Efficacy of different remineralization agents on treating incipient enamel lesions of primary and permanent teeth.

AIMS AND
OBJECTIVES: The aim of this study was to compare the in vitro efficacy of different remineralization (RM) agents on RM of artificial caries by evaluating the density, light reflection, and the crystal size of the newly formed mineral in primary and permanent teeth.
MATERIALS AND METHODS: Caries-free primary and permanent molars were divided into seven groups (n = 20) and treated with: G1 - Deionized water, G2 - Colgate Cavity Protection, G3 - Sensodyne Rapid Relief, G4 - GC MI Paste Plus, G5 - Clinpro Tooth Creme, G6 - Clinpro 5000, and G7 - Sensodyne Repair and Protect. Lesion depths were evaluated using laser fluorescence (DIAGNOdent), and polarized light microscopy (PLM), and the minerals were evaluated using X-ray diffractometry (XRD).
RESULTS: The highest decrease in laser fluorescence readings was observed in G6 for both primary and permanent teeth. No significant difference was observed between G2, G4, G5, and G6 in permanent teeth and G4 and G6 in primary teeth (P > 0.05). In PLM analyses, lesions depths were found to be lower in G6 compared to the other groups. No significant difference was observed between G2, G4, and G6 (P < 0.05). XRD evaluation showed that the newly formed mineral in G6 was denser and highly crystallized compared to the other groups.
CONCLUSION: This in vitro study demonstrated that Clinpro 5000 is more efficient in remineralizing incipient enamel lesions compared to the other agents tested.

INTRODUCTION

Dental caries is a transmittable disease leading to the extensive demineralization (DM) of dental tissues and is related to the oral flora, dental plaque, and consumption of fermentable sugars. In a healthy oral environment, DM and remineralization (RM) occur continuously and are at a balance. However, when the intraoral pH drops below 5.50, mostly due to the diffusion of the bacterial organic acids, the DM exceeds RM because of the increased dissolution of calcium and phosphate in the enamel to the unsaturated plaque fluid.[1]

Many agents to prevent or reverse the formation of carious lesions have been developed or are currently being investigated. To date, fluoride (F) has been the most widely and longest used RM agent. F ions replace the −OH groups in the hydroxyapatite, and the resulting fluorapatite is more resistant to acid challenge.[2]

Next to fluoride, casein phosphopeptide–amorphous calcium phosphate (CPP-ACP) has been the most studied RM agent. Casein is a phosphoprotein, comprising 80% of the milk proteins. It also has a remarkable ability to stabilize ACP.[3] CPP-ACP shows its anticaries activity by being incorporated into the dental plaque and increasing the free calcium and phosphate concentration, thus saturating the tooth surface. It is also suggested that by increasing the concentration of free calcium, CPP-ACP may have bacteriostatic effects.[4]

Tricalcium phosphate (TCP) is another RM agent suggested to play a role in increasing the free calcium concentration in saliva and dental plaque.[5]

Strontium (Sr) is similar to calcium in terms of atomic radius and chemical properties and is found in enamel at trace levels. The observations that Sr-rich enamel has lower caries prevalence have led researchers to investigate the anticarious effects of Sr.[6]

Calcium sodium phosphosilicate (CSP) is a bioactive glass component, which releases the silica, calcium, phosphate, and sodium necessary to remineralize enamel. Since the surface of the bioactive glass is formed of carbonated apatite, it has a high adherence to the tooth mineral, which naturally increases the RM potential.[78]

A survey of the literature has revealed that no studies have been conducted to compare the efficacies of RM agents containing F, Sr, CPP-ACP, TCP, and CSP under identical in vitro conditions. Therefore, this study aims to compare the in vitro efficacies of commercially available at-home use RM products containing F (Colgate Cavity Protection), Sr and F (Sensodyne Rapid Relief), CPP-ACP and F (GC MI Paste Plus), TCP and F (Clinpro Tooth Creme and Clinpro 5000), and CSP + F (Sensodyne Repair and Protect).

MATERIALS AND METHODS

This study has been conducted in full accordance with the World Medical Association Declaration of Helsinki. All the patients have signed to a consent form approved by the ethics committee.

Preparation of the samples and formation of artificial caries

The patients were referred to the Department of Pedodontics, Faculty of Dentistry, Kırıkkale University, the indication of extraction; on the buccal and lingual/palatinal surfaces, visible structural defects such as caries, DM, hypomineralization, and coloration were not included in the study. Seventy primary and 70 permanent freshly extruded molar. Seventy primary and 70 permanent freshly extruded molar teeth were collected. The soft-tissue remnants on the teeth were removed and cleaned with fluoride-free pumice, polishing brush, and nonfluoridated water. The samples were kept in 0.10% thymol solution (ADR Group, Istanbul, Turkey). The crowns of the teeth were cut at the enamel-cementum junction using a diamond wheel under water cooling. Then, the teeth were cut at the mesiodistal direction, and two specimens were obtained from each tooth. Each specimen was embedded in acrylic blocks (Orthocryl EQ, Dentaurum, Ispringen, Germany) with the enamel surface facing outward. On each tooth, approximately 150 μm enamel layer was removed via serial polishing with 500, 1200, 2400, and 4000 grit silicon carbide polishing strips.[9] The specimen surfaces were standardized by polishing with diamond abrasives (DiaDuo-2, Struers, Denmark). Enamel surfaces were covered with 4 mm × 4 mm pieces of scotch tape, and the areas left open were coated with two layers of acid-resistant nail polish (Flormar MATTE, Kocaeli, Turkey). The artificial caries was formed by keeping the specimens in a demineralizing solution containing 2.2 mM Ca(NO3)2, 2.2 mM KH2PO4, 0.1 ppm NaF, and 50 mM acetic acid for 3 days at 37°C.[10] The specimens were demineralized before separating the groups. All specimens were demineralized in the same solution in the same container. The experimental groups were formed by randomly selecting among the demineralized specimens.

Application of the remineralization agents

The prepared primary and permanent teeth were distributed randomly into seven groups. To mimic the intraoral pH changes, the specimens were kept in a DM solution and in a RM solution. The DM contained 1.5 mM CaCl2, 0.9 mM KH2PO4, and 50 mM acetic acid (pH 5.0) and the RM contained 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, and 20 mM HEPES (pH 7.0).[9] The specimens were immersed in freshly prepared DM for 30 min and RM for 2.5 h six times a day and kept in the RM for 6 h during the night. During the 4-week cycling period, the specimens were kept in RM for 48 h during the weekends.[11]

Throughout the 4 weeks' pH cycling period, the RM agents listed in Table 1 were applied on the specimens for 2 min twice a day at 09:00 am and 04:00 pm. After the application, the specimens were cleaned via a toothbrush and distilled water and put back into the respective solutions. At the end of the pH cycling and treatment period, the specimens were kept cold and dry until the time of analysis.

Table 1

Experimental Materials and Ingredients

	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6	Group 7
Product	Deionized water	Colgate Cavity Protection	Sensodyne Rapid Relief	GC MI Paste Plus	Clinpro Tooth Creme	Clinpro 5000	Sensodyne Repaır and Protect
Manufacturer	-	Colgate-Palmolıve, Dublın, Ireland	GlaxoSmithKline, Berks, England	GC Corp. Europe	3M ESPE, St. Paul, USA	3M ESPE, St. Paul, USA	GlaxoSmithKline, Berks, England

Before and after the pH cycle, the artificial caries lesion was evaluated with a DIAGNOdent laser fluorescence device (KAVO, Biberach, Germany). The measurements were performed using the Type B tip designed for flat surfaces and all measurements were performed using the same device and the same tip. Before each measurement, the device was calibrated with the ceramic standard per the manufacturer's instructions. Each measurement was repeated twice per specimen, and the mean value was recorded. The results were interpreted based on the standardized values by the manufacturer.

The depth of the artificial caries was evaluated via polarized light microscopy (PLM). Approximately 200 μm slices were cut via a Micracut 201 precision microtome (Metkon, Bursa, Turkey). The slices were analyzed using a PLM (Euromex, Arnhem, Germany) under ×40 magnification, and the lesion depths and the degree of RM were measured using Image Focus 4.0 (Euromex, Arnhem, Germany).

At the end of the pH cycling, five specimens from each group were randomly taken out of the acrylic blocks, and the 4 mm × 4 mm enamel regions were cut from the borders. The cut pieces were ground using a mortar (Retsch GmbH, Haan, Germany) until approximately 75 μm particle size was obtained. The crystal phase of the obtained powder was analyzed using an APD 2000 PRO X-Ray Diffractometer (GNR, Novara, Italy) with 35 kV and 25 mA CuKa radiation at 10° ≤2 θ ≤70°. The results were analyzed using Economic Value Added 12.0.0 software (Bruker, Germany). The peak width of the apatite phase was measured via full width at half maximum (FWHM). The crystal size was calculated via the Scherrer's formula (D = 0.89 λ/βcosθ) (λ: CuKa wavelength, β: FWHM (211) plane, and θ: Diffraction angle).

Statistical analysis of the data was conducted using IBM SPSS (SPSS Ins., Chicago, USA) statistical software version 16.0 with a significance set at 0.05. All data were examined for normality and homogeneity of variance with Kolmogorov–Smirnov test, and it was determined that the data in all groups had normal distribution. Intergroup comparisons were performed using the one-way ANOVA, post hoc Tukey's Least Significant Difference (LSD), and Dunnett's T3 test (P < 0.05).

RESULTS

DIAGNOdent and polarized light microscopy analyses

The DIAGNOdent and PLM measurements with a statistically significant difference (P < 0.05) in RM of both primary and permanent teeth were observed between Group 1, Group 2, Group 4, and Group 5. However, no statistically significant difference was observed among Groups 2, 4, and 5 (P > 0.05).

No statistically significant difference in RM was observed between the Group 1, Group 3, and Group 7 (P > 0.05).

The highest RM was obtained in TCP + 5000 ppm F (Group 6) both on primary and permanent teeth. However, on permanent teeth, no statistically significant difference was observed between Group 6, Group 2, Group 4, and Group 5 (P > 0.05). On primary teeth, no statistically significant difference was observed between Group 6 and Group 4 (P > 0.05) [Tables 2 and 3].

Table 2

After Remineralization agent applied to DIAGNOdent average values in permanent and primary teeth

DIAGNOdent Values	Permanent teeth			Primary teeth
Groups	n	Average	SD	n	Average	SD
Group 1 (Deionized water)	20	16,2	2,353	20	15,75	1,372
Grup 2 (Colgate Cavity Protection)	20	13,25	1,372	20	13,3	1,75
Grup 3 (Sensodyne Rapid Relief)	20	15,75	1,482	20	14,7	2,155
Grup 4 (GC MI Paste Plus)	20	13,3	1,838	20	12,45	2,305
Grup 5 (Clinpro Tooth Creme)	20	13,5	1,67	20	13,4	1,635
Grup 6 (Clinpro 5000)	20	12,75	1,773	20	11,5	1,277
Grup 7 (Sensodyne Repair and Protect)	20	15,7	2,029	20	14,6	1,569

Table 3

After Remineralization agent applied to PLM average values in permanent and primary teeth (µm)

PLM Values	Permanent teeth			Primary teeth
Groups	n	Average	SD	n	Average	SD
Group 1 (Deionized water)	18	194,14	24,663	19	191,7	19,598
Group 2 (Colgate Cavity Protection)	19	163,91	20,348	18	163,06	19,722
Group 3 (Sensodyne Rapid Relief)	19	183,97	16,486	18	185,64	16,424
Group 4 (GC MI Paste Plus)	20	164,36	23,382	18	167,41	17,698
Group 5 (Clinpro Tooth Creme)	19	170,84	28,63	18	170,15	21,829
Group 6 (Clinpro 5000)	18	145,82	24,042	19	149,99	24,445
Group 7 (Sensodyne Repair and Protect)	19	181,54	18,17	17	184,44	20,02

X-ray diffractometry analysis

Although there were differences between the spectrograms of each group, the main inorganic component of the surface crystals was found to be hydroxyapatite [Figures 1 and 2].

Figure 1

Examples of the permanent teeth 2θ (theta) values

Figure 2

Examples of the primary teeth 2θ (theta) values

In the permanent teeth, the highest crystal density (%I) was observed in Group 6 (75.84%), whereas the lowest density was observed in Group 1 (13.37%). The lowest FWHM was observed in Group 6 (0.197), whereas the highest FWHM was observed in Group 1 (0.274).

In the primary teeth, the highest crystal density (%I) was observed in Group 6 (78.07%), whereas the lowest density was observed in Group 1 (13.16%). The lowest FWHM was observed in Group 6 (0.184), whereas the highest FWHM was observed in Group 1 (0.304) [Table 4].

Table 4

Intensity (I%), FWHM and Crystal Size values obtained by XRD analysis in permanent and primary teeth

XRD Values	Permanent teeth			Primary teeth
Groups	Intensity (I%) Av.	FWHM Av.	Crystal Size Av.	Intensity (I%) Av.	FWHM Av.	Crystal Size Av.
Group 1 (Deionized water)	13,37	0,274	29,988	13,16	0,304	27,28
Group 2 (Colgate Cavity Protection)	66,15	0,207	37,508	71,12	0,196	34,42
Group 3 (Sensodyne Rapid Relief)	35,59	0,255	32,664	37,48	0,262	31,36
Group 4 (GC MI Paste Plus)	56,06	0,247	33,376	48,38	0,214	33,44
Group 5 (Clinpro Tooth Creme)	56,89	0,234	33,568	51,62	0,204	32,5
Group 6 (Clinpro 5000)	75,84	0,197	38,71	78,07	0,184	34,64
Group 7 (Sensodyne Repair and Protect)	48,8	0,25	32,912	45,81	0,236	32,11

DISCUSSION

RM of carious lesions is an important concept for preventive dentistry. Fluoride has been the most widely used agent to prevent or reverse DM. F ions replace the −OH groups in the hydroxyapatite, and the resulting fluorapatite is more resistant to acid challenge.[2] Therefore, the RM activity of fluoride increases only when there are sufficient free calcium and phosphate ions.[12] Due to the calcium-/phosphate-dependent action of fluoride and other concerns, such as toxicity, risk of fluorosis, fluoride-free or fluoride-supplemented products alternative to fluoride have been developed.[13] In this study, we have compared the in vitro efficacy of six products containing different mechanisms of actions and supplemented with fluoride.

The pH cycling model used in this study can mimic the intraoral pH changes and results in more realistic findings in evaluating the RM efficacy of an agent. Jayarajan et al. have reported that DIAGNOdent can produce quantitative measurements by the absorption of laser and reflection of nonabsorbed light by inorganic materials in the infrared spectrum.[14] It has, therefore, been proposed a suitable tool for monitoring the DM–RM processes in in vitro studies.

PLM has often been used to measure the depth of carious lesions.[151617] Since the light is positively diffracted when passing through porous and organic regions and negatively diffracted when passing through inorganic regions, the lesion can be imaged.[18]

The lesion depths measured via PLM have been found to be lower on samples treated with Group 6, Group 2, and Group 4, compared to the other groups.

The lowest lesion depth (highest RM) observed on samples treated with Group 6 can be explained by the high dose of fluoride it contains. This agent can inhibit DM and promote RM by increasing the environmental pH due to the high-dose fluoride it contains. In addition, when the pH increases, the TCP releases calcium and phosphate ions further increases the pH and promotes mineral deposition onto the tooth surface.[19] In a study by Prabhakar et al.,[20] lesion depths have been investigated after a 28 days of pH cycling and treatment with 5000 ppm F, CPP-ACP + F, and TCP + 900 ppm F. The highest decrease in lesion depth has been observed in 5000 ppm F group. The researchers have concluded that the effect of the high fluoride-containing group is a result of the increase of fluoride inside the lesion due to the increase outside the lesion. Although the diffusion of the fluoride into the tissues slows down due to the interaction of fluoride with hydroxyapatite crystals, this effect has been attributed to the movement of the fluoride ions into the lesion.

The lowest RM was observed on samples treated with Group 7 and Group 3. We suggest that the low RM rates of Group 7 and Group 3 are because these agents are designed as antihypersensitivity agents rather than RM agents.[2122]

The crystal intensity evaluations showed that the highest mineral intensity was in Group 6, followed by Group 2, Group 5, and Group 4. The lowest intensity was observed in Group 1. The TCP in the Group 6 serves as a calcium and phosphate ion source and it contains a high dose of F. Therefore, it allows more F to penetrate the pores of the lesion, interact with the porous hydroxyapatite, and deposit new mineral. Both in permanent and primary teeth, FWHM was found to be lowest in Group 6 and highest in Group 1. Similar to our findings, it has been reported that the FWHM is inversely related to the crystal density and quality.[102324]

In both the permanent and primary teeth, the newly formed mineral crystals by the tested agents were larger compared to the native enamel. In accordance with our findings,[25] they have reported that when the application duration of CPP-ACP-containing agents is increased, the Ca/P ratio of the enamel increases, and as a result, the size of the crystals increases.

CONCLUSION

Our findings show that TCP and CPP-ACP combined with F are successful RM agents for treating incipient enamel lesions. For agents used in combination with F, the success rate increases with higher concentrations of F. It can be concluded that in treating initial enamel lesions of adults, high success rate is expected when TCP is used in combination with high-dose (5000 ppm) F. In children, however, due to the risk the high F concentration poses, TCP + 900 ppm F or CPP-ACP (without fluoride) are safer alternatives.

It is thought to be useful in the selection of appropriate and effective RM agent at pediatric patients. It is expected to benefit in preventive dentistry

Our study sample calculation is presented in the appendix. Both primary and permanent teeth in 15 samples (for PLM) and 5 samples (for X-ray diffractometry) were calculated. A total of 20 samples were used in each group.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

The work was supported by Kırıkkale University BAP Coordination Unit.

Conflicts of interest

There are no conflicts of interest.