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 Table of Contents  
Year : 2019  |  Volume : 11  |  Issue : 1  |  Page : 45-49

In vitro comparison of the remineralizing effect of casein phosphopeptide-amorphous calcium phosphate and fluoride varnish on early carious lesions

Research Institute, School of Dentistry, Universidad de San Martín de Porres, Lima, Peru

Date of Web Publication27-Feb-2019

Correspondence Address:
Dr. Rafael Morales-Vadillo
Manuel Scorza 102 St., Apt. 201, San Borja, Lima-41
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jioh.jioh_248_18

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Aims and Objectives: This study aims to compare the remineralizing effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and fluoride varnish (FV) on the body of carious lesions in surface enamel. Materials and Methods: Two 3 mm × 3 mm areas on 37 samples of recently extracted human tooth enamel were demineralized to create artificial early carious lesions on the enamel following the methodology described by ten Cate and Duijsters. The areas were assigned randomly to two groups on which either CPP-ACP or FV were applied, after which they were subjected to a pH-cycling model to simulate intraoral conditions. Then samples were prepared for evaluation under a polarized light microscope. Data were analyzed using Student's t-test for related groups, with the SPSS statistical package. Results: No statistically significant difference was found in the depth of the body of the lesions between groups following 10 days of pH cycling. Conclusion: It was concluded that both therapeutic systems provide preventive and ultrastructural restorative treatment options which could be included in health-care policies for preventing early carious lesions.

Keywords: Casein phosphopeptide-amorphous calcium phosphate, dental caries, polarized light microscopy, sodium fluoride, tooth remineralization

How to cite this article:
Morales-Vadillo R, Guevara-Canales JO, García-Rivera HP, Bazán-Asencios RH, Robello-Malatto JM, Cava-Vergiú CE. In vitro comparison of the remineralizing effect of casein phosphopeptide-amorphous calcium phosphate and fluoride varnish on early carious lesions. J Int Oral Health 2019;11:45-9

How to cite this URL:
Morales-Vadillo R, Guevara-Canales JO, García-Rivera HP, Bazán-Asencios RH, Robello-Malatto JM, Cava-Vergiú CE. In vitro comparison of the remineralizing effect of casein phosphopeptide-amorphous calcium phosphate and fluoride varnish on early carious lesions. J Int Oral Health [serial online] 2019 [cited 2023 Dec 4];11:45-9. Available from:

  Introduction Top

Preserving and maintaining teeth in the oral cavity is one of the principles of dentistry. Dental caries is a disease with multifactorial etiology which develops as a result of countless demineralization and remineralization events which are unidirectionally unbalanced in favor of mineral loss. In the initial stage of dental caries, mineral loss can only be quantified at the ultrastructural level, and at this stage, the caries process is still reversible. It is therefore essential to have the necessary mineral ions to promote remineralization at ultrastructural level.

One mechanism with proven results is the use of casein phosphopeptides (CPPs) such as CPP-amorphous calcium phosphate (CPP-ACP), which are obtained from cow's milk casein.[1],[2],[3] The deposition of ACP s on the tooth surface contributes to maintaining supersaturated mineral ions that reduce demineralization and promote remineralization by forming hydroxyapatite crystals.[4] It has also been reported that the presence of CPP-ACP delays the formation of bacterial plaque, which fosters crystallization of calcium phosphates in mature biofilms.[5] As a result of these properties, CPP-ACP has been included in oral healthcare products such as remineralizing creams, chewing gums, mouthwashes and particularly, toothpastes.[6]

Another strategy to stimulate remineralization is the use of fluorinated agents. Sufficient evidence is available regarding the remineralizing power of fluoride varnish (FV),[7],[8] which, in addition to reducing mineral loss, increases the resistance to acids of the outer enamel surface by forming fluorapatite crystals.

These two therapies have been compared using various methodologies,[9],[10],[11] but we did not find any reports evaluating the deposition of mineral ions in the zone which Silverstone[12] first called the “body of the early carious lesion” on the enamel surface by observation under polarized light microscopy. The aim of this study was therefore to evaluate the remineralizing effect of CPP-ACP and FV on the body of carious lesions in surface enamel.

The null hypothesis considered for the present study is that there are no statistically significant differences in the remineralizing effect of CPP-ACP and FV.

  Materials and Methods Top

This was an experimental in vitro study, approved by the University Institutional Review Board and conducted in accordance with the World Medical Association Declaration of Helsinki on medical research protocols and ethics, where an informed consent was used for the use of the dental piece for research purposes after dental extraction. Informed consent was used in patients for the subsequent use of extracted teeth for research purposes.

The study was designed by pairing two 3 mm × 3 mm surfaces of human dental enamel. The sample size was estimated according to reports from prior experimental studies.[10],[13] Taking the sample size number of some previous studies,[14],[15] 37 pairs of enamel specimens were selected according to the following inclusion criteria: Healthy human dental enamel from incisors, premolars, and molars which had been recently extracted for periodontal, orthodontic or prosthetic reasons; absence of cracks, abrasion, spots, or stains.

To prepare the specimens, surfaces were cleaned using Hu-Friedy periodontal curettes (RJ, Rio de Janeiro-Brazil) and approximately 200 μm of enamel were removed from vestibular or lingual surfaces by grinding with fine-grain sandpaper in 2% formalin at pH 7 to remove surface contamination. Two 3 mm × 3 mm windows were selected on each tooth and the rest of the tooth covered with two layers of acid-resistant nail varnish (Revlon, New York, USA). Then, the teeth were immersed in 10 ml of a demineralizing solution following ten Cate and Duijsters[16] (CaCl2 2.2 mM, KH2 PO42.2 mM, Acetic Acid/Potassium Acetate 50 mM) for 14 days to artificially simulate early carious lesions.

The areas corresponding to the experimental group were used to test the remineralizing potential of CPP-ACP, which was applied once and left in contact for 3 min, according to the manufacturer's specifications (Mi Paste™). The areas corresponding to the control group were brushed with 5% sodium fluoride (NaF) varnish (Duraphat®, Colgate Oral Pharmaceuticals Inc.).

During the next 10 days, the samples were subject to a pH-cycling system following ten Cate and Duijsters,[14] which simulates the intraoral conditions by using a remineralizing solution (CaCl2 1.5 mM, KH2 PO4 0.9 mM, KCl 130 mM, buffer 20 mM, and NaF 2 ppm) at pH 7 for 16 h (representing the protective effect of the saliva) and a demineralizing solution (CaCl2 2.2 mM, KH2 PO4 2.2 mM, and buffer 50 mM) at pH 4.7 for 8 h (cariogenic challenge), both at 37°C. The demineralizing and remineralizing solutions were changed to prevent depletion or saturation by the accumulation of dissolved enamel products. Each time the solutions were changed, the sections of enamel were rinsed for 10 s in distilled water and dried with tissue paper.

Subsequently, the samples were sectioned perpendicularly to the surface and ground to prepare specimens 30 μm thick on which to conduct the histological examination with polarized light microscopy. This involves placing specimens on a slide immersed in distilled water to obtain positive birefringence in the depth of the body of the carious lesion, in contrast to the negative birefringence of the rest of the lesion and the healthy enamel. A second immersion in Canada balsam enabled observation of the dark and translucent areas described in the histology of caries in tooth enamel.

A Leitz Wetzlar Ortholux II Pol-BK polarizing microscope (Wetzlar-Germany) was used with polarized light intensity of 7.5 International System basic units measured in a Leitz voltage stabilizer (Wetzlar-Germany) and a blue light filter at ×100 magnification.

The criterion to determine the depth of the body of the lesion in the enamel was to observe the entire length of the 3 mm isolated window and select the area where the body of the lesion was deepest under ×100 magnification, i.e., the line dividing the body of the lesion from the dark area.

This criterion is based on the histological description of the progression of early enamel lesions provided by Silverstone[17] [Figure 1], where “body of the lesion” represents the area of greatest demineralization, with large pores that allow water to penetrate, showing positive birefringence. When remineralization occurs, the pores grow smaller and are not visible on immersion in water. Silverstone[15] calls this the “dark zone” which forms a clear boundary with the body of the lesion.
Figure 1: Surface enamel caries. Zones of the early carious lesion in enamel (left) compared to remineralized side (right), in which the dark area is much larger, indicating regression in the lesion. (1) Surface zone, (2) Body of the lesion, (3) Dark zone, (4) Translucent zone and (5) Healthy enamel. Adapted from: Silverstone[17]

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Micrometric measurement was performed on microphotographs taken with a Panasonic DMC-FS15 camera (Panasonic AVC Networks Xiamen Co., Ltd - China) with water as an immersion medium. Measurements were taken in microns [Figure 2]. Data were processed with the SPSS statistical package in Spanish (version 22.0, Chicago, USA) with Windows XP® Operative System. P < 0.05 is considered statistically significant.
Figure 2: Photographs of study zones in a sample observed in distilled water. (a) Macrophotograph of a study sample indicating zones analyzed. (b) Microphotograph of a sample observed in distilled water. Note the line marking depth of the body of the lesion and the measurement corresponding to each group: 86 microns for the CPP-ACP group and 127 microns for the FV group. Polarized light microscopy, magnification × 100. CPP-ACP: Casein phosphopeptide-amorphous calcium phosphate, FV: Fluoride varnish

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The normality of the data distribution in both groups was confirmed by Kolmogorov–Smirnov's test and a Student's t-test for related samples were used to compare measurements of the depth of the body of the carious lesion of surface enamel.

  Results Top

At a 95% confidence interval, the average depth of the body of the early carious lesion ranged from 88.83 to 126.09 μm in the CPP-ACP group (mean 107.46 ± 55.8 μm) and from 92.45 to 125.87 μm in the FV group (mean 109.16 ± 50.1 μm) [Table 1].
Table 1: Depth of the body of the early carious lesion on using casein phosphopeptide-amorphous calcium phosphate and fluoride varnish on human dental enamel

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The comparison of depth of the body of the lesions on the enamel surface by polarized light microscopy for both therapies showed a slight difference in favor of the effectiveness of CPP-ACP [Figure 3], notice the smaller depth of the body of the lesion for the CPP-ACP group]. However, there was no statistically significant difference (P = 0.766).
Figure 3: Distribution of depth of the body of the carious lesion in surface enamel after treatment with casein phosphopeptide-amorphous calcium phosphate and fluoride varnish

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  Discussion Top

This study used polarized light microscopy to evaluate the depth of the body of carious lesions on surface enamel treated with two therapies capable of changing demineralization and remineralization equilibrium in favor of incorporating mineral ions into the surface of human dental enamel.

The proposed null hypothesis is confirmed in which there were no statistically significant differences between the mean values found for the two substances, finding similar to that reported in a systematic review[18] that evaluated the in vivo effect of both substances in the long-term observed in white spot lesions.

Both solutions proved their efficacy as remineralizing agents by reducing the depth of the body of the early carious lesion, thus providing solid evidence for recommending their use as a preventive measure and ultrastructural therapy against the progression of caries.

The study design allows potential bias to be controlled by using paired areas of contiguous enamel from the same tooth, equally subjected to the same loss of mineral tissue by means of the decalcification method proposed by ten Cate,[14] which ensures equal baseline conditions.

It is interesting to confirm the protective effect of CPP-ACP considering that it was applied only once for 3 min, and was comparable to the effect of 5% NaF in the form of FV, which increases contact time by several hours, enabling greater incorporation of fluorine ions into the enamel. Savas[19] concludes that a single application of CPP-ACP is adequate for clinical use in remineralization of carious lesions.

Thakkar et al.[20] reports that the preventive property of the CPP-ACP is even greater if it is used as a varnish along with fluorine, a conclusion with which Chandak et al.[21] agrees.

Tao et al.[22] performed a systematic review and meta-analysis comparing the effect of the combination of both substances against the single use of fluoride, reaching the conclusion that there are no statistically significant differences on smooth surfaces, however on occlusal surfaces the combination of both substances seems to work best.

Güçlü et al.[23] reached the same conclusion from a 12-week clinical follow-up of three experimental groups using CPP-ACP, FV, and a combination of both, complementing a standard oral hygiene program which included fluorinated dentifrice, antimicrobial mouthwash, and xylitol chewing gum. That study found no statistically significant difference but did show a significant difference in the appearance and remineralization of white spot lesions in all groups.

The main advantage of using the sodium fluoride as fluorinated varnish compared to other application forms is its contact time with the tooth surface. Patient acceptance and ease of application make fluorinated varnish a measure to be considered in social oral healthcare programs.

The fact that multiple factors are involved in the formation of dental caries highlights the need for educational and preventive measures, including guidance regarding diet and hygiene, in addition to the use of fluoride and/or phosphopeptide- ACP for prevention and treatment.

Bayrak et al.[14] describe the advantage of using fluorinated varnish with CPP-ACP, stating that the combination of the two substances promotes the release of calcium and fluorine ion, which could enhance the enamel remineralization process, providing greater resistance against demineralization. Controlled clinical trials are required to confirm the data obtained in these studies, considering also studying the effect of the presence of dental plaque as a reservoir of free calcium and phosphate ions.

  Conclusion Top

No statistically significant difference was found, so both therapies tested are acceptable options for preventive treatment and ultrastructure restoration by returning constitutive enamel minerals which have been lost during cariogenic challenges. The characteristics of both products warrant their inclusion in health-care policies as effective preventive measures.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Yengopal V, Mickenautsch S. Caries preventive effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP): A meta-analysis. Acta Odontol Scand 2009;67:321-32.  Back to cited text no. 1
Rosen S, Min DB, Harper DS, Harper WJ, Beck EX, Beck FM. Effect of cheese, with and without sucrose, on dental caries and recovery of Streptococcus mutans in rats. J Dent Res 1984;63:894-6.  Back to cited text no. 2
Harper DS, Osborn JC, Clayton R, Hefferren JJ. Modification of food cariogenicity in rats by mineral-rich concentrates from milk. J Dent Res 1987;66:42-5.  Back to cited text no. 3
Reynolds EC. The prevention of sub-surface demineralization of bovine enamel and change in plaque composition by casein in an intra-oral model. J Dent Res 1987;66:1120-7.  Back to cited text no. 4
Rahiotis C, Vougiouklakis G, Eliades G. Characterization of oral films formed in the presence of a CPP-ACP agent: An in situ study. J Dent 2008;36:272-80.  Back to cited text no. 5
Ekambaram M, Mohd Said SN, Yiu CK. A review of enamel remineralisation potential of calcium- and phosphate-based remineralisation systems. Oral Health Prev Dent 2017;15:415-20.  Back to cited text no. 6
Petersson LG, Twetman S, Dahlgren H, Norlund A, Holm AK, Nordenram G, et al. Professional fluoride varnish treatment for caries control: A systematic review of clinical trials. Acta Odontol Scand 2004;62:170-6.  Back to cited text no. 7
Turska-Szybka A, Gozdowski D, Mierzwińska-Nastalska E, Olczak-Kowalczyk D. Randomised clinical trial on resin infiltration and fluoride varnish vs. fluoride varnish treatment only of smooth-surface early caries lesions in deciduous teeth. Oral Health Prev Dent 2016;14:485-91.  Back to cited text no. 8
Krithikadatta J, Fredrick C, Abarajithan M, Kandaswamy D. Remineralisation of occlusal white spot lesion with a combination of 10% CPP-ACP and 0.2% sodium fluoride evaluated using diagnodent: A pilot study. Oral Health Prev Dent 2013;11:191-6.  Back to cited text no. 9
Oliveira GM, Ritter AV, Heymann HO, Swift E Jr., Donovan T, Brock G, et al. Remineralization effect of CPP-ACP and fluoride for white spot lesions in vitro. J Dent 2014;42:1592-602.  Back to cited text no. 10
Hamba H, Nikaido T, Inoue G, Sadr A, Tagami J. Effects of CPP-ACP with sodium fluoride on inhibition of bovine enamel demineralization: A quantitative assessment using micro-computed tomography. J Dent 2011;39:405-13.  Back to cited text no. 11
Silverstone LM. Structure of carious enamel, including the early lesion. Oral Sci Rev 1973;3:100-60.  Back to cited text no. 12
Hattab FN. Remineralisation of carious lesions and fluoride uptake by enamel exposed to various fluoride dentifrices in vitro. Oral Health Prev Dent 2013;11:281-90.  Back to cited text no. 13
Bayrak S, Tuloglu N, Bicer H, Sen Tunc E. Effect of fluoride varnish containing CPP-ACP on preventing enamel erosion. Scanning 2017;2017:1897825.  Back to cited text no. 14
Kamal D, Hassanein H, Elkassas D, Hamza H. Comparative evaluation of remineralizing efficacy of biomimetic self-assembling peptide on artificially induced enamel lesions: An in vitro study. J Conserv Dent 2018;21:536-41.  Back to cited text no. 15
[PUBMED]  [Full text]  
ten Cate JM, Duijsters PP. Alternating demineralization and remineralization of artificial enamel lesions. Caries Res 1982;16:201-10.  Back to cited text no. 16
Silverstone LM. Structural alterations of human dental enamel during incipient carious lesion development. In: Rowe NH, editor. Proceeding of Symposium on Incipient Caries of Enamel. Ann Arbor, MI: University of Michigan, School of Dentistry; 1977. p. 3-49.  Back to cited text no. 17
Indrapriyadharshini K, Madan Kumar PD, Sharma K, Iyer K. Remineralizing potential of CPP-ACP in white spot lesions – A systematic review. Indian J Dent Res 2018;29:487-96.  Back to cited text no. 18
[PUBMED]  [Full text]  
Savas S, Kavrìk F, Kucukyìlmaz E. Evaluation of the remineralization capacity of CPP-ACP containing fluoride varnish by different quantitative methods. J Appl Oral Sci 2016;24:198-203.  Back to cited text no. 19
Thakkar PJ, Badakar CM, Hugar SM, Hallikerimath S, Patel PM, Shah P, et al. An in vitro comparison of casein phosphopeptide-amorphous calcium phosphate paste, casein phosphopeptide-amorphous calcium phosphate paste with fluoride and casein phosphopeptide-amorphous calcium phosphate varnish on the inhibition of demineralization and promotion of remineralization of enamel. J Indian Soc Pedod Prev Dent 2017;35:312-8.  Back to cited text no. 20
[PUBMED]  [Full text]  
Chandak S, Bhondey A, Bhardwaj A, Pimpale J, Chandwani M. Comparative evaluation of the efficacy of fluoride varnish and casein phosphopeptide-amorphous calcium phosphate in reducing Streptococcus mutans counts in dental plaque of children: An in vivo study. J Int Soc Prev Community Dent 2016;6:423-9.  Back to cited text no. 21
Tao S, Zhu Y, Yuan H, Tao S, Cheng Y, Li J, et al. Efficacy of fluorides and CPP-ACP vs. fluorides monotherapy on early caries lesions: A systematic review and meta-analysis. PLoS One 2018;13:e0196660.  Back to cited text no. 22
Güçlü ZA, Alaçam A, Coleman NJ. A 12-week assessment of the treatment of white spot lesions with CPP-ACP paste and/or fluoride varnish. Biomed Res Int 2016;2016:8357621.  Back to cited text no. 23


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1]

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