SRM Journal of Research in Dental Sciences

: 2022  |  Volume : 13  |  Issue : 4  |  Page : 151--156

Assessment of the relative efficacy of fluoridated toothpaste with and without eggshell-derived calcium oxide in the prevention of primary tooth enamel demineralization: An ex vivo study

RV Remi1, Prathibha Rani Shankarappa2, A Anantharaj2, P Praveen2, R Sudhir2,  
1 Senior Lecturer, Department of Pediatric and Preventive Dentistry, Pacific Dental College, Udaipur, Rajasthan, India
2 Department of Pediatric and Preventive Dentistry, D. A. Pandu Memorial RV Dental College, Bengaluru, Karnataka, India

Correspondence Address:
Dr. R V Remi
D/o Shri C. Ravi Raj Kumar, First World Print Pack LLP, Plot No. 5D, 19/6, Mathura Road, Faridabad - 121 006, Haryana


Background: During the cariogenic challenges, the anticaries action of fluoride is limited by the bioavailability of calcium and phosphate in saliva. This mandates the use of additional extrinsic sources of calcium and phosphate to enhance the anticaries potential of fluoride. Aim: This study aimed to assess and compare the efficacy of fluoridated toothpaste with and without eggshell-derived calcium oxide in the prevention of primary tooth enamel demineralization. Materials and Methods: Sixty samples were obtained from 15 extracted primary second molars and allocated to one of four groups: Group A for baseline Vickers hardness testing, Group B was subjected to demineralization only, Group C was treated with fluoridated toothpaste solution, and Group D was treated with fluoridated toothpaste and chicken eggshell powder solution. Surface microhardness and the amount of calcium that was leached into the demineralizing solutions of groups B, C, and D were statistically assessed after 7 days of pH cycling. Results: The mean Vickers hardness values of groups A, B, C, and D were 402.68, 366.28, 392.79, and 395.27, respectively. The mean calcium concentration released into demineralizing solution of groups B, C, and D were 35.52, 29.12, and 27.12, respectively. No statistically significant difference was found between the fluoridated toothpaste with and without eggshell powder. Conclusion: Both fluoridated toothpaste with and without eggshell-derived calcium oxide were equally effective in the prevention of primary tooth enamel demineralization.

How to cite this article:
Remi R V, Shankarappa PR, Anantharaj A, Praveen P, Sudhir R. Assessment of the relative efficacy of fluoridated toothpaste with and without eggshell-derived calcium oxide in the prevention of primary tooth enamel demineralization: An ex vivo study.SRM J Res Dent Sci 2022;13:151-156

How to cite this URL:
Remi R V, Shankarappa PR, Anantharaj A, Praveen P, Sudhir R. Assessment of the relative efficacy of fluoridated toothpaste with and without eggshell-derived calcium oxide in the prevention of primary tooth enamel demineralization: An ex vivo study. SRM J Res Dent Sci [serial online] 2022 [cited 2023 Feb 9 ];13:151-156
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The bioavailability of calcium and phosphate in the saliva is crucial for the anticaries action of fluoride, especially during cariogenic challenges.[1] However, the natural caries protective and remineralizing effects of saliva are not only a slow process but obviously insufficient to protect individuals against caries and remineralize existing lesions.[2] Despite the availability of several remineralizing agents for the treatment of carious lesions such as fluoride, casein phosphopeptide–amorphous calcium phosphate, hydroxyapatite, xylitol, and dental caries prevalence in children continues to increase globally.[3] Therefore, newer caries preventive formulations providing additional calcium and phosphate ions are needed to enhance the preventive potential of saliva.

Chicken eggshell powder (CESP) with rich calcium bioavailability has emerged as a gold mine of opportunities in various fields. Researchers have reported CESP as a safe osteoconductive bone substitute for the regeneration of maxillofacial defects.[4],[5] Calcined CESP with its easy bioavailability and natural source of calcium and phosphate has proved to be a potential remineralizing agent. When the remineralizing potential of CESP solution was compared with Clinpro, both were found to favor remineralization.[6],[7]

However, there is a paucity of studies assessing the efficacy of CESP as anti-demineralizing agent in primary teeth. Hence, the current study aimed at assessing and comparing the effect of child formula fluoridated toothpaste on adding eggshell powder on the demineralization of primary tooth enamel.

 Materials and Methods

Study design

The present ex vivo experimental study was approved by the Research Sustenance and Institutional Review Board Committee on November 07, 2019 (IRB No.: 341/VOL-2/2019). All the participants provided written informed consent for participation in the study. All procedures performed in the study were conducted in accordance with the ethical standards given in the 1964 Declaration of Helsinki, as revised in 2013.

Study setting

Fifteen noncarious primary second molars with intact crown structure were extracted from pediatric patients of the age group 10–12 years, who visited the Department of Pediatric and Preventive Dentistry, D. A. Pandu Memorial R. V. Dental College, Bengaluru.


Sound and noncarious primary teeth were included in this study, whereas those with caries, hypoplasia, discoloration, and white spots were excluded from the study.

Study size

The estimation of sample size was done using the GPower software v. Effect size f, α err prob, and power (1-β err prob) for four groups were 0.56, 0.05, and 0.80, respectively. Noncentrality parameter λ, critical F, numerator df, denominator df, and actual power were 12.5440000, 2.8662656, 3, 36, and 0.8134546, respectively. The sample size has been estimated considering the effect size to be measured (f) at 56%, power of the study at 80%, the margin of the error at 5%, and the total sample size needed for this study was 60. Each group consisted of 15 samples (15 × 4 groups = 60 samples).

Preparation of chicken eggshell powder

The CESP was produced through calcination in accordance with the instructions provided by the World Intellectual Property Organization (WO/2004/105912: Method of generating eggshell powder). To create pure powder devoid of pathogens and raise the alkalinity of the powder, calcination was used. Normal CESP includes 95% calcium carbonate, which on calcination transforms into basic calcium oxide and causes a rise in alkalinity.

The contents of chicken eggs were removed and the eggshells were cleaned in distilled water. The membrane was then taken off the eggshells after they had been in a hot water bath at 100°C for 10 min. These eggshells were calcined in a muffle furnace at the Central Silk Technological Research Institute, Bengaluru, between 300°C and 1000°C at a heating rate of 10°C/min. Calcined eggshells were powdered to small particles and crushed using a sterile mortar and pestle.

Preparation of toothpaste solution

Fluoridated toothpaste solution was prepared by mixing 12 g of child formula fluoridated toothpaste (KIDODENT 500 ppm F in form of sodium monofluorophosphate [SMFP]) with 36 mL of distilled water. The mixture was stirred and agitated for 1 min followed by centrifugation at 3500 rpm for 20 min at room temperature.[8] The sediment of centrifuged slurries was discarded and the supernatant was used as a treatment solution.

A mixture of 1 g eggshell powder and pediatric toothpaste slurry was centrifuged to obtain the solution of fluoridated toothpaste and CESP.

Sample preparation

Before the experiment, the obtained teeth were disinfected with 10% formalin and kept in distilled water. At the cementoenamel junction, teeth were decoronated. With a double-sided diamond disc mounted on a straight handpiece, each decoroned tooth was then longitudinally sectioned, first in a mesiodistal direction and subsequently in a buccolingual direction, yielding four samples from a single tooth as shown in [Figure 1]. Each sample was coated with acid-resistant nail polish, leaving an enamel window of 3 mm × 3 mm. The samples from each tooth were allocated to one of four groups as follows:{Figure 1}

Group A: For baseline measurementGroup B: Demineralization only (negative control)Group C: Treated with fluoridated toothpaste solution followed by demineralizationGroup D: Treated with fluoridated toothpaste and CESP solution followed by demineralization.

The demineralizing solution of pH 4.5 was prepared by the addition of 1.5 mM of CaCl2, 0.9 mM of KH2PO4, 50 mM of acetic acid, and 0.002% of NaN3. 0.9 mM sodium dihydrogen phosphate, 0.15 M potassium chloride, 0.4% carboxymethyl cellulose, 6% sorbitol, 0.2% nipagin, and distilled deionized water were used to create artificial saliva with a pH of 7.4.

The samples in each group were subjected to pH cycling as shown in [Figure 2], which was repeated for 7 days.{Figure 2}

Vickers surface microhardness analysis

After 7 days, the tooth sections in groups A, B, C, and D were analyzed for surface microhardness (SMH) using Vickers SMH tester. Vickers indenter with a load of 100 gF was applied for 10 s on the enamel surface.

Calcium level estimation in the demineralizing solution

The baseline calcium level in the demineralizing solution was evaluated using atomic absorption spectroscopy. After 7 days, the demineralizing solutions of groups B, C, and D were subjected to flame atomic absorption spectroscopy[9] to determine the amount of calcium leached into the demineralizing solution.

Statistical methods

The mean Vickers microhardness values of the four groups and the mean calcium concentration released into the demineralizing solution from the three groups were compared using a one-way analysis of the variance test and Tukey's honest significant difference post hoc analysis. Statistical Package for the Social Sciences for Windows Version 22.0 Released 2013. Armonk, NY, USA: IBM Corp. was used to perform statistical analyses. The level of significance (P) was set at P < 0.05.


The test results demonstrated that enamel sections in Group D (pretreated with fluoridated toothpaste and CESP) had a higher mean Vickers hardness value than Group C (pretreated with fluoridated toothpaste) [Figure 3]. Between groups B and D, there was a highly significant difference (P < 0.001), and between groups B and C, there was a significant difference (P = 0.001). Between groups C and D, there was, however, no statistically significant difference (P = 0.982) [Table 1]. The mean calcium concentration leached from specimens of groups B, C, and D into demineralizing solution did not show statistically significant difference (P = 0.062) [Table 2] and [Table 3].{Figure 3}{Table 1}{Table 2}{Table 3}


The recent trend toward preventive rather than the surgical approach of caries management focuses on the prevention of the occurrence of demineralization to maintain the integrity and function of primary dentition.[10] Fluoride prevents caries at the tooth/plaque interface by encouraging the remineralization of early lesions and lowering the solubility of tooth enamel.[11] In our study, enamel samples pretreated with fluoridated toothpaste showed a significantly higher mean Vickers hardness (392.79 ± 14.48) as compared to demineralized samples (366.28 ± 27.35). The fluorapatite (FAP) and calcium fluoride (CaF2)-like precipitates that form on the enamel and in the plaque are presumed to be the cause for cariostatic potential of the topical fluoride. The fluoride in enamel fluid is highly adsorbed to the surface of carbonated apatite crystals during the cariogenic process, preventing the crystals from dissolving.[12] Crommelin et al. observed almost similar dissolution rate behavior of FAP-coated hydroxyapatite and FAP alone.[13] Therefore, regular low-dose fluoride applications are more efficient than infrequent high-dose applications. Contrarily, during cariogenic challenges, CaF2-like material serves as a pH-driven fluoride and calcium reservoir.[12]

Anticaries efficacy of fluoride is dependent on the bioavailability of calcium and phosphate in saliva, especially at lower pH.[1] To overcome this, a formulation with calcium and/or phosphate releasing and pH buffering properties might be helpful in enhancing the preventive potential of saliva as well as fluoride therapy, thus preventing enamel demineralization.

Few experimental studies have assessed the anti-demineralizing efficacy of fluoride in conjunction with calcium-containing products. CESP with rich bioavailable calcium and increased alkalinity seems to be a feasible option.[6],[7] The protective effect of CESP against erosive enamel loss was evident from the increase in microhardness and reduction in surface roughness.[14],[15] In our study, the use of calcined CESP along with fluoridated toothpaste has resulted in a significantly higher mean Vickers hardness (395.27 ± 14.36) as compared to demineralized ones (366.28 ± 27.35). This can be reasoned by the ability of CESP in maintaining supersaturation of saliva and biofilm fluid with respect to tooth minerals and keeping pH above the critical value of 5.5. Amaechi recommended the use of sound teeth in "demineralization" model of pH cycling to test the ability of investigational products to prevent caries development. Demineralization model of pH cycling was used in the present study.[16]

In our study, the Vickers microhardness test was used to gauge the enamel's surface hardness. The square-shaped indentation left behind by the Vickers indenter on a textured surface is visible and can serve as an indirect indicator of mineral loss or gain.[17],[18] Enamel samples pretreated with fluoridated toothpaste with and without eggshell-derived calcium oxide showed resistance to demineralization, which can be explained by the stable SMFP dentifrice used in our study. The covalent bond between fluoride and phosphate ions in SMFP is more compatible with calcium-containing agents as compared to sodium fluoride and stannous fluoride.[19] Damle et al. found that over 18 months, the mean decayed missing and filled teeth and decayed missing and filled tooth surfaces values in the control (nonfluoride) group increased statistically significantly more than those in the test group (sodium monofluorophosphate + calcium glycerophosphate).[20] In contrast, Parkinson et al. found no statistically significant difference in the percentage of SMH recovery between 927 ppm F with or without 5% calcium sodium phosphosilicate (CSPS), indicating that CSPS had no positive or negative effects on fluoride's ability to affect the mineralization of surface-softened and subsurface caries lesions.[21]

After 7 days of pH cycling, the calcium concentration in the demineralization solution was measured to determine the degree of demineralization. There was no statistically significant difference in the amount of calcium lost between the experimental and demineralized groups in our study. This can be attributed to the fact that at critical pH subsurface dissolution of hydroxyapatite occurs while the fluoride adsorption onto the enamel surface forms fluorhydroxyapatite. With time, fluorhydroxyapatite forms the protective surface layer at the expense of subsurface hydroxyapatite. Lale et al. reported that fluoridated as well as nonfluoridated toothpaste exhibited weaker preventive effects against the occurrence of white spot lesions during orthodontic treatment.[22] In contrast to SMFP alone, Burwell and Greenspan. found that SMFP + calcium sodium phospho solutions aided to prevent microhardness decrease when enamel was exposed to citric acid at pH 2.5.[23] The difference in the methodology used in the above-mentioned studies may be the reason for these conflicting results.

A direct comparison of the present findings cannot be made due to the lack of reports in the literature comparing the products evaluated in our study. Thus, this study could be considered a standalone study evaluating the effect of fluoridated dentifrice with or without CESP pretreatment on microhardness of enamel. The main limitation of our study is that like any other in vitro study, it could hardly imitate the dynamic complex biological system of the oral environment (especially within the dental plaque milieu). Significant changes can be expected from the experimental slurries if carried out for a longer duration in an in vivo model.


Both fluoridated toothpaste with and without eggshell-derived calcium oxide were equally effective in the prevention of primary tooth enamel demineralization. CESP can be considered an option for a routine oral hygiene regimen. Further long-term in vitro studies using qualitative analysis (such as scanning electron microscopy-energy dispersive X-ray) are recommended to confirm these results.


The authors would like to thank the Indian Institute of Science, Bengaluru, for the help provided during the study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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