Malignant bone tumours often require a patient to make the choice between limb salvage surgery and complete amputation. The Ti-6Al-4V alloy is the material of choice for implantable orthopaedic devices as it provides a favourable combination of biocompatibility, corrosion resistance and mechanical properties. The only drawback of titanium is that, owing to its bio-inertness, living tissue struggles to attach, creating an opportunity for bacterial adhesion. The “race for the surface” is the term given for the competition between living tissue and bacteria to colonise the implant surface. If bacterial adhesion occurs at a higher rate than tissue adhesion, the chance of infection rises significantly. It has been shown that there is an opportunity to give tissue adhesion the edge by slowing down the initial colonisation of the implant surface by free-swimming bacteria. Silver has a relatively low toxicity level of 28 mg/kg in the body. Current research has focussed mainly on reducing bio-inertness and improving the antimicrobial properties of titanium via the incorporation of silver. In general, the studies conducted on antibacterial surfaces are limited to testing the final sample directly in contact with bacteria, with no attempt to measure silver release rate profiles. The research in this dissertation aimed to investigate methodologies for the incorporation of silver into a modified surface of Ti-6Al-4V in order to facilitate an antimicrobial effect for use in orthopaedic implants. The methodologies investigated were: anodic oxidation of Ti-6Al-4V, followed by silver ion exchange; Ag-doped TiO2 fused to the surface of Ti-6Al-4V via anodic oxidation; and Ag ion implantation into anodically oxidised and polished Ti-6Al-4V. The generated surfaces and sub-surfaces were characterised microstructurally via SEM, FIB, TEM and AFM and chemically by RBS, XRD, AAS and EDS. Ag+ release rate investigations were conducted with the use of ICP-MS. This study was limited to the use of two anodising electrolytes (i.e. 0.5M H2SO4 and 2.1M H3PO4) and altering the AgNO3 concentration (0.05 - 5.0M) and Ag implantation dosage (0.4 - 1.2x1017 ions/cm2 ), where applicable to the method. Results from the Ag ion exchanged samples showed that, microstructurally, the surface produced via anodising in 0.5M H2SO4 and 2.1M H3PO4 were different in terms of pore morphology, Ra, pore homogeneity across the surface and crystal structure. Sub-surface analysis via FIB/TEM found that the ca. 200nm thick TiO2 samples all contained silver nanoparticles (AgNPs). Samples anodised in 0.5M H2SO4 produced an anatase crystal structure, whilst those anodised in 2.1M H3PO4 produced rutile crystal structures. Silver uptake by samples anodised in 0.5M H2SO4 showed decreases in Ag absorption at high (5.0M) AgNO3 ion exchange concentrations, relative to low (0.05M) concentrations. The opposite effect was observed for samples anodised in 2.1M H3PO4. Ag+ release curves corroborated the absorption data by displaying the same trends in terms of Ag+ release post ion exchange. It was concluded that it was a combination of diffusion bottlenecking and higher reactivity of the anatase phase formed during anodising in 0.5M H2SO4 with Ag+ versus the rutile phase that led to these trends. Synthesis of TiO2 powders showed that increasing the AgNO3 concentration (0.05-5M) resulted in AgTiO2 powders with increasing Ag content. Ag-TiO2 powder was successfully fused to the surfaces via anodic oxidation in 0.5M H2SO4 and 2.1M H3PO4 at 100V. Ag-TiO2 powder fused preferentially in areas where downward pressure was present. Microstructurally, the sub-surfaces produced an anodic oxide approximately 200nm thick, to which a significantly thicker, AgNP-containing, TiO2 was attached. XRD data indicated additional Brookite (020) peaks, owing to the presence of the attached Ag-TiO2 powder on the surfaces. Ag-TiO2 powders attached via 0.5M H2SO4 showed a higher overall Ag+ release at all investigated powder concentrations (0.48 - 76.93 wt% Ag) versus those attached via 2.1M H3PO4. This was concluded to be due to the anatase phase produced by 0.5M H2SO4 having greater oxidative power, thus accelerating oxidative dissolution of the AgNPs. RBS data corroborated these trends. Relative to their Ag ion exchange counterparts, the Ag-TiO2 samples had a lower Ag+ release at 0.05M and 0.5M AgNO3 concentrations. However, at 5.0M AgNO3 the Ag-TiO2 samples had a higher Ag+ release. This was the trend irrespective of the anodising electrolyte. Both the anatase and rutile TiO2s showed a reduction in Ra post Ag ion implantation and the polished Ti6Al4V samples showed an increase in Ra. This was due to preferential erosion of areas with high free surface energy. In the case of both TiO2s these were “high points” in the oxide and for polished Ti6Al4V these were the grain boundaries. Both TiO2s were amorphised during ion implantation. All ion implanted TiO2 showed the presence of AgNPs within the first 50nm of the surface. These AgNPs increased in size as the implantation dosage was increased. Polished Ti6Al4V showed no AgNP formation but EDS mapping confirmed that the silver was also located 50nm within the surface. TiO2 Ag+ release was similar for both implantation dosages because the surfaces had been supersaturated at the low dose, thus an increase in implantation dose had no significant effect on further silver uptake. The release rates were also similar between the oxides because of amorphisation. Polished Ti6Al4V showed an increase in Ag uptake and Ag+ release when the implantation dose was increased. RBS results corroborated the observed Ag+ release results. In comparison, both the ion exchanged samples and the Ag-TiO2 fused samples showed performances in similar ranges of Ag+ release. The Ag-TiO2 samples showed a greater degree of tailorability of the Ag+ release, whereas the ion exchanged samples showed a lesser sensitivity to an increase in AgNO3 concentration. Ag ion implanted samples showed an order of magnitude lower Ag+ release relative to the other studied methods. In comparison to literature, all ion exchanged and Ag-TiO2 samples had the potential to have a 100% antimicrobial effect (AE). Ion implanted oxides had a 55-100% potential, while the polished Ti6Al4V had a 55% AE at low dose and a 100% AE at high dose. In order to achieve maximum silver ion release and the associated antimicrobial effect, the technique of Ag-TiO2 fused to the surface using the 2.1M H3PO4 and 0.5M H2SO4 electrolytes yielded the best results, with a silver ion release of 550 and 600 ppb respectively over two weeks. This technique also satisfied the research aim, in that the methodology offered a combination of tailorability of silver release and commercial scalability.

<p>Silver was used for its antimicrobial effect by the ancient Greeks, long before the existence of microorganisms were first suspected. Nowadays a wide range of antimicrobial dressings containing silver, either incorporated within or applied on the dressings, are available for clinical use. This type of dressings is designed to provide the antimicrobial activity of silver in a more convenient application.</p><p>The aim with this master thesis was to evaluate if silver release and antimicrobial effect of nine silver containing dressings are dependent on the test medium and if there is any relation between silver release and antimicrobial effect.</p><p>Release of silver and antimicrobial effect was evaluated by using a 6-well co-culture system, with inoculated test medium in the wells and dressing pieces in the culture well inserts. Three different test media with increased complexity and nutrient value were inoculated with either</p><p>Results show that release of silver depends on the test fluid used; for phosphate buffered saline (PBS), the silver concentration was as most 1.2 ppm, but for a complex media containing calf serum (SWF), it varied from 9 ppm to 134 ppm. The viable counts in PBS were reduced by at least 3 log units for all dressings and bacteria, whereas in SWF there were no reduction and instead growth was observed. In general, a high release resulted in less bacterial growth. Results also indicated that kinetics of silver release affect the antimicrobial effect. It is likely to assume that it is important for a dressing to release silver quickly.</p><p>It has previously not been possible to correlate silver release of wound care dressings and antimicrobial effect, since the two factors have been measured in different test systems and in different media. Since both factors depend on test medium and method used, it is shown in the present study that it is important to use relevant test medium for in-vitro evaluation. When measuring silver release and antimicrobial effect in the same test system, a relation is found.</p>

Silver was used for its antimicrobial effect by the ancient Greeks, long before the existence of microorganisms were first suspected. Nowadays a wide range of antimicrobial dressings containing silver, either incorporated within or applied on the dressings, are available for clinical use. This type of dressings is designed to provide the antimicrobial activity of silver in a more convenient application. The aim with this master thesis was to evaluate if silver release and antimicrobial effect of nine silver containing dressings are dependent on the test medium and if there is any relation between silver release and antimicrobial effect. Release of silver and antimicrobial effect was evaluated by using a 6-well co-culture system, with inoculated test medium in the wells and dressing pieces in the culture well inserts. Three different test media with increased complexity and nutrient value were inoculated with either Results show that release of silver depends on the test fluid used; for phosphate buffered saline (PBS), the silver concentration was as most 1.2 ppm, but for a complex media containing calf serum (SWF), it varied from 9 ppm to 134 ppm. The viable counts in PBS were reduced by at least 3 log units for all dressings and bacteria, whereas in SWF there were no reduction and instead growth was observed. In general, a high release resulted in less bacterial growth. Results also indicated that kinetics of silver release affect the antimicrobial effect. It is likely to assume that it is important for a dressing to release silver quickly. It has previously not been possible to correlate silver release of wound care dressings and antimicrobial effect, since the two factors have been measured in different test systems and in different media. Since both factors depend on test medium and method used, it is shown in the present study that it is important to use relevant test medium for in-vitro evaluation. When measuring silver release and antimicrobial effect in the same test system, a relation is found.

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Les nanoparticules (NPs) d'Ag sont très utilisées dans le secteur de la santé, dans l'industrie alimentaire et dans les produits de consommation pour leurs propriétés antimicrobiennes. Le grand rapport surface sur volume des NPs d'Ag permet une augmentation importante du relargage d'Ag comparé au matériau massif et donc une toxicité accrue vis à vis des micro-organismes sensibles à cet élément. Ce travail de thèse présente une évaluation des propriétés antimicrobiennes de petites NPs d'Ag (<20 nm) enrobées dans des matrices de silice sur la photosynthèse d'algues vertes. Deux techniques d'élaboration par voie physique ont été utilisées pour fabriquer ces nanocomposites: (i) l'implantation ionique à basse énergie et (ii) la pulvérisation d'Ag couplée avec la polymérisation plasma. Les propriétés structurales et optiques de ces nanostructures ont été étudiées par microscopie électronique à transmission, réflectivité et ellipsométrie. Cette dernière technique, couplée à un modèle basé sur l'approximation quasi-statique de type Maxwell-Garnett, a permis la détection de petites variations dans la taille et la densité des NPs d'Ag. Le relargage d'argent de ces NPs d'Ag enrobées dans des diélectriques a été mesuré par spectrométrie de masse après immersion dans de l'eau tamponnée. La toxicité à court terme de l'Ag sur la photosynthèse d'algues vertes, Chlamydomonas reinhardtii, a été évaluée par fluorométrie. L'enrobage des nanoparticules dans un diélectrique réduit leur interaction avec l'environnement, et les protège d'une oxydation rapide. La libération d'Ag bio-disponible (impactant sur la photosynthèse des algues) est contrôlée par la profondeur à laquelle se trouvent les NPs d'Ag dans la matrice hôte de silice. Cette étude permet d'envisager le design de revêtements à effet biocide contrôlé. En couplant les propriétés antimicrobiennes de ces NPs d'Ag enrobées à leur qualité d'antenne plasmonique, ces nanocomposites peuvent être utilisés pour détecter et prévenir les premières étapes de la formation de biofilms sur des surfaces. Ainsi, une dernière partie de ce travail est dédiée à l'étude de la stabilité et de l'adsorption de protéines fluorescentes Discosoma rouges recombinantes (DsRed) sur ces surfaces diélectriques avec la perspective du développement de dispositifs SERS. / Silver nanoparticles (AgNPs) because of their strong biocide activity are widely used in health-care sector, food industry and various consumer products. Their huge surface-volume ratio enhances the silver release compared to the bulk material, leading to an increased toxicity for microorganisms sensitive to this element. This work presents an assessment of the biocide properties on algal photosynthesis of small (<20 nm) AgNPs embedded in silica layers. Two physical approaches were used to elaborate these nanocomposites: (i) low energy ion beam synthesis and (ii) combined silver sputtering and plasma polymerization. These techniques allow elaboration of a single layer of AgNPs embedded in silica films at defined nanometer distances (from 0 to 7 nm) beneath the free surface. The structural and optical properties of the nanocomposites were studied by transmission electron microscopy, reflectance spectroscopy and ellipsometry. This last technique, coupled to modelling based on the quasi-static approximation of the classical Maxwell-Garnett formalism, allowed detection of small variations over the size and density of the embedded AgNPs. The silver release from the nanostructures after immersion in buffered water was measured by inductively coupled plasma mass spectrometry. The short-term toxicity of Ag to the photosynthesis of green algae, Chlamydomonas reinhardtii, was assessed by fluorometry. Embedding AgNPs reduces their interactions with the buffered water, protecting the AgNPs from fast oxidation. The release of bio-available silver (impacting on the algal photosynthesis) is controlled by the depth at which AgNPs are located for the given host silica matrix. This provides a procedure to tailor the biocide effect of nanocomposites containing AgNPs. By coupling the controlled antimicrobial properties of the embedded AgNPs and their quality as plasmonic antenna, these coatings can be used to detect and prevent the first stages of biofilm formation. Hence, the last part of this work is dedicated to a study of the structural stability and adsorption properties of Discosoma recombinant red (DsRed) fluorescent proteins deposited on these dielectric surfaces with perspectives of development of SERS devices.