Enamel remineralization assessment after treatment with three different remineralizing agents using surface microhardness: An in vitro study.

AIM: The aim of this study is to evaluate the enamel remineralization after treatment with three different remineralizing agents using surface microhardness assessment.
MATERIALS AND METHODS: This in vitro study involves 50 enamel samples divided into five groups of 10 samples each. The positive control group consisted of intact enamel and a negative control group consisted of demineralized enamel samples. All groups excluding the positive control group were subjected to demineralization following which three of these groups were remineralized using remineralizing agents (casein phosphopeptide amorphous calcium phosphate [CPP-ACP] [GC tooth mousse], casein phosphopeptide amorphous calcium phosphate with fluoride [CPP-ACPF] [GC tooth mousse plus], sodium fluoride [phos-flur]). The groups treated with remineralizing agents were subjected to pH cycling over a period of 28 days. This was followed with assessment of surface microhardness (Micro Vickers Hardness tester, Matsuzawa Co., Ltd, Toshima, Japan). STATISTICAL ANALYSIS: One-way analysis of variance test and posthoc Tukey test were conducted for multiple group comparison.
RESULTS: There was an improved enamel remineralization in the group, remineralized using CPP-ACPF in comparison with the other groups.
CONCLUSION: Casein phosphopeptide with fluoride is a promising material for remineralization of enamel subsurface lesions.

INTRODUCTION

Enamel subsurface lesions need to be diagnosed as early as possible for the best possible remineralization of enamel and restoration as well as a function of the tooth. There is evidence based on the remineralization potential of agents such as fluoride, casein phosphopeptide amorphous calcium phosphate (CPP-ACP), casein phosphopeptide amorphous calcium phosphate with fluoride (CPP-ACPF). It has been observed that CPP-ACP remineralized initial enamel lesions and has shown increased remineralization potential when used along with fluoridated toothpaste.[1] APF gel forms more fluoride in enamel than neutral gel and it is more efficient in reducing enamel demineralization of enamel blocks submitted to cariogenic challenge than the neutral one. CPP-ACPF is proven to produce significantly greater mean percentage of remineralization in comparison with CPP-ACP.[2] Mineral deposition throughout the lesion has been proven in relation to use of both CPP-ACP and CPP-ACPF.[2] It has been observed that gums comprising of CPP-ACP nano complexes have a marked remineralization potential.[3]

The CPP-ACP has also been shown to remineralize enamel subsurface lesions in situ when delivered through oral care products. The probable anticariogenic mechanism of CPP-ACP is its ability to localize ACP at the tooth surface, which brings about buffering of calcium and phosphate free ion activities, thereby helping to maintain a state of super saturation with respect to tooth enamel negating demineralization and enhancing remineralization. The CPP-ACP nanocomplexes have shown to localize at the tooth surface. CPP-ACPF due to its added fluoride content has shown improved ability to remineralize initial caries.[456] In incipient enamel caries low concentrations of fluoride are able to penetrate deeper into the body of the lesion and bring about remineralization, hence sodium fluoride (NaF) (200 ppm/0.044%) has been opted for use in this study.[7]

The study aimed at quantitatively evaluating the enamel remineralizing potential of CPP-ACP, CPP-ACPF and NaF using surface microhardness analysis (Vickers hardness test).

MATERIALS AND METHODS

Infection control protocol

The teeth were cleansed of visible blood and gross debris and were maintained in a hydrated state during storage. Extracted teeth were placed in sodium hypochlorite solution diluted with saline in a ratio of 1:10 in the container with a secure lid to prevent leaking and labeled with the biohazard symbol.

Elimination of microbial growth was achieved by using an autoclave cycle for 40 min. Teeth that do not contain amalgam restorations were preferred because they can be safely autoclaved. However, Extracted teeth containing amalgam restorations, were immersed in 10% formalin solution for 2 weeks.[8]

Sample preparation

Fifty freshly extracted human molar teeth were used in this study. Enamel samples (2 mm thickness) were prepared from the buccal and lingual surfaces of the teeth selected, using a double faced diamond disc mounted on a contra-angle hand piece. Following sample preparation windows were created (dimension of 5 mm × 5 mm) using adhesive tape and the sample was made completely resistant to acid attack by coating nail varnish. (Colorama nail varnish, Maybelline).

After drying, the adhesive tape was removed from the enamel using a sharp tipped instrument exhibiting a rectangular area on the enamel surface.

Groups

A total of 50 enamel slabs were randomly divided into five groups of 10 samples in each based on the type of remineralizing agent to be used [Table 1].

Table 1

The division of groups

Lesion preparation

Each of the enamel samples were then immersed in 40 ml of demineralizing solution (acetate 0.1 Mol/L, calcium 0.1 Mol/L, phosphate 0.1 Mol/L, fluoride 0.1 mg/L, pH 5.0)[9] for a period of 4 days at a constant temperature of 37°C, in an incubator to induce artificial caries formation, simulating an active area of demineralization.

Slurry preparation of CPP-ACP, CPP-ACPF and NaF

Slurry of CPP-ACP and CPP-ACP along with fluoride were prepared by agitating the above mentioned preparations in deionized water in the ratio of 1:3.[10]

pH cycling model

pH cycling model was adopted to simulate the dynamic process of demineralization and remineralization that occurs in the oral cavity. Each of the enamel samples were treated with the respective remineralizing agents for a period of 2 min, following which the samples were individually immersed in 20 ml of demineralizing solution (calcium 2.0 mMol/L, phosphate 2.0 mMol/L, acetic acid 75.0 mMol/L, pH 4.4)[10] for a period of 3 h. This was followed-up with treatment of the samples again with slurries of the respective remineralizing agents for 2 min. All the enamel samples were individually immersed in 30 ml of remineralizing solution (1.5 mM calcium, 0.9 mM phosphate, 0.15 M KCl in 0.1 m Tris buffer, pH 7) for a period of 17 h.[10] The remineralizing solution was replaced every 48 h and the demineralizing agent replaced every 5 days. The pH cycling was carried out for a period of 28 days. After the completion of the process of pH cycling, all the groups of enamel samples were assessed for surface microhardness using Vickers hardness test (Micro Vickers Hardness tester, Matsuzawa Co., Ltd, Toshima, Japan).

Surface microhardness

The surface microhardness of the specimens was determined using digital microhardness tester (MATSUZAWA Co., Ltd. Model - MMT X7, Japan) with a Vickers elongated diamond pyramid indenter and a ×40 objective lens. A load of 100 g was applied to the surface for 10 s.[11] Five indentations were placed on the surface and the average value was considered. Precision microscopes of magnification of ×400 were used to measure the indentations. The diagonal length of the indentation was measured by built in scaled microscope and Vickers values were converted to microhardness values.

Statistical analysis

The results were analyzed by one-way analysis of variance (ANOVA). Multiple comparisons between groups were performed by posthoc Tukey test [Tables 2 and 3].

Table 2

One-way ANOVA

Table 3

Posthoc Tukey test

DISCUSSION

In spite of the enamel remineralizing potential of saliva, by itself it fails to initiate the process of increasing the levels of calcium and phosphate.[12]

Calcium and phosphate ions must first penetrate the surface layer of enamel, to bring about deposition of minerals through the body of the lesion, which confirms the reason for the CPP supported metastable calcium phosphate solutions of being such efficient remineralizing solutions.[13] Various compositions of demineralizing solutions are available in the vast literature available. Majority of them are composed of calcium and phosphate with acetic acid or lactate. The main variation lies in concentrations of each component, which influences the final pH and the sample exposure time. The pH employed varies from 3.5 to 5 and the time differs from 2 h to 21 days.[14] In this study demineralization similar to enamel subsurface lesion was sought. An intermediate pH 5.0 was therefore employed for 4 days. The composition similar to the one employed by Featherstone in 1992.[9]

Various studies have used different methods to assess the process of enamel remineralization. The commonly used microhardness tests for evaluating enamel remineralization are Vickers microhardness test and Knoop microhardness test.[11] As this study put the focus on evaluation of surface microhardness of enamel, Vickers surface microhardness test was used.

The values of surface microhardness indicate that remineralization of enamel is more in samples of group III (CPP-ACPF) followed by group IV (NaF) and group II (CPP-ACP). This may be because of the presence of Fluoride in CPP-ACP makes it more capable to remineralize the enamel. The results are in accordance with other studies.[15] However, group IV (F) showed a marginal increase in remineralization compared with group II (CPP-ACP). One-way ANOVA shows that there is a significant difference between all the five groups. However, multiple group posthoc Tukey test showed a significant difference only between groups V with the other groups. No other group showed significance when they were inter-compared.

Adopting the usage of low fluoride concentration as present in CPP-ACPF (0.2%), there is a complex localization of free calcium phosphate and fluoride ion activities, which aid in maintaining a state of super saturation by suppressing demineralization.[16] Even though Fluoride is present in tooth pastes manufactured in India and china, their efficacy is not at par with multinational dentrifices.[17] Research has also proven that APF forms more fluoride and is more efficient in reducing enamel demineralization when compared with neutral gel.[18] Sugar free chewing gums containing CPP-ACP and citric acid result in higher remineralisation when compared with chewing gums without CPP-ACP and citric acid and gums with citric acid alone.[19] Research suggest that fluoride dentifrices in combination with daily applications of Acidulated Phosphate Fluoride (APF) topical thrice and four times a day were the only treatments able to increase the surface microhardness of enamel.[20] The use of fluoride and the CPP-ACP in recent years have been the best possible method in prevention of enamel caries and also in halting the progress of the existent enamel lesions.[2122] CPP-ACP containing remineralising paste has shown remineralization effect within the first three month evaluation period.[23] Conversely, research also suggest that ACP-CPP cream isn’t as effective as fluoride in remineralizing early enamel caries at surface level. Combination of fluoride and ACP-CPP does not provide any additive remineralization potential compared to fluoride alone.[2425] However, It's important to note that compliance of patient in oral hygiene maintenance and in home fluoride use is of utmost importance in prevention of enamel caries.[26] The results of this study showed that CPP-ACPF remineralized enamel subsurface lesion better in comparison with NaF and CPP-ACP. NaF shown to be better than CPP-ACP in enamel remineralization although the comparison between these two agents was insignificant statistically. With this basis we can confirm that CPP-ACPF is an excellent material for remineralization of white spot or initial enamel caries.

CONCLUSION

Under the conditions of this study, these conclusions can be derived.

CPP-ACP effectively remineralizes initial enamel caries, but to a lesser extent in comparison to CPP-ACPF and NaF.

The addition of Fluoride to CPP-ACP shows improved remineralization of initial enamel caries when compared with CPP-ACP and NaF.