Accepted Manuscript

Title: The effect of toothbrush abrasion force on dentine

hypersensitivity in-vitro
Author: Harminder Sehmi Ryan Olley
PII:
DOI:
Reference:

S0300-5712(15)30060-9
http://dx.doi.org/doi:10.1016/j.jdent.2015.10.014
JJOD 2541

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Journal of Dentistry

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19-8-2015
6-10-2015
16-10-2015

Please cite this article as: Sehmi Harminder, Olley Ryan.The effect of
toothbrush abrasion force on dentine hypersensitivity in-vitro.Journal of Dentistry
http://dx.doi.org/10.1016/j.jdent.2015.10.014
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Title page

Title: The effect of toothbrush abrasion force on dentine hypersensitivity in-vitro

Short title: toothbrush abrasion dentine hypersensitivity

Authors
Mr Harminder Sehmi
Department of Prosthodontics
Kings College London Dental Institute

Dr Ryan Olley
Department of conservative dentistry
Kings College London Dental Institute

Corresponding author;
Dr Ryan C Olley PhD BDS Bsc (Hons) MJDF RCS Eng AHEA
Clinical Lecturer and Specialty Registrar in Prosthodontics
King's College London Dental Institute
Department of Conservative Dentistry
Room 301, floor 26, Guys Tower Wing
Guy's Hospital London Bridge SE1 9RT
Tel: 0207 188 1605
Fax: 0207 188 1606
ryan.olley@kcl.ac.uk

Abstract
Objectives: This study investigated the effect of tooth brushing force on changes in dentine
tubule patency in an erosion-toothbrush abrasion model.
Methods: 60 dentine samples prepared with an artificial smear layer and divided randomly
into control (no toothbrush), 100g, 200g or 400g toothbrush groups. They were immersed in
3:1 artificial saliva/NaF 1450ppm and either brushed (p35 soft tooth brush; 120 strokes) or
not brushed. Then samples were subjected to agitated acid challenge (0.3% citric acid pH2.6
for 2minutes). Finally, samples were re-brushed. Calibrated software calculated patent
dentine tubules that cause DH in confocal microscopy images taken of samples at each stage.
Results: At baseline, mean patent tubules in all samples were 188 (SD54) with no significant
inter-group differences. Following first brushing, mean patent tubules decreased using 100g
to 150 (SD32) (p<0.01) and increased using 400g to 215 (SD45) (p=0.02). Following acid
challenge, patent tubules increased to 218 (SD40) in all samples (p<0.01) with no significant
inter-group differences. Following further brushing, mean patent tubules decreased using
100g to 175 (SD72) (p<0.01), but increased with 400g to 232 (SD52).
Conclusions: At higher brushing forces (400g), more tubules were exposed. At lower
brushing forces (100g), tubule patency decreased even post-acidic challenge.
Clinical significance: It is often recommended to our patients with DH to brush using lighter
brushing forces but our understanding of this force on dentine tubule patency is unknown.
The management of DH requires lighter brushing forces to reduce the numbers of patent
dentine tubules.

Keywords
Abrasion force, Toothbrush, Erosion, Dentine hypersensitivity, dentine tubules

Introduction

Dentine hypersensitivity (DH) is a short sharp pain arising from exposed dentine following
gingival recession or tooth wear. Once dentine is exposed, DH initiation involves removal of
the smear layer and exposure of dentine tubules. It is well documented that the presence of an
acid removes the smear layer and causes initiation of a DH lesion 1. However the role of
toothbrush abrasion is less clear-cut. The major modifying factor for the role of tooth
brushing in DH would be the presence of toothpaste, dietary fluids, filament size and
toothbrush force. It has been shown that softer toothbrushes compared to harder (stiffer)
toothbrushes lead to more tooth wear 2. Possible explanations for this have included the fact
that more dentifrice is held between smaller filament sizes. In addition, more deflection can
occur with narrower filaments, increasing contact between the filament and tooth. Current
management strategies for the management of DH including the use of desensitising
dentifrices occluding with modification of tooth brushing habits, to include monitoring of
brushing force, such as use of softer filament toothbrushes and cleaning thoroughly and
gently near the gingiva 3. However it is difficult to define what is a gentle brushing force and
the relationship between this, the effect on the patency of dentine tubules and DH. Moreover,
the force of brushing can vary considerably and the average forced applied during tooth
brushing has been suggested as 102g to 1121g (approximately 1 N-11 N) 4. In addition,
brushing force is often reported as higher using manual, as opposed to electric, toothbrushes
and the amount of tooth wear is also greater 5. Relatively little work has investigated the role
of tooth brushing force and DH. Relationships between tooth brushing force and DH can be
inferred from epidemiological studies looking at the prevalence of DH. Cunha-Cruz et al.
2013, carried out a cross-sectional study looking at the association of DH with different risk
factors 6. In their study group of 787 patients, aggressive tooth brushing habits were not
associated with those patients with DH. However it was unclear how they defined aggressive
tooth brushing in their study, whether toothpaste was used and the type of brush used.
In vitro studies have focused on the effect of brushing forces on dentine or enamel wear and
not DH. The latter is inferred in-vitro from the number and size of dentine tubules. Currently
no work has investigated specifically at the relationship between initiation phase of DH as a
result of dentine tubule patency and tooth brushing force. In order to quantitatively assess the
number of patent dentine tubules that might cause DH, previously software has been
calibrated for use in images taken of dentine samples using Scanning Electron Microscopy
3

(SEM) and Tandem Scanning Microscopy (TSM) 7. TSM requires no sample preparation and
has the advantage that samples can be subjected to various protocols of brushing and erosion
and imaged again after each stage. The aim of this study was therefore to investigate the
effect of tooth brushing force on changes in dentine tubule patency in an erosion-toothbrush
abrasion model. The null hypothesis was that there is no relationship between DH and
brushing force in an abrasion erosion model in-vitro.

Method

Sample preparations
Ethical approval (12/LO/1836) for tooth collection was obtained via the HRA NRES Centre
Manchester. 60 samples of dentine were obtained from un-erupted third molars, just below
the cement enamel junction. All third molars used were caries free and cold sterilized for one
hour in a solution of sodium hypochlorite (20,000ppm) and rinsed in distilled water. Sample
preparation followed previously published protocols 8. The samples were prepared to a size of
2mm x 2mm x 4mm (height, width and length). The samples were then placed (root surface
uppermost) in the centre of a silicone mould and filled with a bisacryl composite (Protemp 4,
3M ESPE, Seefield, Germany). The samples were then polished using sequentially finer
grades of polishing paper of grades 120, 320, 1200, 2400 and 4000 (Versocit, Struers A/S,
Copenhagen, Denmark) for 60 seconds per polishing grit to a smooth flat surface using a
polishing head that applied a constant force of 10N (Vector LC Power Head, Buehler, Lake
Bluff, Illinois, USA), in conjunction with a water cooled rotating polishing machine (MetaServ 3000 Grinder-Polisher, Buehler, Lake Bluff, Illinois, USA) at a rotating speed of 250
rotations per minute. The samples were then randomly allocated to one of the four test groups
(control samples with no brushing and test samples with brushing forces of 100g, 200g and
400g), and numbered 1 to 15 within each group. Samples were stored in a 0.9% saline
solution.

An artificial saliva and toothpaste slurry mix was then made immediately before use. The
toothpaste used was Colgate cavity protection (Colgate Oral Pharmaceuticals, New York).
The artificial saliva was created as previously described (Eisenburger et al, 2001) to a
composition of: CaCl22H2O 0.7mmol/1; MgCl2 0.2 mmol/l; KH2PO4 4.0mmol/l; HEPES

buffer (acid form) 20.0mmol/l; KCl 30.0 mmol/l, mixed with 1 litre of distilled water. The
pH of the solution was buffered to a pH of 7. Dentifrice slurry was then created by a mixing
of 330ml of Colgate cavity protection toothpaste and 660ml of artificial saliva. The slurry
mixture was then thoroughly mixed for 2 minutes to ensure homogenous slurry was created.

Citric acid (0.3%, pH 2.6) was also made immediately before use by dissolving 1.5g of citric
acid in 500ml of distilled water and mixing thoroughly until all citric acid crystals had
dissolved into the distilled water. The citric acid was kept at constant room temperature of 21
degrees.

Sample imaging
All samples (test and control) were initially imaged using Tandem Scanning confocal
Microscopy (TSM) (Noran Instruments, Middleton, USA) in conjunction with an M-Plan 40x
SLWD Brightfield Objective x20/0.35 NA objective).

Samples were gently air dried to

remove excess liquid at the surface then placed in the TSM machine. Initial surface images
were captured digitally using a mounted camera (Andor iXon 885, Andor Technology Ltd,
Belfast, UK), in conjunction with iAndor software (Andor Technology Ltd, Belfast, UK).
Throughout the testing procedure all samples were imaged in the same location.

Control samples
The control group samples were placed into 100 mL of the artificial saliva and toothpaste
slurry mixture for 2 minutes, and then rinsed thoroughly in distilled water. Following this,
samples were imaged a second time with TSM. The samples were then placed in 100ml 0.3%
citric acid for 2minutes and agitated using a mini-orbital shaker (Microtitre plate shaker SS5,
Bibby Scientific, Staffordshire, UK) set at 60 revolutions per minute. They were then
removed and thoroughly rinsed with distilled water and re-imaged a third time using TSM.
The final cycle for the control group was to place the samples into 100 mL of the artificial
saliva and toothpaste slurry mixture for 2 minutes. The samples were then removed and
thoroughly rinsed with distilled water and imaged with TSM a fourth time.

Test samples
For the tests groups that were to be brushed under various tooth brushing force (100g, 200g,
400g), tooth brushing was simulated by the use of an automatic tooth brushing machine
5

(Dentagen, Munich, Germany) and Sensodyne 3.5 toothbrushes. For each test group to
undergo tooth brushing, samples were loaded into the automatic tooth brushing machine and
100ml of the artificial saliva/tooth paste slurry placed into individual reservoirs for each of
the samples. The samples were then subjected to 120 strokes at 1 stroke per second, therefore
totalling 2 minutes of tooth brushing time 10, under the testing loads of 100g, 200g and 400g
using a measuring weight scale (Voltcraft PS 500 Pocket scale, Oldenzaal, Netherlands). A
maximum of 400g brushing force was used in order to avoid over-zealous tooth
brushing. At 400g, the toothbrush bristles deformed and the filaments were more likely
to distort and not engage the tooth at 90 degrees, in contrast to the 100g and 200g
brushing forces.

Samples were then rinsed thoroughly in distilled water and went through a second set of TSM
imaging.
Samples were then exposed to 0.3% citric acid, by placing each group into 100ml of the citric
acid solution and stirring by using a mini-orbital shaker (Microtitre plate shaker SS5, Bibby
Scientific, Staffordshire, UK) at 60 revolutions per minute for 2 minutes. Samples were then
removed, and thoroughly rinsed in distilled water. All samples then went through a third set
of TSM imaging.
The test groups were then exposed to a final round of tooth brushing by placing them back
into the automatic tooth brush machine with fresh artificial saliva and tooth paste slurry of
100ml per reservoir, and the samples brushed for 2 minutes at 120 strokes at their respective
test loads of 100g, 200g and 400g. The samples were then removed and thoroughly rinsed in
distilled water. Samples then went through the fourth and final set of TSM imaging.

Processing of images
The number of dentine tubules in each image was determined quantitatively after each stage
(baseline, 1st tooth brush abrasion, acid challenge and 2nd tooth brush abrasion). This was
achieved by using a previously calibrated computer algorithm

to count the number of

tubules using Image J (version 1.45 s, Wayne Rasband; National Institutes of Health,
Bethesda, Md., USA). The algorithm/software used analysed each TIFF image loaded into
the software and counted the number of patent dentine tubules present in the image.

Statistical analysis
Sample size calculation was calculated using Noordzij et al. 2010

11

, based on an alpha

power 0.05, power level 80%, standard deviation of 50 and estimated difference in the
mean number of patent tubules between samples with and without DH as 52. To
calculate the standard deviation, 10 additional samples were prepared as above. The
mean number of patent dentine tubules was calculated as 180 and standard deviation was 50.
The observed difference between means of groups was based on previously published
data 12. In this study, the mean number of patent tubules per unit area in teeth with DH
was 59 tubules versus teeth that did not have DH, which was 7

12

. Therefore this

difference is 52.
A paired t-test was carries out to determine if there were any differences between the same
samples over abrasion 1, acid challenge and abrasion 2. For inter group comparisons,
ANOVA was and Post Hoc analysis using Tukey were used.

Results

Table 1 shows the mean patent dentine tubules, standard deviations, standard errors
and 95% confidence intervals and statistically significant differences between stages by
group (baseline, 1st abrasion, acid challenge and 2nd abrasion). Figure 1 shows the TSM
images taken of the surfaces of the dentine for control and test groups (100g, 200g and 400g).
Figure 2 shows the mean number of dentine tubules at each stage of the experiment (baseline,
1st abrasion, acid challenge, 2nd abrasion) for each group.
In the control group, the only observed statistically significant change was an increase in
the mean patent dentine tubules between first abrasion (n=183) and acid challenge (n=210),
where p0.03. In figure 1, more dentine tubules are clearly visible between the first abrasion
and acid challenge and a clear increase is shown in figure 2.
For the 100g group, there was a statistically significant decrease in the mean dentine
tubules between baseline (n=196) and first abrasion (n=150), where p0.006. It is
possible to visualise fewer dentine tubules at first abrasion compared to baseline in figure 1
and there is a clear decrease in figure 2. Then, following acid challenge, mean tubules
increased to 215 and was significant (p<0.0001). This is reflected in figures 1 and 2.
7

Finally, after second abrasion, mean patent tubules decreased to 175 and was significant
(p<0.03). It is possible to visualise fewer dentine tubules in 2nd abrasion compared to acid
challenge in figure 1.
In the 200g group, the only observed statistical significance was an increase in the number
of dentine tubules between first abrasion (n=187) and acid challenge (n=222), where p0.03.
In figure 1, more dentine tubules are clearly visible between the first abrasion and acid
challenge.
For the 400g group, a statistical increase was seen between the mean number of dentine
tubules at base line (n=177) and after first abrasion (n=215), where p0.02. Overall, between
baseline and the second abrasion, there was a statistically significant increase in the number
of tubules from 177 to 231 (p0.002). There were no significant changes between 1st
abrasion, acid challenge and 2nd abrasion (p=0.3). This is supported by figure 1, which
shows patent tubules in 1st abrasion, acid challenge and 2nd abrasion, but not at baseline.

When comparing differences in mean patent tubules between groups, significant

changes were seen at first abrasion and second abrasion between the 100g and 400g
brushing groups. At first abrasion, mean tubules for the 100g and 400g groups were 150
and 215 respectively (p<0.001). At second abrasion, mean tubules for the 100g and 400g
groups were 175 and 232 respectively (p<0.001). There were no statistically significant
differences in the mean tubules between all groups at baseline and at acid challenge.

Discussion

In this study, the mean number of patent dentine tubules changed by varying the brushing
force applied to dentine with an artificial smear layer present. Therefore we can refute the
null hypothesis. The 100g brushing force resulted in a decrease in patent dentine tubules
following two minutes brushing pre (p0.006) and post acid challenge (p<0.03). In
contrast, brushing for two minutes with 400g brushing force increased the patent
tubules (p<0.02), but no significant changes occurred post acid challenge (p=0.3). The
100g brushing force reduced the mean number of dentine tubules in contrast to the

400g brushing force pre and post acid challenge (p<0.001). The 200g force had no
significant effect on tubule patency both pre and post acid challenge.

Previous work has not investigated the relationship between brushing force and dentine
tubule patency. The results support anecdotal evidence, which suggests a therapeutic benefit
of a smaller brushing force13. In the case of our study, 100g brushing force with a soft
filament manual brush could more likely recreate a smear layer, causing particulate
deposits to occlude dentine tubules before and even following acid challenge. This might
have occurred as less material was brushed away from the dentine surface.
In contrast, at higher brushing forces (400g), the smear layer was removed as more dentine
tubules were exposed. Following a subsequent acid challenge and 2nd abrasion, no
further statistical differences in mean patent dentine tubules were observed. Therefore,
the overall significant difference in the mean patent tubules between baseline and 2 nd
abrasion (p<0.002) was due to the initial 2 minutes brushing at 400g, rather than the
exposure to the acid challenge. As a result, we might presume that an initial 400g
brushing force has an important role in removing an artificial smear layer and
maintaining the patency of dentine tubules.
The 200g brushing force had little effect on the patency of dentine tubules. The only
significant change within this group occurred following an acid challenge, where more
dentine tubules were exposed (200g) (p0.03), in similarity to the control group (p0.03).
These increases in dentine tubule patency following an acid challenge are supported in the
literature and the use of citric acid and its importance in removing smear layer is well
documented 14. In contrast, in the 400g group, the tubule patency did not increase further post
acid challenge. This reflects the significance of the larger 400g brushing force alone in
exposing patent dentine tubules.

In the context of the management of a patient with DH, high brushing forces (400g) and
acid challenges are important in removing smear layer and exposing dentine tubules. At
very high brushing forces in which the tooth brush bristles bend, the likelihood of
increasing tubule patency is much higher. Therefore, lower brushing forces should be
aimed for. This is difficult to standardise, but if a new soft filament tooth brush is used,
9

the aim is to brush gently in order that the bristles are not deformed. In the case of
electronic toothbrushes, it would be possible to measure and warn the patient if an
excessive force (>200g) was reached.

Other work by Kwon et al. 2012 investigated the timing of brushing post acid challenge
in relation to the exposure of patent dentine tubules in primary teeth15. In this study,
brushing up to 30 seconds immediately post acid challenge resulted in exposure of more
patent dentine tubules, in contrast to brushing at 60 and 120 minutes post acid challenge15. In
contrast, in the present study, it has been shown that 100g of brushing force may have a
therapeutic effect and result in less exposed dentine tubules immediately pre- and postacid challenge. Furthermore, the 200g and 400g tooth brushing forces did not result in
significantly greater increases in the number of dentine tubules post acid challenge. It is
important to note that in the present study, longer brushing times were used and the
brushing force was recorded. Nonetheless, based on the results of Kwon et al 2012, it
can be suggested that a delay exist between brushing time post acid challenge (of one
hour). Other clinical research supports this recommendation and observes an increase
prevalence of DH in patients who consumed an acidic drink within one hour16.

In order to standardise brushing time, two minutes was chosen across samples. This is
recommended by public health England for the whole mouth10 and therefore an
individual tooth would receive a far shorter brushing time. Consequently, the results
represent the effects on the dentine tubules following months (as opposed to single
sessions) of tooth brushing, in order that these effects may be seen in the long term.
Therefore, it is not suggested from this paper that the 100g brushing force is sufficient
to treat DH alone short term, but important in its aetiology and subsequent
management in the long term. A tooth paste of low RDA value was used, Colgate Cavity
Protection has been shown to have an RDA of 70-8017. It contained the ingredient 1450ppm
NaF, but no other active ingredients. Patients with DH often manage the condition using
desensitising dentifrices designed to occlude the dentine tubules and reduce DH in the short
term. The application of a strontium based tooth paste to patients suffering with DH, was
shown to cause a decrease in patent tubules associated with DH

and tubule occlusion

occurred within 5m of the dentine surface18 following 10 seconds of tooth brushing.

10

Therefore, dentine tubule occlusion occurs close to the surface of the dentine. It is
important to modify tooth brushing habits, using a smaller brushing force, in order to
substantiate the effects of the desensitising dentifrice and reduce the possibility of recreating dentine tubule patency over time. This is supported in the literature, which
recommends modification of tooth brushing habits, to include monitoring of brushing
force in the management of DH, in addition to the use of desensitising dentifrices3.

Previous work has not been conducted investigating tubule patency of dentine tubules with
various brushing forces, but they have investigated the effect of brushing force and tooth
wear. Ponduri et al. 2005 investigated a toothbrush force of 200g (with ten seconds
brushing) on the wear of dentine in an erosion abrasion model16. That study found an
increase in surface loss of dentine when samples were brushed at 200g compared to
control16. However, this is in disagreement with Ganss et al. 2007, who showed in-vitro
that there is no loss of surface height with tooth brush forces of 100g, 200g and 400g in
an erosion abrasion model. Here, samples were brushed for 15 seconds twice daily for
nine days after erosion. This difference might be explained by individual erosion times,
which were less in Ganss et al. 2007 in contrast to Ponduri et al. 2005 where erosion time
occurred up to 30 minutes. This reflects the importance of erosion over tooth brush
abrasion in contributing tooth wear in dentine and further work is required.

The limitations of this study were that it was conducted in-vitro as opposed to in-vivo. As a
result, it does not completely replicate the oral environment, the human saliva, variable
dentine surfaces and tooth brushing techniques. Nonetheless, the in-vitro nature of this study
ensured standardisation of tooth samples, brushing regimes and investigation of brushing
forces alone. The testing protocol is based on the dentine disc model in vitro standardised
model 21. At baseline and following an acid challenge, there were no statistically significant
differences in the mean number of patent tubules between all groups and supports
homogenous samples at baseline. All dentine originated from un-erupted third molars and
the sample preparation processes were standardised. The brushing protocol was implemented
via the use of an automatic toothbrush machine to control brushing time, duration and amount
of dentifrice. Finally, the visual analogue scale was simpler than counting individual patent
dentine tubules on images of the surface of dentine and allowed repeated measurement of the
11

same samples. The software removes the subjectivity and is more sensitive at counting
dentine tubules 7.

In conclusion, within the limitations of this study, it appears that tooth brushing force has an
effect on influencing the number of dentinal tubules that are patent at the surface of dentine.
Following two minutes brushing, a tooth brushing force of 400g causes the greatest increase
in the number of tubules after toothbrush abrasion, whereas the 100g brushing force causes
formation of the smear layer, pre and even post acid challenge. The clinical significance of
this is to advise patients to brush using lighter brushing forces in order to reduce the
possibility of DH longer term. If DH is present, brushing using a desensitising dentifrice is
recommended at lighter brushing forces to reduce tubule patency.

Declaration of interests
There are no conflicts of interest

12

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Labahn R, Fahrenbach WH, Clark SM, Lie T, Adams DF. Root dentin morphology
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Kwon E, Choi S, Cheong Y, Park K-H, Park H-K. Scanning Electron Microscopy
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15

Figure 1
Figure 2

16

Baseline

Control
group

100g

200g

400g

1st Abrasion
(excluding control)

Acid challenge

2nd Abrasion
(excluding control)

240

Mean number of
patent dentine
tubules

230
220
210
200
190
180
170
160
150
140
Baseline

First abrasion

Acid Challenge

Stage of experiment

2nd Abrasion

Table 1 Mean patent dentine tubules, standard deviations (SD), standard error (SE), 95% confidence intervals (CI) and statistically significant differences
by group.
1st abrasion

Baseline
Mean
Control 186*
100g

196*A

2nd abrasion

Acid challenge

Significant

SD

SE

CI

Mean

SD

SE

CI

Mean

SD

SE

CI

Mean

SD

SE

CI

differences

51

13

212,160

183

35

201,166

210*

43

11

232,188

210

39

10

231,190

*p<0.003

56

15

225,168

150*B

33

167,134

215*C

31

231,200

175*D

72

19

212,139

*A and B (p<0.006)
*B and C (p<0.0001)
*C and D (p<0.03)

200g

192

60

16

223,161

187*

49

13

213,193

222*

49

13

247,197

213

55

14

241,185

*p<0.003

400g

177*A

48

12

201,153

215*B

45

12

238,192

224*C

39

10

243,204

231*D

52

13

258,206

*A and B (p<0.02)
*A and D (p<0.002)

17