Dental Hypotheses

: 2022  |  Volume : 13  |  Issue : 3  |  Page : 90--93

Spectrophotometric Evaluation of the Color Change of Artificial White Spot Lesions Treated with Three Different Commercially Available Remineralizing Agents: An In Vitro Study

Mohammadreza Malekipoor1, Farzaneh Shirani2, Sayed Mostafa Mousavinasab2, Niloufar Jafari3, Negar Sharifinejad4,  
1 Department of Operative Dentistry, School of Dentistry, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
2 Dental Research Center, Dental Research Institute, Department of Operative Dentistry, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Operative Dentistry, School of Dentistry, Rafsanjan, Iran
4 Faculty of Dentistry, Qom University of Medical Sciences, Qom, Iran

Correspondence Address:
Farzaneh Shirani
Associated Professor, Department of Operative Dentistry, School of Dentistry, Isfahan University of Medical Sciences, Isfahan


Introduction: This study evaluated the color changes induced by the treatment of intact and demineralized enamel surfaces with casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and sodium fluoride. Material and Methods: Ninety-six sound teeth were collected. The samples were divided into “intact” and “demineralized” groups and then subjected to different treatments (CPP-ACFP, sodium fluoride, and CPP-ACP). After the treatment phase, the colors of samples were evaluated and compared with the pretreatment values. Results: Both demineralized and intact enamel samples underwent increased whiteness (L*) and reduced yellowness (b*) upon treatment with any of the agents (P < 0.001). More pronounced results were achieved with CPP-ACP. Conclusion: These treatments generally make the teeth look whiter, brighter, and clearer with a more natural appearance.

How to cite this article:
Malekipoor M, Shirani F, Mousavinasab SM, Jafari N, Sharifinejad N. Spectrophotometric Evaluation of the Color Change of Artificial White Spot Lesions Treated with Three Different Commercially Available Remineralizing Agents: An In Vitro Study.Dent Hypotheses 2022;13:90-93

How to cite this URL:
Malekipoor M, Shirani F, Mousavinasab SM, Jafari N, Sharifinejad N. Spectrophotometric Evaluation of the Color Change of Artificial White Spot Lesions Treated with Three Different Commercially Available Remineralizing Agents: An In Vitro Study. Dent Hypotheses [serial online] 2022 [cited 2022 Sep 26 ];13:90-93
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Dental caries is a biofilm-mediated, diet-modulated, multifactorial, noncommunicable, dynamic disease resulting in the net mineral loss of dental hard tissues. It is determined by biological, behavioral, psychosocial, and environmental factors.[1] Incipient enamel carious lesions are characterized by mineral loss beneath the surface and apparently intact surface layer. As the lesion progresses, the increased porosity within its body causes the characteristic whitish appearance.[2]

The presence of white spot lesions (WSLs) on the buccal surfaces are often associated with areas of plaque accumulation, compromising the esthetic appearance of teeth.[3] If paralyzed, the appearance of these lesions changes from chalky and rough (active) to shiny and smooth (inactive).

WSLs can regress or even disappear through remineralization.[4] These lesions scarcely disappear clinically. Deeper lesions, in particular, do not remineralize completely as the formation of a hypermineralized surface layer may hamper the remineralization of the subsurface lesion body.[5]

Fluoride therapy is one of the most common and oldest treatments that has been used to prevent WSLs.[4] Even though the lesions become hard and shiny after fluoride therapy, the remnants of opaque enamel can still be found.[6] In addition, in recent years, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP, MI Paste, Recaldent, GC America Inc, IL) has become more popular. It is a nanocomplex of calcium ions, phosphate ions, and hydroxide ions stabilized by casein phosphopeptides.[7] The CPP-ACP nanocomplexes release calcium, phosphate, and hydroxide ions at the tooth surface, thereby reducing demineralization and enhancing remineralization. A randomized controlled clinical trial showed that CPP-ACP remineralized the WSLs of enamel, slowed the progression of caries, reduced the tooth sensitivity, and improved the aesthetics of hypomineralized and stained enamel.[8]

Since fluoride is not able to penetrate demineralized areas, other remineralizing agents with a high penetration ability have been introduced, one of which is MI Paste Plus (GC America Inc, IL).[9]It is called casein phosphopeptide-amorphous calcium fluoride-phosphate (CPP-ACFP, MI Paste Plus) and contains 0.2% fluoride. When placed in the oral cavity, it releases phosphate, fluoride, and calcium on biofilms. The CPP-ACFP solutions produce greater remineralization than the CPP-ACP solutions at pH 5.5.[10] However, the effect of CPP-ACP and CPP-ACFP on intact enamel color or on demineralized enamel is not clear. Therefore, the present study was aimed to investigate the effect of fluoride (Jordan, Copenhagen, Denmark), CPP-ACP, and CPP-ACFP on the tooth color change during the treatment of WSLs.

 Materials and Methods

The study protocol was approved by local ethical committee of Islamic Azad University, Isfahan (Khorasgan) Branch (Approval ID:23810201901033). In the present in vitro study, a total of 96 human sound maxillary incisors which were extracted during periodontal treatment were cleaned, disinfected (stored in 0.1% thymol solution for 48 hours), and coated with acid-resistant nail polish (Revlon, New York City, NY) leaving a narrow square “window” of approximately 4 mm width on the intact surface of the buccal enamel.

The prepared samples were randomly divided into six groups (n = 16) using Groups 1, 2, and 3 were kept as “intact” control groups, and groups 4, 5, and 6 were subjected to the demineralization process and considered “demineralized” groups. The initial color of all samples was determined and recorded by Shadepilot spectrophotometer (DeguDent, Hanau, Germany) according to the manufacturer’s instructions. Intact groups directly underwent treatment with CPP-ACFP, CPP-ACP, and fluoride (0.2% NaF), respectively. Simultaneously, groups D4, D5, and D6 were immersed in a demineralizing solution containing 2.2 mM CaCl2, 2.2 mM KH2PO4, and 0.05 M acetic acid, at pH 4.4 with 1 M KOH (all chemicals were from Merck, NJ).[11]

Then, the teeth were exposed to the solution for 96 hours to induce WSLs. The color of these three groups was immediately determined using a DeguDent Shadepilot spectrophotometer after the demineralization process. Next, the demineralized groups were subjected to treatment with CPP-ACFP, CPP-ACP, and fluoride, respectively. Treatment phase for all groups was carried out in pH cycling conditions [Figure S1][12] for 3 weeks[11] to simulate oral conditions.[11] The remineralization solution contained 1.5 mM CaCl2, 0.9 mM NaH2PO4, and 0.15 M KCl, with pH = 7.

The color change was determined and reported according to the CIELAB (Commission Internationale de l’éclairage L*a*b*) color system, which defines the color along the three axes L*, a*, b* using the DeguDent Shadepilot spectrophotometer. In this system, L* represents the lightness, and a* and b* indicate hue numerical axes. In addition, ΔE was used as the indicator of the overall evaluation of color differences and was calculated using the following formula[13]:


Data were analyzed by SPSS-22(IBM Corp, Armonk, NY) software using repeated measures ANOVA, one-way ANOVA, and two-way ANOVA tests. The significance level was set at 0.05.


In the intact group, the a* and b* color indices reduced significantly, and the L* level increased significantly (paired t test, P < 0.001). There was no significant difference in the mean ΔE among the three treatment agents (one-way ANOVA, P = 0.317).

In the demineralization group, the a* and b* color indices reduced and L* increased significantly (repeated measures ANOVA, P < 0.001). There was no significant difference in the mean ΔE among the three treatment agents in the tested intervals (one-way ANOVA, P = 0.23).

The mean ΔE in all three treatment agents in the three intervals was significantly higher than 3.3 (one-sample t test, P < 0.001) [Table 1].{Table 1}

The mean ΔE in the CPP-ACP was significantly higher in the intact group than in the demineralization group (independent t test, P = 0.02). In CPP-ACFP and fluoride, the mean ΔE was higher in the intact group than in the demineralization group, but the difference was not significant (P = 0.07). The overall view of ΔE during different stages of study is shown in Figure S2.[14]


In improving the color characteristics of the demineralized lesions, even though resin infiltration produces better results, the remineralizing agents such as calcium and fluoride formulations are preferred.[8],[15]

Nowadays, remineralization is used for the prevention and treatment of early carious lesions. Fluoride and fluoride-containing products are the most commonly used agents.[11] In recent years, CPP-ACP and compounds derived from it have been added to this class of agents. Some studies have also reported the higher penetration depth and efficiency of CPP-ACP and CPP-ACFP than other remineralization treatments.[16],[17]

In the present study, CIELAB was used to determine the color of samples, which describes the color in three-dimensional environments.[18] Due to the clinical application of color sets in common treatments, color determining sets have been used in some previous studies, but these color sets have more limitations than the applied method. A study showed the intraexaminer agreement on the tooth color can reach 60%.[19] DeguDent Shadepilot spectrophotometer has higher repeatability and reliability than the spectrophotometric reference system, is less dependent on the operator, and has a higher objective value.[13]

In this study, the null hypothesis was rejected, and the utilization of remineralizing agents caused changes in the CIELAB color characteristics in both intact and demineralized dental tissues. In general, regarding the ΔE reported for all the groups, it seems that substantial color changes have occurred due to treatment with all the substances.[20]

Studies have reported varying results for the effect of different agents on the WSLs. Akin and Basciftci showed significantly higher clinical success for CPP-ACP than fluoride and other control groups in reducing the size of WSLs.[21] Cochrane et al. stated that the CPP-ACP and CPP-ACFP-treated teeth showed higher translucency than the opaque demineralized control group,[22] confirming the results of the present study, where CPP-ACP increased the lightness, whiteness, and translucency more than CPP-ACFP and fluoride. Brochner et al. also assessed the effect of CPP-ACP and fluoride toothpaste on the enamel WSLs and found a significant improvement in the color of lesions, but no significant difference between the two compounds,[23]which is consistent with our results.

The results of Kim et al.[24] are in line with the results of the present study regarding significant changes in L* b* indices and slight changes in a* upon treatment with fluoride,[25],[26] indicating reduced yellowness and increased whiteness of the tooth upon fluoride treatment.

Furthermore, another clinically important issue is the effect of these agents on the intact dental tissue. During remineralization, the morphology and structure of minerals change. A mineralized crystalline structure may include different orientations that lead to different visual properties compared to the intact enamel. It is also possible that specific pores in the crystals do not get completely filled upon remineralization.[26]In our study, intact enamel was more appealing, lighter, and whiter after treatment with remineralizing agents, which was more pronounced in CPP-ACP than CPP-ACFP and fluoride groups and significant only in the CPP-ACP group. However, Shirani and Sakhaei reported the same effects for these two compounds as other common fluoride-containing compounds.[27] All the three agents caused considerable color changes in both intact and demineralized tissues, but in terms of esthetics, the convergent or divergent nature of the changes is important.

Based on ΔE, the color difference between the intact and demineralized groups showed increased whiteness (L*) and reduced yellowness (b*) for both groups. However, this is in contrast to some previous reports suggesting the irreversible nature of the lesion appearance.[3],[4] Some previous studies have reported reduced lifeless teeth after the treatment.[2],[9],[28]

In general, changes in the demineralization and treatment processes are relatively high, and it seems that mean ΔE difference does not change significantly between the lesion and surrounding tissue. However, since the trend of all changes is in a common direction, the tooth has a better appearance after the treatment.


All three applied treatment agents, especially CPP-ACP, significantly improved the whiteness and lightness and reduced the redness and yellowness of intact and demineralized teeth. Although WSLs in the demineralized teeth were not disappeared fully by any of the agents, the teeth became whiter, brighter, and clearer with a more natural appearance.

Financial support and sponsorship

This study was financed in part by Isfahan University of Medical Sciences and Dental Material Research Center.

Conflicts of interest

There are no conflicts of interest.


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