Spinal cord injury causes the partial or total loss of the anatomical and functional continuity of the spinal cord tissue, leading to the damage of the organs controlled by nerves that branch off downstream the injury. This thesis analyses the mechanics of two possible treatments based on two different approaches: intraspinal microstimulation (ISMS) and tissue engineering. These two approaches have a common rationale, the delivery of electrical stimuli to the injured spinal cord. In the literature, the feasibility of the electrodes for ISMS is often limited to the analysis of stiffness. The mechanical validation of the device is then focused on the step after the in vivo implantation, considering the interplay with the surrounding tissue. In this work, the mechanical performance of an innovative intraspinal microstimulation device is evaluated thoroughly before the in vivo step, to avoid the waste of material, animals, and time. The study involves the characterisation of the single components (electrodes), prototypes, and possible failure mechanisms. A work on silk fibroin hydrogels for the regeneration of the spinal cord is also presented. Silk fibroin is a highly versatile material for biomedical purposes, and thus largely used in tissue engineering. Moreover, it has piezolectric properties subjected to micro and nanostructure. Given the proven benefits of electrical stimulation in the regeneration of the spinal cord after injury, different approaches studied in literature often require the use of external devices to generate electrical stimuli. This thesis aims to study the mechanical properties of silk fibroin hydrogels obtained by applying an electric field to silk fibroin solutions, to investigate the eventual increase of the microstructure orientation and consequent improvement of the piezoelectric effects of fibroin.

Chronic or degenerative diseases affecting the lumbar spine, commonly referred to as low back pain (LPB), are a major cause of dysfunction, pain, and disability worldwide. According to the Global Burden of Disease (GBD) report of 2019, LPB affects over half a billion people, severely limiting their well-being and lifestyle. Unfortunately, these numbers have been steadily increasing over the last decade, with a rise of more than 15%, mainly due to demographic aging of the population, making it a significant socioeconomic global issue. When conservative treatments such as medications, drugs, and injections fail to alleviate the symptoms, surgical interventions become necessary. Spinal surgeries have become increasingly common and account for 40% of the top ten surgical procedures in the United States alone. As a result, the global market for spinal implants and medical orthopedic devices has been growing at a compound annual growth rate (CAGR) of 5.0% in the United States. Degenerative disc diseases, herniated intervertebral discs, and spondylolisthesis are among the most common problems requiring implant surgery, with lumbar interbody fusion cages or total disc replacements being the most common options. These surgical techniques often utilize a metal endplate or hollow cage as a load-bearing structure to ensure correct load transmission and biomechanical spinal functionality. Currently, endplates for total disc replacement are produced using subtractive manufacturing techniques from bulk biomedical-graded metal alloys like Ti-6Al-4V. The endplates are inserted between two adjacent vertebral bodies, where bone ingrowth and implant fusion are necessary. However, the elastic properties of bulk metals and bone tissue do not match, resulting in stress-shielding phenomena, implant loosening, or implant subsidence. These complications frequently necessitate surgical revision of the implant, which not only impacts the daily activities of the patients but also has a relevant economic impact. Therefore, researchers are exploring alternative design and manufacturing strategies to develop next-generation prosthetic devices that overcome these challenges. Metal additive manufacturing (MAM), particularly Laser-Powder Bed Fusion (L-PBF), has revolutionized the fabrication of specialized components with complex shapes, including architected cellular materials - a novel class of engineered materials with tunable mechanical properties. The biomedical field is a prime example of where lattice application has proved beneficial. MAM provides numerous advantages, including patient-specific customization, a vast design space, and reduced stress shielding. However, issues with structural integrity, lack of AM-specific norms, and the need for fine-tuning process optimizations are still hindering MAM's widespread adoption on the international market. An essential issue that requires resolution is the impact of process-induced flaws on the fatigue behavior of components made of L-PBF lattices. Despite a growing body of scientific literature on the fatigue behavior of lattice unit cells, little attention has been given to the function of fatigue at a millimetric scale, specifically the role of sub-unital lattice elements such as struts and junctions. As fatigue is highly localized, understanding primary fatigue behavior and fracture mechanisms at a strut scale may be critical to addressing the aforementioned problems. Moreover, designing proper prosthetic devices requires fulfilling both biomechanical and biological requirements, leading to a bottleneck in component quality. Proper tuning of osteointegration often requires large porosity and small strut dimensions, approaching the limits of industrial 3D printers. This increases the likelihood of manufacturing lattices with unconnected struts, drosses, parasitic masses, and severe deviations from the nominal as-designed geometries, leading to highly susceptible components under fatigue. To address these challenges, combined approaches with bone tissue engineering may be advantageous. Biopolymers from natural sources, such as silk fibroin and collagen derivatives (i.e., gelatin), are widely used for bone-filler applications due to their exceptional biological properties. These polymers can create highly interconnected biodegradable porous 3D scaffolds suitable for cell differentiation towards an osteogenic phenotype, such as in the form of foams. These foams can be embedded into metal lattice structures, resulting in a hybrid composite device that simultaneously fulfills the load-bearing, fatigue, and osteointegrative requirements that a spinal prosthetic device necessitates. This thesis work covers a range of topics mentioned above. Firstly, an introductory theoretical background is presented in Chapter I, followed by experimental findings which are presented in three different chapters. Chapter II is dedicated to the fatigue behavior of L-PBF Ti-6Al-4V sub-unital lattice elements in the form of miniaturized dog-bone specimens that mimic struts and nodes. This chapter is divided into four sections. The first section investigates the fatigue strength of strut-like specimens based on their building orientations at four different angles with respect to the printing job plate. Morphological features of the miniaturized specimens such as average and minimum cross-section, eccentricity, waviness, and surface texture are correlated with fatigue strength. The role of inner and surface defects, such as lack-of-fusion (LoF) and gas holes, is also considered to explain the main failure mechanisms. The impact of building orientation on the printing quality of the specimens is highlighted, with an increase in surface roughness and defectiveness as the printing angle decreases, resulting in a shorter fatigue life for miniaturized struts. In the second section, the fatigue effect is studied across different fatigue regimes. The role of the mean stress effect is assessed using the Haigh diagram, which reveals an increase in fatigue life moving towards compressive loading regimes. The effect of the printing angle is also investigated, showing different trends according to the different stress ratios, suggesting different fatigue failing mechanisms. The third section introduces strut-junction miniaturized specimens and evaluates their fatigue behavior according to building orientations. Horizontal specimens show an increased fatigue life compared to their thin strut counterparts, and different morphological outcomes are highlighted, including improved surface quality even at lower angles, possibly related to the node acting as an additional supporting structure. The fourth section presents a design-led compensation strategy for sub-unital lattice specimens, aimed at reducing as-designed/as-built deviations. This systematic decrease in geometrical mismatch suggests potential new design strategies for fatigue enhancement. In Chapter III, bone tissue engineering strategies are explored for the design of foam scaffolds as bio-fillers for lattice-based design. The feasibility of the polymer-metal composite is assessed, using an N2O-based gas foaming technique to fabricate silk fibroin and silk fibroin/gelatin porous scaffolds infilled into a cubic L-PBF Ti-6Al-4V lattice structure. The adhesion at the polymer/metal interface is assessed, with simultaneous electrowetting, showing promise for better and more intimate contact on the outermost surface of the lattice struts. A statistical-based analysis of the foam porosity is then carried out, aimed at optimization towards osteointegration improvement. Selected foams are biologically evaluated, revealing good cell adhesion and differentiation towards an osteogenic phenotype. Chapter IV reports on two different strategies for the design of a Ti-6AL-4V L-PBF lattice-based endplate for total disc replacement. The first strategy focuses on homogenized-based topology optimization, designing an octet-truss prosthetic device with a graded structure and a cell size suitable for polymeric infilling. The second strategy aims at optimizing octet-truss lattice components for fatigue, evaluating the optimal building orientation for the specimens. Experimental results reveal an improvement in the fatigue life of three-point bending test specimens, suggesting the potential of the proposed model. In Chapter V, the major takeaways of this thesis work are discussed, highlighting important advancements in understanding the fatigue behavior of lattice structures and the development of novel hybrid strategies for the design of biomedical devices, with a particular focus on spinal orthopedics. Future possible directions for research are also explored.

Tissue engineering (TE) is an interdisciplinary field, in continuous evolution, that possesses as main goal the creation of efficient systems for tissues and organs healing and regeneration. For bone, TE strategies are typically based on the combined use of scaffolds, cells, and bioactive molecules. Different materials were successfully studied and proposed for the fabrication of scaffolds. Among them, silk fibroin (SF) was evaluated as particularly promising for different TE applications, especially for bone tissue regeneration. Silk fibroin, a natural protein forming the structural core of silk filaments, holds biocompatibility, mechanical properties and biodegradation rate suitable for applications in bone regeneration. However, in the past, SF has shown some limitations, especially in terms of bioactivity and effective differentiating ability of hMSCs in regenerating bone tissue. In this work, we wanted to demonstrate that SF, properly processed, chemically modified, and conjugated with selected bioactive species, can be used to prepare different systems: a functionalised scaffold; a bioresorbable material with mineralization ability; an implantable immunomodulatory material. The experimental activities performed and the deep investigation of the properties of the SF-based systems prepared, led to promising results, indicating that SF could be a flexible and powerful platform for the realization of different therapeutic tools. For some of the SF-based systems described in this dissertation, further studies are needed to assess the biological activity of the materials prepared.

Forskningsområdet för bärbar elektronik är fortfarande relativt ungt och det finns ett stort behov av utveckling av nya material inom området. Olika typer av kompositer är mycket intressanta och de ska uppvisa såväl hög hållfasthet som goda ledande egenskaper. I detta avseende är silkes fibroin och MXene mycket intressanta utgångsmaterial eftersom silkestrådarna kan ge en struktur med god jonledningsförmåga och god flexibilitet och MXene kan bidra med hög styvhet och god elektrisk ledningsförmåga. Med detta som bakgrund beslöts att undersöka om kompositer av silkestrådar och MXene kan användas i kompositer som kan användas i bärbar elektronik. 3 olika typer av hydrogeler studerades och de innehöll silkes fibroin med 0, 1 och 5% MXene. De egenskaper som utvärderades var struktur, mekaniska egenskaper, stabilitet i vatten, bionedbrytbarhet och både statisk och dynamisk ledningsförmåga. Resultaten visar att de tillverkade nanokompositerna har lovande förutsättningar inom området eftersom en kombination av silkes fibroin med 5 % MXene har god stabilitet, konduktivitet och en hög och stabil Gauge-faktor. / As the research area of wearable electronics is still relatively new, material science with this focus opens plenty of unexplored fields. That is why a study characterizing the unexplored composite system of silk fibroin and MXene (Silk/MXene) was conducted. These two biocompatible materials are complementary with regard to the requirements for wearable electronics materials. Silk fibroin dispose an ionic conductivity and solid flexibility, while MXene brings mechanical strength and significant increase of electrical conductivity. The reinforced hydrogel materials were studied at two concentrations of fillers, 1% and 5% and compared to pristine silk fibroin. All three materials were studied from the point of view of their structure, mechanical properties, behaviour in aqueous environment, biodegradability and electrical conductivity, both static and dynamic. Nanocomposite systems of silk fibroin and MXene have shown a potential for being used in the intended application area, as Silk/MXene 5% film displays good stability, conductivity with high andstable Gauge factor.

Breast cancer is one of the most common types of cancer affecting women in the world today. To better understand breast cancer initiation and progression modeling biological tissue under physiological conditions is essential. Indeed, breast cancer involves complex interactions between mammary epithelial cells and the stroma, both extracellular matrix (ECM) and cells including adipocytes (fat tissue) and fibroblasts (connective tissue). Therefore, the engineering of in vitro three-dimensional (3D) systems of breast tissues allows a deeper understanding of the complex cell-cell and cell-ECM interactions involved during breast tissue development and cancer initiation and progression. Furthermore, such 3D systems may provide a viable alternative to investigate new drug or drug regimen and to model and monitor concurrent cellular processes during tumor growth and invasion. The development of suitable 3D in vitro models relies on the ability to mimic the microenvironment, the structure, and the functions of the breast tissue. Different approaches to develop a novel 3D breast model have been investigated. Most models use gel scaffolds, including Matrigel® and collagen to generate breast tissue-like structures. However, the physicochemical, mechanical, and geometrical properties of these scaffolds only partially meet the mechanical, physical, and chemical parameters of the breast tissue matrix. In the present studies, we investigated the overall hypothesis that electrospun SF-derived scaffolds promote mammary cell growth and the formation of mammary-like structures depending on the composition and/or coating of the scaffolds with ECM proteins. Through an extensive literature search (1) the importance of 3D modeling of tissues and organs in vivo, (2) 3D modeling of the mammary tissue and currently available models, (3) the properties and applications of SF in tissue modeling and regeneration were reviewed (Chapter 1). Our studies provide evidence of the effects of various concentrations (Chapter 2) of SF along with different electrospinning techniques (Chapter 3) on the structure of electrospun scaffolds and whether those scaffolds provide suitable microenvironments for mammary epithelial cells as determined by MCF10A cell attachment, viability, and structure formation. Further, we investigated the effects of the key ECM proteins collagen I (Chapter 4) and laminin (Chapter 5) used to blend or coat, respectively, SF scaffolds on the attachment, viability and structure formation of mammary epithelial cells. Our studies first highlight the mechanical and physical properties of the different SF-derived scaffolds through various SF concentrations and electrospinning techniques. Second, the biocompatibility of these SF electrospun scaffolds was defined based on MCF10A cell survival and adhesion. Third, our data indicate that scaffolds derived from blended and/or coated SF with collagen I also promoted human mammary cell survival and adhesion. Lastly, our observations suggest that on laminin-coated SF scaffolds MCF10A mammary cells, in the presence of lactogenic hormones, differentiated forming acinus-like structures. Overall, these studies provide evidence that SF electrospun scaffolds closely mimic the structure of the ECM fibers and allow many advantages such as; physical and chemical modification of the microenvironment by varying electrospinning parameters and addition of various proteins, hormones, and growth factors, respectively. Further, coating these SF scaffolds with essential ECM proteins, in particular laminin, promote cell-ECM interactions necessary for cell differentiation and formation of growth-arrested structures, through providing cell integrin binding sites and appropriate chemical cues.

Nowadays, exceptional advantages of silk fibroin over synthetic and natural polymers have impelled the scientists to application of this biomaterial for tissue engineering purposes. Recently, we showed that embedding natural degummed silk fibers in regenerated Bombyx mori silk-based scaffold significantly increases the mechanical stiffness, while the porosity of the scaffolds remains the same. In the present study, we evaluated degradation rate, biocompatibility and regenerative properties of the regenerated 2% and 4% wt silk-based composite scaffolds with or without embedded natural degummed silk fibers within 90 days in both athymic nude and wild-type C57BL/6 mice through subcutaneous implantation. In all scaffolds, a suitable interconnected porous structure for cell penetration was seen under scanning electron microscopy. Compressive tests revealed a functional relationship between fiber reinforcement and compressive modulus. In addition, the fiber/fibroin composite scaffolds support cell attachment and proliferation. On days 30 to 90 after subcutaneous implantation, the retrieved tissues were examined via gross morphology, histopathology, immunofluorescence staining and reverse transcription-polymerase chain reaction as shown in Figure 1. Results showed that embedding the silk fibers within the matrix enhances the biodegradability of the matrix resulting in replacement of the composite scaffolds with the fresh connective tissue. Fortification of the composites with degummed fibers not only regulates the degradation profile but also increases the mechanical performance of the scaffolds. This report also confirmed that pore size and structure play an important role in the degradation rate. In conclusion, the findings of the present study narrate key role of additional surface area in improving in vitro and in vivo biological properties of the scaffolds and suggest the potential ability of these fabricated composite scaffolds for connective tissue regeneration.

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No. of bitstreams: 1 silva_rr_dr_araiq_par.pdf: 1063262 bytes, checksum: cc72dc79773da734cac767490756f56e (MD5) Previous issue date: 2016-05-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Nanoestruturas unidimensionais de telúrio (Te1D) na forma de whiskers, fios e hélices foram preparados com facilidade por uma síntese em etapa única na presença de solução aquosa de Pluronic® F68 à baixas temperaturas (< 100 °C) e pressão ambiente. A forma das nanoestruturas puderam ser controladas de acordo com a cinética da reação. Estruturas empacotadas de nanowhiskers e nanofios de Te foram obtidas via auto-organização em interface líquido-líquido e pela técnica de drop-cast em substrato de Si/SiO2. Estruturas híbridas 1D foram obtidas utilizando nanoestruturas Te1D como molde de sacrifício para anexar nanopartículas metálicas ou mesmo produzir nanoestruturas 1D metálicas. Por exemplo, nanoestruturas híbridas 1D foram preparadas decorando nanofios de Te com nanopartículas de Ag em solução aquosa de poli(vinilpirrolidona). Nanoestruturas 1D de Au com forma de nódulos foram preparados por deslocamento galvânico de íons Au3+ em uma mistura de nanohélices de Te, ácido ascórbico e solução aquosa de poli(vinilpirrolidona). Além disso, nanohélices de Te foram funcionalizadas com uma camada resina resorcinol-formaldeído em condições brandas de síntese. A resina de resorcinol-formaldeído é uma via intermédia para explorar a deposição de compostos opticamente ativos tais como nanopartículas de hidroxicarbonato de Tb3+ ou nanopartículas de Au. Para aplicações práticas é essencial que estas nanoestruturas possam ser suportadas em filmes rígidos ou flexíveis de alta qualidade óptica. Filmes de polímeros naturais puros e filmes híbridos de sol-gel epóxi foram avaliados como potenciais matrizes hospedeiras para luminóforos. A fabricação de híbridos é baseada na incorporação de 3-glicidoxipropiltrimetoxissilano na solução homogênea de polímero natural com posterior secagem sobre uma superfície plana. Particularmente, filmes flexíveis de fibroína da seda e acetato de celulose e os seus híbridos derivados exibiram excelentes propriedades ópticas para hospedar compostos opticamente ativos. Por exemplo, compostos de Eu3+ emissores na região do vermelho e corantes fluorescentes foram incorporados em matriz pura de polímero e híbridos epóxi e suas propriedades ópticas foram investigadas. Laser de corantes por feedback distribuído (DFB) foram fabricados dopando grades de difração de fibroína de seda com Rodamina 6G. Devido a sua capacidade de replicar superfícies padronizadas com resolução nanométrica, grades de fibroina da seda dopadas com corante foram depositadas contra a grade de difração de uma mídia de disco compacto comercial. Lasers modificados de DFB baseados em filmes de fibroina contendo nanopartículas espalhadoras de luz de SiO2 e Ag aleatoriamente distribuídas na grade de fibroina demonstraram aumento da intensidade do laser, além de estreitamento da largura do pico de emissão. Filmes híbridos flexíveis e transparentes (> 85%) de fibroina da seda e acetato de celulose modificados com função epóxi e contendo compostos fluorescentes na região do vermelho como complexos β-dicetonato de Eu3+ e nanopartículas de YVO4:Eu3+ em baixa proporção relativa mássica (<5%) foram preparados. De maneira geral, o resultado são filmes homogêneos com funções epoxi e/ou alcoxissilano não hidrolisados disponíveis para outras modificações químicas. Devido a matéria-prima limitada de polímeros naturais para uma alta demanda de fabricação de dispositivos ópticos, é igualmente importante desenvolver materiais com base na reutilização de polímeros sintéticos. Filmes finos de poliestireno foram concebidos por dissolução de poliestireno expandido (EPS) recuperado de resíduos em D-limoneno, um solvente verde proveniente de óleos cítricos. Filmes transparentes dopados com complexos β-dicetonato de Eu3+ demonstraram excelente transparência e aptos para uso em guias de luz. Estes resultados são motivadores para a) a engenharia de nanoestruturas 1D com propriedades ópticas sintonizáveis bem como, b) desenvolvimento de híbridos flexíveis e transparentes baseados em híbridos de polímeros naturais com alta funcionalidade química ou polímeros sintéticos reciclados como potenciais matrizes hospedeiras ópticas almejadas em aplicações fotônicas. / One-dimensional Te nanostructures (Te1D) in the shape of whiskers, wires and helices were prepared by a facile one-pot synthesis in the presence of aqueous Pluronic® F68 solution at low temperatures (< 100 ºC) and ambient pressure. The shape of Te1D nanostructures could be manuvered according with the reaction kinectics. We evaluate some techniques to assemble Te1D nanostructures on the pursuit for complex nanoarchitectures. Bundles of Te nanowhiskers and nanowires were achieved by self-assembly in liquid-liquid interface or by drop-cast technique in Si/SiO2 substrates. 1D hybrid structures have been conceived by using Te1D nanostructures as sacrificial template to attach metallic nanoparticles or even produce metallic 1D nanostructures. For example, 1D hybrid nanostructures were easily prepared by decorating Te nanowires with Ag nanoparticles in aqueous solution of poly(vinylpyrrolidone). Au 1D nanostructures with nodular-like shape were prepared by galvanic displacement of Au3+ ions in a mixture of Te nanohelices, ascorbic acid and an aqueous solution of poly(vinylpyrrolidone). Furthermore, Te1D nanohelices were functionalized with a layer of resorcinol-formaldehyde resin at mild synthesis conditions. The RF resin allowed us to fashion an intermediate pathway to explore the deposition of optically active compounds like Tb3+ hydroxylcarbonate or Au nanoparticles. Seeking practical applications, these nanostructures should be hosted over rigid or flexible films possessing excellent optical properties. Pure natural polymers and epoxy sol-gel hybrids films were evaluated as potential host for luminophors. The fabrication of epoxy hybrids is based on the incorporation of 3-glycidoxypropyltrimethoxysiloxane on the homogenous solution of natural polymer with subsequent casting over flat surface. Particularly, flexible silk fibroin and cellulose acetate films and their derivative hybrids displayed excellent optical properties to host optically active compounds. For instance, red emitting Eu3+ compounds and fluorescent dyes were hosted on pure natural polymer and hybrid films and the optical features of the luminescent films were investigated thoroughly. Distributed feedback dye-lasers were fabricated by doping silk fibroin diffraction gratings with Rhodamine 6G. Owing its ability to mimic patterned surfaces at nanoscale resolution, dye-doped SF gratings were fabricated using replica-casting patterning against a commercial blank digital versatile disc as template. A modified DFB Laser based on SF films with Ag or SiO2 light scattering particles randomly distributed on the grating unveiled an enhancement of laser intensity withal narrowing of emission peak linewidth. Flexible and highly transparent SF- and CA-epoxy hybrids (> 85%) containing red fluorescent Eu3+ b-diketonate complex and YVO4:Eu3+ nanoparticles at low relative content (< 5 wt%) were tailored. In general, the outcome is homogeneous films with epoxy and/or unhydrolized alkoxysilane functions available for further chemical modification. Owing the limited feedstock of natural polymers for high demanding production of optical devices, it is equally important develop materials based on the reuse of synthetic polymers. Thin films of polystyrene were conceived by dissolving waste-recovered expanded-polystyrene (EPS) in D-limonene, a green solvent from citrus oil. Transparent EPS films doped with Eu3+ b-diketonate complex displayed excellent transparency and light waveguiding, These assertions provide a framework that motivates the research on a) engineering of 1D hybrids nanostructures with tunable optical properties and b) flexible natural polymer/epoxy hybrid with enhanced functionality or plastic recycled as potential optical hosts sought in photonic applications. / FAPESP: 2013/12367-6

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Materials based on polyhydroxybutyrate (PHB), chitosan (CHI) and fibroin (SF) are biocompatible and attractive for applications in bone tissue engineering. In this work, 3D scaffolds of PHB/CHI and PHB/CHI/SF in different proportions was prepared, characterized and evaluated the in vitro behavior: group I: PHB/CHI (50:50 wt.%), group II: PHB/CHI/SF (50:45:5 wt.%) and group III: PHB/CHI/SF (50:35:15 wt.%). The scaffolds were produced by the lyophilization method of the components mixtures. The physical-chemical characterization of the scaffolds was performed by X-ray diffraction, infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis. A highly porous nature was revealed by SEM analysis of the scaffolds. The FTIR analysis revealed that the constituents used in the preparation of the scaffolds interacted chemically with each other. Thermal analysis showed that fibroin increases the thermal stability of the scaffolds. The viability and cell proliferation were assessed by the MTT method and the cytotoxicity results showed that all scaffolds are non- cytotoxic. In addition, the scaffolds stimulated cell proliferation and are promising for tissue engineering applications. / Materiais à base de polihidroxibutirato (PHB), quitosana (QUI) e fibroína (SF) são biocompatíveis e atrativos para aplicações na engenharia tecidual óssea. Neste trabalho foi preparado, caracterizado e avaliado o comportamento in vitro de arcabouços 3D de PHB/QUI/SF em diferentes proporções: grupo I: PHB/QUI (50:50 % em massa), grupo II-PHB/QUI/SF (50:45:5 % em massa) e grupo III- PHB/QUI/SF (50: 35:15 % em massa). Os arcabouços foram produzidos pelo método da liofilização das misturas dos componentes dos três grupos. A caracterização físico-química dos arcabouços foi realizada por difração de raios X, espectroscopia no infravermelho, microscopia eletrônica de varredura e análise termogravimétrica. A análise de MEV mostrou que os arcabouços 3D apresentam uma boa porosidade. Por FTIR observou- se que os componentes utilizados na preparação dos arcabouços interagiram quimicamente entre si. As análises térmicas indicam que a fibroína aumenta a estabilidade térmica dos arcabouços. A viabilidade e a proliferação celular foram avaliadas pelo método do MTT e os resultados de citotoxicidade mostraram que ambos os arcabouços não são citotóxicos. Além disso, os arcabouços estimularam a proliferação celular, sendo promissores para aplicações em engenharia tecidual.

Made available in DSpace on 2017-07-10T18:08:02Z (GMT). No. of bitstreams: 1 Vinicius Muller.pdf: 5054743 bytes, checksum: 4684dd9325f819006bfd84424d666674 (MD5) Previous issue date: 2014-07-04 / Electrospinning technique was applied in the covering of fused silica fibers by regenerated silk fibroin nanofibers (RSF). The parameters of electrospinning process were evaluated through factorial experimental design 22. This study showed the variables flow of solution and capillary-collector distance were statistically significant in the medium diameter response. The model obtained was validated through variance analysis (ANOVA) and response surface methodology. The material was used at solid phase micro extraction (SPME) in the extraction of samples containing a small chain alcohol, applied at gas chromatography (GC). The recovered device was characterized through Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) and thermal analysis (DSC and TGA). The RSF nanofibers covered fibers were morphologic evaluated through scanning electronic spectroscopy (MEV). The results of MEV showed that was possible to obtain fibers in nanoscale, medium diameter around 304 ± 46 nm. The fibers were also submitted to simulated thermal treatment (100°C to 250°C). There was no fusion between the fibers until 250°C (4 hours of treatment), and a little structural damage. The thermal analysis of FRS and covered FRS nanofibers device showed that the electrospun material maintained the thermal stability, with loss due thermal degradation from 250°C. The GC assay demonstrated the standard SPME covered device didn´t interfere on the suggested method to the alcohol isopropyl analysis (AIS). The times of extraction and desorption were optimized in 20 minutes and 10 minutes, respectively. The methodology was evaluated about linearity and specificity. In the concentration between 10 to 500 ppm of AIS, the method was linear, with R2= 0,9927 and selective, with high resolution between peaks of methanol, AIS and ethyl acetate. Electrospinning process of RSF as covering device showed great potential to use in SPME due its high surface area, thermal stability and easiness process, as well as potential to application in extraction of alcohols and GC analysis. / A técnica de eletrofiação foi aplicada no revestimento de fibras de sílica fundida por nanofibras de fibroína de seda regenerada (FSR). Os parâmetros do processo de eletrofiação foram avaliados por meio de delineamento experimental fatorial 2². Este estudo mostrou que as variáveis vazão da solução e distância entre capilar-coletor foram estatisticamente significativas na resposta diâmetro médio. O modelo obtido foi validado através da análise de variância (ANOVA) e metodologia de superfície de resposta. O material foi empregado em Microextração em Fase Sólida (Solid Phase Microextraction SPME ) na extração de amostras contendo um álcool de cadeia pequena, aplicado em cromatografia gasosa (CG). O dispositivo recoberto foi caracterizado por meio de espectroscopia na região do infravermelho (FTIR-ATR) e análises térmicas (DSC e TGA). As fibras recobertas com nanofibras de FSR foram avaliadas morfologicamente através de microscopia eletrônica de varredura (MEV). Por meio dos resultados das análises de MEV, observou-se que foi possível obter fibras em escala nanométrica, com diâmetro médio em torno de 304 ± 46 nm. As fibras também foram submetidas a tratamento térmico em forno (100°C a 250°C). Não houve fusão das fibras até 250°C (até 4 h de tratamento), e poucos danos estruturais. As análises térmicas da FSR e do dispositivo recoberto com as nanofibras de FSR mostraram que o material eletrofiado manteve a estabilidade térmica, com perda por degradação térmica a partir de 250 °C. Os ensaios em CG demonstraram que o branco com o dispositivo de SPME recoberto não interferiu no método proposto para a análise de álcool isopropílico (AIS). Os tempos de extração e dessorção do analito foram otimizados em 20 min e 10 min, respectivamente. A metodologia foi avaliada quanto à linearidade e especificidade. Na faixa de concentração de 10 a 500 ppm de AIS, o método foi linear, com R=0,9927 e seletivo, apresentando alta resolução entre os picos de metanol, AIS e acetato de etila. O processo de eletrofiação da FSR como recobrimento do dispositivo mostrou grande potencial para emprego em SPME devido a sua grande área superficial, estabilidade térmica e facilidade no processo, bem como potencial para aplicação em extração de alcoóis e análise em CG.

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.