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Original Article

A comparative evaluation of the remineralization

potential of three commercially available
remineralizing agents on white spot lesions in primary
teeth: An in vitro study
Pujan Kamath, Rashmi Nayak, Shobha U Kamath1, Deepika Pai
Department of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences Manipal, Manipal University, 1Department of
Biochemistry, KMC Manipal, Manipal University, Manipal, Karnataka, India

ABSTRACT Address for correspondence:

Dr. Rashmi Nayak,
Background: The focus in caries management Department of Pedodontics and Preventive Dentistry,
has shifted to early detection of caries lesions and Room No. 6, Manipal College of Dental Sciences Manipal,
targeted noninvasive management of incipient Manipal University, Manipal ‑ 576 104, Karnataka, India.
lesions using novel remineralizing agents. E‑mail:
Aim: This study aimed to compare and evaluate
the remineralization potential of commercially
available agents containing nano‑hydroxyapatite Access this article online
(nano‑HA), casein phosphopeptide‑amorphous Quick response code Website:
calcium phosphate fluoride (CPP‑ACPF), and
Tricalcium phosphate (TCP) on artificially DOI:
induced white spot lesions in primary teeth. 10.4103/JISPPD.JISPPD_242_16
Settings and Design: This is an in vitro double‑blind PMID:
randomized study. Materials and Methods: Forty ******
extracted or exfoliated primary teeth were selected
and randomized as follows: Group I: FTCP, Group II:
primary teeth, remineralizing agents, tricalcium
fluoridated dentifrice, Group  III: CPP‑ACPF, and phosphate, white spot lesions
Group  IV: Nano‑HA. DIAGNOdent readings
and scanning electron microscope  (SEM) energy
dispersive X‑ray  (EDX) analysis were carried out
at baseline. The samples were subjected to the
test agents after inducing white spot lesions. The
readings were repeated postdemineralization and
The current consensus in caries management is
postremineralization. Statistical Analysis: The that caries should be detected and managed in its
data were analyzed using IBM SPSS version  20 earliest stages when nonsurgical reversal can be
software with one‑way ANOVA, post hoc Tukey’s
HSD, and paired t‑test. Results: SEM evaluation This is an open access article distributed under the terms of the Creative
showed favorable surface changes in all the four Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which
study groups after remineralization therapy. allows others to remix, tweak, and build upon the work non‑commercially,
Intragroup comparison of DIAGNOdent and as long as the author is credited and the new creations are licensed under
EDX readings showed a highly significant the identical terms.
difference between baseline, postdemineralization, For reprints contact:
and postremineralization values. However,
the intergroup comparison was statistically How to cite this article: Kamath P, Nayak R, Kamath SU,
nonsignificant. Conclusion: All test agents were Pai D. A comparative evaluation of the remineralization potential
comparable in their remineralization potential. of three commercially available remineralizing agents on white
spot lesions in primary teeth: An in vitro study. J Indian Soc Pedod
KEYWORDS: Casein phosphopeptide‑amorphous Prev Dent 2017;35:229-37.
calcium phosphate, DIAGNOdent, hydroxyapatite,

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Kamath, et al.: A comparative evaluation of remineralising agents on white spot lesions

achieved. While fluoride is a time‑tested, effective, Teeth with at least one sound tooth surface, without
caries‑preventive agent, the search for compounds any visible caries, hypoplastic lesions, stains, or
with additional and synergistic benefits has yielded a white spot lesions were included in the study. After
plethora of novel agents for targeted remineralization extraction, the teeth were cleaned, washed with water,
therapy.[1] and stored in isotonic saline.

Enamel of primary teeth is less mineralized, exhibits Sample preparation

a greater diffusion coefficient, and consequently The coronal portion of the teeth was separated from
more susceptible to acid dissolution compared to the roots using a diamond disc  (Diatech CH‑9435,
enamel of permanent teeth.[2] Early childhood caries Swiss Dental Instruments, Lot: 9605) mounted on a
which affects the primary dentition frequently Micro Motor Straight Hand Piece  (NSK, Japan) in
manifests as white spot lesions, and aggressive the presence of a water coolant. The samples were
preventive therapy for remineralization of these then embedded into the partially set acrylic resin in
lesions is essential for their reversal. Thus, the aim of cylindrical plastic molds.
the present study was to compare and evaluate the
remineralization potential of commercially available A 3  mm  ×  3  mm sticking plaster was placed on the
agents containing hydroxyapatite  (HA), casein exposed tooth surface and acid‑resistant nail varnish
phosphopeptide‑amorphous calcium phosphate was applied around it to delineate the area to be
fluoride  (CPP‑ACPF), and tricalcium phosphate on studied.
artificially induced white spot lesions in primary teeth.
A null hypothesis was proposed that there will be no
difference in the remineralization potential of the test Baseline recordings
agents. The baseline DIAGNOdent  (Kaltenbach and Voigt
GmbH and Co. KG, Biberach, Germany) readings were
recorded for all the samples. The present study used
Materials and Methods the newly formulated DIAGNOdent cutoff limits given
by Goel et  al.[4] for detection of early carious lesions
The present study was conducted in the Department of in primary teeth. Of the 65 teeth initially collected,
Pedodontics and Preventive Dentistry, in collaboration 25  samples showing a value >6 were discarded,
with the Department of Biochemistry with Institutional establishing a final sample size of 40.
Ethical Committee’s approval (IEC 454/2012).
Test agents used The samples were randomly divided into four groups
• Nano‑HA  –  Reminpro  (VoCo, Germany; Lot No: (I–IV) comprising ten samples each using simple
1106330) randomized sampling  (n  =  10). Individual sample
It is a water‑based cream containing HA and within each group was assigned an alphabetical
fluoride (1450 ppm sodium fluoride) denotation (A–J).
• CPP‑ACPF  –  GC Tooth Mousse Plus  (GC
Corporation, Tokyo, Japan; Lot No: 1108171)
It is a water‑based cream containing CPP‑ACPF Demineralizing regimen
with incorporated fluoride (0.2% w/w, 900 ppm) The freshly prepared demineralization solution
• Tricalcium Phosphate – Clinpro™ (3M ESPE, USA; comprised 2.2 mM CaCl2, 2.2 mM NaH2PO4, 0.05
Lot No: 10021) M lactic acid, and 0.2  ppm fluoride.[5] The pH was
Tooth cream containing tricalcium phosphate and adjusted to 4.5 with 50% NaOH. The solution was
0.21% w/w sodium fluoride maintained at 37°C. Each sample was immersed in
• Fluoridated dentifrice – Colgate (Colgate‑Palmolive, an individual sterile container containing 12  mL of
India) demineralization solution ensuring that the section
Sodium monofluorophosphate with 1000  ppm was completely immersed in the solution. The samples
fluoride. were maintained at 37°C in an incubator  (Autoflow
N‑5510, NuAire, USA) for 72 h.[5] This demineralization
Sample collection procedure was intended to produce a consistent
Sample size of forty was estimated using the power subsurface lesion.
calculation α =  0.05 and β =  0.20 with 80% being the
power of the study. Based on previous findings reported Recording of readings
by Hegde and Moany,[3] a total of 65 primary teeth After demineralization, the samples were thoroughly
indicated for extraction were obtained from patients washed with deionized water and allowed to dry. The
in the age group of 3–14  years. A  written informed samples were evaluated for demineralization using
consent was obtained from the parents at the outset. DIAGNOdent.
Owing to the difficulty in obtaining sound, caries‑free
primary teeth, sound tooth surfaces obtained from Following DIAGNOdent readings, six out of
carious primary teeth were considered in this study. the ten samples from each group  (A–F) were

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Kamath, et al.: A comparative evaluation of remineralising agents on white spot lesions

subjected to scanning electron microscopic  (SEM) Re‑evaluation of test parameters

evaluation  (EVO MA18 with Oxford EDS  [X‑act]) After the 14‑day remineralization regimen, the
to evaluate surface morphology. Estimation of surface was reassessed using DIAGNOdent. The
calcium  (Ca), phosphate, and fluoride  (F)  (weight samples (A–F) were also assessed using SEM‑EDX
percentage) content was done using energy to study the change in surface characteristics and
dispersive X‑ray  (EDX) analysis for which the estimate the mineral content  (Ca, phosphate  [P],
samples were mounted firmly using a graphite and F).
tape on a disc comprising nine specimen holders
and maintained under vacuum. The samples Statistical analysis
were analyzed at 15  kV and  ×  500, ×1500, ×2000, The data were analyzed by IBM Statistical Package
and × 5000 magnifications. for Social Sciences version  20 (IBM corp., Armonk,
New York)  software using one‑way ANOVA, post
Blinding hoc Tukey’s HSD, and paired t‑test. P <  0.005 was
A double‑blind investigation was planned since all considered statistically significant.
the test agents had the same color (white) and similar
consistency. However, since the packaging was not
similar, in order to confirm the double‑blind design
of the study, each of the test agents was coded as I,
II, III, and IV and entrusted to the chief supervisor On comparing the DIAGNOdent readings Paired
of the study. Half pea‑sized quantity of test agent t-test showed a statistically significant increase in
was dispensed half an hour prior to the initiation of the readings from baseline to post-demineralization
treatment in a palette with four wells. The sanctity of [Table 1].
the double‑blind study was strictly maintained and
decoding was done after statistical evaluation of the Paired t‑test showed a statistically significant
data. increase in the readings from baseline to
post‑demineralization  (P  <  0.001) and a statistically
significant decrease postremineralization  (P  <  0.001)
Application of test agents and approaching the baseline values for each of the
The samples in each group were treated with the
experimental groups [Table 2].
allocated remineralizing agent once every 24  h for
14  days. The test agents were continuously applied
onto the tooth surface within the window area with However, post hoc Tukey’s HSD test showed statistically
the help of a disposable cotton tip applicator for nonsignificant difference among the four groups at the
3  min. Samples were then washed with deionized different experimental stages.
water and placed in artificial saliva, maintained at
ambient temperature. Artificial saliva was changed Scanning electron microscopy
every 24  h, just before the immersion of freshly Postdemineralization
treated samples. SEM images [Figures 1-4] taken after 72  h of
demineralization revealed loss of surface integrity.
Following is the composition of artificial saliva used:[5] Loss of aprismatic enamel was evident and it was
• Na3PO4‑3.90 mM, NaCl2‑4.29 mM, KCl ‑ 17.98 mM noticeable that the enamel comprised indistinct,
• CaCl2‑1.10 mM, MgCl2‑0.08 mM, H2SO4‑0.50 mM completely destructed enamel rods with loss of prism
• NaHCO3‑3.27 mM, distilled water, and the pH was cores, wide inter‑rod spaces, and leftovers of fractured
set at 7.2. enamel prism bases suggestive of demineralization.

Table 1: Comparison of DIAGNOdent readings among the four groups

Group Experimental phases Mean SD n P Post hoc test
I Baseline characteristics (a) 2.90 1.29 10 <0.001** b>c>a
DIAGNOdent reading postdemineralization (b) 11.70 3.86 10
DIAGNOdent reading postremineralization (c) 4.00 1.05 10
II Baseline characteristics (a) 3.50 1.08 10 <0.001** b>c>a
DIAGNOdent reading postdemineralization (b) 11.80 4.52 10
DIAGNOdent reading postremineralization (c) 4.40 0.97 10
III Baseline characteristics (a) 3.50 1.51 10 <0.001** b > a, c
DIAGNOdent reading postdemineralization (b) 10.50 2.76 10
DIAGNOdent reading postremineralization (c) 4.10 1.10 10
IV Baseline characteristics (a) 3.10 1.60 10 0.001** b>c>a
DIAGNOdent reading postdemineralization (b) 10.60 4.53 10
DIAGNOdent reading postremineralization (c) 4.40 1.51 10
**Highly significant; *Significant. SD=Standard deviation

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Kamath, et al.: A comparative evaluation of remineralising agents on white spot lesions

Figure 1: Group I  ‑  Postdemineralization  (scanning electron Figure 2: Group I  ‑  Postremineralization  (scanning electron
microscopic image) microscopic image)

Figure 3: Group II  ‑  Postdemineralization  (scanning electron Figure 4: Group II  ‑  Postremineralization  (scanning electron
microscopic image) microscopic image)

Figure 5: Group III  ‑  Postdemineralization  (scanning electron Figure 6: Group III  ‑  Postremineralization  (scanning electron
microscopic image) microscopic image)

Postremineralization [Figures 5‑8] cavities and micropores and re‑establishment of

On observation, the samples demonstrated plugging surface integrity in all the four groups. Distinct surface
of the porous defects with resultant decrease in the coatings deposited by different agents were evident.

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Kamath, et al.: A comparative evaluation of remineralising agents on white spot lesions

Table 2: Intragroup comparison of mean fluoride levels postdemineralization and postremineralization

Group Experimental phases Demineralization Remineralization P
Mean SD Mean SD
I SEM‑EDX postdemineralization ‑ fluoride 4.02 0.50 5.75 0.65 <0.001**
II SEM‑EDX postdemineralization ‑ fluoride 4.20 0.65 5.27 0.42 0.006*
III SEM‑EDX postdemineralization ‑ fluoride 5.21 1.12 7.20 1.86 0.015*
IV SEM‑EDX postdemineralization ‑ fluoride 5.12 3.01 6.59 2.96 0.02*
**Highly significant; *Significant. SD=Standard deviation; SEM=Scanning electron microscope; EDX=Energy dispersive X‑ray analysis

Figure 7: Group IV  ‑  Postdemineralization  (scanning electron Figure 8: Group IV  ‑  Postremineralization  (scanning electron
microscopic image) microscopic image)

However, in comparison to other groups, samples postdemineralization to postremineralization for each

of nano‑HA group showed a superior and uniform of the experimental groups.
re‑establishment of surface integrity.
Thus, the proposed null hypothesis was accepted.
Energy dispersive X‑ray analysis
Calcium and phosphate levels Discussion
The Ca/P content  (weight %) of demineralized and
remineralized enamel was converted into Ca/P ratios The oral cavity is a battlefield of activities of
for each group. demineralization and remineralization, the balance
between them being the crucial determining factor of
The percentage gain of Ca/P was calculated using the the caries process.[6]
following formula:
Demineralization of enamel leads to dissolution of HA
Remineralization ratio − De min eralization ratio and diffusion of Ca/P ions toward the enamel surface.
× 100 Hypersaturation of Ca/P ions on the surface results in
De min eralization ratio
a re‑precipitation of HA forming the intact superficial
layer on the enamel surface. Remineralization of
An intergroup comparison of the mean percentage enamel is enhanced by the presence of fluoride ions.[7]
gain of the four study groups showed statistically
nonsignificant gain posttreatment (P = 0.159) [Graph 1]. The noninvasive treatment of early lesions by
remineralization bridges the traditional gap between
Estimation of fluoride content prevention and surgical procedures.[8] Central to this
One‑way ANOVA test showed statistically vision is the ability to detect caries lesions at an early
nonsignificant difference between the mean stage and correctly quantify the degree of mineral loss,
fluoride content of the four experimental ensuring that the correct intervention is instituted.[9]
groups postdemineralization (P  =  0.275) and
postremineralization (P = 0.494) [Graph 2]. Assessment of in vitro demineralization and
remineralization can be done using different
Paired t‑test demonstrated a statistically significant methods. The methods used are SEM/SEM‑EDX,[10‑12]
difference (P < 0.05) in the mean fluoride content from DIAGNOdent,[10] surface microhardness,[5] and

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Kamath, et al.: A comparative evaluation of remineralising agents on white spot lesions

15.53 fTCP
Percentage Gain

10.01 CPP-ACPF

Experimental Groups Graph 2: Mean fluoride content pre‑ and post‑treatment for the four
Graph 1: Percentage gain of calcium and phosphate for the four experimental groups
experimental groups
in the overall mineral content. Furthermore, decrease
polarized light microscopy. DIAGNOdent was used
[13,14] in values postremineralization can be due to decrease
in the present study with three main objectives. To aid in porosity of the enamel surface.
in the selection of caries‑free, sound enamel surfaces at
the outset of the study, to ensure decalcification prior Artificially produced caries‑like lesions are of
to subjecting the samples to SEM‑EDX, and to detect considerable interest as they can be compared to the
and compare the changes before and after application earliest detectable ultrastructural change in the caries
of the remineralizing agents. DIAGNOdent  (KAVO, process.[26]
Biberach, Germany), a noninvasive method, uses
laser fluorescence to measure early demineralization. To produce and study in vitro enamel demineralization
Various literature reports have shown the value of models, many demineralizing agents have been
DIAGNOdent as a reliable noninvasive caries‑detecting used such as acetic acid,[27] lactic acid,[28] or acidified
device.[15‑17] hydroxyethylcellulose system[29] for different time
periods. In the present study, demineralization was
DIAGNOdent has demonstrated promising results carried out as described by Lata et al.[5] The specimens
for the detection of incipient enamel lesions in studies were kept in the demineralization solution for 72  h
undertaken in vivo and in vitro in permanent teeth, at 37°C, creating a subsurface demineralization of
approximately 150 µ width with an intact surface
but literature regarding its use in deciduous teeth
simulating an early enamel lesion. The concentration
is scanty. Results from permanent teeth cannot be
of both Ca and P, in the demineralization solution, was
extrapolated to primary teeth because these have
at 50% of saturation level, causing dissolution of only
different morphological, anatomical, and physiological
enamel subsurface. Addition of fluoride prevented
characteristics. In view of this, newly formulated cutoff
surface demineralization by forming fluorapatite at the
limits with histological validation developed by Goel surface, which simulated the naturally occurring early
et al.[4] were considered in the present study as against enamel lesions having intact surface layer.
the manufacturer’s cutoff limits.
The present study used paste‑type formulation of
Prior to recording DIAGNOdent readings at baseline, the test agents, applied with disposable cotton tip
postdemineralization, and postremineralization, applicators. This was done in view of replication of
each sample was air‑dried for 10 s. This was in patient convenience in using the tooth creams like a
accordance with Pinelli et  al.[18] who emphasized that toothpaste with toothbrushes or along with cotton
reproducibility of the device increased under dry tip applicators.[30] The remineralization regimen
condition, thereby advising drying to detect caries‑like comprised 3 min daily application for 14 days. This is
lesions on free smooth surfaces. in accordance with the manufacturer’s prescription.
Pai et al.[23] followed a similar regimen of 3 min daily
In the present study, low frequency  (LF) values application for 14  days. In contrast, in a study by
increased postdemineralization and subsequently Shirahatti et  al.,[31] the treatment pastes were applied
decreased postremineralization. Similar results were twice daily for 3 min for 14 days. Hegde and Moany[3]
demonstrated in studies conducted by Al‑Khateeb in their study performed twice daily application of
et al.,[19,20] Eggertsson et al.,[21] Shi et al.,[17] Mendes et al.,[22] CPP‑ACP for 3 min for 7, 14, 21, 28, and 35 days. The
Pai et al.,[23] Bahrololoomi et al.,[24] and Patil et al.[2] who difference in remineralization regimen could be the
also used DIAGNOdent to assess remineralization. possible reason for variation in outcomes in our study.

Mendes et al.[25] attributed the increase in LF values to In the present study, artificial saliva was changed
enhanced porosity in enamel surface due to decrease every 24 h during the 14‑day remineralization regimen

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Kamath, et al.: A comparative evaluation of remineralising agents on white spot lesions

to ensure ionic balance and maintenance of pH. In was greatest in the fTCP group followed by nano‑HA,
contrast, Shirahatti et al.[31] changed the artificial saliva CPP‑ACPF, and fluoride groups, the difference
solution every 72 h in their study. between the groups being statistically nonsignificant.
This is in contrast to the results of the study by Hedge
SEM is one of the most sensitive, time‑tested techniques et al.[42] and Hegde and Moany.[3]
to assess the demineralization and remineralization
of the carious lesions in vitro as reported in earlier The fall in the surface Ca/P ratio following
studies,[23,32,33] while in most studies, using SEM, demineralization did not appreciably increase after
samples are coated with metals such as gold or remineralizing with fluoridated dentifrice. This
palladium to improve image quality.[29,34] Harding finding suggests that fluorides alone bring about only
et al.[35] and Nicolae et al.[36] studied the samples using structural modification of apatite crystal, restricted to
SEM without metal sputtering so that they could be partial hydroxyl group replacement by fluoride ions
observed again, if necessary, once the study ended. without influencing the Ca and P content. Furthermore,
Consequently, this version was considered in our this reinforces the fact that fluoride in the presence of
study as there would not be loss of samples and Ca and P is more effective in driving the mineral for
re‑evaluation of the same samples after treatment remineralization. The specimens treated with nano‑HA
regimen would be possible. A graphite tape was used exhibited surface Ca/P ratio close to that of the biological
to aid in conduction. enamel and synthetic nano‑HA, indicating an apatitic
coating deposition on the demineralized enamel surface.
The demineralized enamel specimens were observed
at different magnifications. At  ×5000 magnification, A study done by Roveri et  al.[43] demonstrated that
numerous depressions in a honeycomb pattern were biomimetic nonsized HA crystals produce an apatitic
revealed which corresponded to the observations coating deposition on the enamel surface, which is
made by other investigators.[37] Correlation of SEM much less crystalline than native enamel apatite, but
observations made it possible to conclude that consists of a new apatitic mineral deposition which
DIAGNOdent is effective in detecting demineralization progressively fills the surface defects as against the
of enamel. Postremineralization, all the test specimens mere structural modification induced by fluoride,
showed re‑establishment of surface integrity. The restricted to a partial hydroxyl group replacement by
potential to induce remineralization of the test agents fluoride ions without affecting the Ca and P structural
was demonstrated as seen by the increase in crystal network appreciably.
size and occlusion of porous defects. However, there
were minor differences in the surface morphology, The present study is in agreement with previous
which may be due to their different mechanisms for studies that demonstrated the capability of the chosen
promoting remineralization.[27] The SEM results were test agents to induce remineralization of early enamel
in accordance with Huang et al.,[27] Roveri et al.,[38] and lesions.[44‑47]
Swarup and Rao.[39]
Although the remineralization potential of fluoride
A superior surface texture of enamel was noted in the in the present study was found to be lower
present study on SEM evaluation postremineralization compared to the other test agents, its utility cannot
with nano‑HA compared to other groups. This could be underestimated by virtue of the nonsignificant
be attributed to similarity of the nano‑sized particles difference in remineralization potential among the test
to the apatitic crystals of tooth enamel in morphology, agents. Judicious use at the target site could facilitate
crystal structure, and crystallinity.[40] remineralization of the early enamel lesions. Sjögren
et  al.[48] observed that the retention of fluoride in the
A correlation of SEM observations and LF readings mouth is site specific and there is minimal transport of
postremineralization indicated that DIAGNOdent was fluoride ions between left and right sides of the mouth
effective in detecting enamel remineralization. and between arches. This explains the development of
localized lesions in patients using fluoride dentifrice
EDX analysis was used to determine the mineral content routinely. Hence, judicious use of fluoride at the
postdemineralization and postremineralization, appropriate localized site could provide an economical
thereby facilitating estimation of alteration in mineral alternative to facilitate remineralization of incipient
content pre- and post-treatment. Eggerath et al., enamel lesions in vivo.
(2011),[41] used EDX elemental analysis to estimate Ca,
P, and F content of enamel after exposure to fl uoride- A limitation of the present study is that SEM‑EDX
containing tablets in vitro.[41] used EDX elemental was done only for postdemineralization and
analysis to estimate Ca, P, and F content of enamel postremineralization samples. SEM‑EDX at baseline
after exposure to fluoride‑containing tablets in vitro. could have facilitated a better comparison of the ability
of the test agents to induce remineralization and their
In the present study, EDX analysis showed that the potential to bring the mineral content closer to baseline
percentage gain of Ca and P postremineralization levels.

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