Effect of Er:YAG and Fluoride Varnishes for Preventing

Primary Enamel Demineralisation

Arlete González-Soteloa / Laura Emma Rodríguez-Vilchisb / Rosalía Contreras-Bulnesb

Purpose: To measure the demineralisation changes on human deciduous enamel produced by pH cycling after pre-
ventive treatment with Er:YAG laser irradiation, fluoride varnish application and a combination of the two.
Materials and Methods: Sixty extracted human anterior primary teeth were assigned to six groups (n = 10). Group
C: untreated; group L: Er:YAG laser; group TCP-NaF: Clinpro White (5% sodium fluoride and modified tricalcium
phosphate); group CPP-ACP-NaF: MI varnish (5% sodium fluoride with casein phosphopeptide-amorphous and cal-
cium phosphate); group L+TCP-NaF: Er:YAG + 5% sodium fluoride + modified tricalcium phosphate; group L+ CPP-
ACP-NaF: Er:YAG + 5% sodium fluoride with casein phosphopeptide-amorphous and calcium phosphate. The
samples were subjected to a 10-day pH-cycling regimen to create caries-like lesions, with 8 h in demineralising so-
lution and 16 h in remineralising solution at 37°C. Enamel demineralisation was evaluated by laser fluorescence
(DIAGNOdent) before and after pH cycling. The Mann-Whitney U-test and Wilcoxon tests were performed with statis-
tical significance set at p ≤ 0.05.
Results: The Wilcoxon test revealed statistically significant differences at baseline and after pH cycling in groups C
(p = 0.02), L (p = 0.034) and L+TCP-NaF (p = 0.025) and the lowest percentage of healthy tooth substance com-
pared to the other groups.
Conclusions: The results suggest that the treatment protocols employed in groups TCP-NaF, CPP-ACP-NaF, and L+
CPP-ACP-NaF had similar effects in terms of preventing demineralisation, as reflected in a higher percentage of
healthy dental structure maintained. Hence, these treatments are recommended for clinical use as an effective
preventive measure.
Key words: demineralisation, Er:YAG, laser fluorescence, primary teeth, remineralisation

Oral Health Prev Dent 2019; 17: 317–321. Submitted for publication: 12.09.17; accepted for publication: 18.03.18
doi: 10.3290/j.ohpd.a42935

D ental caries is a multifactorial disease caused by an

imbalance between demineralisation and remineralisa-
tion.14,16 Modern caries management should be focused on
non-cavited carious lesions and preventing caries progres-
sion.5,16 Thus, several strategies have been suggested to
a Posgraduate Student, Master of Dentistry Programme, School of Dentistry, Au-
tonomous University of the State of Mexico. Idea, hypothesis, experimental
design, statistical evaluation, data analysis and interpretation, wrote and criti-
cally revised the manuscript, final approval of manuscript.
b
Associate Professor, Master of Dentistry Programme, School of Dentistry, Au-
tonomous University of the State of Mexico. Idea, hypothesis, experimental
design, statistical evaluation, data analysis and interpretation, wrote and criti-
cally revised the manuscript, final approval of manuscript.
Correspondence: Dr. Rosalía Contreras-Bulnes, Universidad Autónoma del Es-
tado de México, Facultad de Odontología, Centro de Investigación y Estudios
Avanzados en Odontología, Paseo Tollocan Esq. Jesús Carranza. Toluca, Estado
de México, CP 50130, México. Tel: +52-722-212-4351;
e-mail: rcontrerasb@uaemex.mx
reverse or prevent the development of caries and reminer-
alise early caries lesions.6
Among the methods to prevent enamel caries formation,
topical fluoride varnishes are considered the most valuable
strategies.5,14,16,18 Fluoride varnishes are believed to in-
hibit enamel demineralisation, and the addition of phospho-
peptide-amorphous and calcium phosphate (CPP-ACP) de-
creases demineralisation and facilitates remineralisation.21
Then, fluoride varnishes with added functionalised trical-
cium phosphate (fTCP) enhance the mineralisation on
enamel and dentin surfaces, inhibiting demineralisation.1
On the other hand, laser technology is an alternative sys-
tem proposed for preventing enamel demineralisation, pro-
ducing chemical and morphological changes.4 Moreover,
combined treatments of laser irradiation and topical fluoride
have been described as synergistic.18 Er:YAG laser, with a
wavelength of 2.94 μm, is highly absorbed by water and
enamel,11,13 and transforms hydroxyapatite into fluoridated
hydroxyapatite to inhibit enamel demineralisation and de-
crease enamel permeability.13 Some studies have revealed

Vol 17, No 4, 2019 317

González-Sotelo et al
Table 1 Experimental groups
Groups n Preventive treatment
C 10 Control group (untreated)
L 10 Er:YAG laser (100 mJ, 12.7J/cm2, 3.18 x 104 W/cm2)
TCP-NaF 10 TCP-5% NaF (Clinpro White)
CPP-ACP-NaF 10 CPP-ACP-5% sodium fluoride (MI Varnish)
L+TCP-NaF 10 Er:YAG (100 mJ, 12.7J/cm2, 3.18 x 104 W/cm2 + TCP-5% NaF [Clinpro White])
L+ CPP-ACP-NaF 10 Er:YAG (100 mJ, 12.7J/cm2, 3.18 x 104 W/cm2 + (CPP-ACP-5% NaF [MI Varnish])
TCP: tricalcium phosphate; CCP-ACP: casein phosphopeptide-amorphous and calcium phosphate; NaF: sodium fluoride.
that combined fluoride and laser treatment makes the
enamel more resistant to acid than the fluoride treatment
alone.12
Nevertheless, laser technology has been questioned, be-
cause there are some variables that could alter the final
result.7 Only a few studies23 have focused on primary teeth
treated with a combination of Er:YAG and fluoride for caries
prevention.
One method of detecting early smooth-surface caries le-
sions is by measuring laser fluorescence (LF) with the DIAG-
NOdent device (Kavo; Biberach, Germany), which was intro-
duced to evaluate caries lesions.15 Laser fluorescence has
presented good results for occlusal caries detection and it
has also been used in smooth surface caries lesions.8,9,15
However, few studies have evaluated DIAGNOdent’s perfor-
mance on primary-tooth enamel.
Thus, the aim of this study was to measure the minerali-
sation changes on human deciduous enamel produced by
pH cycling after Er:YAG laser irradiation, fluoride varnish
application, and combined treatment.
MATERIALS AND METHODS
Specimen Preparation
The research protocol was reviewed and approved by the
Research Ethics Committee of the Dental Research and
Advances Studies Center, School of Dentistry at the Autono-
mous University of the State of Mexico.
Sixty human anterior primary teeth (extracted or naturally
exfoliated) were obtained from the mandibular and maxillary
arches with the patients’/parents’ informed consent. Teeth
with obvious caries, restorations, fluorosis, or fractures
were excluded. Immediately after extraction or as soon as
possible after exfoliation, they were kept in 0.2% thymol
solution until preparation.
The specimens were cleaned with deionised water and
traces of soft tissue were removed with a scalpel. Speci-
mens were scanned with the laser fluorescence caries de-
tection system, DIAGNOdent pen (Kavo; Biberach, Germany)
following the manufacturer’s instructions. Only healthy teeth
were included (DIAGNOdent values between 0 and 13).
318
Selected primary teeth were de-coronated, samples were
rinsed with deionised water and stored in 0.2% thymol solu-
tion at 4°C until the experiment was performed.
Surface Treatment
Sixty samples were randomly assigned to six groups (n = 10)
and the buccal enamel surfaces were conditioned as de-
scribed below (Table 1).
Er:YAG laser irradiation
Samples were irradiated using an Er:YAG laser system
(Opus Duo AuaLite EC, Lumenis; Yokneam, Israel) with a
wavelength fixed at 2.94 μm, a pulse repetition of 7 Hz, a
pulse duration of 400 μs at 100 mJ (12.7J/cm2) and a
power density of 3.18 x 104 W/cm2. Energy levels were
calibrated by calibrating the tip of the Erbium fiber with the
special proprietary tip calibration handpiece, according to
the manufacturer’s instructions, and the energy delivered
was measured periodically with a power meter (LaserMate-P,
Coherent; Santa Clara, CA, USA). The surface was scanned
once by hand (13 s) with the sapphire tip (Ø1.0 mm) of the
laser perpendicular to it at a working distance of 1 mm and
irrigated with deionised water spray (5 ml/min).
Fluoride varnishes
Two different fluoride varnishes were employed: tricalcium
phosphate (TCP) + 5% sodium fluoride (Clinpro White, 3M
Oral Care; St Paul, MN, USA) and casein phosphopeptide-
amorphous and calcium phosphate (CPP-ACP) + 5% sodium
fluoride (MI Varnish, GC; Tokyo, Japan).
The control group (group C) was left untreated. Enamel
surfaces of fluoride and laser-plus-fluoride combined groups
(group TCP-NaF, group CPP-ACP-NaF, group L+TCP-NaF and
group L+ CPP-ACP-NaF) were submitted to fluoride varnish
according to the respective group and manufacturers’ in-
structions (Table 1). The fluoride varnishes were all applied
to the buccal surface with a cotton swab for 4 min. After-
wards, the specimens were rinsed with deionised water for
1 min and then air dried. Then the samples were incubated
for 24 h in artificial saliva at 37°C. After incubation for 24
h, the varnishes were carefully removed with a scalpel, and
followed by rinsing with deionised water for 1 min each.
Oral Health & Preventive Dentistry
 González-Sotelo et al

Table 2a DIAGNOdent score percentage (%) at baseline and after pH cycling for groups C, L and TCP-NaF
DIAGNOdent pen score Groups
C L TCP-NaF
Baseline After pH cycling Baseline After pH cycling Baseline After pH cycling
10 days 10 days 10 days
Healthy tooth substance (0–13) 100 A, a 60 A, b 100 A, a 66.66 A, b 100 A, a 93.33 A, a

Initial demineralisation (14–20) 0 A, a 33.3 A, a 0 A, a 26.66 A, a 0 A, a 6.66 A, a

Strong demineralisation (21–29) 0 A, a 6.66 A, a 0 A, a 6.66 A, a 0 A, a 0 A, a

Same capital letters in columns indicate no statistically significant differences in the DIAGNOdent readings at baseline or after pH cycling between the groups.
Same lowercase letters in rows indicate no statistically significant differences between baseline and after pH cycling. Group C: untreated; group L:
Er:YAG laser; group TCP-NaF: Clinpro White (5% sodium fluoride and modified tricalcium phosphate).

Table 2b DIAGNOdent score percentage (%) at baseline and after pH cycling for groups CPP-ACP-NaF, L+TCP-NaF,
and L+ CPP-ACP-NaF
DIAGNOdent pen score Groups
CPP-ACP-NaF L+TCP-NaF L+ CPP-ACP-NaF
Baseline After pH cycling Baseline After pH cycling Baseline After pH cycling
10 days 10 days 10 days
Healthy tooth substance (0–13) 100 A, a 93.33 A, a 100 A, a 66.66 A, b 100 A, a 93.33 A, a

Initial demineralisation (14–20) 0 A, a 6.66 A, a 0 A, a 33.33 A, a 0 A, a 6.66 A, a

Strong demineralisation (21–29) 0 A, a 0 A, a 0 A, a 0 A, a 0 A, a 0 A, a

Same capital letters in columns indicate no statistically significant differences in the DIAGNOdent readings at baseline or after pH cycling between the groups.
Same lowercase letters in rows indicate no statistically significant differences between baseline and after pH cycling. Group CPP-ACP-NaF: MI varnish
(5% sodium fluoride with casein phosphopeptide-amorphous and calcium phosphate); group L+TCP-NaF: Er:YAG + 5% sodium fluoride + modified tricalcium
phosphate; group L+ CPP-ACP-NaF: Er:YAG + 5% sodium fluoride with casein phosphopeptide-amorphous and calcium phosphate.

Combined treatment manufacturer’s instructions. Twice a week for a month, in-

In the first stage, Er:YAG laser irradiation was carried out as
described above, the specimens were rinsed with deionised
water for 1 min and then air dried. Fluoride varnishes were
applied immediately under the respective conditions.
pH Cycling
Following surface treatment and incubation in saliva for
24 h (Incubator RKJ; Tokyo, Japan), the samples were sub-
jected to a pH-cycling regimen for ten days, with 8 h of de-
mineralisation and 16 h of remineralisation at 37°C, as
described by ten Cate et al,19,20 to create caries-like le-
sions. Each group was immersed in 5 ml of a demineralis-
ing/remineralising solution followed by rinsing with deion-
ised water before and between each period. The
remineralisation solution contained 1.5 mM CaCl2, 0.9 mM
KH2PO4, 130 mM KCl, 20 mM HEPES pH 7.0, and the de-
mineralisation solution consisted of 1.5 mM CaCl2, 0.9 mM
KH2PO4, 50 Mm acetic acid adjusted to pH 5.0. The pH-cy-
cling solutions were renewed every 24 h.19,20
dividual calibration before the start of sampling to measure
laser fuorescence was performed in anterior primary teeth
(Kappa 0.95). Three measurements along the longitudinal
axis of the tooth were taken, and the sample’s mean value
was calculated.
Prior to the experiment/treatment, each sample was
taken from the thymol solution, or rinsed immediately after
concluding the pH-cycling process (in after treatment step),
and dried with oil-free compressed air for 5 s.
DIAGNOdent pen measurements for each sample (10
per group) were made using a B probe before treatment and
after pH cycling. The device was calibrated against a propri-
etary ceramic standard before each measurement. The
laser tip was held at an angle of approximately 60 degrees
to the tooth surface, as determined by the geometry of the
laser tip. The cut-off points for the first DIAGNOdent read-
ings were those defined by the manufacturer as healthy
tooth substance (0–13), initial demineralisation (14–20),
strong demineralisation (21–29), and dentin lesions (> 30).

Laser Fluorescence Measurements Statistical Analysis

For observer agreement, sixty human anterior primary teeth All data were analysed using the SPSS 22.0 statistical
were selected from a total of 84 teeth. A blinded operator package (SPSS; Chicago, IL, USA). The Mann-Whitney U-test
performed the laser fluorescence readings according to the was used to evaluate differences between groups at ten

Vol 17, No 4, 2019 319

González-Sotelo et al
days. The Wilcoxon test was performed to compare values
before treatment and after pH cycling (Tables 2a and 2b).
Both tests were applied at a significance level of p ≤ 0.05.
RESULTS
Tables 2a and 2b illustrate DIAGNOdent percentages at
baseline and after pH cycling for each group. The Mann-
Whitney U-test showed no statistically significant differences
between the groups (p > 0.05). However, the Wilcoxon test
revealed statistically significant differences at baseline and
after pH cycling in groups C (p = 0.02), L (p = 0.034), and
L+TCP-NaF (p = 0.025) for the ‘healthy tooth substance’
DIAGNOdent score, which was reduced.
DISCUSSION
Some studies have shown that topical fluorides and fluoride
varnishes decrease dental caries.1,5,14,21,22 Recently, new
fluoride varnishes containing casein phosphopeptide-amor-
phous calcium phosphate (CPP-ACP) and tricalcium phos-
phate (TCP) have been shown to prevent demineralisation
and promote remineralisation of carious lesions.1,14,22 On
the other hand, it has been reported that laser technology
combined with fluoride treatment makes enamel surfaces
more resistant to acid than does fluoride treatment or laser
treatment alone.12,13 However, a few studies have reported
enamel demineralisation with combined treatment as
Er:YAG laser + fluoride varnishes on primary teeth.11,12,18,23
The latter studies are extremely relevant, since the param-
eters established for permanent teeth may not be applica-
ble to the different structure of enamel in deciduous teeth.
A pilot study was carried out on 12 anterior teeth (2 per
group), following the same steps described in above, with
irradiation parameters chosen according to a review of the
literature and after the analysis of pilot study results. The
main problem found in the procedures was the way in which
the varnish products were removed previous to the pH cy-
cling, which was solved by using a scalpel.
The in vitro de- and re-mineralisation technique was devel-
oped to study the effects of caries prevention strategies.
Thus, artificial caries was created using the pH-cycling model
described by Bujis.20 Also, similar works have reproduced
some pH-cycling conditions in their studies;5,16,22 however,
other studies1,5,22 have used lower- or higher-pH solutions,
which could produce variations in the demineralisation rate.
Several techniques have been used to evaluate the ef-
fect of various agents on enamel acid resistance, among
them laser fluorescence, a consistent method for assessing
demineralisation.8,9,15 Mendes et al15 reported that laser
fluorescence was a good way to detect lesion depth in pri-
mary teeth and predict the extension of caries lesions. Rod-
rigues et al17 concluded that laser fluorescence was effec-
tive to detect initial demineralisation on enamel surfaces.
DIAGNOdent has shown good reproducibility, in addition
to being easy to use in dental practice and readily trans-
320
portable. Quantitative laser light-induced fluorescence (QLF)
has been reported as a better method for evaluating min-
eral loss in carious lesions in the enamel; nevertheless, for
quantification of dentin lesions, QLF may be less effective
than DIAGNOdent, since it is suggested only for enamel le-
sions.2 For these reasons, protocols that include the use of
both techniques could provide more information regarding
the preventive effect of the agents employed. Additionally,
it should be taken into account that both techniques have
potential sources of error, such as variations between op-
erators, moist dental tissues, plaque, calculus and/or stain
accumulations.7
The new products and methods described here, e.g.
fluor ide varnishes containing CPP-ACP-NaF(5%), TCP-
NaF(5%), Er:YAG laser irradiation, and combined treatments
(Er:YAG laser irradiation + fluoride varnishes application),
are claimed by the manufacturers to be new approaches for
remineralisation of enamel.5 Alamoudi et al1 compared the
effect of NaF (5%) varnish and TCP-NaF (5%) on the micro-
hardness of primary enamel and found significantly
strengthened structure with the addition of tricalcium phos-
phate. The present study found similar results in group TCP-
NaF and group CPP-ACP-NaF, both of which exhibited low
demineralisation after pH-cycling at ten days. On the other
hand, Memarpour et al14 compared enamel microhardness
produced by NaF (5%) and fluoride treatment (5%) + TCP,
where the latter reached higher microhardness. Further-
more, Tuloglu et al21 evaluated CPP-ACP-NaF vs TCP-NaF
and reported higher microhardness values for CPP-ACP.
Nonetheless, the results of this study showed no significant
differences between laser fluorescence values for CPP-ACP-
NaF and TCP-NaF (groups TCP-NaF and CPP-ACP-NaF, re-
spectively), although it must be borne in mind that a higher-
pH solution was employed.
Regarding Er:YAG laser irradiation alone (group L), re-
sults showed the highest enamel demineralisation, al-
though there were no statistically significant differences in
relation to group L+TCP-NaF; however, this is a combined
treatment group.
Castellan et al3 compared single treatments of Er:YAG
laser (60 mJ, 40J/cm2), Nd:YAG laser or APF (gel containing
1.23% sodium fluoride) in primary molars. They found no
statistically significant difference in mineral loss between
Er:YAG laser- and Nd:YAG-laser irradiated groups. Neverthe-
less, laser Er:YAG and Nd:YAG presented the potential to
inhibit the demineralisation of deciduous enamel equivalent
to that achieved with the use of APF. Nonetheless, in the
current study Er:YAG laser irradiated samples showed more
mineral loss when compared with the fluoride varnishes;
however, higher laser density was used (100 mJ, 12.7J/
cm2). Liu et al11 reported more protection against enamel
demineralisation using an output energy of 100 mJ and
200 mJ on permanent teeth, which have a higher mineral
content.1
The systematic literature search found a high degree of
result heterogeneity due to differences in laser type and
wavelength, laser irradiation parameters, type of deminer-
alisation induced in samples, and method of evaluation.7
Oral Health & Preventive Dentistry

Although Zamudio et al23 reported an increase in the
enamel Ca:P molar ratio when 1.23% APF fluoride was com-
bined with laser irradiation with an output of 100 mJ and
energy density of 12.7J/cm2, the samples treated with
Er:YAG laser + TCP-NaF (group L+TCP-NaF) in this study
showed demineralisation of enamel surfaces. Furthermore,
the Er:YAG + CPP-ACP-NaF protocol (group L+CPP-ACP-NaF)
presented more healthy tooth substance on samples after
treatment than in group L+TCP-NaF.
Several studies agree that when laser irradiation is com-
bined with fluoride treatment, a synergistic effect on acid
resistance is observed. Among them, Fekrazad and Ebra-
himpour7 reported that applying laser without fluoride could
decrease the concentration of calcium compared to the
control group, and the decline was not statistically signifi-
cant. It is important to note that the concentration of the
fluoride product employed, as well as the pH, time and fre-
quency of application can influence its uptake,10 regardless
of the irradiation parameters used. Also, it has been sug-
gested that fluoride played a statistically significantly
greater role in the remineralisation process than in the de-
mineralisation process; an environment with a neutral pH
and high concentration of Ca and P may additionally pro-
mote fluoride precipitation.13
Furthermore, the combination of fluoride and laser could
decrease the frequency of fluoride consumption; neverthe-
less, there are no reports on long-term effects of laser ther-
apy for preventing enamel demineralisation, considering
that this type of therapy cannot be applied periodically, as
opposed to fluorides,7 because of the potentially undesir-
able morphological changes it could produce, as previously
reported.23
Although there were no statistically significant differ-
ences between groups, the by-group analysis demonstrated
better performance in groups TCP-NaF, CPP-ACP-NaF and L+
CPP-ACP-NaF, which retained their ‘healthy tooth substance’
score in more than 90% of samples, which could be clini-
cally relevant.
In this in vitro study, 66% of the samples in groups C, L
and L+TCP-NaF were rated as healthy tooth substance;
thus, more demineralisation than others groups was exhib-
ited. However, the outcomes need to be confirmed clini-
cally.
Furthermore, there is a need to evaluate morphological
and chemical changes on enamel surfaces to compare CPP-
ACP, TCP fluoride varnishes with or without laser Er:YAG
treatment. Furthermore, future studies may be needed to
compare different pH solutions.
CONCLUSION
The results suggest that the preventive protocols employed
in groups TCP-NaF, CPP-ACP-NaF and L+ CPP-ACP-NaF pe-
form similarly with positive preventive results, as reflected
in higher percentages of maintaining healthy dental struc-
ture. Hence, these conditions are recommended for clinical
use as an effective preventive measure.
Vol 17, No 4, 2019
González-Sotelo et al
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