Remineralizing Potential of Various Commercially Available Dentifrices on Artificial Enamel Lesions
Corresponding Author: AJ SaiSankar, Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India, Phone: +91 9346550646, e-mail: email@example.com
Aim: The aim of the present study was to determine the remineralizing potentiality of artificial carious lesions in primary teeth after application of three commercially available dentifrices using pH cycling.
Materials and methods: A total of 24 human primary teeth were randomly divided into three groups. Group I: Clinpro Tooth Creme, Group II: Enafix, and Group III: Amflor. All the teeth in each group were demineralized for 96 hours, sectioned longitudinally using hard tissue microtome and subjected to pH cycling for 7 days. The specimens were viewed under polarized light microscope, depth of demineralization and remineralization was recorded using Micap Microview analyzer software.
Results: The results were analyzed using one way-ANOVA test. All the test groups showed statistically significant decrease in mean depth of lesion 21.62±9.41 µm, 20.75±14.61 µm. 26.13±17.84 µm for group I, II, and III, respectively. However, no statistical difference was noticed on intergroup comparison.
Conclusion: All the tested dentifrices demonstrated remineralizing potential which was elicited by decrease in the lesion depth. Amflor showed better results than other remineralizing agents.
Clinical significance: To emphasize the importance of minimal invasive treatment of incipient carious lesion by remineralizing agents. Opting for remineralizing agents at the early stage can save the time, money and manpower. Usage of dentifrices containing remineralizing agents shown long-term beneficial effects on early caries lesions. The present study demonstrated that all the tested agents can be used to induce remineralization of early enamel lesions, so that complex invasive treatments can be prevented in near future.
How to cite this article: Nalini B, Pranitha K, SaiSankar AJ, et al. Remineralizing Potential of Various Commercially Available Dentifrices on Artificial Enamel Lesions. World J Dent 2022;13(2):116–120.
Source of support: Nil
Conflict of interest: None
Keywords: Demineralization, Dentifrices, Fluorides, Polarized light microscope, Remineralization
Dental caries is the most prevalent oral disease and is considered as the major worldwide health concern. “White spot lesion” is the earliest clinical sign of dental caries.1 In the recent decade, white spot lesions can be prevented through various noninvasive preventive modes, as the routine operative treatment is hard to achieve and may also need special behavior management for treating children.
In children, the demineralization potential at low oral pH is high while the remineralization potential at normal pH is low.2 And also low mineral and high organic content of enamel made the primary teeth more susceptible to caries development. Hence, the progression of caries will be faster and reversal will be slower in children, as they depend upon the balance between demineralization and remineralization.3 Thus, in children while managing the initial caries lesions the most preferred noninvasive intervention is the usage of dentifrices containing remineralizing agents.
The ions liberated upon the application of remineralizing agents will act as diffusion barrier by the formation of thick surface coatings and also deposition of minerals within the enamel crystallites inturn decrease the solubility of enamel.4 Orientation of the remineralized enamel crystals is similar to that of the original enamel crystals and are generally more resistant to decalcification.
Fluoride’s role in remineralization is well established and effective method of remineralization. Fluoride containing dentifrices usually have a dose-response relationship; therefore dentifrices containing 250 to 500 ppm fluoride are used for children to reduce fluorosis risks. In accordance with IAPD Recommendations (2020), using fluoridated tooth paste containing 1000 ppm of fluoride twice a day with age-appropriate amounts of tooth paste on the toothbrush are effective in preventing dental caries in children.5
As science and research advances, newer fluoridated and nonfluoride remineralizing agents have been introduced in the market. The Clinpro Tooth Cremem contains functionalized tri-calcium phosphate ingredient (fTCP) with fluoride range of about 950 ppm. Each gram of it contains 0.95 mg of fluoride ion in a neutral pH base consisting of water, hydrated silica, sorbitol, glycerin, polypropylene glycol, polyethylene-polypropylene glycol, flavor, sodium lauryl sulfate, titanium dioxide, carboxymethyl cellulose, and sodium saccharin.6 This organically modified TCP technology prevents undesirable reactions with fluoride, but may dissolve away when particles contact saliva. This system is stable in aqueous environment and does not affect the fluoride activity.7
Enafix contains anticay 5% which is a complex mix of calcium sucrose phosphate inorganic amorphous calcium phosphate, sorbitol, purified water, silica, glycerin, sodium lauryl sulphate, flavors, sodium methyl cocodyl taurate, sodium carboxy methyl cellulose, titanium dioxide, potassium acesulfame, sodium methyl hydroxyl benzoate, and sodium propyl hydroxyl benzoate. During brushing, calcium sucrose phosphate (anticay) quickly breaks down and releases calcium, phosphate, and sucrose phosphate ions into the saliva. Calcium and phosphate ions rapidly adsorb onto the enamel, decrease the rate of enamel solubility under acidic conditions and at neutral pH and increase the rate of remineralization.8
Amflor contains 1000 ppm amine fluoride, sorbitol, purified water, propylene glycol, silica, cocamidopropyl betaine, titanium dioxide, Hydroxyl ethyl cellulose and sodium saccharin. Amflor toothpaste contains the active ingredient in the form of amine fluoride (organic fluoride) which has caries inhibitory properties due to its antiglycolytic and surface-active properties. It also involves the accumulation of fluoride onto the surface of the tooth, which readily forms calcium fluoride and acts as a labile fluoride reservoir.8, 9
These three remineralizing agents were chosen based on their difference in composition and also studies comparing the above forementioned dentifrices on the primary teeth are sparse. Therefore present in vitro study was undertaken to compare remineralization potential of three commercially available dentifrices (Clinpro Tooth Creme, Enafix, and Amflor) on artificial carious lesions.
MATERIALS AND METHODS
The present in vitro study was performed after obtaining institutional ethical committee approval (pr.171/IEC/SIBAR/2018). The sample size determination was done using G* 126.96.36.199; the effect size is 0.795 and sample size derived is 24. Teeth with preshedding mobility, natural exfoliation, extracted over-retained or orthodontic purpose were included. Whereas the teeth with cracks, white spots and dental caries, or any discolored teeth and hypoplastic teeth were excluded from the study.
Out of total 40 sound human caries-free primary teeth collected, 24 teeth, which fulfilled all the inclusion and exclusion criteria were selected for the study and stored in a 0.1% thymol solution until use.
Sample Preparation and Lesion Formation
The selected teeth were rinsed, hand scaled and thoroughly cleaned and dried. A rectangular window of 2 mm × 4 mm was labeled in the center of the buccal surface and the remaining surface of the tooth was coated with nail varnish. The teeth were randomly divided into three groups with 8 in each using computer randomization technique. The groups are as follows: Group-I: Clinpro Tooth Creme (3M ESPE Dental Products, St.Paul, MN, USA), Group-II: Enafix (Group pharmaceuticals pvt Ltd, Banglore, India), and Group-III: Amflor (Group pharmaceuticals pvt Ltd, Malar, India).
To create artificial caries lesions, all the specimens were immersed for 96 hours in a glass beaker containing 500 mL of demineralizing solution. With a diamond disk mounted on a slow-speed handpiece, the teeth are sectioned longitudinally in buccolingual direction. Each specimen shows a portion of the normal enamel and demineralized area. Out of two sections obtained from each tooth, one section was used to record depth of demineralization and other to record depth of remineralization.
The specimens used to record depth of demineralization in three groups were embedded in self cure acrylic (DPI- PR Cold Cure) resin blocks using a preformed mould. Later these specimens were sectioned using hard tissue microtome (Leica SP 1600 Saw Microtome, Germany) longitudinally through the lesions to produce sections of approximately 100 to 150 μm thickness to examine under polarized light microscope.
Recording the Demineralization Depth
The sectioned samples of each group were immersed in water for a clear demarcation between sound and carious enamel and then viewed under polarized light microsope (Olympus BX51 penta head microscope, Japan) at 40x magnification. After attaining the microscopic images (Fig. 1A, 2A, 3A), the depth of demineralization was noted at three different points from the surface of a tooth to the maximum depth of demineralization and the average of the three was considered as mean value (µm).
Ten Cate and Duijsters pH cycling model was used for all the samples after demineralization.10 It involved 3 hours of demineralization twice daily, with 2 hours of remineralization between periods of demineralization for 7 days. Each section was then treated with dentifrice supernatant for 60 seconds (5 mL/section).
The demineralizing, remineralizing solutions and dentifrice slurries were freshly prepared for each cycle and stored in separate containers for each group throughout the experimental period.11 These solutions were changed daily to prevent depletion or saturation of the solutions and accumulation of enamel dissolution products.3
Recording Depth of Remineralization
Following pH cycling the specimens were embedded in acrylic blocks and sectioned using hard tissue microtome. Each section was visualized under polarized light microscope at 40X magnification to determine the amount of remineralization on microscopic images (1B, 2B, 3B) using Micaps-micro view 3.7 version software.
The entire procedure was performed by a single operator, however to avoid bias, a second operator who is unaware of prior results evaluated the procedural aspect and recorded the values. intraclass correlation coefficient (ICC) values were calculated to examine the reliability. An intraclass correlation value of 0.93 was achieved which indicates good agreement among the investigators, hence the earlier observations were only taken into account.
The de- and remineralization values obtained were tabulated using SPSS and subjected to statistical analysis. Paired t-test was used to calculate mean lesion depth and standard deviations and one-way ANOVA for inter group comparison. The p-value of < 0.05 was considered statistically significant.
The mean lesion depth among the three test groups after demineralization ranged from 167.38 µm to 177 µm which was not statistically significant and after remineralization the values varied between 141.25 µm to 155.38 µm, which were also statistically insignificant. However, a decrease in the mean lesion depth was observed in the all test groups after remineralization.
Coming to reduction in mean lesion depth after remineralization Group III exhibited highest decrease in mean lesion lesion depth (26.13±17.84 µm) followed by Group I (21.62±9.41 µm) and Group II (20.75±14.61 µm) samples. There was statistical significant decrease in mean lesion depth values in all the three groups after de- and remineralization for Group I, II, and III with a p-value of 0.001*, 0.005*, and 0.004*, respectively (Table 1).
|Group||N||Mean ± SD
|Mean ± SD
|Mean difference||p value|
|I||8||177.00 ± 11.88||155.38 ± 16.37||21.62 ± 9.41||0.001*|
|II||8||170.13 ± 14.04||149.38 ± 19.89||20.75 ± 14.61||0.005*|
|III||8||167.38 ± 16.02||141.25 ± 24.4||26.13 ± 17.84||0.004*|
Paired t test; p ≤ 0.5 considered statistically significant
Remineralization is a noninvasive treatment modality for the initial caries lesions that bridge the gap between preventive and invasive dentistry.8 Caries process is said to be dynamic when demineralization periods are far more than the periods of remineralization.1 Saliva’s inherent buffering capacity and the presence of calcium ions, phosphate ions, fluoride, and other components prevent the early tooth demineralization.12 If this natural system of saliva fails, then remineralizing agents are needed to compensate the oral defensive mechanisms at that site and protect the tooth from cavitation. Thus, if remineralizing agents are used at this stage, caries can be arrested or reversed.13
In the present study, artificial carious lesions were produced as they are more homogenously reproducible than natural lesions and showed all the principal histological features of natural caries of enamel in vitro.3 Extracted or naturally exfoliated primary teeth (molars, canines, and incisors) were used for lesion formation. Though there are variations in the morphology of individual teeth, it was hypothesized that these variations among the teeth do not have a significant role in caries formation.7
Single section model proposed by Wefel et al.14 was used for this study as it provides the advantage that the same tissue can be evaluated before and after the exposure period. Thus, any change could only be due to the exposure of the experimental solutions. Similarly, Kiranmayi et al.3 and Malekafzali et al.15 used single section pH cycling models for evaluating the efficacy of fluoride dentifrices on primary teeth.
Ten Cate and Duijsters pH cycling model can mimic the introral pH changes and results in more representative findings in evaluating remineralization efficacy of an agent.16 A 10-day pH-cycling model can be used on the enamel of permanent teeth whereas a 7-day pHcycling or 10-day cycling with added 0.25 ppm fluoride can be used for primary teeth.3,7 In the present study, 7 days pH cycling model without the addition of fluoride was used as incorporation of the same could interfere with the hypothesis being tested.
Polarized light microscope was used in the present study to assess the lesion depth of the artificial caries lesion for all specimens because the characteristic features of enamel can be better visualized due to its birefringence property, which is not well appreciated in a transmitted light microscope.17
The results of the present study quantified that all the tested groups showed better remineralization potential by decreasing the mean lesion depth. However, Group III elicited higher remineralization potential; which could be due to high fluoride content (1000 ppm) and the fluoride compound used (amine fluoride).
The results were similar to the studies conducted by Galuscan et al.18 and Madlena et al.19 who reported that amine (organic) component in amine fluoride (AmF) exhibited an antiplaque effect that resulted in the inhibition of bacterial adhesion. Also, the tensioactive property of the amine component favors the accumulation of fluoride close to the tooth surface, providing a sustained fluoride release. Shetty et al.20 also evaluated the enamel surface microhardness after the application of organic fluoride (AmF) and inorganic fluoride (NaF) dentifrices on permanent teeth and found AmF released a larger amount of fluoride than NaF, which caused the fluoride to be more bioavailable for a longer period of time. Likewise, Prasad et al.21 compared the remineralizing effect of organic fluoride (AmF) and inorganic fluoride (NaF) and found AmF showed better remineralization on the enamel surface of permanent teeth. This might be due to formation of a thick calcium fluoride layer between NaF (inorganic fluoride) and hydroxyapatite of enamel which inhibited the further diffusion of fluoride, thus providing a relatively lower bioavailability of fluoride ions. Arnold HW et al.22 Naumova EA et al.23 also found that more amount of fluoride was deposited on enamel by treating with AmF than sodium fluoride which resulted in enhanced remineralization.
In this study group I (Clinpro Tooth Creme) showed a significant reduction in mean lesion depth thereby promoting remineralization. The reason may be due to the presence of a combination of sodium fluoride with functionalized tricalcium phosphate (f-TCP) which provides a continuous reservoir of ions. As it not only ensures a controlled supply of calcium and phosphate ions, but also enhances the action of fluoride on enamel surfaces. TCP can provide maximum advantages even when administered in a neutral pH environment, whereas other calcium phosphate additions may require an acidic pH.
In accordance to the present study, Rao R et al.24 also found that ClinPro tooth Creme showed better remineralization potential and this could be due to beta-TCP is present in Clinpro tooth creme, which is similar in structure to apatite and has unique calcium environments capable of reacting with fluoride and enamel. While phosphate floats free, the exposed calcium environments are protected, preventing calcium from prematurely interacting with fluoride by providing catalytic amounts of calcium to boost fluoride absorption and promote remineralization.
Similarly, Krishnan et al.25 found Clinpro as a promising agent for remineralizing primary teeth. Likewise, Sreekumar et al.,26Patil et al.1 found that a higher concentration of calcium ion in f-TCP and addition of fluoride was responsible for its superior performance.
On Contrary, study done by Premanth et al.27 found better remineralization with clinpro than Amflor in permanent teeth. This might be due to the synergistic effect of both fluoride compound (NaF) and functionalized tricalcium phosphate (f-TCP). The aqueous-stable functionalized calcium phosphate system has no effect on the fluoride activity added to dentifrices, which increases enamel surface strength. The available evidence, therefore indicated that TCP-containing dentifrices could remineralize teeth in a significant manner.
The significant reduction in mean lesion depth in group II samples could be due to rapid absorption of ions onto enamel surface, make the availability of the sucrose phosphate ion which decreases the rate of acid dissolution of hydroxyapatite that inhibits demineralization and presence of calcium and phosphate ions increase the rate of remineralization by common ion effect. Similarly, Gade28 and George et al.29 in their studies found Enafix as a favorable remineralizing agent for mineralization of artificially demineralized human enamel.
Although remineralizing ability of CaSP with inorganic ACP (Enafix) has shown promising results. However, in comparison to the dentifrices containing fTCP+NaF (Clinpro) and amine fluoride (Amflor), Enafix has shown less effectiveness in promoting remineralization in this study. This might be due to the nonavailability of fluoride ions, as soluble calcium and phosphates were unable to substantially localize at the tooth surface to produce effective concentration gradients for better remineralization. This concept was further supported by studies done by Titty et al.9 Kakkar et al.12 in which Enafix formed a thick layer of calcium fluoride by reaction with hydroxyapatite of enamel that in turn reduced the bioavailability of fluoride ions.
Even though this in vitro experiment provides qualitative research, certain limitations do exist while performing remineralization in in vitro designs such as inadequate simulation of complex and diverse intraoral conditions, lower level of salivary proteins, lack of bacteria in the artificial saliva, control over the salivary flow rate, and lack of pellicle and biofilm formation on the tooth surface. Moreover the specimens were also exposed to multiple cycles of de- and remineralization, which may be severe than actual acid attack in the oral cavity. Hence, further studies need to be conducted with the forementioned points taking into consideration to obtain optimal clinical results.
All the three dentifrices used in the study demonstrated remineralization of artificial carious lesions. Among the three, amine fluoride containing dentifrice exhibited a superior remineralizing potential which might be due to the presence of high fluoride content and organic fluoride component (amine component). Also properties like antiglycolytic, tensioactive property, antiplaque, and anticariostatic effect are further considered for the beneficial effects of remineralization.
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