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Year : 2022  |  Volume : 33  |  Issue : 2  |  Page : 188-192
Influence of two remineralizing agents on bleached enamel surface morphology and mineral composition – An In Vitro study

Department of Conservative Dentistry and Endodontics, Goa Dental College, Bambolim, Goa, India

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Date of Submission11-Sep-2021
Date of Decision19-Apr-2022
Date of Acceptance29-Apr-2022
Date of Web Publication13-Oct-2022


Aim: To investigate the effects of bleaching with 35% hydrogen peroxide on the structure of tooth enamel and the role of two remineralizing agents for their potential to remineralize any damaged regions of enamel. Materials and Methods: Freshly extracted 32 mature permanent central incisors were selected and sectioned at the level of the cemento-enamel junction. The teeth were divided into four groups consisting of eight teeth each: No bleaching (control) [Group 1], bleaching with 35% hydrogen peroxide [Group 2], bleaching with 35% hydrogen peroxide followed by application of casein phosphopeptide amorphous calcium phosphate fluoride paste [Group 3], and bleaching with 35% hydrogen peroxide followed by application of xylitol-coated calcium phosphate fluoride varnish [Group 4]. The enamel surfaces were analyzed under the scanning electron microscope and quantitative energy dispersive X-ray analysis. Results: Results were statistically analyzed by one-way analysis of variance and Tukey's posthoc test. Group 2 revealed changes in enamel surface morphology and a statistically significant decrease in mineral content. Groups 3 and 4 showed statistically significant remineralization potential. Intergroup comparison showed that samples in Group 4 had a higher mineral content compared to Group 3. Conclusions: The application of the tested remineralizing agents following bleaching was effective in repairing the enamel surface morphology with higher efficacy for the fluoride varnish product. Since bleaching regimes with high concentrations of hydrogen peroxide adversely affect the enamel surface, these findings can translate to clinical practice to reduce the long-term damaging effects of tooth bleaching.

Keywords: Bleaching, enamel, hydrogen peroxide, remineralization

How to cite this article:
Godinho M, de Ataide IN, Lambor R, Fernandes M. Influence of two remineralizing agents on bleached enamel surface morphology and mineral composition – An In Vitro study. Indian J Dent Res 2022;33:188-92

How to cite this URL:
Godinho M, de Ataide IN, Lambor R, Fernandes M. Influence of two remineralizing agents on bleached enamel surface morphology and mineral composition – An In Vitro study. Indian J Dent Res [serial online] 2022 [cited 2022 Nov 29];33:188-92. Available from:

   Introduction Top

Tooth bleaching is one of the most sought-after noninvasive esthetic treatment options. The product most widely used today for both vital and nonvital teeth is hydrogen peroxide, in varying concentrations. Hydrogen peroxide on decomposition generates oxygen and perhydroxyl-free radicals, which then oxidize the stained macromolecules, breaking them down into smaller fragments with altered light absorption, thereby reducing or eliminating the stain.[1]

Although the efficacy of in-office bleaching has been well documented, a chief concern is that the enamel structure may be weakened by the bleaching agent, especially in concentrations ranging from 15 to 38%.[2] Hydrogen peroxide, having a low molecular mass, rapidly diffuses into enamel prisms and interprismatic spaces and is capable of being retained in the enamel, thus exerting a prolonged effect.[3] Several studies have reported a reduction in the calcium and phosphorus content of enamel, a change in the enamel surface morphology and texture, and a reduction in the microhardness and wear resistance of enamel.[4],[5],[6]

Over the years, several remineralizing agents have been investigated with the intention of reversing the demineralizing effects of bleaching agents on dental enamel.[7],[8],[9] Recently, a fluoride varnish based on xylitol-coated calcium and phosphate (CXP) technology has been described for the treatment of incipient caries.[10] Owing to the bioavailability of calcium, phosphate, and fluoride combined with the benefit of slow-release properties of varnish, it can be speculated that the application of this varnish immediately after dental bleaching may be favorable for remineralization. Casein phosphopeptide amorphous calcium phosphate with fluoride (CPP-ACPF) paste is another option for remineralization. The calcium and phosphate ions released from the casein phosphopeptide complex penetrate into the enamel rods and increase the density of hydroxyapatite crystals.[11]

The purpose of the in vitro study was to determine the effect of bleaching with 35% hydrogen peroxide on enamel surface morphology and mineral composition and explore the role of two remineralizing agents for their potential to remineralize damaged regions of enamel. The null hypothesis of the study is that bleaching with 35% hydrogen peroxide has no effect on enamel surface morphology and mineral composition; there is no protective effect of the tested remineralizing agents on bleached enamel; furthermore, there is no difference between the two remineralizing agents in relation to enamel surface morphology and mineral composition.

   Materials and Methods Top

Specimen preparation

The protocol of the present in vitro study was approved by the Institutional Ethics Committee (GDCH/IIEC/2019-20). Human maxillary incisors extracted for periodontal reasons were used. All teeth were carefully examined under magnification (10×), and those presenting cracks, caries, or structural enamel defects were excluded. A total of 32 teeth were included in the study. The teeth were stored in 0.1% thymol for 5 days. The crowns were separated from the roots at the cemento-enamel junction, and each crown was sectioned mesiodistally with a double-faced diamond disk (S.S. White Inc., Lakewood NJ, USA) to separate labial and lingual fragments. Only labial sections were used in the study. The specimens were partially embedded in self-cure acrylic resin and stored in artificial saliva (B. N. Laboratories, Mangalore, India) (pH 5.5–6.0, 0.26 g/l Na2HPO4, 6.7 g/l NaCl, 0.20 g/l NaH2PO4, 1.20 g/l KCl, 1.50 g/l NaHCO3, and 0.1 g/l bovine albumin) during the entire experimentation period.

Bleaching procedure and remineralization treatment

The bleaching procedure was performed with one of the most popular professionally used concentration of bleaching agents – 35% hydrogen peroxide (Pola Office, SDI, Victoria, Australia). For the remineralization treatment, two different remineralizing agents were used: Tooth Mousse plus (GC India Dental Pvt. Ltd) and Embrace varnish (Pulpdent Corporation, Watertown, USA). Specimens were randomly assigned to four groups of eight specimens each:

Group 1: Intact enamel (control, no treatment was done).

Group 2: Bleaching with Pola office with no remineralization treatment.

Group 3: Bleaching with Pola office followed by remineralization with Tooth Mousse plus.

Group 4: Bleaching with Pola office followed by remineralization with Embrace varnish.

The control specimens (Group 1) did not receive any treatment. The bleaching treatment with 35% hydrogen peroxide for Groups 2, 3, and 4 was performed according to the manufacturer's instructions. The bleaching agent supplied as a powder and liquid was mixed and applied onto the labial surfaces of the tooth specimens with applicator tips in an approximately 2 mm thick layer. The bleaching agent was wiped off from the surface with lint-free cotton pellets after 8 min from its application followed by rinsing. Four consecutive intervals of the bleaching procedure were carried out at 0, 8, 24, and 36 h for a total of 8 min each time.

In Groups 3 and 4, the remineralizing agents were applied immediately after each bleaching session with hydrogen peroxide at 0, 8, 24, and 36 h. In Group 3, the CPP-ACPF paste was applied for a period of 3 min each time according to the manufacturer's instructions and wiped off, followed by reinsertion in artificial saliva. In Group 4, the fluoride varnish was applied, allowed to air dry each time, followed by reinsertion in artificial saliva.

Scanning electron microscope and energy-dispersive X-ray analysis (SEM-EDX)

The specimens were gently air-dried, dehydrated with alcohol, and dried at the critical point – a method used to minimize specimen distortion due to drying tensions. The samples were mounted on a stub of metal with adhesive and then analyzed under scanning electron microscopy (JEOL JSM 6360 LV SEM with EDS system). Serial SEM microphotographs of the surfaces of each specimen at 5000× to 10,000× magnification were obtained. Simultaneously, the quantitative EDX point analysis was performed on the enamel surface to determine elemental levels (%) of calcium and phosphorus.

Statistical analysis

The statistical analysis was performed by one-way analysis of variance. When statistically significant differences were present (level of significance P < 0.05), the posthoc Tukey's honest significant difference test was applied.

   Results Top

EDX elemental analysis

The mean calcium and phosphorus levels of sound enamel, bleached enamel, and remineralized enamel were calculated. The results obtained from the EDX elemental analysis [Table 1] and [Table 2] confirmed a statistically significant decrease in calcium and phosphorus values occurred in Group 2 with mean calcium and phosphorus values obtained were 19.5 ± 3.5 and 13.29 ± 2.4, respectively, compared to sound enamel in the control group with mean calcium and phosphorus values 30.72 ± 2.8 and 17 ± 1.5, respectively. Following remineralization, the mean calcium and phosphorus values obtained in Group 3 were 25.37 ± 3.4 and 16.42 ± 1.9, respectively, and the mean calcium and phosphorus values obtained in Group 4 were 31.32 ± 3.8 and 16.7 ± 1.3, respectively. Group 4 presented a significantly higher mean calcium ion level when compared to Group 3 (P < 0.05).
Table 1: EDX elemental analysis of enamel surfaces

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Table 2: Posthoc multiple comparisons of calcium and phosphorus levels among different groups

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SEM analysis

SEM images revealed that specimens in Group 1 (control group) presented enamel with a smooth and regular surface morphology [Figure 1]. In Group 2, SEM analysis showed that the bleaching protocol caused a significant increase in surface irregularities [Figure 2]. The remineralizing paste application in Group 3 promoted the formation of a homogeneous layer on the enamel surface; however, the protective effect was minor [Figure 3]a. The calcium phosphate fluoride used in Group 4 was able to regenerate a thick mineral-rich surface layer, obliterating the interprismatic spaces and covering depression areas [Figure 3]b.
Figure 1: SEM image (7000×) of intact enamel (Group 1). A smooth surface morphology with intact prisms and interprismatic structures can be observed

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Figure 2: SEM image (8000×) of enamel exposed to hydrogen peroxide (Group 2). The bleaching agent caused porosities, depressions, and erosions to various degrees

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Figure 3: SEM image (5000×) of enamel exposed to bleaching agent and treated with remineralizing agents. (a) Although the protective paste application was able to regenerate a homogeneous layer on the enamel surface in Group 3, some mineral deficient sites were also evident. (b) Heavy deposition of a mineral-rich layer on the surface was observed in Group 4

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   Discussion Top

The application of a remineralizing agent after a bleaching session is not routinely practiced. With more efforts directed toward increasing tooth whitening efficacy, there is little being done with regard to strengthening the teeth that are being whitened. Since bleaching promotes demineralization and reduction of enamel microhardness, thereby increasing its abrasion susceptibility, incorporating post-bleaching treatments that can remineralize the enamel surface is highly advisable.

The factors associated with ultrastructural changes in enamel are as follows: (a) the pH of the product – those with a lower pH generally cause more changes than those of similar concentration but with a neutral or alkaline pH and (b) the concentration of the product – those of a higher concentration cause more structural changes than those of lower concentration.[12],[13] The morphological changes observed under SEM in enamel post-bleaching in the present study are in accordance with the findings of other studies that have reported porosities, depressions, and erosions on the bleached enamel surface.[14],[15],[16],[17]

The combination of SEM with EDX for studying the mineral composition of enamel and dentin is a precise and nondestructive technique. The technique involves sample bombardment with a high-voltage electron beam that generates different wavelengths for each type of mineral. The change in wavelength of the radiation that is emitted by the sample indicates changes in its mineral concentration.[18] The reduction in calcium and phosphorus observed in Group 2 is attributed to the dissolution of these elements by hydrogen peroxide. This finding is clinically relevant because the loss of minerals reduces the enamel surface hardness as well as increases the enamel permeability to acids produced by cariogenic bacteria, leading to increased susceptibility to carious lesions.[3]

It has been established that fluoride uptake is higher in demineralized enamel compared to sound tissue.[19] Furthermore, bleaching might render enamel porous, which facilitates better diffusion and penetration of the fluoride.[20] Nevertheless, the most effective fluoridation regimen for preventing enamel demineralization due to bleaching has not been indicated in previous research. Some bleaching gels incorporate Ca2+ and F ions in their formulations, as they were thought to be a possible alternative to overcome the adverse effects of bleaching gels on surface enamel since the ions could diffuse into the enamel structure along with the bleaching agent. However, they have not shown any added benefit of remineralization.[21] The use of fluoride compounds, such as gels, solutions, or dentifrices, has also been used after bleaching to prevent tooth sensitivity. However, the disadvantage of the use of remineralizing pastes and gels is low retention and may thus not completely remineralize the enamel to the extent of damage, as was evident in Group 3 using CPP-ACPF paste.

The varnish containing 5% sodium fluoride with CXP technology combines the benefit of sustained time-release properties of a varnish with the deposition of calcium and phosphate ions to ensure successful remineralization. Increased time of contact favors the deposition of more permanently bound minerals. The percentage of calcium and phosphorus in Group 4 approached those of the control group, providing a higher reservoir of bioavailable calcium and phosphate ions. The limitation of the study was that the effect of masticatory forces and chewing on the retention of the remineralizing agents was not evaluated. Additional parameters such as the effect of fluoride varnish application on the color stability of bleached enamel must be evaluated in future studies.

   Conclusions Top

Within the limitations of this study, the following conclusions can be drawn: Hydrogen peroxide at a concentration of 35% produced morphological changes in enamel and reduced the amount of calcium and phosphorus in enamel. The post-bleaching application of the tested remineralizing agents is effective in repairing enamel surface morphology and replenishing the mineral content. The protective effect was different between the different agents with a more superior result obtained with the varnish group.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Correspondence Address:
Dr. Malasha Godinho
Sunshine Bldg, Bernard Guedes Road, Panaji - 403 001, Goa
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdr.ijdr_896_21

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]


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