Este trabalho relata estudos sobre adsorção de carboximetilcelulose (SCMC) marcada com nitróxido (marcador de spin) em hidroxilapatita (HAP), em meio aquoso. São apresentadas isotermas de adsorção, obtidas em tampão acetato, cujo comportamento é descrito pelo formalismo da teoria de LANGMUIR. A quantidade adsorvida é dependente do peso molecular do polímero, sendo observado um incremento de cerca de 20% na adsorbância para um aumento de oito vezes no peso molecular médio. A afinidade independe da temperatura ou peso molecular. O número de sítios por unidade de massa de adsorvente não varia com a temperatura mas diminui com o aumento do tamanho da cadeia polimérica, o que é interpretado como uma consequência da maior restrição estérica oferecida pela molécula de maior peso molecular à aproximação de outras moléculas à superfície. O fenômeno de adsorção estudado tem uma etapa inicial rápida e depois dos primeiros 15 minutos a quantidade adsorvida aumenta muito pouco. Dessorção não é detectável quando o sedimento de carboximetilcelulose/hidroxilapatita é deixado em contacto com o tampão durante 6 horas. A adsorção de SCMC modifica algumas características de HAP dispersa em tampão acetato, diminuindo seu volume de sedimentação a 20% do valor em ausência de SCMC, estabilizando-a em tempos curtos (cerca de 8 horas) e floculando-a efetivamente em tempos maiores de contacto. A SCMC adsorvida não atacada de maneira detectável por celulase, ao contrário de SCMC em solução. Espectros de ressonância paramagnética de elétron de sedimentos de SCMC/HAP não dependem, quanto à forma, da quantidade de polímero adsorvida, de onde se conclue que a conformação do polímero adsorvido independe do grau de cobertura da superfície. É proposto um modelo para a adsorção de SCMC que e compatível com as observações deste trabalho e com um modelo existente para a adsorção de ácidos carboxílicos não poliméricos em HAP. / This is a study on the adsorption of spin-labelled carboxymethyl-cellulose (SCMC) on hydroxylapatite (HAP), in aqueous media. Adsorption isotherms obtained in acetate buffer are presented and their behaviour is formaly described by LANGMUIR theory. The amount adsorbed is dependent on the polymer molecular weight, with an increment of about 20% in the adsorbance for an eightfold increase in the average molecular weight. The affinity is independent of temperature or molecular weight. The number of sites per adsorbent unit mass does not change with temperature but diminishes with the increase of the polymeric chain size, what is interpreted as a result of the larger steric restriction offered by the molecule having the higher molecular weight to the approach of other molecules to the surface. The adsorption phenomena studied have a fast initial step and after the first 15 minutes the amount adsorbed increases too slowly. De-sorption is not detectable when the sediment SCMC/HAP is left in contact with the buffer for 6 hours. The adsorption of SCMC alters some characteristics of HAP dispersed in acetate buffer, diminishing its sedimentation volume to 20% of its value in the absence of polymer. The colloidal stability of HAP is increased if measured in short times after exposure to SCMC but turbidity measurements in times longer than 8 hours show that the polymer flocculates HAP effectively. The adsorbed SCMC is not attacked in a detectable way by cellulase, although dissolved SCMC is a substrate for this enzyme. The shape of the electron paramagnetic ressonance spectra of the SCMC/HAP sediments does not depend on the amount of adsorbed polymer. From these observations we conclude that the conformation of the adsorbed polymer is not affected by the degree of coverage of the surface. We propose a model to the adsorption of SCMC on HAP, which fits the experiments described in this work and is consistent with an existing model for the adsorption of non-polymeric carboxylic acids on HAP.

Este trabalho relata estudos sobre adsorção de carboximetilcelulose (SCMC) marcada com nitróxido (marcador de spin) em hidroxilapatita (HAP), em meio aquoso. São apresentadas isotermas de adsorção, obtidas em tampão acetato, cujo comportamento é descrito pelo formalismo da teoria de LANGMUIR. A quantidade adsorvida é dependente do peso molecular do polímero, sendo observado um incremento de cerca de 20% na adsorbância para um aumento de oito vezes no peso molecular médio. A afinidade independe da temperatura ou peso molecular. O número de sítios por unidade de massa de adsorvente não varia com a temperatura mas diminui com o aumento do tamanho da cadeia polimérica, o que é interpretado como uma consequência da maior restrição estérica oferecida pela molécula de maior peso molecular à aproximação de outras moléculas à superfície. O fenômeno de adsorção estudado tem uma etapa inicial rápida e depois dos primeiros 15 minutos a quantidade adsorvida aumenta muito pouco. Dessorção não é detectável quando o sedimento de carboximetilcelulose/hidroxilapatita é deixado em contacto com o tampão durante 6 horas. A adsorção de SCMC modifica algumas características de HAP dispersa em tampão acetato, diminuindo seu volume de sedimentação a 20% do valor em ausência de SCMC, estabilizando-a em tempos curtos (cerca de 8 horas) e floculando-a efetivamente em tempos maiores de contacto. A SCMC adsorvida não atacada de maneira detectável por celulase, ao contrário de SCMC em solução. Espectros de ressonância paramagnética de elétron de sedimentos de SCMC/HAP não dependem, quanto à forma, da quantidade de polímero adsorvida, de onde se conclue que a conformação do polímero adsorvido independe do grau de cobertura da superfície. É proposto um modelo para a adsorção de SCMC que e compatível com as observações deste trabalho e com um modelo existente para a adsorção de ácidos carboxílicos não poliméricos em HAP. / This is a study on the adsorption of spin-labelled carboxymethyl-cellulose (SCMC) on hydroxylapatite (HAP), in aqueous media. Adsorption isotherms obtained in acetate buffer are presented and their behaviour is formaly described by LANGMUIR theory. The amount adsorbed is dependent on the polymer molecular weight, with an increment of about 20% in the adsorbance for an eightfold increase in the average molecular weight. The affinity is independent of temperature or molecular weight. The number of sites per adsorbent unit mass does not change with temperature but diminishes with the increase of the polymeric chain size, what is interpreted as a result of the larger steric restriction offered by the molecule having the higher molecular weight to the approach of other molecules to the surface. The adsorption phenomena studied have a fast initial step and after the first 15 minutes the amount adsorbed increases too slowly. De-sorption is not detectable when the sediment SCMC/HAP is left in contact with the buffer for 6 hours. The adsorption of SCMC alters some characteristics of HAP dispersed in acetate buffer, diminishing its sedimentation volume to 20% of its value in the absence of polymer. The colloidal stability of HAP is increased if measured in short times after exposure to SCMC but turbidity measurements in times longer than 8 hours show that the polymer flocculates HAP effectively. The adsorbed SCMC is not attacked in a detectable way by cellulase, although dissolved SCMC is a substrate for this enzyme. The shape of the electron paramagnetic ressonance spectra of the SCMC/HAP sediments does not depend on the amount of adsorbed polymer. From these observations we conclude that the conformation of the adsorbed polymer is not affected by the degree of coverage of the surface. We propose a model to the adsorption of SCMC on HAP, which fits the experiments described in this work and is consistent with an existing model for the adsorption of non-polymeric carboxylic acids on HAP.

Transformation of amorphous calcium phosphate (ACP) and brushite into hydroxylapatite, an important biomineral, has been documented. The relationships between synthesis conditions and the formation and transformation of these phases are not comprehensively understood. The metastable nature of ACP has made it historically challenging to investigate, and many analyses attempt to stabilize the phase through drying or including additional ions or proteins in the reaction. In situ investigations provide an incisive approach to examining the structure and transformation of ACP and brushite as a function of synthesis conditions. The first project develops a new method for in situ analyses of the structure of ACP and brushite shortly after reagent mixing, without chemical stabilization. This method was used in the second project to examine how the initial Ca/P affects ACP structure and transformation. Our results identify the first structural differences in types of ACP, controlled by the initial Ca/P. At ratio 0.2 the Ca – P bonding geometry is primarily monodentate, ratio 5.0 produces a coordination that is primarily bidentate, and there is a mix of monodentate and bidentate coordinates at intermediate ratios between the two. These results are independent of system pH between the examined range of 6-11. Further ex situ transformation experiments identified that at ratio 0.2, ACP transformed directly into hydroxylapatite, but at higher ratios the transformation product is brushite. This is a promising mechanism for direct ACP to hydroxylapatite conversion at a biologically relevant pH. In the final project, the statistically significant synthesis parameters (age, pH, temperature, supersaturation, and initial ion ratio) for ACP, brushite, and hydroxylapatite formation are evaluated. Binary logistic regression analysis and nonlinear continuous logistic regression analysis are applied to a dataset compiled from the literature. Equations were developed that predict the percentage of ACP and brushite that will form. The equations and significant variables seem to depend on the transformation pathway of brushite and ACP. The current analysis did not comprehensively describe hydroxylapatite formation when trying to concurrently evaluate the ACP to hydroxylapatite and brushite to hydroxylapatite pathways. Taken together, these studies provide new ways to study and interpret calcium phosphate phases as they form and transform. Experiments identified new relationships between the chemistry and structure of ACP. The new in situ experimental method and the equations we developed can be used to improve future experimental designs towards a comprehensive understanding of the calcium phosphate system. / Doctor of Philosophy / Hydroxylapatite is a mineral made of calcium and phosphate, that is similar to the mineral components of bones and teeth in humans and other mammals and fish. Hydroxylapatite and other calcium phosphate phases can form when solutions, rich in calcium and phosphate, are mixed. Phases without long-range crystal structure, are amorphous calcium phosphates (ACP). Additional calcium phosphate minerals, like brushite can also form. If brushite and ACP are left in solution, they will transform into hydroxylapatite over time. Major questions include the need to learn the short-range atomic structure of ACP and how ACP and brushite transform into hydroxylapatite In this dissertation, I investigate how chemistry and other variables such as age and time impact the calcium phosphate phase to form and how it transforms with aging. The first project develops a new method to study the structure of ACP and brushite without drying the study materials or adding additional chemicals or proteins to prevent them from transforming. The sample forms in a solution and flows directly through an X-ray beam for structural analysis. This method is used in project two to examine how the ratio of calcium and phosphate in the beginning of the reaction affected the structure of ACP and how it transformed. The results identify the first structural differences in types of ACP, controlled by the initial Ca/P. At a ratio of 0.2 a calcium and phosphorus atom mostly share only one oxygen between them, but at Ca/P = 5.0, they mostly share two oxygens. At ratios in between 0.2 and 5.0 they share a mix of one and two oxygens. The results are independent of pH. Additionally, at ratio 0.2, ACP transformed directly into hydroxylapatite, but at all other ratios it transformed to brushite. Investigations of direct ACP to hydroxylapatite transformation are usually performed at a pH above that found in humans, but the transformation at low ratio occurred at a biologically relevant pH. In the final project statistical analysis was used to identify what synthesis conditions (out of age, pH, temperature, supersaturation, and initial ion ratio) had a significant impact on the formation of ACP, brushite, and hydroxylapatite. Equations were developed that can be used to predict the percentage of ACP and brushite that form based on the statistically significant variables. The current analysis did not fully describe hydroxylapatite formation. Results suggest separate equations are needed for hydroxylapatite forming directly from ACP and directly from brushite. Combined, these studies have created new ways to study calcium phosphate phases as they form and transform. This work experimentally identified new relationships between the chemistry and structure of ACP. Both the method and equations will help researchers improve their future experimental designs so investigations can be more directly compared to create a comprehensive understanding of calcium phosphates.

Made available in DSpace on 2017-07-24T19:37:57Z (GMT). No. of bitstreams: 1 Liliam Angelo.pdf: 3335280 bytes, checksum: e85b10f63e1152d525f35139d9abfc4a (MD5) Previous issue date: 2008-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nano-structured hydroxyapatite with crystallinity and composition similar to bone apatite has been widely investigated in last years.Because the bone is a tissue that requires some degree of mechanics tension to keep its vitality, has been used some biopolymers, as the composite HAP/gelatin, in order to minimize this problem. This biomolecule shows a singular characteristic, once that is the main mineral and organic phase bone constituent, respectively. The objective of this work was to consider a route of synthesis in which the hydroxylapatite was gotten by precipitation reaction directly on the collagen and to check out the effect of the gelatin addition in the hydroxylapatite crystal structure. The synthesis used was the wet precipitation through the dripping of a solution contends H3PO4 on the Ca(OH)2 together the addition of gelatin solutions with different concentrations (3 and 4 g/L, named GEL1 and GEL2 respectively). Temperature of the synthesis was controlled in approximately 42oC and pH after the ending of the dripping was kept in 12. After the periods of 1 and 21 days of aging the samples had been characterized. The DRX analyses had shown that the samples with gelatin addition shows bigger unit cell volumes in the two periods of aging studied, when related with the samples controll. Samples (GEL2) shows lesser volumes that samples GEL1, which had the biggest interactions occured between íons Ca2+ of the HAP and íons R___COO- of the gelatin. With the calcite, CaCO3, formation as second phase for these samples, íons Ca2+ are removed of the HAP structure, originating a calcium defficient HAP, and this, probably, makes with that the unit cell volumn diminishes. However, in the thermally treated samples, a unit cell volumn reduction more significant occurs to sample controll (AC) and GEL1, when the samples are aging for more time. For the samples did not treat thermally; it was observed in the same time of aging (21 days), the unit cell volume also is lesser must mainly by the decomposition. mainly the decomposition of carbonate apatites in CaO. On the microstrains of the crystal, were observed that when crystallite size increases there is a consequent microstrains reduction due to reduction of the internal tensions in the crystal while it grows. The FTIR analysis shows that type B carbonate apatite occurs, due the groups PO4 3- are substituted by groups CO3 2-. The substitution was more evident in the controll samples (AC) and GEL1 with lesser aging time, in samples GEL2, the phosphate bands are more evidentes. The SEM (scanning electro microscopy) micrographs show that there are formation of agglomerates and that the crystals are nano-structured. These results confirm that the gelatin presence affects directly the HAP structural properties. The method of synthesis presented in this study is a useful tool to production of nano crystals of the apatites structurally mimicking biological apatite. / Apatitas nanoestruturadas com cristalinidade e composição similar a apatita do osso têm sido amplamente investigadas nos últimos anos. Devido ao osso ser um tecido que requer algum grau de tensão mecânica para manter sua vitalidade, têm sido empregados vários biopolímeros, como o compósito HAP/Gelatina a fim de minimizar este problema. Este biomaterial de característica singular apresenta os principais constituintes da fase mineral e orgânica do osso, respectivamente. O objetivo deste trabalho foi propor uma rota de síntese na qual a HAP fosse obtida por reação de precipitação diretamente sobre o colágeno e verificar o efeito da adição de gelatina na estrutura cristalina da HAP. A sintese empregada foi por via úmida através do gotejamento de uma solução contendo H3PO4 sobre o Ca(OH)2 junto a adição de soluções de gelatina com diferentes concentrações (3 e 4 g/L, designadas, GEL1 e GEL2, respectivamente). Controlou-se a temperatura da síntese em aproximadamente 42oC e o pH após o término do gotejamento foi mantido em 12 Após os períodos de 1 e 21 dias de envelhecimento as amostras foram caracterizadas. As análises de DRX mostraram que as amostras com adição de gelatina apresentavam volumes de cela unitária maiores nos dois períodos de envelhecimento estudados, quando relacionados com as amostras controle. As amostras GEL2 apresentaram volumes menores que as amostras GEL1, devido as maiores interações ocorridas entre os íons Ca2+ da HAP e dos íons R OO- da gelatina. Com a formação da calcita, CaCO3, como segunda fase para estas amostras, íons Ca2+ foram retirados da estrutura da HAP, originando uma HAP deficiente em cálcio, e isso, provavelmente, faz com que o volume da cela unitária diminua. Nas amostras tratadas termicamente, observou-se uma redução do volume da cela unitária mais significativa nas amostras controle (AC) e GEL1, quando se envelheceu por mais tempo as amostras. Para as amostras não tratadas termicamente, observou-se que no mesmo período de envelhecimento (21 dias), o volume da cela unitária também é menor devido principalmente à decomposição das carbonato apatitas em CaO. Quanto às microdeformações da rede cristalina, observou-se que o tamanho de cristalito é diretamente proporcional à redução da microdeformação, devido à diminuição das tensões internas no cristal na medida em que ele cresce. As análises de IVTF mostraram a formação de carbonato apatita do tipo B, devido à substituição dos PO4 3- por CO3 2-. A substituição foi mais evidente nas amostras controle (AC) e GEL1 com menor tempo de envelhecimento; nas amostras GEL2, as bandas de fosfato foram mais evidentes. As micrografias obtidas por MEV revelaram a formação de aglomerados e que os cristais são nanoestruturados. Estes resultados confirmam que a presença da gelatina afeta diretamente as propriedades estruturais da HAP. O método de síntese empregado neste estudo se mostrou eficaz na produção de nanocristais de apatitas mimetizando estruturalmente uma apatita biológica.

Bibliography: leaves [280]-301

This study aimed synthesizing hydroxylapatite (HA) and Zn2+, F- and Cl- doped HA by solution precipitation method. The synthesized compounds were sintered at 1100&deg / C for 1h. For structural characterization, density of the samples were measured by Archimedes&rsquo / method. It was observed that Zn2+ addition increased the density significantly whereas F- caused a decrease and Cl- increased the density with a little amount. XRD was applied to the samples and it was found that co-doping of Zn2+ and F- ions decreased the unit cell volume of HA with F- addition. Other compositions gave fluctuated results in terms of unit cell volumes. HA phase and a little amount of CaO phase were detected in some samples. FTIR spectroscopy was used to detect whether Zn2+, F- and Cl- ions were incorporated to the HA structure or not by observing the bands corresponding to the bonds in the molecules. The amount of addition was also detected by FTIR. Results showed that ion incorporation to the HA structure was done successfully. SEM images were v analyzed and grain sizes of samples were calculated by Rietvelt analysis. Grain sizes of the samples increased by Cl- addition and decreased by Zn2+ and/or F- addition. For mechanical characterization, Vickers microhardness test was applied. Fracture toughness was calculated from Vickers microhardness results. According to the results, the highest microhardness values were found for F- and Zn2+ co-doped samples. It was also shown that fracture toughness decreased by Zn2+ addition. However, Zn2+ and F- co-doped samples gave higher fracture toughness results when compared with pure HA. Cl- addition also decreased the fracture toughness. The best compositions in terms of structural and mechanical properties was chosen as Zn2+ and F- co-doped samples and biological characterization was applied to these samples. Saos-2 cell line was used in biological examinations. For biological characterizations, Alamar Blue&trade / assay to detect viability and alkaline phosphatase activity (ALP) assay to detect differentiation were done. It was observed that 2 mol.% Zn2+ addition increased the cell viability and alkaline phosphatase activity. 1 mol.% F- addition also improved cell viability and alkaline phosphatase activiy. SEM images were analyzed to observe the morphology of the cells on HA and selected doped HA discs. In accordance with Alamar Blue&trade / assay and alkaline phosphatase activity assay, cells showed dendritic shapes on 2 Zn and 2 Zn 1 F sample which was the indicator of good material-cell interaction. Dissolution test was also applied by immersing the samples in simulated body fluid (SBF). pH change and SEM images for Ca2+ deposition were investigated. Increase in pH change with time was observed. F- included samples gave the lowest pH change results, especially 2 Zn 1 F. Dissolution pits and some apatitic formations were observed in SEM images.

Bibliography: leaves [280]-301 / xiv, 317 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1984

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