archives of oral biology 57 (2012) 1176–1182

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Erosive-abrasive tissue loss in dentine under simulated

bulimic conditions

Nadine Schlueter *, Jan Glatzki, Joachim Klimek, Carolina Ganss

Department of Conservative and Preventive Dentistry, Dental Clinic of the Justus-Liebig-University Giessen, Germany

article info abstract

Article history: Objectives: The eroded organic dentine matrix is remarkably resistant to mechanical
Accepted 1 April 2012 impacts. Additional brushing abrasion of eroded dentine has only limited influence on
tissue loss. Digestive enzymes (e.g., pepsin, trypsin) that can reach the oral cavity during
Keywords: reflux or vomiting can partially degrade the matrix. This degradation may have an influence
Abrasion on both the stability of the matrix against mechanical forces and the susceptibility of eroded
Dentine dentine to combined chemo-mechanical impacts. Both were investigated in the present
Erosion study.
Pepsin Methods: Dentine samples of four groups were cyclically demineralised (6  2 min/day, 9
Trypsin days) with an HCl-pepsin-solution (pH 1.6, 1.5 mg/ml pepsin) and treated with a trypsin-
Collagen solution (6  10 min/day, 2000 BAEE units/ml) directly afterwards. One group served as
control; specimens of three groups were additionally brushed (2  15 s/day) directly after
the first and last trypsin treatment with forces of 200 g, 300 g, and 400 g. Loss of deminer-
alised and mineralised tissue was determined profilometrically. Additionally, an SEM
analysis was performed.
Results: Loss of mineralised tissue (mm, mean  SD) was: 135.7  10.9 (control), 165.2  30.8
(200 g), 168.0  16.3 (300 g), and 174.9  17.1 (400 g). Tissue loss was increased significantly
( p  0.001) by brushing independently of the force used (n.s. between brushed groups). SEM
revealed that in all groups, the matrix was equally thinned through enzymatic activity, but it
was still present as a continuous band.
Conclusion: The results indicate that brushing of dentine after impact of acid and enzymes
resulted in an increased tissue loss; however, the matrix persisted on the surface despite
enzymatic treatment and brushing with forces of up to 400 g.
# 2012 Elsevier Ltd. All rights reserved.

clinically relevant acid challenges.3,4 The progression of

1. Introduction
Individuals with chronic reflux or bulimia nervosa are at high
risk for dental erosion because of exposure to gastric juice,
which is highly erosive.1 These patients often suffer from
severe defects involving the dentine.2 In addition to minerals,
dentine contains high amounts of organic materials, which
persist in vitro on the surface of demineralised dentine after
erosive tissue loss is inversely related to the exposure of the
organic structures, i.e., the thicker they become, the slower the
mineral loss progresses.5 Therefore, the organic structures
play a key role in the progression of in vitro dentine erosion.
The present study is a follow-up of two previously
performed studies. The first study showed that demineralised
organic surface material has remarkable stability against both
chemical4 and mechanical impacts.6 Brushing with forces of

* Corresponding author at: Dental Clinic of the Justus-Liebig-University Giessen, Department of Conservative and Preventive Dentistry,
Schlangenzahl 14, D-35392 Giessen, Germany. Tel.: +49 641 99 46173; fax: +49 641 99 46169.
E-mail address: nadine.schlueter@dentist.med.uni-giessen.de (N. Schlueter).
0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.archoralbio.2012.04.001
 archives of oral biology 57 (2012) 1176–1182
up to 400 g led to only a slight compression of the matrix, but
not to its removal.6 Furthermore, brushing in the presence of
the organic matrix had nearly no impact on tissue loss. Thus,
the organic structures act as a barrier for both chemical and
mechanical impacts, hindering erosion or erosion–abrasion
progression. In the second study, the impact of proteolytic
enzymes on the organic matrix was investigated. In contrast
to mechanical or acidic impacts, the matrix can easily be
removed from biochemical challenges such as a specific7 or
non-specific8 enzymatic digestion. Proteolytic enzymes are
omnipresent in the oral cavity9 and generally do not have any
negative impact on healthy structures. However, in the
presence of reflux or chronic vomiting, the content of the
stomach can reach the oral cavity, carrying along proteolytic
digestive enzymes, such as pepsin10,11 or even trypsin from
the duodenum.11 Neither enzymes are present in the oral
cavity under normal conditions, but might affect the demi-
neralised dentine in pathologic situations such as chronic
vomiting. Under in vitro conditions, the isolated impact of
pepsin8,12 or trypsin8 on the organic matrix led to only
minimal degradation of the organic matrix and did not result
in progression of erosive tissue loss. However, the combina-
tion of both enzymes significantly increased erosive tissue loss
compared to non-enzymatically altered dentine specimens.8
Scanning electron microscopy was used and revealed thinning
of the demineralised organic matrix due to the combined
pepsin/trypsin treatment; however, changes in matrix struc-
ture were not found at the magnifications we used.8 Enzymatic
alteration by both digestive enzymes may impair the
ultrastructure of the organic matrix, resulting in reduced
stability. Therefore, the resistance of the demineralised
surface material against mechanical impacts such as brushing
might be reduced, potentially leading to an increase of chemo-
mechanical substance losses.
In the present study, two targets were pursued. First, we
examined whether brushing has an impact on the degree of
erosive tissue loss in enzyme-treated dentine. Second, we
investigated whether brushing removed an enzymatically
altered (combination of pepsin and trypsin) demineralised
dentine matrix. Measurements were performed profilometri-
cally. Additionally, scanning electron microscopy was used for
structural analysis. The null hypothesis was that there is no
difference between the various groups.
2. Materials and methods
2.1. Sample preparation
A total of 80 coronal dentine specimens were prepared from
previously impacted, freshly extracted human third molars
that showed no cracks under a stereo microscope (10-fold
magnification, SMZ-1 Zoom Stereomicroscope, Nikon GmbH,
Düsseldorf, Germany). After removal of the enamel, longitu-
dinal dentine slices with an approximate thickness of 1 mm
were prepared (Exakt Trennschleifsystem, Exakt-Apparate-
bau, Norderstedt, Germany). Specimens were ground flat and
polished under sufficient water flow (50 ml/min, Exakt
Mikroschleifgerät, Exakt-Apparatebau, Norderstedt, Germany;
P800 and P1200 silicon carbide abrasive paper, Leco, St. Joseph,
1177
MI, USA). Specimens were mounted on microscope slides with
light curing acrylate (Technovit 7230 VLC, Kulzer-Exakt,
Wehrheim, Germany). Except for a defined experimental area
(2 mm  2 mm), specimens were covered with the light curing
acrylate. Subsequently, specimens were checked for enamel
remnants, cracks and contaminations under a stereo micro-
scope (10-fold magnification). Then, they were randomly
divided into four groups (n = 20 each) and stored in 100%
humidity until use. Supplementary specimens for scanning
electron microscopy were prepared (n = 5 per group). An
additional five specimens were prepared and were deminer-
alised with HCl, but not treated with enzymes.
2.2. Treatment
Over a period of nine days, all specimens were cyclically
demineralised. Six demineralisation cycles per day, each
lasting 2 min, were performed with an HCl-pepsin solution.
Directly after each erosive impact, all specimens were treated
with a trypsin solution for 10 min, six times a day. Except for
the control group, all specimens were brushed twice a day for
15 s with a fluoride free toothpaste (slurry with remineralisa-
tion solution, 1:3 (w/w), a fluoride-free brand of aronal (Japan),
RDA 77, pH 6.9 for 10% in water; GABA, Loerrach, Germany),
after the first and last trypsin treatments. Brushing forces of
200 g, 300 g and 400 g were used. Brushing was performed with
a powered toothbrush (Oral-B Plak Control Ultra; Braun,
Frankfurt, Germany). The toothbrush was fixed on a movable
clamp that was attached to a stand. To control the brushing
force, the samples were mounted on a scale, and the
toothbrush was lowered until the required value was
achieved, before the toothbrush was activated.
After each intervention, specimens were thoroughly rinsed
for 1 min with distilled water. The specimens were stored in a
mineral salt solution overnight or for 1 h after trypsin
treatment in the control group and brushing treatment in
the remaining group. All procedures were performed under
gentle agitation (30/min) in a water bath at 37 8C.
2.3. Solutions
The mineral salt solution for storage of the specimens was
supersaturated with respect to hydroxyapatite. The solution
contained 4.08 mM H3PO4, 20.10 mM KCl, 11.90 mM Na2CO3
and 1.98 mM CaCl2 (pH 6.7, chemicals from Merck, Darm-
stadt, Germany).13 An HCl-pepsin solution was used for
demineralisation. It was prepared by dissolving 5 mg/ml
NaCl in distilled water and adjusting the pH to 1.6 with
hydrochloric acid (chemicals from Merck, Darmstadt,
Germany).14 Subsequently, 1.5 mg/ml pepsin (4800 U/ml; P-
6887, pepsin from porcine gastric mucosa, 3200 U/mg,
Sigma–Aldrich, Seelze, Germany)15 was added to the HCl
solution. The trypsin solution was prepared by dissolving
2000 BAEE units/ml trypsin (T-9201, trypsin from bovine
pancreas, 7500 BAEE U/mg, Sigma–Aldrich, Seelze, Germany)
in the mineral salt solution.16 All solutions (150 ml per
group) were freshly prepared at the beginning of each
experimental day.
A collagenase-solution was used to remove deminera-
lised organic surface material to permit measurements of
1178 archives of oral biology 57 (2012) 1176–1182
mineralised tissue loss (measurement of the level of the
demineralisation front). Collagenase was also dissolved in
the mineral salt solution (100 U/ml collagenase from Clos-
tridium histolyticum type VII, collagen digestion activity in
the presence of calcium ions: 1680 U/mg solid at 25 8C, pH 7.5,
Sigma–Aldrich, St. Louis, MO, USA).
2.4. Tissue loss measurement
2.4.1. Measurement of loss of organic surface material (first
measurement)
The goal was to measure the level of the demineralised surface
compared to an untreated reference area. Under strict
moisture control, the level of the surface of the demineralised
but not enzymatically or mechanically altered organic matrix
nearly reached the level of the reference area.6,17 This result
may indicate that a measured step height after an enzymatic
or brushing treatment is approximately equivalent to the
spatial loss of the organic matrix due to enzymatic degrada-
tion or due to mechanical removal.
Measurements were performed with a Perthometer S8P
(Perthen-Mahr, Goettingen, Germany) equipped with a non-
contact measuring device (Focodyn, Rodenstock, Germany).18
On each specimen, three tracings with a length of 1.75 mm
were made at 250 mm intervals. Tracings were interpreted
with a special software (Perthometer Concept 4.0; Perthen-
Mahr). The vertical distance between the midpoints of
regression lines constructed on the reference and experimen-
tal areas was defined as spatial tissue loss (mm). The loss of a
given specimen was calculated as the mean of the three
tracings. To avoid drying artefacts, all tracings were made
under standardised moisture control. Specimens’ surfaces
were covered with distilled water for 30 s prior to tracing. The
water was removed with absorbent tissue without touching
the specimens, and surfaces were immediately traced. The
reproducibility (10-fold measurement of a given specimen) for
this method is 1.1 mm.
2.4.2. Measurement of mineralised tissue loss (second
measurement)
The goal was to measure the loss of mineralised tissue by
measuring the demineralisation front, i.e., the border between
the demineralised organic matrix and the underlying miner-
alised dentine. The quantification of this area is only possible
if the totally demineralised organic structures are removed
prior to measurement. The removal was achieved through
immersion of the specimens in collagenase solution.17,18 A
Perthometer S8P (Perthen-Mahr, Goettingen, Germany)
equipped with a contact (FRW-750, Perthen-Mahr, Goettingen,
Germany) pick up was used. All measuring parameters were
the same in terms of loss of the organic surface material,
including moisture control and analysis of traces. The
reproducibility (10-fold measurement of a given specimen)
for this method is 1.0 mm.
2.4.3. Calculation of thickness of remaining organic surface
material
The thickness of the remaining organic surface material was
calculated as the difference between the value obtained from
the measurement of the mineralised tissue loss (second
measurement) and the value obtained from the measurement
of loss of organic surface material (first measurement, level of
the surface of the organic material).
2.5. Scanning electron microscopy
After treatment, the supplementary moist specimens were
fractured into halves, critical point dried (Critical point dryer
CPD 030, Baltec, Witten, Germany) and lightly gold sputtered.
Cross sections of specimens were inspected using a scanning
electron microscope (SEM Type: JSM-6510, Jeol, Tokyo, Japan)
equipped with a tungsten cathode. The acceleration voltage
was set to 5 kV. Images were recorded using a secondary
electron (SE)-detector. The settings of the SEM, including tilt
angle, spot size, and scanning mode, were kept constant for all
sample groups. The SEM-pictures were obtained with a 300-
fold original magnification (reference value 6 cm  8 cm
medium format film). SEM was used to confirm the calculated
matrix thickness values.
2.6. Statistics
All statistical analyses were performed with IBM SPSS
Statistics 19.0 (Armonk, NY, USA). Normal distribution of data
was checked with the Kolmogorov–Smirnov test. Homogenei-
ty of variances was tested with Levene’s test. The groups were
compared using analysis of variance (ANOVA). A significant
deviation from the homogeneity of the variances was found;
therefore, Tamhane’s post hoc test was used, which takes into
account the significant deviation from the homogeneity of the
variances. The level of significance was set at 0.05. Data are
given as mean  standard deviation.
3. Results
3.1. Profilometric measurements (Fig. 1)
3.1.1. Loss of demineralised organic surface material
Values were lowest in the control group and were increased by
24%, 33% and 40% through brushing with 200 g, 300 g and
400 g, respectively (all differences to control group p  0.001).
No significant differences were found between the three
brushed groups.
3.1.2. Loss of mineralised tissue
Tissue loss was again lowest in the control group. The
percentage increase compared to the control group was
22%, 24% and 29% in the 200 g, 300 g and 400 g brushing
group, respectively (all differences compared to control group
p  0.01). Again, no significant differences were found be-
tween the three brushed groups.
3.1.3. Thickness of remaining organic surface material
On all specimens, organic materials remained on the
surface. The thickness of the matrix was 20.2  8.6 mm in
the control group, 21.2  20.8 mm in the 200 g group;
14.3  8.1 mm in the 300 g group and 13.2  13.6 mm in the
400 g group. The differences among groups were not
significant.
 archives of oral biology 57 (2012) 1176–1182
Fig. 1 – Loss (mm, mean W standard deviation) of
demineralised organic surface material (light grey
columns) and of mineralised tissue (dark grey columns) as
well as calculated thickness of residual matrix (hatched
columns, difference between loss of mineralised and loss
of demineralised tissue). Statistical significance between
groups is indicated by different upper case (loss of
demineralised surface material), lower case (loss of
mineralised tissue) and Greek letters (matrix thickness).
3.2. Scanning electron microscopy (Fig. 2)
Scanning electron microscopy revealed only small differences
between the four enzymatically altered groups (B–E). A clear
step between the reference area and the experimental area
was found in all groups, and only a small band of deminer-
alised surface material was found in the experimental area.
This finding indicates digestion of the organic surface
structures by the proteolytic enzymes in B–E. In contrast,
specimens that were only demineralised but not enzymati-
cally treated (A) showed a broad band of demineralised matrix,
with surface levels nearly reaching the level of the reference
area. The vertical distance between the reference area and the
demineralisation front (border between demineralised surface
material and mineralised dentine) was distinctly higher in the
enzymatically treated specimens than in the only deminer-
alised specimen.
4. Discussion
The aim of the present study was to mimic endogenously
caused erosions. We caused erosive demineralisation using
hydrochloric acid and proteolytic digestive enzymes on the
demineralised dentine surface with subsequent brushing. The
pH of the hydrochloric acid (1.6) was adjusted to a value that
can normally be found in the stomach.14 The physiologic
content of pepsin in the gastric juice is approximately 750 mg/
ml; however, after a complete meal, it can be considerably
higher, with reported values of up to 2200 mg/ml.15 Therefore,
we used a concentration of 1500 mg/ml, which has also been
used in a previous study.12 In the duodenum, a trypsin
1179
concentration of 2000 BAEE units/ml can be found,16 which
was used in the present study. Trypsin requires calcium for
optimal activity19; therefore, the enzyme was dissolved in the
mineral salt solution. The immersion times in acid and trypsin
were chosen according to previous studies.6,8 Generally, the
demineralisation period and frequency were relatively high,
mirroring conditions that can occur in patients with eating
disorders such as bulimia. The duration of trypsin treatment
was also chosen according to a previously performed study.8 It
has been found that trypsin can be retained in the oral cavity
of patients with eating disorders after vomiting over a period
of 30 min; however, the activity decreases by approximately
50% within this period.11 Therefore, it can be assumed that
trypsin is present in relevant concentrations for at least
10 min, but may be partially eliminated from the oral cavity
within this time period. The duration of brushing appears
relatively long compared to a previous observational study,
which reported an average brushing duration per sextant of
24 s20 but is still within a clinically relevant range. The
frequency was chosen according to the current recommenda-
tions for proper oral hygiene.21
To the best of our knowledge, this study is the first to
measure degradation of the matrix and the thickness of the
matrix profilometrically by calculating the difference between
the level of the surface of the organic material and the level of
the demineralisation front. This procedure represents an
alternative to the previously applied methods, such as
hydroxyproline analysis (HPA). HPA is a well-established
method for quantification of collagen degradation by means
of quantification of collagen degradation products.22,23 It has
many advantages if the degradation of pure collagen is being
measured. However, dentine consists of both organic materi-
als and minerals; therefore, HPA is limited when used to
quantify the degradation of the demineralised surface
material. The spatial loss of organic material can only be
measured indirectly by converting the measured hydroxypro-
line content (measured in mg/ml test solution) into spatial loss,
assuming a defined mineral content in dentine.12 However,
this procedure is prone to error. Additionally, no statement
can be made about the thickness of the remaining matrix, and
the reproducibility of this method is limited. Therefore, a
method was necessary, which allowed both the measurement
of the surface level of the organic material (representing the
loss of the matrix) and the thickness of the matrix (represent-
ing the residual barrier against mechanical and chemical
impacts). The profilometric method appears to be a good
option, as it showed good reproducibility. Additionally, the
values were compared with the thickness observed on
randomly selected SEM pictures, revealing good concordance
between profilometry and SEM. However, further validation
procedures are necessary before this method can be used as a
standard.
The spatial mineral loss was 136 mm in the control group.
Due to brushing, this value was significantly increased by
approximately 25% independent of the brushing force used.
This result is in clear contrast to a previously performed study
which had the same study design, except for the enzymatic
impact. Here, we investigated the effects of brushing, with
forces of 200 g, 300 g or 400 g, on demineralised dentine in the
presence of the non-degraded organic surface structures.
1180 archives of oral biology 57 (2012) 1176–1182

Fig. 2 – SEM pictures of cross sections of demineralised specimens. (A) shows a specimen, which was only demineralised
with an HCl-solution (pH 1.6). Note that the surface level of the organic matrix nearly reaches the level of the reference area
(white line). (B–E) show specimens, which were demineralised with an HCl-pepsin-solution (pH 1.6) and subsequently
treated with a trypsin-solution. Specimens in (C–E) were additionally brushed twice a day with a fluoride free toothpaste
slurry and a force of 200 g (C), 300 g (D) and 400 g (E), respectively. In comparison to the specimens that were only
demineralised (A), the matrix is thinned by the impact of the digestive enzymes and a clear step between the reference area
and the experimental area can be found. Only small differences were found between the four groups treated with enzymes.
Additionally, the vertical distance between the level of the reference area and the level of the demineralisation front (white
arrows; border between the demineralised matrix and the underlying mineralised dentine) was distinctly higher after
enzymatic treatment (B–E) than after HCl-treatment alone.

Brushing had almost no impact on the loss of mineral in this
study.6 It was hypothesised that the organic matrix, which
was not removed by the brushing procedure, acts as a barrier
against the mechanical forces. However, in the present study,
the matrix was thinned to a residual of only 20 mm or less
through the proteolytic activity of pepsin and trypsin. Due to
the enzymatic thinning process, the potential barrier function
of the organic matrix against mechanical and chemical
impacts was reduced. Therefore, the brushing procedure
was capable of increasing the loss of mineralised tissue by the
25% as mentioned above.
Interestingly, the values of matrix thickness were lower in
the 300 g and 400 g brushing groups than in the control and the
200 g groups. Two possible reasons are worth considering.
First, the enzymatically altered organic matrix could be
partially removed by brushing with higher forces, because
the enzymes may have reduced the matrix’ stability. Second,
the matrix may have been compressed by the higher brushing
forces. This would be comparable to the results of the above
mentioned, previously performed study with the same study
design but without enzymatic impact.6 In this study, the
matrix was compressed at 300 g as seen on SEM pictures.
However, in the present study, neither compression of the
matrix nor indications for a removal by brushing were found
on the SEM pictures. Therefore, we cannot clearly explain
these findings. Studies investigating the tensile strength or the
elasticity of the demineralised and enzymatically altered
dentine could elucidate this question.
Comparing the vertical distance between the reference
area and the level of the demineralisation front of the
 archives of oral biology 57 (2012) 1176–1182
specimens that were only demineralised (Fig. 2A) with the
enzyme-treated specimens (Fig. 2B–E) revealed that the loss of
minerals was distinctly higher after enzymatic digestion. This
finding was expected and confirmed in previously performed
studies, in which the organic surface structures were either
partially8 or completely24,25 removed, showing an acceleration
of erosion progression due to the removal.
Regarding the application of our results in clinical situa-
tions, it is still not clear which conditions exist in the oral
cavity. Various proteolytic enzymes are present in the oral
liquids with a potential for an impact on the demineralised
organic dentine matrix (mainly composed of collagen).26
Enzymes of relevance are various specific collagenolytic
enzymes such as MMPs.27 Different inactive isoforms of MMPs
can be found in the oral cavity,9 which are activated by a
decrease in pH and a subsequent neutralisation of the
surrounding fluids.28,29 This mechanism occurs in the process
of dental erosion; therefore, the presence of active MMPs
during and after an erosive acid challenge is very likely. It is
possible that the digestive enzymes pepsin and trypsin act
synergistically with these specific oral enzymes, leading to an
enhanced increase of erosion as well as erosion–abrasion
progression by a faster thinning or removal of the organic
surface structures. This potential mode of action should be
considered if new therapy strategies are developed specifically
for patients with endogenously caused erosion. In this
context, MMP inhibitors should be considered. The effect of
these inhibitors on the progression of dentine erosion has
recently been shown under in situ conditions, and they appear
quite promising.30,31
In conclusion, the present study showed that thinning of
the demineralised organic dentine matrix through enzymatic
digestion led to higher susceptibility to substance losses due to
combined chemo-mechanic stresses. However, the residual
matrix persisted on dentine surface despite enzymatic
treatment and brushing with forces of up to 400 g, as seen
on the SEM pictures.
Funding
The study was funded by the Justus-Liebig-University,
Giessen.
Competing interests
None declared.
Ethical approval
Not required.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.archoralbio.2012.04.001.
1181
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