*2.7. Enamel Demineralization Assessment*

This experimental study was carried out on the extracted bovine teeth and measured as described previously [28,29] with some modification. Briefly, the baseline surface microhardness (SMH) was measured and bovine specimens in the range from 350 KHN to 550 KHN were collected for the demineralization investigations.

Enamel blocks (4 mm × 6 mm × 2 mm) obtained from bovine incisors were embedded in ethoxyline resin. All samples were cut again and polished by hand plane, using watercooled silicon carbide disks (800–1000 grade paper) on both sides in parallel with a thickness range of approximately 150 nm. The enamel surface outside a 1.5 × 2.5 mm area was covered with nail polish to create the treatment window. Before the demineralization of the bovine enamel block sample, three observation points were randomly made in the above-mentioned area using a microhardness tester (Vickers indenter, 50 gf, 10 s) [30].

The blocks were randomly divided into three groups and disinfected by ethylene oxide before biofilm formation. After 72 h of biofilm incubation, the enamel blocks were collected for transverse microradiography (TMR) imaging.

The tooth slices were put into the exposure box and placed in the TMR cabinet 30 cm directly below the Cu-Kα radiation. The sample collection current was 20 mA, the voltage was 20 kV, and the collection time was 30 min [31]. Then, the image acquisition software TMR 2012 (Amsterdam, The Netherlands) was used to acquire the image of the sample [32]. The images were analyzed by using image analysis software (ImageJ; version 1.42q, Wayne Rasband, NIH, USA) [33] and customized image processing software to calculate the lesion depth (LD, μm) and the integrated mineral loss (vol%·μm, ΔZ). The lesion depth was calculated using a threshold at 90% of the mineral content of sound enamel. The integrated mineral loss was calculated by integrating the difference between the mineral content (Vol.%) in sound and demineralized enamel over the depth of the lesion [34]. Three specimens were tested for each group.
