30 rats (Rattus norvegicus, Wistar) male, weighing approximately 330 grams and 4 months old was used. The animals underwent the surgical procedure for the creation of defect in the left parietal bone of the skull. They were divided into 3 groups of 10 animals each: a control group without biomaterials implantation, another with a blend of collagen and chitosan and the latest with the use of this blend in association with hydroxyapatite. Each group was subdivided into two subgroups of 5 animals according to the sacrifice schedule after surgery. The groups were named G1, G2 and G3 when the sacrifice occurred after 3 weeks postoperative and G4, G5 and G6 after eight weeks. After sacrifice, the calvarias were removed for macroscopic photo documentation and radiographic examinations. Then the samples were subjected to histotechnical procedures for histological evaluation of new bone formation at the surgical site. The study was approved by the ethics committee of the Faculdade de Medicina de Jundiaí (protocol # 301/12). The macroscopic and radiographic analysis demonstrated the biocompatibility of the blends. Histologically, there was a slight bone formation in continuity with the edges of the bone lesion, but the predominant presence of connective tissue in the surgical area. The polymer blends showed biocompatibility with the host tissue but low osteogenic capacity due to the bone defect. There was no osseointegration due to the presence of connective tissue in large quantity adjacent to the implant.

Este trabalho está baseado no desenvolvimento do compósito poli(metacrilato de metila) (PMMA) microfibra de biovidro 45S5® (BV) com porosidade para aplicação direcionada a implantes ósseos. O PMMA é um material do tipo bioinerte amplamente utilizado na fabricação de implantes. O biovidro 45S5® é um biomaterial bioativo classe-A, que significa ser capaz de integração com tecidos moles e ósseos. O presente trabalho objetiva a conjugação das propriedades destes dois materiais em um conceito que introduz microfibra de biovidro 45S5® e porosidade em uma matriz de PMMA na busca de um material com elevada osteointegração e que permita a manufatura de implantes com adequada resistência a carregamentos mecânicos. Foram manufaturados e avaliados fisicamente e in vivo corpos de prova de PMMA com introdução de 20% de microfibra de biovidro (densos e porosos a 80%), corpos de PMMA com introdução de hidroxiapatita (densos e porosos a 80%) e de PMMA (densos e porosos a 80%) para controle. Foram realizados testes mecânicos de compressão e de flexão a três pontos para medidas de resistência e rigidez. Também foram realizadas medidas da microdureza Vickers e da densidade das amostras. Os resultados médios obtidos da tensão de escoamento e módulo elástico para os corpos de prova com introdução de 20% de microfibra de biovidro nos ensaios de compressão foram de 44 MPa e 1403 MPa respectivamente para as amostras densas e de 8 MPa e 13 MPa para as amostras porosas e nos ensaios de flexão foram de 48 MPa e 8129 MPa para os corpos densos e de 5 MPa e 97 MPa para os corpos porosos. A modelagem matemática a partir dos resultados permite a determinação antecipada da formulação para o atendimento específico de cada proposta de implante. Os implantes ensaiados in vivo com adição de microfibra de Biovidro 45S5® foram os que apresentaram os melhores resultados em integração óssea e controle da inflamação local. / This work is based on the development of poly(methyl methacrylate) (PMMA) - bioglass 45S5® microfibers composite with porosity for bone implants application. PMMA is a bioinert material widely used in the manufacture of biological implants. The 45S5® bioglass is a bioactive class-A biomaterial which indicates ability to integrate with bone and soft tissues. The present study aims to combine the properties of these two materials on a concept that introduces bioglass 45S5® microfibers and porosity in a matrix of PMMA in order to find a material with high bone integration and capable of manufacture implants with adequate resistance to mechanical loads. Samples of PMMA with the introduction of 20% of bioglass microfiber (dense and 80% of porous), PMMA with introduction of hydroxyapatite (dense and 80% of porous), and PMMA (dense and 80% of porous) for control were fabricated and evaluated both physically and in vivo. Mechanical tests such as compression and three points bending have been performed for measurements of strain and stiffness. Measurements of Vickers microhardness and density of the samples also were performed. The average results of yield stress and elastic modulus for the samples with the introduction of 20% microfiber bioglass in compression tests were 44 MPa and 1403 MPa respectively for dense samples and 8 MPa and 13 MPa for porous samples and in bending tests were 48 MPa and 8129 MPa for dense bodies and 5 MPa and 97 MPa for the porous bodies. The mathematical modeling based on the results allows the early determination of the formulation to meet the specific needs of each implant proposal. The implants tested in vivo with addition of 45S5® bioglass microfiber presented the best results in bone integration and control of local inflammation when compared with all the other groups with the scaffold presence.