Les microsphères polymériques, biodégradables et biocompatibles sont prometteuses comme échafaudages pour fournir des cellules aux tissus sans dommage et améliorer la réponse des cellules souches. Il a été montré que les microparticules à base de PLGA à base de protéines ECM préparées par une technique d'extraction par solvant en émulsion améliorent l'adhésion et la prolifération des cellules souches. Bien qu'un certain effet biologique du revêtement protéique sur les microparticules de polymère soit établi, il n'est pas bien compris car des informations détaillées sur l'interaction entre les propriétés de surface des particules chimiques et physiques, l'adsorption protéique et la réponse cellulaire restent floues. Le but de cette étude est d'établir une relation entre les propriétés de surface des particules qui proviennent des variables utilisées lors des formulations, l'adsorption des protéines et la réponse des cellules souches pour permettre une conception plus rationnelle des microparticules. Deux types de polymères différents (PLGA et PLGA-P188-PLGA) et deux stabilisants d'émulsion différents; un polymérique PVA et une huile polaire Propylène glycol ont été utilisés pour préparer quatre types de particules dans un procédé en émulsion. Les particules ont été caractérisées en termes de taille, charge, topographie et morphologie et chimie de surface Identification des effets des variables dans la chimie et l'émulsifiant des polymères Des surfaces de polymère plat recouvertes de spins sont générées pour comprendre les interactions entre les polymères et les molécules d'adhésion de la fibronectine et de la poly-D-Lysine pour la fonctionnalisation des microparticule. / Polymeric, biodegradable and biocompatible microspheres are promising as colloidal scaffolds to deliver cells to tissues without damage and to enhance stem cell survival. It has been shown that PLGA (poly(lactic-co-glycolic acid)) based microparticles prepared by an emulsion solvent extraction technique and functionalised with ECM proteins improves stemcell adhesion and proliferation. While it is established that the presence of proteins in these systems has abiological effect, the interplay between microparticle properties and cells is poorly understood because there lationship between chemical and physical particle surface properties, protein adsorption and cell response remain unclear. Protein adsorption on a polymer particle surface is a complex phenomenon that is affected by different interfacial mechanisms/forces (e.g. DLVO and non-DLVO forces) and inherents tructural properties of macromolecules (e.g. polymers, surfactants, peptides and proteins) present on surface, establishing the parameters that involves inprotein/peptide adsorption on microparticles insights the logical design of the particles as a biomaterial useby contributing the understanding of another related complex phenomenon of the colloidal biomaterial-cell interface interaction in tissue engineering where limited study available to fully understand the concept.The purpose of this study is to investigate the impact of different formulation approaches on the physicochemical properties of the microparticles and identify connections between the particle properties, protein adsorption and ensuing cell response on these materials. To examine these, two different polymertypes (PLGA and PLGA-P188-PLGA) and two different emulsion stabilizers; a polymeric surfactant (PVA(Polyvinyl alcohol) and a polar oil propylene glycol were used to prepare four different types of particles in an emulsion process (O/W). Particles were characterized in terms of size, charge, topography, morphology and surface chemistry to identify the effect of the variables of polymer chemistry and use of surfactant on particle properties in first part of the study. Spin coated flatpolymer surfaces were generated to understand the interactions between the polymers and the proteins (fibronectin /poly-d-Lysine).

This work describes the realization of physically crosslinked networks based on gelatin by the introduction of functional groups enabling specific supramolecular interactions. Molecular models were developed in order to predict the material properties and permit to establish a knowledge-based approach to material design. The effect of additional supramolecular interactions with hydroxyapaptite was then studied in composite materials. The calculated properties are compared to experimental results to validate the models. The models are then further used for the study of physically crosslinked networks. Gelatin was functionalized with desaminotyrosine (DAT) and desaminotyrosyl-tyrosine (DATT) side groups, derived from the natural amino acid tyrosine. These group can potentially undergo to π-π and hydrogen bonding interactions also under physiological conditions. Molecular dynamics (MD) simulations were performed on models with 0.8 wt.-% or 25 wt.-% water content, using the second generation forcefield CFF91. The validation of the models was obtained by the comparison with specific experimental data such as, density, peptide conformational angles and X-ray scattering spectra. The models were then used to predict the supramolecular organization of the polymer chain, analyze the formation of physical netpoints and calculate the mechanical properties. An important finding of simulation was that with the increase of aromatic groups also the number of observed physical netpoints increased. The number of relatively stable physical netpoints, on average zero 0 for natural gelatin, increased to 1 and 6 for DAT and DATT functionalized gelatins respectively. A comparison with the Flory-Rehner model suggested reduced equilibrium swelling by factor 6 of the DATT-functionalized materials in water. The functionalized gelatins could be synthesized by chemoselective coupling of the free carboxylic acid groups of DAT and DATT to the free amino groups of gelatin. At 25 wt.-% water content, the simulated and experimentally determined elastic mechanical properties (e.g. Young Modulus) were both in the order of GPa and were not influenced by the degree of aromatic modification. The experimental equilibrium degree of swelling in water decreased with increasing the number of inserted aromatic functions (from 2800 vol.-% for pure gelatin to 300 vol.-% for the DATT modified gelatin), at the same time, Young’s modulus, elongation at break, and maximum tensile strength increased. It could be show that the functionalization with DAT and DATT influences the chain organization of gelatin based materials together with a controlled drying condition. Functionalization with DAT and DATT lead to a drastic reduction of helical renaturation, that could be more finely controlled by the applied drying conditions. The properties of the materials could then be influenced by application of two independent methods. Composite materials of DAT and DATT functionalized gelatins with hydroxyapatite (HAp) show a drastic reduction of swelling degree. In tensile tests and rheological measurements, the composites equilibrated in water had increased Young’s moduli (from 200 kPa up to 2 MPa) and tensile strength (from 57 kPa up to 1.1 MPa) compared to the natural polymer matrix without affecting the elongation at break. Furthermore, an increased thermal stability from 40 °C to 85 °C of the networks could be demonstrated. The differences of the behaviour of the functionalized gelatins to pure gelatin as matrix suggested an additional stabilizing bond between the incorporated aromatic groups to the hydroxyapatite. / Diese Arbeit beschreibt die Entwicklung von durch spezifische physikalische Wechselwirkungen quervernetzten Gelatine-basierten Materialien. Dazu wurden zunächst Computermodelle entwickelt, mit denen Eigenschaften der Materialien vorhergesagt werden sollten, um so eine wissensbasierte Entwicklung zu ermöglichen, um dann die Ergebnisse mit experimentellen Daten zu vergleichen und die Materialien und Modelle als Grundlage für weitere Entwicklungen zu nutzen. Gelatine wurde mit Desaminotyrosin (DAT) und Desaminotyrosyltyrosin (DATT) funktionalisiert, die sich von der natürlichen Aminosäure Tyrosin ableiten. Diese Gruppen können potentiell π-π Wechselwirkungen und Wasserstoffbrückenbindungen auch unter physiologischen Bedingungen eingehen. Es wurden Computersimulationen der Materialien mittels Moleküldynamik durchgeführt, wobei Modelle mit 0.8 Gew.-% und 25 Gew.-% Wassergehalt betrachtet wurden. Die Validierung der Modelle erfolgte durch Vergleich der errechneten mit experimentellen Daten wie z.B. der Dichte, Bindungswinkeln sowie Röntgenstreuungsspektren. Die Modelle wurden dann zur Vorhersage der molekularen Organisation der Polymerketten, Formierung physikalischer Netzpunkte und Berechnung der mechanischen Eigenschaften eingesetzt. Die Funktionalisierung der Gelatine mit DAT bzw. DATT führten wie gewünscht zur Ausbildung physikalischer Netzpunkte durch π-π Wechselwirkungen und Wasserstoffbrücken¬bindungen. Ein Schlüsselergebnis der Simulationen war, dass mit zunehmender Zahl an aromatischen Gruppen auch eine Zunahme der physikalischen Netzpunkte beobachtet werden konnte. Die funktionalisierten Gelatinen konnten durch chemoselektive Reaktion der Aminogruppen der Gelatine mit den freien Carboxylgruppen von DAT und DATT hergestellt werden. Materialien mit 25 Gew.-% Wassergehalt hatten in der Simulation und im Experiment mechanische Eigenschaften derselben Größenordnung (z.B. E-Moduln im unteren GPa-Bereich). Der Quellungsgrad der Materialien im Experiment nahm mit zunehmender Zahl an aromatische Gruppen ab (von 2800 Vol.-% auf 300 Vol.-%), wobei der Elastizitätsmodul, die Bruchdehnung sowie die Zugfestigkeit zunahmen. Die Funktionalisierung der Gelatine ist eine chemische Methode, um die Kettenanordnung auf molekularer Ebene zu beeinflussen, während die genaue Kontrolle der Trocknungs¬bedinguungen von Gelatine-basierten Materialien eine physikalische Methode mit demselben Ziel ist. Es konnte gezeigt werden, dass die Funktionalisierung von Gelatine mit DAT oder DATT zu einer stark verminderten Helixausbildungstendenz, die jedoch durch Variation der Trocknunsgbedingungen noch fein abgestimmt werden konnte. Somit konnten die mechanischen Eigenschaften von Filmen aus funktionlisierter Gelatine mit zwei unabhängigen Methoden eingestellt werden. Komposite der mit DAT oder DATT funktionalisierten Gelatine und Hydroxyapatit (HAp) zeigten deutlich verringerter Quellung. In Zugdehnungsexperimenten und rheologischen Untersuchungen zeigten die Komposite im Gleichgewichtsquellungszustand erhöhte Elastizitätsmoduln (von 200 kPa auf bis zu 2 MPa) und Zugfestigkeit (von 57 kPa auf bis zu 1.1 MPa). Darüber hinaus konnte die Übergangstemperatur Tc deutlich gesteigert werden (von ca. 40 °C auf > 85 °C). Dieses Verhalten ließ sich auf stabilisierende Bindungen zwischen den aromatische Gruppen und dem HAp zurückführen.

This thesis describes a number of novel experiments contributing to the understanding of protein adsorption from both a fundamental and applied perspective. The first three papers involve the use of the localized surface plasmon resonance of gold nanospheres to measure protein conformational dependencies during heat and acid denaturation. Thermal denaturation of BSA is shown to proceed differently depending on the size of nanosphere to which it is conjugated. Activation energies are extracted for thermal denaturing on nanoparticles. These energies decrease with decreasing radius of curvature. Under pH perturbation in the acid region, the multiple transition states of bulk BSA are suppressed, and only one apparent transition around pH 4 is evident. Smaller spheres (diameter < 20nm) do not exhibit any transition. A significant finding of all three studies is that the state and stability of BSA depends strongly upon local curvature. The last two papers investigate protein adsorption relevant to the biomaterial field. Investigation of protein adsorption to polyHEMA hydrogels is carried out using a quartz crystal microbalance. Single and mixed protein adsorption kinetics for BSA, lysozyme and lactoferrin are extracted and interpreted. Selected commercial cleaning solutions are shown to be no more effective than simple buffer solution. Examination of commercial lenses indicates that the morphology of adsorption is material dependent and that siloxane-based hydrogels only deposit low levels of protein. A unique fibril-like morphology is identified on galyfilcon A. Protein morphology is discussed in terms of bare lens morphology, roughness, and surface composition.

This thesis describes a number of novel experiments contributing to the understanding of protein adsorption from both a fundamental and applied perspective. The first three papers involve the use of the localized surface plasmon resonance of gold nanospheres to measure protein conformational dependencies during heat and acid denaturation. Thermal denaturation of BSA is shown to proceed differently depending on the size of nanosphere to which it is conjugated. Activation energies are extracted for thermal denaturing on nanoparticles. These energies decrease with decreasing radius of curvature. Under pH perturbation in the acid region, the multiple transition states of bulk BSA are suppressed, and only one apparent transition around pH 4 is evident. Smaller spheres (diameter < 20nm) do not exhibit any transition. A significant finding of all three studies is that the state and stability of BSA depends strongly upon local curvature. The last two papers investigate protein adsorption relevant to the biomaterial field. Investigation of protein adsorption to polyHEMA hydrogels is carried out using a quartz crystal microbalance. Single and mixed protein adsorption kinetics for BSA, lysozyme and lactoferrin are extracted and interpreted. Selected commercial cleaning solutions are shown to be no more effective than simple buffer solution. Examination of commercial lenses indicates that the morphology of adsorption is material dependent and that siloxane-based hydrogels only deposit low levels of protein. A unique fibril-like morphology is identified on galyfilcon A. Protein morphology is discussed in terms of bare lens morphology, roughness, and surface composition.

The host response toward biomaterial component of tissue-engineered devices has been extensively investigated. The objective of this research was to understand the response of dendritic cells (DCs) to different biomaterials upon contact and identify biomaterials suitable for use in tissue engineering constructs for rheumatoid arthritis (RA) applications. Differential levels of functional DC maturation were observed depending on the type of biomaterial in 2-dimensional films or 3-dimensional scaffolds used to treat immature DCs; Poly(lactic-co-glycolic acid) (PLGA) or chitosan supported higher levels of DC maturation, as compared to immature DCs. Alginate supported moderate levels of DC maturation. Agarose did not support DC maturation whereas hyaluronic acid inhibited DC maturation. Further, these DCs treated with different biomaterials induced differential phenotype and polarization of autologous T cells upon co-culture of DCs and T cells; DCs treated with PLGA induced T helper type I with immunogenic response while DCs treated with agarose did T helper type II with tolerogenic response. Effect of different biomaterials (PLGA and agarose) was assessed in vivo upon implantation of them into the knee joint of RA-induced rabbit. Total leukocyte concentrations in the peripheral blood or in the joint lavage of the left knees (untreated control) were observed in differential levels depending on the biomaterial implant, possibly due to the systemic circulation of the peripheral blood. Furthermore, cartilage and bone healing progression was differentially observed in the osteochondral defect of the knee joint of RA-induced rabbit, depending on type of biomaterial scaffold implanted into the defect. Collectively, these results demonstrate the multifunctional impacts of inherently different biomaterials on in vitro immunomodulation of phenotype and polarization of DCs and autologous T cells. Furthermore, taken together with these immunomodulatory impacts of biomaterials, in vivo effects of different biomaterial scaffolds on RA environment shown in this study can suggest the criteria of selection and design of biomaterials for orthopedic tissue engineering, which may ultimately be best integrated into the diseased cartilage and bone.

Made available in DSpace on 2016-06-02T20:18:11Z (GMT). No. of bitstreams: 1 2456.pdf: 6548607 bytes, checksum: 486173aee7443e35a2910af043a12256 (MD5) Previous issue date: 2009-04-03 / Universidade Federal de Sao Carlos / Bioactive materials have the ability to bond and to integrate with bone tissue by forming a biologically active bonelike apatite layer, which has chemical and structural properties equivalent to the mineral phase of living bone. This process is determined by chemical reactions, whose products also influence the attachment, the proliferation, the differentiation and the mineralizing capacity of bone cells. Cellular responses contribute to the bioactive behavior, which is known for being higher in glass materials. However, as low mechanical properties are also inherent characteristics of glasses, researchers from Federal University of Sao Carlos were stimulated to develop nucleation and growth thermal treatments for the obtainment of the Biosilicate®, a fully-crystallized bioactive glassceramic of the quaternary system P2O5-Na2O-CaO-SiO2. Although a high in vitro osteogenic potential of this novel glass-ceramic has been previously demonstrated, its in vivo effects have not been investigated yet. To contribute to this knowledge, two studies were developed. The first one aimed to investigate the in vivo biological performance of Biosilicate® in bone defects of rat tibias, by means of hystomorphometric and biomechanical analyses 20 days after the surgical procedure. This study revealed that the fully-crystallized Biosilicate® has good bone-forming and bone-bonding properties. Hence, the second study aimed to compare the kinetics of the bone reactions to two different granulometric distributions of this novel glass-ceramic. Although they were both efficient for bone formation, smaller-sized particles of Biosilicate® showed partial reabsortion, which was accompanied by a more pronounced osteogenic activity within the period of time studied. Since positive results were obtained, the search for scaffolds that could serve as supports for the guided bone regeneration had started. A third study preliminarily evaluated cell culture and cocultures in porous structures made of Biosilicate® and of other chemical compositions that were specifically developed for this purpose. The findings suggest that, when in adjusted conditions, the scaffolds can create favorable cellular responses for bone tissue engineering purposes. Taken togheter, these studies point to a promising potential and provide directives for the use of Biosilicate® in bone regenerative processes. / Materiais bioativos possuem a capacidade de se ligar ao tecido ósseo por meio da formação de uma interface apatítica que apresenta similaridade química e estrutural com a fase mineral dos ossos. Esse processo ocorre devido a uma série de reações químicas, cujos produtos também influenciam a adesão, a proliferação, a diferenciação e a capacidade de mineralização da matriz pelas células ósseas. As respostas celulares contribuem para o comportamento bioativo, que é conhecido por ter maiores índices em materiais vítreos. No entanto, como baixas propriedades mecânicas também são características inerentes aos vidros, pesquisadores da Universidade Federal de São Carlos foram estimulados a empregarem nucleação e tratamentos térmicos especiais para o desenvolvimento do Biosilicato®, uma vitrocerâmica biotiva, totalmente cristalina, pertencente ao sistema quaternário P2O5-Na2O-CaO-SiO2. Embora um elevado potencial osteogênico in vitro tenha sido demonstrado para esta vitrocerâmica, seus efeitos in vivo ainda não são conhecidos. Para auxiliar este entendimento, foram desenvolvidos dois estudos. O primeiro teve como objetivo investigar o desempenho biológico in vivo do Biosilicato® particulado em defeitos ósseos em tíbias de ratos, por meio de análises histomorfométricas e biomecânicas 20 dias após o procedimento cirúrgico. Este estudo evidenciou que o Biosilicato® parece favorecer a formação óssea in vivo e o estabelecimento de fortes ligações com o tecido neoformado. Com isso, o objetivo do segundo estudo foi comparar a cinética das reações ósseas frente a duas diferentes distribuições granulométricas desta nova vitrocerâmica. Embora ambas tenham sido eficientes para a formação óssea, as partículas de Biosilicato® com menores diâmetros demonstraram reabsorção parcial no período estudado, que foi acompanhada de uma maior atividade osteogênica. Com os resultados positivos obtidos nestas investigações, iniciou-se uma busca para o desenvolvimento de matrizes porosas que pudessem servir de suporte para a regeneração guiada do tecido ósseo. Um terceiro estudo preliminarmente avaliou monoculturas e coculturas celulares em matrizes porosas de Biosilicato® e de outras novas composições químicas desenvolvidas especificamente para este propósito. Os achados sugerem que, quando em condições adequadas, as matrizes avaliadas podem produzir respostas celulares favoráveis ao seu emprego na engenharia do tecido ósseo. Estes estudos, de maneira conjunta, apontam para um potencial promissor e fornecem diretrizes para o emprego do Biosilicato® no favorecimento de processos regenerativos ósseos.

Made available in DSpace on 2016-06-02T20:19:12Z (GMT). No. of bitstreams: 1 2618.pdf: 1011844 bytes, checksum: acc696873b3edd56b2bb4801a43ed7e8 (MD5) Previous issue date: 2009-08-07 / Financiadora de Estudos e Projetos / Osteoporosis is generally a systematic skeletal disease characterized by low bone density and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility. Osteopenia is characterised by a lower bone density than normal bone tissue but higher than osteoporotic bone tissue. Osteoporosis and osteopenia represent a severe health threat to elderly people and they have recently been recognised as a major public health problem. They are related with some clinic manifestations, mainly the increase of the fractures risk. In this context, there is a critical need to develop technologies able of treating osteoporotic and osteopenic fractures. Promising treatments are the use of biomaterials and the Low Level Laser Therapy (LLLT), which seem to induce osteogenesis and stimulate fracture healing. The aim of this study was to determine the effects of the Biosilicate® and the LLLT on bone consolidation of osteopenic rat. Seventy female Wistar rats (12 weeks-old, ± 250g) ovariectomy (OVX) was carried on. The animals were randomly divided into 7 groups, with 10 animals each: standard control (CP); defect bone control (CD), bone defect treated with Biosilicate® (B), bone defect treated with laser 60J/cm2 (L60), bone defect treated with laser 120J/cm2 (L120), bone defect treated with both treatments Biosilicate® and laser 60J/cm2 (B+L60), bone defect treated with both treatments Biosilicate® and laser 120J/cm2 (B+L120). Sixty days post-OVX the osteotomies were surgically performed on the left tibia. In the Biosilicate® treated animals, the cavities were carefully filled with the biomaterial. An 830nm laser was performed for seven sessions. On day 14 post-osteotomy, rats were sacrificed and the tibias were defleshed. Biomechanical properties of the tibia were determined by two tests: the Indentation Test to a depth of 0-0.5mm, 0-1.0mm and 0-1.5mm and the Three-Point Bending Test. From de load-deformation curve, the maximal load (KN) and energy absorption (J) were obtained. Statistical analyses were performed using Kruskal-Wallis Test and Mann-Whitney U Test, with the level of significance of 5% (p&#8804;0.05). In relation to biomechanical properties of the Indentation Test, the groups CP, B, B+L60 and B+L120 presented higher statistically values (p<0.05) in relation to group CD. The better biomechanical answer among the groups was present by the group with Biosilicate® utilization in association of 120J/cm². In relation to maximal load of the Three-Point Bending Test, the group L60 presented higher statistically values (p< 0.05) in relation to group CP, B, L120, B+L60, B+L120 and the groups had similar values of absorption energy. The Biosilicate® application raised the biomechanical properties of the callus bone, but didn t raise the biomechanical properties of the tibia determined by Three-Point Bending Test. In the two biomechanical tests, the groups with treatment based only on laser irradiation haven t presented significant results in relation to group control fracture. The bones defects treated with both treatments Biosilicate® and laser 120J/cm2 presented higher biomechanical properties of the callus bone in relation to the group based only Biosilicate® application. / A osteoporose é uma doença esquelética sistêmica caracterizada por baixa densidade óssea e deterioração da microarquitetura do tecido ósseo com conseqüente aumento da fragilidade óssea. A osteopenia é caracterizada por apresentar uma menor densidade óssea que o tecido ósseo normal, mas maiores valores do que o tecido ósseo osteoporótico. A osteoporose e a osteopenia representam um grave problema de saúde a idosos e recentemente têm sido reconhecidos como um dos principais problemas de saúde pública, principalmente por aumentar o risco de fraturas. Neste contexto, há uma grande necessidade de se desenvolver tecnologias capazes de tratar fraturas em organismos osteoporóticos e osteopênicos. Existem diversos tratamentos promissores, como o uso do Biosilicato® e da terapia laser, os quais parecem induzir a osteogênese e estimulam o reparo ósseo. O objetivo deste estudo foi determinar os efeitos do o Biosilicato® e da terapia laser sobre o reparo ósseo de ratas osteopênicas. Setenta ratas fêmeas da raça Wistar (12-semanas, ± 250g) foram ovariectomizadas e divididas em sete grupos com 10 animais em cada: controle padrão (CP), controle com defeito ósseo (CD), tratado com Biosilicato® (B), tratado com 60J/cm2 (L60), tratado com 120J/cm2 (L120), tratado com Biosilicato® e 60J/cm2 (B+L60), tratado com Biosilicato® e 120J/cm2 (B+L120). Sessenta dias após a ovariectomia, as tíbias foram osteotomizadas. Nos animais tratados com Biosilicato®, a cavidade do defeito ósseo foi preenchida com o biomaterial. Os animais foram irradiados com 830nm por sete sessões (48- 48h). No décimo quarto dia após a osteotomia, as ratas foram sacrificadas e as tíbias dessecadas. As propriedades biomecânicas das tíbias esquerdas foram determinadas por dois testes: O Teste de Endentação com profundidade de 0-0,5mm, 0-1,0mm e 0-1,5mm que avaliou as propriedades biomecânicas do calo ósseo e o Teste de Flexão a Três Pontos que avaliou as propriedades biomecânicas das regiões ósseas integras do osso osteotomizado, sem considerar a região do calo ósseo que foi utilizada no teste de Endentação. A carga máxima (KN) e a energia de absorção (J) foram obtidas da curva de carga-deslocamento. A análise estatística foi realizada pelo Teste de Kruskal-Wallis e o Teste de Mann-Whitney U, com p&#8804;0,05. Em relação ao Teste de Endentação, os grupos CP, B, B+L60 e B+L120 apresentaram maiores valores estatísticos (p<0,05) em relação ao grupo CD. A melhor resposta biomecânica do Teste de Endentação foi apresentada pelo grupo B+L120. Em relação à carga máxima do Teste de Flexão a Três Pontos, o grupo L60 apresentou maiores valores estatísticos (p<0,05) do que os grupos CP, B, L120, B+L60, B+L120 e todos os grupos tiveram valores semelhantes de energia de absorção. Pode-se concluir que a aplicação do Biosilicato® aumentou as propriedades biomecânicas do calo ósseo e não alterou as propriedades das regiões ósseas integras. A terapia laser não promoveu diferença significativa das propriedades biomecânicas do calo ósseo e das regiões ósseas integras e a associação dos dois tratamentos com fluência de 120J/cm² promoveu um aumento das propriedades biomecânicas do calo ósseo a valores mais elevados do que o grupo que utilizou somente o biomaterial.

A liga Ti-6Al-4V tem sido usada com sucesso como biomaterial com aplicações nos campos da odontologia e ortopedia. As características da liga Ti-6Al-4V que a tornaram um material interessante são sua boa resistência à corrosão em meio biológico, combinada com um excelente grau de biocompatibilidade. Os efeitos biológicos a longo prazo dos íons metálicos resultantes da lenta lixiviação dos implantes de titânio não estão completamente entendidos. É conhecido que os íons titânio são considerados agentes químicos cancerígenos, os íons alumínio causam desordem neurológicas e os íons vanádio estão associados com distúrbios enzimáticos, entre outros problemas. O recobrimento das ligas de titânio por óxido de titânio (TiO2) pode atuar como uma barreira química para os íons lixiviados da superfície metálica da liga, além deste óxido ser um bom osseoindutor. O processo de aspersão térmica é amplamente empregado na aplicação de recobrimentos por óxidos. Uma vez que este processo acontece em altas temperaturas, onde partículas fundidas ou semi-fundidas aderem ao substrato, pode ocorrer difusão localizada. O objetivo deste estudo é caracterizar a interdifusão dos elementos de liga através das camadas superficiais do sistema Ti-6Al-4V/TiO2. Os recobrimentos de TiO2 foram preparados pela técnica de aspersão por plasma. A zona recoberta mostrou pequena porosidade distribuída por toda a camada e algumas trincas radiais. Adicionalmente, uma grande quantidade de buracos foi observada na região entre-camadas. Neste trabalho, a interface Ti-6Al-4V/TiO2 foi estudada por microscopia eletrônica de varredura. Os perfis de difusão do Ti, Al, V e O nas camadas superficiais da amostra recoberta foram obtidos por microanálise semi-quantitativa por energia dispersiva de raios-X. A caracterização química superficial do lado substrato depois da deposição de TiO2 mostrou a formação localizada de TiO. Na região entre-camadas foram observados os perfis de difusão característicos com forma de S para o Ti, V e O. O perfil de difusão do Al mostrou um pico na região entre-camadas indicando um acúmulo de Al2O3 nos buracos presentes nesta zona. O TiO2 foi identificado como o maior constituinte no lado revestimento. Pequenas quantidades de Al e V foram também detectadas homogeneamente distribuídas dentro do depósito. A presença do Al e V (metais tóxicos) na superfície externa do depósito indica que estes metais não competem com a oxidação da camada depositada indicando um efeito desmascarante da superfície da liga metálica pelo óxido superficial TiO2. / Ti-6Al-4V alloy has been used with some success as biomaterial with applications in the field of dentistry and orthopaedics. The features with make the Ti-6Al-4V alloy such an interesting material are its good corrosion resistance in the biological environment, combined with an excellent degree of biocompatibility. The long-term biological effects of the slowly leaching of metal ions from titanium implants are not completely understood. It is known that the titanium ions are considered chemical carcinogen, aluminium ions cause neurological disorders and, vanadium ions are associated with irreversible enzymatic disturbance, among other problems. Titanium oxide (TiO2) coatings on titanium alloys can act as a chemical barrier for ions leaching from the metallic alloy surface, beside the fact that this oxide is a very good osteoinductor. The thermal spray process is widely used to apply oxide coatings. Once this is a high temperature process where molten or semi-molten particles impinge upon the substrate, localized diffusion can occur. The aim of this study is characterize the inter-diffusion of the alloying elements through the surface layers of the system Ti-6Al-4V/TiO2. The TiO2 coatings were prepared by the plasma spray technique. The coated zone showed some porous distributed in all layer and a few radial cracks. Additionally, large amount of holes were observed in the interlayer region. In this work, the interface Ti-6Al-4V/TiO2 was studied by scanning electron microscopy. Diffusion profiles of Ti, Al, V and O in the surface layers of coated samples were measured by semi-quantitative energy dispersive X-ray microanalysis. Chemical surface characterization of the substrate side after the TiO2 deposition showed the localized formation of TiO. At the interlayer region was observed the characteristic S shape diffusion profiles for Ti, V and O. The Al diffusion profile showed a peak in the interlayer region indicating anaccumulation of Al2O3 into the holes present in this zone. TiO2 was identified as the major component in the coating side. Small amounts of Al and V were also detected homogeneously distributed inside the deposit. Presence of Al and V (toxic metals) on the external surface of the deposit indicated that these metals did not compete with the oxidation of the deposited layer indicating a non-masking effect of metallic alloy surface by the TiO2- surface oxide.

Orientador: Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T05:54:56Z (GMT). No. of bitstreams: 1 Rego_PedroBordeaux_M.pdf: 28298145 bytes, checksum: 9790de4962b1c92e7ba928179c03b67a (MD5) Previous issue date: 2011 / Resumo: A lesão cartilaginosa é um problema significante e crescente na área da saúde pública. A terapia com células-tronco adultas têm sido uma alternativa promissora e têm despertado muito interesse dos pesquisadores. As células-tronco mesenquimais derivadas do tecido adiposo são caracterizadas por serem uma população homogênea com morfologia fibroblástica, aderentes e com grande capacidade de proliferação, apresentarem positividade para marcadores celulares CD90+, CD105+, CD29+e CD73+ e por possuírem a capacidade de diferenciação em linhagens mesodérmicas tais como linhagem osteogênica condrogênica e adipogênica. Além da escolha da fonte celular é necessária a utilização de um biomaterial que mimetize um microambiente e que possa dar suporte para as células-tronco possibilitando a restauração do tecido e sua função. Sabe-se que a matriz extracelular da cartilagem hialina é composta pelas proteínas de colágeno, principalmente colágeno do tipo II que tem importância durante a diferenciação e manutenção da cartilagem. O biomaterial de hidrogel de colágeno do tipo II é capaz de dar suporte às células-tronco mesenquimais e permitir a diferenciação dessas células. Descrevemos um método para diferenciação condrogênica das células-tronco mesenquimais do tecido adiposo em um hidrogel de colágeno do tipo II, com o aumento da expressão dos genes relacionados com a formação da matriz extracelular da cartilagem (colágeno do tipo II e agrecana), e relacionado com o controle da diferenciação condrogênica (Sox-9) e o aumento do nível das proteínas glicosaminoglicanas na matriz extracelular. Experimentos em modelo animal para lesão cartilaginosa demonstram que as células-tronco mesenquimais do tecido adiposo e os hidrogéis de colágeno do tipo II são capazes de preencher lesões cartilaginosas e formar um tecido preenchido com células mesenquimais transplantadas. Dessa forma, esse estudo demonstra que hidrogéis de colágeno do tipo II apresentam características apropriadas para o uso em tratamentos para reparo cartilaginoso e que as células-tronco mesenquimais do tecido adiposo podem ser uma fonte alternativa para recuperação de lesões cartilaginosas / Abstract: The cartilage injury is a significant and growing problem of public health. Therapies with adult stem cells have been a promising alternative and have attracted much interest from researchers. The mesenchymal stem cells derived from adipose tissue are characterized as being a homogeneous population with fibroblastic morphology, adherent and with great capacity for proliferation, positive for cell markers CD90 +, CD105 +, CD29 + and CD73 +. They also have the capacity to differentiate into mesoderm lineage such as osteogênica, adipogenic and chondrogenic. Besides the choice of cell source, it is necessary to use a biomaterial that mimics a microenvironment that can support and allow the stem cells to restore tissue and function. It is known that the extracellular matrix of hyaline cartilage is composed of the protein collagen, mainly type II collagen which is important for the differentiation and maintenance of cartilage tissue. The biomaterial collagen type II hydrogel is able to support the mesenchymal stem cells and allow the differentiation of these cells. We describe a method for chondrogenic differentiation of mesenchymal stem cells from adipose tissue in a collagen type II hydrogel, with increased expression of genes related to the formation of the extracellular matrix of cartilage (collagen type II and aggrecan), and Sox-9 (a gene related with the control of chondrogenic differentiation) and increased the level of glycosaminoglycans in the extracellular matrix proteins. Experiments in animal model for cartilage lesions show that mesenchymal stem cells from adipose tissue and collagen type II hydrogel are able to develop a cartilaginous-like tissue filled with transplanted mesenchymal stem cells. Thus, this study demonstrates that collagen type II hydrogel have characteristics suitable for use in treatments to repair cartilage and mesenchymal stem cells from adipose tissue may be an alternative source for recovery of cartilage lesions / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Ciências

Les peptides élastiques (ELP, Elastin-like peptide) sont d'excellents exemples de polymères biomimétiques récemment proposés en médecine régénérative, en particulier dans le domaine de l'ingénierie tissulaire des tissus mous (peau, vaisseaux sanguins, poumons…) pour lesquels la modélisation est complexe car l'instruction correcte des cellules nécessite une élasticité fonctionnelle. L'ajustement précis de la structure primaire des ELP peut moduler voire améliorer les propriétés physico-chimiques, structurales et fonctionnelles de la protéine native. De plus, la capacité des ELP à ajuster leurs caractéristiques physico-chimiques en réponse à des stimuli externes (température, pH), les définit comme des polymères intelligents. Ces polymères bioactifs offrent ainsi une large gamme d'applications très prometteuses encore très peu explorées dans les technologies d'ingénierie tissulaire et les systèmes d'administration de médicaments. Dans ce travail de thèse, nous avons développé, caractérisé et évalué les potentiels thérapeutiques d'une protéine élastique synthétique, Elactiv', inspirée de la structure unique de la tropoélastine humaine, précurseur soluble de l'élastine. Elactiv' conserve les caractéristiques physico-chimiques (comportement thermosensible, propriétés d'autoassemblage) et les fonctions biologiques de la protéine native (prolifération, différenciation et survie des fibroblastes dermiques et kératinocytes humains, sensibilité à la dégradation enzymatique). De plus, Elactiv' possède la particularité in vitro de s'incorporer dans les fibres élastiques néo-synthétisées par des fibroblastes dermiques sains, et d'induire la synthèse de tropoélastine fibrillaire par des fibroblastes pathologiques, syndrome de Williams-Beuren, qui ne synthétisent pas ou très peu de fibres élastiques. Un hydrogel formé exclusivement d'Elactiv' a permis d'accéder aux propriétés mécaniques de l'ensemble et de vérifier sa biocompatibilité in vitro et son innocuité et sa résorption in vivo. Enfin, l'association de la protéine Elactiv' aux dendrigrafts de poly(L-lysine), polymères synthétiques hautement fonctionnalisables, a permis de faire évoluer l'architecture de l'hydrogel vers un biomatériau hybride dans le but d'augmenter ses propriétés mécaniques et biologiques. Ainsi, les potentiels biomimétiques et thérapeutiques de la protéine Elactiv' en font un candidat prometteur pour la régénération des tissus mous / Elastin-like peptides are excellent examples of biomimetic polymers recently proposed in regenerative medicine, particularly for soft tissue engineering (skin, blood vessels, lung ...) for which modeling is a complex task requiring functional elasticity to insctruct cells properelly. Fine-tuning of ELP’s primary structure can modulate or improve physicochemical, structural and functional properties of the native protein. In addition, the adjustment of ELP physicochemical characteristics through external stimuli (temperature, pH) defined them as intelligent polymers. These bioactive polymers thus provide a wide range of very promising applications in tissue engineering and drug delivery, although this has been under-explored until then. In this thesis, we have developed, characterized and evaluated therapeutic potentials of Elactiv', a synthetic elastic protein inspired by the unique structure of the human tropoelastin, the soluble precursor of elastin. Elactiv’ retains physicochemical characteristics (thermoresponsive behavior, self-assembly properties) and biological functions of the native protein (proliferation, differentiation and survival of human keratinocytes and dermal fibroblasts, susceptibility to enzymatic degradation). Besides, Elactiv’ is able to incorporate into neosynthesized elastic fibers by healthy dermal fibroblasts, and to induce fibrillar tropoelastin synthesis by pathological fibroblasts, Williams-Beuren syndrome, which do not synthesize or very few elastic fibres. A hydrogel formed exclusively of Elactiv’ allowed to access to mechanical properties of the scaffold and to verify its biocompatibility in vitro and its safety and resorption in vivo. Finally, the association of Elactiv' protein to poly(L-lysine) dendrigrafts, highly functionalizable synthetic polymers, enabled to evolve the hydrogel's architecture to a hybrid biomaterial in order to increase its mechanical and biological properties for skin tissue engineering. Taken together, biomimetic and therapeutic potentials of Elactiv' protein make it a promising candidate for soft tissue regeneration