Journal of

Dental Research, Dental Clinics, Dental Prospects

Original Article

Prevention of white spot lesions using three remineralizing

agents: An in vitro comparative study
Soodeh Tahmasbi1 • Seyedezahra Mousavi2 • Marjan Behroozibakhsh3 • Mohammadreza Badiee4*
1
Department of Orthodontics, Preventive Dentistry Research Center, Research Institute of Dental Sciences, Dental School, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
2
Dentist, 3Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3
Department of Dental Materials, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
4
Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
*Corresponding Author; E-mail: [email protected]

Received: 27 September 2017; Accepted: 12 February 2019

J Dent Res Dent Clin Dent Prospect 2018; 13(1):36-42|doi: 10.15171/joddd.2019.006
This article is available from: https://joddd.tbzmed.ac.ir

© 2019 Tahmasbi et al. This is an Open Access article published and distributed by Tabriz University of Medical Sciences under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

Abstract
Background. Enamel demineralization around orthodontic brackets is an important clinical problem . This study sought to
compare the efficacy of sodium fluoride (NaF), casein phosphopeptide amorphous calcium phosphate fluoride (CPP-ACP-F;
MI Paste Plus) and a water-based cream (Remin Pro), which contains hydroxyapatite and fluoride for prevention of enamel
demineralization.
Methods. Fifty-six sound human premolars extracted for orthodontic purposes were collected. After cleaning, the crowns
were mounted in acrylic resin and all the surfaces were coated with nail varnish except for a 3×4-mm window on the buccal
surface. The samples were randomly divided into four groups of 14 and subjected to pH cycling for 14 days, during which
the teeth were immersed in artificial saliva for 21 hours and in demineralizing agent for three hours daily. Before transferring
the samples from the saliva to the demineralizing solution, the remineralizing agent (0.05% NaF, MI Paste Plus or Remin Pro
Paste, depending on the group) was applied on the samples once a day for five minutes. No remineralizing agent was used in
the control group. Surface microhardness of samples was measured by Vickers microhardness tester at baseline and after
the intervention. The data were analyzed using one-way ANOVA, ANCOVA, Bonferroni test and Tukey test. Statistical sig-
nificance was set at P<0.05.
Results. The mean microhardness was significantly different between the test and control groups (P<0.0001). Other differ-
ences were not significantly different (P>0.05).
Conclusion. The results showed that 0.05% NaF was more efficient than Remin Pro and MI Paste Plus for prevention of
white spot lesions (WSLs). Remin Pro and MI Paste Plus were not significantly difference from the control group in this
regard.
Key words: Dental caries, fluorides, orthodontics, tooth demineralization.

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 Prevention of White Spot Lesions 37

Introduction and confer acid resistance. Fluoride ions replace the

hydroxyl groups in the formulation of hydroxyapatite
E namel demineralization around orthodontic
brackets is an important clinical problem. The
prevalence of white spot lesions (WSLs) in orthodon-
and result in the formation of fluorapatite.1 Fluoride
inhibits further demineralization of enamel but at the
same time, it prevents the uptake of calcium and phos-
tic patients ranges from 25% to 46%.1-3 These lesions
phate ions, which are required for the repair of deep
most commonly occur in the cervical part of the mid-
lesions.5
dle third of the crowns of first molar, lateral incisor
MI Paste Plus contains amorphous calcium phos-
and canine teeth.4 White spot lesions can be detected
phate (ACP), casein phosphopeptide (CPP) and fluo-
as white opaque lesions after air-drying the teeth.5
ride; ACP is a reactive, super-saturated solution of
These lesions often develop after four weeks if no an-
calcium and phosphate, which can release these ions
ticariogenic agent is used, which highlights their fast
as well as α s1-casein and β-casein. The ACP-CPP
occurrence.6 High prevalence of enamel decalcifica-
nanocomplex can penetrate into the enamel porosities
tion during fixed orthodontic treatment is partly at-
due to the small size of particles. It remineralizes the
tributed to the irregular bracket surface and presence
superficial enamel crystals and prevents deminerali-
of orthodontic wires, bands and other attachments,
zation of tooth structure.10
which enhance plaque retention, complicate oral hy-
Remin Pro, a new remineralizing agent, was re-
giene and limit the self-cleaning capacity of teeth with
cently introduced to the market. It is supplied in the
the salivary flow and movement of oral muscles. Con-
form of a water-based cream, which contains hydrox-
sequently, the plaque pH drops due to the presence of
yapatite and fluoride. It has been suggested for pre-
fermentable carbohydrates, faster accumulation and
vention of enamel demineralization during the course
maturation of plaque and colonization of aciduric bac-
of orthodontic treatment and after dental bleaching to
teria such as Streptococcus mutans and Lactobacilli.7
prevent and control tooth hypersensitivity. It has been
Elimination of microbial plaque after orthodontic
claimed that this product is preferred for use by pa-
treatment is not sufficient for treatment of WSLs be-
tients who are allergic to bovine proteins since it is
cause secondary lesions may develop 5‒12 years after
devoid of bovine proteins present in the composition
completion of orthodontic treatment.8 On the other
of CPP-ACP. In search of the literature, no previous
hand, natural remineralization by the mineral ions
study was found to have compared the efficacy of Re-
present in the saliva only occurs in the superficial
min Pro and MI Paste Plus or fluoride for prevention
layer of WSLs. Moreover, even after remineraliza-
of WSLs.11 Thus, this in vitro study was conducted to
tion, the appearance of the tooth is not esthetically ac-
compare the efficacy of NaF, MI Paste Plus and Re-
ceptable. Thus, remineralizing agents are required for
min Pro for prevention of enamel demineralization.
treatment of deeper lesions and improved esthetics.7
Control of WSLs includes prevention of deminerali-
Methods
zation and enhancement of remineralization of pri-
mary lesions. Obviously, prevention is superior to Ethical approval (code:
treatment.7 No consensus has been reached on an effi- IR.SBMU.RIDS.REC.1394.60) was obtained
cient, predictable and esthetically acceptable treat- from Shahid Beheshti University Medical Ethics
ment for these lesions.4 The most important preven- Committee. Fifty-six sound human premolars (with-
tive measure is to increase enamel resistance against out discoloration or coronal caries) were collected and
acid attacks.9 The ability of bacterial biofilm to absorb cleaned with pumice paste and prophy brush using a
calcium, phosphate and fluoride from the saliva and low-speed handpiece. The crowns were cut at the ce-
extraoral sources results in enamel remineralization mentoenamel junction using a fissure bur and a high-
following demineralization. Efficient remineraliza- speed handpiece. All the samples were stored in saline
tion requires the exposure of enamel to low concen- solution. Using a sticker, a window measuring 3×4
trations of the above-mentioned ions for long periods mm was covered on the buccal surface of the samples
of time. Thus, extraoral sources of calcium, phosphate and the remaining areas were coated with nail varnish.
and fluoride can change the cariogenic potential of The samples were then mounted in auto-polymerizing
dental biofilm.7 Fluoride is the most commonly used acrylic resin (Acropars, Tehran, Iran) and coded from
substance for remineralization.5 The scientific basis 1 to 56. Prior to the intervention, the microhardness of
for the use of fluoride in fight against caries is that samples was measured using a microhardness tester
fluoride ions can penetrate into the crystalline struc- (V-Test, Baresiss, Germany) in three points with a
ture of dental hard tissues, decrease their solubility Vickers diamond indenter under a load of 200 N for

JODDD, Vol. 13, No. 1 Winter 2019

38 Tahmasbi et al.

10 seconds. The resultant value was recorded for each the manufacturer), the paste was wiped off the
sample. Next, each sample was immersed in saline so- surfaces (without rinsing with water) and the
lution in a glass container. The samples were ran- samples were placed in the demineralizing so-
domly divided into four groups of 14 and subjected to lution for three hours.
the intervention for 14 days. The four groups included 4. Remin Pro group: The samples in this group
the control group (group 1), NaF (group 2), MI Paste (#43 to #56) were treated similar to that in the
Plus (group 3) and Remin Pro (group 4). MI Paste Plus group with the exception that
During the experiment, two solutions (manufac- Remin Pro (Voco, Germany) was used as the
tured in the chemical laboratory of Jahad Daneshgahi, remineralizing agent instead of MI Paste Plus.
Tehran, Iran) were used for testing the samples: It should be noted that the samples were not
1. A demineralizing solution with a pH of 4.5 and washed during the process of transfer from one
the following formulation: CaCl2 (2.2 mM) + solution to the other.
NaH2PO4 (2.2 mM) + acetic acid (50 mM) + After 14 days, microhardness of samples was meas-
NaCl (100 mM) + NaF (1 ppm) + NaN3 ured using a microhardness tester (V-Test, Baresiss,
(0.02%). Germany) in three points with a Vickers diamond in-
2. Artificial saliva with a pH of 6.9 with the fol- denter under a load of 200 N for 10 seconds under the
lowing formulation: KCl (0.04) + NaCl (0.04) same conditions. The microhardness values were rec-
+ CaCl2.2H2 (0.09) + NaH2PO4.2H2 (0.069) + orded for each sample.
MgCl2.6H2O (0.008) + urea (0.05) + ascorbic
Statistical Analysis
acid (0.01) + H2O (10 mL + methyl-p hydroxyl
benzoate (0.15) + PEG 6000 (7) + glucose To assess the presence of significant differences
(0.1) among the groups, data were analyzed using one-way
First, the stock solution was prepared from the ANOVA and Tukey test. Statistical analysis of the
above-mentioned compounds. Next, using the for- data was carried out using SPSS 21. Statistical signif-
mula C1V1=C2V2, the required volume of each solu- icance was set at P<0.05.
tion was determined. Afterwards, the samples were
subjected to pH cycling for 14 days as follows: Results
1. Control group: The samples in this group (#1 Assessment of microhardness values after the inter-
to #14) were immersed in 10 mL of artificial vention revealed that the surface microhardness of all
saliva for 21 hours daily and then immersed in the samples in the four groups changed after the inter-
10 mL of demineralizing agent for three hours. vention compared to the baseline values. The mini-
It should be noted that these solutions were re- mum change in microhardness occurred in group 2
freshed daily. samples subjected to NaF mouthwash, which means
2. 0.05% NaF group: The samples in this group that NaF had the greatest efficacy for prevention of
(#15 to #28) were immersed in 10 mL of arti- enamel demineralization, followed by Remin Pro
ficial saliva for 21 hours daily and then im- (group 4), MI Paste Plus (group 3) and the control
mersed in 0.05% NaF (manufactured in the group (group 1). Descriptive statistics of surface mi-
chemical laboratory of Jahad Daneshgahi, crohardness values in the study groups are shown in
Tehran, Iran) for five minutes. Next, the sam- Table 1. The mean changes in microhardness of sam-
ples were retrieved from the mouthwash and ples are presented in Figure 1.
immersed in the demineralizing agent for three Changes in surface microhardness of the samples
hours. after intervention was compared among the four
3. MI Paste Plus group: The samples in this groups using one-way ANOVA, which showed sig-
group (#29 to #42) were immersed in 10 mL nificant differences in microhardness changes be-
of artificial saliva for 21 hours daily. Using a tween two or more groups (P<0.05, Figure 2). Thus,
microbrush, MI Paste Plus (Recaldent, GC pairwise comparisons of the groups were carried out
Corp., Japan) was then applied on their sur- using Tukey tests. The differences in the mean
faces. After five minutes (as recommended by

Table 1. Descriptive statistics (Mean (SD)) of surface microhardness in the four groups
Study groups Baseline microhardness Post-intervention microhardness Change in microhardness Relative change
Control 300.82 (85.25) 57.08 (58.93) 243.73 (85.47) 0.81 (0.15)
0.05% NaF 282.14 (92.05) 194.54 (74.41) 87.6 (91.55) 0.24 (0.31)
MI Paste Plus 276.50 (54.88) 86.25 (39.05) 190.25 (69.04) 0.67 (0.14)
Remin Pro 314.0 (59.41) 148.05 (84.19) 165.95 (81.14) 0.53 (0.26)

JODDD, Vol. 13, No. 1 Winter 2019

 Prevention of White Spot Lesions 39

consensus has been reached on an ideal solution for

this common problem. As a result, WSLs remain a
major concern for orthodontists and patients. Consid-
ering the high prevalence of WSLs in orthodontic pa-
tients and the significance of esthetics in these pa-
tients, occurrence of WSLs must be prevented. Thus,
we evaluated the efficacy of NaF, MI Paste Plus and
Remin Pro for prevention of WSLs to find the most
efficient agent for this purpose. The second objective
of this study was to compare the efficacy of Remin
Pro, a recently introduced agent, with the remaining
two agents. It contains hydroxyapatite and fluoride
and can decrease tooth hypersensitivity, prevent
enamel demineralization and enhance remineraliza-
tion of enamel lesions.13
In a systematic review by Chen et al,8 the efficacy
Figure 1. Comparison of changes in surface microhard-
of remineralizing agents for the treatment of WSLs
ness after the intervention compared to baseline in the
four groups.
after orthodontic treatment, CPP-ACP, NaF and MI
Paste Plus were introduced as effective agents for
changes in surface microhardness were significant be- treatment of enamel lesions. However, comparison of
tween the NaF and control (P<0.0001), NaF and MI the results of the reviewed studies yielded no definite
Paste Plus (P=0.01) and NaF and Remin Pro (P=0.03) conclusion regarding the most ideal and reliable agent
groups; the remaining differences between the groups for treatment of primary enamel lesions.
were not statistically significant (P>0.05). In this study, pH cycling was performed to simulate
the oral environment and assess the preventive effect
Discussion of agents. Duration of immersion of samples in the
White spot lesions are subsurface enamel porosities, demineralizing agent and artificial saliva has been
which develop due to enamel demineralization. WSLs variable in different studies between 5 to 28 consecu-
often occur at the center of tooth surface and have a tive days, 3 to 6 hours a day in demineralizing
distinguishable margin. The WSLs are highly preva- agent.14-16 In this study, we immersed the samples for
lent in patients under fixed orthodontic treatment due 21 hours a day in artificial saliva with a pH of 6.9 and
to the presence of orthodontic wires and bands, which in demineralizing agent with a pH of 4.5 for three
enhance plaque retention, decrease pH and result in hours for a period of 14 days. The pH of demineraliz-
rapid shift of oral microbial flora to a pathogenic ing agent in previous studies varied from 3.5 to 5.17,18
flora.12 Several methods have been used to assess the de-
Although several methods have been proposed for gree of remineralization of samples, including DI-
prevention and control of initial enamel lesions, no AGNOdent,13 quantitative light-induced fluorescence
(QLF), scanning electron microscopy,19 polarized
light microscope,20-21 x-ray spectrophotometer,9
standard photography,4 micro-computed tomogra-
phy22 and transverse micro-radiography.23
Microhardness tester has been used in a number of
studies for assessment of surface microhardness of
samples.5,15,16,24 Similarly, we used Vickers micro-
hardness tester for measurement of surface micro-
hardness of samples in three points; 200-N load was
applied for 10 seconds at baseline and after the inter-
vention for this purpose.
It should be mentioned that many previous studies
on prevention of WSLs have evaluated the efficacy of
different orthodontic bracket bonding agents for pre-
vention of enamel lesions.24-26 A limited number of
studies have compared the caries prevention efficacy
Figure 2. Relative changes in surface microhardness.

JODDD, Vol. 13, No. 1 Winter 2019

40 Tahmasbi et al.

of CPP-ACP and MI Paste Plus with a methodology The majority of previous studies on Remin Pro fo-
similar to ours; however, the therapeutic and reminer- cused on its effects on bleached teeth. For instance,
alizing effects of these products have been well docu- Heshmat et al29 evaluated and compared the effect of
mented.4,5,8 Remin Pro, MI Paste Plus and natural saliva on mi-
Assessment of the microhardness values obtained in crohardness of bleached teeth. They measured the mi-
the current study revealed that minimal changes oc- crohardness at baseline and after bleaching with 35%
curred in microhardness of the NaF group, which in- hydrogen peroxide. Next, they used Remin Pro, MI
dicates that among the study groups, NaF had the Paste Plus and natural saliva for 15 days and measured
greatest efficacy for prevention of enamel demineral- the microhardness of samples again after the interven-
ization. Remin Pro, MI Paste Plus and control group tion. They concluded that the surface microhardness
ranked next. This finding was in agreement with the of samples decreased after bleaching but no signifi-
results of several previous studies. Hamba et al22 re- cant difference was noted among the three materials
ported minimum loss of minerals and shallowest le- for increasing the surface microhardness of samples.
sions in the fluoride group compared to CPP-ACP and Despite the numerous advantages of CPP-ACP and
CPP-ACP-F.22 The preventive effects of CPP-ACP, MI Paste Plus, results of studies on their efficacy for
MI Paste Plus, fluoride varnish and sodium-calcium- prevention of enamel demineralization are controver-
phosphosilicate were compared in another study and sial. However, the therapeutic effects of these agents
it was found that fluoride varnish was the most effec- have been previously confirmed and it has been stated
tive of all.21 Another study confirmed the optimal ef- that materials such as CPP-ACP paste have therapeu-
ficacy of fluoride for prevention of WSLs.27 Several tic efficacy almost similar to that of NaF.5,16,24,27 In the
clinical and in vitro studies have documented that flu- current study, MI Paste Plus ranked third after NaF
oride-containing products have the greatest reminer- and Remin Pro. MI Paste Plus had insignificant effi-
alizing effect compared to other products.15 cacy for prevention of enamel demineralization and
Bergstrand and Twetman28 collected evidence on had no significant difference from the control group
the efficacy of primary and secondary (therapeutic) in this respect. However, in some previous studies, MI
preventive measures for WSLs around orthodontic Paste Plus significantly inhibited the formation of
brackets. In seven out of nine articles related to pre- enamel lesions, although its efficacy was still lower
vention, the mean preventive effect of fluoride was re- than that of fluoride in this respect.21-23 This contro-
ported to be 42.5%. An in vitro study assessed the ef- versy in the results might be attributed to differences
ficacy of four types of bioactive glass bonding agents in methods of assessment of samples since we only
compared to Transbond XT conventional bonding measured the surface microhardness using a Vickers
agent for prevention of WSLs and concluded that flu- microhardness tester while previous studies used po-
oride-containing bonding agents was most effective larized light microscope and micro-computed tomog-
for prevention of WSLs.14 Another study compared raphy for precise quantification of mineral loss and
the efficacy of five different agents for prevention of measurement of depth of lesions.21,23
WSLs and concluded that fluoride-containing glass-
ionomers significantly prevented the occurrence of Conclusion
sub-surface lesions around orthodontic brackets.19 This in vitro study showed that NaF mouthwash had
In the current study, Remin Pro ranked second after the greatest efficacy for prevention of enamel demin-
NaF and prevented the occurrence of enamel lesions eralization compared with Remin Pro and MI Paste
to some extent but had no significant difference from Plus. Although the latter two inhibited enamel demin-
the control group. A search in the literature yielded eralization to some extent, they exhibited no signifi-
only one study on the remineralizing effect of Remin cant difference from the control group in this regard.
Pro, conducted in 2014, which assessed the changes
in mineral content of enamel surface due to the appli- Conflict of Interests
cation of acidulated phosphate fluoride, Remin Pro The authors declare no conflict(s) of interest related to the
and propolis along with CO2 laser irradiation showed publication of this work.
the highest mineral content in samples treated with
Author contributions
acidulated phosphate fluoride combined with laser ir-
radiation.9 This finding was in agreement with our re- 1-Design of the work: Soodeh Tahmasbi
sults, indicating that fluoride products were more ef- 2-The acquisition: Seyedezahra Mousavi, Marjan Beh-
fective than Remin Pro in caries prevention. roozibakhsh
3-Analysis: Mohammadreza Badiee, Soodeh Tahmasbi
4-Drafting the work: Mohammadreza Badiee

JODDD, Vol. 13, No. 1 Winter 2019

 Prevention of White Spot Lesions 41

Acknowledgements bone structure. Scanning. 2007 Jul-Aug;29(4):162-70.

11. Somasundaram P, Vimala N, Mandke LG. Protective poten-
The authors would like to thank the Research Deputy of the tial of casein phosphopeptide amorphous calcium phosphate
Dental School at Shahid Beheshti University of Medical containing paste on enamel surfaces. J Conserv Dent. 2013
Sciences for their grant and financial support. This study Mar;16(2):152-6. doi: 10.4103/0972-0707.108199.
was extracted from the undergraduate thesis of 12. Lapenaite E, Lopatiene K, Ragauskaite A. Prevention and
Seyedezahra Mousavi under supervision of Dr. Soodeh treatment of white spot lesions during and after fixed ortho-
Tahmasbi at Shahid Beheshti Dental School. dontic treatment: A systematic literature review. Stoma-
tologija. 2016;18(1):3-8.
Funding 13. Madhavan S, Nayak M, Shenoy A, Shetty R, Prasad K. Den-
tinal hypersensitivity: A comparative clinical evaluation of
This study was funded by the Research Deputy of the CPP-ACP F, sodium fluoride, propolis, and placebo. J Con-
Dental School, Shahid Beheshti University of Medical serv Dent. 2012 Oct;15(4):315-8. doi: 10.4103/0972-
Sciences. 0707.101882.
14. Manfred L, Covell DA, Crowe JJ, Tufekci E, Mitchell JC. A
Ethics approval novel biomimetic orthodontic bonding agent helps prevent
This study was approved by ethics committee of Research white spot lesions adjacent to brackets. Angle Orthod. 2013
Institute of Dental scinces, Shahid Beheshti University of Jan;83(1):97-103. doi: 10.2319/110811-689.1.
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Medical Sciences and the reference number is ferent topical agents on enamel demineralization around or-
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