UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2023.e3946
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Effect of 10% Proanthocyanidin gel on demineralized organic matrix
degradation: ELISA method
Efeito do gel de Proantocianidina a 10% na degradação da matriz orgânica desmineralizada: método ELISA
Fabrícia CARDOSO1 , Gabriela Guarda DALLAVILLA1 , Ana Paula BOTEON1 , Angélica Feltrin dos SANTOS1 ,
Thiago José DIONÍSIO2 , Heitor Marques HONÓRIO3
1 - Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Dentística, Endodontia e Materiais Odontológicos,
Bauru, SP, Brazil.
2 - Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Biologia Oral, Bauru, SP, Brazil.
3 - Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Odontopediatria, Ortodontia e Saúde Coletiva, Bauru,
SP, Brazil.
How to cite: Cardoso F, Dallavilla GG, Boteon AP, Santos AF, Dionísio TJ, Honório HM. Effect of 10% Proanthocyanidin gel on demineralized
organic matrix degradation: ELISA method. Braz Dent Sci. 2023;26(4):e3946. https://doi.org/10.4322/bds.2023.e3946
ABSTRACT
Objective: This study evaluated Proanthocyanidin protective effect on dentin subjected to erosion and its inhibition on degradation
of the demineralized organic matrix (DOM). Material and Methods: The tested groups were: G1 - 10% Proanthocyanidin
gel (test group), G2 - 1.23% NaF (positive control 1), G3 - 0.012% Chlorhexidine (positive control 2) and G4 – Placebo
(negative control with no active compound) and two methodologies were performed: contact prolometry and ICTP ELISA method.
To quantify dentin wear, prolometry was performed. Data were submitted to Analysis of Variance followed by Fisher’s LSD Test.
To assess the collagen degradation, ICTP ELISA method was performed. Data were submitted to the Kruskal-Wallis followed by
the Dunn´s test. Simple linear regression and Pearson Correlation test were also performed (p<0.05). Results: The prolometry
showed signicantly lower wear of G1 when compared to other groups and G2, G3 and G4, which did not present signicant
difference among them. In the ICTP ELISA analysis, G1 and G4 did not show signicant differences and the same happened between
G2 and G3. However, G1 and G4 had lower values of collagen degradation compared to groups G2 and G3. Data showed that
degraded DOM is a signicant predictor to explain the values obtained through the ICTP ELISA. Conclusions: The results allow
to verify that 10% proanthocyanidin provided less tooth wear and decreased degradation of the DOM, suggesting a good ability to
prevent dentin erosion. The regression analysis also suggests that contact prolometry is a good strategy to quantify dentin wear.
KEYWORDS
Dentin; Tooth erosion; Proanthocyanidins; Dental wear; Preventive health.
RESUMO
Objetivo: Este estudo avaliou o efeito protetor da proantocianidina na dentina submetida à erosão e sua inibição na degradação
da matriz orgânica desmineralizada (MOD). Material e Métodos: Os grupos testados foram: G1 - gel de Proantocianidina
10% (grupo teste), G2 - NaF 1,23% (controle positivo 1), G3 - Clorexidina 0,012% (controle positivo 2) e G4 - Placebo
(controle negativo sem princípio ativo) e duas metodologias foram realizadas: perlometria de contato e método ICTP ELISA.
Para quanticar o desgaste da dentina, a perlometria foi realizada. Os dados foram submetidos à Análise de Variância seguida
do Teste LSD de Fisher. Para avaliar a degradação do colágeno, foi realizado o método ICTP ELISA. Resultados: Os dados foram
submetidos ao teste de Kruskal-Wallis seguido do teste de Dunn. Regressão linear simples e teste de correlação de Pearson
também foram realizados (p<0,05). A perlometria mostrou desgaste signicativamente menor do G1 quando comparado
aos outros grupos e G2, G3 e G4, que não apresentaram diferença signicativa entre si. Na análise ICTP ELISA, G1 e G4 não
apresentaram diferenças signicativas e o mesmo ocorreu entre G2 e G3. No entanto, G1 e G4 apresentaram valores menores de
degradação do colágeno em relação aos grupos G2 e G3. Os dados mostraram que a MOD degradada é um preditor signicativo
para explicar os valores obtidos pelo ICTP ELISA. Conclusão: Os resultados permitem vericar que a proantocianidina a 10%
proporcionou menor desgaste dentário e diminuição da degradação da MOD, sugerindo uma boa capacidade de prevenir a
erosão dentinária. Também sugere que a perlometria de contato é uma boa estratégia para quanticar o desgaste da dentina.
PALAVRAS-CHAVE
Dentina; Erosão dentária; Proantocianidinas; Desgaste dentário; Saúde preventiva.
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
INTRODUCTION
Dentin is composed by 47% inorganic
components (apatite), 33% organic components and
20% water. Among the organic components, 90%
consists of type I collagen and 10% non-collagenous
components: dentin phosphoproteins, proteoglycans
and glycosaminaglycans [1-3]. Demineralization
by erosion causes histological changes in dentin,
starting with an external demineralization [4]. The
dentin demineralization rate decreases when the
amount of degradable collagen increases, thus, the
maintenance of this collagen hinders the diffusion
of acids to the tissue, minimizing erosion [5,6].
However, the organic matrix can be chemically
degraded due to the presence in dentin enzymes
called matrix metalloproteinases (MMPs), which
can be activated by a drop in pH below 4.5 [7].
The degradation of the dentin organic matrix is
only possible after neutralization of saliva pH, since,
although these enzymes are activated at acidic pH,
they do not have the capacity to degrade the dentin
matrix at the same pH [7,8].
In addition to dentin demineralization,
the drop in pH exposes collagen fibrils and
activates MMPs that degrade the demineralized
organic matrix (DOM). This process causes the
progressive loss of dentinal tissue. Thus, the use
of MMP inhibitors could reduce this mechanism
during subsequent erosion challenges, as the
organic matrix would function as a protective
layer, reducing erosion progression [5,6].
Several studies have proven this mechanism
of inhibition, showing a reduction in the
progression of erosion when chlorhexidine [9],
green tea [10] and other substances [11] were
used as MMP inhibitory agents.
Currently, proanthocyanidin (PA) has drawn
the attention of researchers as an alternative
MMP inhibitor agent. It is a natural compound
derived from fruits, vegetables, nuts, among
others [12,13], which has lower toxicity when
compared to synthetic products [14,15]. Studies
have shown that PA has a great affinity with
proline-rich proteins, such as collagen, in addition
to being responsible for increasing the capacity
of cells to synthesize collagen [12]. Other studies
have characterized the behavior of PA on dentin,
showing its effects on the resin-dentin bonding
interface. In these cases, there was a signicant
improvement in adhesion and mechanical
properties, making adhesive restorations more
resistant and more long-lasting [16-18].
A recent
in situ
study demonstrated that PA
can play an important role in preventing erosion
after evaluating the effect t of a mouthwash on
dentin submitted to erosion [19]. The mouthwash
provided signicantly lower wear values compared
to placebo and the control group. This result
may be due to the maintenance of a large part
of the DOM intact after the erosive challenge.
To demonstrate this effect, specic studies are needed
to enable the analysis of the post-demineralization
organic matrix, such as hydroxyproline dosage,
zymography or ICTP Elisa [20].
Analysis by zymography allows visualization of
enzymatic activity, but it is not possible to quantify
the activity of MMPs on type 1 collagen [20]. The
hydroxyproline analysis was used to determine
collagen degradation [11,21], although its validity
is also discussed due to subjectivity [21]. Another
alternative to determine collagen degradation is
the use of specic ICTP and CTX ELISA method for
enzymes such as MMPs and cysteine cathepsins,
respectively. Despite the high cost of the method,
the advantage of this indirect approach is that
one may assay the total protease activities of the
dentin matrix in their natural bound state, and
their responses to activators and inhibitors [20,22].
In addition, ICTP level determination is one of the
most reliable techniques to quantify the activity of
MMPs on type I collagen [20].
Therefore, this study aimed to evaluate
the effect of 10% Proanthocyanidin in the form
of a topical gel to minimize the wear of dentin
submitted to erosion and to inhibit the degradation
of the DOM dentin through contact prolometry
and the specic ICTP ELISA analysis.
MATERIAL AND METHODS
Methodology 1
Experimental design
This cross-sectional simple-blind randomized
study was approved by the Research and Ethics
Committee of the Bauru School of Dentistry, University
of São Paulo (Process: 94400218.8.0000.5417).
Human molar teeth were used in this study and
dentin specimens obtained were randomly allocated into
4 groups: G1 - 10% Proanthocyanidin gel (test group),
G2 - 1.23% NaF (positive control 1), G3 - 0.012%
Chlorhexidine (positive control 2) and G4 – Placebo
(negative control with no active compound).
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
Before the treatment, samples were demineralized by
immersion in 0.87 M citric acid, pH 2.3 (36 h at 4ºC).
Then, the studied gels were applied once over dentin
for 1 minute. Next, the samples were immersed in
articial saliva containing collagenase obtained from
Clostridium histolyticum
for 5 days. The response
variable was depth of dentin loss measured by means
of prolometry.
Blocks preparation
In total, 40 specimens were prepared from
extracted human teeth. Each dentin specimen
(4x4x3 mm) was obtained from a single human
tooth and was stored in 0.1% thymol solution
(pH 7.0) at 4°C. Each sample was obtained by
sectioning the crown longitudinally (Figure 1a)
with 2 parallel diamond discs (XLI 2205, Extec
Corp., Eneld, CT, USA), that allowed the removal
of occlusal enamel, creating a dentin slice. Next,
samples were sectioned to obtain dentin blocks
(4x4 mm) (Figure 1b), which were stored in 0.1%
thymol solution (pH 7.0) at 4oC. The surface of
the specimens was polished (Figure 1c) using
water-cooled carborundum discs (320, 600 and
1200 grades of Al2O3 papers; Buehler, Lake Bluff,
IL, USA) and a 1-μm diamond solution (Buehler).
Prior to treatment, marks were made on the surfaces
of the samples using a scalpel blade for the precise
repositioning on the equipment. Subsequently,
three baseline surface profiles were obtained
from each wet blocks (only the excess of water
was carefully removed with lter paper) using a
prolometer (MarSurf GD 25, Göttingen, Germany)
at certain distances from the edge: 2.0, 1.75, and
1.5μm (Figure 1d). The marks and external dentin
surface were covered with nail varnish (Cosmed
Indústria de Cosméticos e Medicamento
s, S/A,
Barueri, São Paulo, Brazil) (Figure 1e) in order to
allow reference surfaces for wear analysis.
Treatment
Dentin specimens were demineralized by
immersion in 0.87 M citric acid (Figure 1f), pH
2.3 (36 h at 4ºC). Next, samples were thoroughly
rinsed in deionized water (30 sec). Excess
water was removed with absorbent paper. After
demineralization the nail varnish was removed
(Figure 1g) and three profilometric analysis
was performed again at the same sites as the
baseline measurements (2nd measure) (Figure 1h).
In sequence, the nail varnish was applied again
(Figure 1i) and specimens were randomly
allocated into 4 groups, according to the treatment
gel (n = 10 per group), as follows: G1 - 10%
Proanthocyanidin gel (test group), G2 - 1.23% NaF
(positive control 1), G3 - 0.012% Chlorhexidine
(positive control 2) and G4 – Placebo (negative
control with no active compound). The studied
gels were applied once over dentin for 1 minute
(Figure 1j). All gels formulations presented essentially
the same composition (hydroxyethylcellulose,
propyleneglycol, methylparaben, imidazolidinyl
urea, and deionized water, pH 7.0) except for the
active compounds.
Specimens were subjected to collagen
degradation (Figure 1k) by the action of collagenase
obtained from Clostridium histolyticum (Type VII,
Product No. C0773, Sigma-Aldrich, St. Louis,
MO, USA) added in articial saliva (20 mmol/L
HEPES, 0.70 mmol/L CaCl2, 0.20 mmol/L
MgCl2.6H2O, 4 mmol/L KH2PO4, 30 mmol/L KCl,
0.30 mmol/L NaN3) at a concentration of 100 U/mL,
for 5 days (37ºC) [23].
Prolometric analysis
The dentin specimens were maintained wet
until the analysis to avoid shrinkage of the organic
layer. Immediately before the profilometric
measurement, only the excess of water was
carefully removed with lter paper. After the
immersion time, the nail varnish was removed
(Figure 1-l) and three prolometric analysis were
performed again (Figure 1-m) at the same sites
as the baseline measurements (3rd measure).
The dentin blocks were precisely repositioned in
the wells of the prolometer, enabling baseline
proles to match with the nal ones. Then, the
dentin loss was quantitatively determined using
specic software (MarSurf XCR 20, Göttingen,
Germany) by calculating the average depth of
the eroded surface relative to the baseline surface
proles. The response variables were the dentin
wear (difference between 1st and 3rd measures)
and total amount of degradeted DOM (difference
between 2nd and 3rd measures) (Figure 2).
Methodology 2
Evaluation of endogenous matrix-linked proteases
in demineralized dentin
Dentin specimens measuring 6 mm x 2 mm x
1 mm (Figure 1B) were sectioned from the middle
coronal portion. Specimens were completely
demineralized by 0.5 M EDTA (pH 7.4; Sigma,
St. Louis, USA) for 30 days at 4°C (Figure 1C).
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
Every four days, the EDTA solution was changed
for a new one. Then the specimens were washed
with deionized water at 4°C for 72 h for complete
removal of EDTA residues, thus avoiding a
possible inhibition of MMPs.
In sequence, the specimens were treated
according to the treatment gel (n = 10 per group).
The studied gels were applied once over dentin
for 1 minute (Figure 1D) [11,23].
All the specimens were immersed in 0.5 ml
of a buffered medium composed of 5 mM HEPES,
2.5 mM CaCl2.H2O, 0.05 mM ZnCl2 and 120 mM
NaCl adjusted to pH 7.4 (Figure 1E). The sealed
tubes were incubated in a shaking water bath
at 37°C for 3 days. All 0.5 ml of medium was
removed after 3 days. Aliquots of 10 to 20 μL of
the incubation medium were used to measure
ICTP collagen fragments.
Matrix degradation by MMPs was determined
by measuring the amount of solubilized collagen
type I C-terminal telopeptides (ICTP) [20,24,25]
reconnected over the 3-day incubation periods
using the ICTP ELISA method (Human Cross-linked
Carboxy-terminal telopeptide of type I collagen,
Southern California, San Diego - USA) (Figure 1F).
The only source of ICTP telopeptide fragments in
mineralized zones is attributed to the telopeptidase
activity of MMPs [20,25].
Figure 1 - Experimental step flowchart
Figure 2 - Illustrative scheme of wear analysis
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
Statistical analysis
Quantitative data were analyzed by Normality
(Shapiro-Wilk test) and variances homogeneity
(Levene test). In case they met those assumptions,
the data would be compared by a parametric test
(ANOVA followed by Fisher’s test) and if they did
not meet at least one of these assumptions, the
test to be used would be the Kruskal-Wallis Test
followed by Dunn’s test (non-parametric).
Analysis of dentin wear and analysis of the
thickness of the demineralized organic matrix
(DOM) data were submitted to Analysis of
Variance followed by Fisher’s LSD test (p<0.05).
For the Evaluation of Matrix-Linked
Endogenous Proteases in Demineralized Dentin,
there was no normal distribution of data and
these were submitted to the Kruskal-Wallis Test
followed by Dunn’s Test (p<0.05).
Simple linear regression and Pearson
Correlation test were performed to evaluate if
the degraded DOM is a good predictor to collagen
degradation evaluated by the ICTP ELISA.
Statistical analysis was performed with
SigmaPlot 12.0 (Systat Software Inc. San Jose,
CA, USA).
RESULTS
The results of dentin wear and DOM
degradation are shown in Table I. The data
showed that there was no signicant difference
in dentin wear between groups G2, G3 and G4.
However, they showed greater dentin wear when
compared to G1.
In the analysis of DOM degradation evaluated
by ICTP ELISA, the data showed that there was no
signicant difference between groups G2 and G4,
which had lower values of collagen degradation,
showing a statistically signicant difference when
compared to groups G2 and G3 which did not
show signicant differences between themselves.
Simple linear regression analysis was
performed in which the predictor variable was
the degraded DOM evaluated prolometrically
and the dependent variable was the collagen
degradation evaluated by the ICTP ELISA. For
that, the regression equation was: Endogenous
Matrix-Linked Proteases in Demineralized
Dentin = 0.0497 + 0.000793 x degraded DOM.
Figure 3 represents the scatter plot showing
that there is a positive, signicant and moderate
correlation (0.55) where the degraded DOM is a
signicant predictor to explain the values obtained
through the ICTP ELISA.
Table I - Mean (±S.D.) of dentin loss (µm) and DOM degradation for the studied groups. Median (interquartile range) of Endogenous Matrix-Linked
Proteases in Demineralized Dentin (ICTP ELISA)
Groups
Dentin Wear Degradeted DOM ICTP ELISA
(Mean±SD) (Mean±SD) [Median (Interquartile range)]
G1 – PA 10% 156.0 ± 96.8a4.9 ± 119.0a7.0 (4.3-8.0)A
G2 - NaF 1.23% 255.0 ± 89.5b99.0 ± 118.0b147.0 (16.8-445.0)B
G3 – 0.012% Chlorhexidine 293.0 ± 53.0b144.0 ± 63.2b278.0 (126.0-389.0)B
G4 - Placebo 270.0 ± 55.6b83.9 ± 50.8b48.0 (11.0-108.0)AB
Different lowercase letters indicate statistically significant difference between the groups for the dentin wear and DOM degradation
(One Way ANOVA and Fisher’s Test). Different upercase letters indicate statistically significant difference between groups for ICTP ELISA
(Kruskal-Wallis and Dunn’s Test, p<0.05).
Figure 3 - Scatter Plot representing positive correlation between
ICTP/Elisa and Degraded DOM
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
DISCUSSION
Several studies have demonstrated
effective inhibitory feats of various agents on
MMPs [11,26-28]. In this context, chlorhexidine
and uoride have shown the ability to inhibit
collagen degradation and inhibit MMPs [9,23,26]
and that is the reason why they were used
as a positive control. On the other hand,
chlorhexidine has unwanted side effects such as
taste alteration, numbness in mouth and tongue,
pain in mouth and tongue, xerostomia, and
subjective discolouration, throughout 21 days of
chlorhexidine usage [29,30]. Therefore, it may
not be a good strategy to be used in the long term.
Natural products, such as PA, are increasingly
intended for the development of products for
oral health because, at first, they involve a
lower incidence of side effects [31]. The PA
purified from grape seed extract was tested
because previous studies have demonstrated its
protective effect on dentin erosion [19,32,33]
and in this study, the group treated with PA also
had the best results. Thus, since a PA does not
have the side effects of a chlorhexidine, its use
might be more advantageous for dentin erosion
prevention.
Some studies have shown that agents
with a high concentration of uoride are more
effective in preventing dental erosion [11,26,34].
It can form a layer of calcium fluoride that
provides temporary protection against erosive
challenges [35]. NaF group showed no statistical
difference from placebo group. This nding has
already founded [11,33] and could be due to the
response variable used in these studies, which is
dentin wear instead of a direct chemical analysis
of the different gels ability to inhibit collagen
degradation.
In this study, the results of proanthocyanidin
were better than the other groups. Studies
show that proanthocyanidin positively affects
the demineralization and/or remineralization
processes and its remineralizing mechanism
seems to be different from uoride [36] which
might be related to the formation of an insoluble
complex that remains stable in acid pH [37] that
further binds to the Ca2+ ions in saliva, thereby
enhancing remineralization [36]. However, in the
present study, it is most likely to proanthocyanidin
have improved the DOM by its ability to induce
cross-links in dentin collagen and reinforce the
remaining collagen matrix [36,38,39].
In the prolometry analysis, chlorhexidine,
fluoride and placebo group showed lower
results than the PA group, while in the chemical
analysis of collagen degradation performed
using the ICTP ELISA, the PA group did not
show any signicant difference with the placebo
group. This can be directly related to the chosen
demineralizing agent. Citric acid promotes
dentin demineralization and, in this case, caused
a great loss of tooth structure, which made it
difficult to analyze the exact profilometry of
the specimens. Furthermore, in the analysis of
collagen degradation performed with the ICTP
ELISA, there was a large sample loss. These
factors may be related to typing errors in the
results or due to the long storage time of the
teeth before their use. Despite that, it was noted
that both the loss of tooth structure and the
difculty in detecting degraded collagen were
equivalent in all groups, which allowed us to
maintain the comparison between specimens.
Another possible reason for the performance
of the proanthocyanidin’s performance is the
concentration used in this present study (10%).
Studies showed good results in concentrations
of proanthocyanidin less than 10% and its dose-
response effect [39,40].
The selection of a 36-hour demineralization
period was a strategic choice aimed at establishing
a discernible layer of demineralized organic matrix
(DOM) for targeted analysis. This approach, while
divergent from the dynamic acid challenges typical
of erosive cycling studies, enabled the direct
examination of agents’ effects on the organic
matrix. We acknowledge that the thickness of the
resulting DOM layer could potentially lead to an
overestimation of the agents’ effects, given that
clinical scenarios involve frequent acid attacks.
It is important to underscore that the study’s focus
remains on investigating the agents’ direct impact
on the organic matrix, with mineral loss serving
as an ancillary consideration.
Prolometry alone on eroded dentin does
not reect mineral loss, since there is interference
of collagen matrix [41]. Furthermore, contact
profilometers use a diamond-tip stylus moved
across the surface to record the surface prole,
which is simple, but this traditional method has
the potential risk of affecting the reading or even
damaging the sample as a consequence of the
contact. However, despite its limited analysis,
this method showed a very strong correlation
when compared with a non-contact [42].
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Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
Profilometer and a confocal laser scanning
microscope with the same conclusions being
separately drawn from data generated on each
instrument [43]. In this study there were a
signicant difference between PA group gel from
other groups showing less dentin wear compared
to the other groups. Our findings allow us to
understand that there was also a reduction in
DOM degradation by prolometry. In addition,
a positive, signicant and moderate correlation
(0.55) was found between the prolometry results
with those of the chemical analysis (ICTP ELISA).
Another alternative to evaluate dentin wear
and DOM degradation is through collagenolytic
activity by measuring the hydroxyproline
content in articial saliva after incubation with
collagenase [33]. However, this procedure is
prone to error and no statement can be made
about the thickness of remaining matrix and
reproducibility of the method is limited [21].
In this study, a simple linear regression
analysis was performed in which the predictor
variable was the degraded DOM evaluated
prolometrically and the dependent variable was
the collagen degradation evaluated by the ICTP
ELISA. It shows that this regression model is
signicant and that degraded DOM is a signicant
predictor to explain ICTP ELISA’s value. Besides
that, the results show us a signicant correlation
between them (p<0,002). This correlation is a
positive, signicant and moderate (0.55) where the
degraded DOM is a signicant predictor to explain
the values obtained through the ICTP ELISA. For
this reason, these results seem to be more reliable.
CONCLUSION
The results of this study showed that 10%
proanthocyanidin provided less dentin wear
and decreased degradation of the demineralized
organic matrix, suggesting a greater capacity to
prevent dentin erosion.
Furthermore, the contact prolometry can
be an alternative method to measure dentin wear
and estimate the degradation of demineralized
organic matrix.
Author’s Contributions
FC: Experimental design, execution of the
experimental stages and writing of the manuscript.
GGD: Execution of the experimental stages.
APB: Execution of the experimental stages.
AFS: Execution of the experimental stages.
TJD: Execution of the experimental stages.
HMH: Theoretical conceptualization, experimental
design, data analysis and article writing.
Conict of Interest
The authors have no proprietary, nancial,
or other personal interest of any nature or kind
in any product, service, and/or company that is
presented in this article.
Funding
This study was financed in part by the
Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - Brasil (CAPES) - Finance Code 001.
The authors would like to gratefully acknowledge
and FAPESP (Process 2018/05847-5).
Regulatory Statement
This study was conducted in accordance
with all the provisions of the local human subjects
oversight committee guidelines and policies of
Research and Ethics Committee of the Bauru School
of Dentistry, University of São Paulo. The approval
code for this study is: 94400218.8.0000.5417.
REFERENCES
1. Magalhães AC, Wiegand A, Rios D, Honório HM, Buzalaf MA.
Insights into preventive measures for dental erosion. J Appl
Oral Sci. 2009;17(2):75-86. http://dx.doi.org/10.1590/S1678-
77572009000200002. PMid:19274390.
2. Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho
RM, et al. Collagen degradation by host-derived enzymes
during aging. J Dent Res. 2004;83(3):216-21. http://dx.doi.
org/10.1177/154405910408300306. PMid:14981122.
3.
Silverstone LM, Hicks MJ. The structure and ultrastructure of the carious
lesion in human dentin. Gerodontics. 1985;1(4):185-93. PMid:3864713.
4. Ramos-Oliveira TM, Ramos TM, Garbui BU, Hanashiro FS, Freitas
PM. Adhesion to eroded dentin submitted to different surface
treatments. Braz Dent Sci. 2014;17(4):40-7. http://dx.doi.
org/10.14295/bds.2014.v17i4.1029.
5.
Klont B, ten Cate JM. Remineralization of bovine incisor root lesions in
vitro: the role of the collagenous matrix. Caries Res. 1991;25(1):39-45.
http://dx.doi.org/10.1159/000261340. PMid:2070381.
6. Kleter GA, Damen JJ, Everts V, Niehof J, Ten Cate JM. The
influence of the organic matrix on demineralization of bovine
root dentin in vitro. J Dent Res. 1994;73(9):1523-9. http://dx.doi.
org/10.1177/00220345940730090701. PMid:7929987.
7. Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T.
The activation and function of host matrix metalloproteinases
in dentin matrix breakdown in caries lesions. J Dent Res.
1998;77(8):1622-9. http://dx.doi.org/10.1177/0022034598077
0081001. PMid:9719036.
8
Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
8. Pilly V, Brito CHL Jr, Nogueira GR Fo, Prakki A. Protection of tooth
structure by chlorhexidine and natural polyphenols: a review.
Braz Dent Sci. 2012;15(4):3-9. http://dx.doi.org/10.14295/
bds.2012.v15i4.838.
9. Carrilho MR, Geraldeli S, Tay F, de Goes MF, Carvalho RM,
Tjaderhane L,etal. In vivo preservation of the hybrid layer by
chlorhexidine. J Dent Res. 2007;86(6):529-33. http://dx.doi.
org/10.1177/154405910708600608. PMid:17525352.
10. Kato MT, Magalhães AC, Rios D, Hannas AR, Attin T, Buzalaf MA.
Protective effect of green tea on dentin erosion and abrasion.
J Appl Oral Sci. 2009;17(6):560-4. http://dx.doi.org/10.1590/
S1678-77572009000600004 PMid:20027426.
11. Kato MT, Leite AL, Hannas AR, Buzalaf MA. Gels containing
MMP inhibitors prevent dental erosion in situ. J Dent Res.
2010;89(5):468-72. http://dx.doi.org/10.1177/0022034510363248.
PMid:20200409.
12. Han B, Jaurequi J, Tang BW, Nimni ME. Proanthocyanidin: a
natural cross-linking reagent for stabilizing collagen matrices.
J Biomed Mater Res A. 2003;65(1):118-24. http://dx.doi.
org/10.1002/jbm.a.10460. PMid:12635161.
13. Kennedy JA, Taylor AW. Analyses of proanthocyanidins
by high-performance gel permeation chromatography.
J Chromatogr A. 2003;995(1-2):99-107. http://dx.doi.
org/10.1016/S0021-9673(03)00420-5. PMid:12800926.
14. Sabino KC, Gayer CR, Vaz LC, Santos LR, Felzenszwalb I,
Coelho MG. In vitro and in vivo toxicological study of the
Pterodon pubescens seed oil. Toxicol Lett. 1999;108(1):27-35.
http://dx.doi.org/10.1016/S0378-4274(99)00110-1.
PMid:10472807.
15. Ye X, Krohn RL, Liu W, Joshi SS, Kuszynski CA, McGinn
TR,et al. The cytotoxic effects of a novel IH636 grape seed
proanthocyanidin extract on cultured human cancer cells.
Mol Cell Biochem. 1999;196(1-2):99-108. http://dx.doi.
org/10.1023/A:1006926414683. PMid:10448908.
16. Castellan CS, Pereira PN, Grande RH, Bedran-Russo AK.
Mechanical characterization of proanthocyanidin-dentin matrix
interation. Dent Mater. 2010;26(10):968-73. http://dx.doi.
org/10.1016/j.dental.2010.06.001. PMid:20650510.
17. Castellan CS, Bedran-Russo AK, Karol S, Pereira PN.
Long-term stability of dentin matrix following treatment
with various natural collagen cross-linkers. J Mech Behav
Biomed Mater. 2011;4(7):1343-50. http://dx.doi.org/10.1016/j.
jmbbm.2011.05.003. PMid:21783144.
18. Castellan CS, Bedran-Russo AK, Antunes A, Pereira PN. Effect
of dentin biomodification using naturally derived collagen
cross-linkers: one-year bond strength study. Int J Dent.
2013;2013:918010. http://dx.doi.org/10.1155/2013/918010.
PMid:24069032.
19. Cardoso F, Boteon AP, Silva TAPD, Prakki A, Wang L, Honório HM. In
situ effect of a proanthocyanidin mouthrinse on dentin subjected
to erosion. J Appl Oral Sci. 2020;28:e20200051. http://dx.doi.
org/10.1590/1678-7757-2020-0051. PMid:33111880.
20. Osorio R, Yamauti M, Osorio E, Ruiz-Requena ME, Pashley D, Tay
F,etal. Effect of dentin etching and chlorhexidine application
on metalloproteinase-mediated collagen degradation.
Eur J Oral Sci. 2011;119(1):79-85. http://dx.doi.org/10.1111/
j.1600-0722.2010.00789.x. PMid:21244516.
21. Schlueter N, Glatzki J, Klimek J, Ganss C. Erosive-abrasive
tissue loss in dentine under simulated bulimic conditions.
Arch Oral Biol. 2012;57(9):1176-82. http://dx.doi.org/10.1016/j.
archoralbio.2012.04.001. PMid:22554994.
22. Tezvergil-Mutluay A, Mutluay M, Seseogullari-Dirihan R, Agee
KA, Key WO, Scheffel DL,etal. Effect of phosphoric acid on the
degradation of human dentin matrix. J Dent Res. 2013;92(1):87-91.
http://dx.doi.org/10.1177/0022034512466264. PMid:23103634.
23. Kato MT, Leite AL, Hannas AR, Calabria MP, Magalhães AC,
Pereira JC,etal. Impact of protease inhibitors on dentin matrix
degradation by collagenase. J Dent Res. 2012;91(12):1119-23.
http://dx.doi.org/10.1177/0022034512455801. PMid:23023765.
24. Ganss C, Lussi A, Sommer N, Klimek J, Schlueter N. Efficacy of
fluoride compounds and stannous chloride as erosion inhibitors
in dentine. Caries Res. 2010;44(3):248-52. http://dx.doi.
org/10.1159/000314671. PMid:20516684.
25. Garnero P, Borel O, Byrjalsen I, Ferreras M, Drake FH, McQueney
MS,etal. The collagenolytic activity of cathepsin K is unique among
mammalian proteinases. J Biol Chem. 1998;273(48):32347-52.
http://dx.doi.org/10.1074/jbc.273.48.32347. PMid:9822715.
26. Kato MT, Bolanho A, Zarella BL, Salo T, Tjaderhane L, Buzalaf
MA. Sodium fluoride inhibits MMP-2 and MMP-9. J Dent Res.
2014;93(1):74-7. http://dx.doi.org/10.1177/0022034513511820.
PMid:24196489.
27. La VD, Howell AB, Grenier D. Cranberry proanthocyanidins inhibit
MMP production and activity. J Dent Res. 2009;88(7):627-32.
http://dx.doi.org/10.1177/0022034509339487. PMid:19641150.
28. Bedran-Russo AK, Castellan CS, Shinohara MS, Hassan L,
Antunes A. Characterization of biomodified dentin matrices
for potential preventive and reparative therapies. Acta
Biomater. 2011;7(4):1735-41. http://dx.doi.org/10.1016/j.
actbio.2010.12.013. PMid:21167964.
29. Poppolo Deus F, Ouanounou A. Chlorhexidine in
dentistry: pharmacology, uses, and adverse effects. Int
Dent J. 2022;72(3):269-77. http://dx.doi.org/10.1016/j.
identj.2022.01.005. PMid:35287956.
30. Rovai ES, Tera TM, Marco AC, Jardini MAN, Santamaria MP,
Kerbauy WD. Evaluation of dental staining using a dentifrice
containing chlorhexidine and zinc acetate. A doble blind
randomized clinical trial. Braz Dent Sci. 2013;16(2):51-8.
http://dx.doi.org/10.14295/bds.2013.v16i2.884.
31. Khaddam M, Salmon B, Le Denmat D, Tjaderhane L, Menashi S,
Chaussain C,etal. Grape seed extracts inhibit dentin matrix
degradation by MMP-3. Front Physiol. 2014;5:425. http://dx.doi.
org/10.3389/fphys.2014.00425. PMid:25400590.
32. Boteon AP, Prakki A, Buzalaf MAR, Rios D, Honório HM.
Effect of different concentrations and application times
of proanthocyanidin gels on dentin erosion. Am J Dent.
2017;30(2):96-100. PMid:29178771.
33. Boteon AP, Kato MT, Buzalaf MAR, Prakki A, Wang L, Rios D,etal.
Effect of Proanthocyanidin-enriched extracts on the inhibition of
wear and degradation of dentin demineralized organic matrix.
Arch Oral Biol. 2017;84:118-24. http://dx.doi.org/10.1016/j.
archoralbio.2017.09.027. PMid:28987724.
34. Brackett MG, Agee KA, Brackett WW, Key WO, Sabatini C, Kato
MT,etal. Effect of Sodium Fluoride on the endogenous MMP
Activity of Dentin Matrices. J Nat Sci. 2015;1(6) PMid:26052548.
35. Huysmans MC, Young A, Ganss C. The role of fluoride in erosion
therapy. Monogr Oral Sci. 2014;25:230-43. http://dx.doi.
org/10.1159/000360555. PMid:24993271.
36. Xie Q, Bedran-Russo AK, Wu CD. In vitro remineralization
effects of grape seed extract on artificial root caries.
J Dent. 2008;36(11):900-6. http://dx.doi.org/10.1016/j.
jdent.2008.07.011 PMid:18819742.
37. Kosasi S, Hart LA, van Dijk H, Labadie RP. Inhibitory activity
of Jatropha multifida latex on classical complement pathway
activity in human serum mediated by a calcium-binding
proanthocyanidin. J Ethnopharmacol. 1989;27(1-2):81-9.
http://dx.doi.org/10.1016/0378-8741(89)90080-9. PMid:2615430.
38. Bedran-Russo AK, Pauli GF, Chen SN, McAlpine J, Castellan CS,
Phansalkar RS,etal. Dentin biomodification: strategies, renewable
resources and clinical applications. Dent Mater. 2014;30(1):62-76.
http://dx.doi.org/10.1016/j.dental.2013.10.012. PMid:24309436.
9
Braz Dent Sci 2023 Oct/Dec; 26 (4): e3946
Cardoso F et al.
Effect of 10% Proanthocyanidin gel on demineralized organic matrix degradation: ELISA method
Cardoso F et al. Effect of 10% Proanthocyanidin gel on demineralized organic
matrix degradation: ELISA method
39. Castellan CS, Bedran-Russo AK, Karol S, Pereira PN.
Long-term stability of dentin matrix following treatment
with various natural collagen cross-linkers. J Mech Behav
Biomed Mater. 2011;4(7):1343-50. http://dx.doi.org/10.1016/j.
jmbbm.2011.05.003. PMid:21783144.
40. Bedran-Russo AK, Pashley DH, Agee K, Drummond JL, Miescke
KJ. Changes in stiffness of demineralized dentin following
application of collagen crosslinkers. J Biomed Mater Res B
Appl Biomater. 2008;86(2):330-4. http://dx.doi.org/10.1002/
jbm.b.31022. PMid:18161815.
41. Ganss C, Hardt M, Blazek D, Klimek J, Schlueter N.
Effects of toothbrushing force on the mineral content
and demineralized organic matrix of eroded dentine.
Eur J Oral Sci. 2009;117(3):255-60. http://dx.doi.org/10.1111/
j.1600-0722.2009.00617.x. PMid:19583752.
42. Ganss C, Lussi A, Scharmann I, Weigelt T, Hardt M, Klimek J,etal.
Comparison of calcium analysis, longitudinal microradiography
and profilometry for the quantitative assessment of erosion
in dentine. Caries Res. 2009;43(6):422-9. http://dx.doi.
org/10.1159/000252975. PMid:19864904.
43. Paepegaey AM, Barker ML, Bartlett DW, Mistry M,
West NX, Hellin N, et al. Measuring enamel erosion:
a comparative study of contact profilometry, non-contact
profilometry and confocal laser scanning microscopy. Dent
Mater. 2013;29(12):1265-72. http://dx.doi.org/10.1016/j.
dental.2013.09.015. PMid:24209832.
Heitor Marques Honório
(Corresponding address)
Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departmento de
Odontopediatria, Ortodontia e Saúde Coletiva, Bauru, SP, Brasil
Email: heitorhonorio@usp.br
Date submitted: 2023 July 05
Accept submission: 2023 Aug 19
