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Year : 2018  |  Volume : 5  |  Issue : 4  |  Page : 45-49

Comparative study of remineralization potential of three different remineralizing agents on demineralized enamel using light fluorescence and confocal fluorescence microscope: An in vitro study

1 Postgraduate Student, Department of Conservative Dentistry and Endodontics, M.R. Ambedkar Dental College and Hospital, Bengaluru, Karnataka, India
2 Reader, Department of Conservative Dentistry and Endodontics, M.R. Ambedkar Dental College and Hospital, Bengaluru, Karnataka, India
3 Professor and Head of the Deparment, Department of Conservative Dentistry and Endodontics, M.R. Ambedkar Dental College and Hospital, Bengaluru, Karnataka, India

Date of Web Publication29-May-2019

Correspondence Address:
Dr. Trishagni Chaudhury
Department of Conservative Dentistry and Endodontics, M.R. Ambedkar Dental College and Hospital, 1/32 Cline Road, Cooke Town, Bengaluru - 560 005, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/INPC.INPC_6_19

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Aim and Objective: The main objective of this in vitro study was to evaluate the comparative analysis of remineralization potential of three different materials – casein phosphopeptide-amorphous calcium phosphate with fluoride (CPP-ACPF), calcium sucrose phosphate (CaSP), and bioactive glass on demineralized enamel using light fluorescence microscopy and confocal laser scanning fluorescence microscopy.
Materials and Methods: A total of 40 single-rooted maxillary and mandibular premolars were selected; 4 mm × 4 mm window was prepared on the buccal surfaces of the teeth, which was then subjected to demineralization for 96 h at 37°C. Teeth were randomly selected and divided into four study groups of 10 teeth each: Group 1 (artificial saliva), Group 2 (CPP-ACPF), Group 3 (bioactive glass), and Group 4 (CaSP). Each group was treated with respective remineralizing agents and sectioned with Struers Minitom diamond saw. Each section obtained was visualized under light fluorescence microscope for detection of remineralized and demineralized zones and also was visualized under confocal laser scanning fluorescent microscope for the quantification of demineralized and remineralized zones.
Statistical Analysis: Statistical analysis was done using paired t-test, followed by one-way ANOVA, where P ≤ 0.05.
Results: All the groups showed better statistically significant remineralization potential when compared to the control group, but among them Group 4, that is, Toothmin group showed the highest mean remineralized value, followed by Groups 3, 2, and 1, though the values were not statistically significant (P ≤ 0.05). Light fluorescence microscopy was an efficient diagnostic aid in detecting remineralization and demineralization.
Conclusion: CaSP (Toothmin) has got the best remineralization potential when compared to other groups.

Keywords: Bioactive glass, calcium sucrose phosphate, casein phosphopeptide-amorphous calcium phosphate with fluoride, confocal laser scanning fluorescence microscopy, fluorescence microscopy, light, remineralization

How to cite this article:
Chaudhury T, Ananthakrishna S, Kumari R V, Kour S, Syam A. Comparative study of remineralization potential of three different remineralizing agents on demineralized enamel using light fluorescence and confocal fluorescence microscope: An in vitro study. Int J Prev Clin Dent Res 2018;5:45-9

How to cite this URL:
Chaudhury T, Ananthakrishna S, Kumari R V, Kour S, Syam A. Comparative study of remineralization potential of three different remineralizing agents on demineralized enamel using light fluorescence and confocal fluorescence microscope: An in vitro study. Int J Prev Clin Dent Res [serial online] 2018 [cited 2023 Mar 28];5:45-9. Available from: https://www.ijpcdr.org/text.asp?2018/5/4/45/259262

  Introduction Top

Dental caries is an irreversible microbial disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substance of the tooth, which often leads to cavitation. Organic acids produced by the cariogenic bacteria reduce the pH of the saliva below the “critical pH,” leading to abnormal loss of minerals from the enamel surface or subsurface known as demineralization. Thus, the facilitation of regaining new mineral through the natural oral process remains the ultimate goal. Tooth mineral is mainly composed of calcium and phosphorous. Methods for providing mineral constituents or a new constituent to better facilitate remineralization of teeth have been the backbone for this type of research.[1] Along with fluoride, other remineralizing agents were introduced based on mineral ionic technology and protein technology, those are beta-tricalcium phosphate, pronamel, novamin, enamelon, dicalcium phosphate dehydrate, amorphous calcium phosphate (ACP), casein phosphopeptide (CPP) ACP nanocomplex and CPP-ACP with fluoride (CPP-ACPF), xylitol bioactive glass particles, and self-assembling peptides (P114).[2] Thus, the objective of this study is to comparatively evaluate the remineralization potential of CPP-ACPF (GC Tooth Mousse Plus™, RECALDENT TM), bioactive glass (SHY-NM ®), and calcium sucrose phosphate (CaSP) (Toothmin TM paste, Abbott Healthcare, Mumbai, India) using light fluorescence microscopy and confocal laser scanning fluorescence microscopy.

  Materials and Methods Top

Forty maxillary and mandibular noncarious single-rooted premolars were extracted for periodontic and orthodontic reasons from patients between 18 and 25 years of age. All teeth were sterilized and stored in 1% thymol solution for further preparation. The buccal surfaces of all teeth were polished,[3] and all teeth were sectioned 1 mm below cementoenamel junction. They were then embedded in acrylic blocks such that only crown portions were exposed. An area of 4 mm × 4 mm (window) was marked on the buccal surfaces of teeth samples, coated with nail varnish except for the window, which was analyzed for the change in fluorescence values following demineralization and remineralization. The samples were randomly divided into four groups based on the application of the remineralizing agents used as follows:

  • Group 1 (n = 10) – Control group (no remineralizing agent used)
  • Group 2 (n = 10) – Remineralized with CPP-ACP (GC Tooth Mousse Plus™, RECALDENT™)
  • Group 3 (n = 10) – Remineralized with bioactive glass (SHY-NM ®, Group Pharmaceuticals Ltd., Bengaluru, India)
  • Group 4 (n = 10) – Remineralized with CaSP (Toothmin™ paste, Abbott Healthcare, Mumbai, India).

Demineralization procedure

The samples of each individual group were stored in 200 ml of freshly prepared demineralization solution comprising 2.2 mM calcium chloride, 2.2 mM potassium hypophosphate, and 0.05M acetic acid (pH 4.4) with 1 M potassium phosphate [4] for a period of 96 h. The bottles were placed in an incubator at 37°C. The samples were removed, rinsed, and air-dried to detect white spot lesion.[4]

Remineralization procedure

According to the method described by Sato et al., the artificial saliva was prepared.[5] The pH was set at a level of 7.2. Four artificial mouth models were made, one for each group. Each sample was mounted in the base of the individual compartment of a polyvinyl box, such that their buccal surface faced upward; a circular opening was made in the walls of the compartment. Exit hole was made for the exit pipe, which finally drained into a collector. For each compartment, a controlled dropper was fixed and adjusted on the box lid such that it delivers 5 drops of solution/min on the window area. The controlled droppers of each compartment were interconnected using polyvinyl tubes with the help of two-way plastic connectors. All four artificial mouth models were prepared with individual inlet and outlet. The whole setup was placed in the incubator, temperature of which was maintained at 37°C, and there was a continuous flow of saliva with intermittent flow of buffer solution simulating oral conditions.[6] Continuous exposure of the samples to artificial saliva with intermittent exposure to buffer solution 3 times daily for a period of 15 min each for 30 days was maintained, resembling the pH changes occurring in the mouth.[7]

The teeth samples were remineralized by the application of GC Tooth Mousse Plus, SHY-NM and Toothmin using powered toothbrush (oral B) twice daily for 3 min onto the tooth surface window.

Sample preparation

The samples were cross-sectioned horizontally by Struers Minitom low-speed diamond saw in a buccolingual direction (~200 μm) thickness.[8] The sectioned samples were stained with 0.1 mM Rhodamine B dye solution for 24 h and were viewed under light fluorescent microscope and confocal fluorescence microscope, using a ×10 objective for analyzing demineralization and remineralization.

  Results Top

Statistical analysis was done by paired t-test, followed by one-way ANOVA where P ≤ 0.05. The qualitative assessment of the samples was done using light fluorescence microscope. The images were analyzed by Qualitative light fluorescence (QLF) 1.97e, Inspektor™ Research Systems Software (Amsterdam, the Netherlands). This software shows the region of the teeth that emits fluorescence ranging from 0 to 3. The average loss of fluorescence highlighted through the degree of the color. Data obtained through the qualitative assessment of the sample showed presence of demineralised and remineralised areas in every sample. Results revealed that Group 3 (SHY-NM group) showed the higher amount of mean demineralized area, whereas Group 2 (GC Tooth Mousse Plus), Group 4 (Toothmin group), and Group 1 (control group) showed slightly lower amount of mean demineralized area, respectively. The difference in the values of mean demineralized area among the groups was not statistically significant [Table 1] and [Graph 1].
Table 1: Distribution of mean demineralized area versus group

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Difference of mean demineralized area versus group was not statistically significant (P = 0.2931). The mean remineralized area of the samples was 1,659,645.1039 ± 768,501.5703 um 2. Difference of the mean remineralized area versus group was statistically significant (P = 0.0012) [Table 2].
Table 2: Distribution of mean remineralized area versus group

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All the groups showed better statistically significant remineralization potential when compared to control.

However, among them, Group 4, that is, Toothmin group showed the highest mean remineralized value, followed by Groups 3, 2, and 1, although the values were not statistically significant (P ≤ 0.05) [Graph 2].

  Discussion Top

Detection of the early stages of caries lesions and the use of noninvasive treatment for these lesions by remineralization have the potential to be a major advance in the clinical management of the disease.

The purpose of our in vitro study was to simulate the clinical intraoral situation and investigate the correlation between the demineralization depth and the remineralization depth.

Several studies showed that etching of the surface enamel or producing scratches ensure the increased effect of remineralizing agents on the tooth surface.[9] Thus, enamel surfaces of teeth samples were polished. In the previous in vitro studies carried out by Kumar et al. and Lata et al., the pH cycling was carried out for a period of 10 days and 5 days, respectively, to evaluate the effect of CPP-ACP on remineralization of artificial caries-like lesions.[10] However, in this study, the pH cycling was carried out for a period of 30 days with daily twice application of the toothpaste, as it was thought that the effect of remineralization on demineralized enamel is more with extended period of time. Kaveri Baruah et al. reviewed the ideal brushing time and concluded that it should last for 2–3 min. Hence, the remineralizing agents were applied to the teeth surface over the period of 3 min.[11]

An optical method for an objective assessment of early incipient changes in the enamel mineral content is quantitative light-induced fluorescence (QLF), L. Karlsson suggested that QLF (qualitative light fluorescence) is a highly sensitive method, which can be used as a preventive measure to detect the mineral loss.[12] Hence, in this study, QLF was used to detect the loss of mineral in the samples and to identify whether demineralization and remineralization have taken place or not. The amount of demineralized and remineralized area was accessed under confocal fluorescence microscope (Nikon Ti-E A1rSi System, NIKON Corporation; Tokyo, Japan). According to Sundström et al., laser-induced fluorescence can be used as a quantitative method to access remineralizing potential of various agents.[13]

In the present study, CaSP (Group 4)-based dentifrice showed the better remineralization potential than CPP-ACPF (Group 2) and bioactive glass (Group 3), though the difference in the amount of remineralization was not statistically significant; however, this could be due to increased levels of calcium in saliva, which gets deposited onto tooth surface as CaSP increases the salivary calcium level.

Menon et al. concluded that CaSP increases the salivary calcium level significantly causing an increase in the amount of calcium deposition in tooth structures which not only increases the deposition of calcium in the tooth structure but also reduces the deposition of plaque onto the tooth surface in the oral cavity.[14] Gangrade et al. evaluated remineralization efficacy of stannous fluoride, CaSP, and CPP ACPF using microhardness test. The study revealed that there was no statistically significant difference in remineralizing efficacy among the remineralizing agents, although the mean microhardness value of CaSP group was greater than CPP-ACPF group, which was similar to the present study.[15] Kaur et al. (2015) evaluated the remineralization potential of CPP-ACPF and CaSP using surface microhardness test. They concluded that the remineralizing efficacy of CPP-ACPF and CaSP was not significantly different although CaSP showed slightly higher rate of remineralization potential than CPP-ACPF which is in accordance to the present study.[16]

The use of CPP-ACP as a remineralizing agent is time tested over the years.[17] Recently, it has been modified by incorporating low doses of fluoride known as CPP-ACPF complex as studies have shown that remineralization of small lesions with low-dose fluoride therapy is more efficient.[18] There is a complex localization of free calcium phosphate and fluoride ion activities, which help in maintaining a state of supersaturation by suppressing demineralization.[19] In this study, CPP-ACPF (GC Tooth Mousse plus)-based dentifrice showed better results than the control group but showed low remineralization potential than bioactive glass and CaSP-based dentifrices. This is mainly due to the mechanism of action of CPP-ACP, which involves the incorporation of nanocomplexes into dental plaque and onto the tooth surface, which thereby acts as a calcium and phosphate reservoir.[20]

  Conclusion Top

Following conclusions were drawn based on the findings of the present study results:

  • All the three groups (GC Tooth Mousse Plus group, SHY-NM group and Toothmin group) had higher remineralization potential than the control group as they showed higher mean remineralized area value. The application of CaSP, as a remineralizing agent, has a better potential effect on the management of enamel lesions
  • The Toothmin group had the highest remineralization potential as it showed the highest amount of mean remineralized area among the groups, although the difference in remineralization potential among the groups is not statistically significant
  • SHY-NM is slightly better in remineralizing enamel lesions than GC Tooth Mousse Plus as it showed higher mean remineralized area value though the values were not statistically significant
  • Light fluorescence microscopy was potent in establishing the demineralized and remineralized zones, thus it can be used as a diagnostic aid in detecting, qualitatively analyzing enamel lesions and remineralization potential of remineralizing agents, whereas confocal scanning fluorescence microscopy had the potential to analyze quantitatively.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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