Aneropack

Two species of oral bacteria, Streptococcus mutans JCM5705 (RIKEN BioResource Center, Wako, Japan) and Aggregatibacter actinomycetemcomitans JCM2434 (RIKEN BioResource Center, Wako, Japan) were used in this study. Cultures were grown anaerobically using AneroPack (Mitsubishi Gas Chemical Company, Tokyo, Japan) in a brain heart infusion (BHI) broth (Becton Dickinson Labware, Franklin Lakes, NJ, USA) or BHI broth with 10 g/L of yeast extract (BHI-YE) (Oxoid, Hampshire, UK) at 37 °C for 24 h. The prepared bacterial suspension was diluted in sterile saline and contained approximately 1 × 10⁸ colony-forming units (CFUs)/mL, in line with our previous report^{35 (link)}. For the biofilm-related test, the bacterial suspension was seeded on the titanium surface or plastic well and incubated in BHI-YE for Aggregatibacter actinomycetemcomitans or BHI with 1% sucrose (Kanto chemical, Japan) (BHI-S) for Streptococcus mutans at 37 °C for 24 h in anaerobic conditions.

The test strain A. actinomycetemcomitans JCM 2434 was obtained from the Japan Collection of Microorganisms (RIKEN BioResource Center, Wako, Japan). The study design is illustrated in Fig. S5 and biofilm formation was induced using a previously reported method^{65 (link)}. The single-species biofilm model, instead of a multi-species biofilm, was used as a simplified model to evaluate various testing conditions as described below. Bacteria were grown anaerobically using AneroPack (Mitsubishi Gas Chemical Company, Tokyo, Japan) in brain heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10 g/L yeast extract (Oxoid, Hampshire, UK) at 37 °C for 24 h. From this culture, a bacterial suspension was prepared in sterile saline and the concentration was adjusted to approximately 5 × 10⁸ CFU/mL. Autoclaved titanium discs were placed in 48-well plates, and then 1 mL BHI broth supplemented with 10 g/L yeast extract and 100 µL bacterial suspension (inoculum = 5 × 10⁷ CFU) were added to the wells, after which the plates were incubated under anaerobic conditions at 37 °C for 48 h. Thereafter, the specimens were gently washed twice with saline to eliminate non-attached bacteria and used in subsequent assays as Aa biofilm-Ti.