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archives of oral biology 57 (2012) 1176–1182

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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 erosive tissue loss is inversely related to the exposure of the
organic structures, i.e., the thicker they become, the slower the
Individuals with chronic reflux or bulimia nervosa are at high mineral loss progresses.5 Therefore, the organic structures
risk for dental erosion because of exposure to gastric juice, play a key role in the progression of in vitro dentine erosion.
which is highly erosive.1 These patients often suffer from The present study is a follow-up of two previously
severe defects involving the dentine.2 In addition to minerals, performed studies. The first study showed that demineralised
dentine contains high amounts of organic materials, which organic surface material has remarkable stability against both
persist in vitro on the surface of demineralised dentine after 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 1177

up to 400 g led to only a slight compression of the matrix, but MI, USA). Specimens were mounted on microscope slides with
not to its removal.6 Furthermore, brushing in the presence of light curing acrylate (Technovit 7230 VLC, Kulzer-Exakt,
the organic matrix had nearly no impact on tissue loss. Thus, Wehrheim, Germany). Except for a defined experimental area
the organic structures act as a barrier for both chemical and (2 mm  2 mm), specimens were covered with the light curing
mechanical impacts, hindering erosion or erosion–abrasion acrylate. Subsequently, specimens were checked for enamel
progression. In the second study, the impact of proteolytic remnants, cracks and contaminations under a stereo micro-
enzymes on the organic matrix was investigated. In contrast scope (10-fold magnification). Then, they were randomly
to mechanical or acidic impacts, the matrix can easily be divided into four groups (n = 20 each) and stored in 100%
removed from biochemical challenges such as a specific7 or humidity until use. Supplementary specimens for scanning
non-specific8 enzymatic digestion. Proteolytic enzymes are electron microscopy were prepared (n = 5 per group). An
omnipresent in the oral cavity9 and generally do not have any additional five specimens were prepared and were deminer-
negative impact on healthy structures. However, in the alised with HCl, but not treated with enzymes.
presence of reflux or chronic vomiting, the content of the
stomach can reach the oral cavity, carrying along proteolytic 2.2. Treatment
digestive enzymes, such as pepsin10,11 or even trypsin from
the duodenum.11 Neither enzymes are present in the oral Over a period of nine days, all specimens were cyclically
cavity under normal conditions, but might affect the demi- demineralised. Six demineralisation cycles per day, each
neralised dentine in pathologic situations such as chronic lasting 2 min, were performed with an HCl-pepsin solution.
vomiting. Under in vitro conditions, the isolated impact of Directly after each erosive impact, all specimens were treated
pepsin8,12 or trypsin8 on the organic matrix led to only with a trypsin solution for 10 min, six times a day. Except for
minimal degradation of the organic matrix and did not result the control group, all specimens were brushed twice a day for
in progression of erosive tissue loss. However, the combina- 15 s with a fluoride free toothpaste (slurry with remineralisa-
tion of both enzymes significantly increased erosive tissue loss tion solution, 1:3 (w/w), a fluoride-free brand of aronal (Japan),
compared to non-enzymatically altered dentine specimens.8 RDA 77, pH 6.9 for 10% in water; GABA, Loerrach, Germany),
Scanning electron microscopy was used and revealed thinning after the first and last trypsin treatments. Brushing forces of
of the demineralised organic matrix due to the combined 200 g, 300 g and 400 g were used. Brushing was performed with
pepsin/trypsin treatment; however, changes in matrix struc- a powered toothbrush (Oral-B Plak Control Ultra; Braun,
ture were not found at the magnifications we used.8 Enzymatic Frankfurt, Germany). The toothbrush was fixed on a movable
alteration by both digestive enzymes may impair the clamp that was attached to a stand. To control the brushing
ultrastructure of the organic matrix, resulting in reduced force, the samples were mounted on a scale, and the
stability. Therefore, the resistance of the demineralised toothbrush was lowered until the required value was
surface material against mechanical impacts such as brushing achieved, before the toothbrush was activated.
might be reduced, potentially leading to an increase of chemo- After each intervention, specimens were thoroughly rinsed
mechanical substance losses. for 1 min with distilled water. The specimens were stored in a
In the present study, two targets were pursued. First, we mineral salt solution overnight or for 1 h after trypsin
examined whether brushing has an impact on the degree of treatment in the control group and brushing treatment in
erosive tissue loss in enzyme-treated dentine. Second, we the remaining group. All procedures were performed under
investigated whether brushing removed an enzymatically gentle agitation (30/min) in a water bath at 37 8C.
altered (combination of pepsin and trypsin) demineralised
dentine matrix. Measurements were performed profilometri- 2.3. Solutions
cally. Additionally, scanning electron microscopy was used for
structural analysis. The null hypothesis was that there is no The mineral salt solution for storage of the specimens was
difference between the various groups. 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-
2. Materials and methods stadt, Germany).13 An HCl-pepsin solution was used for
demineralisation. It was prepared by dissolving 5 mg/ml
2.1. Sample preparation NaCl in distilled water and adjusting the pH to 1.6 with
hydrochloric acid (chemicals from Merck, Darmstadt,
A total of 80 coronal dentine specimens were prepared from Germany).14 Subsequently, 1.5 mg/ml pepsin (4800 U/ml; P-
previously impacted, freshly extracted human third molars 6887, pepsin from porcine gastric mucosa, 3200 U/mg,
that showed no cracks under a stereo microscope (10-fold Sigma–Aldrich, Seelze, Germany)15 was added to the HCl
magnification, SMZ-1 Zoom Stereomicroscope, Nikon GmbH, solution. The trypsin solution was prepared by dissolving
Düsseldorf, Germany). After removal of the enamel, longitu- 2000 BAEE units/ml trypsin (T-9201, trypsin from bovine
dinal dentine slices with an approximate thickness of 1 mm pancreas, 7500 BAEE U/mg, Sigma–Aldrich, Seelze, Germany)
were prepared (Exakt Trennschleifsystem, Exakt-Apparate- in the mineral salt solution.16 All solutions (150 ml per
bau, Norderstedt, Germany). Specimens were ground flat and group) were freshly prepared at the beginning of each
polished under sufficient water flow (50 ml/min, Exakt experimental day.
Mikroschleifgerät, Exakt-Apparatebau, Norderstedt, Germany; A collagenase-solution was used to remove deminera-
P800 and P1200 silicon carbide abrasive paper, Leco, St. Joseph, 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 measurement) and the value obtained from the measurement
demineralisation front). Collagenase was also dissolved in of loss of organic surface material (first measurement, level of
the mineral salt solution (100 U/ml collagenase from Clos- the surface of the organic material).
tridium histolyticum type VII, collagen digestion activity in
the presence of calcium ions: 1680 U/mg solid at 25 8C, pH 7.5, 2.5. Scanning electron microscopy
Sigma–Aldrich, St. Louis, MO, USA).
After treatment, the supplementary moist specimens were
2.4. Tissue loss measurement fractured into halves, critical point dried (Critical point dryer
CPD 030, Baltec, Witten, Germany) and lightly gold sputtered.
2.4.1. Measurement of loss of organic surface material (first Cross sections of specimens were inspected using a scanning
measurement) electron microscope (SEM Type: JSM-6510, Jeol, Tokyo, Japan)
The goal was to measure the level of the demineralised surface equipped with a tungsten cathode. The acceleration voltage
compared to an untreated reference area. Under strict was set to 5 kV. Images were recorded using a secondary
moisture control, the level of the surface of the demineralised electron (SE)-detector. The settings of the SEM, including tilt
but not enzymatically or mechanically altered organic matrix angle, spot size, and scanning mode, were kept constant for all
nearly reached the level of the reference area.6,17 This result sample groups. The SEM-pictures were obtained with a 300-
may indicate that a measured step height after an enzymatic fold original magnification (reference value 6 cm  8 cm
or brushing treatment is approximately equivalent to the medium format film). SEM was used to confirm the calculated
spatial loss of the organic matrix due to enzymatic degrada- matrix thickness values.
tion or due to mechanical removal.
Measurements were performed with a Perthometer S8P 2.6. Statistics
(Perthen-Mahr, Goettingen, Germany) equipped with a non-
contact measuring device (Focodyn, Rodenstock, Germany).18 All statistical analyses were performed with IBM SPSS
On each specimen, three tracings with a length of 1.75 mm Statistics 19.0 (Armonk, NY, USA). Normal distribution of data
were made at 250 mm intervals. Tracings were interpreted was checked with the Kolmogorov–Smirnov test. Homogenei-
with a special software (Perthometer Concept 4.0; Perthen- ty of variances was tested with Levene’s test. The groups were
Mahr). The vertical distance between the midpoints of compared using analysis of variance (ANOVA). A significant
regression lines constructed on the reference and experimen- deviation from the homogeneity of the variances was found;
tal areas was defined as spatial tissue loss (mm). The loss of a therefore, Tamhane’s post hoc test was used, which takes into
given specimen was calculated as the mean of the three account the significant deviation from the homogeneity of the
tracings. To avoid drying artefacts, all tracings were made variances. The level of significance was set at 0.05. Data are
under standardised moisture control. Specimens’ surfaces given as mean  standard deviation.
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 3. Results
reproducibility (10-fold measurement of a given specimen) for
this method is 1.1 mm. 3.1. Profilometric measurements (Fig. 1)

2.4.2. Measurement of mineralised tissue loss (second 3.1.1. Loss of demineralised organic surface material
measurement) Values were lowest in the control group and were increased by
The goal was to measure the loss of mineralised tissue by 24%, 33% and 40% through brushing with 200 g, 300 g and
measuring the demineralisation front, i.e., the border between 400 g, respectively (all differences to control group p  0.001).
the demineralised organic matrix and the underlying miner- No significant differences were found between the three
alised dentine. The quantification of this area is only possible brushed groups.
if the totally demineralised organic structures are removed
prior to measurement. The removal was achieved through 3.1.2. Loss of mineralised tissue
immersion of the specimens in collagenase solution.17,18 A Tissue loss was again lowest in the control group. The
Perthometer S8P (Perthen-Mahr, Goettingen, Germany) percentage increase compared to the control group was
equipped with a contact (FRW-750, Perthen-Mahr, Goettingen, 22%, 24% and 29% in the 200 g, 300 g and 400 g brushing
Germany) pick up was used. All measuring parameters were group, respectively (all differences compared to control group
the same in terms of loss of the organic surface material, p  0.01). Again, no significant differences were found be-
including moisture control and analysis of traces. The tween the three brushed groups.
reproducibility (10-fold measurement of a given specimen)
for this method is 1.0 mm. 3.1.3. Thickness of remaining organic surface material
On all specimens, organic materials remained on the
2.4.3. Calculation of thickness of remaining organic surface surface. The thickness of the matrix was 20.2  8.6 mm in
material the control group, 21.2  20.8 mm in the 200 g group;
The thickness of the remaining organic surface material was 14.3  8.1 mm in the 300 g group and 13.2  13.6 mm in the
calculated as the difference between the value obtained from 400 g group. The differences among groups were not
the measurement of the mineralised tissue loss (second significant.
archives of oral biology 57 (2012) 1176–1182 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,
Fig. 1 – Loss (mm, mean W standard deviation) of
which reported an average brushing duration per sextant of
demineralised organic surface material (light grey
24 s20 but is still within a clinically relevant range. The
columns) and of mineralised tissue (dark grey columns) as
frequency was chosen according to the current recommenda-
well as calculated thickness of residual matrix (hatched
tions for proper oral hygiene.21
columns, difference between loss of mineralised and loss
To the best of our knowledge, this study is the first to
of demineralised tissue). Statistical significance between
measure degradation of the matrix and the thickness of the
groups is indicated by different upper case (loss of
matrix profilometrically by calculating the difference between
demineralised surface material), lower case (loss of
the level of the surface of the organic material and the level of
mineralised tissue) and Greek letters (matrix thickness).
the demineralisation front. This procedure represents an
alternative to the previously applied methods, such as
hydroxyproline analysis (HPA). HPA is a well-established
3.2. Scanning electron microscopy (Fig. 2) method for quantification of collagen degradation by means
of quantification of collagen degradation products.22,23 It has
Scanning electron microscopy revealed only small differences many advantages if the degradation of pure collagen is being
between the four enzymatically altered groups (B–E). A clear measured. However, dentine consists of both organic materi-
step between the reference area and the experimental area als and minerals; therefore, HPA is limited when used to
was found in all groups, and only a small band of deminer- quantify the degradation of the demineralised surface
alised surface material was found in the experimental area. material. The spatial loss of organic material can only be
This finding indicates digestion of the organic surface measured indirectly by converting the measured hydroxypro-
structures by the proteolytic enzymes in B–E. In contrast, line content (measured in mg/ml test solution) into spatial loss,
specimens that were only demineralised but not enzymati- assuming a defined mineral content in dentine.12 However,
cally treated (A) showed a broad band of demineralised matrix, this procedure is prone to error. Additionally, no statement
with surface levels nearly reaching the level of the reference can be made about the thickness of the remaining matrix, and
area. The vertical distance between the reference area and the the reproducibility of this method is limited. Therefore, a
demineralisation front (border between demineralised surface method was necessary, which allowed both the measurement
material and mineralised dentine) was distinctly higher in the of the surface level of the organic material (representing the
enzymatically treated specimens than in the only deminer- loss of the matrix) and the thickness of the matrix (represent-
alised specimen. 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
4. Discussion values were compared with the thickness observed on
randomly selected SEM pictures, revealing good concordance
The aim of the present study was to mimic endogenously between profilometry and SEM. However, further validation
caused erosions. We caused erosive demineralisation using procedures are necessary before this method can be used as a
hydrochloric acid and proteolytic digestive enzymes on the standard.
demineralised dentine surface with subsequent brushing. The The spatial mineral loss was 136 mm in the control group.
pH of the hydrochloric acid (1.6) was adjusted to a value that Due to brushing, this value was significantly increased by
can normally be found in the stomach.14 The physiologic approximately 25% independent of the brushing force used.
content of pepsin in the gastric juice is approximately 750 mg/ This result is in clear contrast to a previously performed study
ml; however, after a complete meal, it can be considerably which had the same study design, except for the enzymatic
higher, with reported values of up to 2200 mg/ml.15 Therefore, impact. Here, we investigated the effects of brushing, with
we used a concentration of 1500 mg/ml, which has also been forces of 200 g, 300 g or 400 g, on demineralised dentine in the
used in a previous study.12 In the duodenum, a trypsin 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 partially removed by brushing with higher forces, because
study.6 It was hypothesised that the organic matrix, which the enzymes may have reduced the matrix’ stability. Second,
was not removed by the brushing procedure, acts as a barrier the matrix may have been compressed by the higher brushing
against the mechanical forces. However, in the present study, forces. This would be comparable to the results of the above
the matrix was thinned to a residual of only 20 mm or less mentioned, previously performed study with the same study
through the proteolytic activity of pepsin and trypsin. Due to design but without enzymatic impact.6 In this study, the
the enzymatic thinning process, the potential barrier function matrix was compressed at 300 g as seen on SEM pictures.
of the organic matrix against mechanical and chemical However, in the present study, neither compression of the
impacts was reduced. Therefore, the brushing procedure matrix nor indications for a removal by brushing were found
was capable of increasing the loss of mineralised tissue by the on the SEM pictures. Therefore, we cannot clearly explain
25% as mentioned above. these findings. Studies investigating the tensile strength or the
Interestingly, the values of matrix thickness were lower in elasticity of the demineralised and enzymatically altered
the 300 g and 400 g brushing groups than in the control and the dentine could elucidate this question.
200 g groups. Two possible reasons are worth considering. Comparing the vertical distance between the reference
First, the enzymatically altered organic matrix could be area and the level of the demineralisation front of the
archives of oral biology 57 (2012) 1176–1182 1181

specimens that were only demineralised (Fig. 2A) with the references
enzyme-treated specimens (Fig. 2B–E) revealed that the loss of
minerals was distinctly higher after enzymatic digestion. This
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studies, in which the organic surface structures were either potential of gastric juice and a carbonated drink in vitro.
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