![Comparisons between the PCR [19] and the present methodologies for the brightness changes between pre- and post-brushing after brushing with the toothpastes slurries, n = 8, mean value](https://www.researchgate.net/profile/Changxiang-Wang-3/publication/312178811/figure/fig3/AS:962818761297948@1606565289363/Comparisons-between-the-PCR-19-and-the-present-methodologies-for-the-brightness-changes_Q320.jpg)
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R E S E A R C H A R T I C L E Open Access
An in vitro screening assay for dental stain
cleaning
Changxiang Wang
1*
, Robert Lucas
2
, Anthony J. Smith
1
and Paul R. Cooper
1
Abstract
Background: The present study aimed to develop an in vitro model for stain removal from natural enamel for the
assessment and comparison of oral hygiene products.
Methods: Bovine teeth (n= 8 per group) were ground/polished to provide flat enamel specimens and ferric-
tannate deposits were precipitated onto the enamel surfaces. The ferric-tannate stained enamel specimens were
brushed using an in vitro tooth-brushing simulator with slurries containing commercially available toothpaste
products, dental abrasive particles, and sodium tripolyphosphate (STP) solutions of different concentrations. The
colour of the enamel surfaces was measured using a spectrophotometer before and after stain application as well
as after the brushing treatments.
Results: Differences in stain removal efficacy were found between the toothpastes categorised as whitening and
non-whitening comprising of different types of dental abrasives (hydrated silica and alumina). A mean value of 27%
for stain removal was detected for the three non-whitening toothpastes and 59% of stain removal was detected for
the three whitening toothpastes after 1000 strokes. Compared with the slurry with Zeodent 113 abrasive alone, the
addition of STP provided better performance for stain removal under the same brushing conditions (mean value of
62% for Zeodent 113 abrasive alone and 72% with the addition of 5% (w/w) STP after 1000 strokes). No difference
was evident between the STP concentration of 5% (w/w) and 10% (w/w).
Conclusions: The ferric-tannate/bovine enamel model reported here provides good stain retention, is rapidly and
easily prepared, and is shown to be progressively and reproducibly sensitive to toothbrushing using different
toothpastes and surfactant/chelating agent solutions. Importantly, it provides good discrimination between various
oral hygiene products.
The stain removal assay reported here has considerable potential to enable comparative assessments of different
toothpaste types in terms of their cleaning capabilities.
Keywords: Enamel, Stain removal efficacy, Tooth whitening, Aesthetics, Toothpaste, Tooth colour, Surface roughness
Background
The natural colour of permanent teeth is largely deter-
mined by dentine and modified by the thickness and
translucency of the overlying enamel [1]. The appearance
of the teeth, particularly whiteness, is aesthetically import-
ant to individuals and tooth discolouration is a common
dental patient complaint. Personal dissatisfaction with the
appearance of the dentition has been reported to range
from 17.9 to 52.6% [2–6] and the causes of tooth discol-
ouration are multifactorial and are classified as extrinsic,
intrinsic and internalised discolouration [7]. Intrinsic and
internalised discolouration arise generally during tooth de-
velopment or during disease and the more extensive local-
isation deep within the dental tissues constrains its
reversal. Extrinsic staining of the tooth can arise from a
variety of sources, such as smoking, red wine consump-
tion, and the intake of cationic compounds, such as chlor-
hexidine or stannous salts [7–10]. Stain removal can be
challenging as staining compounds can be bound to den-
tal plaque and the acquired pellicle, as well as directly to
the enamel surface [11]. Thus, oral hygiene strategies need
to address both the removal of dental plaque and the ex-
trinsic stain bound in these different ways.
* Correspondence: c.wang@bham.ac.uk
1
Oral Biology, School of Dentistry, University of Birmingham, 5 Mill Pool Way,
Edgbaston, Birmingham B5 7EG, UK
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wang et al. BMC Oral Health (2017) 17:37
DOI 10.1186/s12903-016-0328-3
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
While a variety of stain removal and tooth whitening
procedures are used professionally in the clinic [12–15],
they are relatively costly and labour intensive [16]. Further-
more, there is considerable demand for ‘over-the-counter’
(OTC) tooth-whitening products that can be integrated
into a normal oral hygiene regime, and whitening tooth-
pastes are commonly used for this [17]. In general, tooth-
whitening toothpastes function by abrasive removal of ex-
trinsic stain associated with the dental plaque, the acquired
pellicle and the enamel surface together with the chemical
cleaning action of constituents, such as sodium tripolypho-
sphate (STP), sodium pyrophosphate, sodium hexameta-
phosphate, hydrogen peroxide, as well as by the activity of
the enzymes papain and bromelain [4].
Although randomised controlled trials (RCTs) can pro-
vide a high degree of evidence for cleaning efficacy, de-
velopment of a robust laboratory model to evaluate the
mechanisms of action and screen efficacy of whitening
products or bleaching agents would be of significant
benefit [18]. In vitro models allow rapid screening of a
range of potential products; the most effective can sub-
sequently be clinically assessed in RCTs. Various ap-
proaches have been used to investigate extrinsic stain
removal by oral care products using a wide range of sub-
strates such as polymethylmethacrylate, hydroxyapatite
and enamel (human or bovine). Model stains, including
tea, coffee, gastric mucin, soy broth, bacteria, chlorhexi-
dine, saliva, tea with orange II Rhodamine, blood, cola,
wine, tobacco and ferric-tannate, have all been used
[19–24]. One of the most commonly applied in vitro ap-
proaches uses cut, polished and acid etched bovine en-
amel specimens stained with a solution containing a
combination of coffee/tea/gastric mucin/Sarcina lutea
turtox [19]. The stained specimens are then repeatedly
brushed for a period of time using an automated ap-
proach with toothpaste slurries. Furthermore, a recent
model has utilised a ferric-tannate coating deposited
onto highly polished sintered hydroxyapatite discs and
was reported to mimic the daily control of pellicle
growth, maturation and staining [24].
A significant limitation of current protocols is the
weak bonding between the stain and substrate constrain-
ing discrimination between different cleaning products
and regimes. A further issue is the need for a staining
time of several days necessary to achieve sufficient stain
build-up for high-throughput analysis and discrimin-
ation of cleaning regimes. There is also a need to better
model stain interactions with natural enamel to provide
good clinical relevance. Subsequently, the aim of this
study was to establish a relatively rapid and reproducible
staining protocol with appropriate bonding to enamel,
which would allow high-throughput assessment of the
cleaning ability, and discrimination between oral care
products, following brushing.
Methods
Specimen preparation
Previously, a model has been reported to simulate up-
to-24 h pellicle formation generated by precipitating an
iron (III) complex with tannic acid from aqueous solu-
tion directly onto polished hydroxyapatite discs [24]. In
the present study, this model was modified to investigate
the use of natural enamel specimens and to study the in-
fluence of specimen surface finish, which can influence
stain retention.
Bovine teeth were collected and stored in 0.1% (w/w)
thymol (Sigma-Aldrich, UK) solution at 4 °C prior to
use. Bovine enamel specimens (approximate 18 mm ×
12 mm) were prepared from tooth crowns by dissection
using a diamond-edged saw and embedded in blocks of
epoxy resin (Ø25 mm) (Buehler, UK). To investigate the
effects of surface roughness on the retention and re-
moval of the stain, eight bovine enamel specimens per
treatment group were prepared to either: a) 600-grit SiC
and 3 μm diamond finish (Polished surface group), b)
400-grit SiC ground finish (Partially roughened surface
group), or c) 280-grit SiC ground finish (Roughened sur-
face group) with 5 min ultrasonication in water follow-
ing each treatment for removal of any residual grinding/
polishing materials. A Phoenix Beta Grinder/Polisher
(Buehler, UK) was used with SiC abrasive discs (Buehler,
UK) for sample preparation. Samples were then surface
profiled using a Talysurf Series 2 inductive gauge profil-
ometer (Taylor-Hobson, UK), which has a 1 mm range
in the z-axis and a resolution of 16 nm. The inductive
gauge profilometer uses a conical probe with 2 μm dia-
mond tip to accurately measure surfaces at the sub-
micron level. Linear line profiles (2D) were measured on
the surfaces at a measurement speed of 0.5 mm/s and
with points spacing of 0.25 μm, and the arithmetic mean
surface roughness (Ra) values were calculated (μltra ver-
sion 5.1.14, Taylor-Hobson, UK) prior to staining.
Tooth stain development
Freshly combined solutions (0.1% (w/w) of diammonium
iron (II) sulphate 6-hydrate (Sigma-Aldrich) and 0.1%
(w/w) tannic acid (ACS reagent, Sigma-Aldrich) are ini-
tially colourless, but form a dark colloidal iron (III) tan-
nic acid complex (“ferric-tannate”) on contact with air,
which resembles a dietary tannin staining. The fresh
mixture was applied as successive layers on the enamel
specimens, with each layer being dried at 40 °C in an
oven (D-63450 Hanau, Heraeus Instrument (now Ken-
dro Laboratory Products Ltd), Germany) for 10 mins be-
fore application of the subsequent layer. For the first
layer, a 40 μl aliquot of the solution was pipetted onto
each specimen and allowed to spread evenly over the
specimen surface before drying. For the subsequent
layers (up to a maximum of 9), 10 μl aliquots of the
Wang et al. BMC Oral Health (2017) 17:37 Page 2 of 10
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solutions were applied before drying. A total of 1, 3, 5, 7
and 10 cycles of successive stain deposition layering were
investigated to study in vitro stain removal efficacy. To
mimic the presence of an acquired pellicle layer, one of
four different acidic polymers of Carbopol (Lubrizol Ad-
vanced Materials, Inc., USA), Carrageenan (Cargill, Incor-
porated, USA), Gantrez (Ashland, USA) and Xanthan
(Cargill, Incorporated, USA) was incorporated and applied
to assess stain accumulation and removal. Polymers were
incorporated by either: a) applying a 40 μl aliquot of 0.1%
(w/w) solution of the polymer followed by application of
the ferric-tannate stain, or b) by reducing the amount of
tannic acid in the stain by 50% and replacing it with an
equivalent amount of one of the four acidic polymers as
described above for the application of the stain.
Stain removal
The stained enamel specimens were mounted in two
brushing channels of an in vitro brushing simulator, as
previously reported [25]. Oral B P35 medium tooth-
brushes were used for the brushing. Specimens were
double brushed at a brushing speed of 120 rpm and a
temperature of 20 °C was maintained throughout the en-
tire brushing procedure. 150 g toothpaste/abrasive slurry
was used in each channel and a brushing load of 150 g
was applied. Specimens were double brushed sequentially
for up to 5000 strokes. After brushing for the requisite
number of strokes, specimens were thoroughly rinsed
with water prior to colour evaluation (as described below).
The abrasive slurries examined in the present study in-
cluded a) a range of toothpaste slurries (see Table 1 for a
list of products used) prepared with 25 g toothpaste in
40 ml water, b) 15% (w/w) of Zeodent 113 (Huber Corpor-
ation, USA) silica abrasive in 0.5% (w/w) Hercules 7 MF
Carboxymethyl Cellulose (CMC) (Hercules Incorporated,
USA) containing 10% (w/w) Glycerol (VWR International
BVBA, Belgium), with or without the addition of 5% (w/
w) and 10% (w/w) sodium tripolyphosphate (STP) (Sigma-
Aldrich), c) 10% (w/w) aqueous STP, and d) water as a
control. Zeodent 113 is a precipitated silica, which has
commonly been employed as a toothpaste abrasive [25],
with a 15 μm mean particle size (Mastersizer 2000, Mal-
vern Instruments Ltd, United Kingdom).
The cleaning ability of eight dentifrices to remove
stain pellicle was determined in parallel by a laboratory
testing method (PCR) commonly used within the oral
hygiene industry [19]. The results were compared with
the stain removal of the same dentifrices using the
current stain removal methodology.
Images of the enamel surfaces before staining and
post-stain removal with 5000 brush strokes were also
captured by using a camera (Nikon D7000, Nikon Cor-
poration) to demonstrate stain removal effects.
Colour evaluation
The colour of each tooth specimen (L*, a*, b*) was mea-
sured using a calibrated spectrophotometer (Minolta
CM-2600d) before staining (=initial), after up to 10 cycles
of stain application and after the brushing treatments.
All surfaces were consistently dried by carefully wiping
with a soft tissue prior to colour measurements. The L*
value represents the value of ‘brightness/darkness’of a
colour, such that a perfect black body has an L* value of
zero and the perfect reflecting diffuser has an L* values
of 100. The a* and b* represent two colour axes, with a*
the red-green axis and b* the yellow-blue axis [25].
Removal of the stain was assessed using the following
equation:
%Removal ¼LBrushedðÞ−LStainedðÞ
LInitialðÞ−LStainedðÞ
100
Where L* (Initial) is the brightness before staining; L*
(Stained) is the brightness after 10 cycles of stain
Table 1 Toothpastes studied
Group Toothpaste Type/relevant ingredients Manufacturer
A Aquafresh Multi-Action
Whitening
Whitening/hydrated silica, pentasodium triphosphate GlaxoSmithKline Consumer Healthcare,
Brentford, UK
B Arm and Hammer Advanced
Whitening
Whitening/hydrated silica, sodium bicarbonate Church & Dwight UK Ltd., Kent, UK
C Colgate Cavity Protection Non-whitening/dicalcium phosphate dihydrate,
tetrasodium pyrophosphate
Colgate-Palmolive, Guildford, UK
D Colgate MaxWhite Whitening/hydrated silica, white micro crystals,
tetrasodium pyrophosphate
Colgate-Palmolive, Guildford, UK
E Crest Cavity Protection Non-whitening/hydrated silica, trisodium phosphate Procter & Gamble UK, Weybridge, UK
F Crest Whitening Expressions Whitening/hydrated silica Procter & Gamble, Cincinnati, USA
G Pearl Drops Daily Whitening
Toothpolish
Whitening/hydrated silica, alumina, tetrapotassium
pyrophosphate
Church & Dwight UK Ltd., Kent, UK
H Signal White Now Whitening/hydrated silica, trisodium phosphate Unilever Deutschland, Hamburg, Germany
Wang et al. BMC Oral Health (2017) 17:37 Page 3 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
application and L* (Brushed) is the brightness after
toothbrushing for the requisite number of strokes with
water or toothpaste/abrasive slurry.
Pellicle cleaning ratio (PCR)
The pellicle cleaning ratio (PCR) for the same batch of
toothpastes was undertaken and the details of the meth-
odology have been previously reported elsewhere by
Stookey et al. [19]. The PCR data were compared with
those from the present methodology.
Statistical analyses of the data
Data were analysed by single factor ANOVA with a sig-
nificance level of p≤0.05 applied. Pearson correlation
coefficient was used to determine the linear relationship
between two variables, and is denoted by r. Given a
value between +1 and −1 inclusive, where one is perfect
positive correlation, 0 is no correlation, and −1 is perfect
negative correlation.
Results
Effects of enamel surface finish on stain removal
Enamel specimens of mean surface roughness values of
approximately 0.4 μm (280-grit ground), 0.15 μm (400-
grit ground) and 0.02 μm (diamond polished) were com-
pared with respect to their susceptibility to stain removal
after brushing with either water or commercial tooth-
pastes (Fig. 1). Brushing with water resulted in minimal
stain removal even after 5000 brush strokes with values
of less than 1%, 5% and 10% for the 280-grit ground,
400-grit ground and diamond polished enamel surfaces,
respectively. Stain removal was considerably enhanced
after brushing with the commercial toothpastes with an
inverse relationship between stain removal and specimen
surface roughness. Statistically significant differences (p
< 0.05) were found in the in vitro stain removal efficacy
for the majority of data between 280-grit ground and
400-grit ground, and 280-grit ground and polished en-
amel specimens. However no significant differences (p>
0.05) were found in the in vitro stain removal efficacy
between 400-grit ground and polished enamel specimens
except for brushing with Colgate MaxWhite toothpaste.
The numbers of brush strokes required to remove virtu-
ally all of the stain were 3000 or more for the 280-grit
ground enamel specimens, 1000 or fewer for the 400-
grit ground enamel specimens and 500 strokes or fewer
for the diamond polished enamel specimens, respect-
ively. There was a trend for greater stain removal with
the whitening toothpastes using the same number of
brush strokes. Due to the superior stain retention and
greater ability to discriminate between individual com-
mercial toothpastes, the 280-grit ground finish was used
for all subsequent studies.
The brightness differences for the 280-grit ground bo-
vine enamel surfaces between the pre- and post-
brushing are presented in Fig. 2. Due to the number of
brush strokes (800 for PCR methodology [19], 500 and
1000 strokes for present methodology), an average differ-
ence for the 500 and 1000 brush stroke procedures (de-
noted as 750 strokes in Fig. 2) was used for the
comparisons between the two methods. No statistically
significant differences were detected between the two
test methods when brushed with Arm & Hammer Ad-
vanced Whitening, Crest Whitening Expressions,
Colgate MaxWhite, Pearl Drops Daily Whitening Tooth-
polish, and Signal White Now toothpastes. Statistically
significant differences were however identified when
brushed with Crest Cavity, Colgate Cavity, and Aqua-
fresh Multi-Action Whitening, with more stain being re-
moved by using the PCR method, indicating that better
retention of stain was achieved using the present meth-
odology. Pearson correlation coefficient result (r= 0.82)
showed a strong positive linear relationship between
these two methods.
Figure 3 shows the images of enamel surfaces before
staining and post-stain removal with 5000 brush strokes.
Upon inspection it is apparent that relatively little stain
remained on the roughened enamel surface group after
brushing with the three non-whitening toothpastes, and
no stain remained after brushing with the three whiten-
ing toothpastes. Relatively little stain remained on the
partially roughened enamel surface group only after
brushing with the Colgate Cavity Protection toothpaste,
but no stain was apparent after brushing with the other
tested toothpastes. No stain was apparent on the
polished enamel surface group after brushing with all
the tested toothpastes.
Effects of successive layers of stain deposition
There was a linear decrease in surface brightness of
specimens with deposition of up to 10 successive layers
of stain (r= 0.98). The effects of the number of layers of
stain deposition on stain removal after brushing were
assessed by brushing with a typical toothpaste precipi-
tated silica abrasive slurry (15% w/w Zeodent 113)
(Fig. 4). Significant differences (p< 0.05) in stain removal
efficacy were detected between the first and successive
cycles of stain deposition after more than 100 brushing
strokes, no significant differences (p> 0.05) were de-
tected between the staining cycles of 3, 5, 7 and 10.
However, inter-sample variation tended to decrease with
increasing number of layers of stain deposition.
Modelling the presence of acquired pellicle by application
of acidic polymers
The application of an acquired pellicle prior to stain de-
position (10 layers) was modelled on 280-grit ground
Wang et al. BMC Oral Health (2017) 17:37 Page 4 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
finish enamel specimens using a variety of acidic poly-
mers. Their effects on stain removal efficacy were subse-
quently assessed after brushing with a precipitated silica
(typically present in commercial toothpastes) abrasive
slurry (15% (w/w) Zeodent 113) (Fig. 5a). Significant dif-
ferences (p< 0.05) in stain removal efficacy were ob-
served between the polymers applied with a ranking
order of stain retention of Carbopol and Gantrez as the
best, then no polymer incorporation followed by Carra-
geenan and Xanthan being the poorest for stain reten-
tion. Treatment with the polymers Gantrez and
Carbopol prior to stain deposition resulted in small de-
creases in stain removal efficacy after brushing, while
Carrageenan and Xanthan negatively impacted on stain
retention with greater stain removal observed. Applica-
tion of the polymer by replacing 50% of the Tannic acid
-20
0
20
40
60
80
100
120
140
0 2000 4000 6000
%,ycaciffelavomerniatS
Brushing strokes
Tap water
Colgate Cavity Prote ction
Crest Decay Prevent ion
Aquafresh Fresh & Minty
Colgate MaxWhite
Aquafresh Multi-Action
Whitening
Pearl Drops Daily Whitening
Toothpolish
(a)
-20
0
20
40
60
80
100
120
140
160
0 2000 4000 6000
%,ycaciffelavomernitaS
Brushing strokes
Tap water
Colgate Cavity Protection
Crest Decay Prevention
Aquafresh Fresh & Minty
Colgate MaxWhite
Aquafresh Multi-Action
Whitening
Pearl Drops Daily Whitening
Toothpolish
(b)
0
20
40
60
80
100
120
140
0200040006000
%,ycaciffelavomerniatS
Brushing strokes
Tap water
Colgate Cavity Protection
Crest Decay Prevent ion
Aquafresh Fresh & Minty
Colgate MaxWhite
Aquafresh Multi-Action
Whitening
Pearl Drops Daily Whitening
Toothpolish
(c)
Fig. 1 Effects of initial surface roughness of bovine enamel specimen surfaces on the stain removal efficacy after brushing with the tested
toothpaste slurries and water for up to 5000 brush strokes, n= 8, mean ± standard deviation. aRoughened enamel surface group; bPartially
roughened enamel surface group; cPolished enamel surface group
Wang et al. BMC Oral Health (2017) 17:37 Page 5 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
with the four acidic polymers during stain deposition
generally decreased stain retention following brushing
compared with the control (Fig. 5b). There were signifi-
cant differences in stain removal efficacy between the
presence of the four polymers versus no polymer inclu-
sion (100% tannic acid) in the stain (p< 0.05). Following
brushing with 15% (w/w) Zeodent 113, the rank order of
stain retention was Gantrez and no polymer as the best,
then Carbopol and Carrageenan and Xanthan represent-
ing the worst. Only Gantrez increased stain retention
after brushing for 100, 500 or 1000 strokes, although
such differences were not apparent at 3000 brush
strokes. The application of the other polymers provided
lower or equal levels of stain retention compared with
the control after the various numbers of brush strokes.
STP effect on stain removal
STP is commonly used in oral hygiene products to facili-
tate stain removal [2]. Brushing of stained enamel speci-
mens with STP both in the absence and presence of
silica abrasive (15% (w/w) Zeodent 113) enhanced stain
removal (Fig. 6). Brushing with 10% (w/w) STP alone re-
moved approximately 35% of the stain after 5000 strokes
and addition of Zeodent 113 abrasive further increased
stain removal.
Statistically significant differences in stain removal effi-
cacy were detected between the stained samples brushed
with water (Fig. 1a) and 10% (w/w) STP. There were sig-
nificant differences (p< 0.05) in stain removal when the
stained enamel specimens were brushed with the three
slurries comprising 15% (w/w) Zeodent 113 abrasive parti-
cles (+/−STP), while no significant differences were ob-
served between the slurries comprising either 15% (w/w)
Zeodent 113 abrasive plus 5% (w/w) or 10% (w/w) STP
(p> 0.05). The three slurries with 15% (w/w) Zeodent 113
abrasive particles (+/−STP) exhibited the most effective
stain removal as measured by brush strokes and linear re-
sponses for stain removal were observed during the first
500–1500 brush strokes (r> 0.98). The 10% (w/w) STP
solution alone (without abrasive particles) showed a rea-
sonably linear response for stain removal throughout the
entire range of 500–5000 brush strokes (r=0.97).
Discussion
The in vitro stain methodology developed in this study
provides a relatively inexpensive, easily performed, rapid
and reproducible staining protocol which allows examin-
ation of stain bound to a natural enamel substrate. Of
the many laboratory methods used to evaluate the clean-
ing potential of toothpaste and toothpaste ingredients,
that developed by Stookey et al. [19] (PCR methodology)
Fig. 3 Images of enamel surfaces before staining, after staining and
post-stain removal with 5000 brush strokes. aRoughened enamel
surface group; bPartially roughened enamel surface group; cPolished
enamel surface group. Upper row (left to right): before stain; stain
brushing with water; stain brushing with Colgate cavity Protection
toothpaste; stain brushing with Crest Decay Prevention toothpaste;
Lower row (left to right): stain brushing with Colgate MaxWhite
toothpaste; stain brushing with Aquafresh Multi-Action Whitening
toothpaste; stain brushing with Aquafresh Fresh & Minty toothpaste;
stain brushing with Pearl Drops Daily Whitening Toothpolish toothpaste
0
10
20
30
40
15 20 25 30 35
New method, 750 strokes
PCR, 800 strokes
Brightness Differences ( L*)between Pre-and
Post-brushing
Fig. 2 Comparisons between the PCR [19] and the present
methodologies for the brightness changes between pre- and post-
brushing after brushing with the toothpastes slurries, n=8, meanvalue
Wang et al. BMC Oral Health (2017) 17:37 Page 6 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
is commonly used by the oral care industry. However it
requires a time consuming stain deposition protocol
with opportunities for reproducibility issues when used
at different test sites. The data presented here demon-
strate good agreement between our stain methodology
and the PCR methodology in terms of brightness
changes after brushing using the same batches of tooth-
pastes. The current study has also demonstrated the re-
tention of the stain after toothbrushing in the presence
of either standard toothpastes or whitening toothpastes
0
20
40
60
80
100
120
140
160
180
100 500 1000 3000
Stain removal efficacy, %
Brushing strokes
Stain Removal for Roughened Enamel Surface Group
1
3
5
7
10
Fig. 4 Stain removal efficacy for 280-grit ground enamel specimens with varying numbers of stain layers after brushing with 15% w/w Zeodent
113 precipitated silica abrasive slurry for up to 3000 brush strokes, n= 8, mean ± standard deviation
0
20
40
60
80
100
120
140
100 500 1000 3000
Stain removal efficacy, %
Brushing strokes
Stain Removal for Roughened Enamel Surface Group
Carbopol
Carrageenan
Gantrez
No polymer
Xanthan
(a)
0
20
40
60
80
100
120
140
100 500 1000 3000
Stain removal efficacy, %
Brushing strokes
Stain Removal for Roughened Enamel Surface Group
Carbopol
Carrageenan
Gantrez
Tannic acid
Xanthan
(b)
Fig. 5 Modelling the application of an acquired pellicle prior to stain deposition on 280-grit ground finish enamel specimens using a variety of
acidic polymers, n= 8, mean ± standard deviation. aby application of one layer of acidic polymers followed with 10 layers of tannate stain; bby
replacing half of the tannic acid stain component with acidic polymers for the precipitation of 10 layers of stain
Wang et al. BMC Oral Health (2017) 17:37 Page 7 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
as well as the most common chemically active com-
pound in whitening toothpastes, STP. Furthermore it
demonstrates the ability of the methodology to discrim-
inate between these various oral hygiene products.
Model stains have been previously described [19–23] for
simulation of the extrinsic staining of the teeth. However,
one of the main disadvantages of these staining ap-
proaches has been that the stain properties could not be
directly compared due to the differences in materials used,
i.e., different brand/supplier of reagents used in different
studies. However this ferric-tannate stain now provides a
reliable standardised model for stain removal purposes in
which the stain properties can be controlled due to the
chemically defined nature of the stain deposits.
In the previously reported PCR methodology [19], acid
etching of polished enamel surfaces has been used to ex-
pedite stain accumulation and adherence, although the
reproducibility of the etching process can be difficult to
accurately reproduce. In the present study, stain could
be most readily removed from the diamond polished bo-
vine enamel surfaces, followed by the 400-grit ground
enamel bovine surfaces while the stain on the 280-grit
ground bovine enamel surfaces was retained to the
greatest extent. The surface roughness of enamel speci-
mens can be reproducibly achieved with grinding/polish-
ing protocols contributing to the reproducibility of the
presently reported methodology. The surface roughness
of the tooth is also of clinical importance, particularly in
respect to bacterial retention. A threshold surface rough-
ness for bacterial retention of 0.2 μm has been reported
[26]. Indeed, the mean surface roughness values for 400-
grit ground bovine enamel were approximately 0.15 μm,
which is below the threshold roughness of 0.2 μm and
the stain was more readily removed from the 400-grit
ground surfaces than from the 280-grit ground bovine
enamel surfaces, the latter of which had a mean surface
roughness of approximately 0.4 μm. The polished bovine
enamel surfaces had mean surface roughness values of
approximately 0.02 μm, which is ten-fold less than the
threshold roughness for bacterial retention and the stain
on these polished surfaces was removed much more
readily than that from the 400-grit ground and 280-grit
ground bovine enamel surfaces.
The acquired enamel pellicle (AEP) which occurs in vivo
results from rapid binding of salivary constituents after
saliva contacts a newly exposed enamel surface and re-
portedly contributes to enamel protection [11]. It is gener-
ally understood that the AEP reduces friction between
teeth and between teeth and the oral mucosa. It has also
been reported that the pellicle enables the initial attach-
ment of bacteria to the tooth surface, which is the first
step in plaque formation [11]. In the present study, four
acidic polymers were used to mimic an AEP layer on the
280-grit ground bovine enamel surfaces, by either direct
application of a thin layer of the acidic polymer prior to
application of the ferric-tannate stain or by partly re-
placing tannic acid with acidic polymers for the stain for-
mation. Appreciable differences in stain removal efficacy
were found between the four acidic polymers with a rank
order of stain retention of Gantrez > Carbopol > Carra-
geenan > Xanthan. Gantrez on both occasions showed a
very similar pattern of stain removal, indicating its poten-
tial to mimic an AEP layer prior to the stain deposits.
STP, a sodium salt of triphosphoric acid with surfac-
tant and chelating properties, has been used in whiten-
ing toothpastes for stain removal. In vitro studies with
crystalline hydroxyapatite (HA) powder showed that
STP was effective in removing existing stain and imped-
ing stain formation through inhibition of the adsorption
of salivary proteins or tea stain and the desorption of
existing protein and stain from HA surfaces [16, 27]. In
vivo trials have also reported significant extrinsic stain
0
20
40
60
80
100
120
0200040006000
Stain removal efficacy, %
Brushing strokes
Stain Removal for Roughened Enamel Surface Group
10.0% STP
15.0% Zeodent 113
15.0% Zeodent 113 + 5.0%
STP
15.0% Zeodent 113 + 10.0%
STP
Fig. 6 Stain removal efficacy for stained bovine enamel specimens after brushing for up to 5000 brush strokes with 10.0% (w/w) STP alone, 15.0%
(w/w) Zeodent 113 alone, and 15.0% (w/w) Zeodent 113 with 5.0% (w/w) STP and 10.0% (w/w) STP, n= 8, mean ± standard deviation
Wang et al. BMC Oral Health (2017) 17:37 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
removal efficacy for a STP containing dentifrice versus
the control [28] as well as a reduction in staining by a
chewing gum which contained STP [29]. In the present
study, the effects of STP on in vitro stain removal effi-
cacy were examined both in combination with abrasive
particles and alone using 280-grit ground finish bovine
surfaces. Approximately 35% of the stain was removed
when brushing with 10.0% (w/w) STP alone after 5000
strokes, while a control of water resulted in minimal
stain removal (less than 1%). The addition of 5.0 (w/w)%
or 10.0 (w/w)% STP to a slurry of 15.0 (w/w)% Zeodent
113 abrasive improved stain removal compared with a
15.0 w/w% Zeodent 113 abrasive slurry alone for the
same number of brush strokes. These data also highlight
the efficacy of STP in contributing to stain removal dur-
ing oral hygiene measures. However, no differences were
observed between the STP concentration of 5.0 (w/w)%
and 10.0 (w/w)%. Interestingly, a lack of influence of
STP concentration on stain desorption from HA has also
been reported elsewhere [27]. Compared with the 10.0
(w/w)% STP solution alone (without abrasive particles),
the slurries with 15.0 w/w% Zeodent 113 abrasive parti-
cles (+/−STP) showed significantly higher stain removal
efficacy, demonstrating that the abrasive was mainly re-
sponsible for the removal of extrinsic stain. Similar re-
sults have been reported by others when testing different
types of whitening toothpaste products [30].
The present ferric-tannate/bovine enamel (280-grit
ground finish) methodology provides an easily performed
and reproducible model, which can be readily established
in laboratories. The chemically defined nature of the stain
contributes to reproducibility of the methodology as does
the enamel sample preparation protocol. The rapid stain
deposition (about 2 h for 10 cycles) facilitates the use of
the methodology for rapid screening of large numbers of
oral care products to target those products most suitable
for testing under clinical conditions. Importantly, it allows
good discrimination between various oral care products,
including those used for tooth whitening purposes.
With the growth in the desire for whiter teeth by con-
sumers and patients, toothpaste manufacturers aim to
maximise the cleanability of enamel extrinsic staining
whilst minimising possible damage (wear) to dental hard
tissues. Therefore, it is important to investigate the wear
and roughness in parallel to stain removal when using
the present methodology.
Conclusion
We conclude that this in vitro ferric-tannate stain re-
moval assay provides an easily performed and reprodu-
cible screening assay for a variety of oral care products,
which can be established in most laboratory settings.
Acidic polymers can be applied for the AEP simulation
prior to stain formulations.
Abbreviations
AEP: Acquired enamel pellicle; HA: Hydroxyapatite; OTC: Over-the-counter;
PCR: Pellicle cleaning ratio; RCTs: Randomised controlled trials; STP: Sodium
tripolyphosphate
Acknowledgements
The authors are grateful to Dr Jenny Gordon for helpful discussions and
advice during the course of this study.
Funding
This research was financially supported by GSK Consumer Healthcare. RL is
an employee of GSK, contributed to the design, analysis and interpretation
of the data from this study and contributed to writing of the manuscript.
Availability of data and materials
They are presented in the main paper.
Authors’contributions
RL, AJS, PRC contributed to the design, analysis and interpretation of the
data from this study and contributed to writing of the manuscript. CW
contributed to the design, analysis and interpretation of the data from this
study as well as conducting the laboratory procedures and contributed to
writing of the manuscript. All authors read and approved the final
manuscript.
Competing interests
AJS, PRC and CW have no competing interests. RL is an employee of GSK,
the sponsor of the study.
Consent for publication
Not applicable.
Ethics approval
The research was performed using food industry animal carcass waste and
that ethical approval is not required for waste tissues.
Author details
1
Oral Biology, School of Dentistry, University of Birmingham, 5 Mill Pool Way,
Edgbaston, Birmingham B5 7EG, UK.
2
GlaxoSmithKline Consumer Healthcare,
St. George’s Avenue, Weybridge, Surrey KT13 ODE, UK.
Received: 12 July 2016 Accepted: 21 December 2016
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