Archives of Oral Biology 95 (2018) 118–124

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

In vitro effect of a resin infiltrant on different artificial caries-like enamel T

lesions
Maria Cristina Carvalho de Almendra Freitasa,b, Larissa Vasconcellos Nunesa, Lívia Picchi Comarc,
⁎
Daniela Riosd, Ana Carolina Magalhãesc, Heitor Marques Honóriod, Linda Wanga,
a
Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry, University of São Paulo, Alameda Octávio Pinheiro Brisolla 9-75, ZIP
code: 17012-901, Bauru, SP, Brazil
b
DeVry FACID, DeVry Education Group, Avenida Rio Poti, 2381, ZIP code: 64999-999, Teresina, PI, Brazil
c
Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Alameda Octávio Pinheiro Brisolla 9-75, ZIP code: 17012-901, Bauru, SP, Brazil
d
Department of Pediatric Dentistry, Orthodontics and Collective Health, Bauru School of Dentistry, University of São Paulo, Alameda Octávio Pinheiro Brisolla 9-75, ZIP
code: 17012-901, Bauru, SP, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Objectives: A resin infiltrant was employed for the treatment of active white spot lesions due to its ability to
Dental caries penetrate into the enamel pores and prevent the progression of the lesion. However, limited information is
Demineralization available about its mechanical effect on different artificial enamel lesions as well as on its resistance to further
Dental enamel demineralization. Therefore, this study aimed to evaluate the effects of the Icon® infiltrant on different artificial
Hardness tests
caries-like enamel lesions and its resistance to new acid challenges.
Resin infiltrant
Design: Artificial lesions were produced in bovine enamel using three different protocols (demineralization/
remineralization cycling, DE-RE; 8% methylcellulose gel, MC; and methyl ethyl diphosphonate solution, MHDP;
n = 13). The specimens were treated with Icon® and subjected to a new acid challenge using DE-RE cycling. The
surface and cross-sectional hardness were evaluated in sound, demineralized, treated and further demineralized
enamel areas. Data were statistically analyzed using ANOVA and Tukey’s test (p < 0.05).
Results: All of the demineralizing protocols produced subsurface artificial caries lesions. The infiltrant was able
to partially recover the surface hardness and prevent further surface hardness loss in enamel previously demi-
neralized using the DE-RE and MHDP protocols. In regard to cross-sectional hardness, no positive effect was
found.
Conclusions: The effect of the infiltrant depends on the type of lesion created in vitro, and its action is limited to
the lesion surface.

1. Introduction
The understanding that dental caries is a multifactorial disease that
primarily causes demineralization of dental hard tissues has allowed its
prevention and control in early stages (Kidd & Fejerskov, 2004;
Schwendicke et al., 2016). Stopping or reversing dental lesions as soon
as possible is the main goal to reduce the risk of cavitation and, con-
sequently, the need for any operatory interventions (Kidd, 2011; Meyer-
Lueckel & Paris, 2016; Schwendicke et al., 2016; ten Cate, 1997).
White spot lesion (WSL), which is characterized by an apparently
intact outer surface and a demineralized subsurface, is considered the
first clinical sign of dental caries (De Rooij & Nancollas, 1984). As it is
reversible under favorable conditions, many approaches have focused
on treating it as soon as possible (Cassiano et al., 2017; Han et al.,
2017).
To date, the majority of treatments of WSLs have been based on
chemical mechanisms (remineralization), mainly by the use of fluoride
agents (Cassiano et al., 2017; Tickle et al., 2017) and other topical
agents such as amelogenin-derived peptide products (De Rooij &
Nancollas, 1984; Han et al., 2017).
A mechanical strategy for the treatment of WSLs has been success-
fully introduced by means of a resin-based material (Icon®). It consists

Abbreviations: DE-RE, demineralization/remineralization cycling; MCgel, 8% methylcellulose gel; MHDP solution, methyl ethyl diphosphonate; WSL, white spot
lesion; TEGDMA, triethylene glycol dimethacrylate; TMR, transversal Microradiography; ΔSH, surface hardness; ΔCSH, cross sectional hardness; S, sound; D, de-
mineralized; I, infiltrant; N, new challenge
⁎
Corresponding author.
E-mail address: wang.linda@usp.br (L. Wang).

https://doi.org/10.1016/j.archoralbio.2018.07.011
Received 14 January 2018; Received in revised form 7 July 2018; Accepted 16 July 2018
0003-9969/ © 2018 Elsevier Ltd. All rights reserved.
M.C.C.d.A. Freitas et al.
of a resin component based mainly on triethylene glycol dimethacrylate
(TEGDMA), which has the ability to penetrate the pores of the affected
enamel and prevent caries lesion progression (Kielbassa et al., 2017;
Meyer-Lueckel, Chatzidakis, Naumann, Dörfer, & Paris, 2011; Paris,
Hopfenmuller, & Meyer-Lueckel, 2010; Paris, Bitter, Naumann, Dörfer,
& Meyer-Lueckel, 2011; Schneider et al., 2017). In addition to its ability
to arrest the lesion, this product has the aesthetic advantage of masking
white spot lesions (Kim, Kim, Jeong, & Kim, 2011; Neres et al., 2017;
Torres, Borges, Torres, Gomes, & de Oliveira, 2011).
Though many investigations into its mechanism of action and effects
have been performed, there is still controversy about its ability to pe-
netrate the porous surface and its long-term effect. One of the reasons
might be that in vitro experiments have been based on different stan-
dardized models of artificial enamel caries-like lesions (Meyer-Lueckel,
Paris, Mueller, Cölfen, & Kielbassa, 2006; Paris et al., 2013; Borges,
Caneppele, Luz, Pucci, & Torres, 2014; Araújo, Sfalcin, Araújo, Alonso,
& Puppin-Rontani, 2014; Paris et al., 2013; Min, Inaba, & Kim, 2016).
Magalhães et al. (2009) compared five demineralizing protocols com-
monly used to produce artificial caries-like enamel lesions. They
showed that different protocols promoted distinct levels of deminer-
alization according to hardness testing and transversal micro-
radiography (TMR). Although the hardness analysis could not be
translated in terms of mineral content in their study, it was able to show
the mechanical properties of enamel.
For a more precise interpretation of the properties of resin infiltrant,
the substrate condition is relevant. Its efficacy after acid challenge is
also relevant to determine, as shown by Salomão, Comar, Buzalaf, and
Magalhães, (2016), who assessed the role of fluoride at home and
professional agents on the in situ progress of enamel artificial caries
lesions determined by different protocols.
Usually, infiltrant effectiveness has been assessed by measuring its
depth of penetration by confocal laser scanning microscopy. It can be
supposed that once placed, the effect of the infiltrant could also involve
the hydroxyapatite crystals in the enamel substrate, even though this
has not been evidenced yet (Neres et al., 2017). However, this possi-
bility made us wonder how infiltrant would perform mechanically in
terms of embracing this porous structure and the consequence of such a
phenomenon on resistance.
Thus, there is a lack of information about the mechanical effect of
resin infiltrant on different artificial enamel lesions as well as about its
resistance after further demineralization. Therefore, this study aimed to
investigate the ability of the infiltrant to treat artificial enamel caries-
like lesions created using three different protocols. In addition, this
study evaluated the resistance of the enamel treated with infiltrant to a
new acid challenge, using hardness as the response variable.
The tested null hypotheses were (1) infiltrant does not improve
surface or cross-sectional enamel microhardness regardless of the de-
mineralizing protocol; (2) the demineralizing protocol does not affect
surface or cross-sectional enamel microhardness regardless of the in-
filtrant used.
2. Materials and methods
2.1. Study design
This study evaluated the effect of a resin infiltrant (Icon®) and its
performance over demineralized enamel. The independent variables
were demineralizing protocol in three levels (des re, MC gel and MHDP
solution) and phases in four levels (sound, demineralized, treated with
Icon and further demineralized enamel areas) for surface microhard-
ness analyses (dependent variable). In the cross-sectional assessments,
the depth was also considered as independent variable.
Fig. 1 summarizes the sequence of specimen preparation and the
division of the specimens in accordance with the phase.
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Archives of Oral Biology 95 (2018) 118–124
2.2. Specimen preparation
Forty-five rectangular enamel specimens (6 mm × 4 mm), obtained
from extracted flatter portions of the crowns of bovine incisors, were
prepared using two diamond disks in the Isomet Low Speed Saw
(Buehler, Lake Bluff, IL, USA) and stored in 0.1% thymol solution. They
were then flattened and polished using a metallographic polishing
machine (Arotec APL-4, Cotia, SP, Brazil) with a sequence of # 320,
600 and 1200 ground discs (Buehler, Lake Bluff, IL, USA) and a felt
paper wet with diamond spray (1/4 μm). Specimens were analyzed in
terms of baseline surface hardness to establish a parameter for the
specimens’ selection and for randomized distribution to the groups. For
this analysis, a microhardness tester (Wilson Tukon 1102, Buehler, Lake
Bluff, IL, USA) was used with a Knoop diamond and a load of 25 g
applied for 10 s. Specimens with an average surface hardness (SH) ap-
proximately 350 kg/mm2 were selected, and those showing average
values 20% above or 20% below this were discarded.
2.3. Demineralizing procedures
All specimens were protected with nail varnish and wax (1/4 en-
amel surface, S-sound), exposing 3/4 of the enamel surface to demi-
neralization, which was performed using 3 different demineralizing
protocols (n = 15), which are described in Table 1. This protection with
nail varnish and wax allowed the maintenance of the intact surface to
be used as a reference for the analyses of the next 3 steps. Therefore,
two coats of varnish were applied with a microbrush. Ten minutes after,
wax was also placed to reinforce this protection.
2.4. Application of resin infiltrant
Before treatment, the other ¼ of the enamel surface (now demi-
neralized) was protected with two-layer nail varnish (D-demineralized
surface) as described above. Half of the enamel surface was treated with
the Icon® resin infiltrant (DMG, Hamburg, Germany) according to the
manufacturer's instructions (Meyer-Lueckel & Paris, 2016) as described
in Table 2. Clinically, previously to any dental intervention, the re-
moval of biofilm and acquired pellicle is always recommend, allowing
the improvement of the interaction of the material to the substrate. The
use of a mixture of pumice and water is recommended as it is water-
based, avoiding oil vehicle as it can interfere in the contact of the
material to teeth. Therefore, in this laboratory condition, the same
protocol was followed to be closer to the clinical condition. Nail varnish
was then applied to ¼ of the enamel surface (now demineralized and
treated, I-icon surface).
2.5. New acid challenge
After treatment, all specimens were subjected to a new acid chal-
lenge (1/4 surface, N- new acid challenge) according to the DE-RE
protocol described in Table 1.
2.6. Hardness analysis
The nail varnish protection was gently removed, and the surfaces
were carefully cleaned with 50% acetone solution (Min et al., 2016).
Surfaces were analyzed through visual assessment with optical micro-
scopy (40x), even though previous studies have assured the success of
this removal process (Magalhães et al., 2009; Salomão et al., 2016).
Surface hardness (SH, kg/mm2) was analyzed as previously described
(5 indentations, 25 g/10 s) on different enamel areas (S, D, I, N). The
average hardness was calculated at each area.
Thereafter, all specimens were longitudinally cut using a diamond
disk in the Isomet Low Speed Saw for the cross-sectional hardness
readings, which were performed at the center of each enamel area (S, D,
I, N). After cutting, the specimens were embedded in acrylic resin with
M.C.C.d.A. Freitas et al. Archives of Oral Biology 95 (2018) 118–124

Fig. 1. Fluxogram describing the main steps of this investigation. After bovine tooth selection, samples were prepared from the labial crown surface (6 mm × 4 mm).
Four main areas were determined for the subsequent steps: S: sound enamel; D: demineralized enamel (using the different protocols); I: demineralized and treated
(infiltrant) enamel; N: demineralized and treated enamel subjected to new acid challenge. Surface and cross-sectional microhardness were determined as endpoints.
TRM was performed only for the validation of the solutions/gels to produce subsurface lesions.

the inner face facing the outer surface of the resin and polished using a Table 2
sequence of # 320, 600 and 1200 ground discs in a metallographic Protocol for the application of Icon.
polishing machine. - Prophylaxis with pumice/water and rinse with water;
The cross-sectional hardness (ΔCSH) was determined using three - Application of Icon Etch (15% hydrochloric acid), holding for 2 m;
sequences of seven indentations (25 g/10 s) at distances of 10, 30, 50, - Washing for 30 seconds and drying with air jets;
70, 90, 110 and 220 μm from the outer surface of the enamel. Each - Application of Icon Dry (99% ethanol) for 30 s;
- Air jet drying;
specimen received 12 sequences of indentations (three sequences in
- Application of Icon infiltrant for 3 m;
each area, S, D, I, N). The average hardness was calculated at each - Removal of excess with microbrush;
distance. - Light cure for 40 seconds;
The values of ΔSH or ΔCSH for D, I and N areas were determined as - New application of Icon infiltrant for 1 m;
- Removal of excess;
the percentage of loss compared to the sound area (S).
- Light curing for 40 s;
- Polishing with abrasive disks and abrasive points.

Table 1
Different protocols applied to induce the formation of caries-like enamel lesions.
Protocol Solutions/Gel Exposure conditions

DE-RE (pH cycling) Demineralizing solution: 2.0 mM Ca(NO3)2·4H2O, 2.0 mM Na2HPO4·2H2O, 7 days, renewed daily.
0.075 M acetate buffer, 0.04 ppm F; pH 4.7/ 6 h/day Each specimen was immersed in 30 ml of solution
Remineralizing solution: 1.5 mM Ca(NO3)2·4H2O, 0.9 mM Na2HPO4·2H2O,
150 mM KCl, 0.2 M Tris buffer, 0.05 ppm NaF, pH 7.0/18 h/day
MC 8% Methylcellulose gel; after 12 h, 1.5 ml of 0.1 M lactic acid (Sigma) was Gel covering 0.5 cm of the specimen left to set overnight at 4 °C, then covered
(gel) added, pH 4.6 with an equal volume (1.5 ml) of 0.1 M lactic acid pH adjusted to 4.6 with
1 M KOH and incubated for 14 days/37 °C
MHDP 3 mM CaCl2·2H2O (LabSynth); 3 mM KH2PO4 (Sigma-Aldrich); 0.05 M lactic Each sample was immersed in 30 ml of acid buffer containing 3 mM
(solution) acid (Sigma); 6 μM methyl ethyl diphosphonate (MHDP) (Sigma-Aldrich); CaCl2·2H2O, 3 mM KH2PO4, 50 mM lactic acid, 6 μM MHDP, KOH to adjust
traces of thymol. the initial pH to 5.0 and traces of thymol

All the solutions were prepared in the Laboratory of Biochemistry from Bauru School of Dentistry using Sigma reagents. MC gel was prepared at Bauru Fórmulas
Pharmacy, Bauru, SP, Brazil.

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2.7. Statistical analysis
The SH data were analyzed using a two-way repeated-measures
ANOVA (factors: demineralizing protocol and phase), followed by
Tukey’s test for multiple comparisons. Data referring to CSH were as-
sessed individually for each of the 7 depths (10, 30, 50, 70, 90, 110 and
220 μm) by 2-way RM ANOVA followed by Tukey’s test. To compare the
depths, three-way Repeated Measures ANOVA was performed (factors
protocol, depth and step), followed by Tukey’s test. The software pro-
grams used were Statistica 10 (Statsoft, Tulsa, OK, USA) and SigmaPlot
12.0 (Systat Software Inc., San Jose, CA, USA). For all tests, the sig-
nificance level was set at 5%.
3. Results
Surface hardness loss was significantly affected by the deminer-
alizing protocol (p = 0.009), phase (p < 0.0001), and their interaction
(p < 0.0001). For cross-sectional hardness, demineralizing protocol
(p = 0.469) was not significant, while phase, depth and their interac-
tion were (p < 0.005).
The enamel surface hardness loss (ΔSH D) after the first deminer-
alization was similar among the protocols, although MC induced ap-
proximately 75% of ΔSH D, while the other protocols presented > 90%
(Table 3). The Icon treatment recovered the surface hardness (ΔSH I)
only for enamel previously demineralized using DE-RE and MHDP, with
no differences between these two protocols. ΔSH I was lower for DE-RE
compared to MC, and both did not differ from MHDP. The further
surface demineralization (ΔSH N) was prevented in case of enamel
previously demineralized by DE-RE, while it progressed for the other
types of lesions. However, the progression of demineralization was
higher for MC compared to MHDP and DE-RE (Table 3, p < 0.05).
The enamel cross-sectional hardness loss (ΔCSH D) after the first
demineralization was significantly different among the protocols. MC
and MHDP induced lower ΔCSH D than DE-RE at the first 10 and 30 μm,
respectively (Table 4 and Fig. 5). The Icon treatment was unable to
recover the cross-sectional hardness (ΔCSH I); instead, it induced an
increase in ΔCSH I at the first 50 μm depth for all types of lesions. The
further subsurface demineralization (ΔCSH N) was not prevented by
Icon, regardless of the type of lesion; instead, we noted a progression of
hardness loss (ΔCSH N) from 50 to 110 μm depth. No differences in
ΔCSH N were found among the demineralizing protocols (Table 4,
p < 0.05).
Six additional specimens were similarly prepared and subjected to
the demineralizing protocol (n = 2) to validate them by using TMR
analysis, as shown in Figs. 2–4. The microradiograms were analyzed
using a transmitted light microscope with a 20x objective (Axioplan;
Zeiss, Oberkochen, Germany) and a camera (XC-77CE, Sony, Tokyo,
Japan). The software used was TMR 1.25e (Inspector Research BV,
Amsterdam, The Netherlands). All images showed a subsurface lesion
with a pseudo-intact surface layer (Figs. 2–4).
Table 3
Mean and standard deviation of the surface hardness loss ΔSH (%) in each
phase compared to the sound surface hardness mean.
Protocol ΔSH ΔSH ΔSH
First challenge (D-S) ICON (I-S) Second challenge(N-S)
DE-RE −91.76(9.17) Ba −42.81(20.67) Aa −58.54(8.15) Aa
MC −75.20(9.04) ABa −62.80(20.52) Ab −92.25(15.16) Bb
MHDP −92.50(3.60) Ca −48.46(21.28) Aab −66.81(8.17) Ba
N = 13; p < 0.05, S = sound; D = demineralized; I = infiltrant; N = new
challenge.
Different capital letters mean significant differences between the phases (D–S,
I–S, N–S) under each demineralizing protocol (p < 0.05).
Different small letters mean significant differences between the demineralizing
protocols for each phase (p < 0.05).
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Archives of Oral Biology 95 (2018) 118–124
4. Discussion
Current technologies focus on the use of mechanical agents that fill
the enlarged pores of the carious enamel (de Sousa, Lelis, Figueiredo,
Pires, & Gerlach, 2017; Meyer-Lueckel et al., 2011; Paris et al., 2011).
Several in vitro studies have shown interesting performance character-
istics of resin infiltrants, such as their adequate permeability to into
demineralized enamel associated with improvement of aesthetic con-
ditions (Bergstrand & Twetman, 2011; Torres et al., 2011). However, it
is not completely efficient at filling all pores, as stated by de Sousa et al.
(2017), who found that firmly bound water and air remained in enamel
pores after resin infiltration. Therefore, the use of infiltrant system
seems to be interesting as it is capable of removing/reducing the ac-
cumulation of water and/or air by the previous use of ethanol solution.
However, one of the issues to be considered in in vitro studies is the
type of artificial caries-like enamel lesion used for the infiltrant ana-
lysis, which usually does not simulate real caries lesions (Meyer-Lueckel
et al., 2006 and Paris et al., 2013), but it is important as a first model to
test any preventive or treatment approach. The present study compared
artificial lesions induced using three distinct protocols: DE-RE cycling,
MC gel and MHDP solution. All protocols induced a subsurface lesion,
as expected (Figs. 2–4), but the degree of mineral loss and lesion depth
(< 50 μm) are far from what is found in vivo, as discussed elsewhere
(Meyer-Lueckel et al., 2006 and Magalhães et al., 2009)
To answer the main study question, we applied hardness analysis, as
done in another study on this subject (Paris et al., 2013; Torres, Rosa,
Ferreira, & Borges, 2012). Based on our results, both null hypotheses
can be rejected. Interestingly, no significant differences were found
among the protocols with respect to surface enamel loss, but significant
differences could be detected in the cross-sectional hardness analysis (at
the first 50 μm depth). Generally, DE-RE induced a higher deminer-
alization than MHDP and MC gel, which did not differ from each other.
Magalhães et al. (2009) found that MHDP induced the highest subsur-
face mineral loss, followed by pH cycling and MC gel by using TMR.
However, MHDP provoked the lowest cross-sectional hardness loss in
the cited study, in accordance with our results.
The MHDP protocol has been considered important for the forma-
tion of a surface layer due to the presence of phosphonate, reducing the
progression of demineralization (Buskes, Christoffersen, & Arends,
1985), which may explain the results of this study. The MC gel, on the
other hand, may decrease the progression of demineralization due to
the presence of a dense gel, which reduce the diffusion rate of the acid
and maintain the lost ions near the tooth surface, keeping the surface
more mineralized (Damato, Strang, & Stephen, 1988). The DE-Re cy-
cling is a dynamic process, more similar to what happens in vivo, al-
lowing an intensive ion exchange and, consequently, a high deminer-
alization. Diferently from the study of Magalhães et al. (2009), we
changed the DE solution daily, which might have not allowed the in-
crease of its Ca and P content (from the tooth), and consequently, its
degree of saturation with respect to HA over a period of 24 h. Therefore,
we found higher degree of enamel demineralization by DE-RE protocol
than the former study (Magalhães et al., 2009).
Regarding the mechanical property of the resin infiltrant, higher
recovery of the hardness at the surface was found for the most demi-
neralized lesions (DE-RE and MHDP). Higher demineralized lesions
generally present more pores, allowing better interaction of the mate-
rial with the superficial crystals, enhancing their mechanical properties.
The infiltrant is made of TEGDMA monomer and has improved wetting
power and lower molecular weight compared to other infiltrants, which
may be because the crystals promote a more compact structure even if
the hardness of the resin is not as high as that of the enamel (Calheiros,
Daronch, Rueggeberg, & Braga, 2008; Larsen, Freund, & Munksgaard,
1992; Meyer-Lueckel et al., 2011; Neres et al., 2017; Paris et al., 2010,
2011).
Concerning cross-sectional hardness, the positive effect of infiltrant
was not seen at the subsurface, as Icon did not increase the
M.C.C.d.A. Freitas et al. Archives of Oral Biology 95 (2018) 118–124

Table 4
Mean and standard deviation of the cross-sectional hardness loss ΔCSH (%) in each phase compared to the sound cross-sectional hardness mean.
Protocol Depth (μm) ΔCSH ΔCSH ΔCSH
First challenge ICON (I-S) Second challenge (N-S)
(D-S)

DE-RE 10 −73.42(20.54) Aa −100.00(0.00) Ba −100.00(0.00) Ba

30 −38.33(28.45) Aa −98.65(4.87) Ba −100.00(0.00) Ba
50 −21.40(17.52) Aa −65.83(27.84) Ba −100.00(0.00) Ca
70 −7.00(18.78) Aa −9.44(31.29) Aa −96.89(7.62) Ba
90 −0.10(14.99) Aa 13.52(22.47) Aa −72.69(39.55) Ba
110 5.53(12.95) Aa 19.87(20.39)Aa −51.64(45.13) Ba
220 −4.68(10.72) Aa 4.12(24.90) ABa −0.93(21.69) Ba
MC 10 −33.62(36.96) Ab −100.00(0.00) Ba −100.00(0.00) Ba
30 −37.83(37.77) Aab −94.08(5.88) Bab −98.96(3.37) Bab
50 −30.83(31.37) Aa −66.66(23.00) Ba −95.43(12.06) Ca
70 −11.24(17.65) Aa −4.21(16.24) Aa −79.62(38.54) Ba
90 −3.29(11.84) Aa 3.07(21.72) Aa −59.71(47.61) Ba
110 −6.96(16.46) Aa 2.08(13.91) Aa −36.35(44.55) Ba
220 −0.84(19.60) Aa 10.24(27.06) ABa 6.98(22.05) Ba
MHDP 10 −24.40(30.81) Ab −100.00(0.00) Ba −100.00(0.00) Ba
30 −12.83(27.20) Ab −90.38(17.44) Bb −98.61(5.01) Bb
50 −13.15(13.94) Aa −58.5(34.79) Ba −94.04(21.49) Ca
70 −8.91(13.83) Aa −29.59(39.50) Aa −90.50(23.77) Ba
90 −10.18(13.64) Aa −5.62(30.05) Aa −73.57(35.33) Ba
110 −9.81(14.34) Aa 3.25(17.22) Aa −50.58(49.25) Ba
220 −8.54(14.98) Aa 5.59(24.78) ABa −3.12(32.79) Aa

N = 13; p < 0.05, S = sound; D = demineralized; I = infiltrant; N = new challenge.

Different capital letters indicate significant differences between the phases under the same demineralizing protocol at the same enamel depth (p < 0.05).
Different small case letters indicate significant differences between the demineralizing protocols for each phase at the same enamel depth (p < 0.05).

Fig. 2. Representative TMR image of a specimen demineralized with DE-RE Fig. 3. Representative TMR image of a specimen demineralized with MC gel.
cycling. An intact external surface is preserved, determining the formation of a Also observed is the preservation of an intact external surface to guarantee the
subsurface lesion. determination of a subsurface lesion.

microhardness. Instead, the Icon application increased the subsurface

hardness loss. This undesirable result may have been due to the ap-
plication of hydrochloric acid, which may have penetrated into the
pores, increasing the subsurface demineralization. It can be assumed
that the use of 15% hydrochloric acid for 2 min (following the manu-
facturer's protocol for clinical application) caused erosion wear but
enhanced the infiltration (Meyer-Lueckel, Paris, & Kielbassa, 2007;
Paris, Meyer-Lueckel, & Kielbassa, 2007).
It should be highlighted that the manufacturer’s protocol is applied
to natural caries lesions in human tooth. Natural lesions usually have a
more mineralized surface layer and are more resistant than the artificial
lesions produced in bovine enamel (Meyer-Lueckel et al., 2006). Other
in vitro studies have replaced hydrochloric acid by phosphoric acid to
avoid this side effect, and they found a good effect at the subsurface
(Paris et al., 2013). A recent study proposed that even in natural lesions,
the use of 37% phosphoric acid could increase permeability and mini-
mize the removal of the surface layer without affecting the treatment Fig. 4. Representative TMR image of a specimen demineralized with MHDP
effect of an infiltrant (Yim, Kwon, & Kim, 2014). solution. A subsurface lesion was also obtained.

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Fig. 5. Representative chart showing the differences in CSH hardness loss be-
tween the depths for all demineralizing protocols. Different letters indicate
statistically significant differences.
In the present study, Icon offered some protection against further
demineralization at the surface only for the most demineralized lesion
(DE-RE), in accordance with Torres et al. (2012). For the other lesions,
there was a progression of demineralization similar to the baseline in
case of MC and lower demineralization compared to the baseline in case
of MHDP. Once again, the higher the baseline demineralization, the
lower the progression of demineralization, as previously discussed
(Lippert, Churchley, & Lynch, 2015). On the other hand, we found a
strong progression of demineralization at the subsurface. The lesion
depth at baseline was approximately 50 μm, but after the second de-
mineralization, the lesion progressed at its inner part (50–110 μm) re-
gardless of the baseline demineralization (type of lesion).
The lack of protective effect against further demineralization may
be largely attributed to the infiltrant composition. Despite the favorable
characteristics of TEGDMA as an infiltrant, this monomer is considered
a non-resistant material due to its high hydrophilicity and consequent
degradation in the oral environment (Park, Eslick, Ye, Misra, & Spencer,
2011). Considering that the prisms are surrounded by liquid (Mine
et al., 2010) and that the enamel samples with infiltrant were exposed
to DE-RE for a further 7 days, some degradation could be expected. This
performance has stimulated investigations to propose new formulations
or associations with other products to enhance the properties of the
infiltrant (Araújo et al., 2014; Mine et al., 2010).
5. Conclusions
The mechanical effect of the resin infiltrant on enamel surface, after
its application and further demineralization, depends on the type of
caries lesion previously created in vitro. Furthermore, its mechanical
effect is limited to the lesion surface. These findings highlight the im-
portance of carefully comparing data from in vitro studies with clinical
trials. Despite this unfavorable in vitro result, the use of Icon, even with
its limitations, seems to be a good possibility to minimize WSLs and
their aesthetic appearance through interactionwith crystals.
Funding
FAPESP (The State of São Paulo Research Foundation): #2012/
18579-2 and #2012/13157-2.
Ethical approval
No Ethical appreciation was needed as it did not involve human
tissue/organ.
Conflict of interests
A conflict of interest exists when an author or the author's
123
Archives of Oral Biology 95 (2018) 118–124
institution has financial or personal relationships with other people or
organisations that inappropriately influence (bias) his or her actions.
Financial relationships are easily identifiable, but conflicts can also
occur because of personal relationships, academic competition, or in-
tellectual passion. A conflict can be actual or potential, and full dis-
closure to The Editor is the safest course.
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