Original Paper
Caries Res 2018;52:113–118 Received: March 31, 2017
DOI: 10.1159/000479825
Dynamic Influence of pH on
Metalloproteinase Activity in Human
Coronal and Radicular Dentin
Stella F. do Amaral a, c Polliana M.C. Scaffa d Renata D.S. Rodrigues a
Douglas Nesadal b Marcia M. Marques a Fernando N. Nogueira b
Maria Angela Pita Sobral a
Departments of a Restorative Dentistry and b Biomaterials and Oral Biology, School of Dentistry, University of
São Paulo, and c Department of Dentistry, Cruzeiro do Sul University, São Paulo, and d Department of Biological
Sciences, Bauru School of Dentistry, University of São Paulo, Bauru, Brazil
Keywords
Coronal dentin · Dentin · Hydroxyproline · Matrix
metalloproteinases · Radicular dentin · Tooth erosion
Abstract
The aim of this study was to evaluate the effect of pH on the
activation of matrix metalloproteinases (MMPs) of human
coronal (CD) and radicular dentin (RD). CD and RD were pul-
verized to powder, and proteins were extracted with 1%
phosphoric acid. The extracted proteins and the demineral-
ized powder were separately incubated in the following so-
lutions: 4-aminophenylmercuric acetate (control) or a buf-
fer solution at different pHs (2.5, 4.5, 5.0, 6.0, and 7.0). After
incubation, proteins were separated by electrophoresis to
measure MMP activities by zymography. To assess the solu-
bilized dentin collagen, the demineralized dentin powder
was sustained in incubation buffer, and the amount of hy-
droxyproline (HYP) released was measured. Zymography
revealed MMP-2 gelatinolytic activities for CD and RD in all
experimental groups. For both substrates, the lowest pH so-
© 2018 S. Karger AG, Basel
E-Mail karger@karger.com
www.karger.com/cre
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Accepted after revision: July 26, 2017
Published online: January 4, 2018
lutions (2.5, 4.5, and 5.0) yielded higher gelatinolytic activ-
ity than those obtained by the highest pH solutions (6.0
and 7.0). For HYP analysis, no detectable absorbance values
were observed for pHs of 2.5 and 4.5. The amount of HYP
was higher for pH 7.0 than those of all other groups (p <
0.05), except for pH 6.0. No statistical differences were
found between pHs 6.0 and 5.0 and control (p > 0.05). The
MMP-2 enzyme from human CD and RD is dynamically in-
fluenced by pH: at low pH, the extracted enzyme activates
this latent form, whereas collagen degradation by the ma-
trix-bound enzyme is only observed when pHs are close to
neutral. © 2018 S. Karger AG, Basel
The most frequent exposure of coronal (CD) and ra-
dicular dentin (RD) caused by dental wear and gingival
recession [Hara et al., 2006] renders these substrates sus-
ceptible to erosive acids [Buzalaf et al., 2012]. Therefore,
dental erosion has been considered as a risk factor for
teeth [Lussi et al., 2011].
129.127.145.224 - 1/4/2018 1:39:20 PM
Maria Angela Pita Sobral
School of Dentistry, University of São Paulo
Av. Prof. Lineu Prestes 2227
São Paulo, SP 05508-900 (Brazil)
Univ.of Adelaide
Downloaded by:
E-Mail mapsobra @ usp.br
 Due to structural and compositional differences be-
tween enamel and dentin, the interaction between erosive
agents and dental tissue occurs differently to these sub-
strates [Lussi et al., 2011]. During the erosive process in
dentin, the mineral dissolution by acids occurs while the
organic portion is maintained [Kinney et al., 1995; Bres-
chi et al., 2002; Ganss et al., 2010]. It could be considered
that this organic matrix limits ion exchange into the de-
mineralized area [Klont and Ten Cate, 1991; Kleter et al.,
1994] and hence may be able to reduce the progression of
dentin erosion [Ganss et al., 2004]. However, this demin-
eralized organic matrix, although resistant to mechanical
brushing force [Ganss et al., 2009], can be chemically de-
graded by proteolytic enzymes [Klont and Ten Cate,
1991; Kleter et al., 1994; Schlueter et al., 2010], such as
metalloproteinases (MMPs).
MMPs are a family of endopeptidases with 23 isoforms
in humans [Visse and Nagase, 2003] that participate in
extracellular matrix degradation [Visse and Nagase, 2003;
Hannas et al., 2007]. Various types of these enzymes have
been identified in human dentin [Martin-De Las Heras et
al., 2000], as gelatinases MMP-2 and MMP-9, in their pro
and active forms, both in CD [Martin-De Las Heras et al.,
2000; Mazzoni et al., 2007] and RD [Santos et al., 2009;
Kato et al., 2011]. The great elucidation regarding the par-
ticipation of the host MMPs in the progression of human
dental caries was established when Tjäderhane et al.
[1998] showed that these latent enzymes are functional
when activated in conditions similar to dental caries (pH
4.5 followed by neutralization). These alternating periods
of de- and remineralization resulted in the exposure of
collagen fibers of dentin organic matrix, which were then
degraded by MMPs.
Only recently, some researchers [Buzalaf et al., 2012;
Zarella et al., 2015], have focused on the investigation of
the role of these enzymes in dental erosion. In this con-
text, the few available studies consider that the process of
organic matrix degradation is similar to that found for
tooth decay [Magalhães et al., 2009; Buzalaf et al., 2012].
However, the mechanisms involving caries and erosion
processes are distinct. Regarding caries, the studies con-
sider pH 4.5 for MMP activation as it is described as the
critical pH for mineral dissolution and the development
of carious lesions. Nevertheless, for dental erosion, there
is no defined critical pH for mineral dissolution [Lussi et
al., 2011]. Moreover, the process of demineralization in
dentin caries lesions can last a longer time; while this oc-
curs very quickly in dental erosion, sometimes occurring
in seconds, the degradation of the organic matrix can be
done in a totally different way [Tjäderhane et al., 2015].
114 Caries Res 2018;52:113–118
DOI: 10.1159/000479825
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Recent literature [Kato et al., 2009; Magalhães et al.,
2009; Kato et al., 2010; Zarella et al., 2015; De Moraes et
al., 2016] about the role of these endogenous enzymes in
the progression of dental erosion has focused on the use
of MMP inhibitors in order to prevent this type of lesion.
These studies evaluated the final effects of different MMP
inhibitors regarding the loss of dentin, using indirect
methods of evaluation such as profilometry. However,
MMP activity was not evaluated using the appropriate
and relevant biological methods to confirm their role in
the prevention of dental erosion.
Thereafter, before knowing the protective effect caused
by the MMP inhibitors, it is important to elucidate if
changes in pH occurring during the dental erosion pro-
cess would effectively be able to cause the activation of
dentin MMPs and, subsequently, their role in the pro-
gression of this type of lesion. Thereby, the aim of this in
vitro study was to evaluate the effect of pH on MMP ac-
tivity and function in human CD and RD.
Materials and Methods
Preparation of Dentin Powder
This study was approved by the Ethical Committee of the
School of Dentistry of the University of São Paulo (protocol
#544.544). Twenty human third molars were obtained immedi-
ately after extraction from young volunteers (18–31 years). Or-
ganic debris was immediately removed, and teeth were stored dry
at –80 ° C for no more than 1 month. Then, CD and RD portions
were separated with a low-speed saw (Isomet® 1000 Precision Saw;
Buehler Corporation, Lake Bluff, IL, USA) under water-cooling
and sectioned in 1-mm-thick slices. All pulp tissue was scraped off
with endodontic files, and all enamel and cementum were re-
moved from the CD and RD slices with abrasive disks under water-
cooling using a grinder and polisher (EcoMet®: twin variable
speed; Buehler). CD and RD slices were separately pulverized into
powder, at low temperature, in a mixer mill (MM 400; Retsch,
Newtown, PA, USA). The dentin powder aliquots obtained were
kept frozen until use.
Dentin Protein Extraction
Four 0.5-g aliquots of CD and RD powders, respectively, were
used for the extraction of dentin proteins. The extraction protocol
was performed as described by Breschi et al. [2010]. Briefly, dentin
aliquots were demineralized in 1% aqueous phosphoric acid (pH
1.5) for 10 min. Then, the acid was buffered with 4 M NaOH solu-
tion, centrifuged for 10 min, and the supernatant discarded. The
powder was rinsed with distilled water (DW), centrifuged (1,540 g
for 10 min at 4 ° C) and then resuspended in 1 mL of extraction
buffer (50 mM Tris-HCl, pH 6, containing 5 mM CaCl2, 100 mM
NaCl, 0.1% Triton X-100, 0.1% nonionic detergent P-40, 0.1 mM
ZnCl2, 0.02% NaN3) for 24 h. Samples were ultrasonicated (Thorn-
ton Eletrônica Ltda., São Paulo, Brazil) at 30 W for 10 min at 4 ° C
and centrifuged (1,540 g for 10 min at 4 ° C) to separate the pre-
cipitate from the supernatant. The precipitate (demineralized den-
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Amaral et al.
Univ.of Adelaide
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 tin powder) was lyophilized and stored at –20 ° C until use in the
hydroxyproline (HYP) analysis (matrix-bound enzyme activity).
The supernatant (protein extract) was centrifuged (1,540 g for 10
min at 4 ° C) and concentrated in a Vivaspin centrifugal concentra-
tor (10,000-kDa molecular weight cutoff; GE Healthcare, Little
Chalfont, UK) for 50 min at 4 ° C. The total protein concentration
obtained in the protein extracts (CD and RD) was measured in
triplicate using the protein assay of Lowry et al. [1951] (CD =
6,906.3 ± 0.55 μg/mL and RD = 7,172.8 ± 1.72 μg/mL). These sam-
ples were used in the zymography analysis (extracted enzyme ac-
tivity).
Experimental Groups
To evaluate the pH effect on MMP activity, 0.1 M potassium
phosphate (KH2PO4) buffer at different pHs (2.5, 4.5, 5.0, 6.0, and
7.0) and a solution of 2 mM of 4-aminophenylmercuric acetate
(APMA) was used as experimental groups. APMA, which is an
organomercurial compound commonly used for activating endog-
enous MMPs [Tjäderhane et al., 1998], was chosen as a positive
control.
Zymography Analysis
The samples of dentin protein aliquots (80 μg) were incubated
according to each experimental group, as described above, for 1 h
at 37 ° C. After their dilution in sample buffer at a 1:4 ratio (buffer/
sample), they were subjected to zymography as described else-
where [Breschi et al., 2010] with some modifications. Briefly, the
samples were submitted to electrophoresis under nonreducing
conditions in 10% sodium dodecyl sulfate-polyacrylamide gel
(SDS-PAGE) containing 1 mg/mL gelatin. Prestained molecular
weight standards (Precision Plus Protein® Kaleidoscope®; Bio-
Rad, Hercules, CA, USA) were used as molecular weight markers.
After electrophoresis, the gels were washed for 1 h in 2% Triton
X-100 and incubated in activation solution (50 mM Tris-HCl,
5 mM CaCl2, pH 7.4) for 24 h. The gels were stained with 0.125%
Coomassie blue R-250 for 1 h and destained in a methanol solu-
tion. After that, the zymograms (n = 3) were scanned (Gel Doc XR
System; Bio-Rad) and analyzed using ImageJ software (National
Institutes of Health, Bethesda, MD, USA).
HYP Analysis
The dentin powder precipitates (CD and RD) obtained after
protein extraction were lyophilized and separated into 8 aliquots
of 30 mg for each experimental group. The samples were rinsed
with DW, centrifuged (14,000 g for 10 min at 4 ° C), and then the
supernatant was removed. The pellet was resuspended and incu-
bated in 1 mL of each experimental solution. Subsequently, the
samples were centrifuged, the supernatant was removed, and the
pellet was incubated in 1 mL of AS (50 mM HEPES, 5 mM, CaCl2 · ×
2H2O, 0.001 mM ZnCl2, 150 mM NaCl, 0.3 mM PMSF, and 3 mM
NaN3; pH 7.2) for 24 h at 37 ° C, under constant agitation. After
incubation, AS was analyzed for dentin collagen solubilization by
measuring the HYP content released according to a protocol
slightly modified from that described by DeVito-Moraes et al.
[2016]. Briefly, 500 μL of each AS sample were lyophilized and re-
suspended in 10 μL DW in 2-mL screw-capped test tubes. Then,
2 N NaOH solution was added before hydrolysis obtained by auto-
claving at 120 ° C for 20 min, and 450 μL of chloramine T was add-
ed to the hydrolyzate and gently mixed. This solution was left at
room temperature for 25 min to allow oxidation. The chromo-
pH Influence on MMP Functional
Activity
Slaika Seguias - soicinco.ca@hotmail.com - IP: 190.216.253.12
phore was developed by incubation with 500 μL of Ehrlich reagent
at 65 ° C for 40 min. At the end, the absorbance of the sample was
read for each group at 550 nm in a spectrophotometer (DU 800
Spectrophotometer; Beckman Coulter, Brea, CA, USA) against
standard curves of known HYP concentrations (2, 5, 10, 15, and 25
μg/mL).
Statistical Analysis
The amount of HYP (μg HYP/mg dentin) released was ana-
lyzed using one-way ANOVA complemented by the Tukey post
hoc test. Statistical significance was defined as p < 0.05.
Results
Zymography Analysis
Zymography revealed gelatinolytic activity for both
CD (Fig. 1a, b) and RD (Fig. 1c, d) for all experimental
groups tested. The bands were detected at 66/72 kDa, cor-
responding to active and latent forms of MMP-2, respec-
tively.
Regarding CD, zymograms (Fig. 1a) showed that the
lowest pH solution (pH 2.5) yielded the higher gelatino-
lytic activity of the 66-kDa bands. The other groups with
lower pH values (pHs 4.5 and 5.0) also showed high
MMP-2 activity compared to solutions with pHs close to
neutral (pHs 6.0 and 7.0). These 3 lower pH values showed
higher activity than that observed by APMA (positive
control) activation (Fig. 1b). Regarding the latent form
(72kDa) of MMP-2, less intense bands were found com-
pared to its active form.
Regarding RD, zymograms (Fig. 1c) showed increased
activity of the active MMP-2 form (66 kDa) for the groups
treated with lowest pHs (2.5, 4.5, and 5.0), being greater
at pH 4.5 than in groups with pHs close to neutral (pHs
6.0 and 7.0) (Fig. 1d). The latent form of MMP-2 (72-
kDa) also identified in the zymograms presented less in-
tense bands than those of its active form.
HYP Analysis
Dentin collagen solubilization was evaluated by mea-
suring the amount of HYP released, assuming that type I
collagen contains 10% HYP [Bornstein and Sage, 1980].
The average concentrations of HYP released in the AS
storage medium after different treatments are shown in
Figure 2. At pHs 2.5 and 4.5, groups showed absorbance
values below the detection limits of the method; there-
fore, they were not considered in the statistical analysis.
For the other groups, for both CD (Fig. 2a) and RD
(Fig. 2b), statistically significant differences were found
(p < 0.05). The amount of HYP was higher in the pH 7.0
group than in all other groups (p < 0.05), except for the
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Caries Res 2018;52:113–118 115
DOI: 10.1159/000479825
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 APMA 2.5 4.5 5.0 6.0 7.0 pH APMA 2.5 4.5 5.0 6.0 7.0 pH
72 kDa 72 kDa
a 66 kDa c 66 kDa
25 25

20 20
MMP-2 activity, AU

MMP-2 activity, AU
15 15

10 10

5 5

0 0
b APMA 2.5 4.5 5.0 6.0 7.0 pH d APMA 2.5 4.5 5.0 6.0 7.0 pH

Fig. 1. a, c Representative zymograms obtained after electrophore- b, d Optical density of MMP-2 activity from coronal (b) and ra-
sis of proteins extracted from coronal (a) and radicular dentin (c) dicular dentin (d) extracts after different treatments (control and
in polyacrylamide gels showing enzymatic activity of matrix experimental groups). AU, arbitrary units. Means ± SD. APMA,
metalloproteinase (MMP)-2 (control and experimental groups). 4-aminophenylmercuric acetate.

1.8 1.2
b b
1.6
1.0 a, b
1.4
a
HYP, μg/mg dentin

HYP, μg/mg dentin

a
1.2 a, b 0.8
1.0
a 0.6
0.8
Fig. 2. Hydroxyproline (HYP) concentra- a
0.6 0.4
tion released in the artificial saliva storage
0.4
medium after different pH treatments. 0.2
Means ± SD. Different letters indicate sta- 0.2
tistical differences. a Coronal dentin. b Ra- 0 0
dicular dentin. APMA, 4-aminophenylmer- a APMA 5.0 6.0 7.0 pH b APMA 5.0 6.0 7.0 pH
curic acetate.

pH 6.0 group. No statistical differences were found among tion and pH, showing that the activation of MMP-2 pres-
the pH 6.0, pH 5.0, and control groups (p > 0.05) for both ent in the human CD and RD is pH dependent. Addition-
substrates. ally, dentin solubilization, which is dependent on the ac-
tivity of matrix-bound enzymes, increased when pH
increased. Thus, the proteolytic activity of extracted and
Discussion matrix-bound dentin enzymes was inversely proportion-
al. In fact, it would be expected that once the enzyme was
The mechanisms underlying dentin degradation by bound to the dentin, it was not supposed to be available
dentin MMPs in erosion lesions are still unknown. As low in the extraction medium for zymography analysis.
pH substances are involved in the development of such Based on our results, we can suppose that MMP-2 is
lesions, we hypothesized that pH could be of relevance in activated at low pHs, and its activity was greater at these
the activation and function of endogenous dentin MMPs. pHs in zymography. However, the matrix-bound enzyme
Confirming this hypothesis, the activity of MMPs extract- was active at higher pHs once the HYP released was de-
ed from dentin was affected by pH. Zymographic analysis tected when the pHs increased. Thus, MMP-2 in dentin
revealed an inverse relationship between gelatin degrada- exhibits activation at low pH, whereas its degrading effect
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116 Caries Res 2018;52:113–118 Amaral et al.

DOI: 10.1159/000479825
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Slaika Seguias - soicinco.ca@hotmail.com - IP: 190.216.253.12

 on the exposed collagen fibers will be reached at higher
pHs, similar to what occurs in the activation of salivary
MMPs, as described by Tjäderhane et al. [1998] as a
mechanism of “acid activation.”
The presence and abundance of MMP-2 in the human
CD has been found to be inversely dependent on patient
age [Martin-De Las Heras et al., 2000]. Thus, it is expect-
ed to have higher MMP-2 activity in RD than CD, since
the crown is always formed before the roots, as discussed
by Santos et al. [2009]. Although in the present study zy-
mography of CD and RD was not carried out in the same
gel, the results seemed to show a more marked MMP-2
activity in RD than CD, which corroborates previous
findings [Santos et al., 2009; Kato et al., 2011].
Dentin substrate is more soluble than the enamel sub-
strate, and not only its composition but also a set of other
factors will determine the driving force for mineral dis-
solution during dental erosion [Shellis et al., 2014]. It is
known that the presence of subsaturated solutions with
respect to the tooth structure is an essential factor for de-
mineralization to occur [Lussi et al., 2011]. Regarding the
chemical factors related to erosion, not only the pH but
also the calcium, phosphate, and fluoride contents pres-
ent in acidic foods and drinks will influence the degree of
saturation with respect to the mineral structure of the
substrate. Furthermore, saliva is the main biological fac-
tor in preventing this mineral dissolution and is also im-
portant during dental erosion [Hara et al., 2006]. In fact,
when enamel is dissolved at low pH substances, the neu-
tralization of this pH by saliva will inhibit further enamel
dissolution. However, we believe that in dentin the effect
of neutralization by saliva will make the worst erosion:
when activated by the dissolution of the dentin minerals,
the MMP-2 in a higher pH determined by the neutraliza-
tion of the saliva will be more dangerous to the dentin
substrate. Then, although saliva will dilute the extracted
MMP-2, the establishment of a neutral pH condition will
allow the activity of bound enzymes, which will degrade
the exposed collagen fibrils in the erosion lesion. More
than that, the use of liquids with buffer activity will also
have the same effect: they will provide the conditions for
dentin degradation. Thus, if only the MMPs would be in-
volved in dentin erosion lesions in the mouth, this ero-
sion would always progress, even in the presence of min-
eralizing substances. However, other enzymes and sub-
stances are also involved in the erosion process, e.g., tissue
inhibitors of metalloproteinase, which can inhibit MMP
activity.
In conclusion, although the prevalence of gingival re-
cession increases with age, this condition should not only
pH Influence on MMP Functional
Activity
Slaika Seguias - soicinco.ca@hotmail.com - IP: 190.216.253.12
be seen as a result of the aging process, since other factors
may be involved, such as those related to toothbrushing
[Heasman et al., 2015]. In addition, the prevalence of
tooth wear caused by dental erosion shows an increase
worldwide, mainly in the young population, due to
changes in the diet and lifestyle [Jaeggi and Lussi, 2014].
For this reason, the findings of this study support previ-
ous studies stating the importance of using MMP inhibi-
tors to protect dentin, even following minor MMP expo-
sure to the oral cavity, after ingestions of low-pH food
and drinks.
New studies are required to elucidate the real role of
MMPs in the mechanisms underlying the formation and
progression of erosion lesions in human dentin.
Acknowledgements
S.F.F.A would like to thank CNPq (National Council of Tech-
nological and Scientific Development) for scholarship
(#143072/2011-0). M.M.M. is also supported by CNPq. The
funders had no role in the study design, data collection, and analy-
sis, decision to publish, or preparation of the paper.
Disclosure Statement
The authors report no conflict of interest.
Author Contributions
S.F.F.A., P.M.C.S., M.M.M., N.F.N., and M.A.P.S. conceived
and designed the experiments; S.F.F.A., R.D.S.R., and D.N. per-
formed the experiments; S.F.F.A., M.M.M., R.D.S.R., D.N., and
N.F.N. analyzed the data, and S.F.F.A., P.M.C.S., M.M.M., and
M.A.P.S. wrote the paper.
References Bornstein P, Sage H: Structurally distinct collagen
types. Annu Rev Biochem 1980;49:957–1003.
Breschi L, Gobbi P, Mazzotti G, Falconi M, Ellis
TH, Stangel I: High resolution SEM evalua-
tion of dentin etched with maleic and citric
acid. Dent Mater 2002;18:26–35.
Breschi L, Mazzoni A, Nato F, Carrilho M, Visin-
tini E, Tjäderhane L, Ruggeri A Jr, Tay FR,
Dorigo ES, Pashley DH: Chlorhexidine stabi-
lizes the adhesive interface: a 2-year in vitro
study. Dent Mater 2010;26:320–325.
Buzalaf MA, Kato MT, Hannas AR: The role of
matrix metalloproteinases in dental erosion.
Adv Dent Res 2012;24:72–76.
129.127.145.224 - 1/4/2018 1:39:20 PM
Caries Res 2018;52:113–118 117
DOI: 10.1159/000479825
Univ.of Adelaide
Downloaded by:
 De Moraes MD, Carneiro JR, Passos VF, Santiago
SL: Effect of green tea as a protective measure
against dental erosion in coronary dentine.
Braz Oral Res 2016;30:e13.
DeVito-Moraes AG, Francci C, Vidal CMP, Scaf-
fa PMC, Nesadal D, Yamasaki LC, Nicolau J,
Nascimento FD, Pashley DH, Carrilho MR:
Phosphoric acid concentration affects dentin-
al MMPs activity. J Dent 2016;53:30–37.
Ganss C, Hardt M, Blazek D, Klimek J, Schlueter
N: Effects of toothbrushing force on the min-
eral content and demineralized organic ma-
trix of eroded dentine. Eur J Oral Sci 2009;
117:255–260.
Ganss C, Hardt M, Lussi A, Cocks AK, Klimek J,
Schlueter N: Mechanism of action of tin-con-
taining fluoride solutions as anti-erosive
agents in dentine – an in vitro tin-uptake, tis-
sue loss, and scanning electron microscopy
study. Eur J Oral Sci 2010;118:376–384.
Ganss C, Klimek J, Starck C: Quantitative analysis
of the impact of the organic matrix on the flu-
oride effect on erosion progression in human
dentine using longitudinal microradiogra-
phy. Arch Oral Biol 2004;49:931–935.
Hannas AR, Pereira JC, Granjeiro JM, Tjäderhane
L: The role of matrix metalloproteinases in
the oral environment. Acta Odontol Scand
2007;65:1–13.
Hara AT, Lussi A, Zero DT: Biological factors.
Monogr Oral Sci 2006;20:88–99.
Heasman PA, Holliday R, Bryant A, Preshaw PM:
Evidence for the occurrence of gingival reces-
sion and non-carious cervical lesions as a con-
sequence of traumatic toothbrushing. J Clin
Periodontol 2015;42(suppl 16):S237–S255.
Jaeggi T, Lussi A: Prevalence, incidence and dis-
tribution of erosion. Monogr Oral Sci 2014;
25:55–73.
118 Caries Res 2018;52:113–118
DOI: 10.1159/000479825
Slaika Seguias - soicinco.ca@hotmail.com - IP: 190.216.253.12
Kato MT, Hannas AR, Leite AL, Bolanho A, Za-
rella BL, Santos J, Carrilho M, Tjäderhane L,
Buzalaf MA: Activity of matrix metallopro-
teinases in bovine versus human dentine.
Caries Res 2011;45:429–434.
Kato MT, Leite AL, Hannas AR, Buzalaf MA: Gels
containing MMP inhibitors prevent dental
erosion in situ. J Dent Res 2010;89:468–472.
Kato MT, Magalhães AC, Rios D, Hannas AR, At-
tin T, Buzalaf MA: Protective effect of green
tea on dentin erosion and abrasion. J Appl
Oral Sci 2009;17:560–564.
Kinney JH, Balooch M, Haupt DL Jr, Marshall SJ,
Marshall GW Jr: Mineral distribution and di-
mensional changes in human dentin during de-
mineralization. J Dent Res 1995;74:1179–1184.
Kleter GA, Damen JJ, Everts V, Niehof J, Ten Cate
JM: The influence of the organic matrix on
demineralization of bovine root dentin in vi-
tro. J Dent Res 1994;73:1523–1529.
Klont B, Ten Cate JM: Susceptibility of the collag-
enous matrix from bovine incisor roots to
proteolysis after in vitro lesion formation.
Caries Res 1991;25:46–50.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ:
Protein measurement with the Folin phenol
reagent. J Biol Chem1951;193:265–275.
Lussi A, Schlueter N, Rakhmatullina E, Ganss C:
Dental erosion – an overview with emphasis
on chemical and histopathological aspects.
Caries Res 2011;45(suppl 1):2–12.
Magalhães AC, Wiegand A, Rios D, Hannas A,
Attin T, Buzalaf MA: Chlorhexidine and
green tea extract reduce dentin erosion and
abrasion in situ. J Dent 2009;37:994–998.
Martin-De Las Heras S, Valenzuela A, Overall
CM: The matrix metalloproteinase gelatinase
A in human dentine. Arch Oral Biol 2000;45:
757–765.
Mazzoni A, Mannello F, Tay FR, Tonti GA, Papa
S, Mazzotti G, et al: Zymographic analysis and
characterization of MMP-2 and -9 forms in
human sound dentin. J Dent Res 2007; 86:
436–440.
Santos J, Carrilho M, Tervahartiala T, Sorsa T,
Breschi L, Mazzoni A, Pashley D, Tay F, Fer-
raz C, Tjäderhane L: Determination of matrix
metalloproteinases in human radicular den-
tin. J Endod 2009;35:686–689.
Schlueter N, Hardt M, Klimek J, Ganss C: Influ-
ence of the digestive enzymes trypsin and
pepsin in vitro on the progression of erosion
in dentine. Arch Oral Biol 2010;55:294–299.
Shellis RP, Featherstone JD, Lussi A: Understand-
ing the chemistry of dental erosion. Monogr
Oral Sci 2014;25:163–179.
Tjäderhane L, Buzalaf MAR, Carrilho M, Chaus-
sain C: Matrix metalloproteinases and other
matrix proteinases in relation to cariology:
the era of “dentin degradomics.” Caries Res
2015;49:193–208.
Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Lar-
mas M, Salo T: The activation and function of
host matrix metalloproteinases in dentin ma-
trix breakdown in caries lesions. J Dent Res
1998;77:1622–1629.
Visse R, Nagase H: Matrix metalloproteinases and
tissue inhibitors of metalloproteinases: struc-
ture, function, and biochemistry. Circ Res
2003;92:827–839.
Zarella BL, Cardoso CA, Pelá VT, Kato MT, Tjä-
derhane L, Buzalaf MA: The role of matrix
metalloproteinases and cysteine-cathepsins
on the progression of dentine erosion. Arch
Oral Biol 2015;60:1340–1345.
129.127.145.224 - 1/4/2018 1:39:20 PM
Amaral et al.
Univ.of Adelaide
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