|Year : 2017 | Volume
| Issue : 1 | Page : 19-27
Comparative evaluation of enamel remineralization potential of processed cheese, calcium phosphate-based synthetic agent, and a fluoride-containing toothpaste: An in situ study
Navneet Grewal, Samita Gumber, Nirapjeet Kaur
Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College and Hospital, Amritsar, Punjab, India
|Date of Web Publication||31-Jan-2017|
Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College and Hospital, Amritsar - 143 001, Punjab
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Enamel remineralization potential of variety of products has been established, but there is a lack of evidence of comparison of remineralization potential of natural versus synthetic products. Aim: The aim of this study was to compare the enamel remineralization potential of saliva, cheese, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)-based synthetic agent, and fluoride toothpaste. Design: In situ study was carried out on sixty individuals who wore an intraoral appliance containing demineralized enamel slabs for each agent. One out of six slabs was kept as a control so as to record the baseline values (neither subjected to demineralization nor remineralization). Experimental agents were applied on the designated enamel slabs on day 1, 4, 7, and 10 with a crossover wash out period of 7 days. Quantitative values of mineral content of slab were measured using energy dispersive X-ray and qualitative changes in surface topography of slab were seen under scanning electron microscope at ×20K magnification. Results: Highly significant changes from baseline values were seen in calcium and phosphorus content of slabs treated with cheese and CPP-ACP-based agent whereas levels of fluoride were significantly higher in enamel slabs treated with fluoride-containing toothpaste. Conclusion: Cheese is an organic, economical, and user-friendly option over prescribed synthetic agents. A synergistic effect of fluoride-containing toothpaste with intake of cheese could be a good enamel remineralization protocol.
Keywords: Cheese, casein phosphopeptide-amorphous calcium phosphate crème, fluoride toothpaste, remineralization, saliva
|How to cite this article:|
Grewal N, Gumber S, Kaur N. Comparative evaluation of enamel remineralization potential of processed cheese, calcium phosphate-based synthetic agent, and a fluoride-containing toothpaste: An in situ study. J Indian Soc Pedod Prev Dent 2017;35:19-27
|How to cite this URL:|
Grewal N, Gumber S, Kaur N. Comparative evaluation of enamel remineralization potential of processed cheese, calcium phosphate-based synthetic agent, and a fluoride-containing toothpaste: An in situ study. J Indian Soc Pedod Prev Dent [serial online] 2017 [cited 2017 Mar 23];35:19-27. Available from: http://www.jisppd.com/text.asp?2017/35/1/19/199222
| Introduction|| |
Noncavitated lesions and dental erosions are complex phenomenon that involves a localized mineral loss from tooth surfaces. Typically, the surface layer stays intact and subsurface demineralization occurs which eventually collapses into a full cavity if not controlled in time. Enamel has a unique capacity of natural repair of noncavitated lesions when the demineralized surface is flushed with minerals at neutral or alkaline pH to rebuild a new surface on existing crystal remnants in subsurface lesions.
Natural demineralization of tooth at an early stage is reversed by saliva, which is supersaturated with calcium (Ca), phosphate, buffering agents, fluoride, and other substances. Nevertheless, researches have proved that the net remineralization produced by saliva is minimal and it occurs on the surface layer of the lesion only and new remineralizing systems would be needed to achieve more pronounced lesion regression.
Supported with years of clinically proven research, fluoride has been documented to promote remineralization and can be very easily introduced into the oral environment through personal or professional application. However, its ability to remineralize a lesion is dependent on Ca and phosphorus (P) ions as for every 2 fluoride ions, 10 Ca ions, and 6 phosphate ions are required to form one unit cell of fluorapatite (Ca10 [PO4]6F2). Hence, the availability of Ca and P ions can be the limiting factor for net enamel remineralization to occur.
The relationship between diet and nutrition and dental health has been of great interest for many years. One of the food groups most frequently linked with good oral health is dairy products as they contain high concentration of Ca and Pions and they have been shown to have anticariogenic effects. Recently, the cariostatic properties of cheese have been the subject of intensive research., Processed cheese is hypoacidogenic, prevents demineralization, and enhances remineralization. Remineralization of dairy products is attributed to casein. Casein is one of the two major proteins in milk and accounts for approximately 80% of total protein. Goodness of casein peptide has been commercialized using its extract named casein phosphopeptide (CPP) in various remineralizing agents available in the market.
Although several studies and clinical trials have proved the remineralizing potential and success rate of products based on agents containing CPP-amorphous calcium phosphate (CPP-ACP) with fluoride or without fluoride, very few exist on comparison of naturally occurring Ca-P-based foods which could replenish the lost Ca/P ions from enamel in similar pattern as documented for the above. Hence, the aim of this randomized crossover in situ study was to compare the remineralization potential of saliva, processed cheese, calcium phosphate-based synthetic agent (containing CPP-ACP), and a fluoride-containing toothpaste on demineralized enamel slabs.
| Materials and Methods|| |
The study was conducted through the Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College and Hospital, Amritsar.
After obtaining university/institutional ethical clearance for research, the study was explained to the school authorities, parents/guardians, and children, and written informed consents were taken from school authorities and parents/guardians to carry out the research.
Selection of subjects
The present double-blind, randomized, controlled, four-way crossover in situ study was conducted on sixty individuals selected from a sample of 115 individuals screened from 1000 school-going children aged 7–15 years on the basis of the following criteria:
- Children with healthy oral condition with DMFT <3
- Not using any antimicrobial/antibiotic
- Salivary flow rate of 1 ml/min (unstimulated) and 5 ml/min (stimulated)
- Resting salivary pH between 7.0 and 7.4
- Stimulated salivary pH between 7.0 and 7.4
- Nonallergic to milk/milk proteins/milk products
- Absence of any other medical, physical, or mental condition.
Collection of salivary samples
Individualized salivary samples were collected by making children sit on the chair with their backs straight, feet touching the ground, and their heads slightly tilted downward allowing the saliva to flow into a calibrated plastic cup for 5 min. The collected saliva was labeled as unstimulated saliva. The individuals were then instructed to chew paraffin wax for 2 min and saliva was collected in a separate calibrated plastic cup which was labeled as stimulated saliva. Salivary pH was measured with the help of electronic pH meter. Buffering capacity was checked using Ericsson's method.
Preparation of tooth enamel slabs
Sound permanent molars (indicated for extraction) free from cracks, stains, hypomineralized areas, restorations, and any developmental anomaly stored through proper protocol of sterilization and storage was procured from tooth bank in the Department of Pedodontics and Preventive Dentistry, Punjab Government Dental College, Amritsar, for preparing enamel slabs.
Roots of each of the selected molar were cut at the cementoenamel junction using a low-speed (15,000–20,000 rpm) diamond disk mounted on a straight handpiece (NSK, Japan). The crown of each molar was sectioned mesiodistally into buccal and lingual halves. These halves were further sectioned to obtain six tooth slabs (in which most of the enamel and part of the dentin was available) shaped into a standardized rectangular form (3 mm × 3 mm × 2 mm). Out of the six slabs obtained from each tooth, one tooth slab was kept in an airtight eppendorf and was not subjected to demineralization and remineralization and it was labeled as “P” (positive nondemineralized) control. The rest of the five tooth slabs were immersed in freshly prepared 0.1 M lactic acid buffer solution (0.75 mM CaCl2.2H2O2, 0.45 mM monosodium phosphate) pH 4.8 at room temperature for demineralization. After 2 days, the slabs were removed from the solution, rinsed thrice with distilled water, and placed in fresh demineralizing solution for another 2 days. After a total of 4 days, the slabs were removed from the solution, rinsed with distilled water, and dried with gauze piece. Out of the five demineralized tooth slabs, one tooth slab was kept in airtight eppendorf containing distilled water as a negative control, i.e., it was not subjected to any remineralization procedure and labeled as “D” (demineralized control). The rest of the four demineralized slabs were inserted into a removable midpalatal acrylic Hawley's appliance fabricated on the model made for individual participant.
Preparation of an in situ appliance
A removable midpalatal simple Hawley's appliance was fabricated using self-cure acrylic resin (Pyrex Exports, Roorkee, Dental Products) with retentive stainless steel clasps and a short labial bow (K. C. Smith and Co., UK) and enamel slabs in the premolar region on each individuals cast prepared after taking alginate impressions. Nail varnish was applied on all the slabs. Once the appliance was ready, it was stored in humidified environment (moist gauze piece in a zip lock pouch) till delivered to the participant. The appliance was sterilized in a chemical sterilizing solution (12.5% Alkyl Dimethyl Bezyl Ammonium Chloride by Septodont) as per AAPD recommendations for sterilization of noncritical (intraoral) contact objects before delivering it to the individual.
Every individual was given a code (1–60) and their respective controls (P and D) were tagged with the same code so as to maintain the identification. Participants were instructed not to eat anything for an hour before the insertion of the appliance and were made to rinse with water immediately before inserting the appliance. Nail varnish was removed from one of the slabs and the appliance was worn by participants for 40 min so as to record the effect of saliva on the exposed slab. Participants were instructed to abstain from eating or drinking for these 40 min after which the appliance was removed and kept in a humidified environment (moist gauze in a zip lock pouch). The process was repeated three times after every 60 min on 1st, 4th, 7th, and 10th day.
The exposed enamel slab was removed from the appliance after 10th day, rinsed, and stored in humidified environment in airtight eppendorfs containing distilled water till further scanning electron microscope (SEM) analysis. The eppendorfs were coded 1-S to 60-S (S - saliva-treated slabs).
The above procedure was carried out with three experimental agents vis-a-vis processed cheese (Amul Cheese Slices), calcium phosphate-based crème containing CPP-ACP (Tooth Mousse), and fluoride-containing tooth paste (Colgate Cavity Protection). Each agent was applied individually on each of the coded tooth slab after a crossover wash out period of 7 days. The crème and paste were applied on slabs with the help of a brush extraorally while cheese slices were chewed by participants for 3 min with appliance in the oral cavity. The eppendorfs were coded as 1C-60C (C - cheese), 1T-60T (T - Tooth Mousse), and 1F-60F (F – fluoride-containing toothpaste), respectively.
After completion of the 10th day of application with each of individual experimental agent, corresponding enamel slab was removed, rinsed, and stored in deionized water and sent for the SEM (20.00K magnification) (Hitachi S 4300/M, Japan) and energy dispersive X-ray (EDX) analysis for qualitative and quantitative analysis.
| Results|| |
Scanning electron microscope
Surface topography of all enamel slabs was analyzed using SEM from the center of each slab at ×20.00 K magnification. Microphotographs [Figure 1] of enamel slabs were taken and analyzed.
|Figure 1: Energy dispersive X-ray images showing surface topography of enamel slabs at ×20.00K|
Click here to view
Complete coverage of the surface of enamel slab with a layer of mineral content could be appreciated as diffused globules of Ca and P. No evident loss of prismatic and interprismatic mineral content was seen.
All enamel slabs in this group had irregular surface topography giving it a honey comb appearance. Rough-diffused margins of outlines were seen which may be due to loss of interprismatic substance. A clear difference from baseline group could be appreciated.
Although scattered deposition of globules could be appreciated, complete coverage of enamel surface was not present. The density of deposition was least as compared to other groups.
Surface deposition of minerals could be appreciated in form of tiny granules/globules. The granules represented the mobilization of Ca andP over the enamel surface.
Casein phosphopeptide-amorphous calcium phosphate containing tooth crème
The picture appeared to be similar to that of cheese but supersaturated with granules/globules as compared to enamel slab treated with cheese.
Surface appeared to be homogenously covered by mineral deposition with lesser irregularities in deposition. Although the coverage was regular density of granules appeared to be less compared to the other slabs.
Quantitative analysis of change in mineral content (Ca, P, and F) of enamel slabs was done using electron X-ray dispersive technique. Values obtained were put to statistical analysis using paired t-test using SPSS software version 17.0 (IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp).
Intergroup comparison of mean Ca content (weight%) revealed highly significant increase in Ca content (P < 0.05) [Table 1] in enamel slabs treated with cheese (43.99 ± 2.18) as compared to those treated with CPP-ACP-based crème (48.17 ± 2.21) and fluoride-containing tooth paste (45.07 ± 2.26) and saliva (44.96 ± 2.10) (cheese > CPP-ACP containing tooth crème ≥ tooth paste > saliva > normal > demineralized) [Graph 1].
|Table 1: Intergroup comparison of mean calcium content (in weight %) of normal, demineralized, and remineralized enamel slabs|
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Intergroup comparison of meanPcontent (weight%) revealed highly significant (P < 0.01) [Table 2] increase inPcontent in enamel slabs treated with cheese (21.40 ± 1.33) and those treated with CPP-ACP (21.87 ± 1.37) as compared to those treated with fluoride-containing tooth paste (20.85 ± 1.32) (CPP-ACP containing tooth crème ≥ cheese > tooth paste ≥ saliva > normal > demineralized) [Graph 2].
|Table 2: Intergroup comparison of mean phosphorus content (in weight %) of sound, demineralized, and remineralized enamel slabs|
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Intergroup comparison of mean fluoride content (weight%) revealed highest levels of fluoride in enamel slabs treated with the fluoride-containing tooth paste (3.53 ± 1.35) as compared to enamel slabs treated with cheese (3.16 ± 1.28), CPP-ACP containing crème (3.21 ± 1.24), and saliva (3.12 ± 1.26) (fluoride-containing tooth paste > CPP-ACP containing tooth crème ≥ cheese > normal > saliva > demineralized) with P < 0.05 as compared to all other groups [Graph 3] and [Table 3].
|Table 3: Statistical analysis of intergroup comparison of mean fluoride content (in weight %) of sound, demineralized, and remineralized enamel slabs|
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Ca andP content was converted into Ca:Pratio in each group. Intergroup comparison of Ca:Pratio revealed increase in Ca:Pratio in all remineralized slabs as compared to controls. However, highly significant increase (P < 0.01) [Table 4] was seen on application of cheese (2.21 ± 0.18) as well as CPP-ACP-based crème (2.21 ± 0.19) as compared to other groups, but statistical difference between these two groups was insignificant (P > 0.01) (CPP-ACP containing tooth crème = cheese > toothpaste ≥ saliva > normal > demineralized) [Graph 4].
|Table 4: Intergroup comparison of mean (calcium:phosphorous) ratio of sound, demineralized, and remineralized enamel slabs|
Click here to view
| Discussion|| |
The present study endorses the fact that the remineralization effects are seen with all the experimental agents used in the study.
Out of all commonly consumed dairy products, cheese was chosen to be one of the experimental agents in this study because Ca, P, and pH levels of plaque rise maximum with the use of cheese as compared to other dairy products. Commercially available CPP-ACP-based crème (without fluoride) and a fluoride-containing tooth paste were the other experimental agents.In vitro study designs do not reflect the dynamic conditions of mouth, such as continuous flow and clearance of saliva, even when using artificial saliva as the medium. Moreover, Ca andPions remain in contact with the enamel specimens for 24 h which may not be a representative of normal intraoral conditions where availability of Ca andP can be a limiting factor. Hence, an in situ study design was planned to carry out this research. The best model that could be selected was the individual removable midpalatal appliance which could be removed from the oral cavity once the in situ cycle of experimental agents was completed. The advantage of a removable plate over a fixed appliance is that it makes it possible to have a greater control over experimental variables.
Demineralization of enamel slabs was carried out with the help of 0.1M lactic acid. Lactic acid reacts with enamel hydroxyapatite and produces apatite lactate salts that significantly compromise the strength and resistance of enamel hydroxyapatite  which leads to its easy breakdown. Moreover, lactic acid is the acid produced in maximum amount in human oral cavity, it was chosen as the demineralizing agent of choice to mimic the human oral environment. Quantitative measurements for mineral changes are preferable to qualitative results gained from SEM. The present study made use of one of the most recent techniques available, i.e., SEM with EDX. This microanalytical technique is employed to estimate quantitatively the amount of mineral in a given tooth sample along with the qualitative analysis of its surface topography.
Results of this study revealed that both quantitative and qualitative effects obtained after cheese application were at par with those obtained after CPP-ACP application. However, action of fluoride on demineralized enamel slabs was unique in itself. Although the Ca andP content in fluoride-treated enamel slabs was lesser as compared to cheese and ACP-CPP-treated slabs, but fluoride content was highest in enamel slabs treated with fluoride-containing tooth paste and this rise is undoubtedly appreciable since fluoride makes the enamel surface more resistant to further demineralization by its capacity to improve the crystalline tooth structure with the formation of fluorapatite crystals. The qualitative results showed that surface of enamel slabs treated with fluoride-containing toothpaste had a more homogenous coverage and in an appropriate amount of saturation as compared to cheese and ACP-CPP groups, where supersaturation of deposition of globules was seen.
| Conclusion|| |
Protective food products are gaining attention in modern dentistry because of multiple beneficial effects in oral cavity, but the largest body of evidence is related to the protective action of milk products. Cheese as a protective food has achieved its resonance with evidence of its beneficial effects other than remineralization as it is rich in casein and whey proteins. It protects enamel by its natural CPP, stimulation of salivation, and enhancement of plaque Ca levels and hence acts to reduce demineralization and enhance remineralization. Cheese in diet and fluoride-containing tooth paste are products of daily usage, and hence, their combined use for replenishment of the demineralized surface with Ca and phosphate can be a convenient method of remineralization as compared to usage of synthetic agents available commercially.
This article proves remineralization achieved with cheese and fluoride toothpaste can be a routine home remedy for demineralization of enamel surfaces, especially in high caries risk individuals.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]