Dental caries, one of the two major oral diseases, is a condition where the inorganic components of the tooth are demineralized, and the organic components are destroyed, leading to tooth tissue loss. Caries is a chronic disease caused by biological interactions, with mineral loss being the primary characteristic in the early stages [1].

Dental caries affects a large global population and is associated with risk factors such as the interaction between teeth and microorganisms, dietary habits, and structural characteristics of teeth. Preventing and treating dental caries is significant from both socioeconomic and public health perspectives [2]. It can be prevented through regular dental checkups, balanced dietary habits, and proper oral hygiene practices [3]. However, early-stage caries can be reversed through remineralization of the tooth tissue, and the most effective method is fluoride application [4].

Fluoride can be applied via varnishes, toothpaste, topical fluoride, and water fluoridation [5]. Among these, fluoride varnish is the most effective, but the most accessible method is regular brushing with fluoride-containing toothpaste at home. Fluoride toothpaste has significantly contributed to reducing dental caries worldwide [6]. Fluoride toothpaste was first developed in 1950 and was officially recognized by the American Dental Association (ADA) in 1964 as a therapeutic toothpaste [7]. Fluoride toothpaste is reported to reduce caries incidence by about 24% and caries experience by 15–30% [8]. The World Dental Federation (FDI) has also stated that appropriate fluoride use is safe, effective, and helps prevent caries [9].

ten Cate [10] demonstrated the use of surface microhardness as an objective indicator to measure the remineralization effect of fluoride. As dental caries progress, the mineral structure of the tooth surface weakens, resulting in reduced microhardness. Therefore, comparing surface microhardness can objectively assess demineralization and remineralization [11].

Clinical trials in the United States and Europe that compared caries prevention effects of fluoride concentrations showed that toothpaste containing 1,500 ppm fluoride was more effective in preventing caries than toothpaste with 1,000 ppm [12-15]. Reflecting these findings, the South Korean Food and Drug Administration raised the fluoride concentration limit in toothpaste from 1,000 ppm to 1,500 ppm in 2014 [16].

While many studies have investigated the remineralization effects of high-concentration fluoride on early caries, there is limited information on the surface microhardness improvement in healthy enamel using high-fluoride toothpaste. Therefore, this study aims to assess the preventive effects of fluoride on sound enamel by comparing surface microhardness changes in bovine tooth samples treated with toothpaste containing varying fluoride concentrations.

1.Study subjects

Bovine teeth, which are structurally similar to human enamel and have smooth surfaces, were used as the study samples [17].

1) Selection criteria

①Upper incisors of bovine teeth

②Intact enamel surface without wear or damage

③Bovine incisors with healthy enamel on the labial surface were selected as final samples.

2) Sample preparation

①Bovine teeth were frozen at -20°C until preparation.

②After washing the frozen teeth with distilled water (DW), the crowns were cut into 2 × 2 mm sections using a low-speed handpiece and diamond disk (Southbay Technology, Inc., USA).

③The enamel surface was embedded in acrylic resin to create tooth blocks, leaving the enamel exposed.

④The enamel surfaces were polished with 600, 800, and 1000 grit silicon carbide paper.

2. Study methods

1) Microhardness testing

①The enamel surfaces of the samples were polished and measured using the Microness Vickers Hardness Tester (Mitutoyo, Tokyo, Japan). Five locations (top, bottom, center, left, and right) were measured on each sample, and the average was calculated as the sample’s hardness value.

②Out of 103 prepared samples, 40 were selected with Vickers hardness values between 290 and 400, ensuring no significant differences between groups.

③Fluoride toothpaste solutions were prepared by mixing toothpaste and distilled water in a 1:3 ratio. Brushing was simulated for 3 minutes per session, 3 times daily, and each session consisted of 250 strokes. The samples were tested after 4 weeks (21,000 strokes) and after 8 weeks (42,000 strokes).

2) Statistical analysis

IBM SPSS Statistics 26.0 (IBM Inc., Armonk, New York, USA) was used for statistical analysis. Comparisons between groups were analyzed using the Kruskal-Wallis test, and within-group comparisons were analyzed using Wilcoxon’s signed rank test. Post-hoc analysis was performed using the Duncan test. A significance level of 0.05 was set for statistical evaluation.

The increase in microhardness after treatment was calculated using the following formula:

Hardness Increase Rate (%) = (Post-treatment Hardness – Pre-treatment Hardness) / Pre-treatment Hardness × 100(%)

1. Changes in surface microhardness by group

1) Control group (0 ppm)

As shown in Table 2, the surface microhardness of the control group increased slightly from the baseline, with an average value of 339.03 after 4 weeks and 339.03 after 8 weeks. There were no significant changes after 4 or 8 weeks.

Table 1 . Fluoride concentration of studied toothpastes by group

Group	Fluoride concentration
1	0 ppmF
2	500 ppmF
3	1,000 ppmF
4	1,450 ppmF

Table 2 . Variation of microhardness with time of toothpaste appli-cation in group (Control Group)

	mean ± SD	SE	Min	Max	p-value
Base	338.24 ± 10.85	3.43	323.44	357.84
After 4 weeks	339.03 ± 14.89	4.71	315.22	361.68	0.7989
After 8 weeks	342.50 ± 15.48	4.89	321.20	364.82	0.3329

p-value by Wilcoxon test.

2) Experimental group 1 (500 ppm)

Table 3 shows that the surface microhardness of Experimental Group 1 increased to an average of 360.67 after 4 weeks and 364.03 after 8 weeks, with significant changes compared to baseline (p<0.05).

Table 3 . Variation of microhardness with time of toothpaste appli-cation in group (Experiment 1)

	mean ± SD	SE	Min	Max	p-value
Base	338.59 ± 14.60	4.62	311.74	355.06
After 4 weeks	360.67 ± 21.04	6.65	330.80	392.88	0.0218*
After 8 weeks	364.03 ± 18.27	5.78	338.38	396.58	0.0284*

p-value by Wilcoxon test.

*p<0.05 by between base and after.

3) Experimental group 2 (1,000 ppm)

Table 4 shows that the surface microhardness of Experimental Group 2 increased to an average of 334.51 after 4 weeks and 380.24 after 8 weeks, with significant increases from baseline (p<0.05). A more significant increase was observed after 8 weeks than after 4 weeks (p<0.05).

Table 4 . Variation of microhardness with time of toothpaste appli-cation in group (Experiment 2)

	mean ± SD	SE	Min	Max	p-value
Base	334.51 ± 14.25	4.51	310.32	357.12
After 4 weeks	380.24 ± 28.73	9.09	337.34	412.46	0.0093*
After 8 weeks	382.14 ± 29.40	9.30	351.20	436.62	0.0051*

p-value by Wilcoxon test.

*p<0.05 by between base and after.

4) Experimental group 3 (1,450 ppm)

As shown in Table 5, the surface microhardness of Experimental Group 3 increased to an average of 334.94 after 4 weeks and 383.57 after 8 weeks, with significant increases compared to baseline (p<0.05).

Table 5 . Variation of microhardness with time of toothpaste appli-cation in group (Experiment 3)

	mean ± SD	SE	Min	Max	p-value
Base	334.94 ± 17.05	5.39	304.78	362.54
After 4 weeks	383.57 ± 36.69	11.60	347.22	439.86	0.0051*
After 8 weeks	401.14 ± 45.78	14.48	330.96	478.52	0.0051*

p-value by Wilcoxon test.

*p<0.05 by between base and after.

2.Inter-group comparisons of vickers hardness numbers

As shown in Table 6, there were no significant differences in surface microhardness between groups before toothpaste application. However, after 4 and 8 weeks, significant differences were observed between groups (p<0.05), with more notable differences after 8 weeks.

Table 6 . Variation in microhardness with toothpaste application time by group

Group	ppmF	N	Base		After 4 weeks		After 8 weeks
			Mean ± SD		Mean ± SD		Mean ± SD
1	0	10	338.24 ± 10.85		339.03 ± 14.89a		342.50 ± 15.48a
2	500	10	338.59 ± 14.60		360.67 ± 21.04ab		364.03 ± 18.27ab
3	1,000	10	334.51 ± 14.25		380.24 ± 28.73b		382.14 ± 29.40bc
4	1,450	10	334.94 ± 17.05		383.57 ± 36.69b		401.14 ± 45.78c
p-value			0.941		0.002*		0.001*

p-value by Kruskal Wallis test.

^a,b,cSame letter means no statistical difference.

*p<0.05 by between base and after.

As shown in Table 7, the hardness increase rate after 4 weeks was 0.23% in the control group, 6.52% in Experimental Group 1, 13.67% in Experimental Group 2, and 14.51% in Experimental Group 3. After 8 weeks, the hardness increase rate was 1.25% in the control group, 7.51% in Experimental Group 1, 14.23% in Experimental Group 2, and 19.76% in Experimental Group 3.

Table 7 . Effectiveness rate of microhardness increase as a function of toothpaste application time by group

Effectiveness rate of microhardness increase compared to the base (%)
Group	After 4 weeks	After 8 weeks
1	0.23	1.25
2	6.52	7.51
3	13.67	14.23
4	14.51	19.76

Preventing dental caries by detecting early demineralization and halting progression is a key strategy [18,19]. Strengthening enamel and increasing its resistance to acids before the onset of caries is an effective preventive measure.

Fluoride application prevents demineralization and promotes remineralization, forming fluorapatite, which strengthens the tooth structure [20]. In the United States, fluoride toothpaste has been used since 1964 to prevent caries and treat early caries lesions.

In Korea, fluoride has been widely used in toothpaste since the 1990s. Fluoride has been recognized as an effective ingredient for preventing caries, with pharmacological effects due to fluoride compounds in toothpaste. According to the 2016 report by the Korea Health Promotion Institute [21], fluoride toothpaste was recommended due to the high caries prevalence (over 50%) among 12-year-old children. The fluoride concentration limit for toothpaste was raised to 1,500 ppm in 2014, increasing interest in the effects of high-fluoride toothpaste. This study aimed to evaluate the effects of high-fluoride toothpaste on sound enamel.

A study of 7,422 children aged 5-6 years compared caries prevention between 440 ppm and 1,450 ppm fluoride toothpastes. The study showed that the 1,450 ppm fluoride toothpaste was more effective in preventing caries than the 440 ppm toothpaste [22]. This is consistent with our findings that Experimental Group 3 (1,450 ppm fluoride) showed the greatest increase in surface microhardness, suggesting that higher fluoride concentrations provide better caries prevention.

Previous studies on fluoride toothpaste have focused on remineralization after demineralization. However, this study examined whether high-fluoride toothpaste could increase surface microhardness in sound enamel without prior demineralization.

This study evaluated the caries prevention effect of fluoride concentrations in toothpaste by measuring surface microhardness in sound bovine enamel blocks treated with toothpastes containing 0 ppm, 500 ppm, 1,000 ppm, and 1,450 ppm fluoride over an 8-week period. Microhardness was measured before treatment, after 4 weeks, and after 8 weeks, and the following conclusions were drawn:

Surface microhardness significantly increased after 4 and 8 weeks in Experimental Groups 1, 2, and 3, with greater increases observed after 8 weeks (p<0.05).

The hardness increase rates were 0.23% in the control group, 6.52% in Experimental Group 1, 13.67% in Experimental Group 2, and 14.51% in Experimental Group 3 after 4 weeks. After 8 weeks, the rates were 1.25% in the control group, 7.51% in Experimental Group 1, 14.23% in Experimental Group 2, and 19.76% in Experimental Group 3. The highest hardness increase was observed in Experimental Group 3 (1,450 ppm fluoride).

These results suggest that fluoride toothpaste can increase the surface microhardness of sound enamel and help prevent caries. The higher the fluoride concentration, the greater the effect on increasing enamel hardness and caries prevention.

No potential conflict of interest relevant to this article was reported.