A D
A
ASSESSING DENTIN COLOR CHANGES FROM NIGHTGUARD VITAL BLEACHING
ALSTON J. MCCASLIN, D.M.D.; VAN B. HAYWOOD, D.M.D.; BRAD J. POTTER, D.D.S., M.S.;
GENE L. DICKINSON, D.D.S., M.S.; CARL M. RUSSELL, D.M.D., PH.D.
A B S T R A C T
Background. At-home bleaching with 10
percent carbamide peroxide in a custom-fitted tray
has been reported to change the color of dentin. The
purpose of this study was to validate the color change
of dentin and to determine whether the color change
was uniform or occurred from the outside (the denti-
noenamel junction) to the inside (the pulpal wall).
Methods. The authors sectioned 10 extracted
human teeth incisogingivally through the midfacial
long axis, and sealed their cut surface against glass
microscope slides. Identifying marks were placed on
the glass over the tooth sections to serve as a color
control and in the dentinal areas closest to the denti-
noenamel junction and the pulpal wall. Teeth were
bleached for 10 days with 10 percent carbamide per-
oxide. Photographs were taken from the glass-
covered side of the teeth, digitized and converted to
gray-scale levels (consisting of 256 shades of gray
ranging from black to white). Marked areas were
measured with a National Institutes of Health Image
software program and analyzed statistically for
changes in lightness between the control marks and
the inner and outer dentinal marks over time.
Tooth color is primarily determined by the
dentin, and can be changed with bleaching tech-
niques.1,2 Studies of in-office bleaching with 35
percent hydrogen peroxide have reported that a
color change occurs in both the enamel and
dentin.3-9 However, some authors believe that the
color change occurs in enamel only and masks the
unchanged dentin color.10 Recently, lower concen-
trations of peroxide have been used to bleach
teeth, again raising some of the questions about
the location of the color change. Bleaching of vital
teeth with 10 percent carbamide peroxide
(approximately 3 percent hydrogen peroxide) in a
custom-fitted tray has gained in popularity since
its introduction in 1989.11
Some authors initially reported that only stains
Copyright ©1998-2001 American Dental Association. All rights reserved.
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ARTICLE 3
Results. Paired t-tests and analysis of vari-
ance indicated a significant increase in lightness
(P = .01) for the inner and outer dentinal areas
during bleaching compared with the control areas.
No significant differences were found in the rate of
change for the inner and outer dentinal areas
(P = .89).
Conclusions. The increase in lightness
confirms that a significant color change occurred in
the dentin during bleaching with 10 percent car-
bamide peroxide. This change occurred throughout
the dentin at a uniform rate, rather than from the
outside inward.
Clinical Implications. The results of
this study show that at-home bleaching with 10 per-
cent carbamide peroxide can change the color of
dentin, which is important to treat intrinsic stains
from tetracycline treatment, trauma and aging or
inherited discolorations. The bleaching material
easily penetrates the tooth to change the dentin color
at the same rate throughout, indicating that the type
of stain may be the important factor in determining
bleaching success.
in the enamel were removed.12 However, case
reports on single dark teeth13-18 and studies on
tetracycline-stained teeth19,20 and dentinogenesis
imperfecta discoloration21 have indicated that color
change also occurs in the dentin. To date, no
studies to our knowledge have specifically evalu-
ated internal dentin and the effect of 10 percent
carbamide peroxide on dentin color. Researchers
do not know whether the decoloration of dentin
occurs uniformly or proceeds from the outside of
the dentin to the inside. (If the process is uniform,
then the difficulty in removing some stains may be
due to the type of stain rather than their location.)
The purpose of this study was to validate the color
change in dentin from bleaching with 10 percent
carbamide peroxide, as well as to determine
JADA, Vol. 130, October 1999 1485
BIOMATERIALS/RESTORATIVE DENTISTRY
Figure 1. The cut side of the sectioned tooth is sealed to the glass with
cyanoacrylate cement and identifying marks are placed.
Figure 2. The root surface is sealed to allow viscous 10 percent car-
bamide peroxide solution (Opalescence, Ultradent Products Inc.) to be
applied to the enamel.
whether the dentin changed
color uniformly or from the
outer dentin to the inner dentin.
METHODS AND
MATERIALS
Specimen preparation. We
used a collection of extracted
adult human anterior teeth in
this study. Before collecting the
specimens, we obtained
approval from the Medical Col-
lege of Georgia Human Assur-
ance Committee. The specimens
were stored in tap water to
1486 JADA, Vol. 130, October 1999
Copyright ©1998-2001 American Dental Association. All rights reserved.
avoid contaminants that might
stain or bleach the tooth struc-
ture. Twelve maxillary anterior
teeth were selected from the col-
lection based on the criterion
that some degree of color varia-
tion was present consistent with
intrinsic or extrinsic staining not
removable with prophylaxis.
Teeth were excluded from the
study if obvious developmental
flaws, caries or restorations were
present.
One of the researchers
(A.J.M.) sectioned the 12 speci-
mens in half longitudinally
using a variable-speed
grinder/polisher (Ecomet 3,
Buehler) and removed the distal
half of the tooth. Each specimen
was positioned with the cut side
down onto a glass microscope
slide, and the periphery of the
specimen was sealed with a
cyanoacrylate adhesive (Zapit,
Dental Ventures of America) to
prevent microleakage at the
interface between the glass and
the tooth (Figure 1). The root
from the apex to the cemento-
enamel junction was covered
with three layers of red finger-
nail polish (Figure 2). Thus,
only the coronal enamel por-
tions of the teeth remained
accessible to the bleaching solu-
tion. On the opposite side of the
glass slide, we used a permanent
ink marker to place two marks
in alignment directly on the
glass over the tooth. These
marks would later be used to aid
in gray-level standardization.
To ensure that the specimen
assembly protocol prevented
any significant microleakage,
we evaluated two of the pre-
pared specimens by submerging
them in fuschin dye for 12
hours. The specimens were then
removed and evaluated visually
for signs of dye penetration. We
found that no dye had pene-
trated the seal. Slight traces of
fuschin dye were present in the
dentin, but on closer inspection,
we determined that dye inva-
sion had occurred through
crazes in the enamel and not at
the interface between the tooth
and the slide. The 10 remaining
specimen assemblies were used
for the experiment.
Image acquisition. Before
applying the bleaching solution,
we photographed the cut sur-
face of each specimen through
the glass slide to establish base-

line color. All images were
obtained under standardized
conditions of lighting, distance
and exposure. To avoid reflec-
tion from the glass slide, a flash
was not used. Color transparen-
cies were made with Kodak
Ektachrome Elite II film (ISO
100/21, Eastman Kodak) and a
35-millimeter Yashica camera
(Dental Eye II SLR, Kyocera
Corp.). We selected all film from
the same lot number for stan-
dardization, and processed all
images at the same time to min-
imize processing variability.
Bleaching material. The
bleaching material used in this
study was 10 percent carbamide
peroxide (Opalescence, Ultra-
dent Products Inc.). The thick,
viscous characteristics of this
material allowed it to be placed
directly on the enamel without
the need for a tray. One of the
researchers (A.J.M.) applied the
material directly onto the
exposed coronal portion of each
tooth specimen. On the first day
of the 10-day experiment, the
glass-covered cut dentin was
photographed at baseline and at
two-hour intervals for the first
eight hours after the bleach had
been applied to establish any
immediate color change in the
dentin. During the next nine
days, the teeth were photo-
graphed each morning, rinsed
with water and stored in the
humidor. Each evening, new
bleaching solution was applied
uniformly to the specimens for
the overnight bleaching. On
day 2, we removed specimen
number 2 from the study be-
cause of inadvertent damage.
Digitization and analysis.
The 35-mm color transparencies
of the specimens were placed on
a flat-bed scanner equipped
with a transparency module
(Agfa StudioScan, Agfa-Gevaert
Copyright ©1998-2001 American Dental Association. All rights reserved.
BIOMATERIALS/RESTORATIVE DENTISTRY
N.V.). We used image pro-
cessing software (Photoshop,
Adobe Systems Inc.) and con-
nected the scanner to a com-
puter with an 8-bit color display
(Macintosh 5400, Apple Com-
puter Corp.). The images were
digitized, with an input resolu-
tion of 350 pixels per inch, as
8-bit images, which converted
the contrast resolution into 256
gray levels.
Radiometric density anal-
ysis. We performed radiometric
density analysis of the dentin
using NIH Image software.22
This software produces a his-
togram (frequency distribution
of gray levels) that allows for
quantitative comparisons
between images. Before doing
so, however, we needed to eval-
uate the images to establish
reproducibility of measure-
ments. Variability introduced
by differences in photographic
(such as lighting and exposure)
and processing techniques must
be accounted for in the type of
digital analysis used in this
study. Therefore, the perma-
nent-ink alignment marks that
had been placed on each slide
were used as control standards.
A 4- × 4-pixel matrix region of
interest, or ROI, was drawn
over the identically placed con-
trol mark on each of the digital
images. Mean gray levels, or
MGLs, and frequency distribu-
tions were recorded for the con-
trol dots on each of the 132 dig-
ital images. These values were
then used in the statistical
analyses.
Identically sized square
ROIs, consisting of an 11- × 11-
pixel matrix, were placed on the
two reproducible areas on each
tooth. The outer ROI was
located within the dentin, just
inside the dentinoenamel junc-
tion, and the inner ROI was
located within the dentin, just
lateral to the pulp chamber
(Figure 3). MGLs and frequency
distributions were determined
for each site.
Hydrogen peroxide. At the
end of the study, we placed the
teeth in a watery 3 percent
H2O2 solution for 12 hours to
test whether they had lightened
to their fullest potential.
Although precise data were not
available because of handling
problems, it was apparent from
visual inspection that the teeth
lightened even further during
this 12-hour period.
STATISTICAL ANALYSIS
We conducted a paired t-test to
examine the mean MGL change
in the control dots from day 1 to
day 10. This t-test was also used
to measure the P value for the
outer and inner dentin. P < .05
was considered significant.
A two-way analysis of vari-
ance was used to test for the
differences in MGL changes
between the outer dentin, inner
dentin and control sites, with
each specimen used as its own
control. A multiple comparison
test was conducted to compare
the control dot with the outer
dentin, the control dot with the
inner dentin and the outer
dentin with the inner dentin.
RESULTS
From day 1 to day 10, the mean
change in MGL of the control
dots was 4.2 gray-scale levels
(of 256 gray-scale levels)
(± standard deviation = 11.5),
which was not statistically sig-
nificant (P = .31). The outer
dentin (that is, the dentin
closest to the enamel) demon-
strated a statistically signifi-
cant mean change in MGL
(P = .0036) from day 1 to day 10
(33.42 gray-scale levels;
JADA, Vol. 130, October 1999 1487
BIOMATERIALS/RESTORATIVE DENTISTRY

DISCUSSION

The main purpose of this study

was to assess the color change
Regions of Interest
for Inner and Outer Dentin
in the dentin over time. The
(11x11 Pixels) data indicate that a color
change occurred throughout the
Control Dots dentin. Some people might
argue that this change is caused
solely by the removal of stains
Region of Interest from the dentin rather than
for Control Dot being a change in the basic
(4x4 Pixels)
color of the dentin, since the
original color of the teeth is
unknown. However, clinical sit-
uations involving single dark
teeth indicate otherwise.
Figure 3. Schematic of cut tooth surface against the glass slide with One of the authors (V.H.) has
regions of interest identified. examined young adults with
very light incisors and premo-
lars but markedly discolored
● Control Dot ◆ Outer Dentin ■ Inner Dentin canines. Bleaching has been
shown to lighten the canines to
Mean Gray-Scale Level

0
more closely match the incisors.
40 Certainly, the canines could not
■ have been the only stained
■ ■
■ ■ ■ teeth, but were an inherently
80 ■
◆ ◆ ◆ ◆
■
■ ■ ■ ■ ◆ ◆ ◆ darker color than the adjacent
■ ■
◆ ◆ ◆ ◆ teeth.
120 ◆ ◆ ◆
Similarly, tetracycline
160 staining is contained in the
● ● ● ● ● dentin. Some researchers and
● ● ● ● ● ● ● ●
●
200 clinicians might argue that
0.2 0.4 0.6 0.8 1 2 3 4 5 6 7 8 9 10 tooth enamel becomes more
opaque with bleaching, thus
Time (Days)
masking the dentin color and
giving the appearance of light-
Figure 4. Mean gray-scale levels for the control dots, inner dentin and ening the tetracycline stain.
outer dentin during the 10-day study period. The data show that the
inner and outer dentin changed color at the same rate. However, clinical experience
has shown that the change in
SD = 24.6). The inner dentin found significant differences in color does not result from opaci-
(that is, the dentin closest to the mean MGL changes between fication of enamel, but reflects a
pulp) also demonstrated a sig- the control dot and the outer true change in dentin color.17,19,20
nificant mean change in MGL of dentin (P = .01) and between Barghi23 reported having pre-
32.02 gray-scale levels the control dot and the inner pared teeth for veneers, which
(SD = 24.8) (P = .005). In a clin- dentin (P = .008). The difference involves removing most or all of
ical setting, these changes in mean MGL changes between the enamel; the teeth then were
would represent an almost 13 the outer and inner dentin was bleached and a visible color
percent improvement in visual not significant (P = .89). Figure change was seen in the dentin.
appearance. 4 shows the color changes in In regard to tetracycline-
These mean changes in inner and outer dentin com- stained teeth, several months of
MGLs were used to conduct the pared with those of the control bleaching may be needed to
multiple comparison test. We dots for each time period. achieve a color change because

1488 JADA, Vol. 130, October 1999

Copyright ©1998-2001 American Dental Association. All rights reserved.

of their resistance to bleaching.
Tetracycline is believed to be
tightly bound in the tooth
matrix, requiring additional
time to decolorize the tooth.
Even non–tetracycline-stained
teeth vary in the amount of
time needed to achieve light-
ening. In a 1989 article, Hay-
wood and Heymann11 recom-
mended two to six weeks of
treatment time to achieve a suc-
cessful outcome.
A second purpose of this
study was to determine if the
dentin changed color uniformly
or from the outside to the
inside. Both the inner and outer
dentin showed a dramatic
reduction in MGL from day 1 to
day 10. This reduction demon-
strates that the dentin does
change color uniformly during
nightguard vital bleaching. The
control dots exhibited a slight
change from day 1 to day 10,
which was not statistically
significant.
The control dot is significant
in correcting for photographic
variations as well as for vali-
dating the color change. (Some
could argue that the teeth them-
selves did not change color, but
that the photographs became
lighter as the experiment con-
tinued.) Our analysis of the con-
trol-dot MGLs showed that the
variance from day 1 to day 10
was not significant and that, in
fact, the teeth were actually
becoming lighter, as indicated
by the dentin values. The mul-
tiple comparison test demon-
strated a significant difference
between the control-dot MGLs
and the outer dentin MGLs and
between the control-dot MGLs
and the inner dentin MGLs. The
inner and outer dentin were also
compared, and the findings
showed that the dentin changed
color in a uniform manner.
Copyright ©1998-2001 American Dental Association. All rights reserved.
BIOMATERIALS/RESTORATIVE DENTISTRY
Bowles and Ugwuneri24 and
Cooper and colleagues25
described how various concen-
trations of hydrogen peroxide
and carbamide peroxide diffuse
readily into the pulp in a 15-
minute period. The diffusion is
a concentration-dependent phe-
nomenon (that is, the higher
the concentration, the faster the
diffusion). One hypothesis for
the uniform color change in
dentin is this rapid ingress of
the bleaching material to the
pulp. The easy penetration of
peroxide to the pulp explains
how the dentin color can be
changed as well as how the side
effect of tooth sensitivity from
pulpal irritation can occur.26
At the end of this study, we
placed the teeth in a 3 percent
H2O2 solution to test whether
they had lightened to their
fullest potential. All of the spec-
imens lightened a considerable
amount in the solution, demon-
strating that 10 days may not
be enough time to completely
bleach all teeth. Some discol-
orations may require several
weeks or even several months
to disappear, as in the case of
some tetracycline stains.27 Since
this study was performed on the
laboratory bench, the effects of
the bleaching material may
have been minimized because of
the absence of saliva and other
organic matter to precipitate
the oxidation reaction.
One strength of this study
design is that it enables the
researcher to observe the
dentin surface directly (through
the microscope slide) without
being concerned about the
optical effects of enamel on the
color. We achieved a good seal
between the tooth specimens
and the microscope slides,
which worked well with the vis-
cous bleaching product. How-
ever, care must be taken in
handling glass slides, as
sample number 2 was dropped
and the tooth separated from
the slide. Also, the watery
hydrogen peroxide solution
used at the end of the study
was not as effective as the vis-
cous bleach, partially because
of a delay in storage time
before testing and handling.
The study was also enhanced
by the use of computer analysis
to measure lightness and color
changes.28,29 This computerized
approach to color measurement
holds great promise over the
use of colorimeters30 and shade
guide measurements31 because
of broad technological advances
and support in the computer
industry. It is one of the
methods advocated by the
American Dental Association
for the measurement of color
changes.32
We do not know whether
color regression in teeth
bleached clinically would occur
in the same manner as color
lightening. Researchers and
clinicians do not know why
some patients’ teeth remain the
same lightened color over
extended periods, while other
patients’ teeth gradually
regress in color. Generally, the
color of teeth subjected to
bleaching is expected to be
stable for one to three years,
with gradual darkening occur-
ring.33 However, Leonard and
colleagues34,35 reported that at
seven years the color of some
teeth remained stable; some
color changes may be perma-
nent.36 We also do not know
whether the color regression is
a result of recolorizing of the
lightened centers or whether
the color regression results from
a combination of new stains and
the aging process. More basic
JADA, Vol. 130, October 1999 1489
BIOMATERIALS/RESTORATIVE DENTISTRY
scientific research is needed to
understand which structures or
processes within the dentin pro-
vide the tooth with its inherent
color, and what is being altered
at a microscopic or chemical
level during bleaching.
CONCLUSION
Our analysis of changes in gray-
scale levels demonstrated that a
color change in the dentin
resulted from nightguard vital
bleaching with a 10 percent car-
bamide peroxide solution. This
color change occurred at a uni-
form rate throughout the
dentin. However, 10 days of
nightguard bleaching may not
be an adequate amount of time
for all teeth to achieve the max-
imum desired results. Finally,
computerized analysis of color
changes shows promise for
future research. !
Dr. Russell is an associate professor, Office
of Biostatistics, School of Dentistry, Medical
College of Georgia, Augusta.
Dr. Haywood has been a paid consultant to
Ultradent Products Inc., Colgate Oral Phar-
maceuticals, Discus Dental, Block Drug Co.,
Marion Laboratories and Procter & Gamble.
He has received grant support from Colgate
Oral Pharmaceuticals, Ultradent Products
Inc., Marion Laboratories and Block Drug
Co. In addition, he has been a paid speaker
for Colgate Oral Pharmaceuticals, Ultradent
Products Inc. and Discus Dental. Dr. Hay-
wood acknowledges Ultradent Products Inc.
for providing the 10 percent carbamide per-
oxide bleach used in this study.
The authors thank Dr. Lewis Hinely Jr.,
medical illustrator, Department of Admis-
sions and Academic Support, Medical College
of Georgia, for his assistance with the com-
puter art and graphs.
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