The primary functions of teeth are mastication, phonetics, and aesthetics. While past dental treatments focused mainly on mastication and phonetics, there is now a growing emphasis on aesthetics [1]. Teeth are highly visible during conversations and smiles, playing a crucial role in first impres-sions [2]. Dissatisfaction with one’s appearance can negatively affect self-esteem and interpersonal relationships [3,4]. Factors influencing dental aesthetics include tooth size, shape, alignment, color, surface texture, disease status, facial structure, skeletal structure, and lip color and shape. Among these, discoloration due to staining has the most significant impact [5].

Tooth discoloration can be divided into intrinsic and extrinsic causes. Intrinsic factors include congenital conditions, fluoride intake, nutritional status, diseases, and aging. Extrinsic factors involve frequent consumption of staining substances like wine, tea, coffee, and tobacco [6,7]. The tannins in coffee, which give it a bitter taste, bind to proteins and stain teeth yellow. Similarly, green tea, while reducing caries-causing bacteria, contains tannins that can stain teeth [8]. However, stains from beverages like coffee, tea, and wine are easier to remove than intrinsic stains [9-11].

As aesthetic demands increase, there has been interest in tooth whitening, with attempts to use high-concentration blea-ching agents in dental clinics for rapid satisfaction. However, this can negatively impact enamel mineralization and surface roughness [12,13]. Another approach involves using night guards or custom trays with 10% carbamide peroxide (CP) under dental supervision, which has led to positive outcomes and advancements in new products and techniques [14,15].

Most product development has focused on whitening rather than stain removal. Over-the-counter (OTC) bleaching products containing hydrogen peroxide have been developed for unsupervised use, including whitening toothpaste and strips [16,17]. Various studies have explored these OTC options. Kim et al. [18] investigated the whitening effects of gels used with wraps, while Lee et al. [19] studied self-whitening strips containing 2.6% and 2.9% hydrogen peroxide. Kim et al. [20] researched self-whitening gels in manicure form containing hydrogen peroxide. These studies have resulted in a variety of whitening products available today.

This study aims to enhance safety and efficacy by using two dentifrices instead of one for tooth whitening. According to the Korean Ministry of Food and Drug Safety’s standards for dentifrices, the amounts of active ingredients such as calcium carbonate (not exceeding 50%), dicalcium phosphate (15%), and colloidal silica (15%) are regulated. This study uses a two-step approach: first, a dentifrice containing 35% calcium carbonate and 15% dicalcium phosphate for effective stain removal; second, a dentifrice containing 7% colloidal silica and 2.14% hydrogen peroxide for whitening and polishing effects. This research aims to evaluate the whitening efficacy of these formulations.

1. Study subjects

(1) Main ingredients

The study involved two types of dentifrices, and the main active ingredients of each are listed in Table 1.

Table 1 . Main ingredient and content of toothpaste

Division	Main ingredient	Content (%)
Step 1	Calcium anhydride	15
	Calcium carbonate	35
Step 2	Hydrogen peroxide 35%	2.14
	Colloidal silicon oxide	7

(1) Extraction of bovine teeth

Healthy bovine anterior teeth were used for the experiment. Teeth were extracted from the alveolar bone using an extrac-tion elevator and forceps. Soft tissue attachments around the teeth were removed using an Orban knife and periosteal elevator, and the teeth were stored in saline at 2℃.

(2) Preparation of specimens

Teeth with existing stains were selected. The teeth were sectioned using a disc bur mounted on a low handpiece into 5×5 mm pieces. Each specimen was embedded in acrylic resin with the labial surface exposed. Three specimens were prepared from each tooth by dividing each bovine anterior tooth into three parts (Figure 1).

Figure 1. The process of making specimens.

To prevent drying and simulate an oral environment, STEP 1 and STEP 2 dentifrices were diluted with sterile distilled water (40 ml) and toothpaste (60 ml) in a 2:3 ratio. The mixture was stirred at 150 rpm for about 25 minutes (Figure 2, 3).

Figure 2. Dilution process of denti-frice.

Figure 3. Dentifrice specimen. (A) Step 1 dentifrice, (B) Step 2 denti-frice.

2. Methods

The study was conducted over two months from October 26, 2020, to December 25, 2020. A total of 124 specimens were used, each labeled with a number and alphabet for identification.

Before the main experiment, a preliminary test was conducted to set the conditions and application time for the polishing device (Endo A class Apex wireless reciprocating motor & canal measuring device - Seyang Co.). Three stained bovine teeth were selected for the preliminary test. The polishing device was applied vertically on the labial surface with STEP 1 dentifrice diluted 2:3 with distilled water.

(1) First preliminary experiment for setting polishing conditions and application time

The first preliminary experiment aimed to determine the time required to remove stains under two conditions using STEP 1 dentifrice. The first condition involved polishing stained teeth at 400 rpm and 3.0 N/cm torque for five intervals of one minute each. The second condition involved polishing under the same rotational speed and torque for three intervals of one and a half minutes each.

(2) Second preliminary experiment for setting polishing conditions and application time

The second preliminary experiment aimed to determine the degree of stain removal and brightness change by applying both STEP 1 and STEP 2 dentifrices. Four conditions were tested, as detailed in Table 2, Figure 4.

Table 2 . Endo A Apex mechanical conditions used in the second preliminary experiment

Category	Step 1	Step 2	Application time
Condition 1	400 rpm, 3.0 N/cm	200 rpm, 2.0 N/cm	Step 1: 90 seconds×3 times Step 2: 90 seconds×3 times(total 9 minutes)
Condition 2	400 rpm, 3.0 N/cm	200 rpm, 3.0 N/cm
Condition 3	400 rpm, 3.0 N/cm	300 rpm, 2.0 N/cm
Condition 4	400 rpm, 3.0 N/cm	300 rpm, 3.0 N/cm

Figure 4. Polishing equipment and rubber cup.

During STEP 1 application, the rubber cup rotated in the clockwise direction, while during STEP 2 application, it rotated counterclockwise.

Based on the conditions established in the preliminary experiments, the main experiment compared the degree of stain removal and whitening effect over time using STEP 1 and STEP 2 dentifrices.

(1) Verification of polishing effect in stages

Twenty-four specimens were used to compare the stain removal and whitening effects of STEP 1 and STEP 2 dentifrices in ten stages using an optical microscope and shade guide (Table 3).

Table 3 . Polishing rotation speed and dentifrice application time

Category	Step 1	Step 2	Application time
Condition 1	400 rpm, 3.0 N/cm	200 rpm, 2.0 N/cm	Step 1: 1 minute×5 times Step 2: 1 minute×5 times(total 10 minutes)
Condition 2	400 rpm, 3.0 N/cm	200 rpm, 3.0 N/cm
Condition 3	400 rpm, 3.0 N/cm	300 rpm, 2.0 N/cm
Condition 4	400 rpm, 3.0 N/cm	300 rpm, 3.0 N/cm

(2) Measurement using 100 specimens

One hundred specimens were subjected to five applications each of STEP 1 and STEP 2 dentifrices, totaling ten applications. The results were measured with Easyshade^Ⓡ V.

Tooth color changes before and after dentifrice application were measured using a Vita Lumin vacuum shade guide (Vident Inc., California, USA), a visual comparison method used in dental clinics and laboratories (Figure 5).

Figure 5. Lightness value of shade guide VITA classical color classi-fication.

Shade Guide Measurement (visual comparison)

Value: Select the guide tip closest in brightness (value) to the measured tooth.

Chroma: Determine the chroma using the selected guide tip.

Hue: Identify whether the determined tooth color is red or yellow.

Tooth colors were classified using the Vita Shade Guide, which categorizes colors into 16 stages based on hue (A-D) and value (1-4). The lightness value for each stage was referenced (Figure 5), and scores were assigned to evaluate the difference before and after treatment. A score closer to 1 indicated a lighter color, while a score closer to 16 indicated a darker color. For the 24 specimens, non-parametric statistical methods were applied, and for the 100 specimens, the mean and standard deviation of lightness value changes were calculated for each condition.

1. Preliminary experiment results

In both the first and second experiments, stain removal became visibly noticeable after 3 minutes of application. When applying for 1 minute and 30 seconds three times, it was observed that a significant amount of dentifrice was displaced, making accurate comparison difficult. Therefore, in the main experiment, a duration of 1 minute applied five times was set as the baseline time for measuring stage-by-stage progress.

The results of the experiment conducted under the conditions specified in Table 4 are shown in Figure 6. Changes in tooth brightness over time were measured using the VITA classical shade guide, with variations in rotational speed and torque, as detailed in Table 5 (Figure 6).

Table 4 . Polishing rotation speed and dentifrice application time

Category	Step 1	Step 2	Application time
Condition	400 rpm, 3.0 N/cm	200 rpm, 3.0 N/cm	Step 1: 1 minute×5 times Step 2: 1 minute×5 times(total 10 minutes)

Table 5 . The second preliminary experiment result (shade guide comparison)

Category	Control group	After applying both Step 1 and Step 2 dentifrices
Condition 1	A4	A2
Condition 2	A2	B2
Condition 3	A4	B1
Condition 4	A3	C2

Figure 6. Second preliminary expe-riment result (comparison between control and experimental group).

2. Main experiment results

The experiment was conducted under the conditions specified in Table 3 to compare the degree of stain removal. The results indicated that the lightness changes at each step were significant according to the Wilcoxon signed rank test (Table 6).

Table 6 . Step by step lightness changes

Group	Lightness value						Base			Step 1+Step 2		p-value1 (Z)
	BASE	After Step 1	After Step 2	Delta Step 1	Delta Step 2		Mean	SD		Mean	SD
Condition 1	11	3	5	8	−2		7.25	3.76		3.50	2.51	0.027 (−2.207)
	5	2	1	3	1
	5	2	2	3	0
	5	2	2	3	0
	5	1	2	4	−1
Condition 2	5	5	1	0	4
	2	2	2	1	0		6.20	2.68		2.40	1.52	0.039 (−2.060)
	5	2	2	3	0
	12	11	7	1	4
	11	9	5	2	4
	11	9	5	2	4
Condition 3	14	3	6	11	−3		6.50	3.02		2.63	1.51	0.018 (−2.375)
	3	2	2	1	0
	9	5	5	4	0
	11	7	5	4	2
	9	3	5	6	−2
Condition 4	5	2	2	3	0		9.20	4.02		4.60	1.52	0.042 (−2.032)
	7	1	2	6	−1
	7	5	2	2	3
	3	1	1	2	0
	8	4	5	4	−1
	11	5	5	6	0
	9	5	2	4	3
	2	2	2	0	0

¹p-value by paired t-test.

The experiment was conducted under the conditions specified in Table 4 to compare the degree of stain removal. Brightness analysis using the VITA SYSTEM 3D-MASTER^Ⓡ method showed that applying type 1 dentifrice five times resulted in a decrease of 0.47 from the base (4.28 to 3.81), and applying type 2 dentifrice resulted in a decrease of 0.90 from the base (4.28 to 3.38), which is 0.43 lower than type 1 (Table 7).

Table 7 . The changes of lightness value by VITA SYSTEM 3D-MASTER^Ⓡ checking

Lightness value	N
	Base	Type 1	Type 2
1	0	0	1
2	3	20	29
3	16	16	20
4	31	27	31
5	50	37	19
Mean	4.28	3.81	3.38
SD	0.84	1.14	1.13
p-value1		<0.001	<0.001
p-value2			<0.001

¹p-value by paired t-test base and type 1/2.

²p-value by paired t-test type 1 and type 2.

Additionally, brightness analysis using the VITA classical A1-D4^Ⓡ shade checking method showed that applying type 1 dentifrice five times resulted in a decrease of 0.69 from the base (14.82 to 14.13), and applying type 2 dentifrice resulted in a decrease of 1.54 from the base (14.82 to 13.28), which is 0.85 lower than type 1 (Table 8).

Table 8 . The changes of lightness by VITA classical A1-D4^Ⓡ shade checking

Lightness value	N
	Base	Type 1	Type 2
9	1	1	3
10	0	0	0
11	4	15	29
12	16	20	20
13	0	0	0
14	0	0	0
15	27	25	26
16	52	39	22
Mean	14.82	14.13	13.28
SD	1.72	2.06	2.20
p-value1		<0.001	<0.001
p-value2			<0.001

¹p-value by paired t-test base and type 1/2.

²p-value by paired t-test type 1 and type 2.

With the improvement in economic income, modern individuals are increasingly concerned about health and appea-rance. As spending on facial aesthetics, which greatly influence first impressions, increases, the demand for tooth whitening naturally grows [1]. The concern over tooth discoloration caused by popular beverages like tea, coffee, and wine has led to the development of dentifrices that can achieve whitening effects through daily brushing at home without visiting the dentist [21].

Traditionally, dentifrice development focused primarily on whitening [22]. This study aimed to enhance efficiency by developing a 2-step dentifrice. Step 1, containing abrasive agents like calcium carbonate and dicalcium phosphate, was designed for stain removal. Step 2, containing hydrogen peroxide, was formulated for whitening effects.

To create optimal experimental conditions and simulate the oral environment, the dentifrices were diluted with sterile distilled water (40 ml) and toothpaste (60 ml) in a 2:3 ratio and mixed at 150 rpm for about 25 minutes. Preliminary experiments were conducted before the main experiment.

Previous studies used whitening toothpaste, patches, and night guards. This study utilized a polishing device that ensures consistent rotational speed (rpm) and uniform torque, along with a rubber cup to minimize enamel damage, thereby enhancing the consistency and stability of the experiment. After conducting experiments on 24 specimens to compare stain removal and whitening effects using an optical microscope and shade guide, optimal polishing speed and application time were determined. These conditions were then applied to 100 specimens, and measurements were taken with Easyshade^Ⓡ V.

The mean standard deviation of lightness value changes for each condition was calculated. Significant differences in brightness (p<0.001) were observed after applying the Step 1 dentifrice five times, and similarly, significant differences in brightness (p<0.001) were noted after applying the Step 2 dentifrice five times. The use of the 2-step dentifrice resulted in increased brightness, demonstrating its effectiveness in stain removal and tooth whitening.

This study had some limitations. First, it was challenging to replicate the oral environment’s temperature and the various components of saliva. Second, using a polishing device and rubber cup, which are not easily accessible to the general public, may limit the applicability of the results. Third, convincing individuals accustomed to using a single dentifrice to adopt a two-step process may be difficult.

Future studies should focus on clinical trials to determine the appropriate frequency of dentifrice use and the method of using these tools. Additionally, research on the impact of residual abrasives from the Step 1 dentifrice on periodontal health is necessary.

This study aimed to develop two dentifrices that are safer and more effective than pumice powder used in dental clinics for managing stained teeth caused by smoking, coffee, and tea. The dentifrices are designed for home use with a personal polisher. The study was conducted over two months using a total of 124 specimens prepared by sectioning the anterior teeth of bovine teeth.

The study involved two types of dentifrices with 24 specimens and 100 specimens. For the 24 specimens, the polishing conditions for dentifrice application in Step 1 were standardized at 400 rpm and 3.0 N.cm, while Step 2 was tested under four different conditions (Table 4). For the 100 specimens, Step 1 dentifrice was applied at 400 rpm and 3.0 N.cm for 60 seconds per session, repeated five times. Step 2 dentifrice was applied at 200 rpm and 3.0 N.cm for 60 seconds per session, repeated five times. Shade measurements were taken using Easyshade^Ⓡ V.

Changes in brightness before and after treatment were assessed using a shade guide. The degree of stain removal was evaluated with an optical microscope. Tooth color was categorized using the Vita Shade Guide, which includes 16 stages of color based on hue (A-D) and value (1-4). Lightness values for each stage were referenced from Figure 5, and scores were assigned to evaluate the difference before and after treatment.

The results showed that improvements in tooth brightness after Step 2 were greater when the polisher was set to 200 rpm compared to 300 rpm. Additionally, a rotational torque of 3.0 N.cm was more effective than 2.0 N.cm. The optimal condition was found to be 200 rpm with 3.0 N.cm torque.

These findings confirm that using Step 1 and Step 2 dentifrices effectively removes stains and improves tooth whitening.

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