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VOLUME 40 NUMBER 3 MARCH 2009 195
QUINTESSENCE INTERNATIONAL
Since its introduction by Haywood and
Heymann,1 home dental bleaching has been
the most commonly used method for tooth
whitening. The original technique, published
in 1989, used 10% carbamide peroxide in a
vacuum-formed custom tray. Nightguard vital
bleaching is an effective and simple method
of lightening extrinsically stained or discol-
ored teeth.2 One of the changes in the origi-
nal technique has been the use of carbamide
peroxide in concentrations greater than 10%,
claiming greater effectiveness and less time.3
However, several studies have reported
important adverse effects, such as tooth sen-
sitivity,48 soft tissue changes,9,10 hard tissue
changes,11,12 genotoxic effect in bacteria and
cultured cells,1315 cytotoxic effects,16,17 and
gingival irritation,3,79,1821 due to multiple and
prolonged exposures of bleaching agents.
According to Haywood et al,2 approximately
two-thirds of patients who undergo bleaching
treatment with carbamide peroxide experi-
ence tooth and/or gingival sensitivity. Gingival
Effect of reservoirs on gingival inflammation after home dental bleachingGiovanna A. Kirsten, DDS1/Andrea Freire, DDS2/
Antonio Adilson S. de Lima, DDS, MDS, PhD3/
Sergio Aparecido Igncio, DDS, MDS, PhD4/
Evelise M. Souza, DDS, MDS, PhD4
Objective: To evaluate the influence of reservoirs on the gingival mucosa of patients sub-
mitted to at-home bleaching with 16% carbamide peroxide. Method and Materials:
Nineteen nonsmoking male patients, 18 to 25 years of age, were submitted to home
bleaching with a 16% carbamide peroxide gel for 2 consecutive hours for 21 days. The
custom-made mouth trays were made with a reservoir on only the left side and cut anatom-
ically 1 mm beyond the gingival margin. Smears of the gingival mucosa were obtained by
the exfoliation cytology in liquid media technique before (control), immediately after, and
30 and 45 days after treatment. The samples were processed in the laboratory and evalu-
ated according to Papanicolaous criteria of malignity. Statistical analysis was carried out
by McNemar test, 2 proportions test, and Wilcoxon test with a level of significance of 1%.
Results: The presence of a reservoir in the custom tray resulted in an increase of inflam-
mation only immediately after the bleaching procedure. After 30 and 45 days, the differ-
ence between inflammation on the sides with and without a reservoir was not statistically
significant. Significant differences were found in the degree of inflammation, classified as
predominantly mild on the nonreservoir side and moderate on the reservoir side (P < .01).
Conclusions: A 16% carbamide peroxide bleaching gel caused gingival inflammation
immediately after the procedure and persisted until 45 days after the bleaching treatment.
The use of a reservoir in the custom tray for home bleaching resulted in higher rates and
higher intensity of gingival inflammation. (Quintessence Int 2009;40:195202)
Key words: carbamide peroxide, dental bleaching, exfoliative cytology, gingival
inflammation, reservoir, tray
1MDS student, School of Dentistry, Pontifical Catholic University
of Paran, Curitiba, Paran, Brazil.
2PhD student, School of Dentistry, Pontifical Catholic University
of Paran, Curitiba, Paran, Brazil.
3Professor, School of Dentistry, Pontifical Catholic University of
Paran, Curitiba, Paran, Brazil.
4Adjunct Professor, School of Dentistry, Pontifical Catholic
University of Paran, Curitiba, Paran, Brazil.
Correspondence: Dr Evelise M. Souza, Programa de Pos-gradu-
ao em Odontologia, School of Dentistry, Pontifical Catholic
University of Paran, R. Imaculada Conceio, 1155, Curitiba
PRBrazil 80215-901. Fax: 55 41 3271-1405. Email: evesouza@
yahoo.com
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irritation can be associated with the chem-
istry of the bleaching agent5 or the design of
the tray.22,23 However, Haywood and Heyman24
claim that most irritations appear to involve
the nightguard itself and are rarely due to
chemical irritation.
Clinical trials have evaluated gingival
health by measuring the Bleeding Index,
Gingival Index, and Plaque Index.3,21
However, these methods are visual and
demand an adequate calibration of the
examiners involved in the study. Numerical
scales2527 and questionnaires3,9,28 are also
used for the assessment of gingival irrita-
tion, but these methods rely on the
patients perception of discomfort and are
dependent on the pain threshold of each
individual. Histopathologic studies29,30 are
more accurate but too invasive, because a
biopsy of the tissue must be obtained for
cell evaluation. However, exfoliative cytology
has also been used for evaluation of
changes in the cells of oral epithelium.3034
This method is noninvasive and painless
and can be more accurate because of the
standardization of the technique and cali-
bration of the examiner.
The liquid-based cytology has been con-
sidered more advantageous than the con-
ventional technique because it produces
more homogeneous samples, due to the
reduction in the amount of mucus and blood
in the preparation, and less clumping of
epithelial cells.35 Thereby, the liquid-based
cytology technique reduces the proportion
of specimens classified as technically unsat-
isfactory for evaluation,36 decreasing false-
negative or false-positive results.33 Also, the
liquid-based cytology method allows for the
preparations of more than 1 slide per sample
collected, and because of the long storage
life of the liquid-fixative solution, the material
can be preserved and is therefore available
for additional analyses.30
The aim of this in vivo study was to evalu-
ate the effect of using reservoirs on the gin-
gival inflammation of patients undergoing
at-home dental bleaching treatment with
16% peroxide carbamide. The null hypothesis
of this study was that there would be no
difference in the gingival inflammation with
or without the use of reservoirs.
METHOD AND MATERIALS
The experimental protocol of the present
study was approved by the Ethics Committee
of the Pontifical Catholic University of Parana.
SubjectsMale university dental students 18 to 25
years old were invited to participate in the
study. For inclusion, subjects had to be non-
smokers, not submitted to dental bleaching
before the study, and without extensive
restorations in anterior teeth. For exclusion,
the subjects were submitted to a cytologic
evaluation by the liquid-based cytology to
assure that the selected subjects did not
have gingival inflammation prior to the study.
A total of 19 subjects were selected for the
dental bleaching procedure.
Custom tray fabricationAlginate impressions of the maxillary arches
of the subjects were made with Jeltrate Plus
(Dentsply), and casts were fabricated from
dental stone (Herodent, Vigodent). The
Fig 1 Stone cast with reservoirs made on only the left sideof the maxillary arch with a light-activated gingival barrier.
Fig 2 A silicone tray trimmed anatomically on the labialsurface 1 mm beyond the gingival margin.
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reservoirs were made on only the left side of
each subjects cast with a light-activated
gingival barrier (Top Dam, FGM Dental
Products). The material was applied so that
the labial surface was covered with the
exception of 1 mm mesially, distally, cervical-
ly, and incisally (Fig 1). The trays were made
by a vacuum-formed process with a silicone
soft sheet (FGM Dental Products). The
excess was trimmed anatomically on the labial
surface 1 mm beyond the gingival margin
(Fig 2).
Bleaching procedureThe fit of the tray was carefully inspected,
and adjustments were made to ensure that
the tray did not abrade the tissue and was
well-adapted. Subjects were instructed ver-
bally and via hands-on practical demonstra-
tion in the use of the bleaching gel.27 The
16% carbamide peroxide gel (Whiteness
Perfect 16%, FGM Dental Products) was
used 2 hours per day for 21 days. The sub-
jects were asked to return the trays at the
completion of treatment.
Cells collectionExfoliated cells of the gingival margin
between the maxillary canine and premolar
were obtained by liquid-based exfoliative
cytology (Fig 3). Initially, the mouth was
rinsed with water to remove excess debris
and bacteria. The squamous epithelial cells
were collected using a cytobrush and uni-
versal collection medium kit (UCM)
(Universal Collection Medium of DNA-Citoliq
System, Digene Brasil). The cells were col-
lected before, immediately after, and 30 and
45 days after the bleaching treatment. To
compare the degree of inflammation after
dental bleaching, the cells were collected
bilaterally at the sides with and without
reservoirs.
Cytologic preparationsAn aliquot of 200 L UCM was filtered
through Filtrogene polycarbonate membrane
filters (Digene Brasil) with a pore size of 5 m
and diameter of 25 mm. The filter was placed
in prepgene press (Digene Brasil), which was
attached to glass slides. The former were
immediately fixed in absolute alcohol for 20
minutes, and smears were then stained with
routine Papanicolaou stain.
Morphologic analysisEach slide was assessed using a binocular
light microscope Olympus BX50 (Olympus)
at 400 magnification. All cellular features
were coded according to Papanicolaous
classification37: (0) insufficient smear, (1) nor-
mal smear, (2) normal smear with inflamma-
tory changes, (3) dysplastic smear, (4) smear
suggesting neoplastic changes, and (5) neo-
plastic smear. Inflammatory cytologic results
were categorized as absent (0 cells/field),
mild (1 to 5 cells/field), moderate (6 to 20
cells/field), or severe (> 20 cells/field). The
type of predominant cells (cellularity) in each
smear was also analyzed.
Statistical analysisAll data were tabulated, and statistical tests
were performed with SPSS for Windows
13.0 (SPSS). The McNemar test, two propor-
tions test, and Wilcoxon test were used in
this study with a level of significance of 1%.
Fig 3 A brush collecting cells onthe gingival margin between themaxillary canine and premolar forexfoliative cytology.
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RESULTS
The McNemar test found statistically signif-
icant differences in the type of epithelial
cells among the control group (before) and
other periods of evaluation after the bleaching
procedure (immediately after and 30 and
45 days after), characterized by an increase
in the amount of inflammation cells (Table 1).
The presence of a reservoir in the custom
tray resulted in an increase of inflammation
only immediately after the bleaching procedure
(P = .00758), as detected by the two propor-
tions test. At 30 and 45 days after bleaching,
the difference in the amount of inflammatory
cells on the sides with and without reservoirs
was not statistically significant.
The Wilcoxon test detected statistically
significant differences in the inflammation
rates on the reservoir side immediately after
bleaching when compared to 30 and 45
days. However, these were not statistically dif-
ferent from each other. On the other side of
the tray, without a reservoir, the 3 observation
periods showed no statistical differences in
the inflammation prevalence (P > .01).
Figure 4 demonstrates the amount of
inflammatory cells before bleaching (control)
and immediately after bleaching on the sides
without and with a reservoir of the tray.
Statistically significant differences were
found in the inflammation intensity between
both sides of the tray immediately following
and 45 days after bleaching (P < .01). At
Table 1 No. and percentage of patients submittedto home bleaching with 16% carbamide peroxide with inflammation during differentperiods of observation (n = 19)
Period of observation Reservoir Inflammation (%)
Before (control) 0 (0)a
Immediately Without 13 (68.0)b
following With 19 (100.0)c
30 days Without 5 (26.3)b
With 3 (15.8)b
45 days Without 8 (42.1)b
With 4 (21.1)b
Groups connected by the same letter are not statistically different (P > .01).Fig 4 Liquid-based cytology (Papanicolaou, origi-nal magnification 400). (a) Normal aspect of thecells before bleaching (control). (b) Epithelial andinflammatory cells immediately after bleaching onthe nonreservoir side of the tray. (c) Epithelial andinflammatory cells immediately after bleaching onthe reservoir side of the tray.
a b
c
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these periods of evaluation, there was a
prevalence of mild inflammation at the side
of the tray without a reservoir (Fig 5) and
moderate inflammation at the side with a
reservoir (Fig 6).
DISCUSSION
The inflammatory response is closely related
to the process of repair. Inflammation serves
to destroy, dilute, or isolate the injurious
agent, but in turn, it sets into motion a series
of events that, as far as possible, heal and
reconstitute the damaged tissue. On the
other hand, inflammation and repair may be
potentially harmful. Chronic inflammation is
considered to be inflammation of prolonged
duration (weeks or months) in which active
inflammation, tissue destruction, and attempts
at healing are proceeding simultaneously.
This kind of inflammation frequently begins
insidiously, as a low-grade and often asymp-
tomatic response.38
The signs of gingival inflammation after
dental bleaching treatments may not be
observed by visual inspection. Therefore, an
accurate method, such as exfoliative cytology,
must be used to confirm the presence or
absence of inflammatory cells, as well as the
intensity of the inflammation.3436
Although some authors19,25 claimed that
patients experience mild gingival sensitivity
that lasts for a few days after dental bleach-
ing, the present study demonstrated that
16% carbamide peroxide gel, used 2 hours
per day for 3 weeks, was capable of causing
morphologic changes in the human gingival
epithelium not only immediately following
the treatment but also up to 45 days after.
Immediately after treatment, the reservoir
side exhibited a prevalence of moderate
inflammation, whereas on the nonreservoir
side, a greater incidence of mild inflammation
was found. At the 30-day observation, the
intensity of inflammation was similar for both
sides of the tray. By contrast, at the 45-day fol-
low-up, inflammation was largely absent on
the nonreservoir side, while a prevalence of
70
60
50
40
30
20
10
0
% o
f in
flam
ed c
ells
Immediatelyafter
30 days 45 days
Absent
Mild
Moderate
Severe
Immediatelyafter
70
60
50
40
30
20
10
0
% o
f in
flam
ed c
ells
30 days 45 days
Absent
Mild
Moderate
Severe
Fig 5 Inflammation intensity on the nonreservoirside after different periods of observation.
Fig 6 Inflammation intensity on the reservoir sideafter different periods of observation.
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moderate inflammation was seen on the
reservoir side. Additionally, the reservoir in
the tray resulted in even more inflammation in
45 days than the nonreservoir side immedi-
ately after the bleaching procedure.
One possible explanation for these find-
ings could be related to custom-made trays.
The anatomic design, 1 mm beyond the gin-
gival margin, and the flexibility of the silicone
tray could have allowed extrusion of the
bleaching gel. Perhaps a more rigid material
and/or a trimming parallel to the incisal or
occlusal plane could have avoided or
reduced the gingival irritation. Although tray
designs seek to avoid covering the attached
gingiva, the interdental papillae are still
exposed to the bleaching gel.7 Therefore,
total avoidance of soft tissue contact is
impossible.
According to Haywood et al,8 the pres-
ence of reservoirs decreases the retention of
the tray, allowing more room for the gel but
also reducing the adaptation of the tray. This
could explain why the presence of a reservoir
caused more gingival inflammation in this
study. Accordingly, the examiners of the pres-
ent study observed clinical signs of inflamma-
tion in the subjects after 1 week of bleaching
on only the reservoir side of the tray.
In a clinical investigation by Matis et al26 no
difference was found in gingival sensitivity
between areas bleached with and without
reservoirs. However, this parameter was evalu-
ated based on a daily record and classification
of tooth and gingival sensitivity into categories
attributed by the patients.
The presence of severe inflammation 45
days after bleaching was observed only in 2
patients, 1 in the reservoir side and the other
in the side without reservoir. This fact could
be explained by the possible residual effect
of the bleaching agent or the plaque accu-
mulation due to patients negligent hygiene.
Additionally, there is an individual factor
involved in the gingival tissue response to
toxic agents, since the concentration of sali-
vary modulators varies among patients. The
role of saliva and its modulators must be
taken into account: They act as protective
agents of gingival tissue. Tipton et al39
demonstrated that whole saliva, lactoperoxi-
dase, and catalase, at sufficient concentra-
tions, could provide complete or nearly com-
plete protection from the toxic effects of car-
bamide peroxide, removing the hydrogen
peroxide generated during its degradation.
Bleaching agents are cytotoxic to human
gingival fibroblasts, increasing the effects on
cell viability and morphology, and on the pro-
liferation and production of fibronectin and
collagen.39 In an in vitro study, Koulaouzidou
et al17 investigated the cytotoxic effect of a
bleaching agent on 2 fibroblast cell lines and
found that both were sensitive to urea perox-
ide. These authors17 suggested that the
potential damage to oral tissues in vivo may
be considerable because of the direct and
long-term exposure of the tissues to the
bleaching agents.
Therefore, indiscriminate, frequent, or
prolonged treatments, even under profes-
sional supervision, might increase the potential
damage of bleaching agents to the
periodontal tissues, leading to chronic
inflammation. Because the presence of
inflammatory cells was observed 45 days
after the bleaching procedure, this treatment
must not be given frequently, so as to allow
healing of the injured tissues. The dental cli-
nician must be careful to indicate and super-
vise bleaching procedures and be conscious
about the damage that they could cause to
their patients.
CONCLUSIONS
The 16% carbamide peroxide home bleach-
ing caused gingival inflammation not only
immediately after the procedure but also until
45 days following the bleaching treatment.
The use of a reservoir in the custom tray for
home bleaching resulted in higher rates and
higher intensity of gingival inflammation.
ACKNOWLEDGMENTS
The authors thank FGM Dental Products for the supply
of bleaching agents and trays.
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