PET imaging, using the bone tracer Na18F, allows the non-invasive location of atherosclerotic plaques that are at risk of rupture. However, the spatial resolution of PET is only 4-5 mm, limiting the mechanistic information this technique can provide. In this thesis, the use of fluorescence and Raman imaging to elucidate the mechanism of micro-calcification within atherosclerotic plaques has been investigated. A number of fluorescent probes to detect fluoride and calcium have been synthesised. One of the fluoride probes has been shown to be selective for fluoride however, the concentration of fluoride required to activate the probe is order of magnitudes higher than the amount of Na18F used for PET imaging making it problematic to use for future studies. On the other hand, a calcium probe has been shown to: selectively bind to hydroxyapatite (HAP); permit visualisation and quantification of HAP in both vascular and bone cell models; and effectively stain cultured aortic sections and whole mouse aorta for OPT imaging. Building on these preliminary data, fluorescence imaging and immunohistochemistry (IHC) imaging of both healthy and atherosclerotic tissue that were previously subjected to PET imaging, were successfully carried out showing the ability of the probe to detect HAP in human vascular tissue. IHC staining for Osteoprotegerin (OPG) and Osteopontin (OPN), two bone proteins recently detected in vascular tissue, showed the co-localization of OPG with the probe. Conversely, the OPN was shown to localize in areas surrounding high OPG and probe signal. To determine the exact composition of vascular calcification, Raman spectroscopy was also used. It is believed that the biosynthetic pathway to HAP passes through a series of transitional states; each of these has different structural characteristics which can be studied using Raman spectroscopy. In particular, HAP has a strong characteristic Raman peak at 960 cm-1. An increase in HAP concentration has been detected by Raman in both calcified cell models and aortic sections. When human vascular tissue was analysed, an additional peak at 973 cm-1 was present suggesting the presence of whitlockite (WTK) in this tissue as well as HAP.

Devido as limitações relacionadas ao emprego do enxerto autólogo, o uso dos biomateriais poliméricos naturais tornou-se uma opção viável em terapias regenerativas do tecido ósseo. O objetivo desse trabalho é avaliar de forma qualitativa e quantitiva a contribuição da membrana de elastina utilizada isoladamente ou em associação a hidroxiapatita e proteína morfogenética óssea, no reparo de defeitos ósseos no crânio de ratos. Foram utilizados 49 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para a criação do defeito ósseo no osso parietal esquerdo e divididos em 7 grupos com 7 animais cada. Os grupos foram implantados com os seguintes bioamateriais: grupo 1 controle (G1-C) sem implante, grupo 2 (G2-E24h) membrana de elastina 24 h, grupo 3 (G3-E24h/HA) membrana de elastina 24 h com hidroxiapatita, grupo 4 (G4-E24h/BMP) membrana de elastina 24 h com proteína morfogenética óssea, grupo 5 (G5-E96h) membrana de elastina 96 h, grupo 6 (G6-E96h/HA) membrana de elastina 96 h com hidroxiapatita, grupo 7 (G7-E96h/BMP) membrana de elastina 96 h com proteína morfogenética óssea. Após a morte indolor induzida em 6 semanas, as calotas cranianas foram retiradas para análise macroscópica, radiográfica, histológica e morfométrica. As análises macroscópicas, radiográficas e histológicas demonstraram a biocompatibilidade dos biomateriais utilizados. As médias e desvios-padrão do volume percentual relativo de osso neoformado nos defeitos cranianos dos grupos G1 a G7 foram 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectivamente. Os biomateriais mostraram-se biocompatíveis e o grupo 7 (G7-E96h/BMP) resultou na maior neoformação óssea. / Due to the limitations related to the use of autologous grafts, the use of natural polymeric biomaterials has become a viable option in regenerative therapies of bone tissue. The objective of this dissertation is to evaluate in qualitative and quantitative way the contribution of the elastin matrice used alone or in combination with hydroxyapatite and bone morphogenetic protein in the repair of bone defects in the skull of rats. Were use 49 Mices (Rattus norvegicus, Wistar), weighting approximately 330 grams and 4 months of age, were used. The animals were submitted to the surgical procedure to create the bone defect in the left parietal bone and divided into 7 groups with 7 animals each. The groups were implanted with the following biomaterials: group 1 control (G1-C) without biomaterial, group 2 (G2-E24h) 24 h elastin membrane, group 3 (G3-E24h/HA) 24 h elastin membrane with hydroxyapatite, Group 4 (G4-E24h/BMP) elastin membrane 24 h with bone morphogenetic protein, group 5 (G5-E96h) elastin membrane 96 h, group 6 (G6- E96h/HA) elastin membrane 96 h with hydroxyapatite, group 7 (G7-E96h/BMP) 96 h elastin membrane with bone morphogenetic protein. After painless death induced at 6 weeks, the skull caps were removed for macroscopic, radiographic, histological and morphometric analysis. Macroscopic, radiographic and histological analysis demonstrated the biocompatibility of the biomaterials used. The mean and standard deviations of the relative percentage volume of newly formed bone in the cranial defects of the G1 to G7 groups were 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectively. The implanted biomaterials were shown to be biocompatible and the group 7 (G7-E96h/BMP) resulted with greater bone neoformation.

Este trabalho testou a hipótese nula de que a utilização de uma cerâmica bifásica porosa (29-33% poros) (GenPhos, Baumer S.A., Mogi Mirim, Brasil) não tem efeito significativo na perda de volume depois da remodelação óssea 4 meses após a elevação da parede do seio maxilar, em comparação ao osso autógeno. Treze pacientes com altura de osso alveolar entre 1 a 5 mm na região mais estreita entre o assoalho do seio maxilar e a crista alveolar foram inclusos neste estudo clínico randomizado. Em um mesmo indivíduo, a diferença foi o tipo de material utilizado em cada um dos seios maxilares, a saber: cerâmica bifásica porosa (experimental, HA+&#x3B2;-TCP, Baumer S.A.) e osso autógeno. Quatro meses após a cirurgia, numa avaliação tomográfica computadorizada, a perda de volume na remodelação óssea foi mensurada. Os dados foram analisados pelo teste t pareado (p<0.05). A porcentagem de manutenção do volume enxertado foi significativamente maior para o GenPhos poroso (91,9±20,5%) quando comparado ao osso autógeno (69,3±5,1%). Estes resultados indicam que o material avaliado é promissor, devido à alta previsibilidade de manutenção do volume no momento da instalação dos implantes. / This study tested the null hypothesis that the use of a porous biphasic ceramics (29-33% pores) (GenPhos, Baumer S.A., Mogi Mirim, Brazil) has no significant effect on loss of bone remodeling volume after 4 months after the maxillary sinus augmentation, compared to autogenous bone. Thirteen patients with alveolar bone height between 1 - 5 mm at the narrowest region between the maxillary sinus floor and the alveolar crest participated in this randomized clinical trial. In the same individual, the difference was the type of material to be used in each of the maxillary sinuses, namely porous biphasic ceramic (experimental, HA + &#x3B2;-TCP, Baumer S.A) and autogenous bone. Four months after surgery, through a computer tomography evaluation, volume loss in bone remodeling was mesured. Data were analyzed by paired t test (p<0.05). The percentage of maintenance of the graft volume was significantly higher for porous GenPhos (91.9± 20.5%) when compared with autogenous bone (69.3±5.1%) These results indicate that the material evaluated is promising, due to the high predictability of maintanence of volume when the implants are installed.

Background: Directing autogenous Mesenchymal Stem Cell (MSC) to defect sites has a great promise in bone regeneration. We designed a MSC specific, bone affinity peptide (E7HA7) by conjugating E7 with a polyglutamate hydroxyapatite (HA) binding motif. We sought to characterize the in-vivo releasing pattern and bioactivity of E7HA7. Methods: HA discs were coated with fluorescent labeled peptides E7HA7, E7HA2 or E7 were subcutaneously implanted in Sprague Dawley rats. In an ectopic bone formation model was used to test the in-vivo bioactivity of E7HA7 conjugated to DBM. Results: E7HA7 showed slower peptide release from scaffolds in comparison to other groups, being statistically significant at week 2 compared to E7, and to E7HA2 at week 4 and 8. In ectopic model, the medians for new bone formation in each group were: iDBM=0.041mm3, iDBM-E7=0.071mm3, aDBM=0.138mm3, and aDBM-E7=0.192mm3. Conclusions: Conjugation of E7 to polyglutamate bone binding domain showed slow releasing kinetics and osteoinductive potential.

In the field of maxillofacial surgery, the treatment for the recovery of the fracture at condyle region of the mandible has been carried out using Bio-metals such as Titanium, Cobalt, Stainless-Steel because they were considered the standard materials for Implant plate devices for fracture fixation. Using these materials have led to undesired disturbances where the patient must undergo secondary surgery after recovery leading to exposure of the fracture site to the surrounding, metal-ion release into the human system, stress-shielding and interruption during imaging i.e. (Computerized tomography scans). The healing of the mandible requires a delicate and stable fixation procedure for the bone structure to heal. Bio-resorbable materials are the renovation for substituting metals for recovery of the fracture. The main dis-advantage using resorbable plates is absence of mechanical strength and stability. Bio-composites are the innovation for the treatment of the fractures the main study for this thesis is comparing the combination bio-ceramic and bio-resorbable materials using Finite Element Analysis software.

Le développement économique et l'industrialisation pendant les dernières années ont créés de nombreuses zones de déchets dangereux qui contribuent à une contamination généralisée des eaux souterraines à travers le monde. Les chercheurs ont tourné leur attention à trouver de méthodes d'efficace et moins coûteux pour l’assainissement, prenant en compte que les principaux polluants (les métaux lourds) ne sont pas biodégradables et qu’ils ont tendance plutôt à s'accumuler dans les organismes vivants, en déterminant des troubles des différentes fonctions vitales et des graves maladies. En raison de ces inconvénients il est envisagé le développement des technologies permettant la restauration de l'environnement d’une manière efficace à la fois écologique et biologique ainsi qu’en termes de coûts impliqués. Dans le cadre de cette thèse, nous avons proposé d'obtenir des matériaux avec une grande efficacité pour la décontamination des eaux continentales et océaniques. La structure du travail présent comprend quatre chapitres et présente des propriétés générales et spécifiques de matériaux nanométriques. Le premier chapitre présente des concepts généraux des matériaux céramiques et oxyhydriques utilisés pour remédier l'environnement. Le deuxième chapitre présente des méthodes générales pour la préparation des matériaux céramique et oxyhydriques ainsi que des méthodes adaptées au laboratoire pour obtenir ces matériaux à l'échelle nanométrique afin d'améliorer les propriétés d'adsorption des différents polluants. Dans le cadre de ce chapitre sont présentées aussi les techniques pour la caractérisation des matériaux à l'échelle nanométrique qui fait l’objectif de notre étude. Les deux chapitres suivants sont consacrés à la présentation des résultats expérimentaux originaux obtenus suite à la caractérisation physico-chimique, cinétique et biologiques des matériaux céramiques et oxyhydriques utilisés dans l'assainissement des eaux contaminées. / He economic development and industrialization from recent years have created many areas of hazardous waste that contributed to the contamination of groundwater worldwide. The researchers turned their attention to finding cost-effective methods for decontamination, taking into account that the main pollutants (heavy metals) are not biodegradable and they tend to accumulate in living organisms, determining disorders of different vital functions and serious illness. Because of these drawbacks the development of technologies for environmental restoration effectively both ecologically and biologically are of a great interest. As part of this thesis, we proposed to obtain materials with high efficiency for the decontamination of continental and oceanic waters. The structure of this work consists of four chapters and provides general and specific properties of nanoscale materials. The first chapter presents the general concepts of ceramic materials and iron oxide based materials used in environmental applications. The second chapter provides general methods for the preparation of ceramic materials and iron oxides and the adapted laboratory methods used for obtaining these materials at the nanoscale in order to improve their adsorption properties of different pollutants. This chapter also presents the techniques used for the characterization of the obtained materials. The next two chapters are devoted to the presentation of original experimental results obtained from the physico-chemical characterization, kinetics and biological investigations of the ceramics and iron oxide based materials used in the remediation of contaminated waters.

<p>Loosening of implants is a frequent complication in orthopaedic surgery. The aim of this thesis was to evaluate hydroxyapatite coating of pedicle screws with mechanical and histological methods and to correlate the results with the radiological findings.</p><p>Hydroxyapatite (HA) coating of pedicle screws was investigated in experimental studies in sheep. Nine sheep were operated on with destabilising laminectomies at L2-L3 and L4-L5 and stabilised with pedicle screw instrumentations, where HA coated and uncoated screws were used in a randomised fashion. After both 6 and 12 weeks of implantation, the HA coated screws demonstrated a higher bone-to-implant contact and a higher amount of bone in the area close to the screw. The pull-out resistance, stiffness and total energy to failure was higher for the HA coated screws after 12 weeks of implantation.</p><p>HA coating of pedicle screws was examined in two clinical studies. In the first series, the patients were stabilised with pedicle screw instrumentations, where HA coated screws were used in either the upper or lower instrumented level in a randomised fashion. After 10-22 months of implantation, the extraction torque was markedly higher for the HA coated screws. In the second series, instrumentations with uncoated, partly HA coated or fully HA coated screws were used. After 11-16 months implantation, the extraction torques were recorded. There were significant differences between all three groups, with the lowest extraction torques for the uncoated screws and the highest torques for the fully HA coated screws. The frequency of radiolucent zones surrounding the screws was higher for the uncoated screws than for the HA coated screws.</p><p>Radiographs from both experimental and clinical studies were examined. Screws demonstrating radiolucent zones were compared to screws without zones with respect to pull-out resistance, extraction torque, bone-to-implant contact and amount of bone surrounding the screws. All these variables demonstrated higher values for the screws without radiolucent zones. The frequency of radiolucent zones surrounding uncoated screws in the clinical study was 53%.</p><p>Conclusions: Radiolucent zones are good predictors of screw loosening. The frequency of radiolucent zones is higher than previously described. Hydroxyapatite coating improves the purchase of pedicle screws and reduces the frequency of screw loosening.</p>

Taking inspiration from nature, where composite materials made of a polymer matrix and inorganic fillers are often found, e.g. bone, shell of crustaceans, shell of eggs, etc., the feasibility on making composite materials containing chitosan and nanosized hydroxyapatite were investigated. A new preparation approach based on a co-precipitation method has been developed. In its earlier stage of formation, the composite occurs as hydrogel as suspended in aqueous alkaline solution. In order to get solid composites various drying procedures including freeze-drying technique, air-drying at room temperature and at moderate temperatures, between 50oC and 100oC were used. Physicochemical studies showed that the composites exhibit different properties with respect to their structure and composition. IR and Raman spectroscopy probed the presence of both chitosan and hydroxyapatite in the composites. Hydroxyapatite as dispersed in the chitosan matrix was found to be in the nanosize range (15-50 nm) and occurs in a bimodal distribution with respect to its crystallite length. Two types of distribution domains of hydroxyapatite crystallites in the composite matrix such as cluster-like (200-400 nm) and scattered-like domains were identified by the transmission electron microscopy (TEM), X-ray diffraction (XRD) and by confocal scanning laser microscopy (CSLM) measurements. Relaxation NMR experiments on composite hydrogels showed the presence of two types of water sites in their gel networks, such as free and bound water. Mechanical tests showed that the mechanical properties of composites are one order of magnitude less than those of compact bone but comparable to those of porous bone. The enzymatic degradation rates of composites showed slow degradation processes. The yields of degradation were estimated to be less than 10% by loss of mass, after incubation with lysozyme, for a period of 50 days. Since the composite materials were found biocompatible by the in vivo tests, the simple mode of their fabrication and their properties recommend them as potential candidates for the non-load bearing bone substitute materials. / Inspiriert von Natur, bei der Kompositmaterialien aus Polymermatrices und anorganischen Füllstoffen z.B. in Knochen, Krustentieren und Eierschalen vorzufinden sind, wurde die Herstellungsmöglichkeit von Kompositmaterial aus Chitosan und Hydroxyapatitdispersionen untersucht. Basierend auf einem Kopräzipitationsverfahren wurde eine neue Herstellungsmethode entwickelt, die als flexibler Zugang zu einem Spektrum von Komposittypen führt. In den frühen Phasen der Kompositbildung entsteht ein in der wässrigen alkalischen Lösung suspendiertes Hydrogel, das durch Filtration und Zentrifugation isoliert werden kann. IR und Ramanspektroskopie klären das Vorhandensein von Chitosan und Hydroxyapatit im Kompositmaterial. Hydroxyapatit ist als Nanopartikel der Größe von 15-50 nm in bimodaler Verteilung in der Chitosanmatrix dispersiert, und in durch Transmissionselektronenmikroskopie (TEM), X-Ray Diffraction (XRD) und Konfokaler Laserscanmikroskopie (CSLM) nachweisbaren 200-400 nm großen Clustern assembliert. NMR-Relaxationsmessungen an Hydrogelkompositmaterial decken die Existenz zweier Klassen vorkommenden Wassers im Netzwerk auf, gebundenes und freies Wasser. Mechanische Tests zeigen, dass die mechanische Festigkeit etwa eine Größenordnung unter der von massivem Knochen liegt, der Festigkeit von porösem Knochen aber gleichkommt. Enzymatische Abbauraten des Kompostimaterials sind als langsam einzuschätzen. Eine 50-tägige Einwirkzeit von Lysozym führte zu einem Abbau von 10 % der Kompositmasse. Die sich durch in vivo Tests herausstellende Biokompatibilität, die einfachen Herstellungsmöglichkeiten und die physikochemischen Eigenschaften empfehlen dieses Material als vielversprechenden Kandidaten für Knochenersatzmaterial in mäßig belasteten Bereichen.

Loosening of implants is a frequent complication in orthopaedic surgery. The aim of this thesis was to evaluate hydroxyapatite coating of pedicle screws with mechanical and histological methods and to correlate the results with the radiological findings. Hydroxyapatite (HA) coating of pedicle screws was investigated in experimental studies in sheep. Nine sheep were operated on with destabilising laminectomies at L2-L3 and L4-L5 and stabilised with pedicle screw instrumentations, where HA coated and uncoated screws were used in a randomised fashion. After both 6 and 12 weeks of implantation, the HA coated screws demonstrated a higher bone-to-implant contact and a higher amount of bone in the area close to the screw. The pull-out resistance, stiffness and total energy to failure was higher for the HA coated screws after 12 weeks of implantation. HA coating of pedicle screws was examined in two clinical studies. In the first series, the patients were stabilised with pedicle screw instrumentations, where HA coated screws were used in either the upper or lower instrumented level in a randomised fashion. After 10-22 months of implantation, the extraction torque was markedly higher for the HA coated screws. In the second series, instrumentations with uncoated, partly HA coated or fully HA coated screws were used. After 11-16 months implantation, the extraction torques were recorded. There were significant differences between all three groups, with the lowest extraction torques for the uncoated screws and the highest torques for the fully HA coated screws. The frequency of radiolucent zones surrounding the screws was higher for the uncoated screws than for the HA coated screws. Radiographs from both experimental and clinical studies were examined. Screws demonstrating radiolucent zones were compared to screws without zones with respect to pull-out resistance, extraction torque, bone-to-implant contact and amount of bone surrounding the screws. All these variables demonstrated higher values for the screws without radiolucent zones. The frequency of radiolucent zones surrounding uncoated screws in the clinical study was 53%. Conclusions: Radiolucent zones are good predictors of screw loosening. The frequency of radiolucent zones is higher than previously described. Hydroxyapatite coating improves the purchase of pedicle screws and reduces the frequency of screw loosening.

The unique features of the Spark plasma sintering (SPS) were used to investigate the sintering and deformation behaviour of titanium and titanium–titanium diboride composites, and to control the sintering and grain growth of ferroelectric Na0.5K0.5NbO3 (NKN) and of hydroxyapatite (HAp). In the SPS the samples experience a temperature different from that recorded by the thermocouple (pyrometer) used and this temperature difference has been estimated for Ti and NKN.   Sintering and deformation of titanium was investigated. Increasing heating rate and/or pressure shifted the sintering to lower temperatures, and the sintering and deformation rates changed when the α→β phase transition temperature was passed. Fully dense Ti/TiB2 composites were prepared. The Ti/TiB2 composites could be deformed at high temperatures, but the hardness decreased due to the formation of TiB.    The kinetic windows within which it is possible to obtain fully dense NKN and HAp ceramics and simultaneously avoid grain growth are defined. Materials have a threshold temperature above which rapid and abnormal grain growth takes place. The abnormal grain growth of NKN is due to a small shift in the stoichiometry, which in turn impairs the ferroelectric properties. Fully transparent HAp nanoceramics was prepared, and between 900 and 1050 oC elongated grains are formed, while above 1050 oC abnormal grain growth takes place.NKN samples containing grains of the sizes 0.35–0.6 µm yielded optimum ferroelectric properties, i.e. a high remanent polarization (Pr = 30 µC/cm2) and high piezoelectric constant (d33= 160 pC/N). The ferroelectric domain structure was studied, and all grains exhibited a multi-domain type of structure. / At the time of doctoral defense the following articles were unpublished and had a status as follows: Article 4: Manuscript; Article 5 : Manuscript