**5. Conclusions**

Ag- and Cu-incorporated porous titanium dioxide layers were formed on Ti surfaces by MAO treatment. The chemical states of P, Ca, and Ti in the Ag- and Cu-incorporated oxide layer were phosphate, Ca2+, and TiO2, respectively. These chemical states did not change upon incubation in saline for 28 days. Moreover, the chemical state of Ag changed from Ag2O to metallic Ag, and that of Cu did not change by incubation in saline for up to 28 days. The concentrations of Ag and Cu were dramatically decreased by incubation for up to 7 days, and remained a slight amount until 28 days. The antibacterial e ffect of Ag-incorporated specimens changed, and the e ffect of Cu was maintained even after incubation in saline. The findings highlight the importance of the time-transient e ffects of antibacterial elements on their antibacterial properties.

**Author Contributions:** Conceptualization, M.S. and T.H.; formal analysis, M.S., Y.T., K.N., T.M. and P.C.; investigation, M.S., K.N., A.H. and M.A.; methodology, M.S., K.N., P.C. and A.H.; project administration, M.S., Y.T., A.N. and T.H.; supervision, A.N. and T.H.; validation, M.S., Y.T., K.N. and A.H.; writing—original draft, M.S.; writing—review and editing, T.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This study was supported by the Research Center for Biomedical Engineering, Tokyo Medical and Dental University, Project "Creation of Life Innovation Materials for Interdisciplinary and International Researcher Development" and project "Cooperative project amount medicine, dentistry, and engineering for medical innovation-Construction of creative scientific research of the viable material via integration of biology and engineering" by the Ministry of Education, Culture, Sports, Science and Engineering, Japan.

**Conflicts of Interest:** The authors declare no conflict of interest.
