A contaminação dos implantes e seus componentes protéticos por patógenos específicos, ainda é um problema a ser resolvido na Implantodontia. Nanoestruturas associadas à prata, aplicadas em superfície de titânio, têm sido investigadas e vêm demonstrando inibir significativamente a adesão, crescimento e colonização de uma vasta gama de microrganismos patogênicos, inclusive os resistentes a antibióticos. O objetivo deste experimento foi verificar a presença de toxicidade de nanoestruturas associadas à prata, aplicadas à superfície de titânio, frente a cultura de osteoblastos, visando desenvolver um material biocompatível, promissor na corrida pela prevenção da colonização bacteriana dos implantes. Discos de titânio comercialmente puro foram revestidos com três tipos diferentes filmes nanoestruturados. As amostras foram expostas a culturas de linhagens imortalizadas de osteoblastos (MC3T3-E1) e submetidas a ensaios de toxicidade direta (MTT), após 24h, 48h e 72h de incubação, a fim de determinar a taxa de proliferação e redução da viabilidade celular. Além disso, para verificar adesão e morfologia celular, foi realizada Microscopia Eletrônica de Varredura (MEV) após 24h de incubação. No ensaio de toxicidade celular MTT, após 48h de incubação, as superfícies nanoestruturadas, nomeadas TiAg, TiAA e SiO2, comportaram-se de maneira muito similar ao grupo controle de titânio puro (Ti) (p<0,05). O grupo SiO2 registrou maior absorbância em 24h, com diferença relevante em relação a todos os outros grupos (testes e controles). Em MEV foram verificadas uma morfologia celular compatível com viabilidade e presença de adesão dos osteoblastos em todas as superfícies testadas, similar aos grupos controles. Dessa forma, verificou-se que os três filmes nanoestruturados demonstraram biocompatibilidade a osteoblastos. / Contamination of implants and their prosthetic components by specific pathogens is still a problem to be solved in Implantology. Nanostructures associated to silver applied on titanium surface have been investigating, and have showing to inhibit significantly the adhesion, and growth of a broad range of pathogenic microorganisms, including antibiotic-resistant ones. The aim of this experiment was to verify the presence of toxicity in nanostructures associated to silver applied to titanium surface against osteoblast culture, in order to develop a biocompatible promising material in the race for prevention on implants bacterial colonization. Each commercially pure titanium disk received one of three different nanostructured films: SiO2, TiAg, and TiAA. Samples have exposed to cultures of immortalized lines osteoblasts (MC3T3-E1), and submitted to direct toxicity assay (MTT) after 24h, 48h and 72h of incubation to determine the proliferation rate and cell viability reduction. Furthermore, to verify adhesion and cell morphology, Scanning Electron Microscopy (SEM) has performed after 24h incubation. The 48h MTT cell toxicity assay showed similarity in results among test surfaces and pure titanium control group (Ti) (p <0.05). The SiO2 group registered higher absorbance in 24h, with significant difference regarding to all other groups (tests and controls). SEM exhibited cell morphology indicative of viability, and presence of osteoblast adhesion on all test surfaces, similar to control groups. Thus, it found of that three nanostructured films demonstrated osteoblasts compatibility.

Objetivo: Avaliar as modificações estruturais do pericárdio bovino após hidrólise seletiva de grupos carboxiamidas em carboxílicos e resposta biológica em defeitos ósseos reparados com as matrizes de colágeno aniônico. Material e métodos: Amostras das matrizes modificadas foram comparadas com amostras de pericárdio bovino nativo através de microscopia óptica e de microscopia eletrônica de varredura. A resposta biológica foi avaliada implantando fragmentos das matrizes em trepanações criadas em tíbias de ratos. O reparo dos defeitos foi estudado 3, 7, 15 e 30 dias pós-implantação usando-se microscopia óptica. Resultados: O processo de hidrólise seletiva de grupos carboxamidas em carboxílicos removeu completamente as células intersticiais do pericárdio bovino e transformou a estrutura compacta e fibrilar do colágeno nativo em uma estrutura esponjiforme. Além disso, foi observado que as matrizes modificadas permitiram o reparo das trepanações ósseas com a formação de osso trabecular, sem induzir nenhuma reação adversa. Conclusões: A hidrólise utilizada aplicada ao pericárdio bovino produz uma matriz acelular com poros distribuídos na sua estrutura. Essas matrizes são biocompatíveis, não induzem reação inflamatória e permitem o reparo de trepanações ósseas experimentalmente induzidas. / Objective: Evaluate the structural modifications in the bovine pericardium after selective hydrolysis of carboxiamides groups into carboxilic and the biological response in bone defects repaired using the anionic collagen matrices. Material and methods: Samples of the modified matrices were compared to samples of native bovine pericardium using light microscopy and scanning electron microscopy. The biological response were evaluated implanting fragments of the matrices into defects made in rat tibias. The repair of the defects were evaluated 3, 7, 15, and 30 days after implantation using light microscopy. Results: The process of selective hydrolysis of carboxiamides groups into carboxilic completely removed the interstitial cells of the bovine pericardium and turned the compact and fibrilar structure of native collagen into a sponge-like structure. Besides, it was observed that the modified matrices allowed the repair of the bone defects through the formation of cancellous bones, without any side reaction. Conclusions: The used hydrolysis applied to bovine pericardium produces a acellular matrix with pores distributed along its structure. These matrices are biocompatible, they do not induce any inflammatory reaction and allow the repair of experimentally induced bone defects.

Nos últimos anos as cerâmicas à base de zircônia, além de outras aplicações, têm sido empregadas em próteses. Isso se deve as suas características como, biocompatibilidade, boa resistência à corrosão e ao desgaste, e tenacidade á fratura. Suas propriedades ópticas permitem reproduzir as características estéticas dos dentes naturais. A resistência da zircônia está relacionada à sua microestrutura, que por sua vez esta relacionada com as características dos pós iniciais. Nesse trabalho foi obtido pó de zircônia estabilizada com ítria pelo processo sol-gel. O tamanho de partículas obtidas foi aproximadamente 2,5 ?m, com zircônia predominantemente na fase tetragonal. O pó foi compactado e sinterizado a 1550°C por 120 minutos. Nestas condições as amostras apresentaram densidade relativa 98%, tamanho médio de grão 0,56 ?m, e predominantemente na fase tetragonal. Foram realizados ensaios em amostras sinterizadas e sinterizadas e tratadas em líquido corpóreo simulado. O módulo de ruptura obtido foi aproximadamente 482 e 560 MPa, a dureza 8,4 e 9,8 GPa e tenacidade á fratura 9,7 e 8,5 MPa.m1/2, para as amostras sinterizadas e sinterizadas e tratadas respectivamente. Também, foi estudado o comportamento à molhabilidade da zircônia sintetizada e sinterizada com água e com fluido corpóreo, mostrando-se excelentes resultados para aplicações biomédicas. Com os resultados obtidos pode-se supor que a zircônia sintetizada é um material promissor para produção de cerâmicas para aplicações biomédicas. / Zirconia based ceramic has been used, besides other applications, in implants due to good strength, good fracture toughness, high wear and corrosion resistance and excellent biocompatility. Its properties permit to reproduce the esthetic characteristics of natural teeth. The zirconia resistance is related with microstructure, that in turn this related with the characteristics of the initials powders. In this work was obtained powder of yttria-stabilized zirconia by sol-gel process. The particles average size 2,5 ?m, in the tetragonal phases. The powders were compacted and sintered at 1550° for 120 minutes. Considering this condition, relative density 98% was reached, the samples presented average grain size 0,56 ?m, and predominantly in the tetragonal phases. Sintered samples and sintered and treated in the simulated body fluid were tested. The flexural strength obtained were 482 and 560 MPa, the hardness 8,4 and 9,8 GPa and fracture toughness 9,7 e 8,5 MPa.m1/2, for sintered samples and sintered and treated respectively. Also, was studied the wetability behavior of the sintered zirconia with water and simulated fluid, showing excellent results for biocompatibility application. With obtained results can be supposed the sintetized zirconia is promissory material to biomedical applications.

O uso de biomateriais para acelerar reparos de fraturas ósseas tem aumentado nos últimos anos. Neste estudo, as propriedades piezelétricas do fluoreto de polivinilideno foram utilizadas com a finalidade de mimetizar os potenciais bioelétricos associados à deformação óssea, que são diminuídos quando ocorre fratura óssea. Estudos histomorfométricos foram efetuados para verificar a influência do polímero. Testes de biocompatibilidade e de esterilização permitiram avaliar os riscos da utilização do polímero como material de implante. O polímero piezelétrico associado à placa de osteossíntese resultou em uma maior quantidade de matriz óssea mineralizada na lesão quando comparado ao implante da placa sem o polímero. / The use of biomaterials to promote accelerated bone fracture repair has been improved in the recent years. In the present study, the piezoelectric properties of poly(vinylidene fluoride) were used with the aim of mimetizing bioelectrical potentials associated to bone strain which decrease when a bone fracture occurs. Histomorphometric studies were accomplished to verify the influence of the polymer. Biocompatibility and sterilization tests assessed any risk related to the use of a polymer as an implantable material. The piezoelectric polymer associated to an osteosynthesis plate resulted in a larger amount of mineralized bone matrix in the lesion when compared to a plate implanted without the polymer.

Tese de Mestrado. Mestrado em Engenharia Biomédica. Faculdade de Engenharia. Universidade do Porto. 2012

Lower respiratory diseases are currently the third leading cause of death worldwide. For many end-stage patients with these diseases, there is no cure and a shortage of donor organs available for transplant. A promising solution is to design regenerative scaffolds or complete bioengineered lungs, using decellularized lung tissues as a template for regeneration. Recent advances in the field have made significant strides towards developing a transplantable lung. However, the current technology has not produced a functional lung for in vivo transplant due to immature gas exchange barriers. The mechanisms driving alveolar barrier maturation and role that extracellular matrix (ECM) plays within the strengthening of each type of junction are not fully understood. This research has characterized and tailored a decellularized ECM (dECM) coating for the in vitro study of dECM component depletion and potential effects on cell barrier function, attachment, and survival. Adjustments to dECM digestion duration drastically changed the resulting structural and biochemical properties for each cellular microenvironment. Shorter digestion time resulted in a dense branching of the ECM architecture and biomimetic mechanical properties needed for epithelial culture. Also, through systematic supplementation of essential basement membrane (BM) proteins to dECM, we have found that supplementation with laminin enhanced barrier strength by ZO-1 junction stabilization. This indicates that dECM can promote barrier formation but may have lost vital proteins that need to be replenished. Laminin-mediated barrier function was determined to be caused by the upregulation of the Epac/Rap1 pathway. This pathway has previously been implicated in lung endothelial barriers but not alveolar epithelial junction strengthening. Finally, to establish the translatability of these findings to whole lung recellularization, the dECM coating was used to pre-treat the airways of decellularized lungs for recellularization. Culture of MLE12 mouse epithelial cells into dECM-coated lungs increased cell survival and distribution. In combination with dECM coatings, rotational cell seeding improved cell dispersal and viability. Altogether, these techniques, devised to promote healthy alveolar barriers, are vital to enhancing current lung recellularization strategies and the treatment of many edema-associated pulmonary diseases.

<p>A novel strategy for synthesising segmented poly(urethane urea) (PUU) without using a chain extender but nevertheless with the opportunity to vary the hard segment content has been developed. The strategy is based on amine formation from isocyanate upon reaction with water. By adding a dissolved soft segment to an excess of diisocyanate followed by the addition of water in the gas phase, amines are formed <i>in situ</i>. Urea linkages are then formed when these amines react with the excess of isocyanate groups. The gas phase addition facilitates addition in a slow and continuous manner. The hard segment content can easily altered by varying the diisocyanate/soft segment ratio. Even though the strategy is shown to be applicable to different diisocyanates, the focus has been on the potentially biodegradable methyl-2,6-diisocyanatehexanoate (LDI) and 1.4-butanediisocyanate (BDI) and various well known biodegradable polyesters and polycarbonates. </p><p>All the synthesised materials exhibited pronounced phase separation and hydrogen bonding within the hard domains. However, a major increase in hydrogen bonding strength was seen when a symmetric diisocyanate was used instead of an asymmetric. Based on FTIR measurements, PUUs with BDI and a polydisperse hard segment can exhibit the same degree of phase separation and hydrogen bonding as the monodisperse product.</p><p>The elastic properties of this new group of PUUs were exceptional with an elongation at break from 1600% to almost 5000% and the elastic modulus could be varied from a few MPa up to a couple of hundreds. </p><p>Hydrolytic degradation was greater in the polyester-based than in the polycarbonate-based PUUs due to the more reactive ester bonds. Low mass loss but a considerable loss in molecular weight was seen in the polyester PUUs. The tensile strength decreased dramatically due to the loss of strain hardening.</p><p>An MTT seeding assay using human fibroblasts and an in vivo biocompatibility study were performed and no signs of cytotoxicity were seen and the inflammatory response was comparable to other inert polymers.</p><p>A biodegradable PUU with properties that can be tailored through an easy synthesis is here presented. </p>

Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed. In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair. In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

A novel strategy for synthesising segmented poly(urethane urea) (PUU) without using a chain extender but nevertheless with the opportunity to vary the hard segment content has been developed. The strategy is based on amine formation from isocyanate upon reaction with water. By adding a dissolved soft segment to an excess of diisocyanate followed by the addition of water in the gas phase, amines are formed in situ. Urea linkages are then formed when these amines react with the excess of isocyanate groups. The gas phase addition facilitates addition in a slow and continuous manner. The hard segment content can easily altered by varying the diisocyanate/soft segment ratio. Even though the strategy is shown to be applicable to different diisocyanates, the focus has been on the potentially biodegradable methyl-2,6-diisocyanatehexanoate (LDI) and 1.4-butanediisocyanate (BDI) and various well known biodegradable polyesters and polycarbonates. All the synthesised materials exhibited pronounced phase separation and hydrogen bonding within the hard domains. However, a major increase in hydrogen bonding strength was seen when a symmetric diisocyanate was used instead of an asymmetric. Based on FTIR measurements, PUUs with BDI and a polydisperse hard segment can exhibit the same degree of phase separation and hydrogen bonding as the monodisperse product. The elastic properties of this new group of PUUs were exceptional with an elongation at break from 1600% to almost 5000% and the elastic modulus could be varied from a few MPa up to a couple of hundreds. Hydrolytic degradation was greater in the polyester-based than in the polycarbonate-based PUUs due to the more reactive ester bonds. Low mass loss but a considerable loss in molecular weight was seen in the polyester PUUs. The tensile strength decreased dramatically due to the loss of strain hardening. An MTT seeding assay using human fibroblasts and an in vivo biocompatibility study were performed and no signs of cytotoxicity were seen and the inflammatory response was comparable to other inert polymers. A biodegradable PUU with properties that can be tailored through an easy synthesis is here presented.

Transverse mechanical properties of wood are important in many practial applications and an interesting scientific subject. A very low transverse shear modulus has been identified in spruce, which causes large strain concentrations in wood structures. In this thesis, experimental characterization of local density variations as well as local strain fields are carried out using the SilviScan apparatus and digital speckle photography, respectively. This is combined with micromechanical modeling based on hexagonal wood cells in combination with finite element analysis. Problems addressed include the moduli in the transverse plane, including variations at the scale of individual annual rings. The relative importance of cell wall bending and stretching deformation mechanisms is analysed as a function of wood cell geometry, relative density and direction of loading (radial, tangential and shear). Transverse anisotropy is also analyzed, including its dependency of earlywood and latewood characteristics. The wood cell shape angle variation and density effects are sufficient to explain transverse anisotropy in softwoods (no ray effects), and the influence of earlywood/latewood ratio is explained. As a practical test method for shear modulus measurements, an off-axis compression test with full-field strain determination is proposed. The advantage is a simple fixture and large region of representative strain required for a heterogeneous material such as wood. As an alternative, the single cube apparatus (SCA) for shear tests is evaluated. The SCA is used to determine the shear strain distribution within the annual rings. Based on the density distribution of the shear test specimen and a micromechanics model, a finite element model is developed, and predictions are compared with the measured shear strains. The agreement between predicted and measured shear strains at the annual ring scale are remarkably good. It shows that the low GRT of spruce is due to the low earlywood density and the large cell wall bending deformation resulting from shear loading. Furthermore, it illustrates the need for improved understanding of annual ring scale effects. For example, fairly low transverse global loads will lead to lage local shear strains. / Transversella mekaniska egenskaper hos trä är viktiga i många praktiska tillämpningar och är av vetenskapligt intresse. Gran har exemplevis mycket låg transversell skjuvmodul, vilket leder till stora lokala töjningskoncentrationer i trästrukturer. I den här avhandlingen utförs experimentella mätningar av densitetsfördelning och lokal töjningsfördelning med hjälp av SilviScan utrustning (röntgen) och digital speckelfoto grafi (DSP). Det kombineras med mikromekanisk modellering med hexagonala cellmodeller som utgångspunkt, ibland i kombination med finita elementberäkningar. Transversella moduler bestäms liksom töjningseffekter på skalan individuella årsringar. Den relativa betydelsen av böjning och sträckning av cellväggen analyseras som funktion av relativ densitet och belastningsriktning (radiell, tangentiell och skjuvning). Stor andel böjdeformation ger låg modul och proportionerna mellan de båda mekanismerna styr graden av anisotropi. Transversell anisotropi analyseras därför, inklusive dess beroende av karakteristiken hos vårved och sommarved. Formvinkeln på vedcellen och inverkan av densitet är tillräckliga för att förklara graden av anisotropi (utan inverkan från märg- strålar). Inverkan av förhållandet mellan mängden vårved och sommarved på anisotropin analyseras särskilt. En enkel tryckbelastningsmetod (“off-axis metod”) används för att bestämma transversell skjuvmodul hos trä. Metoden kombineras med DSP. Fördelen är en enkel fixtur i kombination med det stora område av ren skjuvdeformation som uppstår i provstaven. Som ett alternativ utvärderas också en metod baserad på kubiskt prov (SCA). Metoden används för att bestämma lokala skjuvtöjningar på skalan individuella årsringar. Baserat på densitetsfördelningen i provet och en mikromekanisk modell så utvecklas en finita element-modell. Den utnyttjas för att beräkna lokala skjuvtöjningar. Jämförelsen mellan beräkningar och uppmätta skjuvtöjningar ger enastående god överensstämmelse. Det visar att den låga skjuvmodulen för gran orsakas av låg densitet i kombination med att böjning av cellväggarna dominerar som deformationsmekanism. Det illustrerar också att vi behöver förbättra vår förståelse för deformationsfält på årsringsnivå. En praktisk konsekvens är t ex att relativt låga globala laster ger upphov till mycket hög lokal skjuvdeformation. / QC 20100830