*2.7. Acid-Neutralizing Ability*

To estimate the acid-neutralizing ability, disk specimens were immersed in individual plastic vials containing 10 mL of acetic acid solution (pH = 4.5) at 37 ◦C. The time-dependent changes in the pH of the solutions were measured (model SI600; Sentron, Roden, The Netherlands) with a micro-pH electrode (model 9070-008; Sentron) over 24 h (*n* = 5).

#### *2.8. Enamel Remineralization Ability and Changes in the Mechanical Properties*

A total of 38 human non-carious premolars, obtained by extraction from patients undergoing orthodontic treatment, were cut with a slow-speed, water-cooled diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) so that they were divided into mesial and distal halves; the sectioned specimens were then encapsulated in epoxy resin (Epofix; Struers, Copenhagen, Denmark). This in vitro study was approved by the ethics committee of the Health Sciences University of Hokkaido. After 24 h, the specimens were lightly ground with 600-grit sandpaper, and polished progressively using diamond suspensions with particle sizes of 3, 1, and 0.25 μm to obtain a surface suitable for nano-indentation. This polishing procedure removed approximately 200 μm of the tooth surface; a total of 75 polished-surface enamel specimens with an approximate area of 4 × 4 mm2 were finally obtained. The specimens were divided into five groups of 15 specimens. Embedded human enamel specimens were etched with 35% phosphoric acid gel (Transbond XT Etching Gel; 3M Unitek, Monrovia, CA, USA) for 15 s, washed for 20 s, and dried in an air stream. Each etched enamel and disk specimen was fixed on a specimen stage and then immersed in a plastic vial containing 10 mL of artificial saliva at 37 ◦C for 3 months, with the solution changed weekly. Nano-indentation testing of the enamel surfaces was carried out at 28 ◦C (model ENT-1100a; Elionix) using two different loads (10 and 100 mN), before and after etching and during immersion periods. The hardness and elastic modulus of the buccal enamel surfaces were calculated.
