Antibacterial silver- and copper-based surfaces on additively manufactured titanium implants

Background: The increase in orthopaedic implants, as a result of an ageing population, is accompanied by increasing numbers of implant associated infections (IAI). Current prevention and treatment procedures are not sufficient to prevent IAI, with a tremendous impact on patients’ quality of life. Preventing IAI at the source of the infection, increases the effectiveness while simultaneously the quantity of the antibacterial agents can be reduced. By synthesising an antibacterial surface, the implant prevents bacteria from adhering to the implant surface as well as infection of the surrounding tissue. Silver and copper nanoparticles have limited reports of resistant bacteria. Combining both agents could aid in the combat against antibacterial resistance by attacking the bacteria with multiple mechanisms. Combining this novel prevention technique with additive manufactured implants, allow it to be compatible with the advancements in orthopaedic implant production. This study demonstrates the synthesis of self-defending implants by simultaneously incorporating silver and copper nanoparticles in a titanium oxide surface grown on additively manufactured Ti6Al4V implants. Methods: Porous Ti6Al4V implants were designed and manufactured using selective laser melting (SLM). Using plasma electrolytic oxidation (PEO) a titanium oxide layer was grown on the entire implant surface. By addition of silver and copper nanoparticles along with calcium and phosphate in the PEO electrolyte, multifunctional surfaces were formed on the implants. The nanoparticles were added in silver to copper ratios of 0/100, 25/75, 50/50, 75/25, 100/0 and 100/100 to obtain several implant groups. The implant surface morphology was analysed using a scanning electron microscope (SEM). The composition of the oxide layer was analysed using energy dispersive X-ray spectroscopy (EDS), to demonstrate the incorporation of both silver and copper nanoparticles in the different silver to copper ratios. silver- and copper-ions release of the implants was measured in a biomimetic environment to characterise the in vitro ion release activity over one month. Subsequently, in vitro antibacterial experiments were performed, against Methicillin-resistant Staphylococcus aureus, a resistant and recurring pathogen in IAI. First, the antibacterial leaching activity was measured using a zone of inhibition assay. Secondly, the minimum inhibitory concentration (MIC) of silver-, copper-ions and the extent of synergy between the silver and copper ions was measured using a checkerboard assay. Finally, the bactericidal activity of the implants was quantified with a colony forming units (CFU) assessment. Results: The PEO process resulted in the formation of an oxide layer with simultaneous incorporation of silver and copper nanoparticles. SEM and EDS analysis showed that the silver to copper ratio added to the electrolyte determined the nanoparticle ratio present in the oxide layer. The ion release showed silver- and copper-ions releasing over one month. The implants with both silver and copper nanoparticles showed a lower release of silver ions and a higher release of copper ions with respect to the implants with a single nanoparticle. The antibacterial leaching activity assessment showed limited leaching for the copper implants, good leaching for the silver implants and a decreasing leaching area when silver was combined with copper. The MIC of silver ions was 0.016 mM, of copper ions, 10 mM and combined 0.002 mM silver- and 5 mM copper-ions. Finally, the CFU assay showed adhering bacteria on all implants after 24h incubation and no inhibition against non-adhering bacteria. Conclusions: The PEO process allowed for the simultaneous incorporation of silver and copper nanoparticles in the formed oxide layer, without affecting the porous geometry of the SLM manufactured implants. Combining silver and copper nanoparticles in the oxide layer promotes the cumulative Cu2+ and inhibits the Ag+ release. The Ag and Cu nanoparticles present in the implants did not prevent bacteria from adhering to the implants.Biomedical Engineerin