Silver-based antibacterial surfaces for bone implants

Total hip arthroplasty (THA) is the most effective and safest method for treating severe degenerative, post-traumatic and other diseases of the joints. With an aging population that is increasingly active, the use of biomedical implants will continue to rise. It is estimated that more than 1,000,000 THAs are performed each year globally. Consequently, the threat posed by implant associated infections (IAI) will affect a large percentage of this populace. IAI are serious complications, caused by infectious bacteria that colonize the implant surface, rapidly proliferate and secrete a matrix of polysaccharides known as a biofilm. Once formed, the biofilm is extremely resistant to host-defense mechanisms or antibiotics, and ultimately will lead to implant loosening and revision surgery with devastating effects on the patient. Currently, no solutions are clinically available to prevent IAI on cementless implants. Therefore, intense research efforts are focused on development of antibacterial surfaces that will kill any adherent bacteria and thus, prevent the bacteria from generating a biofilm. The aim of this PhD thesis was to develop a novel antibacterial surface for cementless implants. The research was focused on the synthesis, physicochemical characterization and biological evaluation of an Ag-based antibacterial surface on the Ti6Al7Nb biomedical alloy. Using the plasma electrolytic oxidation (PEO) process, various concentrations of Ag nanoparticles were incorporated into a microporous TiO2 layer grown on the Ti6Al7Nb alloy to render its surface antibacterial. Following synthesis, thorough characterization of the novel oxide layers with regard to Ag nanoparticles distribution, chemical and phase composition, surface roughness, coating porosity, pore density, pore size distribution, and surface free energy was performed. Furthermore, their antibacterial effectiveness and toxicity to human bone cells were assessed in vitro. Finally, a feasibility study assessing whether Ag-based antibacterial surfaces can be applied on commercially available bone implants such as plasma sprayed titanium hip components was also performed. The thesis comprises six chapters starting with an introduction in Chapter 1 that provides the background, as well as the state of the art information on implant associated infections and current biomaterial-based strategies to prevent this problem. Furthermore, a description of the PEO process, as a possible process for generating antibacterial surfaces for bone implants, and the motivation of using Ag nanoparticles as antibacterial agent are also presented. Finally, the aim of the research and the thesis outline are defined. The experimental conditions and the required equipment for the synthesis and characterization of novel Ag-based antibacterial layers on the Ti6Al7Nb alloy are presented in Chapter 2. In addition, the main results on surface characterization are reported in this chapter. During the PEO process, the Ti6Al7Nb disk was immersed in an aqueous calcium acetate/calcium glycerophosphate-based electrolyte bearing dispersed Ag nanoparticles and connected to a high-voltage power supply. A stainless steel plate served as the counter-electrode. When the applied voltage exceeded a certain critical value, dielectric breakdown of the anodic barrier TiO2 layer occurred on the surface of the Ti6Al7Nb alloy thereby resulting in a modified surface. The morphological investigations by scanning electron microscopy (SEM) revealed the presence of a porous TiO2 layer with well-separated round/elongated pores, ranging from a few nanometers up to 10 ?m in size. The presence of Ag nanoparticles in the porous TiO2 layers was confirmed by high-resolution SEM coupled with the back-scattering and energy dispersive X-ray spectroscopy (EDX) detectors. Except the presence of different concentrations of Ag nanoparticles in the TiO2 layers, the other surface characteristics such as chemical and phase composition, surface roughness, porosity, pore density, pore size distribution, mean pore size and surface free energy remained unchanged as compared with the Ag-free TiO2 layers. Therefore, the effect of Ag nanoparticles embedded in the TiO2 coatings in various concentrations, on the bacteria killing ability and viability of the bone cells could be evaluated without the interference of the other factors. The challenge addressed in Chapter 3 was the investigation of the mechanism of Ag nanoparticles incorporation in the TiO2 layers during the PEO process. How and where are the Ag nanoparticles incorporated in the porous TiO2 layer were the main research questions addressed. Thus, the Ag-bearing TiO2 layers were grown at different oxidation times i.e., 10, 30, 60, 90, 120, 180, 240 and 300 seconds and thoroughly studied in plan view and cross-section using state of the art imaging techniques such as high-resolution transmission electron microscopy and SEM coupled with EDX analyses for chemical composition. It was observed that Ag nanoparticles could be incorporated in the growing TiO2 layers starting with very initial stages of oxidation (i.e., 10 seconds) with further incorporation as the PEO process was continued for longer durations. Thorough investigation of the coatings for evidence of particles revealed three different locations of Ag within the oxide: (i) fused on the surface of the oxide (with some particles protruding from the layer), (ii) fused into the pore walls (both open and closed pores), and (iii) embedded across the thickness of the dense oxide layer (starting just above the barrier-like layer). The morphology of the Ag nanoparticles found in the layers seemed to match that of the nanoparticles used in the electrolyte. As far as the mechanism of Ag nanoparticles incorporation is concern, four main steps were proposed: (i) transport of Ag nanoparticles at the TiO2/electrolyte and Ti6Al7Nb/TiO2 interfaces by mechanical agitation and electrophoretic mobility through short-circuit channels, open pores and cracks; (ii) attachment/adsorption of Ag nanoparticles to the sites of coating growth, where under the local heating generated by the sparking events the layer is relatively soft; (iii) fusion of Ag nanoparticles into the soft oxide once the sparks extinguish and the site is cooled by the electrolyte; (iv) encroachment of the already embedded Ag nanoparticles during the coating growth as the new coating formed close to the Ti6Al7Nb/TiO2 interface might be extruded through the breakdown channels causing the molten material to fill the previously created pores and cracks moving along the Ag nanoparticles that were fused in a previous discharge event. The particular focus of Chapter 4 was to perform in vitro tests to evaluate the antibacterial activity of the TiO2 layers bearing different concentrations of Ag nanoparticles. The main challenge was to find and use the proper antibacterial assays to investigate the antibacterial activity of solid surfaces. Considering that the current available assays do not reflect the precise conditions under which the bacteria will come in close contact with a potentially bactericidal surface, new assays for surface antibacterial activity as well as leachable antibacterial activity were proposed and tested. The pathogen used for the tests was methicillin-resistant Staphylococcus aureus (MRSA), one of the most prevalent and virulent microorganism responsible for implant associated infections. Surface antibacterial activity, tested by a direct contact assay, revealed 98% and 99.75% reduction in bacteria colony forming units (CFU) for 0.3Ag and 3.0Ag surfaces, respectively. In contrast, the untreated Ti6Al7Nb and Ag-free (0Ag) surfaces showed a 1000-fold increase in bacterial CFU. Testing of leachable antibacterial activity revealed a well-defined inhibition zone around the Ag-bearing samples due to the release of Ag ions after 24 hours of incubation with MRSA. Quantification of Ag ions release and determination of total Ag content in the layers, together with the antibacterial tests, led to the conclusion that both Ag nanoparticles per se as well as Ag ions release contributed to the bactericidal activity of the TiO2 surfaces. The next step in the development of antibacterial surfaces for bone implants was to check their toxicity to human cells. Whether or not the Ag-bearing TiO2 layers were toxic to osteoblast cells was described in Chapter 5. For this, a Simian Virus 40 (SV40)-immortalized human fetal osteoblast (SV-HFO) cell line was used. Cell viability, cell morphology and spreading, and the actin cytoskeletal organization and nucleus were studied in vitro using the Alamar Blue assay, SEM and fluorescence microscopy, respectively on TiO2 surfaces bearing different concentrations of Ag nanoparticles (i.e., 0.3Ag, 0.8Ag, 1.6Ag and 3.0Ag). The results showed that the viability of osteoblast cells was strongly dependent on the concentration of Ag nanoparticles in the layers. The 3.0Ag surfaces, because of the higher concentration of Ag nanoparticles in the TiO2, were found to be toxic to osteoblast cells. In contrast, the osteoblasts viability on the 0.3Ag surfaces, assessed by Alamar Blue, SEM and fluorescence microscopy, was not inhibited after 2, 5 and 7 days of culture. The testing of the TiO2 surfaces bearing intermediate concentrations of Ag nanoparticles appeared to follow the same trend. A significantly lower number of bone cells survived on the 0.8Ag and 1.6Ag samples after 2 days of culture. Chapter 6 covers a feasibility study to see whether the PEO process, used to render the surface of a Ti6Al7Nb alloy antibacterial, can be applied on commercially available cementless bone implants such as plasma-sprayed titanium hip components. Such implants have very complex geometries and the main challenge was to create an Ag-bearing antibacterial TiO2 layer that covers accurately the topography of such implants. A titanium plasma-sprayed acetabular cup was chosen as a substrate and PEO was performed using the Ag-bearing calcium acetate/calcium glycerophosphate electrolyte. The superimposed layers were then characterized with respect to surface morphology and chemistry using SEM and EDX. The surface morphology results showed the successful creation of the TiO2 layer, with its fine network of interconnected, round or elongated pores, superimposed on the macro-porous structure of the plasma-sprayed coating. The PEO antibacterial layer followed accurately the surface topography of the plasma-sprayed sample. Furthermore, the Ag nanoparticles were found to be present on top as well as in the porous structure of TiO2 layer. The work performed in this thesis leads to the final conclusions that (i) TiO2 layers bearing Ag nanoparticles can be produced on titanium biomedical alloys and on titanium-based bone implants using the plasma electrolytic oxidation process and (ii) the resultant novel layers can be a suitable engineering solution for the clinical problem of implant associated infections of cementless bone implants