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 Table of Contents  
Year : 2020  |  Volume : 7  |  Issue : 1  |  Page : 29-34

Effect of varnish containing casein phosphopeptides-amorphous calcium phosphate and fluoride on surface microhardness of enamel – An in vitro study

1 Department of Dentistry, Hassan Institute of Medical Sciences, Hassan, Karnataka, India
2 Department of Pedodontics and Preventive Dentistry, The Oxford Dental College, Hospital and Research Centre, Bengaluru, Karnataka, India

Date of Submission15-May-2019
Date of Decision30-Jun-2019
Date of Acceptance16-Sep-2019
Date of Web Publication05-Feb-2020

Correspondence Address:
Dr. Priya Subramaniam
Professor and Head, Department of Pedodontics and Preventive Dentistry, The Oxford Dental College and Hospital, Bangalore - 560 068, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sjos.SJOralSci_43_19

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Background: Fluoride varnish is one of the professionally applied topical fluoride agents. The ease of application of fluoride varnishes has led to its popularity in pediatric dentistry.
Aim: The aim of the study is to evaluate and compare the effect of varnish containing casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) and fluoride (MI Varnish®) with that of varnish containing only fluoride (Fluor Protector®) on surface microhardness (SMH) of enamel.
Materials and Methods: Enamel blocks were cut from the 90 premolar teeth samples. The samples were divided into three Groups (A, B, and C) consisting of 30 blocks each. Varnish containing CPP-ACP with fluoride was applied on samples of Group A and varnish containing only fluoride was applied on the samples of Group B. Group C was used as control group; without any varnish application. After varnish application, these samples were subjected to pH cycling. Following, SMH was assessed using SMH tester machine.
Results: The mean values of SMH for Group A were 488 ± 6 vickers hardness number (VHN), Group B were 485 ± 12 VHN, and Group C were 448 ± 12 VHN.
Conclusion: There was no significant difference in the SMH of enamel between the varnish containing CPP-ACP and fluoride, with that of varnish containing only fluoride.

Keywords: Demineralization, dental caries, enamel, VHN

How to cite this article:
Babu K L, Subramaniam P, Teleti S. Effect of varnish containing casein phosphopeptides-amorphous calcium phosphate and fluoride on surface microhardness of enamel – An in vitro study. Saudi J Oral Sci 2020;7:29-34

How to cite this URL:
Babu K L, Subramaniam P, Teleti S. Effect of varnish containing casein phosphopeptides-amorphous calcium phosphate and fluoride on surface microhardness of enamel – An in vitro study. Saudi J Oral Sci [serial online] 2020 [cited 2020 Jul 14];7:29-34. Available from: http://www.saudijos.org/text.asp?2020/7/1/29/270534

  Introduction Top

Fluoride is a preventive agent that has almost mesmerized the field of dentistry. It is one of the elements categorized as strongly cariostatic in nature. Over the last few decades, the worldwide reduction in dental caries in both primary and permanent dentition is largely due the extensive use of fluoride.

Topical fluoride delivered via various vehicles is an effective anticaries treatment. Fluoride varnish is a standard remineralizing agent which is professionally applied. The ease of application of varnishes has led to its popularity in pediatric dentistry. Fluoride varnish works by increasing the concentration of fluoride in the outer surface of teeth, thereby acting as a slow-releasing reservoir of fluoride which is released during the early stages of demineralization.

Although fluoride has a profound anticaries effect, it is far from complete cure of the carious lesion. It is unlikely that any concentration of fluoride that will eliminate caries totally. High fluoride strategy cannot be followed in most instances to avoid potential for adverse effects due to overexposure to fluoride. This need has redirected research to develop novel preventive agents that can act as an adjunct to fluoride or independent of it.

Casein phosphopeptides-amorphous calcium phosphate (CPP-ACP) is one such agent that has been proposed to have anticariogenic properties. CPP is a sticky protein that binds and stabilizes calcium and phosphate ions in an amorphous state. The CPPs are multiphosphorylated peptides derived from enzymatic tryptic digestion of milk protein, casein, from cow's milk. Through phosphoseryl residues, CPP stabilizes calcium and phosphate ions in solution as an ACP. CPPs can bind up to 25 calcium ions, 15 phosphate ions, and 5 fluoride ions per molecule. CPP-ACP has been shown to localize to tooth surfaces. During acidogenic challenges, it might release calcium, phosphate, and fluoride, thus maintains the supersaturation of calcium and phosphate ions in the vicinity of the tooth surface and depresses demineralization and enhances remineralization. It readily binds to saliva pellicle, plaque, soft tissues, and even to the hydroxyapatite component of enamel.[1]

Commercially, CPP-ACP has been incorporated into chewing gums, dentifrices, pastes, crèmes, lozenges, and rinses. Studies that reported the benefits of the synergistic effect of CPP-ACP with fluoride were carried out on dentifrices.[1],[2],[3] Recently, a combination of CPP-ACP with fluoride has been introduced as a dental varnish – MI Varnish®. An earlier study evaluated the ion release of this varnish.[4] Hence, the aim of the present study was to assess the efficacy of newly introduced varnish containing CPP-ACP and fluoride (MI Varnish®). The null hypothesis was that there is no difference in the efficacy of both the varnishes. It was tested by evaluating surface microhardness (SMH) of enamel following application of varnish containing CPP-ACP and fluoride (MI Varnish®) and comparing it with that of varnish containing only fluoride (Fluor Protector®).

  Materials and Methods Top

Ethical clearance to conduct the study was obtained from the institutional review board prior to commencement of the study. Premolar teeth samples extracted for the purpose of orthodontic treatment were collected from normal, healthy children aged 13–15 years. Written informed consent was obtained from patients as well as parents for the use of these teeth in this particular study. Teeth with intact enamel surfaces and no signs of decalcification were included in the study. Teeth with caries, developmental defects, and signs of fracture were excluded. A total of 90 teeth were collected. The soft-tissue deposits and calculus were removed from the teeth with a surface scaler. Teeth were cleaned using slurry of pumice. All teeth were then stored in distilled water containing 0.2% thymol to inhibit the microbial growth until the study was carried out.

Estimation of surface microhardness of enamel

All the 90 samples were sectioned using Silverstone–Taylor hard-tissue microtome (Scientific Fabrications, Littleton, CO, USA) to obtain enamel blocks (3 mm × 3 mm) from the most prominent portion on the buccal surface of crown. The blocks were serially polished and flattened using polishing grits no. 800, 1000, and 1200. These blocks were embedded in acrylic blocks and smoothened to achieve a flat surface.[5]

All the samples were divided into three groups, namely, Group A, B, and C consisting of 30 enamel blocks each. On the enamel blocks of group A, a thin layer of varnish containing CPP-ACP with fluoride (MI Varnish®, GC India Dental Pvt Ltd., India) was applied according to manufacturer's instructions using a soft sponge pellet provided by the manufacturer. On the enamel blocks of Group B, a thin layer of varnish containing only fluoride (Fluor Protector®, Ivoclar Vivadent, Amherst, and NJ, USA) was applied according to manufacturer's instructions using a soft-bristled applicator tip provided by the manufacturer [Table 1]. Enamel blocks of Group C were used as control group and no varnish was applied. The samples were stored in deionized water till further processing.
Table 1: Composition of the varnishes used in the study

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After 24 h, the varnish was removed carefully from enamel a block of Groups A and B. Removal was completed with cotton swabs soaked in acetone. Acetone was used, as it is a good solvent for many plastics and some synthetic fibers and used as one of the volatile components of some paints and varnishes. Then, the blocks were washed with deionized water for 1 min. Demineralization and remineralization solution was prepared in our department according to Rodrigues et al.[6] All the samples from each group were subjected to a demineralization-remineralization cycle simulating a high caries challenge.[6]

The enamel blocks were immersed in demineralizing solution (2.0 mmol/L calcium, 2.0 mmol/L phosphate in 0.075 mol/L acetate buffer, 0.02 μmol F/mL, pH 4.7) for 3 h (35.5 mL per block) for 3 h.[5],[7] After 3 h, all the samples were removed from demineralization solution and dried using a blotting paper. Following, all the samples were immersed in remineralizing solution (1.5 mmol/L calcium, 0.9 mmol/L phosphate, 150 mmol/L KCl in 0.1 mol/L Tris buffer, 0.03 μmol F/mL, pH 7.0) for 21 h.[5] This cycle was repeated every day for 7 days.[5],[7] On 8th day, all the samples were taken out of the solution and dried using blotting paper and SMH was assessed.[5],[7],[8],[9],[10] The modified pH-cycling model allowed the evaluation of changes on the outermost enamel layer during caries development.[9] On 8th day, all the samples were taken out of the solution and dried using blotting paper.

The SMH of enamel samples of Group A, B, and C was assessed using the microhardness tester machine (Shimadzu HMV-2000/Shimadzu Corporation, Kyoto, Japan). SMH of each sample was assessed by making an indentation on enamel by applying 25 mg of load for 10 s.[5],[11] The value displayed on the machine was noted. Five such indentations were made on left upper, left lower, central, right upper, and right lower part of the enamel block. The average SMH of five indents was calculated for each sample. The values were expressed in VHN.

The data obtained for SMH of enamel were tabulated and subjected to statistical analysis.

Statistical analysis

Student's t-test was used for the comparison of two means. SPSS software 19.0 (IBM Corp. Released 2010, IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY, USA) was used for the analysis of the data.

  Results Top

The mean values of VHN for Group A were 488 ± 6, Group B were 485 ± 12, and Group C were 448 ± 12 [Table 2]. On intercomparison between three groups, the mean difference of SMH of enamel between Group A and B was not statistically significant (P = 0.35). However, the mean difference between Group A and C and between Group B and C were statistically significant [Table 3].
Table 2: Mean surface microhardness of enamel of Groups A, B, and C

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Table 3: Comparison of surface microhardness of enamel between all three groups

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  Discussion Top

The growing emphasis on prevention of dental caries has led to rapid development of newer agents and more innovative delivery systems modalities, aimed at early prevention. In this context, fluoride varnishes are fast becoming an integral component of prevention-based programs along with patient and parent education. Fluoride varnishes have replaced topical gel treatments in many countries.

The fluoride ions released by fluoridated varnish has a greater interaction with enamel and provide less mineral loss with reduced enamel surface demineralization and decreased depth of carious lesion.[5] Fluoridated varnishes are more effective in inhibiting erosive enamel loss than APF gel.[12] They are also effective in reducing or arresting the white spot lesion.[13] Other studies have also shown that fluoride varnish is clinically effective.[14],[15],[16]

The fluoride retention on enamel and subsurface lesion remineralization depends on the availability of the calcium and phosphate ions. However, combining calcium phosphate and fluoride ions can lead to loss of bioavailable fluoride ion due to a reaction between the calcium phosphate phase and the fluoride ion. In an approach to overcome this incompatibility of calcium phosphates and fluoride ions, various calcium phosphate technologies have been developed. One such approach is incorporating stable CPP and unstable ACP into dental products. The presence of the CPP stabilizes the ACP phase to deliver bioavailable calcium, phosphate, and fluoride ions to the tooth surface and promotes remineralization of carious lesions.[17]

Using this technology, many tooth creams have been marketed. Dentifrice containing CPP-ACP with and without fluoride have shown the remineralization of carious lesion, occlusion of the dentinal tubules, and reduction in depth of carious lesion.[18] Treatment of softened enamel surface with CPP-ACP rehardens the enamel surface significantly when compared to fluoride-treated enamel surfaces.[19] Cai et al. demonstrated that CPP-ACP containing rinsing solution produced higher remineralization than the controls.[20] A study reported that the combination of CPP-ACP and photoactivated disinfection proved to be very effective for stabilizing root surface caries in clinical practice.[21] CPP-ACP paste decreases lesion depth and has higher remineralization potential when used in combination with fluoride toothpaste than when used alone.[22] Despite all, the beneficiary effects of CPP-ACP containing toothpastes, its effect is largely dependent on compliance of patient. Professionally applied remineralizing agents have an upper hand when compared to home-based oral health-care products.

The pH cycling protocol followed in the present study was as described by Featherstone et al.[23] This protocol is most commonly used for human enamel, which is a modification of protocol proposed by ten Cate and Duijsters.[24] The model is of particular interest because it simulatesin vivo high caries risk condition and simultaneously measures the net result of the inhibition of demineralization and the enhancement of remineralization. In this model, the dynamic cycles of de- and remineralization are simulated by sequentially immersing enamel specimens in acidic (demineralizing) and supersaturated (remineralizing) buffer solutions. These de- and remineralization solutions approximate the mineral ion composition and supersaturation of saliva.[25]

Measuring the surface characteristics of teeth by means of microhardness test is a common method for studying enamel surface changes after remineralization and demineralization cycles. To rule out the initial SMH variations of enamel samples, SMH in all three groups was measured and found to have no statistically significant difference. Hence, changes in microhardness values after intervention would be attributed to the fluoride therapy procedure.

Various studies have used different methods to assess the SMH of enamel. The commonly used microhardness tests are Vickers microhardness test and Knoop microhardness test. The instrument used in Vickers microhardness test is designed for rapid microhardness tests of all types and shapes of metallic and nonmetallic materials.[19] A diamond indentor provides diagonal measurements of the indentations and resultant hardness values.[11] In tooth hardness studies, the Vicker indenter is more useful, as Vickers is less prone to specimen curvature and can be used on more demineralized specimens than Knoop. Therefore, the authors have proposed to use the Vicker indenter in the tooth hardness studies.[26] As this study focused on evaluation of SMH of enamel, Vickers SMH test was used.

In the present study, comparison of SMH of enamel of Groups A and B with that of Group C showed a statistically significant difference. This could be because the enamel blocks were polished and made sufficiently flat area to assess the enamel SMH. Thus, the area subjected in both cycles was not the original surface of the enamel. Removal of the outer layer of the enamel (the layer often containing fluorapatite) could have made the enamel more susceptible to the softening process. Whereas, the fluoride ions present in the varnishes applied to Groups A and B, after coming into contact with free calcium and phosphate ions present in both solutions, would have led to the rapid formation of fluorapatite on the surface layer of enamel.[2]

Fluor protector contains 0.9% difluorsilane in a polyurethane varnish base with ethyl acetate and isoamyl propionate solvents. The fluoride content is equivalent to 0.1% or 1000 ppm in solution.[27] As the solvents evaporate, the fluoride concentration at the tooth surface will increase to much higher values (nearly ten times higher). Bi Fluorid 10® contains 5% sodium fluoride equal to 22,600 ppm of fluoride and 5% calcium fluoride. It permits an immediate high fluoride release through sodium fluoride. Fluor Protector® has been shown to exhibit higher demineralization inhibitory effect, in comparison to Bi Fluorid 12®.[28] Formation of fluorhydroxy apatite or fluorapatite could be the probable reason because fluorapatite is harder and more resistant to acid dissolution than hydroxyapatite.[11] An increase in mineral content following application of both studied varnishes, Fluor Protector® and MI Varnish®, could be responsible for higher VHN values obtained in this study.

The SMH of enamel in Group A was higher than that of B, but the difference was not statistically significant. This could be because at the enamel surface of Group B, the fluoride ions come in contact with free calcium and phosphate ions of deremineralizing solutions and fluorapatite is rapidly formed. Whereas, in Group A, the presence of CPP-ACP in the varnish could have prevented the rapid transformation of the calcium phosphate phase. The calcium and phosphate ions will form a reservoir like deposits on the surface of enamel. These ions stabilize calcium and phosphate phase furthermore and will drive the diffusion gradients into the subsurface lesions. Therefore, CPP has the ability to localize and stabilize these ions at the tooth surface in correct molar ratio (Ca: PO4: F =5:3:1).[6] Similar to our study, Santos et al. also showed that there is similar increase in SMH of enamel by calcium nano phosphate paste as compared to high concentrated fluoride agents.[15]

However, CPP-ACP tooth cream showed a lower SMH of enamel compared to fluoridated varnish in anin vitro study.[11] Mehta et al. also reported a lower hardness value with CPP-ACP than bioactive glass. This difference could be due to the use of only CPP-ACP without fluoride in their tested products. Furthermore, both the CPP-ACP products were in tooth cream form. Tooth cream being creamy inconsistency may not properly wet the surface.[29]

It is an effective way to increase the concentration of fluoride in enamel by coating varnish on the surface of deciduous teeth. Shorten the coating period could increase the concentration of fluoride in enamel effectively, but the concentration of fluoride in enamel does not go up along with the coating dose increase.[30] The varnish Duraphat was more effective in prevention of dental caries than fluorprotector in a 3 years follow-up study.[31] However, on comparison of dental caries increments in the Duraphat and Fluor protector, the caries reduction of Fluor protector was 35%. However, children with the highest dental caries increment were benefitted from the applications of Duraphat.[32]

The effect of fluoride ion on surface hardness of dental enamel has been investigated in numerous studies. Argenta et al. studied the effect of fluoride on the surface hardness of teeth and found that higher concentrations of fluoride ion were correlated with decrease in loss of inorganic content.[9] Wiegand et al. also showed that increasing fluoride concentration results in higher microhardness of enamel samples.[33] Various forms of fluoridated products have been compared in former studies. Lee et al. in 2010 investigated the effectiveness of three local fluoride therapy agents (NaF 2% solution, APF foam, and fluoride varnish) and observed that changes in microhardness as a result of the application of these agents were not significantly different.[34]

The results of the present study showed varnish containing CPP-ACP and fluoride is as effective as fluoride varnish. Although fluoride has a profound effect on the level of caries progression, it is unlikely that there is any concentration of fluoride that will eliminate caries totally. High fluoride strategy cannot be followed due to its adverse effects. In addition, patients are instructed not to brush their teeth for 24 h following varnish application. During this time, fluoride is ingested and not expectorated. In addition, overapplication is a common occurrence. Thus, there is a need for nonfluoridated remineralizing agents or remineralizing agents with less fluoride. Hence, varnish containing CPP-ACP and fluoride can be effectively used as an alternative for fluoride varnish.

As this study was carried out underin vitro conditions on premolar teeth, the results may not be transferred completely to anin vivo situation. The process of dental caries is dependent on several biological factors that are present in the oral environment. Furthermore, the porous nature of primary enamel may facilitate increased incorporation of fluoride, calcium, and phosphate ions into the lesion. Therefore,in vivo studies on primary and permanent teeth needs to be carried out in order to further evaluate the effectiveness of varnish containing CPP-ACP with fluoride.

  Conclusion Top

The mean difference of SMH of enamel between Group A (varnish containing CPP-ACP with fluoride) and B (varnish containing only fluoride) was not statistically significant. The varnish containing CPP-ACP and fluoride can be effectively used as an alternative for fluoride varnish as high fluoride strategy cannot be followed due to its adverse effects.

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3]


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