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Year : 2021  |  Volume : 39  |  Issue : 1  |  Page : 67-73

Remineralization efficacy of nonfluoride versus herbal-based pediatric dentifrice in demineralized primary teeth

Department of Pedodontics and Preventive Dentistry, Sri Siddhartha Dental College and Hospital, Sri Siddhartha Academy of Higher Education, Tumkur, Karnataka, India

Date of Submission17-Feb-2021
Date of Decision01-Mar-2021
Date of Acceptance02-Mar-2021
Date of Web Publication22-Apr-2021

Correspondence Address:
Dr. Balamurugan Suba Ranjana
Department of Pedodontics and Preventive Dentistry, Sri Siddhartha Dental College and Hospital, Sri Siddhartha Academy of Higher Education, Tumkur - 572 017, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jisppd.jisppd_62_21

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Background: Dental caries is one of the most prevalent posteruptive bacterial infections worldwide, characterized by a progressive demineralization process that affects the mineralized dental tissues. Although the decline of dental caries prevalence can be attributed to the widespread use of dentifrices that contain fluoride, yet there is a need for an advanced alternative nonfluoride remineralizing dentifrice. Yet, there is a need for an advanced alternative nonfluoride remineralizing dentifrice. Aim: The aim of this study was to evaluate and compare the remineralizing effect of nonfluoride-based and herbal-based pediatric dentifrice in demineralized primary teeth with an ideal in vitro method of pH cycling and evaluating the values under Polarized Light Microscope (Olympus BX43) using image analysis software (ProgRes, Speed XT core3). Materials and Methods: A total of 30 tooth samples were collected and placed in the demineralizing solution for 96 h to produce a demineralized lesion of approximately 100 μm, and then cut longitudinally into 60 sections that were randomly assigned to two groups with 27 samples each, Group A – nonfluoride-based dentifrice (Mee Mee®), Group B – herbal-based dentifrice (Mamaearth), after which they were subjected to pH cycling for 7 days along with dentifrice slurry preparation. The sections were evaluated under the polarizing light microscopy for remineralizing efficacy. The lesion depth was measured and tabulated to be sent for statistical analysis. Results: The mean demineralization value for nonfluoride and herbal-based dentifrice groups were 7.8730 μm and 28.3174 μm, respectively. Hence, it can be inferred that since lesion depth measured was lesser in nonfluoride than herbal-based dentifrice, remineralization has occurred in the nonfluoride-based dentifrice group. Conclusion: Nonfluoride-based dentifrice showed significant results in remineralizing the demineralized lesion, while herbal-based dentifrice showed poor efficiency in remineralizing the demineralized lesion.

Keywords: Nonfluoride, pediatric dentifrice, Polarizing light microscope, remineralization, tricalcium phosphate

How to cite this article:
Ranjana BS, Chowdhary N, Kiran N K, Chaitan S M, Reddy VR, Prabahar T. Remineralization efficacy of nonfluoride versus herbal-based pediatric dentifrice in demineralized primary teeth. J Indian Soc Pedod Prev Dent 2021;39:67-73

How to cite this URL:
Ranjana BS, Chowdhary N, Kiran N K, Chaitan S M, Reddy VR, Prabahar T. Remineralization efficacy of nonfluoride versus herbal-based pediatric dentifrice in demineralized primary teeth. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2022 Jun 29];39:67-73. Available from: https://www.jisppd.com/text.asp?2021/39/1/67/314372

   Introduction Top

Dental caries is one of the most prevalent posteruptive bacterial infections worldwide, responsible for high morbidity rate among the population. The caries process involves a continuum of demineralization and remineralization cycles.[1] When the critical pH level of 5.5 is breached, calcium and phosphate exit the enamel, which weakens it and eventually causes the chalky white spot lesion that may further lead to cavity.[2] The calcium content of enamel is about 30 moles per liter. The calcium content in the saliva is about 1 mmol/L and about 2 mmol/L in an optimal calcifying solution. To replenish the lost mineral, it would be necessary to supply 10,000 mmol/L of saliva or a calcifying solution to reform one volume of remineralized enamel.[3] Whether demineralization and remineralization are proceeding at any one time is determined by the balance between pathological factors and protective factors.[4]

According to the World Health Organization expert committee, the decline of dental caries prevalence can be attributed to the widespread use of dentifrices that contain fluoride.[5] However, the use of fluoride dentifrices containing 1000–1100 ppm in high fluoridated belt can cause fluoride toxicity. Hence, to avoid the toxic effect of fluoride in case of overdosage and for its restricted usage in children below the age of 6 years, alternative dentifrices were searched for.[6]

Recently, a new nonfluoride-based dentifrice has been introduced containing tri-calcium phosphate (TCP) Ca3(PO4)2. It is a “smart” calcium phosphate that controls the delivery of calcium and phosphate ions to the teeth, works synergistically with fluoride to improve the plaque pH and thereby initiating remineralization.[7] The traditional medicinal method for the prevention and treatment of oral disease and caries is the use of phytochemicals that are isolated from plants. Aloe barbadensis miller (aloe vera), a tropical plant from which mucilaginous tissue found in the center of aloe vera leaf is extracted and is used for various cosmetic and medicinal purposes that has a strong bactericidal property against  Streptococcus mutans Scientific Name Search ptococcus mitis.[8] With added benefits of xylitol, a nonfermentable, nonacid producing sugar alcohol substitute, it enhances the protective barriers of saliva as it increases the concentration of bicarbonate and phosphate in stimulated saliva resulting in increase in plaque pH and salivary buffering capacity.[9]

Since there are less studies on comparison of remineralization efficacy of calcium-phosphate compound and herbal infused dentifrices, the present study was conducted to compare the remineralization effect of nonfluoride-based (Mee Mee®) and herbal-based (Mamaearth) pediatric dentifrices in demineralized primary teeth with an ideal in vitro method of pH cycling and evaluating the values under Polarized Light Microscope (Olympus BX43) using image analysis software (ProgRes, Speed XT core3).

   Materials and Methods Top

The study was started after taking the approval of the ethical committee, Sri Siddhartha Dental College and Hospital, Tumkur, Karnataka, India. Thirty sound extracted or naturally exfoliating primary anterior teeth were collected after getting an informed consent from the participants who reported to the OP of the Department of Pedodontics and Preventive Dentistry, Sri Siddhartha Dental College. Teeth were stored in sterile distilled water until use.

Preparation of specimens

A total of 30 freshly extracted retained or exfoliating primary maxillary anterior teeth were used in this study. After careful selection and excluding teeth with cracks, hypoplasia, caries, and white spot lesions, they were rinsed in normal water and stored in distilled water until use. Teeth samples were dried, and a 3 mm × 3 mm narrow window was prepared on the middle third of the labial surface by sticking adhesive tape and covering the remaining surface with two coats of acid-resistant nail varnish.


The demineralizing solution was prepared using 2.2 mM calcium chloride, 2.2 mM potassium hydrogen orthophosphate, unstirred solution of 0.05 M acetic acid, and 1 M potassium hydroxide (KOH) pH at 4–5. Preparation of demineralizing solution is done by taking 1050 ml of distilled water in a beaker and 2.2 g of calcium chloride was added to it. To this, 2.2 g potassium hydrogen orthophosphate, 3 g of acetic acid, and 56 g of KOH were added. The specimens were subjected to demineralization cycle by immersing in prepared demineralizing solution with a final pH of 4.5 for 96 h at normal room temperature to produce demineralized lesions.[5],[6]

The study samples were then longitudinally sectioned equally using a diamond disc (Dentsply, India) in labio-lingual direction, that obtained 60 sound sections which was then randomly divided into 27 sections each in Group A – nonfluoride-based pediatric dentifrice (MeeMee® Santacruz, Mumbai) and Group B – herbal-based pediatric dentifrice (Mamaearth Sihor, Gujarat) [Figure 1] for the remineralization procedure. The rest three sections from each group were kept to determine the demineralization lesion depth without undergoing the pH cycling.
Figure 1: Pediatric dentifrices. Mamaearth™ - Herbal dentifrice. Mee Mee® - nonfluoride dentifrice

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Remineralization solution was prepared using 0.5 mM calcium chloride, 0.9 mM sodium dihydrogen phosphate, and 0.15 M potassium chloride pH adjusted at 7. Preparation of remineralizing solution was done by taking 152 ml of distilled water and 0.1665 g of calcium chloride, 0.108 g of sodium hydrogen phosphate, and 11.25 g of potassium chloride were added.[5] Dentifrice solution was freshly prepared, by adding 17 g of dentifrice to 51 ml of deionized water (3:1 ratio) and stirred with stirring rod until thoroughly mixed. Fresh solution for both the dentifrice was prepared before each pH cycle and was stored in separate containers throughout the study.[6]

Groups A and B samples were then subjected to demineralization, remineralization, and dentifrice solution of nonfluoride- and herbal-based dentifrices, respectively, for a specific period of time in a pH cycle. The pH cycle used followed Ten Cate and Duijsters pH cycle model, and was as follows:

  • Step 1: Dentifrice supernatant solution (5 ml/section) for 60 s
  • Step 2: Demineralizing solution (10 ml/section) for 3 h
  • Step 3: Remineralizing solution (10 ml/section) for 2 h
  • Step 4: Dentifrice supernatant solution (5 ml/section) for 60 s
  • Step 5: Demineralizing solution (10 ml/section) for 3 h
  • Step 6: Dentifrice supernatant solution (5 ml/section) for 60 s
  • Step 7: Remineralizing solution (10 ml/tooth) overnight
  • Step 8: This pH cycle continued for 7 days.

After following the pH cycle for 7 days, the remineralized sections were then again reduced to size of 100 μm thickness using Arkansas stone and were mounted on the microscope glass slide with D. P. X Mountant and Coverslip [Figure 2] to be analyzed for lesion depth under the Polarized light microscope (Olympus BX43). Specimens were photographed using polarized light microscope (Olympus BX43). These photomicrographs were then measured for lesion depth using image analysis software (ProgRes, Speed XT core3) by an independent observer who was blinded for study. This was done by taking lesion depth at three different points for each lesion and taking the mean of three measurements obtained. The lesion depth was measured, tabulated, and sent for statistical analysis.
Figure 2: Sectioned tooth sample postremineralizing treatment reduced to 100 μm

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   Results Top

The mean demineralized lesion depth in nonremineralized samples measured 101.3417 μm. Nonetheless, the specimens were sectioned from different teeth; the demineralization lesion depth variation did not show paramount effect on the progression of the lesion.

The result from the lesion depth measurements after 7 days of pH cycle showed that the mean posttreatment lesion depth from the two groups nonfluoride-based and herbal-based dentifrice ranged from 7.8730 to 28.3174 μm, respectively, and the standard deviation being 3.81204 μm for nonfluoride-based dentifrice and 15.49191 μm for herbal-based dentifrice [Table 1]. The evaluated values measured as lesion depth indicated that the lesser the value more the remineralization efficacy and higher the value lesser the remineralization efficacy.
Table 1: Mean value and standard deviation for demineralized areas

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Posttreatment lesion depth among the two groups was then analyzed by one-way ANOVA to compare the remineralization efficiency [Table 2]. The t - 6.659 for the mean difference between nonfluoride-based dentifrice and herbal-based dentifrice was significant (P = 0.000).
Table 2: One way ANOVA to compare the remineralization efficiency between two groups

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The mean difference between the pre- and postlesion depth was Group A – nonfluoride-based dentifrice 93.4687 μm, Group B – herbal-based dentifrice 73.0234 μm [Figure 3], [Figure 4], [Figure 5]. Graphical representation [Graph 1] of the two groups for the mean demineralization value revealed that the least measured lesion depth was seen in Group A – nonfluoride-based dentifrice than Group B – herbal-based dentifrice. Hence, it can be inferred that, since lesion depth measured was lesser in nonfluoride-based dentifrice (Mee Mee®) than herbal-based dentifrice (Mamaearth), remineralization has occurred in the nonfluoride-based dentifrice.
Figure 3: Maximum depth of demineralization noted in the control group (arrow shows the area of demineralization)

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Figure 4: Maximum depth of demineralization noted in Group A (nonfluoride-based dentifrice) (arrow shows the area of demineralization)

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Figure 5: Maximum depth of demineralization noted in Group B (herbal-based dentifrice) (arrow shows the area of demineralization)

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   Discussion Top

Dental caries is a worldwide chronic noncommunicable infectious disease that affects people of all races and ages. Even though the prevalence of caries among children and adults has been stabilized in the past decade, these rates remain at a constant high for specific subgroups.[10]

The early signs of caries activity are by the appearance of white opaque spot lesions that are less harder than adjacent sound enamel and are termed as “incipient carious lesion,” which are capable of being reversible by the remineralization process both by occurring natural or be induced by therapies.[11] Remineralization is the therapy for an active incipient lesion, aimed to reverse or arrest the progression of partially demineralized tooth structure, whereby calcium and phosphate ions from external source deposit into the crystal voids of demineralized enamel, to produce a net mineral gain.[12]

The cariostatic efficacy of fluoride-containing dentifrice has been convincingly demonstrated in numerous clinical trials and the recent decline in carious prevalence is primarily attributed to the increase use of fluoride agents.[13] But because of growing concerns regarding fluoride ingestion by children and risk of dental fluorosis, there is a need to identify newer approaching novel dentifrice that would inhibit demineralization and enhance remineralization. As the “minimal intervention” concept unfolds, various products with tooth surface protection, anticariogenic, or remineralizing effects are gaining an increasing attention by targeting the basic principle in remineralizing early carious lesion.

The application of β tri-calcium phosphate in dentifrice, mouthrinse, varnish was executed by combining fluoride and functionalized TCP. fTCP is tailored, low-dose calcium phosphate system that is incorporated into a single-phase aqueous or nonaqueous formulation, which enhance fluoride-based nucleation “seeding” activity with subsequent dietary in bounding salivary calcium and phosphate.[14] TCP is milled with simple organic materials to create a functionalized TCP ingredient, that ensures that the active calcium sites are protected from the premature interaction with fluoride. Thereby tri-calcium phosphate serves two major roles: First, it provides a barrier that prevents premature fluoride interaction, and second, it provides target delivery of calcium and phosphate.[15]

On the other hand, aloe vera (Aloe barbadensis) used as dentifrice has complex mixture of chemical composition. Its gel is composed of 98%–99% of water and 1%–2% of active components such as anthraquinones, naftoquinones, flavonoids, polysaccharides, essential amino acids, and vitamins, which are highly variable. Among the essential amino acids, aloe vera gel has arginine in abundant. It also contains salicylic acid, uronic acid and galacturonic acid; fructose, mannose, glucose, and other hydrolysable sugars; enzymes as oxidase, amylase, and catalase; sodium, potassium, calcium, and magnesium. The hard and mineralized enamel surface is porous and may enable the passage of ions, such as sodium, potassium, magnesium, and calcium. Therefore, the remineralizing capacity of the aloe vera-based dentifrice could be attributed to the deposition of arginine associated with the calcium on the enamel surface.[16] The other component xylitol cannot be converted into harmful lactic acid by cariogenic bacteria making its effects and metabolic products to significantly reduce, thereby allowing the mouth to naturally remineralize tooth structure.[17]

This study focused on comparing the remineralizing efficacy of commercially available nonfluoride (Mee Mee®) and herbal (Mamaearth™) pediatric dentifrices in demineralized primary teeth as there are no studies comparing these two commercially available agents. The demineralization solution used here was 2.2 mM calcium chloride (CaCl2), 2.2 mM potassium dihydrogen phosphate (KH2PO4), and 50 mM acetic acid adjusting the pH at 4.5 by adding 10M KOH as the previous studies by Ten Cate et al.,[18] Gujarathi et al., and[6] Kiranmayi et al.,[19] have shown demineralization of the tooth with this solution.[20] The pH cycling model refers to in vitro experimental exposure of substrates, enamel, or dentin in combination with demineralization and remineralization. This is created to imitate the dynamic differences in mineral saturation and pH associated with natural caries process for both mechanistic studies and for profile evaluation of dentifrices and mouthrinses.[21] It has been suggested that the use of a continuous exposure in the acidic environment, as in this study, would simulate a scenario of demineralization, magnifying the effects of each treatment on enamel.[19]

While using the polarized light microscope the samples need to be sectioned labio-lingually to a 100 μm thickness so as to observe under the microscope. So a control group was kept to assess the depth of demineralization with the demineralizing solution alone. Enamel is uniform with adequate thickness in the central portion of the tooth. So depth measurement was standardized to be measured in this specific area and also to minimize sampling errors due to inadequate thickness of enamel. Since remineralization cannot be directly measured, it was measured indirectly by the decrease in the depth of demineralized areas.

Polarized light microscopic analysis is a very sensitive technique for showing changes in hard tissues. With respect to demineralization and remineralization, birefringence experiments can qualitatively show mineral loss and mineral gain.[17] Readings of the total path difference were recorded at various intervals along the transverse axis running from the outer surface through the lesion into sound enamel. Polarized light measurements can provide quantitative information on the pore volume in demineralized and remineralized enamel, and on lesion characteristics, as stated by Wefel and Harless et al.[22] Keeping in mind all these factors, polarized light microscopy was used.

Maximum depths of demineralization were assessed in each sample. The mean remineralization was assessed by subtracting the mean demineralization depth of experimental groups from the mean demineralization depth of the control group. As far as nonfluoride-based dentifrice is concerned, studies have concluded that TCP provided superior and subsurface remineralization compared with 5000 ppm fluoride and CPP-ACP.[23] The results of the present study with nonfluoride pediatric dentifrice measuring the mean demineralization value 7.8730 μm, make it a promising agent for remineralizing primary teeth.

As far as herbal pediatric dentifrice is concerned, there is little or no prior studies on the efficacy of remineralization of the primary teeth using aloe vera. Although the antimicrobial effect is the same as the conventional toothpaste with fluoride content as stated by Silva et al.,[16] no significant clarification on the efficacy of remineralization was stated. Hence, the present study with herbal pediatric dentifrice measuring mean demineralization value 28.3174 μm, shows an insignificant result making it a not so practical remineralizing agent for primary teeth.

The limitations in our study are the varying pH levels in the mouth that cannot be duplicated in-vitro i.e the lack of oral microflora in the artificial salivary solution, control over salivary flow rate, and harsher acidogenic challenges used in intermittent period and the remineralization used in the study was 7 days. Although promising, studies on nonfluoride caries preventive agents showed low evidence and no reliable benefits. According to panelists, in such cases, clinicians and patients should understand the instability in the evidence as well as any potential risks of using or not using a particular intervention.[24]

In summary, according to the present study, the nonfluoride pediatric dentifrice (Mee Mee®) was the most effective agent for the intervention of incipient carious lesion than the herbal pediatric dentifrice (Mamaearth) making it an effective alternative to fluoride-based dentifrice. Yet, direct extrapolation to clinical conditions must be exercised with caution because of the obvious limitations of in vitro studies, and further elaborate, well-designed clinical trials are to be researched upon to determine the most effective remineralizing agents in each situation.

   Conclusion Top

The nonfluoride pediatric dentifrice promises to be a better alternative than herbal-based dentifrice. The findings of this in vitro study should be extrapolated to the oral cavity with caution and in relation to recent guidelines for prescribing dentifrices. Child formula dentifrice with no fluoride can remineralize or decrease the progression of early enamel carious lesions. Henceforth, the knowledge about various child dentifrice formulations will help pedodontists to prescribe the appropriate formulations for children who are at high risk of caries.


The authors would like to thank Dr. Ganganna Kokila, Professor and Head of the Department, Department of Oral Pathology, Sri Siddhartha dental college and hospital, Sri Siddhartha academy of higher education, Tumkur, for her timely guidance and support throughout the research work.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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García-Godoy F, Hicks MJ. Maintaining the integrity of the enamel surface: The role of dental biofilm, saliva and preventive agents in enamel demineralization and remineralization. J Am Dent Assoc 2008;139 Suppl: 25S-34S.  Back to cited text no. 2
Cury JA, Tenuta LM. Enamel remineralization: Controlling the caries disease or treating early caries lesions? Braz Oral Res 2009;23 Suppl 1:23-30.  Back to cited text no. 3
Featherstone JD. Dental caries: A dynamic disease process. Aust Dent J 2008;53:286-91.  Back to cited text no. 4
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]


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