Les travaux de cette thèse portent sur la caractérisation fine des cristaux d’apatite osseuse, principalement par résonance magnétique nucléaire à l’état solide. Leur originalité majeure a été de se tourner vers l’étude d’un échantillon d’os frais et intact, analysé dans les deux heures après son extraction de chez l’animal (i.e. une brebis âgée de deux ans). Cette démarche évite toute altération de l’échantillon d’os, lequel se trouve proche de son état d’hydratation naturel. Une telle rigueur expérimentale a permis de mettre en lumière le fort caractère hydrophile des cristaux d’apatite du minéral osseux. Un même caractère hydrophile a également été rapporté pour des analogues biomimétiques de synthèse étudiés dans un état humide artificiel. Il est prouvé ici que ce caractère hydrophile est fourni par la présence d’un domaine minéral particulier se trouvant en surface des cristaux d’apatite osseuse et biomimétique, appelé domaine non-apatitique. Les signatures RMN (des noyaux 31P et 1H) ainsi que les propriétés d’hydrophilie de ce domaine non-apatitique ont été comparées à celles d’un échantillon de phosphate de calcium amorphe de synthèse, et se sont avérées similaires. Une étude sur la composition chimique de ce domaine non-apatitique a également été entreprise, réalisée parfois sur un échantillon d’os intact et parfois sur des analogues biomimétiques de synthèse enrichis en certains isotope (i.e. 13C et 43Ca). Il a de cette manière été montré que ce domaine non-apatitique est principalement composé d’espèces divalentes : Ca2+, HPO42- et CO32-. Enfin, des expériences de diffraction des rayons X et de cryo-microscopie électronique en transmission ont permis d’étudier le comportement en solution des cristaux d’apatite. Il a de cette manière été prouvé, de manière inattendue, que les molécules d’eau rigidement adsorbées sur le domaine de surface des cristaux d’apatite peuvent favoriser l’adhésion entre les cristaux ainsi que leur organisation 3D. / The work of this thesis is focused on the characterization of bone apatite crystals, mainly by solid-state nuclear magnetic resonance. The originality of this work was to study a fresh bone sample, analyzed within two hours after its extraction from the animal (i.e. a two years old sheep). This approach avoids any alteration of the bone sample, and keeps its natural state of hydration. Such experimental rigor led to the evidence that the apatite crystals of bone own a high hydrophilic character. Furthermore, a analogous hydrophilic character has also been reported for two samples of biomimetic apatites, both studied in a artificial wet state. It is shown here that this hydrophilicity is given by the presence of a particular mineral domain located at the surface of the bone apatite (and biomimetic apatite) crystals, the so called non-apatitic domain. The NMR signatures (1H and 31P nuclei) and the hydrophilic properties of the non-apatitic domain were compared with those of a synthetic amorphous calcium phosphate, and all were found to be similar. The chemical composition of the non-apatitic domain was also studied, sometimes performed on a bone sample and sometimes on labeled (13C and 43C) biomimetic apatites. It has been shown that this non-apatitic domain is mainly composed of divalent species : Ca2+ , CO32- and HPO42-. Finally, some X-ray diffraction and cryo-electron transmission microscopy experiments were done to study the behavior of apatite crystals in aqueous media. It has been shown, surprisingly, that the water molecules strongly adsorbed on the non-apatitic domain at the surface of apatite crystals, can promote the adhesion between crystals and their 3D organization.

[pt] O câncer é a segunda maior causa de morte no mundo inteiro e estima-se que até 2030, serão 27 milhões de novos casos e 17 milhões de mortes. Devido a esse fato, o desenvolvimento de novos sistemas de liberação (Drug delivery systems - DDS) que permitam a distribuição de um agente bioativo no seu sítio terapêutico na dose certa, numa taxa específica, acarretando menor toxicidade, e que possa ser utilizado tanto para o tratamento como para o diagnóstico de enfermidades. Atualmente, as sílicas mesoporosa surgem como potenciais DDS ou carreadores em radiofármacos, devido a sua estrutura organizada, alta área superficial e tamanhos e volumes de poros ajustáveis. Nesse trabalho foram sintetizados três potencias sistemas de liberação ou carreadores para radiofármacos para exames de imagem em medicina nuclear. A SBA-15 e sua associação com a hidroxiapatita (HAP) por dois métodos diferentes: adição dos precursores durante formação da mesofase (SBA-15/HAP_1) e o segundo por impregnação na estrutura da SBA-15 já sintetizada e calcinada (SBA-15/HAP_2). Os materiais foram marcados com o radioisótopo 99mTc e tiveram suas marcações e biodistribuições avaliadas. A SBA-15 também foi conjugada com o aptâmero anti-MUC1 para realização de direcionamento ativo para células tumorais. Os materiais foram caracterizados por: espectroscopia de emissão ótica por plasma acoplado indutivamente (ICP-OES); análise termogravimétrica (ATG); fisissorção de nitrogênio; difração de raios-X (DRX); infravermelho (IV); microscopia eletrônica de transmissão e varredura (MET e MEV); e difração a laser. As avaliações da marcação e da biodistribuição foram realizadas por contador de raios gama e gama-câmara respectivamente. A SBA-15 apresentou uma estrutura mesoporosa altamente ordenada, com alta área superficial. Os materiais impregnados com hidroxiapatita apresentaram diferenças estruturais: Na SBA-15/HAP_1 houve bloqueio quase total dos mesoporos com nanopartículas de hidroxiapatita, enquanto na SBA-15/HAP_2, houve um bloqueio parcial dos mesoporos, apresentando também agregados de hidroxiapatita. A metodologia de marcação com o 99mTc apresentou estabilidade de 8 horas. A conjugação da SBA-15 com o aptâmero anti-MUC1 apresentou maior reconhecimento das partículas de SBA-15 por células tumorais. / [en] Cancer is the second major leading cause of death worldwide and is estimated till 2030, 27 million new cases and 17 million of deaths. So, the development of new drug delivery systems (DDS) that allows a bioactive agent distribution in its therapeutic site in the right dose, in a specific rate, leading lower toxicity, and that can be used for treatment and diagnosis of several diseases. Recently, mesoporous silicas arise as promising DDS or carriers for radiopharmaceuticals, because of its highly organized structure, high superficial area and tunable pore size and volumes. In this work, were synthesized three potential DDS or carriers for radiopharmaceuticals for imaging in nuclear medicine. SBA-15 and its association with hydroxyapatite (HAP) by two distinct methodologies: precursors addition during mesophase formation (SBA-15/HAP_1); and precursors impregnation in SBA-15 previously synthesized and calcined (SBA-15/HAP_2). The materials were labelled with tecnecium-99m radioisotope and its labeling methodology and biodistribution were evaluated. SBA-15 was also conjugated with the aptamer Anti-MUC1 for active delivery to tumoral cells. The materials were characterized by: inductively coupled plasma optical emission spectroscopy (ICP-OES); thermogravimetric analysis (TGA); nitrogen physisorption; x-ray diffraction (DRX); infrared (IR), transmission and scanning electronic microscopy (TEM and SEM); and laser diffraction. The labeling and biodistribution evaluation were performed by gamma-ray counter and gamma-camera respectively. The materials were synthesized and have shown peculiar characteristics. SBA-15 has highly ordered mesoporous structure with high superficial area. The materials impregnated with hydroxyapatite showed structural differences: In SBA-15/HAP_1, hydroxyapatite nanocrystals almost blocked the entire mesopores, while in SBA-15/HAP_2, there were a partially blocking of the mesopores and also presented hydroxyapatite nanocrystals aggregates. The tecnecium-99m labeling methodology of the materials showed an 8 hours stability. SBA-15 conjugation with Anti-MUC1 for active delivery has shown a better recognition by tumor cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22367号 / 医博第4608号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Caledonian alum shales of Sweden host a vast number of economically interesting metals. In Fetsjön, Västerbotten, the shales contain significant amounts of rare-earth elements, vanadium, molybdenum and uranium. As metals with a multitude of high-technological applications, the former rare-earth elements (REEs) are particularly attractive in a world where the supply may be exhausted as the demand continuously increase while new deposits are not being discovered fast enough. Meanwhile, the latter uranium notably constitutes as an unwanted secondary product during the extraction of rare-earth elements. As the mineral association of the REEs in Fetsjön is unknown, the intent of this study is to analyze and thus determine their mineralogical expression. The assumed REE-bearing mineral apatite was confirmed to host the rare-earths in the Fetsjön shales after microscopy and spectrometry analyses.

This work presents new method for determination of geological provenance based on the statistical analysis of cathodoluminescence spectra of marbles and carbonate rocks from various localities in the Bohemian Massif. Analysis is based on the combination of data from seven different measures of spectral similarity and provides results that are more robust than those obtained by approach based only on a single measure of similarity. These results may be further improved by employing meta-analysis that evaluates behaviour of spectra during the individual steps of analysis. Analysis was performed on the original cathodoluminescence spectra, as well as on the residual spectra in which relative variability was amplified by the subtraction of Gaussian trend present in all spectra of carbonates. By combining results from analysis and meta-analysis of both original and residual spectra it was possible to almost unambiguously determine provenance of samples from all studied localities. Agglomerative hierarchical clustering was employed as a supplementary method for classification of spectra. Out of 72 different methods for clustering, one was selected for both original and residual data based on the number of correctly classified spectra. Second part of this work presents analysis of cathodoluminescence...

Tooth enamel is the hardest and most resistant highly mineralized inorganic component in mammalian bodies that significantly affects both the life quality and expectancy of an indi- vidual. Its specific qualitative properties are given by the biomineralization process responsible for its formation. In this process the mineralization of hydroxyapatite (HAp), the only inor- ganic phase composing the mammalian hard tissues, is controlled by activity of enamel-forming cells ameloblasts and their products. Over the past years, the studies of enamel matrix pro- teins, their structure, composition and function has become the prevalent field of experimental investigation. However, unique enamel qualities, which enable the teeth to withstand high pressure and stress demands, cannot be accurately assessed without the thorough systematical study of its mineral compound. In this thesis, I focus on the crystallographic and compositional characteristics of enamel hydroxyapatite and their influence on the mechanical properties of teeth. Obtained results are discussed in context of developmental and adaptation dynamics of mammalian species. The main aspect of the work is to extend our knowledge about the protein-mediated mineraliza- tion process from the perspective of inorganic compound and its contribution to the...

[pt] Em todas as atividades cirúrgicas, que se ocupam dos tecidos estruturais do ser humano, existem situações em que se faz necessário o emprego de tecido ósseo adicional. As indicações mais comuns para esse emprego de preenchimento ósseo são pequenas e médias reconstruções. O estudo de tecido ósseo é uma parte da engenharia de biomateriais que tem a finalidade básica de formar substitutos ósseos. O presente trabalho teve como objetivo principal caracterizar dois materiais, sendo um de origem bovina (Pro-Bone) e o outro de origem sintética (TCP-trifosfato de cálcio), como materiais de preenchimento em defeitos ósseos. A difração de raios x e a espectroscopia de infra vermelho revelaram que o osso bovino é altamente cristalino e composto por hidroxiapatita - HA, carbonatos e compostos minoritários. Em relação ao TCP os resultados demonstraram razão atômica Ca/P igual a 1,5 e alta cristalinidade. As partículas de ambos os materiais, foram analisadas a partir de imagens digitais (MO e MEV), utilizando o programa KS-400. Desta forma, foram medidas as dimensões relacionadas ao tamanho e à forma das partículas: área, perímetro, eixo maior, eixo menor, diâmetro médio, circularidade e convexidade. Observou-se que a área das partículas de Pro-Bone é, em média, cerca de 10 vezes menor do que a área das partículas de TCP. O menor tamanho das partículas de Pro-Bone pode levar a uma reabsorção muito mais rápida do material do implante, gerando fibrose e limitando a osteo-integração. Do ponto de vista da forma, não foram encontradas diferenças significativas entre os dois materiais. / [en] In all the surgical activities that are occupied with structural tissue of the human being, we have situations presented where it is necessary the use of additional bone tissue. The most common indications to use bone fulfilling are: small reconstructions and average reconstructions. The patient, who needs bone reconstruction surgery of small and medium average, can make use of allopathic material, with a cost well more accessible than bone graft alogenic. The study of a bone tissue is a part of engineering with basic purpose to form bone substitutes. This work had as priority the objective to characterize two materials, one with bovine origin e another one with synthetic origin, for future applications as material of fulfilling in bone defects. The X-Ray diffraction and the infra red spectroscopy had disclosed that bovine bone is highly crystalline and composed for hidroxiapatite - HA, carbonates and composites minority and the same with TCP. The bone particles were measured from digital images (MO and the MEV), using program KS- 400. The dimensions related to the particle size and form had been measured based on: area, perimeter, bigger axle, lesser axle, average diameter, factor of form, convexity and diameter of Ferret. It was observed that the particles area of Pro-Bone is, in average, about 10 times smaller than TCP particles area. The smaller particle size of Pro-Bone can lead to a much faster reabsortion of the implant material, generating fibroses and limiting the osteo-integration. No significant differences in shape between the two materials were found.

[pt] A hidroxiapatita está entre os biomateriais cerâmicos de maior interesse na área médica, pois, além da sua semelhança com a fase inorgânica dos ossos e dentes, apresenta exelente biocompatibilidade e bioatividade. Devido a sua ampla aplicação e aos gastos inerentes a utilização de implantes em processos cirúrgicos, torna-se necessário o desenvolvimento de processos mais viáveis encomonicamente e de materiais com melhores propriedades, com isso, este trabalho teve como objetivo a síntese de hidroxiapatita (HAp) pelo método de precipitação utilizando cascas de ovos de galinha como fonte de cálcio na presença de diferentes concentrações de sacarose, para avaliar sua influência nas características dos materiais sintetizados. A utilização da casca de ovo surge como um insumo atrativo, uma vez que é composta majoritariamente por carbonato de cálcio, um material abundante e barato. A casca de ovo foi calcinada à 1000 graus Celsius para obtenção do CaO que foi hidrolisado para a obtenção do Ca(OH)2.Os materiais foram obtidos pela reação de precipitação entre o Ca(OH)2 e H3PO4 a temperatura ambiente e com controle de pH, utilizando diferentes quantidades de sacarose. Os materiais obtidos foram caracterizados pelos métodos de Difração de Raios X (DRX), Microscopia Eletrônica de Varredura com emissão de campo (MEV-FEG), adsorção de N2 a 77K (BET), Microscopia Eletrônica de Transmissão (MET), espectroscopia de infravermelho com transformada de Fourier (FTIR), análise termogravimétrica (TG) e Fluorescência de Raios X (FRX). Os resultados de DRX confirmaram a formação da HAp. O FTIR apresentou as bandas correspondentes a HAp, corroborando os resultados de DRX, identificando também bandas de carbonato. Os resultados de EDXS confirmaram a composição química das amostras, sendo esta de P, O e Ca. A caracterização por MEV permitiu a avaliação da mudança da morfologia com o para as diferentes da quantidade de sacarose utilizada, sendo observada uma tendência a formação de partículas esferoidais. Os resultados de BET evidenciaram um aumento de superfície específica com o aumento da quantidade de sacarose, passando de 36 para 93 m2/g, decorrente do aumento de porosidade e diminuição do tamanho de partícula, resultado confirmado pelo MET, com tamanho de partícula passando 50 nm para valores em torno de 10-20 nm. Para avaliação da citotoxicidade foi realizado o ensaio da redução da resazurina para o período de 24h. Os resultados obtidos indicaram que as amostras são não citotóxicas. O ensaio de bioatividade foi realizado em meio McCoy para o período de 3 e 7 dias, apresentando resultados de dissolução das amostras compatíveis com caracteríticas de superfícies bioativas, porém, para o período de da análise, não foi observada a formação da camada bone like apatite. Os materiais sintetizados apresentaram propriedades semelhantes aos obtidos a partir de fontes de cálcio comerciais, apresentando promissor potencial em aplicações da engenharia de tecido ósseo, além de contribuir para a reciclagem desses bio-resíduos que é a casca de ovo. Os resultados obtidos indicam que a sacarose pode alterar as características do material, sendo um promissor aditivo/template de fácil obtenção e baixo custo, proporcionando materiais com elevada superfície específica e redução do tamanho de partícula. / [en] Hydroxyapatite is among the ceramic biomaterials of greatest interest in the medical field, because, in addition to its similarity with the inorganic phase of bones and teeth, it presents excellent biocompatibility and bioactivity. Due to its wide application and the expensive surgical processes, it is necessary to develop more viable processes and materials with better properties, therefore, the work aimed the hydroxyapatite synthesis (HAp) by precipitation method using hen’s eggshells as a calcium source in the presence of different sucrose concentrations, to evaluate its effect on the characteristics of the synthesized materials. A comparative study was also carried out with HAp samples obtained without sucrose and with commercial Ca(OH)2. The use of eggshells appears as an attractive raw material since it is mainly composed of calcium carbonate and because it is an abundant and cheap material. The eggshell was calcined at 1000 Celsius to obtain CaO, which was hydrolyzed to obtain Ca(OH)2. The materials were obtained by the precipitation reaction between Ca(OH)2 and H3PO4 at room temperature with pH control, using different amounts of sucrose. The obtained materials were characterized by X-Ray Diffraction (XRD), Scanning Electron Microscopy with field emission (SEM-FEG), N2 to 77K adsorption (BET), Transmission Electron Microscopy (TEM), infrared spectroscopy with Fourier transform (FTIR), thermogravimetric analysis (TG) and X-Ray Fluorescence (XRF). The XRD results confirmed the formation of HAp. The FTIR showed the bands corresponding to HAp, corroborating the results of DRX, in addition to the presence of carbonate bands. The results of EDXS confirmed the composition of the samples, being of P, O and Ca. The SEM characterization allowed the evaluation of the morphology changes for the different amounts of the sucrose used, forming spheroidal particles. The BET results showed an increase in specific surface area with the rise in the sucrose amount, from 36 to 93 m2/g, due to the rise in porosity and decrease in particle size, a result confirmed by TEM, with particle size changing from 50 nm to values around 10-20 nm. The resazurin reduction assay was performed for the 24h period to evaluate of cytotoxicity. The results obtained indicate that the samples were non-cytotoxic. The bioactivity assay was carried out in McCoy medium for 3 and 7 days, showing dissolution results of the samples compatible with characteristics of bioactive surfaces. The formation of the bone-like apatite layer was not observed for the analysis period. The synthesized materials showed properties similar to those obtained from synthetic calcium sources. Therefore, it presents promising potential in bone tissue engineering applications, besides contributing to the recycling of this bio-residue, the eggshell. The high specific surface area and small particle size results obtained indicate that sucrose can be a promising additive/template that is easy to obtain at low cost.

Whole-rock and mineral compositions of three eruptive deposits from the Twin Falls caldera, associated with the Yellowstone hotspot, provide a window into melt generation and evolution for hot, dry, A-type rhyolites. Three rhyolitic units were sampled via the Kimberly drill-core as a part of project HOTSPOT, a study focused on mantle plume and continental lithosphere interaction. Previous work has been done to collect high resolution U-Pb zircon ages, and Hf- and O-isotopic compositions. This study examined the geochemistry of apatite and zircon along with host rock compositions in the context of this previous work. The Kimberly core sampled the Shoshone Rhyolite (6.06 Ma, 120 m thick), Kimberly Member (7.70 Ma, 169 m thick), and Castleford Crossing Member (7.96 Ma, >1400 m thick). Apatite compositions more closely reflect the composition of their whole rock hosts than zircons. SiO2 content is higher in apatite of the Kimberly Member at (1.1 ± 0.75 wt.%), vs (0.72 ± 0.47 wt.%) for the Castleford Crossing and (0.84 ± 0.27 wt.%) for the Shoshone Rhyolite. REEs compensate for Si substitution in these apatites, with the Kimberly Member most enriched. Volatile contents in the apatites are typical of metaluminous A-type rhyolites, with very low Cl and high F concentrations. Average Ti-in-zircon crystallization temperatures were highest in the Castleford Crossing Member (847 ± 68°C), followed by the Shoshone Rhyolite (806 ± 78°C), and then the Kimberly Member (804 ± 70°C). Oxygen fugacity calculated from zircons has average ΔQFM values for the Shoshone (0.8), Kimberly (-0.2), and Castleford Crossing (0.2). Hf concentrations and Eu anomalies are comparable in zircons from all three units. REE patterns in zircons are also similar and concentrations of REEs in the Shoshone and Kimberly units are similar even though the whole rock compositions of all three units are distinct. Less than 15% of zircons in the Kimberly and Castleford Crossing rhyolites have CL-dark cores enriched in several REEs, U, and Th. These CL-dark cored zircons are likely xenocrysts entrained from chemically evolved granite and then overgrown with less enriched rims prior to eruption. There are several apatite grains with Si-LREE enriched rims in the Kimberly Member, which serves as further evidence of assimilation of silicic igneous rock by the Kimberly Member before eruption. Principal component analysis of the geochemical data distinguishes between the units using both whole-rock and apatite compositions. However, zircon compositions are not statistically distinguishable using PCA. A global comparison of Ti, U, Th, Yb, and Nb concentrations in zircons show that the zircons in the Central Snake River Plain are similar to zircons in Hawaiian basalts, while younger zircons from Yellowstone formed in cooler more differentiated magma. We propose that the zircon and apatite chemical patterns and trends confirm the A-type origin of Snake River Plain rhyolites and make it unlikely that they represent partial melts of felsic continental crust but are instead derived in large part from partial melts of young mafic crust--the midcrustal sill.

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