Odontology (2015) 103:274279

DOI 10.1007/s10266-014-0163-4

ORIGINAL ARTICLE

Effect of various tooth whitening modalities on microhardness,

surface roughness and surface morphology of the enamel
So Ran Kwon Steven R. Kurti Udochukwu Oyoyo
Yiming Li

Received: 15 January 2014 / Accepted: 8 June 2014 / Published online: 28 June 2014
The Society of The Nippon Dental University 2014

Abstract The purpose of this study was to evaluate the

effect of four whitening modalities on surface enamel as
assessed with microhardness tester, profilometer, and scanning electron microscopy (SEM). Whitening was performed
according to manufacturers directions for over-the-counter
(OTC), dentist dispensed for home use (HW) and in-office
(OW) whitening. Do-it-yourself (DIY) whitening consisted
of a strawberry and baking soda mix. Additionally, negative
and positive controls were used. A total of 120 enamel
specimens were used for microhardness testing at baseline
and post-whitening. Following microhardness testing specimens were prepared for SEM observations. A total of 120
enamel specimens were used for surface roughness testing at
baseline and post-whitening (n = 20 per group). Rank-based
Analysis of Covariance was performed to compare microhardness and surface roughness changes. Tests of hypotheses
were two-sided with a = 0.05. There was a significant difference in Knoop hardness changes (DKHN) among the
groups (KruskalWallis test, p \ 0.0001). Significant
hardness reduction was observed in the positive control and
DIY group (p \ 0.0001). Mean surface roughness changes
(DRa) were significantly different among the groups (KruskalWallis test, p \ 0.0001). Surface roughness increased in
the OTC group (p = 0.03) and in the positive control
(p \ 0.0001). The four whitening modalitiesDIY, OTC,
HW and OW induced minimal surface morphology changes
when observed with SEM. It can be concluded that none of

S. R. Kwon (&)
University of Iowa College of Dentistry and Dental Clinics,
Iowa City, IA, USA
e-mail: soran-kwon@uiowa.edu
S. R. Kurti
U. Oyoyo
Y. Li
Loma Linda University, Loma Linda, CA, USA

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the four whitening modalities adversely affected enamel

surface morphology. However, caution should be advised
when using a DIY regimen as it may affect enamel microhardness and an OTC product as it has the potential to increase
surface roughness.
Keywords Tooth whitening
Enamel
Microhardness

Surface roughness
Scanning electron microscope

Introduction
The demand for tooth whitening is increasing and is
reflected by a wide variety of whitening options available.
Current tooth whitening products range from professionally
applied in-office whitening (OW) and professionally
supervised patient applied home whitening (HW) to nonsupervised over-the-counter products (OTC) and do-ityourself (DIY) regimens [1].
Several studies have investigated the effect of different
whitening regimen on tooth whitening efficacy [24].
According to a clinical study that compared three different
whitening techniques with respect to the whitening times
required to achieve a defined level of whiteningthe
cycles required increased from office whitening, home
whitening to OTC products [3]. Whereas a recent in vitro
study showed that the whitening efficacy of Crest Whitestrips, an OTC product was comparable to professionally
dispensed home whitening and professionally applied in
office whitening for up to 3 months post whitening, while a
DIY regimen composed of a strawberry mixture did not
produce any significant color change [1].
The availability of OTC products and various DIY
whitening regimens have provided better access to whitening for the public [5]. However, the ADA Council on

Odontology (2015) 103:274279

Table 1 Active ingredient, pH
and application regimen by
group

CA citric acid, CHNaO3 Sodium

bicarbonate, HP hydrogen
peroxide

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Group

Concentration and pH

Application regimen

NC: water of grade 3

pH 7.0

1 Application (60 min), at 35 C

DIY: strawberry mix

CA, CHNaO3, pH 7.2

3 Applications (5 min each) at 5-day intervals

OTC: crest 3D intensive

9.5 % HP, pH 6.0

1 Daily application (2 h) for 7 days

HW: opalescence PF

10 % CP, pH 7.4

1 Daily application (6 h) for 14 days

OW: zoom whitespeed

25 % HP, pH 7.2

3 Applications (45 min each) at 5-day intervals

PC: citric acid

1.0 % CA, pH 3.9

1 Application (60 min), at 35 C

Scientific Affairs has raised concerns regarding the longterm safety of unsupervised whitening procedures, due to
possible undiagnosed or underlying oral health care problems [69]. There have been numerous studies evaluating
the effect of home whitening and in-office whitening on
surface morphology and microhardness changes [1018].
However, the use of DIY whitening regimens have not
been investigated in terms of effects on microhardness,
surface roughness and surface morphology changes.
The purpose of this study was to evaluate the effect of four
different whitening modalities: in-office whitening, dentist
dispensed home whitening, over-the-counter products and
do-it-yourself whitening on surface enamel as assessed with
microhardness tester, profilometer, and scanning electron
microscopy following guidelines of International Standards
Organization (ISO) 28399 [19]. The null hypotheses tested
were that there would be no differences in the three tested
parameters, microhardness, surface roughness and surface
morphology, after whitening among the test groups.

Materials and methods

Sample selection and preparation
Extracted sound human third molars without identifiers (240)
were collected and stored in 0.1 % buffered Thymol solution
at 4 C. Loma Linda University Institutional Review Board
(IRB) approved the use of the extracted human teeth with no
identifiers as a non-human subjects study. Teeth were
cleaned of gross debris and placed in artificial saliva for 24 h
at 37 C prior to initiating the experiment. The artificial
saliva was prepared according to the modified Fusayama
solution and adjusted to pH 6.0 as described in ANSI/ADA
Specification 41 [20]. Teeth were sectioned and the crowns
embedded in self-curing polyacrylic cylinders (2 cm wide
and 3 cm high) to expose a flat 4 9 6 mm window of enamel
surface for microhardness, surface roughness measurements
and SEM observations. The surface was ground using a
sequence starting at P400 and sequentially increasing to
P1200 silicone carbide paper under a constant flow of water.
A slurry of aluminum oxide with a mean particle size of
0.3 lm was used for the final polishing.

Experimental groups
Table 1 shows the composition, pH, manufacturer, and
regimen of the whitening protocols used in the study.
Group NC was the negative control group, treated with
water of grade 3 for 60 min at 35 C. Group DIY
received a puree of strawberry (15 gm) mixed with
baking soda (2.5 gm) (Arm & Hammer Baking Soda,
Church & Dwight Co., Inc, Princeton, NJ, USA) for
5 min followed by a final brush with a soft toothbrush
(Colgate Oral Pharmaceuticals, Inc., New York, NY,
USA) for 30 s. The procedure was repeated two more
times at 5-day intervals [21]. Group OTC received daily
applications of whitening strips (Crest 3D Intensive,
Crest Pro Health, P&G, OH, USA) for 2 h for 7 days,
according to manufacturers instructions. Group HW was
treated daily with a 10 % carbamide peroxide gel (Opalescence, Ultradent Products Inc, UT, USA) placed in a
custom fabricated tray for 6 h/day for 14 consecutive
days. Group OW represents the professionally applied
group and was treated with 25 % hydrogen peroxide
(Philips Zoom White Speed, Philips Oral Healthcare,
LA, CA, USA) for 45 min using light activation. The
whitening material was replenished every 15 min
according to manufacturers instructions. Group PC, the
positive control group, was treated with 1.0 % citric acid
for 60 min at 35 C. Any remaining material of the
treatment session was rinsed off with distilled water and
specimens stored in artificial saliva at 37 C throughout
the experiment.
Microhardness testing
A total of 120 enamel specimens were used for microhardness testing (n = 20 per group). Measurements were
taken with a microhardness tester (M-400-H1 Hardness
Testing Machine, LECO, Joseph Charter Township, MI,
USA) at baseline and 24 h after the last whitening session
(post-treatment). Specimens were positioned perpendicular
to the long axis of the diamond indentor to record the
Knoop hardness (KHN). A load of 0.49 N was applied for
15 s. Three indentations were obtained from each specimen
and averaged.

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Table 2 Knoop hardness and
surface roughness parameters
(Mean/SD) by group at baseline
and after the whitening
treatment

* p \ 0.05, ** p \ 0.0001

Odontology (2015) 103:274279

Group

KHN

Ra

Baseline

After whitening

Baseline

After whitening

NC

333.13/27.51

334.34/27.26

0.045/0.017

0.046/0.016

DIY

324.45/32.23

275.06**/25.01

0.047/0.014

0.047/0.017

OTC

326.00/31.66

324.28/27.75

0.040/0.015

0.045*/0.018

HW

329.40/30.77

329.36/32.87

0.036/0.012

0.037/0.009

OW

332.80/34.80

330.77/31.03

0.045/0.013

0.048/0.015

PC

333.63/32.32

100.75**/12.33

0.042/0.011

0.076**/0.016

Surface roughness testing

A total of 120 enamel specimens were used for surface
roughness testing (n = 20 per group). A contact profilometer
(Mitutoyo Surftest, SV-2000, Andover, UK) was used to
measure the roughness at baseline and after the last whitening
session (post-treatment). Surface roughness was measured
along the X-axis with a preset evaluation length of 4 mm.
Three scans were conducted from at least 100 z values across
the scan as calculated by software and averaged (Ra).

summarized in Table 2 and illustrated in Figs. 1 and 2.

There was no significant difference of the two measured
parameters (KHN & Ra) among the six different groups at
baseline. There was a significant difference in Knoop
hardness changes (DKHN) among the groups (Kruskal
Wallis test, p \ 0.0001). There was a significant drop in
hardness in the positive control and in the DIY group
(p \ 0.0001) whereas the other groups showed no difference when compared to baseline values. The reduction in
Knoop hardness after treatment was more than 10 % in the

Scanning electron microscopic (SEM) evaluation

Enamel specimens used for the microhardness testing were
removed from the acrylic block and prepared for observation
with the scanning electron microscope (Hitachi High Technologies America, Inc, USA). Specimens were dried with a
series of ethanol solutions and sputter coated with gold in a
vacuum evaporator. Photomicrographs of representative
areas were taken at 2,0009 magnifications. The enamel
changes were classified as no alterations (0), mild or slight
alteration as demonstrated by slight alterations in surface
roughness and irregular patterns of conditioning (1), moderate when distinct etching patterns were observed demonstrating loss of superficial structure (2), and severe surface
alterations including pitting of the enamel surface (3).
Fig. 1 Boxplot of change in Knoop hardness (DKHN) by group

Statistical analysis
Measurements of interest included Knoop hardness and
surface roughness values. Rank-based Analysis of
Covariance (ANCOVA) and the KruskalWallis procedure
were performed to compare microhardness and surface
roughness changes among the different treatment groups.
Tests of hypotheses were two-sided with an alpha level of
0.05. Analysis was conducted with SAS v 9.2 (SAS Institute, Cary, NC, USA).

Results
Mean Knoop hardness (KHN) and surface roughness (Ra)
by group at baseline and after whitening treatment are

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Fig. 2 Boxplot of change in surface roughness (DRa) by group

Odontology (2015) 103:274279

277

Fig. 3 Surface morphology as observed by scanning electron

microscopy

Fig. 5 Representative SEM image exhibiting mild surface alterations

Fig. 4 Representative SEM image exhibiting no surface alterations

DIY group, thus failing to meet the requirements of ISO

standard 28399.
Mean surface roughness changes (DRa) were significantly different among the groups (KruskalWallis test,
p \ 0.0001). Mean surface roughness significantly
increased in the OTC group (p = 0.03) and in the positive
control (p \ 0.0001). However, it is noteworthy to point
out that surface roughness changes in group OTC did not
exceed three times the level of the positive control thus
meeting the requirements based on ISO 28399. All other
groups showed no significant change in surface roughness
when compared to baseline values.
Enamel surface morphology changes as observed by
scanning electron microscopy are summarized in Fig. 3.
No changes imply a surface that shows the scratching
pattern associated with the polishing of surface enamel
with polishing discs (Fig. 4). A few mild changes were
seen in most groups exhibiting mild interprismatic dissolution on the surface (Fig. 5). The positive control

Fig. 6 Representative SEM image exhibiting moderate surface

alterations

exhibited mostly moderate changes in the surface morphology as observed by the presence of distinct type I and
II etching patterns (Fig. 6). Severe surface alterations that
are usually accompanied by pitting of the enamel surface
could not be observed in this study.

Discussion
The tooth surface interface exhibits a continuous dynamic
ion exchange between the oral biofilm and the apatite
crystals in both directions to maintain a proper mineral
balance [22]. This physiologic process in the oral cavity

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makes it difficult to interpret the clinical relevance of

laboratory measurements of changes in enamel hardness,
roughness, and morphology. Thus within the limitations
of this in vitro study the first null hypothesis was
rejected. There was a significant difference in Knoop
hardness changes among the four different whitening
modalities with DIY exhibiting a decrease of more than
10 %. Based on the results the second null hypothesis
was also rejected. Surface roughness significantly
increased in the OTC group. However, the increase did
not exceed three times the level of the positive control.
The third null hypothesis was retained. All whitening
modalities exhibited none or only a mild change in surface morphology as observed by scanning electron
microscopy.
Surface hardness measurement is a relatively simple
method to determine the mechanical property of enamel
and dentin to resist plastic deformation from a standard
source, and is closely related to a loss or gain of mineral
component [16]. However, despite its simplicity there is a
great inconsistency in the outcome of studies evaluating the
effect of tooth whitening on micro hardness changes. A
review on studies applying microhardness testing for
structural enamel defects after whitening showed that those
studies, which simulated intraoral conditions closely by
using human saliva and fluoride, and evaluation after a
post-treatment phase, the risk of enamel microhardness
drop seemed to be reduced as compared to the remaining
studies [16]. Our study is in agreement with a study that
compared the effect of strip, tray, and office bleaching
systems on enamel hardness and showed that bleaching did
not affect hardness values in vitro [23]. This may be
attributed to the fact that artificial saliva was used
throughout the study and a post-treatment phase of 24 h
was allowed following the protocol of ISO standards. It is
interesting to note the pH change throughout the preparation of the strawberry mixture in the DIY group. The fresh
strawberry mixture when measured initially exhibited a pH
of 3.4 and changed to neutral by adding baking soda into
the mixture. The drop in Knoop hardness that was seen
only in the DIY group may be explained by the fact that
there were still small chunks of strawberry present in the
mixture that contributed to a low pH in localized area and
caused a drop in Knoop hardness.
Profilometry is a widely used methodology that provides quantitative data on surface roughness and profile
changes. The inconsistency reported in microhardness
studies is unfortunately also seen in the outcome of
studies evaluating the effect of tooth whitening on surface
roughness. While several studies showed an increase in
surface roughness after bleaching [2426] others reported
no effect of whitening on surface roughness [5, 23, 27,
28]. The erosive potential of acids is influenced by

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Odontology (2015) 103:274279

numerous factors such as pH, titratable acidity, degree of

saturation, kind of acid, chelating properties, and viscosity [29]. Among these variables pH is an important factor
in the degree of tissue loss by erosion [30]. This is also
reflected by our study that showed a slight increase in
surface roughness in the OTC group, which had a measured pH of 6.0. However, the clinical relevance of this
increase needs to be determined since the increase in
roughness did not exceed three times the level of the
positive control.
Scanning electron microscopy has been widely used in
analyzing the surface morphology of enamel and dentin
following tooth whitening. Depending on the study design,
type and concentration of peroxide compound, pH and
exposure time, different results have been reported using
SEM ranging from no changes in surface morphology [14,
15], mild surface pitting at localized areas and some
enamel porosity to significant surface alterations [10, 11].
A recent study comparing neutral and acidic hydrogen
peroxide (HP) on the surface morphology showed that
neutral HP did not affect the surface morphology whereas
acidic HP resulted in significant enamel surface changes
[17]. Thus it has been suggested that studies which use
whitening agents with relatively low pH are probably
describing primarily demineralization effects caused by
acidic erosion processes rather than adverse effects by
peroxide per se [16]. Based on our SEM results all whitening modalities did not adversely affect the enamel surface that may be attributed to the neutral pH of whitening
agents employed. It was interesting to observe that Group
DIY that exhibited a significant decrease in Knoop hardness did not alter the surface morphology. Decrease in
Knoop hardness reflects loss of mineral in the enamel but
may have been not enough to induce visible changes in
surface morphology.
This is the first study that evaluated the effect of four
different whitening modalities on surface enamel. Within
the limitations of this study it can be concluded that,
1.

2.

None of the four whitening modalities induced adverse

surface morphology changes when observed with SEM
and can be considered safe
However, caution should be advised to the general
public when using a DIY regimen made of strawberry
mixture as it may adversely affect enamel microhardness and an OTC product as it has the potential to
increase surface roughness

Acknowledgments The authors would like to thank Ultradent

Products Inc and Philips Oral Healthcare for kindly providing the
bleaching materials used in this study.
Conflict of interest
of interest.

The authors declare that they have no conflict

Odontology (2015) 103:274279

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