Study Design

Enamel demineralization of the control and experimental group was monitored over 12 weeks in vitro (Figure 1). Oral conditions were simulated by thermocycling and tooth brushing. After 2, 4, 8, and 12 weeks, pH cycling was conducted, and enamel demineralization was evaluated using the FluoreCam system. Representative samples were examined at the end of the study using a polarized light microscope (PLM).

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Sample Size and Power Analysis

Assuming an effect size of 0.72, which was based on published estimates,²³ a sample size of n = 40 per group was necessary to achieve a type I error rate of 5% and a power of 99%.²⁴ Forty sound human permanent molars and premolars were cut into halves so that both buccal and lingual surfaces could be evaluated.

FluoreCam Imaging

The specimens were randomly assigned to either a control or experimental group. Pretreatment images of the enamel surfaces were recorded under standardized conditions using the FluoreCam.²³ The FluoreCam device was positioned a fixed distance from a mounting table. A mold was prepared for each specimen using a vinyl polysiloxane impression material (Exaflex Putty, GC America, Inc, Alsip, Ill), into which the root of each specimen was inserted horizontally. The FluoreCam was placed touching the enamel, and the impression material was molded around the tip, producing an indentation. The indentation served to ensure the same FluoreCam position and orientation for subsequent imaging. An image of each specimen was captured, and the baseline areas (mm²), light intensities (pixels), and impacts of demineralization (pixel.mm²) were recorded. FluoreCam imaging was repeated for each specimen after 2, 4, 8, and 12 weeks.

Treatment Application

The specimens were covered with an acid-resistant nail polish (Revlon, New York, NY), except for a 2- × 4-mm window of exposed enamel. Each specimen was photographed to record the position of the enamel window for later use. MI Varnish (GC America Inc) was applied to the enamel windows of the experimental group. It was scraped from the enamel windows on the following day because, clinically, varnish usually starts to abrade off of smooth enamel surfaces during the first 24 hours. The control group received no treatment. Both groups were kept in 10 mL of artificial saliva (1.5 mmol/L Ca, 0.9 mmol/L P, 150 mmol/L KCl, 0.05 μg F/mL in 0.1 mol/L Tris buffer [pH 7.0]) for 24 hours.

Thermocycling and Toothbrushing

To simulate the thermal conditions of the oral environment, both groups were thermocycled.²⁵ The specimens were immersed alternately into two baths of distilled water (5°C and 55°C) for 15 seconds (LAUDA-Brinkmann LP, Delran, NJ),²⁵ with a 5-second transfer time at 23°C. A total of 150, 300, 600, and 1200 cycles were performed, representing 2, 4, 8, and 12 weeks, respectively (10 cycles/d).²⁵ To simulate the mechanical effect of toothbrushing, the groups were placed in a toothbrushing simulator (Proto-Tech Oral Wear products, Portland, Ore).²⁶ The specimens were individually placed in customized rubber molds (Exaflex Putty, GC America, Inc) mounted in the brushing simulator. Medium-bristled toothbrushes (Deluxe Denta-Brite, Eagle, NY) were centered over the exposed enamel windows and oriented to brush in a mesiodistal direction.²⁶ To simulate a normal manual brushing force, a constant force of 280 g and 20 strokes/d was applied. A total of 300, 600, 1200, and 1800 strokes were applied for simulating 2, 4, 8, and 12 weeks, respectively.²⁶ A slurry of fluoridated toothpaste (Crest, Procter and Gamble, Cincinnati, Ohio), with a 1:3 paste to water ratio, was constantly circulated during the brushing procedure.²⁶ New brushes and fresh slurry solution were used for each group.

pH Cycling

The two groups were subjected to 9 days of pH cycling (8-day de/remineralization +1-day remineralization).²⁷ Falcon tubes (VWR, Radnor, Pa) were prepared filled with demineralizing solution (50 mL) and remineralizing solution (25 mL). The specimens were immersed alternately in demineralizing solution for 4 hours and remineralizing solution for 20 hours over an 8-day period.²⁷ The tubes were kept in an incubator at 37°C and under constant agitation (Excella E24 Incubator Shaker Series, New Brunswick Scientific Co, Inc, Enfield, Conn). The solutions were replaced every 4 days. On day 9, the specimens were kept in the remineralizing solution for 24 hours. The demineralizing solution consisted of 0.05 M acetate buffer containing 1.28 mmol/L Ca, 0.74 mmol/L P, and 0.03 μg F/mL (pH 5), prepared from Ca (NO₃)₂.4 H₂O, KH₂PO₄, and NaF, respectively. The proportion of demineralizing solution per area of exposed enamel was 6.25 mL/mm². The remineralizing solution consisted of 1.5 mmol/L Ca, 0.9 mmol/L P, 150 mmol/L KCl, 0.05 μg F/mL in 0.1 mol/L Tris buffer (pH 7.0). The proportion of remineralizing solution per area of exposed enamel was 12 mL/mm². Following pH cycling, the nail polish was removed because it could affect the FluoreCam readings. The nail polish was reapplied, leaving the same enamel window exposed, and the subsequent thermocycling and toothbrushing were conducted.

PLM Sample Preparation

To ensure consistency of pattern, five specimens from each group were randomly selected and prepared for PLM assessments. The roots were removed, and the crowns were serially sectioned perpendicular to the 2- × 4-mm enamel windows. The slices were ground and polished to a thickness of approximately 100 μm and soaked in distilled water overnight. The demineralized enamel lesions were examined under the PLM attached to a camera (Olympus-BX51, Olympus Corp, Center Valley, Pa). The mean lesion depths of the demineralized enamel were measured in microns using CellSens standard software (Olympus Corp). Three lines were drawn, one at the center and one on each side of the lesion (ie, enamel window). Each line was perpendicular to the outer enamel surface and extended to the depth of the lesion.

Statistical Analysis

The data were normally distributed. Independent t tests were used to evaluate pretreatment differences. Repeated-measure analysis of variance, with Greenhouse-Geisser Correction, was used to determine the effects of time and varnish. The effects of time within each group were then tested using paired t tests with Bonferroni corrections. Between-group differences at 2, 4, 8, and 12 weeks were conducted using independent t tests with Bonferroni corrections.

FluoreCam

At pretreatment, there were no statistically significant between-group differences for area, intensity, or impact. Posttreatment, there were statistically significant (P < .001) time and varnish effects for area, intensity, and impact. In the control group, area, intensity, and impact of demineralization showed statistically significant increases at every time point (Table 1; Figures 2–4).

Table 1 Mean Changes in Area, Intensity, and Impact of Enamel Demineralization Over Time for the Control and Varnish Group, Along With Probabilities From Paired t Test

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In the experimental group, the area of demineralized enamel decreased significantly after 2 weeks. After 4 weeks, there was no significant difference in area compared with pretreatment. There was an increase in area (P = 0.001) after 8 weeks, which continued until 12 weeks (P < .001; Figure 2).

The intensity of demineralization in the experimental group showed no statistically significant changes after 2 weeks or 4 weeks. There was a statistically significant increase in the intensity of demineralization after 8 and 12 weeks (Figure 3).

The impact of demineralization in the experimental group followed the same pattern as the area of demineralization. The impact decreased significantly after 2 weeks. After 4 weeks, there was no significant difference compared with pretreatment, followed by significant increases at 8 and 12 weeks (Figure 4).

There were significant between-group differences in area, intensity, and impact at each of the time periods (Figures 2, 3, and 4).

Polarized Light Microscopy

The PLM images revealed typical WSLs in the untreated control representative samples. The mean lesion depth was 190 ± 34 μm. The experimental group showed more limited areas of enamel demineralization, with a mean lesion depth of 37 ± 9 μm.