This study protocol was approved by the ethical committee of Osaka University (H26.E-36).

SEM analysis

The SEM analysis was performed as previously described. The decontaminated implant samples were fixed with 2% glutaraldehyde-RPMI 1640 immediately for 1 h at room temperature and washed with distilled water. Then, the samples were dehydrated with 100% t-butyl alcohol and freeze-dried. Finally, the samples were coated with platinum and examined using an emission-scanning electron microscope (JSM-6390LVZ; JEOL Ltd., Tokyo, Japan). An ordinal scale with the following variables was set to facilitate the evaluation of cleansability qualitatively by each method.

• No effect: Surface was cleansed ineffectively and covered with an enormous amount of amorphous material, debris, and bacteria.

• Fair: Surface was cleansed partially well but was far from the ideal and covered with a certain amount of amorphous material, debris, and bacteria.

• Good: Surface was cleansed effectively but not perfectly and covered with a little amount or partially no amount of amorphous material, debris, and bacteria.

• Excellent: Surface was cleansed ideally and covered with no or only a small amount of amorphous material, debris, and bacteria.

Bacterial CFU counts

To examine bacterial amounts on the implant surfaces, the samples were vortexed at maximum power for 30 s in 1 ml phosphate-buffered saline solution. Resuspended bacteria were serially diluted in a phosphate-buffered saline solution and plated on Brain Heart Infusion agar (Becton Dickinson, Sparks, MD, USA). The number of CFUs was counted after overnight growth on the BHI agar at 37 °C in a candle jar.

Statistical analysis

Statistical significance of differences in bacterial CFU counts among the six groups, including the control group, was analyzed using the Steel-Dwass test (R version 3.4.0 (R Foundation for statistical Computing, Vienna, Austria)). Significant differences between rough and machined surface implants were analyzed using the Mann-Whitney U test (SPSS 23.0 (SPSS Inc., Chicago, IL)). p < 0.05 was considered statistically significant.