Orientador: Antônio Carlos Guastaldi / Banca: André de Souza Tarallo / Banca: Luiz Ricardo Menani / Banca: Vanderlei Salvador Bagnato / Banca: Jonas de Carvalho / Resumo: A reabilitação oral utilizando-se implantes dentários está dentre os procedimentos necessários para a manutenção da qualidade de vida do cidadão. No Brasil, são instalados aproximadamente 1 milhão de implantes por ano. Atualmente o procedimento para a instalação de implantes e, subsequentemente, a reabilitação com próteses sobre implantes tem seus custos determinado com os materiais utilizados e pelo tempo que o profissional dispende neste tipo de procedimento. As metodologias modernas, não acompanham a demanda crescente por este tipo de procedimento. É importante salientar que devido ao grande avanço nos equipamentos utilizados para obtenção de radiografias digitais odontológicas, surge a necessidade do desenvolvimento de novas metodologias computacionais para o auxílio no diagnóstico. Essa tese apresenta o início do desenvolvimento de novas metodologias computacionais para processamento e análise de radiografias digitais odontológicas, metodologias essas de suma importância na área odontológica, pois devido ao envelhecimento da população e cada vez mais a necessidade de reabilitação oral com implantes dentários, demonstra que é de suma importância o desenvolvimento de novas metodologias para auxiliar os profissionais de odontologia em seus diagnósticos. / Abstract: Oral rehabilitation using dental implants is among the procedures necessary to maintain the quality of life of the citizen. In Brazil, approximately 1 million implants are installed per year. Currently, the procedure for the implant installation and, subsequently, the rehabilitation with implants has its costs determined with the materials used and the time that the professional spends on this type of procedure. Modern methodologies do not accompany the increasing demand for this type of procedure. It is important to note that due to the great advance in the equipment used to obtain dental digital radiographs, the need arises for the development of new computational methodologies to aid in diagnosis. This thesis presents the beginning of the development of new computational methodologies for the processing and analysis of dental digital radiographs, which are extremely important methodologies in the dental area, since due to the aging of the population and increasingly the need for oral rehabilitation with dental implants, the development of new methodologies to assist dentistry professionals in their diagnosis is of paramount importance. / Doutor

Over 15 million root canal treatments (RCT) are performed yearly in the United States to treat deep caries and dental pulp infection. This procedure however, removes both the diseased and healthy pulp, leading to tooth devitalization. Furthermore, RCTs are associated with a high incidence of re-infection and dentin fracture, reduced sensitivity and eventual tooth loss. Thus there is an unmet clinical need for alternative endodontic therapies that can preserve tooth vitality and ensure long term dental health. The strategy of vital endodontic therapy explored in this thesis centers on the design of a bioactive scaffold that guides host cell homing while providing antibiotic release, in effect harnessing the intrinsic repair potential of the native pulp while simultaneously eliminating residual bacteria that can cause recurrent infection. Specifically, a bioactive polyethylene glycol fibrinogen (PEG-fibrinogen) hydrogel is optimized to support host cell infiltration, maintain dental pulp cell phenotype, and enable pulp regeneration. Ciprofloxacin, a clinically relevant antibiotic for RCT, is incorporated into PEG-fibrinogen to prevent infection. The scaffold and culturing parameters optimized in vitro using either explant or a tooth slice model includes fibrinogen, poly(ethylene glycol) diacrylate (PEGDA) and photoinitiator concentration, as well as cell source and density. In addition, dose-dependent antibiotic effects on both anaerobic bacteria isolated from deep caries and healthy pulp cells are evaluated. The collective findings of this thesis demonstrate that a cell-instructive hydrogel comprised of a fibrinogen backbone and cross-linked with difunctional poly(ethylene glycol) side chains supports pulp cell viability, phenotypic morphology, and host cell migration. Furthermore, increasing pulp cell density promotes cell biosynthesis and a higher fibrinogen concentration is found to enhance collagen deposition. Photoinitiator and PEGDA concentrations have been optimized to enhance hydrogel mechanical properties and gel degradation, while supporting pulp cell phenotype. An optimal antibiotic dosage in the hydrogel has been identified that significantly reduces bacteria count from infected dental pulp without harmful side effects on dental pulp cell phenotype and host cell migration. In summary, this thesis focuses on the design of a bioactive hydrogel-based scaffold with antibiotic release that can induce dental pulp regeneration without the addition of cells and stimuli such as growth factors and minimize post-therapy infection. The innovative scaffold design strategy presented here lays the foundation for the development of vital endodontic therapy that harnesses pulp self-repair and sustains long-term tooth function.

This thesis focuses on the study of nanotechnology and its applications in immunology and mechanosensing using micro- and nano-scale topographies, such as gratings, grids, and pillar substrates. In the past five years, we have developed three types of platforms and explored the influence of nano-patterned substrates on cell morphology, proliferation, protein secretion, and mechanosensing. I will introduce the three generations of Integrated Mechanobiology Platform (IMP) for T cell study, including the fabrication process of each generation of IMP, their advantages and disadvantages, and the comparison with existing High Throughput Screening System (HTSS). For the applications of IMP, I will focus on grating and grid topographies with IMP generation 3 format, and study how these nano-patterned substrates affect T cell morphology, expansion, cytokine secretion, drug-topography combination effects on T cells and long-term expansion for adoptive immunotherapy. I will demonstrate how IMP enables such studies in a high throughput manner. I also will discuss how Multiple Stiffness Pillar Platform (MSPP) facilitates the study of mechanosensing in cells spanning across different rigidities. First, I will talk about how MSPP is different from existing dual stiffness platforms. Differences include flexibility in distribution of different rigidities, consistency in pillar dimensions and ease of controlling the stiffness fold increase. In the sections of MSPP fabrication and characterization, I will focus on measurements of stiffness change and surface chemistry uniformity. I will then discuss the Mouse Embryonic Fibroblast (MEF) mechanosensing study on dual stiffness pillar substrates, including the preferential localization of rigidity sensing associated proteins (myosin IIA, phosph-myosin, paxillin, and p130CAS), MEFs actomyosin network building, and adhesion formation. These studies revealed previously undiscovered results in MEF mechanosensing, and demonstrate the great potential of MSPP in this research discipline. In the last part of this thesis, I will present on the mass production of thermoplastic nanopatterned molds. The demonstrated technology can produce large batches of nanostructured molds with decreased fabrication time and expense. In this chapter, I will discuss the necessity of developing such a technology and platform, as well as the design, fabrication, and characterization of the thermoplastic nano-patterned molds.

Cells sense and interact with their microenvironment to retrieve signals which include cell-matrix and cell-cell contacts. These signals account for the influence of culturing conditions and often control the local cellular phenotype and global functions of tissues. Here, I sought to understand if there is any information processed by cells in guiding cellular phenotype given the control of cell shapes and substrate rigidities. If there is, would these phenotypic changes achieve biomedical purposes? What is the strategy to engineer platforms that can handle the longstanding challenges in those fields? In this dissertation, the first chapter serves as an introduction which involves the origin of motivations, which mainly came from current challenges in biomedical researches of kidney podocytes. I have attempted to understand if it is possible to control podocyte differentiation through cell shape control which mimics their in vivo morphology. On the other hand, I have tried to reveal if it is possible that tissue stiffness can affect podocyte phenotype as a result of stiffness sensing. These two topics were rarely investigated for kidney podocytes, which is the critical component of human filtration barrier to perform renal functions. The effort that addresses the question how shape and substrate rigidity as in- formation repositories affect kidney podocytes phenotype has profound meaning in the understanding of renal physiological system and pathological mechanisms. The second chapter will focus on the methods to achieve successful long-term shape control on cells. Engineered cell-device interface using cross-linking biomaterial SU-8 plays a key role in this study. Compared with other previously used approaches summarized in this chapter, SU-8 provides various advantages both in the fabrication of micro- pattern architecture as well as its sustaining effectiveness in controlling cell shape. This approach has been proved very efficient and economic to facilitate single cell level manipulation. The chapter will describe in details the interface micro-fabrication and encountered technical challenges. The results that kidney podocytes were in good compliance with the micro-pattern proved the successful application of this technique. The third chapter will then transfer from micro-fabrication to biological experiments, which discusses in details how in intro kidney podocytes responded to their shapes by enforcing protein localization which characterizes a phenotype found in vivo. This phenotype among in vitro podocytes was further verified that it may contribute to podocytes differentiation and physiological functions. The information processed by shape was proved independent of tension-related processes and thus shape and tension could be regarded as separate contributors in cellular development. The interpretation of shape’s contribution could be referred to my previous publication in the journal of Cell: ”Decoding Information in Cell Shape”. In this study, the motifs of research were applied to other cell lines (Human vascular smooth muscle cell) as a step to generalize the ubiquity of shape’s contribution to cell differentiation. The study here was to differentiate shape and tension through investigating the difference between two major mechanosensors: β3 and β1 integrin receptors. The difference in cell phenotypes through integrin inhibition experiments demonstrated critical but unique role of integrin-based shape sensing in vitro. This chapter in dissertation covers most of the content in a previously submitted paper to Nature Cell Biology. In the fourth chapter, I further carried out a study that investigated if stiffness sensing can influence kidney podocyte phenotype. The fourth chapter will basically review the techniques in the fabrication of hydrogel-based cell culture platforms. In a similar manner to previous study using biomimetic shape for podocytes and find its phenotype, the target of this analysis was to use hydrogel-based biomimetic substrate with renal physiological stiffness and find if there is a differentiation phenotype. Since numerous materials have been reported in hydrogel studies, I will focus on the introduction to representative ones that have been most widely used. Their characteristics will be compared with the demands of kidney podocyte reasearch. Methodologies were the key to a successful research, and in this chapter I will describe in details what choices I made in choosing experimental methods that improved the efficiency and quality of cell culture platforms. A natural enzyme (microbial transglutaminase) cross-linked gelatin hydrogel was adopted here to provide ideal substrate rigidity control for podocytes. This method has demonstrated high efficiency and stability in making large cell culture surface. Moreover, it provides the hydrogel platform with an ideal range of elastic moduli suitable for renal tissue culture. The results will be discussed in detail in the fifth chapter. I successfully found a differentiation phenotype for podocytes cultured on the hydrogel platforms with a physiological stiffness. Similar phenotype, on the contrary, were not found in podocytes on platforms which were either too soft or too stiff. These resutls have formed one of my submitted paper to Scientific Report. The differentiation phenotype for kidney podocytes was characterized by up-regulation of differentiation markers. These findings were in a similar manner to a series of stem cells differentiation guided by regulated substrate stiffnesses. This phenotype of kidney podocytes was verified by microarray technique which confirmed the stiffness sensing using transcription factors. The enrichment analysis of kinases also showed significant response of Src, Fyn etc, of which the activities have been shown critical for podocytes to preserve their physiological functions. These results have successfully suggested the close relations between stiffness changes of glomeruli basement membrane (GBM) and progressive podocyte dysfunction. In summary, this dissertation covers interdisciplinary researches that decoded the information processed by cells from two separate aspects: shape and stiffness sensing. The details in each chapter cover a broader scope than the content selected for publications. Through this dissertation, readers will get in touch with the technique developed for plat- form and their applications to biomedical researches. I wish this will help people new in the field to get my hands-on experience.

Avaliou-se a taxa de degradação de elásticos ortodônticos de látex de diferentes fabricantes e diâmetros em diversos intervalos de tempo. Grupos de 15 elásticos de força média dos fabricantes AmericanOrthodontics (Sheboygan, Wis, EUA), Tp (La Porte, IN, EUA), Morelli (Sorocaba, SP, Brasil) e Uniden (Sorocaba, SP, Brasil); de diâmetros 3/16, 1/4 e 5/16 foram analisados nos intervalos de 0,1,3,6,12 e 24 horas, totalizando-se 1080 espécimes. Os elásticos foram estirados individualmente à distância de 30mm, respeitando-se o intervalo de um minuto entre cada estiramento. Os materiais foram acondicionados imersos em água deionizada à 37C. Realizou-se leitura das forças na máquina de ensaios Emic DL 500 (Emic Co, Sao Paulo, Brasil) à velocidade de 30 mm/min, com uma célula de 2Kg (Emic Co, Sao Paulo, Brasil). A leitura de cada elástico consumiu aproximadamente um minuto. Teste Kruskal-wallis com correções por Dunns aferiu significância estatística dos resultados. Foram observadas diferenças entre os grupos analisados, exceto às marcas Morelli e Tp. Foram significativas as inferências das variáveis tempo e marca comercial. Em 0 hora, a relação entre as forças geradas foi Morelli>AO>Uniden>Tp para elásticos 3/16 (p=0,0016) e 1/4 (p=0,0016) e, de Morelli>AO>Tp>Uniden para elásticos 5/16 (p=0,0087). Após 24 horas, as porcentagens de degradação dos elásticos foram AO>Morelli>Uniden>Tp no diâmetro 3/16; AO>Tp>Morelli>Uniden no diâmetro 1/4 e Tp>AO>Uniden>Morelli para elásticos de diâmetro 5/16. O comportamento no intervalo de 0-24 horas demonstrou uma queda acentuada no período de 0-3 horas, um ligeiro aumento em 3-6 horas, seguido de uma queda progressiva no intervalo de 6-24 horas. / The purpose of this study was to evaluate the force relaxation of different brands and diameters of latex elastics occurring during different times. Groups of 15 medium forces elastics from manufacturers: American Orthodontics (Sheboygan, Wis, USA), Tp (La Porte, IN, USA), Morelli Orthodontics (Sorocaba, SP, Brazil) and Uniden Orthodontics (Sorocaba, SP, Brazil) with diameters: 3/16, 1/4 and 5/16 inch size were tested at 0,1,3,6,12,24 hours, making a total of 1080 specimens. An apparatus was designed to simulate oral environments during elastics stretching. The forces were read after 1,3,6,12 and 24-hour periods in Emic testing machine (Emic Co, Sao Paulo, Brazil) with 30 mm/min cross-head speed and load cell of 2Kg Emic force (Emic Co, Sao Paulo, Brazil). Kruskal-Wallis and Dunns tests were used to identify statistical significance. Overall, there were statistically differences among the different manufacturers at all observation intervals, except between Morelli and Tp. Significantly variability were seen in Morelli and Uniden force delivery. The relationships among loads at 0-hour time period were as follows: Morelli>AO>Uniden>Tp for 3/16 (p=.0016) and 1/4 elastics (p=.0016) and Morelli>AO>Tp>Uniden for 5/16 elastics (p=.0087). The forces relaxation over the 24-hour time period were AO>Morelli>Uniden>Tp for 3/16 elastics; AO>Tp>Morelli>Uniden for 1/4; and Tp>AO>Uniden>Morelli for 5/16 elastics.The force decay pattern show a notable drop-off of forces during 0-3 hours, a slight increase in force values from 3-6 hours and a progressive force decay was seen over 6-24 hours.

Orientador: Ana Paula Rosifini Alves Claro / Banca: Claudia Eliana Bruno Marino / Banca: Carlos Roberto Grandini / Resumo: Titânio e suas ligas são amplamente empregados em aplicações biomédicas, devido a sua biocompatibilidade e excelentes propriedades de volume, como resistência mecânica. As ligas de titânio tipo β com baixo módulo de elasticidade, como Ti10Mo8Nb, são indicadas para amenizar o efeito "stress shielding", característico da interface implante/osso. No entanto, quando materiais bioinertes são inseridos no corpo humano não induzirão uma resposta específica. As técnicas de modificação de superfície, como crescimento de nanoestruturas de TiO2, são utilizadas para alterar as propriedades de superfície desses materiais permitindo melhor resposta. O objetivo dessa pesquisa é a modificação de superfície da liga experimental Ti10Mo8Nb a partir do crescimento de nanoestruturas de TiO2. Os lingotes da liga experimental Ti10Mo8Nb foram obtidos a partir da fusão dos metais puros em forno a arco voltaico. A oxidação anódica potenciostática foi realizada utilizando um eletrólito formado por glicerina e H2O (1:1, em vol.) com adição de 2,7 % (m/v) de NH4F sob 20 V durante 3 h, a temperatura ambiente. Após o tratamento as amostras foram calcinadas. A superfície da liga foi analisada por microscopia eletrônica de varredura (MEV), difração de raios X (DRX), ângulo de contato, medidas da energia de superfície, espectroscopia de raios X por dispersão em energia (XPS). Estudos in vitro, como adesão celular e bacteriana, foram realizados para avaliar a resposta desse tratamento. A liga Ti10Mo8Nb apresen... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Titanium and its alloys have been widely used as biomedical material due their biocompatibility and excellent bulk properties, such as mechanical strength. β titanium alloys with low modulus of elasticity, such as Ti10Mo8Nb, are suitable to relieve the stress shielding effect that occurs in the interface implant/bone. However, when these materials are inserted inside human body the surface properties will not induce a specific response. Surface modifications techniques can be used for change the inert surface of these alloys, e.g. TiO2 nanostructure growth by using anodic oxidation. The purpose of this research is the surface modification of Ti10Mo8Nb experimental alloy by using TiO2 nanostruture growth. Ingots of Ti10Mo8Nb experimental alloy were produced by fusion from sheets of molybdenum, niobium and titanium commercially pure in arc melting furnace under argon atmosphere. The potentiostatic anodic oxidation was performed using an electrolyte formed of glycerol and H2O (1: 1 by vol.) with addition of 2.7% (w/v) NH4F under 20 V for 3 h at room temperature. After the surface treatment, the samples were annealing. The surface of the alloy was analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), contact angle, surface energy measurements, energy dispersive X-ray spectroscopy (XPS). In vitro studies, such as cell and bacterial adhesion, were performed to evaluate the response of this treatment. The Ti10Mo8Nb alloy exhibited beta phase after the processing, and also occurred a decrease in β-transus temperature. After the surface treatment a nanoporous layer of TiO was obtained. The anatase phase was found in the annealed samples (450 °C for 3 hours). In vitro analyzes showed cell growth viability and decreased bacterial proliferation due to larget specific surface area / Mestre

Orientador: Ana Paula Rosifini Alves Claro / Banca: Laís Regiane da Silva Concílio / Banca: Andrea Carvalho de Marco / Resumo: Titânio e suas ligas são considerados materiais bioinertes e dessa forma técnicas de modificação de superfície podem ser empregadas para uma melhor interação osso/biomaterial. Assim, o presente estudo teve por objetivo avaliar a modificação da superfície da liga experimental Ti25Ta25Nb3Sn a partir do emprego do tratamento alcalino, com vistas à obtenção de uma superfície bioativa. Os lingotes das ligas foram obtidos em forno a arco voltaico e posteriormente foram submetidos a tratamento térmico e forjamento a frio, de tal forma que discos com 10 mm de diâmetro e 3mm de espessura fossem obtidos. Para o tratamento alcalino as amostras foram imersas em solução de NaOH na concentração de 0,5, 1,0 e 1,5M. A morfologia das superfícies das amostras foi avaliada por meio do microscópio eletrônico de varredura e a composição avaliada por espectroscopia de energia dispersiva (EDS). As fases presentes após tratamento foram avaliadas por técnicas de difração de raios-X e a molhabilidade das superfícies a partir do ângulo de contato. Estudos in vitro foram realizados para avaliar a adesão bacteriana e proliferação celular. Os resultados evidenciam a produção de uma estrutura porosa na superfície da liga quando ela foi submetida ao tratamento com NaOH em todas as molaridades usadas. A molhabilidade da superfície apresentou caraterística hidrofílica, o difratograma revelou picos mais intensos após o tratamento alcalino. Verificou também maior quantidade de bactérias nas superfícies porosas ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Titanium and its alloys are considered bioinert materials and thus surface modification techniques can be employed for better bone / biomaterial interaction. Thus, the present study aimed to evaluate the surface modification of the experimental alloy Ti25Ta25Nb3Sn to obtain a bioactive surface, from the use of alkaline treatment. The ingots of the alloys were obtained in an arc furnace and afterwards they were subjected to thermal treatment and cold forging, in such a way that disks with 10 mm of diameter and 3 mm of thickness were obtained. For the alkaline treatment the samples were immersed in NaOH solution at the concentration of 0.5, 1.0 and 1.5M. The morphology of the surfaces of the samples was evaluated by scanning electron microscopy and the composition evaluated by dispersive energy spectroscopy (EDS). The phases present after treatment were evaluated by X-ray diffraction techniques and the wettability of the surfaces from the contact angle. In vitro studies were performed to evaluate bacterial adhesion and cell proliferation. The results evidenced the production of a porous structure on the alloy surface when it was subjected to NaOH treatment in all the molarities used. The wettability of the surface presented a hydrophilic character, the diffractogram showed more intense peaks after the alkaline treatment. It also verified a greater amount of bacteria in the porous surfaces resulting from the alkaline treatment and greater cell adhesion with viability without variation between the treatments. It is concluded that the treatment with 1.0M NaOH provides a bioactive surface / Mestre

University of Macau / Faculty of Science and Technology / Department of Electromechanical Engineering

No description available.

The microstructure and mechanics of collagen and elastin protein fiber networks dictate the mechanical responses of all soft tissues and related organ systems. In this project, we endeavored to meet or exceed native tissue mechanical properties through mimicry of these extracellular matrix components with synthetic collagen fiber and elastin analogues. Significantly, these studies led to the development of a framework for the design and fabrication of protein-based soft tissue substitutes that reproduced many aspects of native biomechanics. A scalable process was developed for production of synthetic collagen microfibers at a rate of 60 m/hr. Fiber properties and ultrastructure were characterized by uniaxial mechanical testing, differential scanning calorimetry, transmission electron microscopy, and second harmonic generation analysis. In vivo responses to synthetic fibers were evaluated in a murine model. A scalable, semi-automated process was designed for the fabrication of multilamellar membranes comprised of sheets of an elastin analogue reinforced with synthetic collagen fibers. Fibers could be organized in a precisely defined three-dimensional hierarchical pattern. The structure of these fiber composites was analyzed by scanning and transmission electron microscopy, and digital volumetric imaging. The effects of fiber orientation and volume fraction on uniaxial mechanical responses were evaluated. Increased fiber volume fraction and alignment increased Young's modulus, resilience, and yield stress. Highly extensible, elastic tissues display a functionally significant transition from low to high modulus deformation at a transition point strain dictated by the crimped collagen microstructure. This response was replicated by the fabrication of dense arrays of microcrimped synthetic collagen fibers embedded in an elastin analogue. The degree of microcrimping could be varied, and generated a transition point mechanical response. Cyclic tensile deformation did not substantially alter microcrimp morphology. A series of small-diameter vascular grafts consisting of an elastin-like protein reinforced with controlled volume fractions and orientations of synthetic collagen fiber was designed and prototyped. The optimal design satisfied target properties with suture retention strength of 173 ± 4 g-f, burst strength of 1483 ± 143 mm Hg, and compliance of 5.1 ± 0.8 %/100 mm Hg.