|Year : 2015 | Volume
| Issue : 1 | Page : 28-34
Evaluation of remineralizing potential of commercially available child formula dentifrices: An in vitro study
Apurva Jagdish Gujarathi, Santosh M Sholapurmath, Praveenkumar S Mandroli, Deepa B Benni
Department of Pedodontics and Preventive Dentistry, Maratha Mandal's, Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India
|Date of Web Publication||9-Jan-2015|
Dr. Apurva Jagdish Gujarathi
Maratha Mandal's NGH Institute of Dental Sciences and Reasearch Centre, R.S No 47 A/2, Bauxite Road, Near K.S.R.P Ground, Belgaum - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of this in vitro study is to evaluate the remineralizing potential of commercially available low fluoride child formula dentifrice on primary teeth. Materials and Methods: Total 30 primary teeth were placed in demineralizing solution for 96 hours to produce artificial carious lesions of approximately 100 μm depth, and then cut longitudinally into 30 sections of 100-150 μm thickness and randomly assigned to three groups. Sections were treated with dentifrices containing Colgate ® (anti tooth decay) 500 ppm NaF, Cheerio gel ® 458 ppm MFP and Vicco ® non-fluoridated dentifrice. Lesions were evaluated using polarized light microscopy. Results: Colgate ® (anti tooth decay) 500 ppm NaF sections exhibited a statistically significant decrease in lesion depth (P < 0.05, paired t-test), whereas those in Cheerio gel ® 458 ppm MFP showed a decrease in lesion depth but was not statistically significant. Vicco ® non-fluoridated dentifrice showed increase in lesion depth. Statistics: A paired t-test is used to evaluate pre- and post-treatment lesion depth measurements, and Newman-Keuls multiple post hoc procedures was carried out to compare pair-wise difference of pre- and post-treatment lesion depth. Conclusion: The Colgate ® (anti tooth decay) 500 ppm NaF dentifrice and Cheerio gel ® 458 ppm MFP demonstrated remineralization of carious lesions by virtue of decrease in lesion depth, whereas Vicco ® non-fluoridated dentifrice showed increase in lesion depth.
Keywords: Child formula dentifrice, fluoride, primary teeth, polarized light microscope, remineralization
|How to cite this article:|
Gujarathi AJ, Sholapurmath SM, Mandroli PS, Benni DB. Evaluation of remineralizing potential of commercially available child formula dentifrices: An in vitro study. J Indian Soc Pedod Prev Dent 2015;33:28-34
|How to cite this URL:|
Gujarathi AJ, Sholapurmath SM, Mandroli PS, Benni DB. Evaluation of remineralizing potential of commercially available child formula dentifrices: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2015 [cited 2019 May 25];33:28-34. Available from: http://www.jisppd.com/text.asp?2015/33/1/28/148971
| Introduction|| |
In the oral cavity, there is a delicate balance of de-/remineralization of the enamel surface. The interruption of this balance results in dental caries and is most common disease of childhood worldwide. Remineralization is defined as the process whereby calcium and phosphate ions are supplied from a source external to the tooth to promote ion deposition into crystal voids in demineralized enamel to produce net mineral gain. Demineralization begins at the atomic level on the crystal surface inside the enamel or dentin and can continue unless halted with the end point being cavitation. 
Various newer systems are developed for remineralization such as complex of casein phosphopeptides and amorphous calcium phosphate, sodium calcium phosphosilicate (bioactive glass), calcium carbonate carrier - SensiStat, xylitol carrier, nano-hydroxyapatite, the trimetaphosphate ion, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, novamin, enamelon, and ion exchange resins.  Zero DT in 2006 gave requirements of an ideal remineralization material, which states novel materials should show a benefit over fluoride. 
Many studies have shown that early carious lesions can be remineralized, with fluoride being one of the most significant agents for promoting remineralization. , Fluorides are added to community and school water supply and also available as dietary supplements, mouth washes, and dentifrices. The most common source of topical fluoride for majority of children is dentifrice.  The effectiveness of fluoridated dentifrices in caries prevention has been accepted for nearly four decades and researchers now speculate that the use of such toothpastes may be contributing significantly to the large decline in dental caries. Various fluoride compounds are tested for caries inhibitory properties among these sodium fluoride (NaF) and sodium monofluorophosphate are two fluoride agents most widely used in toothpaste. 
However, the negative side of wide spread use of fluorides has been highlighted in a number of studies, which have identified early, or excessive use of fluoride dentifrices as an important risk factor for dental fluorosis. , The risk of fluorosis is a function of both the amount of dentifrice ingested and the fluoride concentration. Young children ingested greater amounts of fluoride through tooth brushing than the older ones; mainly due to inadequate control over their swallowing reflexes.  Although small quantities of fluoride dentifrice carry a lower risk of fluorosis, this must be balanced against the reduction in cariostatic effects.  One means of reducing the amount of fluoride ingested is to minimize the amount toothpaste placed on the toothbrush. Another approach has been used is to reduce the levels of fluoride in dentifrices.
In vitro de-remineralization studies using pH cycling model provide a valuable tool to investigate fluoride efficacy.  There are only a few studies conducted to evaluate remineralizing potential of low concentration fluoride dentifrices on primary teeth. Consequently the effectiveness of such dentifrice on primary teeth remains unclear. Thus, the aim of this in vitro study is to evaluate and compare the remineralizing potential of Cheerio gel ® , Colgate ® (anti tooth decay toothpaste) and Vicco ® dentifrices.
| Materials and Methods|| |
Thirty sound extracted or naturally exfoliated primary anterior teeth were collected and were inspected for cracks, hypoplasia, caries, and white spot lesion. Teeth were stored in sterile distilled water till use.
Dentifrices used in study were
Group 1: Cheerio gel ® with 458 ppm of sodium monofluorophosphate.
Group 2: Colgate ® (anti tooth decay toothpaste) with 500 ppm of sodium fluoride.
Group 3: (control group) - Vicco ® non-fluoridated toothpaste.
Dentifrice slurry preparation
Dentifrice slurry was prepared in 3:1 ratio of deionized water to dentifrice. To achieve this, 17 gm of dentifrice was dispensed using Wensar highprecision balance weighing machine from the respective tube and then transferred into three tubes to which 51 ml of deionized water was added and to stir with stirring rod until it was well mixed. Fresh slurry for all three toothpastes was prepared before each pH cycle, and it was stored in separate containers throughout the study.
Demineralizing solution preparation
The demineralizing solution contained 2.2 mM CaCl 2 , 2.2 mM NaH2PO4, and 0.05 M acetic acid. 1 M KOH was used to adjust the pH to 4.5. The remineralizing solution, which contained 1.5 mM CaCl 2 , 0.9 mM NaH 2 PO 4, and 0.15 M KCl with a pH of 7.
Fresh demineralizing and remineralizing solutions were prepared before each pH cycle, and pH of the both solutions was checked by AZ ® 86501 pH meter before use.
Thirty sound extracted teeth were cleaned to remove soft tissue debris and were inspected for cracks, hypoplasia, and white spot lesions. Teeth were coated with acid resistant nail varnish (Lakme® true color nail wear) leaving 1-mm-narrow window on sound intact buccal surface. Teeth were then immersed in demineralizing solution for 96 hours to produce 100-μm lesions. Approximately 100-150 μm longitudinal enamel sections were prepared by using Leica SP 1600 hard tissue microtome. After discarding the damaged sections, 10 sections were randomly assigned to each of the three experimental groups.
pH cycling model
Specimens were placed in pH cycling system on an orbital shaker (Sakoba scientific co) for 7 days. Each cycle involved 3-hour demineralization twice daily and 2 hour of remineralization in between demineralization. One-minute treatment with dentifrice slurry before 1 st demineralization and before and after 2 nd demineralization. Then, sections were placed in remineralizing solution overnight. Demineralizing solution, remineralizing solution, and dentifrice slurry were freshly prepared for each cycle and separate containers were used for each group throughout the study. The pH level of the demineralizing and remineralizing solutions was measured before every cycle. After 7 days, nail varnish was removed with acetone before post-treatment evaluation under polarized light microscope microscope.
Polarized light microscopy measurements
Specimens were photographed using polarized light microscope (Olympus BX 51 research microscope), both before and after the pH cycling to evaluate the lesion depth in the enamel samples. This was accomplished by imbibing the section in water, which normally shows a clear demarcation between sound enamel and the initial lesion. Any changes between pre- and post-treatment lesions could be obtained from the same magnification photomicrographs taken before and after the pH cycling, (4× camera lens) under the polarized light microscope. These photomicrographs were then measured for lesion depth using Pro plus Image analysis software version 4.1.0 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 mean of three measurements obtained.
| Results|| |
The mean pre-treatment lesion depth from each group ranged from 126.4589 μm to 149.1567 μm with lesion depth of 126.4589 μm for Cheerio gel ® , 149.1567 μm for Colgate ® , and 129.4023 μm for Vicco ® with standard deviation of 50.2151 μm, 31.5169 μm, 42.1542 μm, respectively [Table 1]. Pre-treatment lesion depths among three groups were analyzed by one way ANOVA. No statistically significant difference among these pre-treatment lesion depths was noted. (F-value = 0.8637, P = 0.4329 ANOVA) [Table 2]. This implies that even though all the specimens were sectioned from different teeth, the variations among the teeth did not show a major effect on the progression of demineralization. It was reasonable, therefore, to ignore these variations when the de/remineralization efficacy of different dentifrices were evaluated and compared after pH cycling.
|Table 1: The pre-treatment lesion depth of all the three toothpastes and their standard deviation|
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|Table 2: Comparison of three toothpastes (Cheerio gel®, Colgate® and Vicco®) with respect to pretreatment lesion depth by one way ANOVA|
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The result from the lesion depth measurements after the 7 days pH cycle showed that mean difference between pre- and post lesion depth was Group 1- 11.0059, Group 2- 32.2629, Group 3- −28.5995 with lesion depth reduction by 8.7032% for Group 1 (Cheerio gel ® ) [Figure 1] and [Figure 2] and 21.6302% for Group 2 (Colgate ® ) [Figure 3] and [Figure 4]. Although there was increase in lesion depth by 22.1012% for Group 3(Vicco ® ) [Table 3] [Figure 5] and [Figure 6].
|Figure 1: Before pH cycling polarized light photo-micrograph of a representative enamel lesion in Cheerio gel® group|
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|Figure 2: After 7 days' pH cycling, polarized light photomicrograph of a representative enamel lesion in Cheerio gel® group (Note decrease in the lesion depth)|
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|Figure 3: Before pH cycling polarized light photo-micrograph of a representative enamel lesion in Colgate® group|
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|Figure 4: After 7 days' pH cycling polarized light photomicrograph of a representative enamel lesion in Colgate® group (Note decrease in the lesion depth)|
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|Figure 5: Before pH cycling, polarized light photo-micrograph of a representative enamel lesion in Vicco® group|
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|Figure 6: After 7 days' pH cycling polarized light photomicrograph of a representative enamel lesion in Vicco® group (Note increase in lesion depth)|
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|Table 3: Comparison of pre- and post-treatment (after 7 days' pH cycling) lesions depth in Cheerio gel®, Colgate® , and Vicco® toothpaste by paired t-test|
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A paired t-test confirmed a statistically significant difference (P < 0.05) between pre- and post-treatment lesion depth measurements in Group 2 (colgate ® ) with P-value of 0.0005, whereas no statistically significant difference seen between pre- and post-lesion depth measurements in Group 1 (Cheerio gel ® ) and Group 3 (Vicco ® ) with P-value of 0.3175 and 0.0708, respectively [Table 3].
When pair-wise comparison was done using Newman-Keuls multiple post hoc procedures with respect to difference of pre- and post-treatment lesion depth for Cheerio gel ® and Colgate ®, there was no statistically significant difference between both groups with P-value of 0.1691. Although pair-wise comparison between Cheerio gel ® group and Vicco ® group showed statistically significant difference with P-value of 0.0140*, also pair-wise comparison between Colgate ® group and Vicco ® group showed statistically significant difference with P-value of 0.0012* [Table 4].
|Table 4: Pair-wise comparison between three toothpastes (Cheerio gel®, Colgate® and Vicco®) with respect to difference of pre- and posttreatment lesion depth by Newman-Keuls multiple post hoc procedures|
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| Discussion|| |
Clinical trials of effectiveness of fluoride toothpaste began in 1960 and have shown effect of fluoride in reducing caries and remineralization of carious lesion, so fluoride toothpaste along with water fluoridation play very important part in dental health. There is always issue raised with the fluoride toothpaste ingestion and risk of fluorosis in children , and risk is more in first 7 years of life as compared to later years.  When similar amount of toothpaste is placed, due to less control over their swallowing reflexes younger children ingest more fluoride as compared with older children.  As fluoride dentifrices have a dose response relationship  ; this is important because dentifrices for children usually contain between 250 and 500 ppm fluoride. Although small quantities of fluoride dentifrice carry a lower risk of fluorosis, this must be balanced against the reduction in cariostatic effects.  The anticariogenic efficacy of a 550 ppm fluoride designed for children was found to be similar to that of 1100 ppm fluoride dentifrice.  However, studies have shown that remineralization of enamel by a 1000 ppm is achieved more rapidly than with a 250 ppm fluoride. , In this study, we have compared effectiveness of Colgate ® anti-tooth decay toothpaste with 500 ppm of sodium fluoride and Cheeriogel ® with 458 ppm sodium monofluorophosphate and Vicco ® is taken as non-fluoride group dentifrice, which can be taken as positive control.
To evaluate efficacy of fluoride, an in vitro model is developed termed as pH cycling model. A 'pH cycling' is applied to an in vitro experiment involving the exposure of specimens (enamel and/or dentine) to a combination of remineralization and demineralization.  Since then many researchers have adopted and modified such pH cycling model to fit their protocols. The solution concentration and pH should be kept within the range that exists in the oral fluid. In this study, to avoid the risk of the solutions becoming saturated, fresh demineralizing and remineralizing solutions were prepared daily before each cycle, and the pH was checked every time before use.
Ten Cate and Duijisters et al., in 1982 and Featherstone et al in 1986 described pH cycling model, which are most commonly used. Two modified models for re/demineralizing experiments on primary teeth have emerged. A 7-day pH cycling experiment and a 10-day pH cycling with 0.25 ppm fluoride added to the remineralizing and demineralizing solutions. Thaveesangpanich et al. conducted a pH cycling experiment on primary teeth, without fluoride in the remineralizing or demineralizing solutions and found that all of the sections were eroded by the 8th day of the experiment and hence rendered the sections inappropriate for evaluation.  As the addition of fluoride to the demineralizing solution have an inhibitory effect on the rate of demineralization it will interfere with the re/demineralization carried out in study and alter the results. In this study, 7 days' pH cycling is used instead of 10 days' pH cycling. However, this shortening of the period of the pH cycling might produce results that inadequately represent the natural process of re/demineralization.
There have been very few studies on the efficacy of fluoride dentifrices on primary teeth especially using single section pH cycling models. The basic components of the single-section model were described by Wefel et al in 1987.  The single-section model used in this study has the advantage that the same tissue can be measured before and after the experiment; thus any changes due to exposure to the treatment regimen can be evaluated.
Depth-related properties of artificial caries lesions can be described quantitatively by mineral content and hardness profile  and qualitatively by polarized microscopy  , scanning electron microscopy  , transverse microradiography, and recently microcomputed topography.  In this study, polarized light microscopy was used to qualitatively evaluate the lesion depth of the artificial caries lesion in enamel before and after pH cycling in same specimens.
The means and the standard deviations of the pre-treatment lesion depths formed after 96-hour demineralization for each group were not statistically different from each other where P-value is 0.4329 (P < 0.05). This proves that even though the specimens were sectioned from different teeth, the variations among the teeth did not show a major effect on the progression of demineralization. So, it was reasonable to disregard these variations when the de/remineralizing efficacy of different dentifrices were evaluated and compared after the pH cycling.
From the presented results, it can be hypothesized that both Colgate ® with 500 ppm of sodium fluoride and Cheerio gel ® with 458 ppm sodium monofluorophosphate showed decrease in lesion depth after pH cycling. But as compared to Cheerio gel ® , Colgate ® showed more decrease in lesion depth. Between these two groups statistically significant difference is not seen as P-value obtained by Newman-Keuls multiple post hoc procedure is P = 0.1691 (P < 0.05). Vicco ® non-fluoride toothpaste showed no remineralizing action where lesion depth after pH cycling samples increased. When comparison was made between Colgate ® and Vicco ® and between Cheerio gel ® and Vicco ® by Newman-Keuls multiple post hoc procedure statistically significant result is seen with P-value of 0.0012 and P = 0.0140 where P < 0.05.
Also from the present study results, when pre and post-treatment lesion depth were compared for all three toothpastes, by students paired t-test only Colgate ® showed statistically significant reduction in lesion depth with P-value of 0.0005 where as for Cheerio gel ® and Vicco ® difference of pre- and post-treatment lesion depth was not statistical significant with P-value of 0.3175 and 0.0708 where P < 0.05.
Present study result is in accordance with study conducted by Ittagarun A. and S.H.Y. Wei, Wafel J.S 1997,  Ittagarun A., S.H.Y. Wei, Wafel J.S 2000  and Ekamabarm M, Ittagarun A, King NM 2011.  Also Stookey and Beiswanger 1989 suggested that sodium fluoride provided consistently improved anti-caries efficacy relative to sodium monofluorophosphate.  This was further supported by Rank where 9 of 10 studies favored sodium fluoride over sodium monofluorophosphate, and in meta-analysis by Proskin and Leverett 1991 result showed advantage of sodium fluoride over sodium monofluorophosphate.  The critical review by Stookey et al in 1993 demonstrated that sodium fluoride was significantly more effective than sodium monofluorophosphate in preventing caries, and authors of this study recommend that sodium fluoride be used as the active system in fluoridated dentifrice whenever feasible. 
The present study result showed that fluoride ion from sodium fluoride was more effective as compared to sodium monofluorophosphate. This might be due to the remineralizing solution used in present study contained only inorganic solution and enzyme system required for sodium monofluorophosphate was absent.  It is important to use natural saliva to make dilution for sodium monofluorophosphate preparation  as salivary enzymes help in the hydrolysis of the covalently bound sodium monofluorophosphate to release fluoride ions.  In study conducted by Faller R. et al 1997 to eliminate this limitation of in vitro study, they used the pooled human saliva as diluents while making treatment slurries, and each slurry was used within 5 minutes after preparation. Even under these conditions, sodium monofluorophosphate was shown to be less efficient than sodium fluoride at incorporating fluoride into demineralized enamel. 
The findings of this in vitro study should be interpreted and extrapolated to oral cavity in caution and in relation to recent guidelines for prescribing fluoride dentifrices. Based on the results of present study, sodium fluoride containing dentifrices can be recommended in high caries risk child.
| Conclusion|| |
Based on the data obtained from present study, it can be concluded that A Child formula dentifrice containing Colgate ® (anti tooth decay toothpaste) 500 ppm of sodium fluoride and Cheerio gel ® 458 ppm MFP has shown caries-preventive effect in vitro. Child formula dentifrices with low fluoride can remineralize or decrease the progression of early enamel carious lesions. So knowledge about various fluoride dentifrice formulations will help a paediatric dentist to prescribe the appropriate formulation for children who are at high risk of caries.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]