Orientador: José Angelo Camilli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:42:16Z (GMT). No. of bitstreams: 1 Domingues_JulianaAlmeida_M.pdf: 6712230 bytes, checksum: abfef192b69eeb8cc178a08e435692fd (MD5) Previous issue date: 2013 / Resumo: Cimentos de fosfato de cálcio (CFC) a base de ?-TCP, que originam como processo final de cura uma hidroxiapatita deficiente em cálcio (CDHA) são muito utilizados como biomateriais para osso, por possuírem similaridade estrutural e química à porção inorgânica do tecido ósseo. Porém o uso dos CFC se restringe a pequenos defeitos bucomaxilofaciais e recobrimento de próteses metálicas, em função do déficit considerável de suas propriedades mecânicas. Visando melhorar as propriedades mecânicas dos CFCs estudos têm sido direcionados à aplicação de fibras de vários materiais, como as fibras de carbono, as fibras de vidro, as de carboneto, entre outras. No entanto, muitas dessas fibras introduzidas se mostraram tóxicas às células, impedindo sua aplicação clínica. Recentemente, os "whiskers" (fibras curtas de monocristal) de biocerâmicas vêm sendo estudados como outro tipo de reforço, cujos resultados têm se mostrado promissores por melhorarem as propriedades mecânicas e biológicas dos materiais. Nesse trabalho, a resposta biológica das CDHA pura e contendo 5 e 10% de "whiskers" de wollastonita, foram estudadas através de um sistema de cultivo celular com células osteoblásticas, o qual tem sido muito utilizado para elucidar a resposta celular em biomateriais. Porém cultivar células em CDHAs não é uma tarefa fácil, pois o processo de hidrólise sofrido por esses materiais promove a liberação de íons para o meio de cultura, o que acarreta na mudança de pH e concentração iônica do meio. Essas mudanças promoveram a morte das células após 48h de cultivo. Foi possível manter as células viáveis ao longo do tempo realizando a lavagem dos discos de CDHA com DMEM (Dulbecco's Modified Eagle Medium) antes da semeadura e também com a troca diária do meio de cultura. Nossos resultados demonstraram que as CDHA contendo "whiskers" foram capazes de estimular a proliferação celular quando comparadas com a placa de poliestireno e a CDHA pura. No ensaio de MEV foi possível observar uma densa camada celular na superfície da CDHA contendo 10% de "whiskers". A atividade da fosfatase alcalina (FAC) foi significativamente maior nas CDHAs contendo "whiskers", sendo proporcional a concentração de "whiskers" wollastonita nas amostras, o ensaio de mineralização corroborou com o ensaio de FAC, no qual a CDHA contendo 10% de "whiskers" apresentou maior produção de matriz mineralizada. Tendo-se em vista os presentes resultados pode-se dizer que as CDHA contendo "whiskers" de wollastonita são biomateriais promissores para engenharia tecidual óssea, pois foram capazes de estimular a proliferação e diferenciação celular, além da resistência mecânica melhorada pela adição dos "whiskers" / Abstract: Calcium phosphate cements (CFCs) are widely used as biomaterials for bone because they have chemical and structural similarity to the inorganic portion of bone tissue. But the use of CFCs is restricted to maxillofacial small defects and coating metallic prostheses, due to the considerable deficit of its mechanical properties. In order to improve the mechanical properties of CFCs studies have focused on the application of fibers of many materials such as carbon fibers, glass fibers, carbide fibers among others. However, many of these fibers are shown toxic to the cells, preventing their clinical application. Recently, the "whiskers" (short crystal fibers) of bioceramics have been studied as another type of reinforcement and the results have shown promise for improving the mechanical and biological properties of materials. In this work the biological response of hydroxyapatites deficient in calcium (CDHA) pure and containing 5 and 10% "whiskers" of wollastonite, were studied using a cell culture system with osteoblastic cells, which has long been used to elucidate the cell behavior in biomaterials. However cultivating cells in CDHAs is not an easy task because the hydrolysis process undergone by such materials promotes the release of ions to the culture medium, which results in the change of pH and ionic concentration of the medium. These changes promoted cell death after 48 h of cultivation. Cell viability was maintained for 14 days by a simple washing of CDHA discs with DMEM before cell culture and also with daily changes of culture medium. Our results demonstrate that CDHA containing "whiskers" were able to stimulate cell proliferation when compared to the polystyrene plate and CDHA pure. In the assay by SEM was possible to see a dense cell layer on the surface of the CDHA containing 10% "whisker." The alkaline phosphatase activity (ALP) was significantly higher in CDHAs containing "whiskers" being proportional to the concentration of "whiskers" wollastonite samples. The mineralization assay corroborated with ALP assay in which the CDHA containing 10% " whisker "showed higher production of mineralized matrix. In summary the CDHAs containing "whiskers" of wollastonite are excellent biomaterials for bone tissue engineering, because they were able to stimulate cell proliferation and differentiation, as well as mechanical strength improved by the addition of "whiskers" / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Orientador: Cecília Amélia de Carvalho Zavaglia / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-24T06:22:47Z (GMT). No. of bitstreams: 1 Saito_Lia_M.pdf: 10338325 bytes, checksum: 36e37affce64dade6095d6f2d7761537 (MD5) Previous issue date: 2013 / Resumo: A necessidade de substituintes ósseos está crescendo rapidamente no campo da cirurgia reconstrutiva. Os cimentos à base de fosfato de cálcio são largamente empregados no reparo do tecido ósseo devido a uma série de propriedades atrativas, como excelente bioatividade e biocompatibilidade. São muitos os estudos feitos em busca da otimização das propriedades desses materiais ou a análise do comportamento biológico desses substitutos, entretanto esses estudos analisam essas propriedades isoladamente, sem se atentar para que as duas variáveis devem ser analisadas simultaneamente. O objetivo deste trabalho foi avaliar a influência da distribuição granulométrica do pó de ?-TCP no comportamento biológico do cimento ósseo que utiliza este pó precursor através de uma análise simultânea de propriedades físico-químicas, mecânicas e morfológicas e do comportamento biológico do material. Para isso, o pó de ?-TCP foi obtido em laboratório a fim de se alcançar elevada pureza de fase. Este mesmo pó passou por diferentes protocolos de moagem a fim de se obter duas faixas de distribuição granulométrica. Finalmente, os cimentos que apresentaram potencial para aplicação como implantes de acordo com as caracterizações prévias do material passaram por avaliação das suas propriedades biológicas através de testes in-vitro e in-vivo. Ambos as amostras apresentaram pureza de fase, livres de contaminações. Além disso, obtiveram valores de resistências mecânicas semelhantes ou maiores do que consta na literatura e resultados positivos em relação à biocompatibilidade / Abstract: Bone substitutes are largely used in reconstructive medicine, especially calcium phosphate cements to repair bone tissue due to its attractive properties as excellent bioactivity and biocompatibility. There are several studies about the increase of these materials¿ properties or the analysis of biological behavior from these substitutes, however the researches only show the properties separately, instead of analyze the two variables simultaneously. The aim of this project was to analyze the influence of two different particle size distribution of ?-TCP powder on biological behavior of its bone cement through simultaneous analysis of physical-chemical, mechanical and morphological properties and biological behavior of the material. Thus, ?-TCP powder was obtained in laboratory to reach the maximum purity level. The same powder was processed by different milling protocols to obtain two distinct ranges of particle size distribution. Finally, the cements that presented high potential application as implants from its characterization methods were evaluated by in vitro and in vivo essays. Both samples presented phase purity without contamination, mechanical resistance similar or superior than described on literature and positive results in biocompatibility tests / Mestrado / Materiais e Processos de Fabricação / Mestra em Engenharia Mecânica

The increasing population with its associated rising food demand requires more agricultural fertilizers to maintain the harvest for food security. However, the natural calcium carbonate phosphate mineral (carbonate apatite) used to produce phosphorus (P) fertilizer is a non- renewable ore. Therefore, in this study, the objective is to extract the inorganic polymeric phosphate (polyphosphate: polyP) P-component in waste activated sewage sludge (WAS) from Ottawa’s municipal wastewater plant (ROPEC). Once extracted, the goal was to break down the polyP to inorganic phosphate (Pi) to produce carbonate apatite. PolyP is a P-component in WAS because a group of wastewater microorganisms called polyphosphate-accumulating organisms uptake phosphate and generate intracellular polyP stores during aerobic digestion in municipal wastewater treatment plant processes. The total acidic, oxidative P content of WAS was measured. PolyP was extracted and quantified to estimate the fraction of total P as polyP in WAS. Different polyP extraction methods were undertaken. It was determined that the complicated composition and weight fractions of TWAS, including iron phosphate, complicated polyP extraction and Pi measurement. Lessons learned were applied towards preliminary batch and continuous precipitation of carbonate apatite with the product slurry from the anaerobic digestion process at ROPEC. Limestone was tested as an inexpensive calcium carbonate source for carbonate apatite precipitation. The dissolution of calcium and carbonate from limestone was assessed, and it was determined that further optimization is required. Preliminary work indicated that the calcium- carbonate solution from limestone could precipitate synthetic carbonate apatite from anaerobic digester material. This synthetic carbonate apatite product may lead to mitigating the impending limitations on natural carbonate apatite availability for P-fertilizer production.

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Bone is currently the second most transplanted tissue, second only to blood. However, significant hurdles including graft supply and implant failure continue to plague researchers and clinicians. Currently, standard clinical procedures include autologous and allogeneic grafting. Autologous grafts may achieve functional repair; yet, they are available in limited supply and are associated with donor site morbidity. Allogeneic grafts are available in greater supply, but have a higher risk of infection. To overcome the disadvantages of current grafts, tissue engineering has become a major focus for the regeneration of bone. The goal of tissue engineering is to use a multidisciplinary approach to create biomimetic constructs that stimulate osteogenic regeneration to heal bone defects and restore tissue function. Biodegradable scaffolds are used in tissue engineering strategies as an interim template for tissue regeneration. The scaffold architecture provides mechanical support for cell attachment and tissue regeneration. Biocompatible poly(lactic-co-glycolic acid) (PLGA) has been processed through a number of techniques to create porous 3D architectures. Hydroxyapatite (HAP) and tricalcium phosphate have been used in conjunction with polymer scaffolds due to their osteoconductivity and biocompatibility, but they often lack osteoinductivity and are resistant to biodegradation. Conversely, amorphous calcium phosphate (ACP) is a mineral that solubilizes under aqueous conditions, releasing calcium and phosphate ions, which have been postulated to enhance osteoblast differentiation and mineralization. Controlled dissolution can be achieved by stabilizing ACP with divalent cations such as zinc or copper. Furthermore, incorporation of such osteogenic ACPs within a biodegradable PLGA scaffold could enhance the osteoconductivity of the scaffold while providing calcium and phosphate ions to differentiating osteoprogenitor cells, thereby stimulating osteogenesis when implanted in vivo. In this research, the effect of zinc on the differentiation of osteoprogenitor cells was investigated. Zinc supplementation of the culture media had no stimulatory effect on cell proliferation or differentiation. ACPs were synthesized using zirconium (ZrACP) and zinc (ZnACP) as stabilizers to achieve sustained ion release. Elevated concentrations suggested sustained ion release over the course of 96 hours and enhanced solubility of ZrACP and ZnACP. X-ray diffraction analysis showed a conversion of ZrACP to a semi-crystalline material after 96 hours, but ZnACP showed no conversion after 96 hours. Composite scaffolds were fabricated by incorporating HAP, zirconium-stabilized ACP (ZrACP), or zinc-stabilized ACP (ZnACP) into a sintered PLGA microsphere matrix and then characterized to determine the effect of the minerals on the in vitro differentiation of MC3T3-E1 cells. Scanning electron microscopy revealed a porous microsphere matrix with calcium phosphate powders distributed on the surface of the microspheres. Measurements of mechanical properties indicated that incorporation of 0.5 wt% calcium phosphates resulted in a 30% decrease in compressive modulus. When cells were cultured in the scaffolds, composite ACP scaffolds stimulated proliferation and ALP activity, while HAP scaffolds stimulated osteoblast gene expression. Overall, the results of this work indicate the addition of calcium phosphate minerals to PLGA scaffolds supported cell growth and stimulated osteogenic differentiation, making the scaffolds a promising alternative for bone tissue regeneration. / Ph. D.

This dissertation is comprised of three independent but interconnected studies with the scope of further understanding uranium and iodine partitioning between apatite and fluid. The studies herein presented investigated: 1) brushite to apatite crystallization method; 2) the degree of uranium incorporation into apatite; 3) the degree of iodine incorporation into apatite. The importance of this work is assessing the role of apatite in immobilizing these elements, where uranium is a major component of spent nuclear fuel and iodine is a chemical analog of its radioactive isotope (129I). Once we understand the incorporation mechanisms, we will provide data that can be used in development of engineering barrier systems via add-on of phosphate minerals. In the first chapter we evaluate a method for the crystallization of apatite (Ca10(PO4)6(OH, F, Cl)2) using brushite (CaHPO4·2H2O) as initial material. The solutions evaluated for this transformation were NaCl, NaF, and KOH. Result yielded 100% apatite transformation from brushite when pH is 3.5 or greater. At a pH lower than 3.5, transformation yields monetite-apatite mixtures. Crystal size is reduced during the transformation from ~10 micrometer to ~1 micrometer. In the second chapter, degree of uranium uptake by apatite was evaluated. Phosphate minerals were crystallized from U-bearing NaCl solutions at 25-350°C. After experimental runs uranium concentrations in experimental solids and fluids were analyzed using Inductively Coupled Plasma – Mass Spectrometry. Additionally, characterization of the solids was performed via X-Ray Diffraction, Scanning Electron Microscopy and Electron Microprobe to confirm the brushite to apatite conversion. Results show that >90% of uranium was extracted from solution. Moreover, that the partitioning of uranium between apatite and fluid decreases with increasing temperature. In the third chapter apatite was crystallized from iodine-bearing solutions. The crystallization was evaluated at 39 and 200°C. Iodine concentration in solids were acquired via Electron Microprobe Analysis (EMPA) whereas iodine concentration in fluids were acquired via UV-Visible Spectrophotometry. Iodine concentrations in fluids yielded minimum depletion (0.1M) from initial iodine added to the system (0.1M). Partitioning data suggest that iodate (the oxidized form of iodine) is more compatible with apatite compared to iodide (the reduced state of iodine).

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24836号 / 医博第5004号 / 新制||医||1068(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 安達, 泰治, 教授 森本, 尚樹, 教授 上杉, 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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