Fresh bovine incisors were obtained from a local slaughter house (Yokohama, Japan). After cleaning the bovine teeth of debris and soft tissue, enamel blocks () () were cut from the incisors using a low speed diamond saw (Isomet; Buehler, Lake Bluff, Illinois) under running water, and embedded in acrylic resin (Unifast Trad; GC, Tokyo, Japan). The outer enamel surface was polished to a mirror finish using wet polishing papers (800, 1000, 1200, 1500, and 2000 grit lapping papers; 3M, St. Paul, Minnesota). Three areas, namely sound (SND), demineralized (DEM), and remineralized (REM), were assigned on the polished enamel surface of each block as follows; first, one-third of the surface on each block was covered with a nail varnish (nail POP; Chamon, Gyeonggi, South Korea), which served as the SND portion. Second, subsurface lesions were then formed on the remaining surface using the two-layer demineralization method with 8% methylcellulose gel (Methocel MC; Fluka, Everett, Washington) and 0.1 M lactate buffer (pH 4.6) at 37°C for 14 days.27 The ratio of gel to lactate buffer was 3:5, respectively. After demineralization, the blocks were sterilized by ethylene oxide gas sterilizer (Steri-Gas; 3M, St. Paul, Minnesota). Third, the middle-third of the surface was kept as a DEM area, and the remaining third of enamel surface, which was cut away by low speed saw, served as REM area as described below.