Dental erosion and caries are conditions characterized by the destruction of tooth structure due to acidic exposure. Dental erosion is the demineralization of the outermost surface of hard dental tissues caused by direct contact with acidic substances or environments with a pH lower than 4.5. In contrast, dental caries results from bacterial activity, where acids with a pH between 4.5 and 5.5 are produced, leading to demineralization of the tooth just beneath the surface due to an imbalance between demineralization and remineralization processes [1].

Acid exposure impacts tooth solubility. The critical pH for enamel dissolution is around 5.5, while the risk of dental erosion increases with exposure to acidic foods and drinks with a pH below 4 [2]. Furthermore, for every 1.0 decrease in oral pH from the normal value of 6.5, the solubility of tooth enamel increases by 7 to 8 times [3]. This relationship between tooth solubility and acid exposure has been the focus of extensive research.

Enamel is composed of 96% inorganic material, 1% organic material, and 3% water, making it a highly mineralized tissue. The inorganic component is primarily composed of calcium and phosphate in the form of hydroxyapatite (HA), tricalcium phosphate (TCP), octacalcium phosphate (OCP), and dicalcium phosphate dihydrate (DCPD), among which hydroxyapatite is the most stable and acid-resistant form [4-6]. Given its biocompatibility and inert nature, hydroxyapatite has been widely studied and used in medical applicationssuch as implants, drug delivery systems, and bone substitutes [7-9]. The high crystalline structure of hydroxyapatite, which composes over 90% of enamel, makes it a potential substitute for enamel in erosion studies [10,11].

However, despite the chemical similarities between enamel and synthetic hydroxyapatite, their microstructures are quite different [12]. Enamel has a highly organized, patterned microstructure, consisting of bundles of tightly packed crystallites, whereas synthetic hydroxyapatite has a more compact and uniform structure. Previous studies have also shown that enamel, due to its high mineral content and minimal organic matter, is more prone to fracture than hydroxyapatite discs [13]. Moreover, the dissolution rate of hydroxyapatite was found to be significantly slower than that of enamel across all pH levels [14]. Therefore, it was anticipated that under identical conditions, the dissolution rate of synthetic hydroxyapatite would differ from that of enamel.

This study aims to determine the required exposure time for synthetic hydroxyapatite to experience a similar degree of hardness reduction as enamel when subjected to an artificial caries-inducing solution. Both enamel and hydroxyapatite blocks were exposed to an artificial caries-inducing solution at pH 4.4, and surface microhardness was measured at various time intervals to compare their rates of hardness reduction.

1. Materials

1) Hydroxyapatite

The experimental hydroxyapatite blocks used in this study were sterile hydroxyapatite discs with a diameter of 9.5 mm and a thickness of 1.6 mm (HA Disc, 3D Biotek LLC, HA48-3, USA).

2) Teeth samples

Bovine teeth, which have similar composition to human teeth, were used as test specimens in this study.

(1) Selection criteria

The following criteria were used to select the bovine teeth specimens:

Maxillary incisors from cattle.

Intact enamel with no damage or wear on the surface.

Based on these criteria, bovine maxillary incisor teeth with a minimum of 2 mm of intact enamel on the labial surface were selected for this experiment.

(2) Sample preparation

The preparation process for the tooth samples was as follows:

Bovine teeth were stored at −20°C until use.

After thawing at room temperature, the teeth were cleaned with distilled water, and the crowns were sectioned into six pieces using a low-speed bur.

Tooth blocks were prepared by embedding the specimens in acrylic resin molds with an innerdiameter of 8 mm, an outer diameter of 12 mm, and a height of 5 mm.

The enamel surfaces were polished using 600 to 1,500 grit silicon carbide paper.

3) Artificial caries-inducing solution

An artificial caries-inducing solution with a pH of 4.4 (Artificial caries inducing solution, Biosesang Co., Seoul, Korea) was used to artificially induce demineralization. The composition of the solution is shown in Table 1.

Table 1 . Composition of tooth artificial caries inducing solution

Ingredient	Content
Calcium chloride	2.2 mM
Potassium phosphate, monobasic	2.2 mM
Acetic acid	50 mM
pH	4.4

2. Methods

1) Baseline hardness measurement and group assignment

(1) Baseline hardness measurement

The microhardness of the enamel and hydroxyapatite blocks was measured using a Microness Vickers Hardness Tester (HM series, Mitutoyo, Tokyo, Japan).

For each sample, 10 measurements were taken under a load of 300 gf for 10 seconds. The average of the 10 readings was used as the representative hardness value for each sample.

The Vickers hardness number (VHN) of the enamel and hydroxyapatite blocks was measured before and after immersion in sterilized distilled water for 24 hours.

(2) Group assignment

Bovine enamel samples with VHN values between 310 and 400 were selected.

A total of 20 enamel specimens and 15 hydroxyapatite blocks were used for the experiment.

2) Application of artificial caries-inducing solution

The bovine enamel and hydroxyapatite blocks were immersed in the artificial caries-inducing solution (pH 4.4). The enamel specimens were exposed to the solution for 1 hour, repeated four times, for a total of 4 hours. The hydroxyapatite blocks were exposed for 1 hour, repeated four times, followed by four additional exposures of 4 hours each, for a total of 20 hours.

3) Post-treatment hardness measurement

After each exposure, the microhardness of the enamel and hydroxyapatite blocks was measured using the Microness Vickers Hardness Tester. For each sample, 10 measurements were taken under a load of 300 gf for 10 seconds, and the average value was used as the representative hardness. For the enamel specimens, VHN was measured after each 1-hour exposure. For the hydroxyapatite blocks, VHN was measured after each 4-hour exposure (at 8, 12, 16, and 20 hours).

1. Microhardness changes in hydroxyapatite blocks over time

The average microhardness values of the hydroxyapatite blocks over time are shown in Table 2. Before storage in distilled water, the average hardness was 412.85, which decreased to 398.52 after 24 hours in distilled water. After exposure to the caries-inducing solution, the average hardness values were 397.39 after 1 hour, 370.14 after 2 hours, 368.84 after 3 hours, 348.52 after 4 hours, 350.76 after 8 hours, 313.63 after 12 hours, 299.79 after 16 hours, and 254.07 after 20 hours. The percentage decrease in hardness compared to the initial value was 96.3% after 1 hour, 89.7% after 2 hours, 89.3% after 3 hours, 84.4% after 4 hours, 85.0% after 8 hours, 76.0% after 12 hours, 72.6% after 16 hours, and 61.5% after 20 hours (Figure 1, 2).

Figure 1. Average microhardness of hydroxyapatite blocks according to caries-inducing solution treatment time. The average microhardnessof the hydroxyapatite blocks treated with the caries-inducing solution for 16 hours shows a decrease of approximately one-third, reaching 299.79.

Figure 2. The decrease rate of average microhardness value of hydroxyapatite blocks according to the application time of a caries-inducing solution. The decrease rate of the average microhardness value of hydroxyapatite blocks, according to the application time of a caries-inducing solution for 16 hours, shows a reduction of approximately 72.6%, indicating a decrease of about one-third.

Table 2 . Mean microhardness measurements of hydroxyapatite blocks as a function of application time (unit: VHN)

Sample	Dry	Wet	1 hour	2 hours	3 hours	4 hours	8 hours	12 hours	16 hours	20 hours
HA_01	423.04	428.74	434.31	399.01	376.53	386.95	355.09	321.92	237.93	240.13
HA_02	337.35	333.27	335.32	309.26	287.10	307.54	309.55	239.68	199.00	190.01
HA_03	396.85	372.14	369.83	362.05	352.19	316.98	381.34	294.99	308.18	240.13
HA_04	443.16	452.93	418.52	410.85	410.46	395.20	452.90	394.39	398.23	369.89
HA_05	426.69	456.00	420.05	420.23	400.27	349.58	404.88	339.21	277.22	221.80
HA_06	398.13	376.50	383.46	350.58	355.70	325.94	335.42	315.08	270.42	236.46
HA_07	438.39	437.00	397.69	387.59	387.73	388.23	379.57	383.01	360.89	263.24
HA_08	374.03	373.52	394.64	372.67	353.19	316.98	320.61	260.40	217.87	-
HA_09	462.07	416.54	455.19	400.87	412.42	397.92	370.62	410.42	348.78	288.83
HA_10	366.00	385.93	360.48	372.40	381.07	340.16	367.56	324.13	329.32	263.10
HA_11	405.19	357.22	347.93	273.67	307.66	307.97	291.98	234.15	461.55	239.03
HA_12	420.60	429.89	427.31	355.43	382.41	351.14	372.65	319.59	319.26	276.89
HA_13	428.06	394.29	408.51	377.20	353.03	363.72	303.22	270.39	278.30	200.93
HA_14	404.63	367.07	372.62	378.97	328.39	334.78	310.81	307.46	261.39	264.53
HA_15	468.54	396.75	434.93	381.25	444.42	344.64	305.22	289.63	228.47	261.95
mean	412.85	398.52	397.39	370.14	368.84	348.52	350.76	313.63	299.79	254.07
%	100.0	96.5	96.3	89.7	89.3	84.4	85.0	76.0	72.6	61.5

2. Microhardness changes in bovine enamel over time

The average microhardness values of the bovine enamel specimens are shown in Table 3. Before storage in distilled water, the average hardness was 362.91, which decreased to 353.31 after 24 hours in distilled water. After exposure to the caries-inducing solution, the average hardness values were 246.70 after 1 hour, 182.36 after 2 hours, 126.65 after 3 hours, and 100.29 after 4 hours. The percentage decrease in hardness compared to the initial value was 68.0% after 1 hour, 50.2% after 2 hours, 34.9% after 3 hours, and 27.6% after 4 hours (Figure 3, 4).

Figure 3. The average microhardness of tooth enamel according to the treatment time with a caries-inducing solution. The average microhardness of the tooth enamel treated with the caries-inducing solution for 1 hour shows a decrease of approximately one-third, reaching 246.70.

Figure 4. The decrease rate of average microhardness value of tooth enamel according to the application time of a caries-inducing solution. The decrease rate of the average microhardness value of tooth enamel, according to the application time of a caries-inducing solution for 1 hour, shows a reduction of approximately 68.0%, indicating a decrease of about one-third.

Table 3 . Mean microhardness measurements of tooth enamel as a function of application time (unit: VHN)

Sample	Dry	Wet	1 hour	2 hours	3 hours	4 hours
CT-01	384.00	378.63	264.15	178.68	130.13	111.71
CT-02	372.10	356.70	238.83	186.49	99.24	98.39
CT-03	367.32	341.00	212.75	154.69	101.68	95.17
CT-04	386.50	371.87	223.23	183.13	139.40	104.12
CT-05	342.62	347.96	237.07	154.31	104.42	93.87
CT-06	387.50	373.14	294.20	231.33	155.94	115.62
CT-07	392.46	380.01	255.28	189.98	166.50	106.04
CT-08	338.72	342.27	231.61	193.07	117.18	99.65
CT-09	368.22	369.30	256.95	176.63	134.35	118.00
CT-10	375.46	368.29	274.46	186.85	119.85	115.76
CT-11	395.24	363.52	304.09	223.93	161.88	124.66
CT-12	358.42	322.27	222.82	139.70	116.84	99.80
CT-13	348.48	355.50	234.21	190.24	142.88	71.57
CT-14	345.58	334.34	246.06	222.21	143.11	99.94
CT-15	379.86	346.61	243.50	132.33	128.14	97.61
CT-16	334.78	342.38	252.78	181.49	116.10	86.98
CT-17	380.90	374.56	275.66	222.57	144.73	114.92
CT-18	318.66	321.80	221.78	170.84	92.46	76.97
CT-19	323.24	350.01	232.62	207.98	127.83	91.65
CT-20	358.16	325.94	211.85	120.73	90.39	83.33
mean	362.91	353.31	246.70	182.36	126.65	100.29
%	100.0	97.4	68.0	50.2	34.9	27.6

Dental caries involve the dissolution of minerals such as calcium and phosphate within the tooth structure due to acids produced by bacterial biofilms [15]. The demineralization process disrupts the integrity of the hydroxyapatite crystals in the enamel, leading to structural changes. As a result, the spaces between the crystals increase, allowing ion exchange and further demineralization [16]. In this study, we hypothesized that dental caries would result in a reduction in enamel microhardness, and we compared this with hydroxyapatite blocks as a substitute for enamel.

Although bovine teeth and human enamel have similar hardness in their healthy states, previous studies have shown that bovine teeth are more susceptible to artificial demineralization and acid erosion than human teeth [17,18]. To replicate the acid-erosion conditions found in the oral cavity, we polished the enamel surface, which may have exaggerated the response to acid exposure compared to unpolished enamel [19].

Hydroxyapatite discs offer several advantages as substitutes for enamel. First, pure hydroxyapatite crystals of 99% purity are readily available for experimental use. Second, these discs can be produced with consistent quality and standardized properties, which allows for more reproducible experiments [20]. Third, the use of hydroxyapatite discs reduces the need for animal testing, addressing ethical concerns. However, previous studies have shown that the dissolution rate of hydroxyapatite is significantly slower than that of enamel and dentin [14]. This was confirmed in our study, where the hydroxyapatite blocks exhibited a much slower rate of dissolution than enamel, with a 15-hour delay in comparable hardness reduction.

Both bovine teeth and hydroxyapatite blocks were stored in distilled water for 24 hours, as teeth are generally recommended to be stored in fluid to prevent dehydration and maintain their properties [21]. However, hydroxyapatite can undergo some demineralization in solution, which may have affected the hardness values measured in this study [22,23].

This study aimed to compare the microhardness reduction of bovine enamel and hydroxyapatite blocks when exposed to an artificial caries-inducing solution. Bovine enamel was exposed for a total of 4 hours, and hydroxyapatite blocks were exposed for up to 20 hours. The results demonstrated that hydroxyapatite exhibited a hardness reduction of approximately one-third after 16 hours, while enamel exhibited a similar reduction after just 1 hour. Based on these findings, we recommend using hydroxyapatite for approximately 16-20 hours when simulating acid erosion in future studies.

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