Etching treatment of enamel. To simulate early caries lesions, tooth enamel windows were etched with H3PO4 (37 wt %) for 30 s. To both simulate early caries lesions and remove the prism-less enamel of the outermost tooth surface (49), the whole tooth samples were etched with H3PO4 (37 wt %) for 10 min. All of the samples were ultrasonicated in deionized water for 20 min to ensure the removal of any residual contaminants and air-dried.

Enamel repair using CPICs. For comparison, half of the enamel surface was covered with acid-resistant nail varnish, and the remaining area was used for repair by using CPICs. The CPIC ethanol solution (100 μl; 2 mg/ml) was dropped onto the enamel surface. Subsequently, the samples were air-dried at 25°C for 5 min, resulting in the formation of a gel-like CPIC-coated layer on the enamel; at 15 min, the CPIC layer spontaneously turned into an ACP layer. Then, the enamel windows were immersed into modified simulated oral fluid (m-SOF; 10 ml per sample; containing 1.5 mM CaCl2, 0.9 mM K2HPO4, 15 ppm F−1, 130 mM KCl, 1 mM NaN3, and 20 mM Hepes buffer) at pH 7.00 ± 0.03 at 37°C for the designated time. The total duration of the incubation process was 48 hours, which was a sufficient time period that allowed full crystallization of the amorphous phase. To increase the thickness of the repaired layer, the above-mentioned CPIC treatment on enamel was applied repeatedly to establish the layer-by-layer repair. After incubation, the repaired enamel windows were sonicated in water for 20 min, rinsed with water, and air-dried at 25°C before examination.

Enamel repair using ACP. An ACP nanoparticle ethanol solution (100 μl; 2 mg/ml) was dropped onto the acid-etched enamel window, and the sample was air-dried for 15 min at 25°C and a relative humidity of 60%. Then, the windows were immersed in m-SOF (10 ml per sample) at pH 7.00 ± 0.03 at 37°C for 48 hours to ensure maturation. After incubation, the repaired enamel windows were sonicated in water for 20 min, rinsed with water, and air-dried at 25°C before examination.

Repair of whole tooth enamel using CPICs. For the purpose of comparison, half of a tooth surface was covered with acid-resistant nail varnish as the control zone, and the remaining was left as an experimental zone for repair by using CPICs. A CPIC ethanol solution (1 ml; 2 mg/ml) or calcein (1 μM)–containing CPIC ethanol solution (1 ml; 2 mg/ml) was dropped onto the whole tooth enamel surface. Then, all of the samples were immersed in m-SOF (50 ml per sample) at pH 7.00 ± 0.03 at 37°C for 48 hours to ensure maturation. After incubation, the whole teeth were sonicated in deionized water for 20 min, rinsed with water, and air-dried before examination. To obtain a clear fluorescence picture of the whole tooth, we rinsed the varnish using acetone three times and took a photograph through an optical filter under UV light with a light intensity of 1200 mW/cm2 (±10%) and a utilizable wavelength range of 430 to 480 nm.

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