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 Table of Contents  
Year : 2015  |  Volume : 7  |  Issue : 6  |  Page : 583-586  

Evaluation of remineralization capacity of casein phosphopeptide-amorphous calcium phosphate on the carbamide peroxide treated enamel

1 Department of Preventive Dental Sciences, College of Dentistry, Salman Bin Abdul Aziz University, Alkharjh 11942, Kingdom of Saudi Arabia
2 Department of Pediatric Dentistry, Al-Farabi College, Riyadh 11691, P. O. Box 85184, Kingdom of Saudi Arabia
3 Department of Restorative Dental Sciences, Al-Farabi College, Riyadh 11691, P. O. Box 85184, Kingdom of Saudi Arabia
4 Department of Restorative Dentistry, IBN Sina National College of Dentistry, Al Mahjar, Jeddah 22421, Kingdom of Saudi Arabia

Date of Submission28-Apr-2015
Date of Decision28-Apr-2015
Date of Acceptance22-May-2016
Date of Web Publication1-Sep-2015

Correspondence Address:
Dr. Raja Rajeswari Kaminedi
Department of Pediatric Dentistry, Al-Farabi College, Riyadh 11691, P. O. Box 85184
Kingdom of Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.163556

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Objective: The aim of this study was to evaluate the potential of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) in remineralizing the bleached enamel surface using micro-hardness. Materials and Methods: Thirty human enamel slabs were randomly divided into three groups (n = 10). Groups A and B were exposed to 20% carbamide peroxide and 35% carbamide peroxide gel, respectively. After the exposure to the bleaching agent, the slabs were kept in artificial saliva for 1-week. Group C (control group) were kept in artificial saliva for 1-week. Vickers micro-hardness test was performed by Leica VMHT-Mot micro-hardness tester. CPP-ACP (Gc Tooth Mousse, Melbourne, Australia) was then applied to specimens of Groups A and B for 3 min for 2 weeks. Micro-hardness values of postbleach Group A (Ar) and Group B (Br) were recorded and statistically analyzed by paired t-test and one-way analysis of variance at the significance level of α =0.05. Results: There was a significant decrease in micro-hardness of enamel in carbamide peroxide bleached groups. However, there was a significant increase in micro-hardness after the remineralization by CPP-ACP and the extent of remineralization is more for the Group B. Conclusions: That bleaching agents reduced enamel micro-hardness and the use of CPP-ACP after bleaching can significantly enhance the micro-hardness of bleached enamel.

Keywords: Bleaching, hardness, remineralization

How to cite this article:
Penumatsa NV, Kaminedi RR, Baroudi K, Barakath O. Evaluation of remineralization capacity of casein phosphopeptide-amorphous calcium phosphate on the carbamide peroxide treated enamel. J Pharm Bioall Sci 2015;7, Suppl S2:583-6

How to cite this URL:
Penumatsa NV, Kaminedi RR, Baroudi K, Barakath O. Evaluation of remineralization capacity of casein phosphopeptide-amorphous calcium phosphate on the carbamide peroxide treated enamel. J Pharm Bioall Sci [serial online] 2015 [cited 2022 Dec 9];7, Suppl S2:583-6. Available from:

Cosmetic dentistry is very important part of today's restorative dental practice. Discoloration of anterior teeth is an esthetic problem that requires effective treatment. Vital bleaching is a viable option to consider when treating intrinsically stained or discolored teeth whose form and integrity are deemed acceptable. Carbamide peroxide introduced by Haywood and Heyman [1] was most widely used for tooth whitening (bleaching) both in professional and in self-administered products.

Though the decrease in discoloration has been satisfactory with the application of bleaching agents, there were also adverse effects as the consequence of this treatment. [2] Prominent side effects of concern included pulpal sensitivity, [3] surface compositional changes, [4] mineral loss from the tooth structure [5] and decreased hardness of the enamel. [6] The decrease in the micro-hardness number has a linear relationship with the mineral loss under the conditions of demineralization. [7]

To overcome this loss of minerals and subsequent micro-hardness remineralizing solutions are recommended. [8] Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is a derivative of milk protein casein found in cow's milk. CPP-ACP is produced from a tryptic digest of milk protein casein by aggregation with calcium phosphate and purification by ultra-filtration. The casein phosphopeptide (CPP), by stabilizing calcium phosphate in solutions maintains high concentration gradients of calcium and phosphate ions and ion pairs into the demineralized lesion [9] and thus affects high rates of enamel remineralization when compared to other calcium phosphate remineralization solutions. [10]

The more the concentration of the bleaching gel the more the adverse effects were reported. Studies showed that the adverse effects were not so much in the clinical situation due to the buffering and remineralization capacity of saliva.

Hence, this study was conducted using artificial saliva to simulate clinical conditions.

The aim of this study was to evaluate the effect of higher concentrations of 20% and 35% carbamide peroxide on enamel surface micro-hardness and to determine remineralizing potential of CPP-ACP on this bleached enamel.

   Materials and Methods Top

This study was comprised of 30 noncarious premolar teeth extracted for orthodontic purpose.

Bleaching agents: Opalescence 20% (20% carbamide peroxide Gcsscorp, Asia) and Clinex gel 35% (35% carbamide peroxide Vishal Dento Care, India).

Remineralizing agent: GC Tooth Mousse (CPP-ACP, Gc crop, Asia).

Specimen preparation

The extracted teeth were washed thoroughly under running tap water to remove blood, saliva, and other debris. Soft tissue attached to tooth was removed using hand scaler. The teeth were stored in artificial saliva at room temperature till the experiment was started.

Preparation of artificial saliva


  • 0.053% tricalcium phosphate
  • 0.01 n HCl
  • 15 ml of sorbitol
  • 0.6 gm KCl
  • 0.42 gm NaCl
  • 0.026 gm magnesium lactate
  • 0.05 M NaOH
  • 5 ml of 0.2 M sodium phosphate
  • Distilled water.


100 ml of 0.053% tricalcium phosphate in 0.01n HCl is mixed with 100 ml of a mixture containing 15 ml of sorbitol, 0.6 g KCl, 0.42 g NaCl and 0.026 g magnesium lactate. After mixing the two at room temperature the pH was adjusted to 7.4 by 0.05 M NaOH and 5 ml of 0.2 M sodium phosphate, was added and made to volume 500 ml. Exact pH was verified using digital pH meter and the mixture was sterilized in an autoclave and stored at 4°C.


The teeth were buccolingually sectioned with the help of double faced diamond disk mounted on micro motor with the straight handpiece. The buccal surface portions of the crowns were mounted using cold-cure acrylic resin. The specimen were subjected to polishing sequentially using 80 μm, 100 μm, 200 μm, 400 μm, grit size of sand papers and finally with diamond polishing paste. Baseline micro-hardness values were recorded and the blocks having the average surface micro-hardness 10% above and 10% below the overall average were discarded.

The specimens were randomly divided into three equal groups; the grouping of specimens is as follows:

  • Group A: Ten specimens, exposed to 20% carbamide peroxide gel (Opalescence 20%, Gc corp, Asia) for 2 h daily for 1-week
  • Group B: Ten specimens, exposed to 35% carbamide peroxide gel (Clinex Gel 35% Vishal Dento Care, India) for 30 min daily for 1-week
  • Group C: (Control group) Ten specimens, exposed to artificial saliva
  • Group AR: Postbleached specimens of Group A treated with CPP-ACP for 3 min daily for a period of 2 weeks
  • Group BR: Postbleached specimen of Group B treated with CPP-ACP for 3 min daily for a period of 2 weeks.

Groups A and B were exposed to respective concentrations of carbamide peroxide gel daily for the time periods recommended by the manufacturer for 1-week. After the recommendation time of exposure, daily the teeth were removed from bleaching gels, washed with de-ionized distilled water and placed in artificial saliva for the reminder of 24 h. The procedure was carried out for all specimens for a period of 1-week. The control group teeth were kept in artificial saliva for the entire period of 1-week.

The micro-hardness test was done using Leica VMHT-Mot micro-hardness tester with Vicker's diamond indenting tool after a period of 1-week under a load of 100 g for a period of 12 s per indentation. Three indentations were made for each specimen to get average micro-hardness value and mean and standard deviation (mean ± SD) for each group was calculated. Percentage of micro-hardness change after the blocks being bleached was calculated.

Gc Tooth Mousse was then applied to specimens of Groups A and B according to manufacturers instructions for a period of 2 weeks. After the recommended time period, 3 min daily, the teeth were removed from the solution washed with de-ionized distilled water and placed in artificial saliva for the reminder of the 24 h. After a period of 2 weeks micro-hardness tests were done, and the values were subjected to the variance of analysis of variance and paired t-test. All statistical analyses were conducted at a significance level of α =0.05.

   Results Top


The mean ± SD value of control Group C is 417.02 ± 10.39; the mean ± SD value of Group A is 382.42 ± 12.14, the mean ± SD value of Group B is 346.65 ± 30. There is significant reduction of micro-hardness values in both Group A (P = 0.001) and Group B (P = 0.001) after bleaching when compared to control Group C. Group B micro-hardness values are lower than Group A. Percentage of surface hardness in Group A was 8.39% and for Group B was 17.02% calculated by formula % surface hardness change = 100 (base surface hardness-surface hardness)/base surface hardness.

After remineralization of bleached tooth

The mean ± SD value of Group Ar is 386.45 ± 10.43; the mean ± SD value of Group Br is 372.69 ± 15.61. There is significant increase in mean micro-hardness value in Group Br (P = 0.03) when compared to postbleach (Group B) and slight increase, but not significant in Group Ar (P = 0.44) when compared to postbleach (Group A) and the values after bleaching are remained less than control Group C [Graph 1].

Percentage of surface change is calculated. Group Ar was 1.07% and for Group Br was 7.51%. The extent of remineralization for Group BR was more than the Group Ar [Table 1] denoting that the more demineralization, the more remineralization can occur after CPP-ACP application [Graph 2].
Table 1: Mean VHN values of microhardness postbleach and application of CPP-ACP

Click here to view

   Discussion Top

Bleaching is an easy and conservative method for treating discolored teeth than veneers and laminations. Introduction of night guard vital bleaching has rekindled a resurgence of interest in tooth bleaching. Bleaching is a decolorization or whitening process that can occur in a solution or on the surface. The color producing materials are typically organic compounds that possess extended conjugated chains of alternating single or double bonds and often include hetero atoms, carbonyl, and phenyl rings in the conjugated system and are often referred to as chromophore. Bleaching or decolorization of the chromophore can occur by destroying one or more of the double bonds in the conjugated chain or by oxidation of other chemical moieties in the conjugated chains. [11]

Carbamide peroxide is an adduct of urea and hydrogen peroxide which on contact with water breaks down to urea and hydrogen peroxide. The chemistry of this agent is based primarily on its ability to generate free radicals in most solvents. The hydroxyl radicals lack one electron, are extremely electrophilic and unstable and will attack most other organic molecules to achieve stability generating other radicals. [12] Hydrogen peroxide bleaching can proceed via the perhydroxyl anion or free radicals depending on pH. As the peroxide diffuses into the tooth, it can react with organic color materials found within the tooth structure leading to a reduction in color. These oxidation reactions can cause alterations in the enamel structure, [13] calcium and phosphate mineral loss, [14] and pulpal sensitivity. The results of these studies which are in accordance with the studies done by Majeed et al., [15] Flaitz and Hicks [16] showed clearly that the application of carbamide peroxide bleaching gel lowered the micro-hardness of the enamel and also showed the changes in enamel are directly proportional to concentration of bleaching agent which are in similar to the studies done by Hegedus et al., [17] The results are in contrast with the study of Basting et al. [18]

The oral environment can naturally rebuild enamel through a process called as remineralization, to some extent on its own, but the extent of remineralization is controlled by pH and the amount and availability of calcium and phosphate ions. [19] The minimum thermodynamic requirement for remineralization to occur is simply that the ion activity product is greater than the solubility product constant for tooth mineral. Solubility constraints limit the level of supersaturation and forms calcium phosphate mineral, therefore, limit mineral ion concentration in remineralizing solutions to a relatively low level. [20] A considerably large volume of remineralizing solution is required to supply the needed mineral ions, suggesting that remineralization in vivo is a relatively slow kinetic reaction.

Casein phosphopeptides are multi phosphorylated from an enzymatic digest of the bovine milk protein casein. These peptides have remarkable ability to stabilize the calcium phosphate in solution as amorphous calcium phosphate (ACP), through their multi phosphoryl residues CPP bind to forming nanoclusters of ACP in metastable solution, preventing their growth to critical size required for nucleation and phase transformation. CPP-ACP nanoclusters have shown to localize at the tooth surface, which buffers free calcium and phosphate ion activities by helping to maintain a state of supersaturation with respect to tooth enamel depressing demineralization and enhancing remineralization. [21],[22] The results of this study showed that there was a definitive increase in microhardness when compared to the postbleach hardness even after the specimens are maintained in artificial saliva. At normal oral pH and temperature, the addition of ACP components to the salivary system can cause the hydrolysis of ACP into apatite within a few minutes, which is roughly 20,000 times faster than normal. [23] The susceptibility of bleached enamel to caries is still a topic of study, but precautions should be taken as remineralization is a slow process in vivo. This study result recommends the application of CPP-ACP for remineralization after bleaching.

The advanced whitening technologies containing CPP-ACP as component are marketed, but the efficacy of the agents has to be determined. Milk protein casein can cause allergic reactions in sensitive people, so judicial use of this material is recommended keeping this matter of concern in view.

   Conclusions Top

Higher concentrations of carbamide peroxide bleaching gels had a definite effect of reducing the surface micro-hardness of sound enamel to significant levels. Decrease in micro-hardness was directly proportional to the peroxide concentration. CPP-ACP application enhanced remineralization of bleached enamel. The enhancing potential of CPP-ACP is in direct correlation with a degree of demineralization of enamel. The importance of bleaching as a conservative treatment option for the discolored teeth when compared to veneers and laminates cannot be undervalued, but must be used with caution and postbleaching regimen with CPP-ACP are worth consideration. Further research for the better remineralizing material should be done as the micro-harness values had not reached the baseline values.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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Tredwin CJ, Naik S, Lewis NJ, Scully C. Hydrogen peroxide tooth-whitening (bleaching) products: Review of adverse effects and safety issues. Br Dent J 2006;200:371-6.  Back to cited text no. 2
Amengual J, Forner L. Dentine hypersensitivity in dental bleaching: Case report. Minerva Stomatol 2009;58:181-5.  Back to cited text no. 3
Soares DG, Ribeiro AP, Sacono NT, Loguércio AD, Hebling J, Costa CA. Mineral loss and morphological changes in dental enamel induced by a 16% carbamide peroxide bleaching gel. Braz Dent J 2013;24:517-21.  Back to cited text no. 4
Elfallah HM, Swain MV. A review of the effect of vital teeth bleaching on the mechanical properties of tooth enamel. N Z Dent J 2013;109:87-96.  Back to cited text no. 5
Attin T, Vollmer D, Wiegand A, Attin R, Betke H. Subsurface microhardness of enamel and dentin after different external bleaching procedures. Am J Dent 2005;18:8-12.  Back to cited text no. 6
Feagin F, Koulourides T, Pigman W. The characterization of enamel surface demineralization, remineralization, and associated hardness changes in human and bovine material. Arch Oral Biol 1969;14:1407-17.  Back to cited text no. 7
Alkhtib A, Manton DJ, Burrow MF, Saber-Samandari S, Palamara JE, Gross KA, et al. Effects of bleaching agents and Tooth Mousse(™) on human enamel hardness. J Investig Clin Dent 2013;4:94-100.  Back to cited text no. 8
Cross KJ, Huq NL, Reynolds EC. Casein phosphopeptides in oral health - Chemistry and clinical applications. Curr Pharm Des 2007;13:793-800.  Back to cited text no. 9
Reynolds EC. Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions. J Dent Res 1997;76:1587-95.  Back to cited text no. 10
Kihn PW. Vital tooth whitening. Dent Clin North Am 2007;51:319-31, viii.  Back to cited text no. 11
McCaslin AJ, Haywood VB, Potter BJ, Dickinson GL, Russell CM. Assessing dentin color changes from nightguard vital bleaching. J Am Dent Assoc 1999;130:1485-90.  Back to cited text no. 12
Sulieman M, Addy M, Macdonald E, Rees JS. The bleaching depth of a 35% hydrogen peroxide based in-office product: A study in vitro. J Dent 2005;33:33-40.  Back to cited text no. 13
Grobler SR, Majeed A, Moola MH. Effect of various tooth-whitening products on enamel microhardness. SADJ 2009;64:474-9.  Back to cited text no. 14
Majeed A, Grobler SR, Moola MH, Rossouw RJ, van Kotze TJ. Effect of four different opalescence tooth-whitening products on enamel microhardness. SADJ 2008;63:282-4, 286.  Back to cited text no. 15
Flaitz CM, Hicks MJ. Effects of carbamide peroxide whitening agents on enamel surfaces and caries-like lesion formation: An SEM and polarized light microscopic in vitro study. ASDC J Dent Child 1996;63:249-56.  Back to cited text no. 16
Hegedüs C, Bistey T, Flóra-Nagy E, Keszthelyi G, Jenei A. An atomic force microscopy study on the effect of bleaching agents on enamel surface. J Dent 1999;27:509-15.  Back to cited text no. 17
Basting RT, Rodrigues AL Jr, Serra MC. The effect of 10% carbamide peroxide, carbopol and/or glycerin on enamel and dentin microhardness. Oper Dent 2005;30:608-16.  Back to cited text no. 18
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