archives of oral biology 55 (2010) 294–299

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Influence of the digestive enzymes trypsin and pepsin in

vitro on the progression of erosion in dentine

Nadine Schlueter a,*, Martin Hardt b, Joachim Klimek a, Carolina Ganss a

a
Department of Conservative and Preventive Dentistry, Dental Clinic of the Justus-Liebig-University Giessen, Schlangenzahl 14,
D-35392 Giessen, Germany
b
Central Biotechnology Unit, Justus-Liebig-University Giessen, Giessen, Germany

article info abstract

Article history: Objectives: In patients with eating disorders, gastric and pancreatic enzymes could possibly
Accepted 4 February 2010 reach the oral cavity during vomiting and could perhaps degrade the organic matrix of
eroded dentine. This in vitro study sought to investigate whether pepsin, trypsin or the
Keywords: combination of both, have an influence on erosive mineral loss in dentine and whether they
Dentine are able to degrade the organic matrix.
Erosion Methods: Sixty-four human dentine specimens were prepared and randomly divided into
Pepsin four groups. Specimens were cyclically de- and remineralised for six days. Demineralisation
Trypsin was performed with an HCl-solution (6 5 min daily, pH 1.6) in groups 1 and 3; in groups 2
Collagen and 4 the demineralisation solution additionally contained pepsin (750 mg/ml). After demi-
neralisation, specimens of groups 3 and 4 were treated with a trypsin solution (6 10 min
daily, 2000 BAEE/ml). After each day, mineral content (mm) was determined microradio-
graphically, and the matrix degradation was determined by hydroxyproline analysis.
Results: After six days, treatment with pepsin (group 2) or trypsin (group 3) had no significant
influence on mineral loss. The combined impact of pepsin and trypsin led to significantly
higher mineral loss (group 4: 202.5  37.4) compared to all other groups (group 1:
139.1  29.5, p  0.001; group 2: 108.8  34.7, p  0.001; group 3: 157.8  37.2, p  0.05). Hy-
droxyproline was found in all pepsin-solutions but in no trypsin- or HCl-solutions.
Conclusion: The combined impact of pepsin and trypsin intensified dentine erosion progres-
sion in vitro. This could be one reason for the fast proceeding of dental erosion in patients
with chronic vomiting.
# 2010 Elsevier Ltd. All rights reserved.

1. Introduction extrinsic acids can lead to dental erosion particularly in

patients with regular vomiting who often show severe and
Eating disorders are psychosomatic diseases with a preva- generalised defects reaching the dentine.3–5 Similar applies for
lence in western societies of 0.5–2% in 16–35-year-old patients with gastro oesophageal reflux disease (GERD). In
females.1,2 Bulimia nervosa, one form of these disorders, is these patients the gastric juice reaches the oral cavity with
characterised by repeated episodes of intake of large quanti- either high frequency or with long duration,6 resulting in
ties of high caloric food and self-induced vomiting and higher risk for dental erosion.7
episodes of restrictive ingestion oftentimes in combination In enamel, erosion is a surface phenomenon where the acid
with frequent consumption of fruit, vegetables and acidic impact leads to a centripetal mineral loss.8 The rapid progress
beverages during the restrictive period. All these intrinsic and of enamel loss in patients with bulimia nervosa can be

* Corresponding author. Tel.: +49 641 99 46173; fax: +49 641 99 46169.
E-mail address: [email protected] (N. Schlueter).
0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2010.02.003
 archives of oral biology 55 (2010) 294–299 295

explained by the high acidity and erosivity9 of the hydrochloric 7230 VLC, Kulzer-Exakt, Wehrheim Germany), leaving the
acid that is contained in the gastric juice.10 experimental area undisturbed, and fixed with acrylate on
However, in dentine, the erosive tissue loss is not a simple special holders for longitudinal microradiography. Speci-
surface process, and the acid impact leads to an exposure of the mens were checked for enamel remnants, cracks and
organic structures.11 As the matrix is not soluble by clinically contaminations under a stereo microscope (10-fold magnifi-
relevant acid impacts, it remains on the surface whilst mineral cation). A total of 64 specimens were made, which were
loss continues. The progression of erosive tissue loss is randomly divided into four groups (n = 16 each) and stored in
inversely related to the exposure of the organic matrix; the 100% humidity until use.
thicker the matrix becomes the slower the mineral loss
proceeds because it acts as a diffusion barrier.12,13 However,
in case of an enzymatic removal of the organic structures the 2.2. Solutions
erosion progression rate increases.12,14,15 Therefore, it can be
Mineral salt 4.08 mM H3PO4, 20.10 mM KCl, 11.90 mM
reasoned that erosion progression is influenced, at least in vitro, solution: Na2CO3, 1.98 mM CaCl2, pH 6.721
by the presence of the organic matrix. HCl-solution: 0.5 g NaCl solved in 100 ml of distilled
The organic matrix (mainly composed by collagens) is water, adjusted to pH 1.6 with
protected against enzymatic degradation as long as it is hydrochloric acid22
mineralised, but after demineralisation it can be assailed by Pepsin–HCl-solution: 750 mg/ml of pepsin (2400 U/ml)
(P-6887, pepsin from porcine
enzymes.12,15–17 During gastro oesophageal reflux disease
gastric mucosa, 3200 U/mg,
(GERD), the gastric enzyme pepsin can reach the oral cavity.18
Sigma–Aldrich, Seelze, Germany)10
Therefore, it is quite conceivable that during vomiting pepsin added to the HCl-solution
or even enzymes from pancreas, like trypsin, could also reach Trypsin-solution: 2000 BAEE units/ml of trypsin
the oral cavity and impact the dental tissues. It has been (T-9201, trypsin from bovine
previously shown that pepsin is able to degrade the isolated pancreas, 7500 BAEE U/mg,
organic dentine structures completely after a harsh and less Sigma–Aldrich, Seelze, Germany)
added to the mineral salt solution23
physiologic incubation time of more than three days.16,17
Additionally, our own studies with a simulated physiologic
situation have shown that pepsin is capable of degrading the 2.3. Treatment
eroded dentine matrix by approximately 25%.19,20 However,
the degradation was shown to have no influence on mineral All specimens were subjected to a cyclic de- and reminer-
loss.20 The remaining organic layer was probably sufficient to alisation procedure for six days.
act as the above mentioned diffusion barrier. However, Groups were defined as follows:
nothing is currently known about the impact of trypsin on
Group 1: No-Enzyme-group (control)
the eroded dentine matrix. Group 2: Pepsin-group
The aim of this in vitro study was therefore to investigate Group 3: Trypsin-group
the influence of pepsin, trypsin and the combination of both Group 4: Pepsin–Trypsin-group
on erosive mineral loss in dentine. Further, the extent of
degradation of the organic matrix was investigated by Six demineralisation periods were performed per day for
detection of collagen degradation products. The null-hypoth- 5 min each. Specimens of the No-Enzyme-group and the
esis was that there is no difference between the various Trypsin-group were demineralised with the HCl-solution and
treatments in mineral loss and collagen degradation. specimens of the Pepsin-group and the Pepsin–Trypsin-group
were demineralised with the Pepsin–HCl-solution. Addition-
ally, specimens of the Trypsin- and Pepsin–Trypsin-group
2. Materials and methods were treated with the Trypsin-solution for 10 min directly
after the demineralisation. Between treatments, specimens
2.1. Sample preparation were rinsed for 1 min with tap water. After demineralisation
(groups 1 and 2) and after trypsin-treatment (groups 3 and 4),
Dentine specimens were prepared from the coronal part of respectively, specimens were stored for 1 h as well as
previously impacted, freshly extracted human third molars overnight in a mineral salt solution. All procedures were
without cracks when inspected under a stereo microscope performed at 37 8C under gentle agitation. To achieve the same
(10-fold magnification, SMZ-1 Zoom Stereomicroscope, immersion time, specimens were put onto plastic sieves,
Nikon GmbH, Düsseldorf, Germany). Coplanar longitudinal which were transferred to containers filled with 150 ml of the
dentine slices were prepared (Exakt Trennschleifsystem, respective solution. All solutions were renewed at the
Exakt-Apparatebau, Norderstedt, Germany), ground flat to a beginning of an experimental day. At the end of each
thickness of 750 mm, and polished under sufficient water flow experimental day, 10 ml of the HCl-, Pepsin- and Trypsin-
(Exakt Mikroschleifgerät, Exakt-Apparatebau, Norderstedt, solutions were frozen at 20 8C until further analysis.
Germany; P800 and P1200 silicon carbide abrasive paper, Leco,
St. Joseph, USA). From these slices, specimens with a defined 2.4. Mineral loss measurement
surface area were prepared with a hollow drill (outer diameter
5 mm, Rio Grande, Albuquerque, USA). Then, specimens were Mineral loss was measured with longitudinal microradiogra-
embedded in light curing, acid resistant acrylate (Technovit phy.24 For this purpose, an X-ray-projection (Cu-K-radiation,
296 archives of oral biology 55 (2010) 294–299

20 kV, 50 mA, duration of exposure 2.5 min) of each sample Eindhoven, The Netherlands) equipped with a LaB6 cathode.
slice was made together with an aluminium-calibration step- The acceleration voltage was set to 5 kV. Images were recorded
wedge on a high resolution film (high speed holographic film, using a secondary electron (SE)-detector with the voltage of
Kodak SO-253, Kodak, Stuttgart, Germany). Radiographs were the collector grid biased to +300 V in order to improve the
made before the experiment (baseline) and after each signal to noise ratio and to reveal optimal topographical
experimental day. Films were developed under standardised contrast. The settings of the SEM including tilt angle, spot size,
conditions following the manufacture’s instructions. The scanning mode, etc. were kept constant for all sample groups.
mineral content was determined with a computer controlled The SEM-pictures were made with a 500-fold original
microdensitometer (Leitz MPV compact Ortholux II, Leitz, magnification.
Wetzlar, Germany). Mineral content values output from the
computer program were based on the assumption that the 2.7. Statistics
sample consisted of pure hydroxyapatite. Bulk tissue loss of a
given sample was calculated assuming that the mineral The statistics are given for data concerning mineral loss. All
content of dentine was 47 vol%.25 Cumulative tissue loss statistical procedures were performed with SPSS 15.0 for
(mm) at each day was defined as the difference to the values at Windows. Normal distribution of data was checked with the
baseline. Kolmogorov–Smirnov-test. For comparison of groups, analysis
of variance (ANOVA with Tukey’s post hoc) was used. Level of
2.5. Detection of collagen degradation significance was set at 0.05. Data are given as mean  standard
deviation.
Collagen degradation was measured with hydroxyproline
analysis after each experimental day in the HCl-, Pepsin-
and Trypsin-solutions. The amino acid hydroxyproline is 3. Results
specific for collagens26,27 and can be used as a quantitative
value for collagen degradation, which is determined photo- 3.1. Tissue loss
metrically in a test solution. Each solution (2 ml) was
hydrolysed together with 1 ml of 6 M HCl for 24 h at 105 8C Results are displayed in Table 1. In all groups, an increase of
in air-tight closed reaction vessels (Scintillation vessel 20 ml, cumulative mineral loss over time was measured. Between
MAGV, Rabenau, Germany). Afterwards, 0.8 ml of citrate– groups, differences become visible as of day 3 and become
acetate buffer was added to 0.2 ml of the hydrolysate to raise clearly apparent at day 6.
the pH to 5.5. Then, 0.5 ml of Chloramine-T reagent was added
and incubated for 20 min at room temperature. Next, the 3.2. Hydroxyproline content
solution was incubated with 0.5 ml of perchloric acid for
12 min at room temperature, and finally 0.5 ml of p-dimethyl- Each solution was hydrolysed three times. The results are
amino-benzaldehyde was added and incubated for 20 min at shown in mg/ml as the mean of the three hydrolysates (Fig. 1).
60 8C. A resulting colour shift from yellow to red indicated the No hydroxyproline was found in the HCl- and in the Trypsin-
presence of hydroxyproline. Content was determined photo- solutions. However, in the Pepsin-solutions relatively high
metrically within 1 h after last incubation (Visible UV amounts of hydroxyproline were found on each experimental
Spectrometer ‘‘Ultrospec 3000’’, Amersham Pharmacia Bio- day. In the Pepsin-solution of the Pepsin-group (group 2), the
tech, Cambridge, Great Britain; Measuring cells, No. 27111, mean content per day was 0.51  0.17 mg/ml. In the Pepsin-
Sarstedt, Nümbrecht, Germany) and expressed in mg/ml. solution of the combined Pepsin–Trypsin-group (group 4),
Unless otherwise noted, all chemicals were obtained from approximately 25% more hydroxyproline per day was found
Merck, Darmstadt, Germany. (0.64  0.12 mg/ml). Cumulatively, after six experimental days
in the Pepsin-solution of the Pepsin-group (group 2) and of the
2.6. Scanning electron microscopy Pepsin–Trypsin-group (group 4), 2.66 mg/ml and 3.31 mg/ml of
hydroxyproline was found, respectively.
After treatment and tissue loss measurement, moist speci-
mens were fractured into halves, critical point dried (Critical 3.3. Scanning electron microscopy
point dryer CPD 030, Baltec, Witten, Germany) and lightly gold
sputtered. Sample surfaces were inspected using a scanning On cross sections from all groups, a zone of totally
electron microscope (SEM/Type: XL20, Philips Electron Optics, demineralised dentine (organic matrix) was found [HCl-

Table 1 – Tissue loss (mm, mean W SD) in all groups and after each experimental day. Statistical significance between
groups within one experimental day is indicated by different superscript letters (ANOVA with Tukey’s post hoc; p = 0.05).
Group Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
a a a a,b a,b
HCl 48.1  14.5 62.9  20.8 83.1  25.1 102.0  23.6 127.4  26.0 139.1  29.5a,b
Pepsin 38.7  15.8 a 60.7  10.4 a 86.1  15.6 a 91.4  32.7 a 111.8  36.4 a 108.8  34.7 a
Trypsin 35.3  20.7 a 69.6  31.5 a 96.1  28.2a,b 118.8  24.1a,b 149.9  25.6 b 157.8  37.2 b
Pepsin–Trypsin 44.8  18.4 a 61.6  21.6 a 108.2  21.1 b 125.4  30.4 b 155.7  36.1 b 202.5  37.4 c
 archives of oral biology 55 (2010) 294–299 297

pH-optimum at pH 7–8 and needs calcium ions for an optimal

activity.31 Hence, it was added to the mineral salt solution at
physiological concentrations (2000 BAEE).23 Six demineralisa-
tion periods of 5 min each appears relatively harsh. Patients
suffering from bulimia nervosa reported that vomiting
frequencies of 6 times per day are not a rarity. Saliva is
capable of neutralising the acidic content of the stomach
within a few minutes,32,33 and therefore 5 min per erosive
challenge appeared meaningful. In patients with GERD the
conditions would probably be milder, since smaller volumes of
gastric juice reach the oral cavity and the neutralising
properties of the saliva have more effect. Trypsin can be
reversibly inactivated at low pH (i.e., during vomiting) and is
reactivated with an increase of pH34 in the presence of calcium
ions, i.e., during neutralising by saliva. Hence, the trypsin
Fig. 1 – Cumulative hydroxyproline content per day in the solution was applied after the erosive challenge to mimic
pepsin solutions of the Pepsin- and Pepsin–Trypsin-group these processes. An application duration of 10 min was chosen
(mg/ml). In the HCl- and trypsin-solutions, no because it was expected that trypsin is removed within this
hydroxyproline was found. time by saliva.
Regarding the results of the present study, the values for
mineral loss in the control group were comparable to a
previously performed study using the same erosive medium.20
(Fig. 2a), Pepsin- (Fig. 2b), Trypsin- (Fig. 2c) and Pepsin– Similar to the previously mentioned study, in the present
Trypsin-group (Fig. 2d)]. A relatively sharp border between the study the treatment with pepsin actually led to a slight
organic matrix and the sound dentine (dotted line) was found. decrease rather than an increase in substance loss, even
The thickness of the matrix was nearly the same in the HCl- though considerable amounts of hydroxyproline were found
and the Trypsin-group, and the matrix was only slightly in the pepsin solutions of this group. This can be explained by
thinner in the Pepsin-group. However, in the Pepsin–Trypsin- means of the SEM pictures. Even if the matrix is partially
group the organic matrix was considerably less thick than in degraded by pepsin, the residual matrix on the surface of the
the specimens from the other groups. specimens had considerable thickness and was only margin-
ally thinner (Fig. 2b) than in the HCl-group (Fig. 2a). The slight
decrease in mineral loss can possibly be explained by a change
4. Discussion in ultra-structure of the dentine matrix induced by the
enzymes, which was perhaps not depicted by SEM. This
Until now, the impact of various enzymes on the eroded would make the matrix more homogenous or denser, which
organic dentine matrix has been only rarely a focus of would form a more efficient diffusion barrier.
research; in particular, very little is known about the impact Hydroxyproline was not detectable in any of the trypsin
of different digestive enzymes on these organic structures and solutions. This was not expected because trypsin is a potent
their influence on erosive tissue loss. The experimental design proteolytic enzyme. Although it is known that trypsin can only
of this study should simulate the situation that could possibly degrade the native collagen matrix in eroded dentine to a
occur in patients with eating disorders in combination with limited extent,35,36 one would expect that after denaturation of
vomiting (bulimia nervosa). Even if nothing is published about the collagen by the strong hydrochloric acid or by pepsin,
the occurrence of digestive enzymes in the oral cavity in these trypsin would have higher collagenolytic activity, similar to
patients, it is all but certain that the gastric enzyme pepsin the study by Kleter et al.16 Why no hydroxyproline was found
reaches the oral cavity during vomiting. Additionally, also in the trypsin solutions cannot currently be explained, but this
intestinal enzymes (e.g. trypsin) can be present in the stomach is most likely the reason for the lack of difference between the
due to reflux of duodenal content.28 Therefore, it is also most HCl- and the Trypsin-group.
likely that this enzyme is as well regurgitated. Not only in case Interestingly, the combined impact of pepsin and trypsin
of eating disorders, but also in patients with GERD the led to an increase in mineral loss (45%) and in hydroxyproline
presence of digestive enzymes in the oral cavity can be content in the pepsin solutions (25%, Fig. 1) of the Pepsin–
relevant. Pepsin has been found in the mouth of these Trypsin-group, albeit no hydroxyproline was found in the
patients.18 And also a reflux of duodenal secretion, containing trypsin solution. It could be that the trypsin changed only the
bile acids and trypsin, into the stomach is often a symptom of structure of the organic matrix on an ultra-structural level or
severe cases of GERD.29 The duodenal secretion even often destabilised the tertiary structure of the collagen molecule
reaches the lower oesophagus30 and therefore potentially the without splitting the amino acid chain itself. This procedure
oral cavity. possibly opened more binding sites for pepsin and made the
Pepsin has its pH optimum between pH 1 and 3. Therefore, matrix more accessible for this enzyme, resulting in increased
it was added to the HCl-solution at a physiological concentra- degradation. This was supported by the findings from the SEM
tion (750 mg/ml)10; the demineralisation solution had a pH pictures (Fig. 2), showing that the matrix in the Pepsin–
comparable to this of gastric juice.22 However, trypsin has its Trypsin-group (Fig. 2d) was considerably thinner than in all
298 archives of oral biology 55 (2010) 294–299

Fig. 2 – Cross sections of specimens from the HCl-group (a), the Pepsin-group (b), the Trypsin-group (c) and the Pepsin–
Trypsin-group (d) after cyclic de- and remineralisation and enzyme treatment for 6 days. In the upper part, the
demineralised organic matrix is clearly visibly. Below, a relatively sharp boarder between the organic matrix and the sound
dentine can be found (dotted line). The matrix from the Pepsin–Trypsin-group is clearly thinner than the matrix in all other
groups (white bar = 50 mm).

other groups (Fig. 2a–c). This thinning diminished the function reach the oral cavity during vomiting and affect the eroded
of the matrix as a diffusion barrier, which normally slows dental hard tissue.
down the progression of erosive mineral loss,14 and led to a
rise in mineral loss in the Pepsin–Trypsin-group. These results Funding
were in concordance with findings from previous studies that
showed an increase of erosive tissue loss after a (total) There was no funding.
degradation of the organic matrix by collagenase.12,15
In conclusion, the digestive enzyme pepsin, but not trypsin,
was able to degrade the eroded organic dentine matrix. Conflict of interest
Additionally, pepsin alone, similar to trypsin, was not able to
increase erosive mineral loss. However, the combination of There is no conflict of interests.
both pepsin and trypsin led to an increase of erosive mineral
loss, which was probably a result of increased matrix
digestion. This mechanism could play a role in patients with Ethical approval
eating disorders in combination with vomiting who suffer
from dentine erosion because both enzymes could possibly Not relevant.
 archives of oral biology 55 (2010) 294–299 299

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