|Year : 2020 | Volume
| Issue : 2 | Page : 217-223
|Comparative evaluation of three different toothpastes on remineralization potential of initial enamel lesions: A scanning electron microscopic study
TP Chandru1, M Bazanth Yahiya1, Faizal C Peedikayil1, N Dhanesh1, N Srikant2, Soni Kottayi1
1 Department of Pediatric and Preventive Dentistry, Kannur Dental College, Kannur, Kerala, India
2 Department of Oral Pathology, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Karnataka, India
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|Date of Submission||02-Oct-2018|
|Date of Decision||28-Jan-2019|
|Date of Acceptance||22-Feb-2019|
|Date of Web Publication||19-May-2020|
| Abstract|| |
Background: The early enamel lesions are reversible as it is a process involving mineral transactions between the teeth and saliva. Aim: To evaluate the efficiency of three different tooth pastes on remineralization potential of initial enamel lesions using Vickers Micro hardness Test and Scanning electron microscopy. Materials and Methods: Artificial carious lesions were prepared in human enamel with demineralizing solution. The treatment agents included were Colgate sensitive plus® toothpaste, Regenerate enamel science™ toothpaste, BioRepair® toothpaste and control as Deionized water. All the samples were subjected to treatment solutions as per the pH cycling model for 12 days to simulate the daily oral environment's acid challenge. The remineralization parameters-surface hardness and surface roughness of enamel blocks were evaluated with Vickers indenter and Scanning electron microscope respectively. Statistical Analysis: ANOVA test was used to check mean differences between the groups. Post hoc analysis was done using Tukey's post hoc test. SEM images were graded according to Bonetti et al grading criteria. Results: As per statistical analysis, maximum remineralization of enamel blocks occurred after applying Colgate Sensitive Plus® tooth paste followed by BioRepair® tooth paste and Regenerate enamel Science™ toothpaste. Least remineralization potential was shown by control group. Conclusion: Colgate sensitive plus tooth paste with Pro Argin™ formula can be regarded as a potential remineralising agent. It can be concluded as a noninvasive means of managing early enamel carious lesions.
Keywords: Biorepair ® toothpaste, Colgate sensitive plus ® toothpaste, pH cycling, Regenerate enamel science™ toothpaste, surface microhardness
|How to cite this article:|
Chandru T P, Yahiya M B, Peedikayil FC, Dhanesh N, Srikant N, Kottayi S. Comparative evaluation of three different toothpastes on remineralization potential of initial enamel lesions: A scanning electron microscopic study. Indian J Dent Res 2020;31:217-23
|How to cite this URL:|
Chandru T P, Yahiya M B, Peedikayil FC, Dhanesh N, Srikant N, Kottayi S. Comparative evaluation of three different toothpastes on remineralization potential of initial enamel lesions: A scanning electron microscopic study. Indian J Dent Res [serial online] 2020 [cited 2020 Aug 11];31:217-23. Available from: http://www.ijdr.in/text.asp?2020/31/2/217/284583
| Introduction|| |
Dental caries is a chronic, infectious oral disease present worldwide that progresses slowly in most individuals. Even though we are aware about the drastic increase in the prevalence and complications in association with dental caries, it is an innate human tendency to get treated at a later stage than focusing on the preventive aspect. This motivated the dental researchers and the clinicians to develop strategies for fighting dental diseases in their initial stages.
Enamel is the visible outermost layer of the tooth known to be the hardest tissue in the human body., It is composed of 96 wt% of inorganic components that are mainly calcium and phosphorus as hydroxyapaptite and 4 wt% organic material and water. The formation of enamel is limited by its inability to function after the crown is complete, rendering it incapable of further growth and repair., According to Featherstone, newly calcified teeth and bone were described as carbonated hydroxyapatite and is considered as an impure form of hydroxyapatite. Further calcification increases the mineral weight from 45 wt% to 96–98 wt%, ultimately forming the most mineralized tissue found in the body.,
The oral bacteria adhere to the pellicle to form a bacterial mass called plaque. Plaque bacteria produce weak organic acids decreasing the surface pH. The acids diffuse through the plaque and into the tooth, consequently seeping calcium and phosphate from the enamel (demineralization) and lowering the plaque pH to 4.0–4.5.
An interventional approach for the elimination of demineralized area was developed almost a century ago. This approach was necessary at that point of time, because there was no valid alternatives. The focus on caries management has then recently shifted to the development of methodologies for the detection of caries lesions at an early stage and the use of noninvasive treatment which includes various remineralizing toothpastes for these lesions.,,
Fluoride was the mainstay for many years when it comes to noninvasive management of noncarious lesions. Demerits of fluoride paved a way for the development of newer remineralizing agents.
This study evaluated the remineralization potential of three different commercially available toothpastes, namely, Colgate Sensitive Plus®, Regenerate Enamel Science™ toothpaste, and BioRepair® toothpastes using Vicker's microhardness test and scanning electron microscopy (SEM).
| Materials and Methods|| |
Anin vitro study was designed and conducted in the department of pediatric and preventive dentistry of a dental institution. Ethical clearance was obtained to use human extracted teeth. In all, 62 permanent maxillary and mandibular incisors extracted for therapeutic purpose were collected.
The inclusion criteria and sampling technique include the following:
- Teeth devoid of any visible surface cracks
- Teeth without any kind of white spot lesions
- Absence of developmental defects
- Teeth without hypoplastic enamel surface.
Stereoscopic examination was used for surface examination. Simple random sampling technique was done to divide the samples among four groups (sample size: n = 15 per each group) [Table 1]. In addition to 15 samples in each group, two more samples were included in the study. These samples were used for SEM evaluation before and after demineralization.
Preparation of enamel blocks
Samples were subjected to ultrasonic and then transferred to 0.1% thymol solution. The crowns of all the incisors were separated from the roots at the cementoenamel junction. Enamel blocks of 3 × 3 × 2 mm were prepared from flatter labial surface and embedded in polymethyl methaacrylate. The superficial surface of the enamel was ground flat with water-cooled carborandum disc (1200 grit; waterproof silicon carbide paper; Struers, Germany) and polished with a polishing paste (15 μm diamond paste; Struers), thereby removing approximately 100 μm of the outermost enamel layer and yielding a flat surface.
Pre-demineralization Vicker's microhardness test and SEM examination
All the samples were then subjected to Vicker's microhardness test to record the baseline microhardness values. Hardness was recorded under 500 g of load for 15 s. Baseline microhardness values were recorded as SMH1.
One sample was subjected for SEM evaluation to get the image of an intact enamel surface. Before the procedure, the sample was kept in hot air oven for 15 min to obtain complete dehydration. Considering the fact that the enamel is a nonconductive material, it was made conductive by giving a coating of gold through a process known as sputtering. Gold-coated sample was finally loaded into the sample chamber of SEM.
Preparation of demineralizing solution
Demineralizing solution was prepared with 0.05 M lactic acid, 2.2 mM of calcium chloride, and 2.2 mM of sodium dihydrogen orthophosphate. Potassium hydroxide pellets were added to adjust the pH of the solution to 4.5.
Post-demineralization Vicker's microhardness test and SEM examination
After preparing the demineralizing solution, all the samples were kept in the solution for 72 h after which the samples were subjected for Vicker's microhardness test (recorded as SMH2). Of 62 samples, 1 sample was again observed under SEM.
Preparation of artificial saliva
Artificial saliva was prepared by adding 3.90 mM of sodium phosphate, 4.29 mM of sodium chloride, 17.98 mM of potassium chloride, 1.1 mM of calcium chloride, 0.08 mM of magnesium chloride, 0.05 mM of sulfuric acid, and 3.27 mM of sodium bicarbonate. The pH of the artificial saliva was set at 7.2.
Initiation of pH cycling
The cycling schedule was set in such a way that all the samples remained in demineralizing solution for 2 h in a day followed by washing the samples in deionized water. An applicator brush was used to coat all the samples with experimental toothpastes. The samples were left undisturbed for 3 min. All the samples were then transferred into artificial saliva. The entire procedures were repeated on the next day. On the third day, the samples were subjected to Vicker's microhardness test (to obtain SMH3). The demineralizing solution and artificial saliva were freshly prepared every third day.
The above-mentioned procedures were repeated for different time intervals, that is, 6th, 9th, and 12th days. All the samples were again subjected to Vickers microhardness test during 6th, 9th, and 12th days of pH cycling (SMHn).
Calculation of Percentage surface microhardness recovery (%SMHR)
It was calculated using the formula %SMHR = 100 (SMHn− SMH2)/(SMH1- SMH2), where n is the mean microhardness value at 3rd, 6th, 9th, and 12th days of pH cycling model, SMH1 is the mean baseline microhardness value, and SMH2 is the mean microhardness value after demineralization for 72 h.
Post-treatment scanning electron microscopic examination
At the end of pH cycling, SEM image of one randomly selected sample from each group was taken to compare the surface roughness.
Statistical analysis and scoring of SEM images
The results obtained were tabulated and subjected to statistical analysis. Descriptive and analytical statistics were done. The normality of data was analyzed using Shapiro–Wilk test. As the data followed normal distribution, parametric tests were used to analyze the data. One-way analysis of variance test was used to check the mean differences between the groups. Post hoc analysis was done using Tukey's post hoc test. SEM images were graded according to Bonetti et al.'s grading criteria [Table 2].
| Results|| |
The mean baseline surface microhardness (SMH) values before demineralization were statistically nonsignificant among the groups [Table 3]. The difference in the mean SMH value after demineralization was also statistically nonsignificant among the groups [Table 3]. SMH value in all the treatment groups had comparatively increased after 12 days of pH cycling when compared with SMH values after demineralization [Table 3].
|Table 3: Enamel thickness of different groups at baseline, after demineralization, and during pH cycling at different time intervals|
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Colgate Sensitive Plus® toothpaste had a significantly greater %SMHR than others at each interval of pH cycling model [Table 4]. Colgate® and BioRepair® toothpaste showed no significant difference between them except for the third day of pH cycling where Colgate® showed better remineralization than BioRepair® toothpaste [Table 5]. The %SMHR of Regenerate Enamel Science™ toothpaste was minimal [Table 4]. Regenerate™ and BioRepair® toothpastes showed significant difference between them except for ninth day of pH cycling where BioRepair® was more effective than Regenerate Enamel Science® toothpaste. Regenerate Enamel Science™ toothpaste showed significantly higher %SMHR than the control group except on third day of pH cycling [Table 5]. The least %SMHR was shown by the control group [Table 4].
|Table 4: ANOVA test for percentage microhardness recovery (%SMHR) of each treatment materials at different time interval (P≤0.001)|
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|Table 5: Tukey's post hoc analysis for intergroup comparison of percentage surface microhardness recovery (%SMHR) at different intervals of pH cycling|
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SEM observation showed a smooth and intact surface on the normal anatomical enamel surface before demineralization [Table 6] and [Figure 1]. After demineralization, uneven and rough surface with marked increased porosities was evident [Figure 2]. Distinct surface coatings deposited by different agents were evident by SEM on the treated anatomical enamel surfaces of the specimens [Figure 3], [Figure 4], [Figure 5], [Figure 6]. After pH cycling, in the Colgate Sensitive Plus® group, crystals sedimented on the enamel surfaces and the cavities and defects of the enamel surface had decreased [Figure 3]. Wrinkles and demineralization of prismatic/interprismatic enamel were evident in the Regenerate™ group [Figure 4]. Presence of surface irregularities on enamel surface, without demineralization of prismatic and/or interprismatic enamel, was visible in the BioRepair® group [Figure 5]. Multiple porosities and irregular surface with slight amorphous surface precipitation were visible in the control group [Figure 6].
|Table 6: Comparison of the four materials under scanning electron microscope using Bonetti et al.'s rating scale|
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|Figure 1: (a) Sem image of enamel before demineralization (under 200 magnification). (b) Sem image of enamel before demineralization (under 3500 magnification)|
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|Figure 2: (a) Sem image of enamel after demineralization for 72 hrs (under 200 magnification). (b) Sem image of enamel after demineralization for 72 hrs (under 3500 magnification)|
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|Figure 3: (a) Enamel surface after application of colgate sensitive plus toothpaste (under 200 magnification). (b) Enamel surface after application of colgate sensitive plus toothpaste (under 3500 magnification)|
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|Figure 4: (a) Enamel surface after application of regenerate enamel science toothpaste (under 200 magnification). (b) Enamel surface after application of regenerate enamel science toothpaste (under 3500 magnification)|
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|Figure 5: (a) Enamel surface after application of biorepair toothpaste (under 200 magnification). (b) Enamel surface after application of biorepair toothpaste (under 3500 magnification)|
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|Figure 6: (a) Enamel surface after treating with de-ionized water (under 200 magnification). (b) Enamel surface after treating with de-ionized water (under 3500 magnification)|
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| Discussion|| |
Caries progression occurs very slowly and intermittently in normal populations as each period of demineralization is followed by a period of rest or even remineralization. The literature reveals that the progression of incipient caries may take 3–48 months. If we are able to intervene during this time period, remineralization is possible. Remineralization of noncavitated lesions has been reported from early twentieth century, when demineralized enamel was observed to harden in the presence of saliva. The immersion of white spot lesions in supersaturated solutions of calcium and phosphate, eventually leading to its reversal, has been well-documented in the literature.
The objective of this study was to determine the effects of three toothpastes and its specific content on the early enamel lesions over a range of time periods under dynamic pH cycling conditions. This remineralization study has allowed demineralization and remineralization to occur in a dynamic equilibrium. In this study, analysis of SMH and surface roughness has been done for the assessment of remineralization potential using Vickers microhardness test and SEM.
Enamel blocks of 3 × 3 × 2 mm were made out of the teeth followed by embedding it in polymethyl methaacrylate. The surface aprismatic layer was removed with a carborandum disc to create a more uniform surface to compare demineralization and remineralization effect on enamel. This protocol followed in this study was in accordance to the study conducted by F Cai et al. (2003) and Huang et al. (2009). In this study, Vickers microhardness was preferred over Knoop hardness considering the fact that Vickers microindenter penetrates into the white spot lesion twice as that of Knoop hardness indenter and allowed to assess the SMH without extensive cracking. Baseline microhardness values were between 270 and 320 VHN (similar to that of sound enamel). The Vickers hardness number was re-recorded after 72 h of demineralization to ensure the reduction in microhardness which ranged from 60 to 70 VHN. The demineralization process on the sample surface was reassured with an SEM image of a randomly selected sample.
The pH cycling model followed in this study was according to the study reported by White. It involved exposure of the enamel samples to various study materials, artificial saliva, and demineralizing solution which simulated the decrease in pH occurring in the oral environment every day, which in turn offered high level of scientific control, lower variability, and smaller sample size requirement., In the interest of standardization, all the samples were subjected to 12 days of pH cycling.
In this study, maximum %SMHR recovery was shown by Colgate Sensitive Plus® with Pro-Argin™ formula followed by BioRepair® toothpaste. Minimal percentage microhardness recovery was shown by Regenerate Enamel Science® toothpaste.
The reason attributed to maximum %SMHR by Colgate Sensitive Plus® group could be due to the presence of arginine, calcium carbonate, and sodium monofluorophosphate. Arginine alone may be insufficient to remineralize demineralized lesions or to provide resistance to further acid challenge. Therefore, a combined use of arginine and fluoride in calcium carbonate base might have triggered the uptake of fluoride, creating more acid-resistant enamel resulting in maximum percentage microhardness recovery by this group.
There was no significant difference in %SMHR shown by Colgate Sensitive Plus® toothpaste and BioRepair® toothpaste on 6th, 9th, and 12th days of pH cycling. The protein matrix which comprises ~1% of the organic part of a mature enamel probably acted as a scaffold for ion exchange of nano hydroxy apatite crystals which is the ingredient in this experimental toothpaste. The enamel proteins situated in the interprismatic space were possibly capable of capturing the mineral from the solutions and allowed for penetration of calcium and phosphorous along the sides of these crystallites, causing an increase in SMH.,,,
Regenerate Enamel Science™ toothpaste with mainly calcium silicate, sodium phosphate, and sodium monoflurophosphate showed the least %SMHR when compared with Colgate Sensitive Plus® and BioRepair® toothpastes. It could be due to the specificin vitro study protocol followed here which is predominantly controlled by basic chemistry fundamentals such as pH, ion concentrations, and (enamel) solubility. This study did not include organic components such as oral bacteria or plaque. Therefore, antimicrobial effects were not active.
The extent of remineralization from exposure to the deionized water would be limited due to the relatively poor mineral nucleation ability of calcium and phosphate of the artificial saliva relative to other groups. Robinson et al. have substantiated that when there is no effective nucleation, the development of acid resistant mineral does not result, leading to leaching of apatite constituents such as calcium, phosphate, and carbonate from the enamel tissue.
In this study, the enamel surfaces in different treatments were examined by an SEM. The different enamel surface morphologies after the corresponding treatments may be due to different mechanisms for promoting remineralization. Following 72 h of demineralization [Figure 2], initial enamel lesion was formed on the surface which showed significantly more porosity than the sound enamel. This allows a greater penetration of solution ion constituents and a larger surface area for a subsequent reaction of enamel mineral. Colgate Sensitive Plus® group [Figure 3] showed maximum reduction in surface irregularities and it can be due to a combination of arginine and calcium carbonate in the experimental toothpaste which favors calcium and phosphate ions to deposit dentin-like minerals within dentin tubules and on the dentin surface. BioRepair® toothpaste [Figure 5] containing zinc carbonated nano hydroxy apatite is said to penetrate the enamel pores acting as a template in the precipitation process and attracting a large amount of Ca2+ and PO43− from the remineralization solution to the enamel surface to fill the vacant positions of the enamel calcium crystals. SEM picture with Regenerate Enamel Science™ toothpaste [Figure 4] showed minor honeycomb pattern of demineralization indicating that very little occurred on the surface which was probably due to theirin vitro protocol followed here. Oral environment and saliva, perhaps, promote surface remineralization along with subsurface remineralization in case of Regenerate Enamel Science™ toothpaste, Deionized water failed to show any significant surface remineralization when observed under SEM [Figure 6].
| Conclusion|| |
All the experimental toothpastes had the potential to remineralize initial enamel lesions, but Colgate Sensitive Plus® toothpaste had the maximum effect on demineralized enamel surface. Colgate Sensitive Plus toothpaste with Pro-Argin™ formula can be regarded as a potential remineralizing agent. It can be concluded as a noninvasive means of managing early enamel carious lesions.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. M Bazanth Yahiya
Department of Pediatric and Preventive Dentistry, Kannur Dental College, Kannur - 670 612, Kerala
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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