|Year : 2010 | Volume
| Issue : 4 | Page : 264-270
In vivo remineralization of artificial enamel carious lesions using a mineral-enriched mouthrinse and a fluoride dentifrice: A polarized light microscopic comparative evaluation
K Bansal1, K Gauba2, A Tewari3, HS Chawla3, A Sahni4
1 Professor and Head, Department of Pedodontics and Preventive Dentistry, SGT Dental College, Gurgaon and Ex-Junior Resident, Oral Health Sciences Center, Post Graduate Institute of Medical Education and Research, Chandigarh, India
2 Principal, Dr HS Judge Institute of Dental Sciences, Post Graduate Institute of Medical Education and Research, Chandigarh;Former Professor, Oral Health Sciences Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3 Former Professor, Oral Health Sciences Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India
4 Former Professor, Department of Geology, Panjab University, Chandigarh, India
|Date of Web Publication||25-Jan-2011|
Professor and Head, Department of Pedodontics and Preventive Dentistry, SGT Dental College, Budhera, Gurgaon, Haryana
| Abstract|| |
Background: Remineralization process is accelerated by the presence of fluoride ions in the oral environment, but this mechanism of caries reversal will be further enhanced if the concentration of calcium, phosphate and fluoride ions is supersaturated with respect to that of oral fluids. Aim: This in vivo study was carried out to evaluate and compare the remineralizing efficacy of a urea-based mineral-enriched mouthrinse and a fluoridated dentifrice using an in vivo intraoral appliance model and polarized light microscopic evaluation technique. Materials and Methods: The specimens were prepared from sound teeth and artificial caries was produced using an artificial caries medium in vitro and enamel specimens were inserted in removable orthodontic appliances that were to be worn by 14 children of 10-15 years of age. They were divided into three groups - nonfluoridated dentifrice, fluoridated dentifrice and mineral-enriched mouthrinse groups. After the 6-month experimental period, during which the enamel specimens inserted in the intraoral appliance were subjected to one of the agents (either fluoride, nonfluoride dentifrice or mouthrinse) in vivo, the specimens were retrieved from the patients and were evaluated using the polarized light microscopic technique. Observations and Results: On analysis, mineral gain occurred in all groups, viz. nonfluoride dentifrice group, fluoride dentifrice and mineral-enriched mouthrinse group. However, it was found to be complete in the mouthrinse group, i.e. both at the surface and at the subsurface (67%), while in the fluoridated dentifrice group, 43% of the samples showed mineral gain in both zones. In the nonfluoridated dentifrice group also, remineralization occurred but was limited either to the surface or the subsurface zone. Conclusions: Urea-based mineral-enriched mouthrinse was shown to be more efficacious in the process of remineralization of artificial carious lesions.
Keywords: Artificial caries, polarized light microscopy, remineralization, urea-based mouthrinse
|How to cite this article:|
Bansal K, Gauba K, Tewari A, Chawla H S, Sahni A. In vivo remineralization of artificial enamel carious lesions using a mineral-enriched mouthrinse and a fluoride dentifrice: A polarized light microscopic comparative evaluation. J Indian Soc Pedod Prev Dent 2010;28:264-70
|How to cite this URL:|
Bansal K, Gauba K, Tewari A, Chawla H S, Sahni A. In vivo remineralization of artificial enamel carious lesions using a mineral-enriched mouthrinse and a fluoride dentifrice: A polarized light microscopic comparative evaluation. J Indian Soc Pedod Prev Dent [serial online] 2010 [cited 2014 Jul 28];28:264-70. Available from: http://www.jisppd.com/text.asp?2010/28/4/264/76156
| Introduction|| |
The natural repair of early carious lesions, also known as lesion remineralization, has received considerable attention during the last decade. The concept of remineralization has been known for almost a century.  The chemistry, including the mechanism of remineralization, was studied in detail by many investigators. ,,,,
Dental caries is a dynamic process comprising of alternating periods of demineralization and remineralization. The process of remineralization begins right from the earliest stage of dental caries initiation. Further, if the reservoir of fluoride ions is maintained at the plaque-enamel interphase of incipient (white spot) lesion, the latter never progresses to cavitations and becomes arrested.  During the caries process, the enamel mineral ions such as calcium and phosphate get lost due to acidic pH and result in porosities between the crystallites.  When the supersaturation of calcium and phosphate ions is elevated than that of oral fluids, redeposited enamel mineral is formed either due to regrowth of existing crystallites or de novo formation of crystallites. , This forms the basis of mechanism of remineralization. The concept of remineralization has been documented in several studies, clinical, experimental and using intraoral in vivo models. ,, Most of these studies have emphasized on the beneficial effects of fluoride. Urea is another constituent of saliva known to raise the pH of plaque, which has not been studied much in remineralization studies.  On breakdown, urea releases ammonia, which raises the plaque pH and causes the reprecipitation of calcium and phosphate and fluoride ions in the plaque-enamel interphase.  The present study was planned to investigate the remineralizing efficacy of a urea-based mineral-enriched mouthrinse (containing calcium, phosphate and fluoride and urea) and to compare it with that of a fluoridated dentifrice using the polarized light microscopic technique.
| Materials and Methods|| |
A total of 14 sound teeth, premolars and third molars extracted due to orthodontic reasons and impaction were collected and stored in thymol solution till use. Three enamel blocks were prepared in the longitudinal direction out of each tooth with the help of carborundum discs attached to the micromotor handpiece. All the three enamel blocks were first exposed to a demineralizing medium containing 2 mM calcium, 1.8 mM phosphate, 0.1 mM lactic acid, sodium carboxy methyl cellulose and pH 4.0.  One of the three prepared enamel blocks was kept as control and did not receive any in vivo treatment while the other two, designated as experimental, were inserted in the orthodontic appliance of a child undergoing orthodontic therapy. Informed consent was taken from a total of 14 participants who were divided into three groups, i.e. nonfluoridated dentifrice group, fluoridated dentifrice and mineral-enriched mouthrinse group.
During the experimental period, which continued for a duration of 6 months, all the participants were followed-up regularly on a monthly basis in the dentifrice groups and on a fortnightly basis in the mouthrinse group. They were instructed to brush twice daily with paste in the dentifrice group and rinse twice daily with mouthrinse in the mouthrinse group. The mineral-enriched mouthrinse was especially prepared for this investigation and contained 20 mM calcium, 12 mM phosphate, 4.72 mM fluoride (in the form of monofluorophosphate), 500 mM urea and spearmint, vanilla and saccharin were added for taste and flavor. These dentifrices and mouthrinse were provided to the participants by the investigator herself. In the mouthrinse group, the children were also provided with nonfluoride dentifrice.
After the experimental period, samples were retrieved from the patient's appliances and were subjected to evaluation by polarized light microscopy.
For analysis, each sample was prepared on a glass slab by grinding with abrasive powder having a mesh size no. 800. The experimental enamel samples were then imbibed in quinoline for 24 h, whereas controls were examined after water imbibation for 24 h. The same controls were then dried and imbibed in quinoline for 24 h and again examined for comparison with the experimental samples. Further, these samples were subjected to quantitative birefringence analysis using the method given by Darling.  The form birefringence values for two arbitrarily selected points on the enamel samples, designated as A (surface) and B (subsurface), were calculated and the comparison was made between the control and the experimental samples.
| Results|| |
Artificial caries technique
In the previous investigation,  where scanning electron microscopy (SEM) was used as the evaluation technique, the enamel samples, both control and experimental, were subjected to demineralization periods of 16 h after standardization. However, when 16 h had elapsed, the lesion was examined under polarized light microscope, and it was found to be insignificant in depth [[Figure 1]a and b]. Hence, the enamel samples, both control and experimental, were exposed to various time periods such as 60, 132 (~five and a half days) and 240 h (~10 days) to demineralize the medium. A uniform and desirable body of lesion was evident in the enamel samples after as 5.5 days, the demineralizing period as shown in [Figure 1]c. After 10 days of exposure, it was found that the surface enamel got fractured during sample preparation because it had become too brittle to bear the stresses of grinding, i.e. a cavitation had formed [[Figure 1]d]. As a result, all the samples during the polarized light microscopy Polarised Light Microscopy (PLM) experiment were demineralized for a standard period of 132 h.
|Figure 1 :Polarized light micrographs of longitudinal ground sections of artificially demineralized enamel samples (x50) imbibed in water showing: (a) at 16 h demineralization period - initiation of carious lesions in the form of isolated dark areas of positive birefringence (b) at the surface of the enamel, (b) at 60 h - initial lesion in the form of a narrow body of lesion (b) with lobulated inner advancing front and intact surface layer, (c) At 132 h - broad, positively birefringent dark body of the lesion (b) with smooth inner advancing front and intact surface layer, (d) At 240 h - a narrow, dark, positively birefringent body of lesion with the evident surface breakdown exhibited by step-like formation at the junction of sound and carious enamel|
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The photographic result of intraoral remineralization of artificial carious lesions using various agents is presented in [Figure 2],[Figure 3],[Figure 4]. Under PLM, the control enamel samples revealed demineralization in the form of body of lesion, which was distinct, dark and extended well beneath the surface of the enamel when examined after imbibation in water. The in vivo effect of various test agents was found to be as follows:
|Figure 2 :Polarized light micrographs of longitudinal ground sections of control (a) and experimental (b) enamel samples (at x50) after in vivo nonfluoride dentifrice treatment as seen after quinoline imbibation: (a) control section showing translucent body of lesion (b) and translucent zone (tz), (b) experimental section showing a broad, positive dark zone at the advancing front of the lesion|
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|Figure 3 :Polarized light micrographs of the longitudinal ground sections of the enamel (at x50) after in vivo fluoride dentifrice treatment, as seen after quinoline imbibation: (a) control section showing the body of the lesion as a translucent area and a very faint band at the inner border of the lesion, (b) experimental section showing the body of the lesion with laminations and a broad, dark, positive birefringent dark zone.|
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|Figure 4 :Polarized light micrographs of longitudinal ground sections of enamel (at x50) after in vivo mineral-enriched mouthrinse treatment as seen after quinoline imbibation: (a) control section - translucent body of lesion (b), (b) experimental section showing well-developed dark body of lesion (dz) on advancing front as well as on the surface of the lesion.|
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Nonfluoridated dentifrice group
Qualitative analysis of the in vivo treated enamel samples in this group revealed mineral precipitation in the form of dark zones, but they were confined to the subsurface areas [Figure 2] in 66% of the samples.
On quantitative analysis, it was found that form birefringence, which is a measure of pore volume due to carious dissolution, was reduced in the surface zone in 33% of the experimental samples as compared with the respective controls and in the subsurface in 66% of the samples [Graph 1].[Additional file 1]
Fluoridated dentifrice group
Qualitative analysis: Enamel samples treated in vivo with fluoridated dentifrice showed the presence of well-defined prominent dark zones in 70% of the experimental samples [Figure 3].
On quantitative analysis, it was found that form birefringence got reduced in 66% of the experimental samples in subsurface zones. In addition to this, 43% of the samples showed a decrease in form birefringence both at the surface and at the subsurface zones, thus indicating a complete remineralization. Overall analysis showed that 85% of the experimental enamel samples had net mineral gain as shown in Graph 2.[Additional file 2]
Mineral-enriched mouthrinse group
Qualitative analysis of the enamel samples in this group revealed remineralization in the form of dark zones in all samples analyzed. However, there was variation in the site of the dark zone. In 50% of the samples, the dark zones were present in the surface as well as the subsurface zone encircling the body of the lesion.
Quantitative analysis [Graph 3][Additional file 3] revealed complete remineralization, i.e. on both surface as well as subsurface, in 67% of the samples as compared with the control, while in the remaining samples, this was seen either on the surface or subsurface zone, Therefore, overall analysis showed positive remineralization in 100% of the samples.
| Discussion|| |
There is a lot of evidence to state that the potential of remineralizing solutions like saliva is markedly increased by the presence of F' ions in concentration as low as 1 ppm or even lower.  Moreover, the balance between the demineralization and the remineralization depends on the levels of saturation of tooth mineral ions, i.e. calcium and phosphate in the oral fluids. If the salivary calcium and phosphate are supersaturated with respect to the tooth mineral ions, further remineralization is enhanced. In addition to this, saliva also contains urea, which can be metabolized by plaque bacteria to liberate ammonia that further neutralizes the acids produced by carbohydrate metabolism.  Fasting plaque has been reported to have a high pH, which has been attributed to salivary urea breakdown.  Based on this, the role of urea in raising plaque pH, Pearce  proposed the use of urea-based mineral-enriched mouthrinse along with other tooth mineral ions that can cause remineralization. The present study aimed to investigate the remineralizing efficacy of this mineral-enriched mouthrinse. An in vivo intraoral model was used to assess the remineralization. A large number of in vitro studies have studied remineralization, but in vivo studies are more close to real-life situations.
Each tooth was cut longitudinally into three samples; one of them served as control and the other two as experimental. Artificial caries was produced in all samples as it was difficult to obtain a large number of uniform, natural white spot lesions. The technique of producing artificial caries was standardized to obtain early caries-like lesions that had almost uniform depth of lesion on which effect of remineralization was to be assessed. The controls were stored in number-coded vials and experimental samples were inserted in removable orthodontic appliances for undergoing the effect of oral environment. Thus, each of the controls had two corresponding experimental samples. Because there are chances of loss of sample from the appliance as well, to have an additional experimental sample, two samples from the same tooth were inserted into the appliance. Further, there were three groups of children who received one of the following test regime: (1) nonfluoride paste, (2) fluoride paste and (3) mineral-enriched mouthrinse. All the children were informed about the enamel specimens and were instructed according to the group distribution. Consent was taken from all the patients.
Several analytical techniques have been used in remineralization experiments like SEM, , microradiography,  quantitative light-induced fluorescence,  microhardness, , polarized light microscopy  and fluoride uptake by enamel. , The polarized light microscopy is a sensitive technique for assessing de- and remineralization in in vivo studies. It requires only a polarized light microscope with a Bereck Compensator to study the enamel samples quantitatively. All other mentioned techniques either need expensive equipment like microradiography or would need a flat surface of enamel sample, which is unsuitable for in vivo evaluation. Keeping in mind all these factors, polarized light microscopy was feasible. In the previous study,  SEM was used to assess the efficacy of mouthrinse and fluoride dentifrice, but it was a qualitative analysis only.
Histological appearance of early enamel caries
The early enamel caries has been divided into four zones based on its histological appearance when longitudinal ground sections are examined under polarized light microscope-translucent zone, body of lesion and dark zone and surface zone. There is a translucent zone at the inner advancing front of the lesion, while a dark zone may be found superficial to this. The body of the lesion is the third zone lying between the dark zone and apparently undamaged surface enamel. This is the major part of the lesion. The unaffected surface zone lies above on the outer side. Because all the zones are not visible in one imbibation media, the control were imbibed in water for 24 h to view the body of the lesion. The experimental samples were seen after imbibation in quinoline for 24 h. Dark zone is the positively birefringent zone and a sign of remineralization and is visible only in quinoline.  Thus, controls were photographed after both media imbibation separately whereas experimental samples were examined only after quinoline imbibation.
The technique for producing the artificial caries was standardized and the results were found to be similar to those of Featherstone. 
All the three groups exhibited remineralization but to a varying degree. For quantitative analysis, the form birefringence, the measure of pore volume of the enamel, of both the control and the experimental samples was determined and compared.
The formula used to calculate form birefringence is:
- Observed birefringence = intrinsic birefringence + form birefringence
where, intrinsic birefringence = 0.003 (Darling 1958)
- Observed birefringence = retardation of sample(r)/thickness of sample (t)
when both t and r are expressed in the same units, i.e. um.
Calculation of retardation of samples was carried out with the help of a Bereck Compensator, as has been described by Winchell 1968.  The values of form birefringence in control with their respective experimental is represented in histograms 1-3 at points A and B. This can be said from the observations that net remineralization occurred more so in the mouthrinse group, followed by the fluoride dentifrice group, and least in the nonfluoride dentifrice group. This confirms the findings of studies that saliva in itself has a remineralization potential, , but to a limited extent. Similarly, fluoride through tooth paste and chewing gums has been shown to enhance the remineralizing efficacy of saliva, as shown by polarized light microscopic studies of Joyston-Bechal and Kidd  and Hattab et al.  On the basis of data, in the mouthrinse group, net remineralization occurred in all samples. The rationale for better efficacy could be that mineral-enriched mouthrinse maintains the supersaturation of plaque fluid with respect to enamel mineral thus available for remineralization whenever plaque pH falls during sugar exposure. Pearce,  under Transmission Electron Microscopy (TEM) visualized the location and distribution of fluor hydroxyapatite deposited in plaque by in vivo treatment with mineral-enriched mouthrinse. SEM findings of the similar kind of experiment also showed an amorphous globular mineral precipitate in experimental samples after in vivo treatment with mineral-enriched mouthrinse. 
Sjogren et al., in an intraoral experiment, compared fluoride and urea chewing gum with controls (placebo and no gum) using transversal microradiography. The data revealed little or no difference between the products, except for an inhibitory effect after using fluoride chewing gums. Similarly, Dames and Dibdin  in their study using urea chewing gum also supported that urea-based gum is likely to inhibit caries when chewed after consumption of carbohydrates than before.
| Conclusions|| |
On the basis of these observations, keeping in view the limitations of an in vivo study, it can be concluded that urea-based mineral-enriched mouthrinse has a better anticaries action on artificial carious lesions than a fluoride vehicle alone. However, to document the long-term clinical performance of this mouth rinse, well-designed clinical trials are required using other sophisticated evaluation techniques that quantify the extent of remineralization, such as polarization-sensitive optical coherence tomography and quantitative light fluorescence.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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