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Year : 2015  |  Volume : 6  |  Issue : 6  |  Page : 163-166  

Evaluation and comparison of the microhardness of enamel after bleaching with fluoride free and fluoride containing carbamide peroxide bleaching agents and post bleaching anticay application: An in vitro study

Department of Conservative Dentistry and Endodontics, Annoor Dental College, Muvatupuzha, Ernakulam, Kerala, India

Date of Web Publication8-Oct-2015

Correspondence Address:
Liza George
Department of Conservative Dentistry and Endodontics, Annoor Dental College, Muvatupuzha, Ernakulam, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0976-237X.166835

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Aims and Objectives: The purpose of the study was to evaluate and compare the microhardness of enamel after the application of anticay on bleached enamel with fluoride containing and fluoride free bleaching agent. Materials and Methods: Twenty freshly extracted teeth decoronated and divided mesiodistally into two halves were randomly divided into five groups with 10 samples in each group. The enamel surface was treated as follows: Group 1 - no treatment, Group 2 - fluoride free bleaching agent, Group 3 - fluoride containing bleaching agent, and Group 4 - fluoride free bleaching agent followed by anticay application. The samples were subjected to indentation to test the microhardness using Vicker's hardness analyzer. Conclusion: Enamel microhardness significantly increased in samples where anticay was used after the application of bleaching agent.

Keywords: Anticay, bleaching, microhardness

How to cite this article:
George L, Baby A, Dhanapal T P, Charlie K M, Joseph A, Varghese AA. Evaluation and comparison of the microhardness of enamel after bleaching with fluoride free and fluoride containing carbamide peroxide bleaching agents and post bleaching anticay application: An in vitro study. Contemp Clin Dent 2015;6, Suppl S2:163-6

How to cite this URL:
George L, Baby A, Dhanapal T P, Charlie K M, Joseph A, Varghese AA. Evaluation and comparison of the microhardness of enamel after bleaching with fluoride free and fluoride containing carbamide peroxide bleaching agents and post bleaching anticay application: An in vitro study. Contemp Clin Dent [serial online] 2015 [cited 2020 May 28];6, Suppl S2:163-6. Available from:

   Introduction Top

Bleaching although not new to dentistry has gained much popularity in recent years. It is the most preferred conservative method to lighten discolored teeth.[1] Bleaching agents contain either hydrogen peroxide or carbamide peroxide (CP). One of the popular bleaching techniques is night guard bleaching that utilizes 10% CP in a customized tray that is worn by the patient at night. CP is formed from hydrogen peroxide and urea and about one-third of CP is released as hydrogen peroxide.[2] The color change in enamel and dentin is the result of penetration of hydrogen peroxide through the enamel and break down of high molecular weight organic molecule into simpler low molecular weight molecules with lesser color reflectance.[3] However, its action is not restricted and could also contribute to protein damage and mineral loss.[4]

The concentration of CP in night guard bleaching technique is less and its bleaching action is made effective by increasing its duration of contact with the teeth. Morphological changes have been observed in enamel with the use of CP bleaching agents.[5],[6],[7],[8],[9],[10],[11] Also, there can be alteration in microhardness of enamel following treatment with 10% CP bleaching material.[12]

The loss of mechanical properties of enamel after the use of bleaching agents could be regained by the incorporation of agents that can compensate for the mineral loss. Studies have reported that the use of agents such as fluoride and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) promote remineralization of tooth structure. CPP-ACP enhances remineralization by stabilizing the level of calcium and phosphate in saliva.[13],[14],[15],[16],[17],[18] In a study conducted by Panich andPoolthong it was found that CPP-ACP increased the microhardness of enamel in eroded teeth.[19] Anticay (ToothMin) that contains calcium sucrose phosphate (SP) releases calcium and phosphate ions to remineralize the enamel.

The purpose of this study was to evaluate and compare the microhardness (Vicker's hardness analysis) of enamel after bleaching with fluoride free, fluoride containing CP bleaching agents, and also to compare the microhardness after the application of anticay (ToothMin) following the use of fluoride free CP bleaching agent. The null hypothesis states that the microhardness of enamel does not increase after the use of anticay.

   Materials and Methods Top

Twenty non carious freshly extracted human premolars for orthodontic purpose were obtained as samples and visually inspected to exclude those with cracks or defects. After cleansing, the samples were stored in distilled water until use. The roots of the teeth were decoronated at the level of cemento-enamel junction using diamond disc (Horico, Berlin, Germany) with copious water spray. Crowns were vertically sectioned into two equal halves. Two halves from each tooth were embedded in acrylic resin of standard dimension of 10 mm × 10 mm × 5 mm such that the enamel surface faced upward. The samples were kept in cold water until complete curing of the resin to avoid thermal effects generated by the resin during curing process. The enamel surface of the teeth was ground flat using water-cooled abrasive wheel in a sequence of fresh 120, 240, and 400-grit silicon carbide papers to obtain a flat surface.

The samples were randomly divided into the following five treatment groups:

  • Group 1 - Control group: Samples not treated with any bleaching agents (n = 10)
  • Group 2: Samples treated with fluoride free bleaching agent (opalescence 10%) (n = 10)
  • Group 3: Samples treated with fluoride containing bleaching agent (opalescence PF 10%) (n = 10)
  • Group 4: Samples treated with fluoride free bleaching agent (opalescence 10%) and an additional ToothMin application for 1 min after each bleaching treatment (n = 10).

Bleaching agent [Table 1] was applied onto the exposed enamel surface of the samples 8 h a day for 7 consecutive days in the respective groups. The samples were rinsed with air water spray for 30 s and air dried after each treatment. In Group 4, samples were subjected to additional application of ToothMin paste for 1 min after each bleaching treatment. All the samples were immersed in distilled water for the remaining hours.
Table 1: Bleaching agents and remineralization agents used in this study

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Determination of microhardness

The surface hardness of each sample was determined with a Vickers hardness tester (Matsuzawa MMT7, Matsuzawa SEIKI Co., Ltd., Tokyo, Japan) with 200 g load, which was used to make indentations on the enamel surface. The loaded diamond was allowed to sink and rest on the enamel surface for 15 s and the Vickers hardness number was determined and recorded.

Statistical analysis

The inter-group difference was statistically analyzed by one-way

analysis of variance (ANOVA) test followed by Tukey's post-hoc test. Statistical significance was set at P < 0.05.

   Results Top

The mean enamel microhardness was shown to be maximum in Group 4 (opalescence and ToothMin) followed by Group 1 (control), Group 3 (opalescence PF), and Group 2 (opalescence) [Table 2] and [Graph 1]. The standard deviation of enamel microhardness was maximum in Group 1 followed by Group 4, Group 2, and Group 3 [Table 2].
Table 2: Descriptive statistics of enamel microhardness

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From the ANOVA test [Table 3], it was observed that the mean enamel microhardness (F (3, 36) = 63.205, P = 0.000) for the four groups was significantly different. Tukey's post-hoc analysis [Table 4] further showed that except Group 1 and Group 4 all other groups had statistically significant difference between them.
Table 3: Level of significance of enamel microhardness using one.way ANOVA test

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Table 4: Level of significance of enamel microhardness using Tukey's post-hoc test

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   Discussion Top

Mechanical properties of enamel depend largely on the degree of mineralization [20] and alignment of enamel prisms with respect to the crack orientation.[21] Cuy et al. hypothesized that there is a strong correlation between degree of mineralization of enamel and its mechanical properties and that prism alignment and loading direction have limited influence.[22] Bleaching agents have been found to affect the mechanical properties of enamel by decreasing the microhardness. In a study done by Senghi and Denry human enamel microhardness decreased after application of 10% CP.[23] Asefzadehand Hoseini, in their study found that 56 h application of 10% CP from two different brands, led to a decrease in the enamel hardness.[24] The microhardness decreased over a period of two weeks when enamel slabs were subjected to 10% CP bleaching agent as observed by Shannon et al.[6] In the current study also, opalescence containing 10% CP was found to decrease the enamel microhardness compared to the control group.

Factors such as concentration and time duration of the bleaching application has got a direct effect on the enamel hardness.[25] Other factors influencing the enamel hardness are the fluoridation of drinking water, age, etc. In this study random sampling was done to nullify such effects. All the samples were stored in distilled water to study the direct effect of CP without the effect of demineralization and remineralization.

Incorporation of fluoride in dentrifice remineralizes artificial caries lesion by forming a layer of calcium fluoride.[26] Further, it has been shown that post bleaching application of fluoride in the form of fluoride varnish prevented loss of enamel hardness.[27] Addition of fluoride as a protective mechanism in a CP bleaching agent may raise concerns regarding its whitening efficiency. This may be attributed by the calcium fluoride layer formed on the surface which impairs the whitening efficiency of CP. However in a study done by Chen et al., scanning electron microscopy evaluation of the enamel specimens treated with fluoridated bleaching agents showed only minor erosive patterns without crystal deposition on the enamel surface and the whitening efficiency was not altered.[28] Fluoridated bleaching agents preserved the microhardness of enamel more effectively than post bleaching fluoride application.[28] Caries resistance further increased in teeth treated with fluoridated bleaching agents.[29] In the current study also, incorporation of fluoride in the bleaching agent increased the microhardness.

Anticay consists of calcium SP and inorganic calcium phosphate. It is completely soluble in water at all pH values and hence able to provide high concentrates of calcium and phosphate ions in the oral cavity. It has been proposed that one percent anticay solution contains about 30 × 10−3 M calcium (Ca) and 10 × 10−4 M SP ions. Saliva contains about 1.4 × 10−3 M calcium and 4 × 10−3 M phosphate (P) ions. High concentration of calcium and phosphate in the presence of anticay facilitates rapid remineralization of enamel. This has been demonstrated in vitro by rapid hardening of acid-softened enamel.[30] In the current study, application of anticay significantly improved the microhardness of enamel by helping to regain the minerals lost during the bleaching procedure.

   Conclusion Top

Within the limitations of the study, it was concluded that:

  • Fluoride containing bleaching agent increased the microhardness of enamel than when bleached with agents without the incorporation of fluoride
  • The application of remineralizing agent containing calcium SP/anticay after bleaching increased the microhardness of enamel.

Further clinical trials supplemented with in vitro studies are needed to authenticate the results for future clinical applications.

Source of Support:


Conflict of Interest:

None declared.

   References Top

Haywood VB, Heymann HO. Nightguard vital bleaching. Quintessence Int 1989;20:173-6.  Back to cited text no. 1
Kelleher MG. Dental Bleaching. London: Quintessence Publishing Co.; 2008. p. 6.  Back to cited text no. 2
Goldstein GR, Garber DA. Complete Dental Bleaching. Chicago: Quintessence Books; 1995. p. 165.  Back to cited text no. 3
Rotstein I, Dankner E, Goldman A, Heling I, Stabholz A, Zalkind M. Histochemical analysis of dental hard tissues following bleaching. J Endod 1996;22:23-5.  Back to cited text no. 4
Goldstein GR, Kiremidjian-Schumacher L. Bleaching: Is it safe and effective? J Prosthet Dent 1993;69:325-8.  Back to cited text no. 5
Shannon H, Spencer P, Gross K, Tira D. Characterization of enamel exposed to 10% carbamide peroxide bleaching agents. Quintessence Int 1993;24:39-44.  Back to cited text no. 6
Ben-Amar A, Liberman R, Gorfil C, Bernstein Y. Effect of mouthguard bleaching on enamel surface. Am J Dent 1995;8:29-32.  Back to cited text no. 7
Akal N, Over H, Olmez A, Bodur H. Effects of carbamide peroxide containing bleaching agents on the morphology and subsurface hardness of enamel. J Clin Pediatr Dent 2001;25:293-6.  Back to cited text no. 8
Spalding M, Taveira LA, de Assis GF. Scanning electron microscopy study of dental enamel surface exposed to 35% hydrogen peroxide: Alone, with saliva, and with 10% carbamide peroxide. J Esthet Restor Dent 2003;15:154-64.  Back to cited text no. 9
Cavalli V, Arrais CA, Giannini M, Ambrosano GM. High-concentrated carbamide peroxide bleaching agents effects on enamel surface. J Oral Rehabil 2004;31:155-9.  Back to cited text no. 10
Yeh ST, Su Y, Lu YC, Lee SY. Surface changes and acid dissolution of enamel after carbamide peroxide bleach treatment. Oper Dent 2005;30:507-15.  Back to cited text no. 11
Basting RT, Rodrigues Júnior AL, Serra MC. The effect of 10% carbamide peroxide bleaching material on microhardness of sound and demineralized enamel and dentin in situ. Oper Dent 2001;26:531-9.  Back to cited text no. 12
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. 13
Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res 2003;82:206-11.  Back to cited text no. 14
Reynolds EC. Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review. Spec Care Dentist 1998;18:8-16.  Back to cited text no. 15
Cross KJ, Huq NL, Stanton DP, Sum M, Reynolds EC. NMR studies of a novel calcium, phosphate and fluoride delivery vehicle-alpha(S1)-casein(59-79) by stabilized amorphous calcium fluoride phosphate nanocomplexes. Biomaterials 2004;25:5061-9.  Back to cited text no. 16
Grewal N, Kudupudi V, Grewal S. Surface remineralization potential of casein phosphopeptide-amorphous calcium phosphate on enamel eroded by cola-drinks: An in-situ model study. Contemp Clin Dent 2013;4:331-7.  Back to cited text no. 17
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Shetty S, Hegde MN, Bopanna TP. Enamel remineralization assessment after treatment with three different remineralizing agents using surface microhardness: An in vitro study. J Conserv Dent 2014;17:49-52.  Back to cited text no. 18
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Panich M, Poolthong S. The effect of casein phosphopeptide -amorphous calcium phosphate and a cola soft drink onin vitro enamel hardness. J Am Dent Assoc 2009;140:455-60.  Back to cited text no. 19
Kodaka T, Debari K, Yamada M, Kuroiwa M. Correlation between microhardness and mineral content in sound human enamel (short communication). Caries Res 1992;26:139-41.  Back to cited text no. 20
Xu HH, Smith DT, Jahanmir S, Romberg E, Kelly JR, Thompson VP, et al. Indentation damage and mechanical properties of human enamel and dentin. J Dent Res 1998;77:472-80.  Back to cited text no. 21
Cuy JL, Mann AB, Livi KJ, Teaford MF, Weihs TP. Nanoindentation mapping of the mechanical properties of human molar tooth enamel. Arch Oral Biol 2002;47:281-91.  Back to cited text no. 22
Seghi RR, Denry I. Effects of external bleaching on indentation and abrasion characteristics of human enamel in vitro. J Dent Res 1992;71:1340-4.  Back to cited text no. 23
Asefzadeh F, Hosseini F. A comparative study of the effects of Kimiya bleaching-gel (An Iranian brand) and opalescence on human enamel micro-hardness. Daneshvar, Scientific-research Journal of Shahed University 2006;13:7-1.  Back to cited text no. 24
Sunil ChR, Sujana V, Choudary TM, Nagesh B.In vitro action of various carbamide peroxide gel bleaching agents on the micro hardness of human enamel. Contemp Clin Dent 2012;3:193-6.  Back to cited text no. 25
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White DJ, Featherstone JD. A longitudinal microhardness analysis of fluoride dentifrice effects on lesion progression in vitro. Caries Res 1987;21:502-12.  Back to cited text no. 26
Attin T, Kielbassa AM, Schwanenberg M, Hellwig E. Effect of fluoride treatment on remineralization of bleached enamel. J Oral Rehabil 1997;24:282-6.  Back to cited text no. 27
Chen HP, Chang CH, Liu JK, Chuang SF, Yang JY. Effect of fluoride containing bleaching agents on enamel surface properties. J Dent 2008;36:718-25.  Back to cited text no. 28
Al-Qunaian TA. The effect of whitening agents on caries susceptibility of human enamel. Oper Dent 2005;30:265-70.  Back to cited text no. 29
Rogerson MJ. The role of a calcium sucrose phosphate-calcium orthophosphate complex in the reduction of dental caries. Aust Dent J 1973;18:160-6.  Back to cited text no. 30


  [Table 1], [Table 2], [Table 3], [Table 4]

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