Photomedicine and Laser Surgery
Volume 25, Number 1, 2007
© Mary Ann Liebert, Inc.
Effects of Treatment for Manipulation of
Teeth and Er:YAG Laser Irradiation on Dentin:
ARaman Spectroscopy Analysis
LUÍS EDUARDO SILVASOARES, D.D.S., M.Sc.,1,2ALDO BRUGNERAJUNIOR, D.D.S., Ph.D.,3
FÁTIMAANTÔNIAAPARECIDAZANIN, D.D.S., Ph.D.,3
MARCOS TADEU TAVARES PACHECO, Ph.D.4andAIRTON ABRAHÃO MARTIN, Ph.D.1
Objective: The main purpose of this study was to evaluate the utility of Raman spectroscopy analysis as a re-
search tool to study the effects of Er:YAG laser etching on dentin mineral and organic components. Asecondary
aim was to study the effects of the decontamination process and the storage procedure on dentin components.
Background Data: There are no spectroscopy reports relating the effects of Er:YAG laser irradiation as an al-
ternative to acid etching and the manipulation process on the dentin structure. Methods: Twelve non-carious
human third molars were divided in two main groups: stored in thymol solution (group A, n = 6) or autoclaved
(group B, n = 6). The specimens were either etched with 37% phosphoric acid (control subgroup) or irradiated
with Er:YAG laser. Irradiated samples were divided into the following subgroups: I, II, and III (80 mJ, 3 Hz,
30 sec; 120 mJ, 3 Hz, 30 sec; and 180 mJ, 3 Hz, 30 sec, respectively). Samples were analyzed by Raman spec-
troscopy. Results: The mineral and organic dentin contents were more affected in autoclaved teeth than in the
specimens stored in thymol. Peak area reduction in group A specimens treated with phosphoric acid and pulse
energy of 80 mJ were the most conservative surface treatments regarding changes in the peak area of organic
and inorganic dentin components. Conclusion: The autoclaving process and pulse energies of 120 and 180 mJ
produced greater reduction of organic and inorganic contents in dentin, associated with greater reduction in
the areas of 968, 1077, 1460, and 1670 cm–1Raman peaks.
and formation of marginal gaps.1Formation of an adequate
dentin-adhesive bond depends on the diffusion of adhesive
throughout the demineralized layer and into the unaffected
dentin. If the adhesive does not penetrate into the demineral-
ized dentin, the naked protein within this layer will be exposed
to oral fluids and becomes vulnerable to degradation by bacterial
STRONG AND DURABLE bond between composite resin re-
storation and the tooth is needed to prevent microleakeage
enzymes. Such degradation can ultimately lead to premature
failure of the composite restoration.2
Conventional adhesive systems involve enamel and dentin
etching with phosphoric acid before application of an adhesive
resin. Acid etching removes the smear layer, opens dentin
tubules, increases dentin permeability, and demineralizes
peritubular and intertubular dentin. After water rinsing,
approximately 70% of the volume of demineralized dentin, or
50% of the intertubular area, is filled by water, which replaces
the removed minerals.3
1Biomedic Vibrational Spectroscopy Laboratory, Research and Development Institute, (Instituto de Pesquisa e Desenvolvimento [IP&D])
2Operative and Restorative Dentistry, School of Dentistry, 3Center of Laser in Biomedicine, IP&D, and 4IP&D, Universidade do Vale do
Paraíba (UNIVAP), São José dos Campos, Brazil.
Manipulation of Teeth and Laser Irradiation on Dentin51
Recently, the possibility of replacing conventional acid etching
treatment by Er:YAG laser irradiation has been investigated.4–8
This laser is able to condition the dental surface, resulting in a
rough micro-retentive pattern.8According to Hibst and Keller,6
Er:YAG laser irradiation on dental tissues promotes a partial re-
moval by means of vaporization and microexplosions, depending
on the energy used in such irradiation.7Consequently, the use of
this laser could produce a microretentive pattern in enamel and
dentin, revealing open tubuli in the latter.7
The effectiveness of Er:YAG laser as a dentin conditioning
agent depends on several parameters, such as energy output,
frequency, pulse mode, focal distance, irradiation time, and
water cooling.8Those parameters have been extensively studied
using in vitro samples.
However, the manipulation methods for extracted human teeth
used in scientific research have received little attention. It is
known that in vitro investigations of dental materials require a
significant number of extracted human teeth.9Moreover,
extracted teeth may harbor microorganisms, which can lead to
cross-contamination. Thus, infection control procedures must be
considered before manipulating extracted human teeth to elimi-
nate the risk of disease transmission and sample contamination.9,10
Inadequate manipulation and decontamination of teeth may
modify the results of an in vitro tensile bond strength test. Cur-
rently, the intra-oral model for in vitro studies uses teeth speci-
mens that have been disinfected using several methods.
However, these aspects make it difficult to compare results
among studies. Autoclave sterilization induced a loss of min-
eral and collagen components from the surface, and changed
Moreover, the autoclaving process is still used in some stud-
ies to prepare the specimens 12while in other studies, teeth de-
contamination or storage treatment is not discussed.5,13–16
Thymol solution is commonly used to prepare teeth specimens
for in vitro studies without damaging the teeth components.17–22
However, because of increasing emphasis on infection control,
it is important to identify an effective way to manipulate these
specimens without altering their structure. There are no defini-
tive answers at the molecular level yet as to how these proce-
dures may affect subsequent etching or bonding to the dentin.
Chemical characteristics after laser irradiation or teeth ma-
nipulation are also important. Raman analysis gives informa-
tion about the chemical state without causing damage.14
Raman spectroscopy is a technique used for the molecular
analysis of dental tissues and is able to characterize the spatial
distributions of organic and inorganic compounds,15,23reveal-
ing their chemical composition.15
The vibrational modes of PO4
groups can be studied by Raman spectroscopy. In addition, the
relative intensities of the bands between normalized spectra
can lead to quantitative estimate of these constituents. Thus,
Raman spectroscopy can provide complementary information
The available literature on the Er:YAG laser presents diverse
irradiation parameters and results, using morphological and
mechanical tests to evaluate the influence of laser irradiation
on dentin without clarifying its effect on inorganic structure
and collagen fiber network. Another point that needs to be
tested is the influence of the teeth manipulation process on
dentin structure at the molecular level.
3–, OH–, HPO4
2–, and CO3
Considering all these factors, the main purpose of this study
was to evaluate the utility of Raman spectroscopy analysis as a
research tool to study the molecular level effects of Er:YAG
laser etching on the dentin mineral and organic components. The
secondary aim was to study the effects of the decontamination
process and the storage procedure on dentin components at the
The specimens used in this study were prepared from 12 ran-
domly selected, erupted, non-carious human third molars. The
teeth used had been recently extracted (less than 3 months) from
patients whose extractions were part of their dental treatment
and stored in saline solution (Farmavale & Cia, LBS Laborasa
Ind. Farm. Ltd., Brazil) at 9ºC. The research protocol was ap-
proved by the UNIVAP Ethics Committee on Human Research
(protocol no. 01/14384–8). After extraction, the remaining soft
tissue was removed from the tooth surface with a dental scaler
(7/8 Duflex, Brazil). The teeth were polished with a pumice
paste (S.S. White, Brazil) and water using a Robinson brush
(Viking, KG Sorensen, Brazil) in a low-speed handpiece (KaVo,
Brazil). Teeth were washed and then divided into two major
groups, according to the manipulation treatment (sterilization or
storage): in group A, six teeth were stored in 0.1% thymol aque-
ous solution at 9ºC for 1 week;17–22in group B, six teeth were au-
toclaved at 121ºC for 15 min (Biodont, Alpax, Brazil) in a flask
filled with sterile saline (Farmavale & Cia, LBS Laborasa Ind.
Farm. Ltd., Brazil), closed tightly, and stored at 9ºC.
The teeth stored in aqueous thymol solution were washed
with filtered water for 24 h to eliminate thymol residues. The
occlusal one-third of the sample crowns were sectioned per-
pendicular to the long axis of the teeth (Fig. 1A) by means of a
water-cooled low-speed diamond disc at 250 rpm and with a
100g load (Isomet 1000, Buehler, USA). The deep dentin sur-
face was ground on wet 600-grit silicon carbide paper (3M,
Brazil) at 150 rpm (Knuth Rotor, Struers, Brazil) for 1 min,
under constant water cooling in order to produce a standard
smear layer (Fig. 1B).3,24
Ultrasonic cleaning (Cole-Parmer 8891, USA) with distillated
water to remove the excess debris (Fig. 1C) was performed for
5 min, and then specimens were washed and stored in saline so-
lution in a refrigerator at 9°C. Roots were removed with a water-
cooled low-speed diamond disc producing one 4-mm-thick
dentin disc-shaped slice for each tooth (Fig. 1D,E). All slices
were obtained from the same tooth depth approximately.
Digital images of each dentin sample were obtained using a
CCD color video camera (JVC, TK-C1408E, Thailand), using
a macro-focusing lens (Vivitar 100 mm) and a filter (ND2X,
49 mm) to identify each sample surface before and after treat-
ments. To obtain the images, the samples were illuminated
with a halogen lamp (Ram Optical Instrumentation [ROI],
Model 150 Illuminator, USA).
A reference point was created with a diamond bur (no. 2, KG
Sorensen, Brazil) in the buccal enamel of the samples with a
52Soares et al.
high-speed turbine (KaVo, Brazil; Fig. 2). The surfaces of the
slices were schematically divided into four areas, with each one
corresponding to a treatment group (Fig. 2). Every sample re-
ceived all the studied treatments in order to avoid individual bio-
chemical differences among teeth and to standardize the surface
to be treated. Each quadrant of the sample received a different
treatment, generating four subgroups, as described in Table 1.
Specimens were removed from the saline solution and laser
irradiation was performed in a non-contact mode by an
Er:YAG laser (KaVo Key Laser II, Germany, (? = 2.94 µm,
beam diameter = 1 mm) with the no. 2051 handpiece at a focal
distance of 12 mm, with water spray cooling (2 mL/min) and
energy density value of 15 J.
TABLE 1.DESCRIPTION OF THE EXPERIMENTAL TREATMENTS
Group AManipulation process: 0.1% aqueous
thymol solution (1 week)
Manipulation process: autoclaving 121ºC
37% phosphoric acid (15 sec)a
Er:YAG laser (80 mJ, 3 Hz, 15 J,
Er:YAG laser (120 mJ, 3 Hz, 15 J,
Er:YAG laser (180 mJ, 3 Hz, 15J, 86 pulses)a
Sequence of dentin sample discs preparation.
point (marker) and the divisions of the experimental groups.
Digital image of one dentin disc with the reference
Manipulation of Teeth and Laser Irradiation on Dentin53
Irradiation of the control group quadrant was avoided, and a
space was left between the sides of each group. After the irra-
diation procedure, acid etching was performed in the control
group area using 37% phosphoric acid gel (FGM, Brazil) for
15 sec. The etched surface was then rinsed with air-water spray
for 15 sec.
Dispersive Raman spectroscopy analysis
The dentin surfaces (before and after treatment with either
the phosphoric acid or Er:YAG laser) were analyzed by disper-
sive Raman spectroscopy. For the Raman spectra calibration,
the spectrum of the indine (C9H8) substance was obtained.25
Raman data of each sample before the treatment was recorded
as references. The samples were placed on a precision X-Y-Z
stage to obtain four spectra per area group, totaling 192 spectra.
The samples were excited in the near-infrared region by a
Ti:Sapphire laser (model 3900S, Spectra-Physics, (? = 785 nm,
beam diameter = 1.5 mm) pumped by an Argon laser (Stabilite
2017/Spectra-Physics, ? = 514 nm). The schematic description
of the near-infrared Raman spectroscopy system used to evalu-
ate the dentin components is shown in Figure 3. A notch filter
was used to block the laser line to the spectrograph (Cromex
250IS. The power of the Ti:Sapphire laser was limited to 80
mW in the sample holder. The spectral slit was set to 200 µm. A
CCD detector cooled by liquid nitrogen collected the Raman
spectra of the samples. For each measurement, four spectra per
each area group were accumulated with 10 readings of 1 sec, to-
taling 192 spectra. The data collection was controlled by the
Averages of the 192 Raman spectra were obtained from each
dentin group and manipulation treatment. The fluorescence
from the 48 average spectra was removed with a polynomial
fitting of varying degrees using the Microcal Origin 5.0®soft-
ware. Relative areas of the peaks were calculated by the Microcal
Evaluation of changes in mineral and organic structures was
performed by comparing the relative intensity ratio of the 968,
1077, 1460, and 1670 cm–1peaks to the 1046 cm–1peak. This
procedure is based on the assumption that the ratio of the inte-
grated intensities of the antisymmetric and symmetric stretching
modes of the phosphate ion should not change.26
Statistical analysis of the Raman results was performed by
one-way analysis of variance (ANOVA) at a 95% level of con-
fidence. The Tukey-Kramer Multiple Comparisons post-hoc
test was also performed using the Instat®software to assess the
significance of the relative area evaluation between the normal
and treated dentin data. The post-hoc test was done comparing
the two manipulation methods and each dentin treatment. The
Kolmorogov and Smirnov test verified the normal distribution
of the sample data. The standard deviations were assessed by
the Bartlett’s test using the Instat®software.
Digital images of the dentin after the treatments showed that
the laser produced an evident thermomechanical ablation of
the dentin surfaces on groups II and III with a more irregular
surface in group III. The surface area of the group I was similar
to the control group and smoother than the ones in groups II
and III. Laser energy of 180 mJ produced an irregular layer on
the dentin surface (Fig. 4).
The typical Raman spectra for the normal and treated human
dentin in the region of 1800–1000 cm–1and 1020–900 cm–1are
shown in Figures 5 and 6. Except for the absolute change of the
intensity in the Raman peaks, no other effect was observed in
the Raman spectrum. The strongest peak at 968 cm–1(Fig. 5) is
associated with the phosphate (PO4
the mineral apatite component of dentin. The peak at 1077 cm–1
(Fig. 6) is due to carbonate (CO3
at 1460 and 1670 cm–1(Fig. 6) are attributed to the organic
components of dentin: CH3-CH2and C=O (amide I) vibrations,
The results of the peak area evaluation showed reduction in
the relative area of the inorganic phase (968 and 1077 cm–1)
3– v1) stretching vibration in
2– v1) vibrations.20Weak bands
spectroscopy system used to evaluate the dentin components.
Schematic description of the near-infrared Raman
Er:YAG laser irradiation.
Digital image of dentin surface after acid etching and
54Soares et al.
in the relative area after the surface treatment for groups A–C,
A-I and A-II. However, the groups A-III, B-C, B-I, B-II, and B-
III presented significant reduction in the relative area after the
The requirements of an effective dentin adhesive system in-
clude the ability to thoroughly infiltrate the collagen network in
the partially demineralized zone, to comingle and encapsulate
the thymol (A) and autoclaved (B) specimens after the acid
etching (C) and Er:YAG laser irradiation with 80 (I), 120 (II),
and 180 (III) mJ of laser energy.
Dentin Raman spectra with the peak at 968 cm–1(1) of
1460 (2) and 1077 cm–1(3) of the thymol (A) and autoclaved
(B) specimens after the acid etching (C) and Er:YAG laser irra-
diation with 80 (I), 120 (II), and 180 (III) mJ of laser energy.
Dentin Raman spectra with the peaks at 1670 (1),
and in the organic component (1460 and 1670 cm–1) for all
tested groups (Figs. 7–10).
The Tukey-Kramer multiple comparisons, post-hoc test results
are shown in Figures 8–10. For the peak at 968 cm–1(Fig. 7),
non-significant statistical differences (p > 0.05) were found
after the surface treatment for the groups A–C and A–I. Groups
A-II, A-III, B-C, B-I, B-II, and B-III presented significant
reduction in the relative area after the treatments.
The area comparisons for the peak at 1077 cm–1(Fig. 8)
showed reduction with statistical significance for all tested
Analysis of the relative area reduction for the peak at 1460
cm–1(Fig. 9) showed non-significant statistical differences
(p > 0.05) after the surface treatment for groups A–C and A–I.
Groups A-II, A-III, B-C, B-I, B-II, and B-III presented signifi-
cant reduction in the relative area after the treatment.
The area comparisons for the peak at 1670 cm–1(Fig. 10)
demonstrated non-significant statistical differences (p > 0.05)
the phosphate (PO4
process (A, thymol; B, autoclaved) of the dentin treatments:
GC, GI, GII, and GIII for normal (N) and treated (T) speci-
mens (*,**,***significant statistical difference; nsnonsignificant
Relative area of the peak at 968 cm–1associated to
3– v1) according to the teeth manipulation
the carbonate (CO3
process (A, thymol; B, autoclaved) of the dentin treatments:
GC, GI, GII, and GIII for normal (N) and treated (T) speci-
mens (*, **, ***significant statistical difference; nsnon-significant
Relative area of the peak at 1077 cm–1associated to
2– v1) according to the teeth manipulation
Manipulation of Teeth and Laser Irradiation on Dentin55
Teeth manipulation has also been the subject of several
studies.10Most groups use gamma irradiation to sterilize the
teeth and it does not influence the bond strength to dentin.9
However, in some countries, gamma irradiation is not an
Another point to consider is that due to manipulation proce-
dures, the evaluation of structural or chemical alterations at a
molecular level, has not been fully studied yet. Moreover, in
spite of the damaging effects, the autoclaving process is still
used to prepare specimens12in some groups and in some stud-
ies the teeth decontamination or storage treatment is not even
informed.5,13–16Thymol solution is an alternative method com-
monly used to prepare teeth specimens for in vitro studies.17–22
In the present study, the utility of Raman spectral analysis
was evaluated as a complementary method to the mechanical
tests (bond strength) to obtain chemical information on
Er:YAG laser–irradiated human dentin as an alternative to
chemical etching. Additionally, chemical information about the
effects of the sterilization and storage procedures was obtained.
Reductions in the inorganic and organic components were
indirectly observed by changes in the relative area of the peaks
at 968, 1077, 1460, and 1670 cm–1(Figs. 5 and 6). The auto-
claved specimens showed significant statistical reduction in
the relative area of the peaks at 968, 1460, and 1670 cm–1in all
tested groups. However, the same reduction was not verified in
the specimens treated by thymol in some tested groups. This is
probably due to the thermal effect promoted by autoclaving
sterilization process at 121°C and 15 lb psi.
Considering other treatments for teeth manipulation,
Sperandio et al. reported that the shear bond strength was not
affected by gamma radiation. Nevertheless, the authors suggested
that further investigation is required to establish comparisons and
agreements.9 Furthermore, Cheung et al. reported that collagen
molecules in tissue are readily altered by gamma radiation.30
The mineral content changes associated to the phosphate
peak (968 cm–1/PO4
fected in the autoclaved specimens than in the specimens
treated with thymol. This is probably also related to the ther-
mal effect of the treatment. Therefore, the choice of dentin ma-
nipulation method prior to the treatment may compromise the
Teeth storage in thymol and subsequently treatment with either
Er:YAG laser with 80 mJ or phosphoric acid etching by 15 sec,
removed less phosphate than the autoclaved treatment and,
therefore, proved to be a more conservative treatment. Higher
laser energies (120 and 180 mJ) affected more the phosphate
content than the phosphoric acid and 80 mJ laser treatments.
The decrease of the relative intensity for the CO3
(1077 cm–1) was greater than that for the PO4
mode in all tested groups, indicating that the removal of car-
bonate either by acid or Er:YAG laser is greater than the
changes in phosphate content.
The organic component evaluated in the peak area at 1460
cm–1(CH3–CH2) presented reduction without statistical signif-
icance when the dentin was decontaminated by thymol and
treated with phosphoric acid or irradiated with pulse energy of
80 mJ. This could indicate a more favorable substrate to the ad-
hesion process, where the collagen fibrils were less affected
than the group of the autoclaved specimens. Higher pulse ener-
gies (120 and 180 mJ) affected the organic content of the
3– v1) showed that the dentin was more af-
the collagen and hydroxyapatite crystallites at the front of the
demineralized dentin, and to produce a well-polymerized
durable layer.1Previous reports claimed that there is an im-
provement of adhesion effectiveness of lased dentin.29
After Er:YAG laser irradiation the adhesive area is enlarged
due to the typical scaly and flaky structure exhibits by dentin.
This is caused by the microexplosions produced by the laser,
due to its thermo-mechanical ablation.5,6Other studies demon-
strated that the laser irradiation adversely affected the adhesion
compared to the traditional acid etching.5,7
the C=O (amide I) according to the teeth manipulation process
(A, thymol; B, autoclaved) of the dentin treatments: GC, GI,
GII, and GIII for normal (N) and treated (T) specimens
(*,**,***significant statistical difference; nsnonsignificant statis-
Relative area of the peak at 1670 cm–1associated to
the CH3-CH2 vibrations according to the teeth manipulation
process (A, thymol; B, autoclaved) of the dentin treatments:
GC, GI, GII and GIII for normal (N) and treated (T) specimens
(*,**,***significant statistical difference; nsnonsignificant statis-
Relative area of the peak at 1460 cm–1associated to
56Soares et al.
specimens treated with thymol, but the autoclaved specimens
were more affected by these laser energies, the reducing the or-
Carmelingo et al. also reported chemical changes in laser
treated dentin detected by micro-Raman spectroscopy.28The
conventional mechanical technique (drill) was compared with
the laser treatment with different laser pulse times, and higher
frequencies and energies. However, only changes in the spectra
intensity28 were reported.
In the present study, a more complete evaluation of changes
in the relative area of inorganic and organic dentin components
was performed. The conventional acid etching was used as a
standard treatment to be compare with the laser treatment. The
effects of Er:YAG laser irradiation on dentin and additionally
the effects of a sterilization and storage treatment were reported.
The type I collagen (1670 cm–1/C=O) dentin component was
not significantly reduced by acid etching or by laser energies of
80 and 120 mJ in the specimens treated with thymol. Only the
Er:YAG laser energy of 180 mJ affected the collagen content.
However, in the autoclaved specimens, the collagen was af-
fected by the acid etching and by all laser energies. It is impor-
tant to consider this fact since 90 wt% of the organic phase in
dentin is collagen, which is almost exclusively type I.31
Our findings confirm the previous results obtained in mor-
phological studies using scanning electron microscopy
(SEM)32and transmission electron microscopy (TEM).5,33In
the previous SEM study, the Er:YAG laser irradiation with
80 mJ and 2 Hz produced open dentin tubuli without smear
layer and in the TEM study, the irradiation with Er:YAG laser,
energy pulse of 180 mJ and 2 Hz frequency melted and vapor-
ized the dentin collagen fibrils.5,33The presence of this fused
layer resulted in lower shear bond strength values.5
Gonçalves et al. showed that Er:YAG laser dentin irradiation
with 140 mJ and 4 Hz did not improve the tensile bond resis-
tance.7In the present study, lower laser energies were used to
verify their effects on the dentin components. We chose 3 Hz
frequency for the laser because in the previous study the use of
2 and 4 Hz associated with higher laser energies adversely af-
fected the adhesion to the dentin, not representing an alterna-
tive to the acid etching in the adhesion process.5,7
Finally, our findings complement the results of the previous
morphological and mechanical studies. The chemical informa-
tion obtained by Raman spectroscopy showed that higher laser
energies (180 mJ) affected more the phosphate, carbonate and
the organic components of the dentin than in groups treated
with phosphoric acid and laser with 80 and 120 mJ. The teeth
manipulation method used to prepare the specimens for in vitro
tests should be carefully chosen. Raman spectra showed more
chemical changes in the autoclaved specimens than those
caused by thymol treatment, which can influence the adhesion
process. These results contributes to a better understanding of
the laser irradiation effects on human dentin and help provide
more information to develop a new class of materials to restore
the laser-irradiated teeth.
Raman spectral analysis proved to be an adequate research
tool to study the changes that occurred in dentin produced by
the manipulation process and by laser and chemical etching.
Sample preparation is fairly simple, since it does not require
specific size or translucency. Raman measurements can be
carried out under normal atmospheric conditions without the
need for a high vacuum. Finally, since this technique is non-
destructive, samples can be used in multiple analyses.
Raman spectroscopy measurements showed that the mineral
and organic dentin contents of teeth submitted to the autoclav-
ing process were more affected than those of specimens treated
with thymol. Phosphoric acid etching for 15 sec and Er:YAG
laser irradiation with pulse energy of 80 mJ were the most con-
servative procedures with respect to changes in the phosphate
and organic dentin components. Pulse energies of 120 and
180 mJ produced significant reduction in the carbonate, phos-
phate, and organic content associated with reduction in the
peak areas at 968, 1077, and 1460 cm–1. Type I collagen was
more affected by 180 mJ laser energy.
We thank Marianna Dirickson for a critical reading of the
manuscript. This study was supported by FAPESP (01/14384–8)
and CNPq (grant 302393/2003–0).
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Av. Shishima Hifumi, 2911, Urbanova
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