BACKGROUND.Implant-associated infection (IAI) is a rising complication in bone-related medical treatments using metal implants. Preventive measures, such as implant surfaces with integrated antibacterial properties, are hence required. The emergence of antibiotic-resistant pathogens has led to a growing interest in inorganic nanoparticles as antibacterial agents. In addition, implants should simultaneously stimulate bone tissue regeneration and integration to improve implant longevity. Additive manufacturing (AM) of orthopedic implants has attracted interest as improved bone tissue regeneration and integration was demonstrated by AM porous implants. AM porous implants, however, are at enhanced risks of IAIs; these porous implants require integrated antibacterial properties. This study focused on a systematic comparison of the antibacterial properties of AM porous Ti6Al4V implant surfaces with silver nanoparticles (Ag NPs), copper nanoparticles (Cu NPs) and/or zinc nanoparticles (Zn NPs) against methicillin resistant Staphylococcus aureus (MRSA). METHODS.Medical grade porous Ti6Al4V implants were additively manufactured using selective laser melting (SLM). Subsequently, the SLM Ti6Al4V implants were biofunctionalized by plasma electrolytic oxidation (PEO) using an electrolyte that consisted of Ca/P-based species and ratios of Ag NPs, Cu NPs and/or Zn NPs. Implants with 0, 25, 75 and 100% of Ag NPs, Cu NPs and/or Zn NPs were synthesized. After biofunctionalization, the surface morphology, structure and chemistry of the implants were investigated using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). The antibacterial properties of the implants against MRSA were studied in vitro as well as ex vivo. The leachable antibacterial activity was studied in an agar diffusion assay, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Ag+, Cu2+ and Zn2+ ions was determined by a micro-dilution assay and the bactericidal activity of the implants was quantified by a colony-forming unit (CFU) count in vitro against surface-adherent and non-adherent MRSA. Furthermore, ex vivo antibacterial properties were determined using a femoral murine infection model mimicking the in vivo environment. RESULTS.Biofunctionalization of the porous SLM Ti6Al4V implants by PEO resulted in a uniform and homogeneous micro-/nano-porous oxide layer that covered the entire surface of the implants. SEM and EDS analysis demonstrated the presence of Ag NPs, Cu NPs and Zn NPs in the TiO2 surface layer. The agar diffusion assay demonstrated a strong leachable antibacterial activity for the implants with Ag NPs and Cu NPs or Zn NPs, while no leachable antibacterial activity was observed for implants without Ag NPs. Antibacterial testing showed that Ag+, Cu2+ and Zn2+ ions, as well the combinations of the ions, were growth inhibitory and bactericidal to MRSA. In addition, implants with ≥ 50% of Ag NPs and combinations of ≥ 75% of Ag NPs and Cu NPs or Zn NPs, fully eradicated surface-adherent and non-adherent MRSA within 24 h and prevented biofilm formation up to 24 h. Moreover, strong ex vivo bactericidal properties against MRSA were demonstrated for these implants within 24 h. CONCLUSION. Biofunctionalized surfaces with Ag NPs and Cu NPs or Zn NPs on porous SLM Ti6Al4V implants showed strong in vitro and ex vivo antibacterial properties against MRSA. The implants with 50% of Ag NPs and combinations of 75% of Ag NPs and 25% of Cu NPs or Zn NPs are promising for further development, to prevent IAIs and improve the longevity of the implants in clinical applications.