Tese de doutoramento. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 2007

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.

Dissolved compounds and ions, including mineral elements and products of microbial metabolism, are present in many cheeses in relatively high concentrations. These dissolved substances may precipitate from the aqueous phase of cheese to form sparingly soluble crystals that can impart a crunchy, gritty, or sandy texture on the cheese. In the present work, optical and diffractometric methods were optimized for use with cheese samples to identify crystal phases in several cheese varieties. These techniques, which included powder X-ray diffractometry (PXRD), single crystal X-ray diffractometry (SCXRD), and petrographic microscopy (PM) have traditionally been used on geological specimens that are quite different from the cheese samples used in the present study. Nonetheless, these techniques were successfully used to gain valuable insight into crystal development in cheese. Powder X-ray diffractometry was optimized to minimize the occurrence of artifacts that may occur due to the high water content and low crystallinity of some cheese samples. The use of enhanced sample preparation techniques facilitated the identification of organic and inorganic crystal phases such as tyrosine, leucine, brushite (CaHPO4·2H2O), and calcite (CaCO3) in hard and soft cheeses. SCXRD was used to determine the crystal structures of ikaite and struvite, which had been tentatively identified in washed-rind cheese using PXRD. PM was used to observe morphological and optical properties of crystals in white mold cheese and washed-rind cheese. In two subsequent aging studies, PXRD was used to determine the approximate timing of crystal nucleation in the rinds of white mold cheese and washed-rind cheese. These observations were paired with inductively coupled plasma-atomic emission spectroscopy (ICP-AES) data to demonstrate that the onset of crystallization in the rinds coincided with a diffusion phenomenon in which mineral ions diffused from the center of the cheese and became concentrated in the rind. PM observations demonstrated that maximum crystal size in the rinds generally increased as aging progressed. These observations will be useful in future work that investigates the impact of crystallization on sensory properties of cheese.

Calcium phosphate cements are used in medicine to fill bone defects or give support to screws and plates in fracture fixation. The cements are formed via mixing a powder with water and the mixture harden through a dissolution-precipitation reaction. Today the cement mixing is performed in the operating room and consists of several complicated steps that need to be performed under sterile conditions. This renders the mixing a risk factor, potentially leading to harm for the patient e.g. unsatisfactory healing or infection. To reduce this risk, premixed cements have been developed using glycerol as mixing liquid. The premixed cement sets when it is exposed to body liquids. Therefore, premixed cement can be delivered to the operating room in prefilled syringes ready for use, thus eliminating the mixing step. The aim of this thesis is to describe differences between premixed and water-mixed cements and their advantages and drawbacks. The differences will be discussed based on results obtained from bench testing of specific cement properties as function of cement formulations as well as in vitro and in vivo studies. Several cement formulations were evaluated e.g. the influence of powder to liquid ratio (P/L), powder particle size and addition of water on key properties. The results showed that premixed cements have excellent handling properties and have mechanical properties similar to water-based cements. Both P/L and particle size can be used to control these properties. It was shown that small amounts of water improve certain cement properties while dry raw materials were important for long shelf life. To better understand the setting of premixed cements new methods for evaluating working time and setting of premixed cements were developed. In vivo studies showed that the formulations developed in this thesis are biocompatible, resorbable and show good tissue response in bone. This thesis concludes, that the premixed cements are a promising biomaterial with excellent handling properties and good biological response. The most important challenge for the premixed cements, in order to become commercially successful, is to obtain clinically relevant setting time and shelf life simultaneously. An increasing use of premixed cements in the clinics should shorten operation times and reduce infection rates to the benefit of both patients and medical staff.

Yes / Archaeological burial contexts may include a variety of white substances, but few analyses have been published. This study reports on the physico‐chemical characterization of such residues from seven archaeological sites. It is often assumed that white materials from burial contexts are lime. Our findings demonstrate that they can be gypsum, calcite (chalk), aragonite, brushite, degraded metal, natural (gum) resins or synthetic polymer–based products. These may be present as the result of diagenetic processes, funerary practices or modern contamination. This paper provides an analytical approach for the holistic investigation of white materials encountered in burial contexts. / Investments for the future’ (IdEx Bordeaux ANR‐10‐IDEX‐03‐02). Grant Number: ANR‐10‐IDEX‐03‐02; Collaborative Projects of the France‐Stanford Center for Interdisciplinary Studies; Collaborative Projects of the France–Stanford Center; French State. Grant Number: IdEx Bordeaux ANR‐10‐IDEX‐03‐02; Northern Archaeological Associates Ltd; PACEA; Wessex Archaeology; INRAP; Mersea Island Museum Trust; Vatican's Pontifical Commission for Sacred Archaeology; University of Reading; IRAMAT-CRP2A; University of Bradford; CEREGE

Ce travail vise à étudier l’hydratation et les propriétés des ciments brushitiques à base de wollastonite dans la perspective d’une application au traitement et au conditionnement de déchets radioactifs de faible ou moyenne activité acides et/ou contaminés en strontium. Une étude des processus conduisant à la prise et au durcissement d’un ciment commercial est d’abord menée. L’influence de la composition de la solution de gâchage sur les réactions mises en jeu est ensuite précisée. Enfin, une première évaluation du potentiel de ce matériau pour la décontamination ou l’immobilisation d’effluents aqueux contaminés en strontium est réalisée.Les pâtes de ciment brushitique à base de wollastonite sont préparées à partir de wollastonite broyée et d’une solution d’acide phosphorique concentrée contenant des cations métalliques (Zn2+ et Al3+) ainsi que du borax. La wollastonite réagit selon un processus de dissolution/précipitation qui conduit à la formation de brushite, de silice amorphe et d’un aluminophosphate de calcium et de zinc amorphe dont la structure a été caractérisée par RMN-MAS du 31P et 27Al. La précipitation de brushite est précédée par la formation transitoire de phosphate monocalcique monohydraté. La simulation thermodynamique à l’aide d’un code de spéciation géochimique de la réaction de la wollastonite avec une solution d’acide phosphorique reproduit bien la séquence de précipitation des phases cristallines observée expérimentalement.Il apparaît que la concentration optimale de l’acide phosphorique dans la solution de gâchage est comprise entre 9 et 10 mol.L-1. Le bore retarde la prise du ciment tandis que le zinc l’accélère. L’ajout d’aluminium, qui permet la précipitation massive d’aluminophosphate de calcium amorphe, s’avère nécessaire pour obtenir un matériau avec de bonnes performances mécaniques. Une étude de surfaces de réponses par plan d’expériences, suivie d’une optimisation multicritères, a permis d’identifier un domaine de compositions de solutions de gâchage conduisant à un matériau possédant les propriétés recherchées (en terme de temps de prise, auto-échauffement et résistance mécanique) pour une matrice de conditionnement de déchets.Une étude cristallographique montre par ailleurs que la brushite peut incorporer au moins 30 % (mol/mol) de strontium en substitution du calcium dans sa structure. Des essais de piégeage du strontium par une pâte de ciment broyée, menés en suspension diluée, conduisent à une isotherme de sorption de type S, témoignant de l’existence de plusieurs mécanismes de rétention. Enfin, un essai de lixiviation sur un monolithe contenant du strontium révèle le bon confinement de ce dernier, avec un coefficient de rétention Rd plus de 100 fois supérieur à celui obtenu avec une pâte de ciment Portland. / This work aims at studying the hydration process and the properties of wollastonite-based brushite cements. These binders may indeed offer new prospects for the treatment or conditioning of low- or intermediate- level radioactive wastes characterized by a strong acidity and/or a contamination by strontium. First, a study of the hydration process is carried out with a commercial cement. Then, the influence of the mixing solution composition on the setting and hardening process is determined. Finally, a first assessment of the potential of this material for the decontamination and immobilization of strontium-containing aqueous effluents is performed.Wollastonite-based brushite cement pastes are prepared by mixing ground wollastonite and a phosphoric acid solution containing metallic cations (Al3+ and Zn2+) and borax. Wollastonite reacts through a dissolution/precipitation process, which leads to the formation of brushite, amorphous silica and amorphous zinc and calcium aluminophosphate whose structure has been investigated by 31P and 27Al MAS-NMR. The brushite precipitation is preceded by the transient formation of monophosphate calcium monohydrate. The thermodynamic simulation of the wollastonite reaction with a phosphoric acid solution, using a geochemical speciation code, fairly well reproduces the precipitation sequence of crystalline phases observed experimentally.It appears that the optimum phosphoric acid concentration in the mixing solution is comprised between 9 and 10 mol.L-1. Boron retards the cement setting whereas zinc accelerates it. The addition of aluminium, which leads to the massive precipitation of amorphous calcium aluminophosphate, is proved to be necessary to obtain a material with high mechanical strength. Thanks to response surface methodology and multi-criteria optimization, a composition domain of the mixing solution is pointed out, leading to a material with good properties (in terms of setting time, self-heating and mechanical strength) for waste conditioning.A crystallographic study also shows that brushite is able to incorporate at least 30 % (mol/mol) of strontium in substitution for calcium in its structure. Strontium retention tests by a ground cement paste in diluted suspension lead to a type S sorption isotherm, meaning that several retention mechanisms are involved. Finally, a leaching experiment performed on a cement monolith containing strontium shows that this species is well confined within the cement matrix, with a retention coefficient at least two orders of magnitude higher than that of a Portland cement paste.

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No / Raman spectroscopy was evaluated as a non-destructive analytical tool for the characterisation of white substances in burials. In addition, Fourier transform Raman spectroscopy was used to assess the conversion of hydrated lime into calcium carbonate. Fourteen samples of white substances from archaeological and forensic sites were analysed and characterised. The results show that not all white residues in burials are lime. Lime can easily be mistaken for other building materials (gypsum), for minerals (brushite) or degraded metal (cerussite). This study highlights the need for chemical analysis of white residues when encountered in burials. Analytical information derived from Raman spectra of white substances can further assist in the interpretation of the taphonomic processes of burials and their funerary context. Copyright (c) 2014 John Wiley & Sons, Ltd.

Calcium phosphate cements (CPCs) are used as bone void fillers and as complements to hardware in fracture fixation. The aim of this thesis was to investigate the possibilities and limitations of the CPCs’ mechanical properties, and find out if these ceramic bone cements can carry application-specific loads, alone or as part of a construct. Recently developed experimental brushite and apatite cements were found to have a significantly higher strength in compression, tension and flexion compared to the commercially available CPCs chronOS™ Inject and Norian® SRS®. By using a high-resolution measurement technique the elastic moduli of the CPCs were determined and found to be at least twice as high compared to earlier measurements, and closer to cortical bone than trabecular bone. Using the same method, Poisson's ratio for pure CPCs was determined for the first time. A non-destructive porosity measurement method for wet brushite cements was developed, and subsequently used to study the porosity increase during in vitro degradation. The compressive strength of the experimental brushite cement was still higher than that of trabecular bone after 25 weeks of degradation, showing that the cement can carry high loads over a time span sufficiently long for a fracture to heal. This thesis also presents the first ever fatigue results for acidic CPCs, and confirms the importance of testing the materials under cyclic loading as the cements may fail at stress levels much lower than the material’s quasi-static compressive strength. A decrease in fatigue life was found for brushite cements containing higher amounts of monetite. Increasing porosity and testing in a physiological buffer solution (PBS), rather than air, also decreased the fatigue life. However, the experimental brushite cement had a high probability of surviving loads found in the spine when tested in PBS, which has previously never been accomplished for acidic CPCs. In conclusion, available brushite cements may be able to carry the load alone in scenarios where the cortical shell is intact, the loading is mainly compressive, and the expected maximum stress is below 10 MPa. Under such circumstances this CPC may be the preferred choice over less biocompatible and non-degradable materials.