Effect of different remineralization agents on the optical properties of bleached enamel

Abstract

Background

The aim of this study was to evaluate the effect of different remineralization agents applied immediately after bleaching on the colour stability, whiteness index and whiteness index difference of enamel.

Methods

One hundred enamel samples from bovine incisors were divided into two groups for at-home and in-office bleaching. The samples were further divided into five subgroups: (1) Acidulated phosphate fluoride (APF), (2) Casein phosphopeptide amorphous calcium phosphate fluoride (CPP-ACPF), (3) Calcium glycerophosphate, fluoride (CaGP, F), (4) Hydroxyapatite, fluoride (HAp, F), and (5) control. After treatment, all samples were stained with red wine. Color measurements were taken at baseline, after bleaching, after remineralization, and after staining using a dental spectrophotometer. Optical parameters included L*, a*, b*, CIEDE2000 color difference (ΔE₀₀), whiteness index for dentistry (WI_D), and whiteness index difference (ΔWI_D). Statistical analyses were performed with Kruskal Wallis, Friedman, and Dunn tests.

Results

Color and whiteness differences after bleaching were statistically similar between in-office and at-home bleaching (p > 0.05). Remineralization protocols had no significant effect on enamel color or whiteness across all groups (p > 0.05), but significant differences were found between the CPP-ACPF, CaGP, F, HAp, F, and control groups (p < 0.05).

Conclusion

Application of remineralization agents after bleaching was found to be ineffective on color and whiteness change of bleached enamel. Remineralization agents containing HAp, CPP-ACP and CaGP, respectively, were found to be more successful in terms of color stability.

Background

In recent years, as individuals’ expectations regarding their aesthetic appearance have increased there has been an increase in the number of patients applying to the Department of Restorative Dentistry with a request for tooth bleaching [1]. Bleaching is widely performed by clinicians because it is a minimally invasive, low-cost and safe procedure [2]. Bleaching can be performed by the dentist in the clinic or by the patient at home, in accordance with the dentist’s application recommendations.

In office whitening, agents containing approximately 30–40% hydrogen peroxide are applied to the teeth by the dentist for several sessions. Despite low patient compliance and an instantly visible whitening effect, it creates disadvantages such as high office cost, long application time and tooth sensitivity [3].

In at-home bleaching system, whitening gel containing approximately 10–22% carbamide peroxide is applied in a customized plate. It is simple to apply and less costly. Although it is a more reliable method due to the application of gels containing low concentrations of peroxide, it has disadvantages such as requiring patient compliance and taking longer to achieve the desired whitening result [4].

Today, the active ingredient in the gels used in both office and home bleaching systems is hydrogen peroxide [5]. Hydrogen peroxide causes the formation of free reactive oxygen molecules. These free molecules present whitening activity by breaking pigmented molecules into small pieces. In this way, the enamel tissue, whose molecular structure changes, absorbs less light and appears lighter in color [5].

Hydrogen peroxide has been reported to cause a decrease in the mineral content and surface hardness of enamel and an increase in the surface roughness [6]. The decrease in minerals such as calcium, phosphate and fluoride from the enamel surface increases the porosity of the surface, which can facilitate the adhesion of various color pigments to the tooth surface [7]. Therefore, repairing the weakened enamel structure is important in preserving the color obtained as a result of whitening.

To repair bleached enamel, remineralization agents with different contents can be applied [8, 9]. However, there is no clear procedure on this issue. It is also important that these agents do not have a negative effect on the enamel color obtained after whitening. However, there is a lack of scientific studies on the effect of these agents on the optical properties of bleached enamel.

For these reasons, the aim of this study was to evaluate the effect of post-bleaching use of remineralization agents with different components such as fluoride, casein phosphopeptide-amorphous calcium phosphate, calcium glycerophosphate and hydroxyapatite on the optical properties of enamel. The null hypothesis of the study were: (1) Immediate post-bleaching application of remineralization agents would not affect color and whiteness change (2) Immediate post-bleaching application of remineralization agents would not have any effect on color stability after staining.

Methods

Power analysis was performed to determine the number of samples (G* Power 3.1.9.4). Considering that the effect size that could be obtained in the study would be hypothetically strong (f = 0.4), it was calculated that 80% power could be achieved with 95% confidence when at least 100 enamel blocks (at least 10 samples for each group) were included in the study.

Sample preparation

The tooth samples to be used in the study were obtained from 2 to 3 year old bovine sacrificed for consumption. These animals were slaughtered at a slaughterhouse in Turkey, where they were processed for human consumption, and their teeth were collected as by-products. The deposits and discolorations on the surface of the teeth were removed using peridontal scaler, polishing brush and polishing paste. Color measurements of the teeth were made and 100 central incisors with L* values between 85 and 87 were included in the study to ensure standardization.

A hundred enamel blocks in 6 × 6 × 3 mm sections were obtained from the middle third of the crowns. The surfaces of the enamel blocks were examined under a light microscope (Zumax OMS 2360, Zumax Medical Co, Ltd, Jiangsu, China) to ensure that there were no breaks, cracks, rots or any defects on the surfaces. The obtained samples were embedded in autopolymerized transparent acrylic resin blocks (IMICRYL SC, Konya, Turkey) leaving the enamel surface outside. The prepared blocks were randomly distributed into subgroups according to the bleaching and remineralization treatment to be applied.

Application of bleaching treatments

Office bleaching (1) was applied to half of the randomly distributed samples and home bleaching (2) was applied to the other half. Product, manufacturer, content and Lot number information of the applied whitening gels are shown in Table 1.

Table 1.

Manufacturer, content and lot number information of the bleaching agents

Groups	Material	Manufacturer	Content	Lot Number
1	Office Bleaching	Opalescence Boost, Ultradent, South Jordan, UT, USA	40% Hydrogen Peroxide, 3% Potassium Nitrate, 1.1% Sodium Fluoride,	BM147
2	At-home Bleaching	Opalescence PF, Ultradent, South Jordan, UT, USA	16% Carbamide Peroxide, Glycerin, Urea, Xylitol, Carbomer, PEG-6, Sodium Hydroxide, Aroma, EDTA, Potassium Nitrate, Sodium Fluoride	BM54W

For office bleaching, bleaching gel containing 40% hydrogen peroxide (Opalescence Boost, Ultradent Products Inc. South Jordan, UT, USA) was applied to the clean surfaces of the samples in a thick layer. The application was carried out in a total of 3 sessions and 2 applications of 20 min per session on the 1st, 8th and 15th days, in accordance with the manufacturer’s application instructions. At the end of the application period, samples were brushed with a soft toothbrush for 20 s and stored in artificial saliva. Between treatments, samples were kept at 37° in artificial saliva which was changed daily. The artificial saliva solution used was 2.560 mg NaCl, 332.97 mg CaCl2, 250 mg MgCl2(6H2O), 189.48 mg KCl, 3.015 mg CH3COOK, 772 mg K3PO4(3H2O) and 0.1 ml H3PO4 (85%) of the formulation in distilled water [10].

For home whitening, bleaching gel containing 16% carbamide peroxide (Opalescence PF, Ultradent Products Inc. South Jordan, UT, USA) was applied to the enamel surfaces and left on the surface for 6 h according to the time recommended in the manufacturer’s instructions. Home whitening treatment was applied 6 h a day for 14 days. Between treatments, samples were kept at 37° in artificial saliva that was changed daily.

Application of remineralization treatments

The bleached enamel samples were randomly divided into five subgroups according to remineralization treatments described below (n = 10). Product, manufacturer, content and Lot number information of the remineralization agents used in the study are shown in Table 2.

Table 2.

Manufacturer, content and lot number information of the remineralization agents

Groups	Material	Manufacturer	Contents	Lot Number
1B-2B	APF gel	İonite APF Gel, Dharma Research, Miami, USA	2.09% Sodium Fluoride, Citric acid, Hydrofluoric acid, Phosphoric acid, Polysorbate 20, Water, Sodium benzoate, Sodium saccharin, Titanium dioxide, Xylitol, Tocopheryl acetate, Xantham gum, Aroma	D210918C
1–2 C	CPP-ACPF	MI Paste Plus, GC, Europe N.V. Leuven, Belgium	CPP-ACP, Sodium Fluoride (900 ppm Fluoride), Purified water, Glycerol, D-Sorbitol, CMC-Na, Propylene Glycol, Silicon dioxide, Titanium Dioxide, Xylitol, Phosphoric acid, Sweetener, Sodium saccharin, Ethyl p-hydroxybenzoate, Propyl p-hydroxybenzoate, Butyl p-hydroxybenzoate	201214B
1D-2D	CaGP, F	Fluor Protector Gel, İvoclar Vivadent, Schaan, Liechtenstein	Calcium glycerophosphate, Water, Xylitol, Potassium fluoride (1450 ppm Fluoride), Hydroxyethylcellulose, Alcohol, Panthenol, Methyl paraben, Sodium saccharin, Laureth-23, Aroma	Z040W6
1E-2E	HAp, F	Remin Pro, Voco GmbH, Cuxhaven, Germany	Sodium fluoride (1450 ppm fluoride), hydroxyapatite, Xylitol, Aroma	101881

Groups 1 A and 2 A: Control groups were kept in artificial saliva that was changed every day for 14 days.

Groups 1B and 2B: APF gel (Ionite APF Gel, Dharma Research, Miami, USA) was applied to the whitened enamel surface twice in total, on the 1st and 7th days. The gel was applied to the surface with an applicator and left for 4 min. Since the user instructions recommend not eating or drinking anything for 30 min after gel application, the samples were placed in artificial saliva after 4 min without washing. After a 30-minute waiting period, the surface of the removed samples was cleaned by brushing for 20 s with a soft toothbrush and re-placed in renewed artificial saliva. Samples were stored in artificial saliva that was changed daily between treatment sessions.

Groups 1 C and 2 C: Remineralization agent containing CPP-ACPF (MI Paste Plus, GC, Europe N.V. Leuven, Belgium) was applied with an applicator and left on the surface for 3 min. Since it is recommended not to eat or drink anything for 30 min after gel application in accordance with the manufacturer’s instructions, the samples were placed in artificial saliva without washing after 3 min. After a 30-minute waiting period, the surface of the removed samples was cleaned by brushing for 20 s with a soft toothbrush and re-inserted into the renewed artificial saliva. This application was repeated twice a day for 14 days.

Groups 1D and 2D: CaGP, F (Fluor Protector Gel, Ivoclar Vivadent, Schaan, Liechtenstein) was applied using an applicator and left on the surface for 3 min. Since it is recommended not to rinse the gel after application according to the manufacturer’s instructions, the samples were placed in artificial saliva after 3 min without washing in order to ensure standardization with other processes. After a 30-minute waiting period, the surface of the removed samples was cleaned by brushing for 20 s with a soft toothbrush and re-inserted into the renewed artificial saliva. This application was repeated twice a day for 14 days.

Groups 1E and 2E: Remineralization agent containing HAp, F (Remin Pro, Voco GmbH, Cuxhaven, Germany) was applied to the samples with an applicator and left on the surface for 3 min. Since it is recommended not to eat or drink anything for 30 min after gel application according to the manufacturer’s instructions, the samples were placed in artificial saliva without washing after 3 min. After a 30-minute waiting period, the surface of the removed samples was cleaned by brushing for 20 s with a soft toothbrush and re-inserted into the renewed artificial saliva. This application was repeated twice a day for 14 days.

Staining

Remineralized enamel samples were soaked in red wine (Yakut, Kavaklıdere, Ankara, Turkey) for 10 min a day for 9 days. At the end of the immersion period, the removed samples were washed, brushed with a soft brush for 20 s, and placed in artificial saliva that was changed every day.

Color measurements

Color measurements were performed with a dental spectrophotometer (VITA Easyshade V, VITA Zahnfabrik, Bad Säckingen, Germany) in four measurement steps: Immediately after sample preparation (baseline), after bleaching, after remineralization and after staining.

At each measurement step, evaluations were done three times and the average values of these measurements were recorded. Measurements were made at the same hour of the day and on a gray back ground, using a transparent mold prepared in appropriate dimensions with the 6 mm diameter tip of the spectrophotometer in order to standardize the measurement from the same region of each sample. The dental spectrophotometer was calibrated in every 5 measurements.

Measurements were defined with the CIE L*a*b* system. For each sample, the measurement was repeated three times and the average L*, a*, b*, c*, h* values were recorded. The chroma (c) and hue (h) values of the samples were also obtained numerically. CIEDE2000 color difference (∆E₀₀) was calculated using the following formula:

In this study, the detectability and acceptability thresholds for ΔE₀₀ are accepted as 0.8 and 1.8, respectively [11]. Additionally, the degree of whiteness was calculated using the WI_D formula based on the CIE L*a*b* color system as follows: WI_D = 0.55 L* − 2.32a* − 1.100b*.

The change in whiteness index between measurement values at two different times was calculated as ∆WI_D. The detectability and acceptability thresholds for ∆WI_D are accepted as 0.72 and 2.62, respectively [12].

Statistical analyses

Data were analyzed with IBM SPSS V23 program. Compatibility with normal distribution was analyzed by Shapiro-Wilk Test. Kruskal-Wallis test and Dunn’s test were used for multiple comparisons of three or more groups that did not conform to normal distribution. Friedman’s test and Dunn’s test for multiple comparisons were used to compare measurements that did not conform to normal distribution. Significance level was taken as p < 0.05.

Results

Results of L* values

L* values obtained from the groups at different measurement stages are shown in Table 3 (The statistical tables have been placed at the end of the manuscript). L* values were statistically significantly increased after bleaching in all groups (p < 0.001, Friedman Test, Dunn Test, Table 3). The increase in L* values after in office and at home bleaching was statistically similar (p = 0.778, Kruskal Wallis Test, Dunn Test, Table 3). There was no statistically significant change in L* values in all groups after remineralization (Friedman Test, Dunn Test, Table 3).

Table 3.

Intergroup and intragroup comparison of L* values obtained from groups

	L* VALUES
	n/N	Baseline L0	Bleaching L1	Remineralization L2	Staining L3	T-statistic	P**
1 A	10/100	86.6 (85–87)a	92.3 (91–93)b	92.8 (91.6–93.6)b	80 (78–80.3)cB	30.000	< 0.001
1B	10/100	86.5 (85–87)a	92.2 (91.2–93.1)b	92.7 (91.6–93.7)b	80 (78.6–80.1)cB	30.000	< 0.001
1 C	10/100	86.3 (85–86.8)a	92.2 (91.2–92.8)b	93.1 (92–93.8)b	84.8 (83.9–85.2)aA	30.000	< 0.001
1D	10/100	86.3 (85–86.8)a	92.2 (91.2–92.9)b	93 (92.3–93.8)b	83.1 (81.3–84.2)aAB	30.000	< 0.001
1E	10/100	86.2 (85.1–87)a	91.9 (91.2–93.1)b	93 (92.3–93.7)b	84.3 (83.8–86.6)aA	30.000	< 0.001
2 A	10/100	86.7 (85–87)a	92.7 (91–93.1)b	92.9 (91.6–93.7)b	80.1 (78–81.3)cB	30.000	< 0.001
2B	10/100	86.8 (85.3–86.9)a	93 (91.2–93.1)b	93.5 (91.6–93.6)b	79.9 (79.5–81.8)cB	30.000	< 0.001
2 C	10/100	86.4 (86–87)a	92.2 (91.7–93.1)b	93 (92.7–93.7)b	86.1 (84,1–87)aA	28.080	< 0.001
2D	10/100	86.3 (86–86.5)a	92.2 (91.7–92.9)b	93.1 (93–93.5)b	84.2 (82.8–85.2)aA	30.000	< 0.001
2E	10/100	86.3 (85.1–87)a	92.2 (91.2–93.1)b	93 (92.3–93.7)b	86.4 (83.8–87.1)aA	27.000	< 0.001
T-statistic		6.938	5.616	7.081	78.860
P*		0.644	0.778	0.629	< 0.001

*Kruskal Wallis test, Dunn test **Friedman test, Dunn test; data are expressed as median (min-max); a-c: There is no difference between those with the same letter in each group row; A-B: There is no difference between groups having the same letter in each column

*1A: Office-Control, 2A: Home-Control, 1B: Office-APF, 2B: Home-APF, 1C: Office-CPP-ACPF, 2C: Home-CPP-ACPF, 1D: Office-CaGP, F, 2D: Home-CaGP, F, 1E: Office-HAp, F, 2E: Home-HAp, F

After staining, L* values were statistically significantly decreased in all groups (p < 0.001, Friedman Test, Dunn Test, Table 3). While the lowest L* values were observed in the control and APF gel groups in the office and home whitening groups after staining, the highest L* values were obtained in the gel groups containing CPP-ACPF and HAp, F (p < 0.001, Kruskal Wallis Test, Dunn Test, Table 3).

Results of a* values

The a* values obtained from the groups at different measurement stages are shown in Table 4 (The statistical tables have been placed at the end of the manuscript). In all groups, a* values were statistically significantly decreased after bleaching treatments (p < 0.001, Friedman Test, Dunn Test, Table 4). The decrease in a* values after in office and at home bleaching was found statistically similar (p = 0.616, Kruskal Wallis Test, Dunn’s Test, Table 4). There was no statistically significant change in a* values in all groups after remineralization applications (Friedman Test, Dunn’s Test, Table 4). After staining, a* values were significantly increased in all subgroups (p ≤ 0.001, Friedman Test, Dunn Test, Table 4). After staining, the highest a* values were observed in the control and APF gel groups, while the lowest a* values were obtained in the office bleaching, CPP-ACPF and HAp, F containing gel groups, and in the at home bleaching, CPP-ACPF, CaGP, F and HAp, F containing gel groups (p = 0.001, Kruskal Wallis Test, Dunn Test, Table 4).

Table 4.

Intergroup and intragroup comparison of a* values obtained from groups

	a* Values
	n/N	Baseline a0	Bleaching a1	Remineralization a2	Staining a3	T-statistic	P**
1A	10/100	1 (0.3–1.4)a	0.67 (0–1.1)b	0.57 (0.1–1.1)b	0.9 (0.1–1.2)aB	20.091	< 0.001
1B	10/100	0.9 (0.6–1.2)a	0.7 (0.5–1)b	0.63 (0.4–0.7)b	0.93 (0.7–1.2)aB	27.551	< 0.001
1C	10/100	0.83 (0.6–1)c	0.63 (0.5–0.9)ab	0.5 (0.4–0.8)b	0.7 (0.5–0.9)acA	25.347	< 0.001
1D	10/100	0.8 (0.6–0.9)a	0.67 (0.5–0.8)b	0.6 (0.4–0.7)b	0.8 (0.5–0.9)aAB	26.375	< 0.001
1E	10/100	0.8 (0.6–0.9)c	0.53 (0.5–0.8)ab	0.48 (0.4–0.7)b	0.63 (0.4–0.8)acA	19.469	< 0.001
2A	10/100	0.8 (0.6–1.3)a	0.63 (0.5–1.1)b	0.52 (0.4–0.9)b	0.9 (0.7–1.2)aB	27.120	< 0.001
2B	10/100	0.8 (0.6–1.1)a	0.6 (0.5–0.9)b	0.53 (0.4–0.8)b	0.9 (0.7–1)aB	26.082	< 0.001
2C	10/100	0.7 (0.6–0.9)c	0.58 (0.5–0.8)ab	0.47 (0.4–0.7)b	0.63 (0.4–0.8)acA	19.469	< 0.001
2D	10/100	0.7 (0.6–0.9)c	0.53 (0.5–0.8)ab	0.49 (0.4–0.6)b	0.6 (0.5–0.9)acA	25.250	< 0.001
2E	10/100	0.73 (0.6–0.9)c	0.59(0.5–0.8)ab	0.5 (0.4–0.7)b	0.67 (0.4–0.8)acA	15.866	0.001
T-statistic		8.348	7.205	14.202	27.898
P*		0.499	0.616	0.115	0.001

*Kruskal Wallis test, Dunn test; **Friedman test, Dunn test; data are expressed as median (min-max); a-c: There is no difference between those with the same letter in each group row; A-B: There is no difference between groups having the same letter in each column

*1A: Office-Control, 2A: Home-Control, 1B: Office-APF, 2B: Home-APF, 1C: Office-CPP-ACPF, 2C: Home-CPP-ACPF, 1D: Office-CaGP, F, 2D: Home-CaGP, F, 1E: Office-HAp, F, 2E: Home-HAp, F

B*Results of b* values

The b* values obtained from the groups at different measurement stages are shown in Table 5. (The statistical tables have been placed at the end of the manuscript). After whitening applications, b* values were statistically significantly decreased in all groups (p < 0.001, Friedman Test, Dunn Test, Table 5). The decrease in b* values after office and home bleaching was found to be statistically similar (p = 0.139, Kruskal Wallis Test, Dunn Test, Table 5).

Table 5.

Intergroup and intragroup comparison of b* values obtained from groups

	b* Values
	n/N	Baseline b0	Bleaching b1	Remineralization b2	Staining b3	T-statistic	P**
1A	10/100	29.5 (27–31)a	23.8 (22.2–26.1)b	19.85 (17.3–22.2)c	26.2 (23.8–28.3)abB	30.000	< 0.001
1B	10/100	28.6 (27–30.1)a	22.9 (21.2–24.4)b	19 (17–20.1)c	23.2 (22.1–24.2)bA	27.120	< 0.001
1C	10/100	28.6 (28–30)a	23.3 (22.7–24.8)b	19 (18–20)c	23.4 (22.8–25)bA	28.080	< 0.001
1D	10/100	28.6 (28–30)a	23.3 (22.8–24.7)b	19.1 (18.1–20)c	23.3 (22.8–25.3)bA	27.120	< 0.001
1E	10/100	29 (28.5–30)a	23.5 (23.1–24.7)b	19.1 (18.5–20,1)c	23.6 (21.6–24)bA	28.041	< 0.001
2A	10/100	29.4 (27–31)a	24.05 (22.2–25.8)b	19.6 (17.3–21.6)c	25.6 (23.8–28.1)abB	30.000	< 0.001
2B	10/100	28.6 (27.1–29.1)a	23.2 (21.2–24)b	19 (17–19)c	23.2 (21.9–25)bA	28.041	< 0.001
2C	10/100	29 (28.5–30)a	24 (23.1–24.8)b	19.1 (18.6–20)c	23.9 (23.5–24.6)bA	28.250	< 0.001
2D	10/100	30 (28.6–30)a	24.4 (23.3–24.7)b	19.8 (19–20)c	23.7 (23.2–25)bA	28.041	< 0.001
2E	10/100	28.85 (28.5–30)a	23.5 (23.1–24.7)b	19.05 (18.5–20.1)c	23 (21.6–24)bA	29.510	< 0.001
T-statistic		7.050	13.568	11.321	39.955
P*		0.632	0.139	0.254	< 0.001

*Kruskal Wallis test, Dunn test; **Friedman test, Dunn test; data are expressed as median (min-max); a-c: There is no difference between those with the same letter in each group row; A-B: There is no difference between groups having the same letter in each column

*1A: Office-Control, 2A: Home-Control, 1B: Office-APF, 2B: Home-APF, 1C: Office-CPP-ACPF, 2C: Home-CPP-ACPF, 1D: Office-CaGP, F, 2D: Home-CaGP, F, 1E: Office-HAp, F, 2E: Home-HAp, F

A statistically significant decrease in b* values was determined in all groups that underwent remineralization (p < 0.001 Friedman Test, Dunn Test, Table 5). There was no statistically significant difference in b* values between subgroups after remineralization (p = 0.254, Kruskal Wallis Test, Table 5). After staining, b* values increased statistically significantly in all groups (p < 0.001, Friedman Test, Dunn Test, Table 5). This increase was found to be statistically similar in all subgroups to which remineralization agents were applied.

Results of WI_D values

WI_D values obtained from the groups at different measurement stages are shown in Table 6 (The statistical tables have been placed at the end of the manuscript). After bleaching, WI_D−1 values increased statistically significantly in all groups (p < 0.001, Friedman Test, Dunn Test, Table 6). It was determined that there was no statistically significant difference between the WI_D−1 values obtained after office and home whitening (p = 0.024, Kruskal Wallis Test, Dunn Test, Table 6).

Table 6.

Intergroup and intragroup comparison of WI_D values obtained from groups

	WID Values
	n/N	Baseline WID−0	Bleaching WID−1	Remineralization WID−2	Staining WID−3	T-statistic	P**
1A	10/100	9.9 (7.1–11.9)c	19.59 (16.4–20.5)ab	23.73 (21.6–26.3)b	10.29 (7.3–11.8)acC	28.080	< 0.001
1B	10/100	11.16 (8.3–11.9)c	20.56 (18–21.9)ab	25.98 (23.2–26.9)b	12.85 (11.8–13.8)acBC	30.000	< 0.001
1C	10/100	10.23 (9.3–11.2)c	19.56 (18.3–20.3)ab	25.07 (24.6–26.3)b	15.43 (14.5–16.2)acA	30.000	< 0.001
1D	10/100	10.23 (9.3–11.5)c	19.57 (18.6–20.4)ab	25.07 (24.6–26.4)b	14.54 (13.2–15)acAB	30.000	< 0.001
1E	10/100	10.28 (9.3–11.2)c	19.78 (18.6–20.6)ab	25.21 (24.5–25.7)b	16.18 (15.2–17.4)acA	30.000	< 0.001
2A	10/100	10.47 (7.1–11.9)c	19.58 (16.4–20.6)ab	25.08 (21.6–26.3)b	11.12 (7.3–12.7)acC	28.080	< 0.001
2B	10/100	10.83 (9.8–11.9)c	20.3 (19.9–21.9)ab	25.7 (24.7–26.9)b	13.25 (12.8–14.6)acBC	30.000	< 0.001
2C	10/100	10.44 (9.3–11.6)c	19.78 (18.3–20.6)ab	25.21 (24.6–25.7)b	16.18 (14.5–17.4)acA	30.000	< 0.001
2D	10/100	9.81 (9.3–10.5)c	19.47 (18.5–19.6)ab	25.05 (24.6–25.1)b	14.63 (14.4–15.4)acAB	30.000	< 0.001
2E	10/100	10.44 (9.3–11.6)c	19.8 (18.6–20.6)ab	25.58 (24.5–25.7)b	17.06 (16.4–18.5)acA	30.000	< 0.001
T-statistic		9.727	19.100	8.370	85.419
P*		0.373	0.024	0.497	< 0.001

*Kruskal Wallis test, Dunn test; **Friedman test, Dunn test; data are expressed as median (min-max); a-c: There is no difference between those with the same letter in each group row; A-C: There is no difference between groups having the same letter in each column

*1A: Office-Control, 2A: Home-Control, 1B: Office-APF, 2B: Home-APF, 1C: Office-CPP-ACPF, 2C: Home-CPP-ACPF, 1D: Office-CaGP, F, 2D: Home-CaGP, F, 1E: Office-HAp, F, 2E: Home-HAp, F

There was no statistically significant change in WI_D−2 values in all groups after remineralization (Friedman Test, Dunn Test, Table 6). No statistically significant difference was detected between the WI_D−2 values obtained from the groups after remineralization (p = 0.497, Kruskal Wallis Test, Dunn Test, Table 6).

A statistically significant decrease was observed in WI_D−3 values after staining in all groups (p < 0.001, Friedman Test, Dunn Test, Table 6). In the office and at home whitening groups, the lowest WI_D−3 values after staining were seen in the control and APF gel applied groups, while the highest WI_D−3 values were obtained in the CPP-ACPF and HAp, F containing gel applied groups (p < 0.001, Kruskal Wallis Test, Dunn Test, Table 6).

Results of ΔE₀₀ values

ΔE₀₀ values obtained from the groups are shown in Table 7 (The statistical tables have been placed at the end of the manuscript). There was no statistically significant difference between the ΔE_00 − 1 values (bleaching-baseline) obtained from all groups (p = 0.157, Kruskal Wallis Test, Dunn Test, Table 7). Similarly, no statistically significant difference was observed between the ΔE_00 − 2 (remineralization-bleaching) values obtained from the groups (p = 0.097, Kruskal Wallis Test, Dunn Test, Table 7).

Table 7.

Intergroup and intragroup comparison of ΔE₀₀ values obtained from groups

	ΔE00 Values
	n/N	ΔE00 − 1 (bleaching-baseline)	ΔE00 − 2 (remineralization-bleaching)	ΔE00 − 3 (staining-bleaching)	T-statistic	P**
1A	10/100	4.48 (4.2–4.6)ab	2.34 (1.7–2.6)b	8.13 (8–8.6)aB	20.000	< 0.001
1B	10/100	4.58 (4.4–4.8)ab	2.31 (2–2.3)b	8.31 (7.9–8.4)aB	20.000	< 0.001
1C	10/100	4.45 (4.3–4.7)a	2.43 (2.3–2.6)b	4.75 (4.7–4.9)aA	16.800	< 0.001
1D	10/100	4.48 (4.4–4.7)ab	2.44 (2.2–2.5)b	5.8 (5.5–6.4)aAB	20.000	< 0.001
1E	10/100	4.53 (4.2–4.6)a	2.39 (2.3–2.5)b	4.81 (3.9–4.9)aA	15.200	0.001
2A	10/100	4.51 (4.3–4.6)ab	2.33 (1.7–2.6)b	8.13 (7.5–8.6)aB	20.000	< 0.001
2B	10/100	4.69 (4.3–4.7)ab	2.32 (2–2.6)b	7.4 (7.3–8.8)aB	20.000	< 0.001
2C	10/100	4.46 (4.2–4.6)a	2.43 (2.3–2.6)b	4.08 (3.8–4.9)aA	15.200	0.001
2D	10/100	4.45 (4.3–4.7)ab	2.34 (2.2–2.5)b	4.86 (4.7–6)aA	20.000	< 0.001
2E	10/100	4.53 (4.4–4.6)a	2.37 (2.3–2.6)b	3.92 (3.7–4.8)aA	15.200	0.001
T-statistic		13.136	16.486	84.176
P*		0.157	0.097	< 0,001

*Kruskal Wallis test; **Friedman test; data are expressed as median (min-max); a-b: There is no difference between those with the same letter in each group row; A-C: There is no difference between groups having the same letter in each column

*1A: Office-Control, 2A: Home-Control, 1B: Office-APF, 2B: Home-APF, 1C: Office-CPP-ACPF, 2C: Home-CPP-ACPF, 1D: Office-CaGP, F, 2D: Home-CaGP, F, 1E: Office-HAp, F, 2E: Home-HAp, F

There was a statistically significant difference between the ΔE_00 − 3 (staining-bleaching) values obtained from the groups (p < 0.001, Kruskal Wallis Test, Dunn Test, Table 7). The highest ΔE_00 − 3 values in office and home whitening groups; While it was seen in the control and APF gel applied groups, the lowest ΔE_00 − 3 values were obtained from the CPP-ACPF and HAp, F containing gel applied groups. (p < 0.001, Kruskal Wallis Test, Dunn Test, Table 7).

Results of ΔWI_D values

ΔWI_D values obtained from the groups are shown in Table 8 (The statistical tables have been placed at the end of the manuscript). No statistically significant difference was detected between the ΔWI_D−1 (bleaching-baseline) and ΔWI_D−2 (remineralization-bleaching) values obtained from the groups (p < 0.001, Kruskal Wallis Test, Dunn Test, Table 8). A statistically significant difference was found between the ΔWI_D−3 (staining-bleaching) values obtained from the groups (p < 0.001, Kruskal Wallis Test, Dunn Test, Table 8). In the office and home whitening groups, the highest negative ΔWI_D−3 values were seen in the control and APF gel applied groups, while the lowest ΔWI_D−3 values were obtained in the CPP-ACPF group.

Table 8.

Intergroup and intragroup comparison of ΔWI_D values obtained from groups

	ΔWID Values
	n/N	ΔWID−1 (bleaching-baseline)	ΔWID−2 (remineralization-bleaching)	ΔWID−3 (staining-bleaching)	T-statistic	P**
1A	10/100	9.2 (8.6–11.2)b	5.69 (4.9–5.9)ab	-8.9 (-10.3 - -8.8)aC	20.000	< 0.001
1B	10/100	9.35 (9.1–9.6)b	5.59 (5.1–5.9)ab	-7.88 (-8.2 - -6.1)aBC	20.000	< 0.001
1C	10/100	9.23 (9–9.5)b	5.76 (5.5–6.2)ab	-4.1 (-4.4 - -3.9)aA	20.000	< 0.001
1D	10/100	9.31 (8.9–9.7)b	5.85 (5.3–6)ab	-5.03 (-5.4 - -5)aAB	20.000	< 0.001
1E	10/100	9.34 (9.2–9.5)b	5.78 (5.1–6)ab	-3.35 (-3.6 - -2.7)aA	20.000	< 0.001
2A	10/100	9.35 (8.2–11.2)b	5.42 (4.2–6.1)ab	-8.87 (-10.3 - -7.5)aC	20.000	< 0.001
2B	10/100	9.45 (8.3–9.8)b	5.47 (4.9–6)ab	-7.05 (-7.2 - -6.8)aBC	20.000	< 0.001
2C	10/100	9.23 (8.4–9.4)b	5.73 (5.1–6.2)ab	-3.53 (-3.9 - -3.2)aA	20.000	< 0.001
2D	10/100	9.31 (9–9.7)b	5.6 (5.3–6.1)ab	-4.43 (-5 - -4.1)aAB	20.000	< 0.001
2E	10/100	9.34 (8.4–9.5)b	5.59 (5.1–5.9)ab	-2.67 (-3.5 - -2)aA	20.000	< 0.001
T-statistic		14.217	17.217	94.553
P*		0.112	0.076	< 0.001

*Kruskal Wallis test; **Friedman test; data are expressed as median (min-max); a-b: There is no difference between those with the same letter in each group row; A-C: There is no difference between groups having the same letter in each column

*1A: Office-Control, 2A: Home-Control, 1B: Office-APF, 2B: Home-APF, 1C: Office-CPP-ACPF, 2C: Home-CPP-ACPF, 1D: Office-CaGP, F, 2D: Home-CaGP, F, 1E: Office-HAp, F, 2E: Home-HAp, F

Discussion

Bleaching treatments are favored for their ease, effectiveness, and non-invasiveness compared to advanced restorative procedures. With increasing demand and material advancements, research is essential to evaluate their efficacy, safety, and long-term outcomes [1, 2].

Studies report that bleaching agents can weaken enamel, affecting its mineral content, surface morphology, and roughness [13, 14].

Mineral loss in enamel creates porosity, allowing pigment reattachment and making bleaching stability unpredictable. Reinforcing and smoothing enamel is crucial, often achieved by applying remineralizing agents post-bleaching [15, 16].

Fluoride-based agents, particularly the APF used in our study, are commonly employed for their efficacy [17, 18]. However, newer products enhance fluoride’s effectiveness through synergistic components. CPP-ACPF (MI Paste Plus) was included for its proven remineralization potential, combining fluoride with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) [19, 20]. Fluor Protector Gel, containing fluoride and calcium glycerophosphate, provides an alternative remineralization method, though its post-bleaching efficacy is less explored [21]. Finally, Remin Pro, containing hydroxyapatite and fluoride, was chosen for its comparable efficacy to CPP-ACPF and its suitability for those with milk protein allergies [20, 22].

In bleaching treatments, 35-40% hydrogen peroxide is typically used for in-office procedures, and 10-16% carbamide peroxide for at-home treatments [23–25]. This study used 40% hydrogen peroxide-based Opalescence Boost (Ultradent Products Inc, South Jordan, UT, USA) for in-office and 16% carbamide peroxide-based Opalescence PF (Ultradent Products Inc, South Jordan, UT, USA) for at-home bleaching.

Bovine teeth were used for enamel sample preparation in this study. Both human and bovine teeth are commonly used in in vitro bleaching studies, as bovine teeth offer a reliable and effective alternative due to their similar physical and chemical properties, yielding comparable results in whitening studies [26]. Previous research has shown that, despite natural color differences, bovine and human enamel respond similarly to bleaching and staining procedures [23].

A study on in-office and at-home bleaching found that 35% hydrogen peroxide and 16% carbamide peroxide gels significantly increased L* values while reducing a* and b* values [27]. Using Paravina et al.‘s [11] perceptibility (0.8) and acceptability (1.8) thresholds, ΔE₀₀ values were calculated via the CIEDE2000 formula. In our study, ΔE_00 − 1 (bleaching-baseline) exceeded 1.8 in all groups (4.45–4.69), with no statistical difference between treatments, indicating effective and perceptible color change. Similarly, Moghadam et al. [28] and Mounika et al. [27] found no significant difference in bleaching efficacy between comparable agents.

In bleaching studies, assessing the level of whiteness and color change is essential. The Whiteness Index for Dentistry (WI_D), developed by Perez et al. [29], is the only CIE L*a*b* based index for whiteness evaluation. High WI_D values indicate greater whiteness, while low values indicate less whiteness. Perez et al. [12] reported a perceptibility threshold of 0.72 and an acceptability threshold of 2.62 for ΔWI_D. In our study, ΔWI_D−1 (bleaching-baseline) exceeded 2.62 in all groups (9.2–9.45), with no significant difference between at-home and in-office bleaching, indicating an effective and perceptible whiteness change with both agents, regardless of concentration or method. ΔWI_D−1 was consistent with ΔE_00 − 1 values.

In vitro research mimics oral conditions, with samples stored in artificial saliva to simulate in vivo environments for remineralization studies [30, 31]. To ensure consistency, all procedures were conducted by the same operator in a controlled clinical setting. Samples were kept in an incubator at 37 °C with artificial saliva, and the solution was changed daily. In our study, all groups were maintained in artificial saliva in the incubator when not receiving remineralization agents post-bleaching, while the control group received no treatment.

This setup enabled the comparison of color values from samples treated with different remineralization agents to the control group, allowing the evaluation of the agents’ effects on bleaching efficacy.

In both in-office and at-home bleaching groups, L* values remained similar to those at the end of bleaching, regardless of the remineralization agents used. No significant difference in L* values was observed compared to the control group. Few studies assess the impact of different remineralization agents on L* values post-bleaching. Carneiro et al. [32] found similar L* values in groups treated with calcium silicate, sodium phosphate, fluoride, or stored in artificial saliva and distilled water after bleaching with 35% hydrogen peroxide, aligning with our study. A 2022 study using 37.5% hydrogen peroxide gel, NaF, CPP-ACPF, and HAp found no significant ΔL difference between groups, and no significant impact on Δa* and Δb* values, consistent with our findings [33].

Research shows that the b* parameter, in addition to L*, is an important indicator of bleaching efficiency [34]. In our study, only b* values decreased across all groups, including the control, possibly due to ongoing whitening in the b* parameter. Within two weeks post-bleaching, peroxide-apatite forms as free radicals are released from hydrogen peroxide, and studies suggest these radicals dissociate from the tooth structure after about two weeks [35].

Özdemir et al. [36] evaluated color using a spectrophotometer at 24 h, 7 days, and 14 days after in-office bleaching. Using CIEDE2000 and WI_D formulas for color and whiteness, they found WI_D values increased at all intervals, concluding whitening continued for two weeks. However, in our study, whitening continuation is attributed only to a decrease in b*, with no change in L* values. This could be due to the higher initial L* values of bovine teeth. Kwon et al. [37] similarly found that bovine teeth had significantly higher L* values than human teeth, likely due to simpler diets and shorter lifespans.

In a study by Malekipour et al. [38], the effects of CPP-ACPF, Remin Pro, and 0.05% NaF mouthwash on tooth discoloration after 20% carbamide peroxide bleaching were evaluated. Contrary to our findings, they reported no significant difference in ΔL between the CPP-ACPF and NaF groups, but both agents increased ΔL more than the Remin Pro group. This difference could be due to their use of a one-month pH cycling process before remineralization, which was not done in our study. However, due to limited research on this topic, further studies are needed to assess the impact of these agents on L*, a*, and b* values.

No significant differences were found when comparing the ΔE_00 − 2 and ΔWI_D−2 values between groups, indicating that remineralization treatment does not affect color or whiteness change in office and at-home bleaching efficacy. Therefore, the null hypothesis that the tested remineralization agents have no effect on color and whiteness change immediately after bleaching is accepted.

Crastechini et al. [25] evaluated the effect of a remineralizing gel combining fluoride with calcium silicate and phosphate, as well as sodium fluoride gel, on bleached enamel. They concluded that the gels did not affect color change in enamel bleached with 40% hydrogen peroxide. Similarly, Armenio et al. [39] found that 1.23% sodium fluoride gel applied after bleaching with 16% carbamide peroxide did not alter bleaching efficacy. Misilli et al. [33] also found no impact on bleaching efficacy from the application of 2% NaF gel or remineralization agents (CPP-ACPF and HAp) mixed with or applied after bleaching gel.

Bleached enamel may be more prone to staining than unbleached enamel, as bleaching alters its mineral content and increases surface roughness, promoting pigment adhesion [7]. Beverages like tea, coffee, chlorhexidine, and red wine have been used to assess staining susceptibility of bleached teeth [15, 40]. While all these substances cause staining, some studies suggest bleached teeth are more susceptible to red wine stains [41]. Thus, red wine was used for tooth staining in this study. Samples were immersed in red wine for 10 min daily over 9 days, as done by Ley et al. [42].

This study aimed to evaluate the protective potential of remineralization agents against restaining, given that bleaching increases enamel roughness and susceptibility to staining, which can be improved by remineralization.

In our study, L* values significantly decreased after staining across all groups. The most considerable reduction in L* value was observed in the control and APF gel groups, while the least affected groups were those treated with CPP-ACPF and HAp, F gels.

Our study found that the APF gel was less effective in maintaining L* values compared to other agents. L* indicates lightness and brightness, which is influenced by surface roughness. Rough surfaces reflect light irregularly, reducing brightness [43]. Melo et al. [44] compared various agents, and including 1.23% fluoride group showed less remineralization.

Another study applied 1.23% APF gel and 2% neutral fluoride gel after in-office bleaching with 35% hydrogen peroxide. It was reported that enamel morphology worsened with the 1.23% APF gel due to its lower pH compared to the 2% neutral fluoride gel [45].

The acidic structure of acidulated phosphate fluoride gel facilitates fluoride accumulation on the enamel surface. However, in our study, the APF gel applied post-bleaching did not protect the L* value. This is consistent with previous findings, likely due to its acidic nature increasing surface roughness after bleaching and promoting pigment adherence.

The literature lacks studies on the protective effects of remineralization agents on re-staining after bleaching, particularly regarding a* and b* parameters. Our study found significant increases in a* and b* values post-staining in all groups, with the control and APF gel groups showing the highest a* values, while remineralization groups showed similar b* values. Further research is needed to assess the effects on a* and b* parameters.

In this study, the highest color change values (ΔE_00 − 3) after staining were observed in the control and APF groups, while the lowest values were found in the CPP-ACPF and HAp, F groups. Similarly, ΔWI_D−3 values were consistent with ΔE_00 − 3 values. As a result, the hypothesis that the application of the tested remineralization agents immediately after bleaching would not have any effect on color stability was rejected.

According to Reynolds et al. [46], the capacity of fluoride to promote remineralization depends on the availability of calcium and phosphate ions. In this study, the greater color stability observed with agents other than APF may be attributed to the synergistic effect of fluoride in restoring lost minerals, reducing surface roughness, and preventing the attachment of color pigments to the enamel surface.

CPP-ACP in MI Paste Plus, which showed success in our study, acts as a calcium and phosphate reservoir, promoting remineralization by maintaining supersaturation [47]. Liena et al. [47] found that applying CPP-ACPF for five minutes after bleaching restored lost calcium. Similarly, Mohammed et al. [48] reported that CPP-ACPF improved color stability and surface smoothness after bleaching and tea staining.”

Remin Pro, which was effective in maintaining color stability in our study, contains hydroxyapatite, a biocompatible and low-solubility mineral naturally found in enamel and bone [49]. Valian et al. [50] reported that CPP-ACP and Remin Pro reduced surface roughness more effectively than artificial saliva after bleaching. Similarly, Melo et al. [44] found that CPP-ACP and HAp-containing agents enhanced remineralization more than fluoride or arginine-based agents. Another study also showed that Tooth Mousse Plus and Remin Pro were superior to fluoride-only agents in reducing enamel roughness post-bleaching [51].

A study evaluating the effects of various remineralizing agents on color stability and surface properties post-bleaching found similar color changes in groups treated with 1.23% APF gel, Remin Pro, and MI Paste Plus after staining with coffee [52]. In contrast, Sign et al. [53] applied CPP-ACP and 1.23% APF gel after bleaching with 10% carbamide peroxide and staining with tea. Both remineralization treatments provided greater color stability compared to untreated samples. However, in our study, APF was ineffective in maintaining color stability after both in-office and at-home bleaching, possibly due to differences in bleaching agents, application methods, and staining solutions used.

While no data exist on the post-bleaching use of CaGP-containing Fluor Protector gel, which was more effective than the control and APF gels in our study, various studies have shown that CaGP plays a key role in remineralization due to its pH buffering effect and calcium phosphate source [54]. In our study, this agent, recommended for post-bleaching use, demonstrated protective potential against staining without compromising bleaching effectiveness. However, further research is needed to confirm these findings and examine its effects on enamel surface changes and staining susceptibility.

Conclusion

Within the limitations of this current study, it was concluded that:

• In-office and at-home bleaching procedures showed similar color and whiteness changes.

• The application of remineralization agents containing fluoride, CPP-ACPF, CaGP, F, and HAp, F immediately after in-office and at-home bleaching procedures had no effect on color and whiteness change.

In terms of the efficacy of remineralization on color stability after bleaching, the results of this study indicated that:

• Remineralization agents containing HAp, F, CPP-ACPF, and CaGP, F, respectively, were found to be more successful in terms of color stability.

Abbreviations

APF

Acidulated Phosphate Fluoride

CaGP

Calcium Glycerophosphate

CIE

Comission Internationale de I’Eclairage

CPP-ACP

Casein Phosphopeptide Amorphous Calcium Phosphate

F

Fluoride

HAp

Hydroxyapatite

NaF

Sodium Fluoride

WI_D

Whiteness İndex for Dentistry

∆E₀₀

CIEDE 2000 Color Difference

∆WI_D

Whiteness Index Difference

IBM SPSS V23

Statistical Package for the Social Sciences version 23

Author contributions

A.E. and B.Y. contributed equally to all stages of the study (conceptualization, data collection, analysis, and writing). All authors read and approved the final manuscript.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

All statistical data used in this study are presented within this article.

Declarations

Ethics approval and consent to participate

This in vitro study was carried out at Department of Restorative Dentistry, Pamukkale University Faculty of Dentistry. Ethical approval for the study was received from Pamukkale University Non-Interventional Clinical Research Ethics Committee (date:15/11/2022, no: E-60116787-020-289510). The animal teeth used in this study were obtained from a slaughterhouse in Turkey, where the animals were processed for human consumption under veterinary supervision. The teeth were requested from the slaughterhouse with permission from the owner. Since the animals were already designated for food production, no additional ethical approval was required beyond our institutional ethics committee’s permission.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.