In this study, 3D porous anodic coatings containing 1.92 wt% copper (Cu-1.92 wt%) are developed on Ti-6Al-4V alloys via micro-arc oxidation (MAO). The in vitro long-term antibacterial activity of the MAO-treated samples is systematically evaluated. The results show that after 14 days’ immersion in Hanks’ solution, the antibacterial rate of Cu-1.92 wt% against Staphylococcus aureus (S. aureus) remains at 100%, while that against Escherichia coli (E. coli) decreases to 92% and 12% after 8 and 14 days, respectively, suggesting that Cu-1.92 wt% still exhibits good long-term antibacterial properties even after 14 days. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis and reactive oxygen species (ROS) fluorescence imaging reveal that the antibacterial efficiency of Cu-1.92 wt% is higher than those of Cu-1.92 wt%-8D, Cu-1.92 wt%-14D, and the untreated control. The enhanced antibacterial properties of the MAO samples are attributed to ROS-mediated DNA damage prompted by the presence of Cu in the coatings. In addition, Cu-1.92 wt% favors good initial cell adhesion and high cell viability. These results reveal that the fabricated biocompatible copper-doped 3D porous coatings exhibit good in vitro long-term antibacterial properties and have potential to be applied in the orthopaedics clinic in the future.
