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Year : 2014  |  Volume : 25  |  Issue : 4  |  Page : 464-469
Sodium fluoride and casein phosphopeptide-amorphous calcium phosphate cream plus sodium fluoride efficacy in preventing enamel erosion in a simulated oral environment study model

Department of Restorative Dentistry, Analytical Laboratory of Restorative Biomaterials, LABiom-R, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil

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Date of Submission13-Jun-2013
Date of Decision31-Mar-2014
Date of Acceptance27-Jun-2014
Date of Web Publication10-Oct-2014


Aim: The aim of this study was to evaluate the effectiveness of dentifrices containing high concentrations of sodium fluoride (NaF) and casein phosphopeptide-amorphous calcium phosphate cream plus fluoride (CPP-ACPF) in prevention of the erosion in a simulated oral environment study model.
Subjects and Methods: Fifteen flat human enamel specimens were polished and half of the surfaces were protected with adhesive tape. Initial Knoop microhardness (KHN) and surface roughness (SR) were measured, and specimens were assigned to four groups: Control (placebo toothpaste - G1); CPP-ACPF (G2), NaF 1450 ppm (G3), and NaF 5000 ppm (G4). Enamel surfaces were brushed 3 times daily in association with demineralization-remineralization cycles (5s in cola drink + 5s in artificial saliva/10 cycles/twice daily) and the specimens were maintained in a salivary flow simulator apparatus. After 14 days, KHN and SR were measured again, and the enamel surfaces were analyzed by scanning electronic microscopy (SEM).
Statistical Analysis Used: Data were analyzed using the two-way ANOVA and Student-Newman-Keuls multiple range test (α =0.05).
Results: All the tested groups presented a decrease in KHN after 14 days (P < 0.05). There was no statistical significance among materials tested. Significant increase in SR was observed for all groups. SEM analysis showed morphological alterations with honeycomb structures in enamel surfaces in the four experimental groups.
Conclusions: It was concluded that tooth brushing with dentifrices with high concentration of NaF and CPP-ACPF cream was not able to prevent enamel erosion in simulated oral environment.

Keywords: Erosion, caseins, dental enamel, fluorides, toothpastes

How to cite this article:
Amaral CM, da Silva NG Miranda MM, Correa DS, Silva EM. Sodium fluoride and casein phosphopeptide-amorphous calcium phosphate cream plus sodium fluoride efficacy in preventing enamel erosion in a simulated oral environment study model. Indian J Dent Res 2014;25:464-9

How to cite this URL:
Amaral CM, da Silva NG Miranda MM, Correa DS, Silva EM. Sodium fluoride and casein phosphopeptide-amorphous calcium phosphate cream plus sodium fluoride efficacy in preventing enamel erosion in a simulated oral environment study model. Indian J Dent Res [serial online] 2014 [cited 2023 Mar 31];25:464-9. Available from:
Dental erosion is a complex phenomenon that involves a localized mineral loss from tooth surfaces, without the involvement of microorganisms. [1] Several published studies have shown an increasing prevalence of enamel erosion among young patients around the world, [2],[3] and the relationship between dental erosion and excessive consumption of soft drinks has been reported. [4]

One of the ways proposed for diminishing this deleterious effect of soft drinks on tooth erosion is by modifying their composition in order to reduce their demineralizing power. [5],[6] However, the effectiveness of product modification will depend on many factors, including concentration and solubility of the additives, complex formation, position of the equilibrium point, pH, and temperature. [5] Another strategy for protecting the enamel against acid erosion is the use of solutions and dentifrices with different concentrations of fluorides. [7],[8],[9] Fluorides influence the equilibrium between de- and re-mineralization of teeth in two ways. First, the fluoride ions inside the bacterial biofilm increase the critical pH for Ca and PO 4 dissolution. [10] Second, the fluorides form chemical-stable fluorapatite crystals in the enamel structure, thereby reducing its acid solubility. [10]

Now-a-days, toothpastes or topical creams containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) are available as an approach to counterattack the effects of dental erosion. [1],[11],[12],[13] The nanocomplex CPP-ACP is a bioactive agent that increases the level of Ca 2+ and PO 4 3− ions in the bacterial biofilm. During an erosive attack, the CPP-ACP could release Ca 2+ and PO 4 3− ions, supersaturating the media with these ions and creating an environment favorable to enamel remineralization. [13]

Since there are few reports on effectiveness of CPP-ACP plus sodium fluoride (NaF) [1],[11],[14] and conflicting results have been shown, it seems relevant to investigate its performance with regard to dental erosion when used as a dentifrice, and to compare its effectiveness with the use of dentifrices containing high concentrations of NaF. For this, an experimental model was developed to simulate the oral environment, in which the erosion and treatment phases are short, comparable to those seen in vivo event.

Therefore, the purpose of this in vitro study was to evaluate the effectiveness of dentifrices with high concentrations of NaF and a CPP-amorphous calcium phosphate cream plus fluoride (CPP-ACPF) cream in preventing enamel erosion by cola drink in simulated oral environment. The experiment was designed such that we could assess the effect of the treatments on the microhardness, surface roughness (SR), and surface characteristics of human enamel.

   Subjects and methods Top

0Specimen preparation

Ten freshly extracted human molars (approved by Ethics Committee CEP/HUAP CAAE-0159.0.258.000-08) were cut through the cemento-enamel junction. Labial and lingual crown surfaces were wet-ground in a polishing machine (Panambra Tec. Imp. Exp. Ltda, Sγo Paulo, Brazil) with 600 and 1200 grit SiC papers to obtain flat enamel areas. Enamel fragments of 5 Χ 5 mm were sectioned and embedded in epoxy resin (Resilab, Rio de Janeiro, Brazil). After 24 h, the flatted enamel surfaces were polished in a polishing machine with 2500 and 4000 grit SiC papers. After this, half of each enamel surface was protected with adhesive tape leaving an area of 5 Χ 2.5 mm exposed. The study design is illustrated in [Figure 1].
Figure 1: Schematic design of the demineralization-remineralization-brushing cycle

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Initial Knoop microhardness and surface roughness measurement

Five indentations were made in unprotected enamel surfaces of each specimen at intervals of 500 μm, using a Knoop diamond at a 50 g load and 20s dwell time [15] (Micromet 2003, Buehler, Lake Bluff, USA).

The initial SR was evaluated using profilometer (Surftest SJ51, Mitutoyo, Tokyo, Japan) at a constant speed of 0.1 mm/s [16] and the cut-off was set at 0.25 mm. Three tracings were performed on each specimen at different locations, and the average roughness (Ra) was recorded.

The specimens were divided into four groups (n = 5) according to the materials tested [Table 1].
Table 1: Materials tested in this study

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Demineralization-remineralization-brushing cycle

After immersion in artificial saliva (KCl, NaCl, MgCl 2 , CaCl 2 , K 2 HPO 4 , NaF, nipagin, carboxy methyl cellulose, xylitol, sorbitol, and deionized water - pH 6.8) [17] for 24 specimens was subjected to manual brushing with slurry (3:1, water: dentifrice [w/w]). The tooth brushing was performed for 10 s with a soft nylon bristle toothbrush (Colgate 360° Sensitive, Colgate-Palmolive Ind. Com. Ltda., Sγo Paulo, Brazil) and the total time of immersion in the slurry was 2 min. [12]

Afterwards, the specimens were placed in a salivary flow simulator apparatus with continuous artificial saliva dripping (0.4 mL/min) to simulate the unstimulated human salivary flow. [1] After 4 h, [18] the specimens were submitted to a demineralization-remineralization-brushing cycle (DRB-C). [12] DRB-C consisted of 10 alternated exposures to cola drink (5s) and artificial saliva (5s) dripping, followed by rinsing with deionized water and a toothbrushing (as described above). After 8 h in a salivary flow simulator apparatus, a new RDB-C was carried out. Finally, the specimens were maintained in the salivary flow simulator apparatus overnight (12 h). Therefore, the specimens were rinsed continuously with artificial saliva between the DRB-C cycles, a total of approximately 23 h and 50 min. [12] This cycling regimen was repeated for 14 days. [12] Then, Knoop microhardness (KHN) and SR were carried out again.

Scanning electronic microscopy analysis

For the scanning electronic microscopy (SEM) analysis, two specimens of each group were randomly selected. The specimens were ultrasonicated, cleaned with ethanol, and air dried for 24 h in a desiccator containing freshly dried silica gel. Dried specimens were sputter-coated with Au-Pd (EMITHEC model K550, Ashford, Kent, UK) and observed by SEM (JSM 5310, Jeol Ltd., Akishima Tokyo, Japan). Photomicrographs were taken at Χ1000 and Χ4000 magnification.

Statistical analysis

The microhardness and roughness data were checked to ensure their homogeneity of variance and normal distribution by Levene's test and Shapiro-Wilk's test, respectively. Afterwards, the microhardness and roughness data were submitted to two-way ANOVA and Student-Newman-Keuls test (α = 0.05). The analyses were performed with Statgraphics 5.1 software (Manugistics, Rockville, MD, USA).

   Results Top

The mean KHN values of the enamel surfaces before and post-DRB-C are given in [Table 2]. Student-Newman-Keuls's test showed that all groups presented a significant decrease in KHN after 14 days (P < 0.05). The factor dentifrice was not significant (P = 0.3203).
Table 2: Before and post-DRB-C microhardness of each group

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The mean Ra values of the enamel before and after DRB-C are given in [Table 3]. Two-way ANOVA showed statistical significance for the factor time (P < 0.05). Student-Newman-Keuls test showed a significant increase in the roughness of all groups after DRB-C.
Table 3: Mean roughness of each group before and post-DRB-C

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[Figure 2], [Figure 3], [Figure 4], [Figure 5] show representative SEM micrographs of specimens after 14 days of DRB-C. In (a), the limit between the protected and unprotected area of the specimens is shown (Χ1000, magnification) and in (b), a detailed area of the specimens submitted to DRB-C (Χ4000, magnification). The presence of honeycomb structures suggesting demineralization of enamel prisms in all groups. Figures show that none of the tested products was able to prevent enamel erosion after 14 days of DRB-C.
Figure 2: Representative scanning electronic microscopy micrograph of the specimen treated with placebo toothpaste

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Figure 3: Representative scanning electronic microscopy micrograph of the specimen treated with casein phosphopeptide-amorphous calcium phosphate cream plus fluoride

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Figure 4: Representative scanning electronic microscopy micrograph of the specimen treated with sodium fluoride 1425 ppm

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Figure 5: Representative scanning electronic microscopy micrograph of the specimen treated with sodium fluoride 5000 ppm

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   Discussion Top

The effects of dentifrices on dental erosion have been exhaustively studied. [1],[8],[11],[12],[15],[19] While some studies have shown that products with bioactive agents such as fluoride, CPP-ACP, and calcium sodium phosphosilicate have the potential to prevent enamel demineralization, [1],[11],[13],[14],[20] other studies have shown no favorable effects of these agents. [9],[12],[15]

Differently from the studies that have used methods such as pH cycling, [11],[19] immersion in cola drink, [1],[21] immersion or rinsing in citric acid, [8],[15],[22] topical dentifrice application, [1],[12] and immersion in dentifrice slurry, [15],[19],[23] the experimental protocol of the present study was conducted in attempt to better simulate the daily habits of soft drink consumption and toothbrushing. According to Jensdottir et al. [24] to predict the erosive potential of a soft drink, the method used should simulate what happens in vivo when the drink enters the mouth. For this reason, the method used in the present study (10 cycles of cola drink (5s) and artificial saliva (5s) dripping twice daily) was considered to mimic, as closely as possible, the natural consumption of cola drink during the main daily meals. During the entire experimental protocol, the specimens were maintained under a condition of fresh artificial saliva dripping in order to simulate the washing effect of saliva and to prevent the accumulation of dentifrice residues on the specimen surfaces. Among the soft drinks, Cola drink has the highest erosive potential [24],[25] and this was the rationale for using it in the present study. Some studies [12],[26],[27] have related that the toothbrushing time is approximately 2 min for 2 times/day. Therefore, the time of toothbrushing of 10 s was calculated: If 2 min is the total time of toothbrushing, each group of teeth (molars, premolars, for example) of each dental arch may be brushed for about 10s.

According Rao et al., [20] dentifrices would be the preferable mode of delivering topical CPP since they are used routinely as an oral hygiene measure. These aspects were the rationale for use  GC MI Paste Plus (GC Corporation, IL, EUA) as a dentifrice in the present study. Although its manufacturer indicates that this topical cream should be applied using process was effective, and irrespective of the product applied, demineralization of the enamel occurred. This was confirmed by SEM images that showed enamel prism demineralization. Therefore, toothbrushing with toothpastes with high concentrations of NaF and CPP-ACPF topical cream did not prevent enamel demineralization. These results are in disagreement with several studies [1],[11],[14] that showed significant hardening of enamel by topical application of CPP-ACP [1] and a protective effect of CPP-ACP paste on human enamel demineralization [11],[14] under erosive conditions.

It should be noted that the conditions used in the present study were somewhat different from those applied in some of the above-mentioned studies. In the study of Tantbirojn et al., [1] bovine enamel specimens were immersed in Cola drink (Rio de Janeiro Refrescos, RJ, Brazil) for 8 min before being placed under uninterrupted saliva-substitute solution dripping at 0.4 ml/min and being treated with CPP-ACP topical paste for 3 min at baseline and after 8, 24, and 32 h. This means that the specimens were in contact with the bioactive agent (CPP-ACP) for 12 min without suffering a demineralizing acid attack and stayed in remineralizing solution for 48 h. Moreover, in the study of Poggio et al., [14] the specimens were submitted to four consecutive intervals of 2 min in 6 ml of Coca Cola and then treated with CPP-ACP for 3 min at 0, 8, 24, and 36 h. In the study of Oshiro et al., [11] bovine enamel blocks were also stored in a diluted solution of CPP-ACP for a long period (10 min), followed by 10 min immersion in a demineralizing solution (0.1 M lactic acid).

It would be reasonable to claim that during immersion of the specimens in Coca Cola or demineralizing solution, the Ca 2+ and PO4 3- ions dissolved from the enamel surfaces had buffered these substances, thereby decreasing their erosive potential. [1],[14] Moreover, it is possible that due to the topical applications, the longer time in which the CPP-ACP remained in contact with enamel favored remineralization of the specimens in these studies. Jager et al. [21] showed that different exposure times to acid beverages also result in very different estimates of erosive potential, and that effect of the choice of study methodology may affect the results of the study.

On the other hand, as in this study, other studies have also shown that enamel erosion cannot be prevented or repaired by CPP-ACP or CPP-ACP plus F cream. [29],[30] In the study of Wang et al., [30] the paste slurry (1:1 CPP-ACP plus F cream: Artificial saliva) was also prepared and the treatment was performed for 3 min, only. Although Turssi et al. [29] made topical application of CPP-ACP or CPP-ACP plus F cream, these authors performed cycles of alternating erosive challenge and remineralization in artificial saliva. In the studies of Turssi et al. [29] and Wang et al., [30] the authors also attempted to use clinically relevant parameters, as in this study. Other studies have shown low levels of enamel fluoride deposition, low enamel remineralization, and did not show any effect on the inhibition of lesion progression with the use of CPP-ACP with or without NaF (MI Paste Plus). [12],[19],[23]

It has been hypothesized that there is poor affinity of CPP-ACP to enamel in an erosive challenge. Under acidic condition, casein carries a positive charge as its isoelectric point 4.6, and consequently its affinity to enamel may be reduced. [30]

As regards SR, all groups showed significant increase in roughness in this study. Enamel demineralization, evidenced by decreased microhardness and SEM images, may be responsible for increased enamel SR. In the study of Poggio et al., [14] the roughness of the enamel specimens submitted to Coca Cola and treated with CPP-ACP tooth mousse also increased significantly (from 0.06 to 0.14).

Considering that the enamel specimens in the present study were submitted to Cola drink dripping and then maintained under constant dripping of artificial saliva, it is possible that the Ca 2+ and PO4 3- ions derived from the enamel demineralization may have been washed off by artificial saliva, preventing the buffering effect on the Coca Cola and maintaining its erosive potential throughout the 14 days of the experiment. [1] Second, the constant and uninterrupted dripping of artificial saliva could have prevented the accumulation of CPP-ACP on the enamel specimen surfaces as well as lixiviating the partially demineralized enamel minerals. Consequently, the medium was not supersaturated with Ca 2+ and PO4 3- ions, and did not create a favorable environment for enamel remineralization. [13] The similarity between [Figure 2], [Figure 3], [Figure 4], in which honeycomb structures suggesting demineralization of enamel prisms after 14 days of DRB-C would seem to reinforce the idea that the tested products were ineffective in counterattacking the erosive potential of Coca Cola.

Dentifrices with high concentration of NaF were also unable to prevent enamel erosion. This is in agreement with other studies that observed toothpastes with high concentration of NaF were not able to fully recover the microhardness of human enamel after citric acid erosion [15] and were not capable of controlling the enamel erosion caused by HCl. [31] However, a preventive effect has been observed when toothpastes or solutions with high concentration of NaF were applied before the erosive attack. [15],[22],[32] The mechanism responsible for the beneficial effects of fluoride against erosive lesions is the deposition of calcium fluoride-like material (CaF2 ) on the dental surface. [33] The topical application of fluoridated dentifrice leads to a CaF2 layer formation that supplies available fluoride and minimizes the demineralization in a subsequent erosive challenge. [34] It was also been suggested that high concentration of NaF can cause some precipitation of fluorhydroxyapatite within the previously softened surface enamel [22] and this can result in both an increase of hardness and a reduce susceptibility to subsequent dissolution. [22]

Wegehaupt and Attin [9] showed that tooth wear after erosive attacks could be reduced significantly by daily application of NaF or amine/NaF gels (12,500 ppm), whereas the application of CPP-ACP-containing cream was not effective. Finally, several studies have demonstrated that highly concentrated fluoride agents for topical application are able to protect enamel against erosion and toothbrushing abrasion, [8],[9] and promising results have been found with TiF 4 solutions. [7],[35]

Based on the results, it may be concluded that toothbrushing with dentifrices with high concentrations of NaF and CPP-ACPF cream were unable to prevent the enamel erosion and increase in enamel roughness caused by cola drink in a simulated oral environment study model for 14 days. Since CPP-ACPF can bind the components of oral biofilm, in situ or clinical studies are needed to clarify this.

   References Top

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Correspondence Address:
Cristiane Mariote Amaral
Department of Restorative Dentistry, Analytical Laboratory of Restorative Biomaterials, LABiom-R, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.142536

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3]

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