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.2024.e4263
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Braz Dent Sci 2024 July/Sept;27 (3): e4263
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
Papel de peptídeo antimicrobiano no biofilme cariogênico e na desmineralização do esmalte dentário
Priscila Vieira da SILVA1 , Tatiana PEREIRA-CENCI2 , Juliana Lays Stolfo UEHARA2 , Aurélio Rosa da SILVA JÚNIOR1 ,
Maximiliano Sérgio CENCI2 , Andreza Maria Fábio ARANHA1
1 - Universidade de Cuiabá-UNIC, Faculdade de Odontologia, Departamento de Ciências Odontológicas Integradas. Cuiabá, MT, Brazil.
2 - Universidade Federal de Pelotas, Faculdade de Odontologia. Pelotas, RS, Brazil.
How to cite: Silva PV, Pereira-Cenci T, Uehara JLS, Silva Júnior AR, Cenci MS, Aranha AMF. Role of antimicrobial peptide on cariogenic
biolm and dental enamel demineralization. Braz Dent Sci. 2024;27(3):e4263. https://doi.org/10.4322/bds.2024.e4263
ABSTRACT
Objective: To investigate the effect of antimicrobial peptide (AMP) D1-23 on antibiolm activity and enamel
demineralization by using a microcosm biolm model. Material and Methods: Human saliva biolm was grown
on bovine enamel discs, which were divided into 4 groups according to the 24-hour growth treatment: 0.2 mM
AMP D1-23; 1 mM AMP D1-23; Dened Medium with Mucin (DMM; negative control); and 2% chlorhexidine
(CHX; positive control). The biomass and number of cultivable cells of a 4-day-old biolm were evaluated for
antibiolm activity. For analysis of enamel mineral loss by the 7-day-old biolm and investigated treatments, the
Knoop surface microhardness method was performed. ANOVA and Tukey tests were used to analyze data from
biomass and enamel surface microhardness. Culturable cells data were analyzed by a pairwise Kruskal-Wallis test.
A signicance level of 5% was considered for all tests. Results: Although the two concentrations of AMP D1-23 had
similar effects on the amount of biolm biomass (p>0.05), a reduction in biomass was observed when compared
to the control group (p < 0.05). The antimicrobial activity of 1 mM AMP D1-23 was similar to that of 2% CHX
against
Streptococcus mutans
, whereas against
Streptococci sp
and
Candida sp
it was lower (
p
<0.05). Also, the
1 mM AMP D1-23 signicantly reduced enamel demineralization (
p
< 0.05). Conclusion: Under the limitations
of this study, the 1 mM AMP D1-23 appears to have positive effect on controlling dental enamel demineralization
and may be considered a potential treatment for non-cavitated carious lesions.
KEYWORDS
Antimicrobial cationic peptides; Dental caries, Dental plaque; Public health; Tooth remineralization.
RESUMO
Objetivo: Investigar o efeito do peptídeo antimicrobiano (AMP) D1-23 na atividade antibiolme e na desmineralização
do esmalte usando um modelo de biolme microcosmo. Material e Métodos: Biolme de saliva humana foi
cultivado em discos de esmalte bovino, que foram divididos em 4 grupos: AMP D1-23 0,2 mM; AMP D1-23 1 mM;
Meio Denido com Mucina (DMM; controle negativo); e clorexidina 2% (CHX; controle positivo). A biomassa e o
número de células cultiváveis do biolme com 4 dias foram avaliados quanto à atividade antibiolme. Para análise
da perda mineral do esmalte um biolme com 7 dias foi cultivado e realizado pelo método de microdureza supercial
Knoop. Os testes ANOVA e Tukey foram utilizados para analisar dados de biomassa e microdura supercial. Os
dados de células cultiváveis foram analisados por um teste de Kruskal-Wallis pareado. Foi considerado nível de
signicância de 5% para todos os testes. Resultados: Embora as duas concentrações de AMP D1-23 tenham tido
efeitos semelhantes na quantidade de biomassa do biolme (p>0,05), foi observada uma redução na biomassa
quando comparado ao grupo controle (p <0,05). A atividade antimicrobiana de AMP D1-23 1 mM foi semelhante
à de CHX 2% contra
Streptococcus mutans
, enquanto contra
Streptococci
sp e
Candida
sp foi menor (p<0,05).
Além disso, o AMP D1-23 1 mM reduziu signicativamente a desmineralização do esmalte (p < 0,05). Conclusão:
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Silva PV et al.
Role of antimicrobial peptide on cariogenic biofilm and dental enamel demineralization
Silva PV et al. Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
INTRODUCTION
Although preventable, dental caries is a
disease of major public health concern, that
imposes an expensive burden on health systems.
The importance of maintaining a healthy lifestyle
and controlling oral biolm through moderate
refined sugar consumption is important for
long-term disease control. However, behavioral
changes in diet are difcult to achieve and to
maintain, especially current eating habits [1].
There is no doubt that uoride will continue
to be the pillar of any caries prevention protocol, as
it still remains the most effective and cost-effective
protective agent against tooth decay [2,3].
However, uoride alone does not offer complete
protection against caries disease, and its efcacy
can be improved when in combination with
additional cariostatic agents [1,2]. Silver diamine
uoride (SDF) has been recommended in the
treatment of early childhood caries (ECC)
because of its interesting role in the control of
enamel demineralization [3,4]. Nonetheless,
SDF promotes dentin discoloration, which has
negative aesthetic effects [3]. New preventive
therapies involving salivary peptides have
been proposed [5,6]. Antimicrobial peptides
(AMPs) have been investigated since they are
responsible for modulating the host’s immune
response, keeping the microbiota balanced in
different niches (Wiesner and Vilcinskas, 2010).
The main AMPs in saliva/crevicular uid are α-
and β-defensins (hBD) and cathelicidins [7,8].
The role of AMPs in dental caries control has
raised interest, as these molecules are identied
as a new class of antibiotics. The long length
of their amino acid chains has hampered the
production of peptides for therapeutic uses;
resulting in the interest of investigating peptide
fragments against cariogenic bacteria [9].
The peptide Defb14-1CV(1-23) or D1-23, derived
from Defb14 (the mouse ortholog of human
β-defensin-3), showed promising preliminary
results against planktonic microbial strains and
in monospecies biolms [9,10].
No evidence has yet been found regarding
the antimicrobial potential of AMP D1-23 in
complex biolms and its effect on tooth enamel
demineralization. Based on the need for a more
effective treatment to control the progression
of dental caries disease, this study aimed to
evaluate antibiofilm activity and the ability
to inhibit dental enamel demineralization of
AMP D1-23. The hypotheses tested were: the
concentration of AMP D1-23 does not inuence:
(1) the antibiofilm activity and (2) enamel
demineralization.
METHODS
Ethical aspects
This study was approved by the Research
Ethics Committee (Protocol number [CAAE]
87670818 9 0000 5165), and written informed
consent was obtained from the saliva donor.
Experimental design
The microcosm biolm model was used in
this
in vitro
study. To provide a multispecies
biolm, human stimulated saliva was used as
the inoculum. Biolms were grown on bovine
enamel discs, which were divided into 4 groups
according to the 24-hour treatment [11]: 1 mM
AMP D1-23, 0.2 mM AMP D1-23, defined
medium with mucine (articial saliva/DMM;
negative control), 2% chlorhexidine (positive
control). The experiments were performed in
triplicate.
Biofilm inoculation on enamel discs was
performed with human donor saliva and
DMM as the culture medium. The DMM was
renewed twice a day, and enamel-biofilm
discs were subjected to cariogenic challenge
through cyclic exposure to sucrose, A 4-day-old
biolm and a 7-day-old biolm were used for
microbiological tests [11] and microhardness
test [12], respectively. After that, the enamel
discs were treated for 24 hours according to the
different groups. The microhardness test was
performed to assess mineral loss, which was
O AMP D1-23 1 mM parece ter efeito positivo no controle da desmineralização do esmalte danicado e pode ser
considerado uma alternativa de tratamento em potencial para lesões de cárie não cavitadas.
PALAVRAS-CHAVE
Cárie dentária; Peptídeos catiônicos antimicrobianos, Placa dentária; Saúde pública; Remineralização dentária.
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Silva PV et al.
Role of antimicrobial peptide on cariogenic biofilm and dental enamel demineralization
Silva PV et al. Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
recorded as the percentage change in enamel
surface microhardness (%SMH).
Preparation of antimicrobial agents
AMP D1-23 (FLPKTLRKFFARIRGGRAAVLNA),
derived from Defb14 (the mouse ortholog of
human β-defensin-3) (AminoTech Research and
Development Ltda, Diadema, SP, Brazil) [10],
was resuspended in sterilized deionized water at
20 mM and stored at -20°C. Two concentrations of
AMP D1-23 were investigated: 0.2 mM and 1 mM.
DMM (Interlab, São Paulo, SP, Brazil) and a 2%
chlorhexidine solution (GeneralMed, São Paulo,
SP, Brazil) were used as negative and positive
control, respectively.
Preparation of bovine enamel discs
Thirty two enamel discs (5 mm in diameter
x 2 mm in thickness) were prepared from the
mid-height of the dental crown of bovine incisors.
The enamel surfaces were sequentially polished
with 100/600/1200 grit silicon carbide paper
discs. Next, the bovine disc specimens were
covered with nail polish, exposing only the tooth
enamel surface.
Microcosmos biolm growth
Thirty milliliters of saliva stimulated by
parafn lm (Paralm “M”, American National
CanTM, Chicago, IL, USA) were collected from a
good general health human donor [11].
The enamel discs were inoculated with
400 µL of homogenized saliva and were
incubated for 1 hour at 37°C. For the cariogenic
challenge, 1.8 mL of articial saliva (DMM)13 was
supplemented with 1% sucrose (Interlab, São
Paulo, SP, Brazil) for 6 hours under anaerobic
conditions (5-10% CO2 < 1% O2) at 37° C,
without shaking.
The cariogenic challenge was repeated
daily, for 4 or 7 days [13], depending on the
experiments, with two changes: DMM enriched
with 1% sucrose for 6 hours and DMM without
sucrose for 18 hours. After 4 and 7 days of biolm
growth, for antibiolm evaluation and enamel
surface microhardness evaluation, respectively,
the discs were divided into four groups, according
to the investigated treatment.
In order to detach the biofilm from the
enamel discs, the samples were sonicated
(Ultrasonic Cell Tip-Disruptor Sonicator, Model:
DES500, Unique, São Paulo, SP, Brazil).
Total biomass and microbial composition of
the biolm
The total biofilm biomass was evaluated
by the dry weight method [11], determined by
the weight difference between the microtube
containing the biolm and the empty microtube.
Biolms were dehydrated with ethanolic solutions
(99% and 75%) and stored in a glass desiccator
(LaborQuimi Vidrolabor - Poá, SP, Brazil) for
48 hours.
The quantification of viable cells was
performed after disaggregation of the treated
biofilm. Serial dilutions of the biofilm (from
0 to 10-6) were inoculated in Petri dishes with
CHROMagar culture medium (Difco Laboratories,
Franklin Lakes, New Jersey, USA) for
Candida sp
cultures; Mitis Salivarius agar culture medium
(Difco Laboratories) supplemented with 0.2 U/mL
bacitracin (Sigma-Aldrich, St. Louis, MO, USA)
for
Streptococcus mutans
cultures; and Mitis
Salivarius agar culture medium for streptococci
cultures.
S. mutans
and
Streptococci
cultures
were incubated under anaerobic conditions for
72 hours, while
Candida
sp was grown under
aerobic conditions. The number of colony forming
units (CFU) in each culture was determined by a
trained researcher and the results were expressed
in CFU/mg of biolm (dry weight).
Microhardness change analysis
The microhardness test was performed
to assess mineral loss, which was recorded
as the percentage change in enamel surface
microhardness (% SMH).
Enamel demineralization was analyzed by
the surface microhardness (SMH) Knoop test [12]
at two different times, before biolm formation
(healthy enamel; initial microhardness) and
after biofilm growth and treatment (final
microhardness).
The initial SMH was determined based on the
SMH measured with a Knoop diamond (Future-
Tech Corp., Tokyo, Japan) under a 25 g load for
5 seconds, using three equidistant indentations in
the center of the enamel discs (150 µm). The mean
microhardness was calculated and analyzed by
ANOVA test to select samples that were statistically
equivalent at baseline (
p
> 0.05). The selected
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Silva PV et al.
Role of antimicrobial peptide on cariogenic biofilm and dental enamel demineralization
Silva PV et al. Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
specimens were subjected to cariogenic challenge
by the biolm model.
After 7 days of biolm growth, the specimens
were treated for 24 hours according to the
investigated groups. Final microhardness was
evaluated using three equidistant indentations
(150 µm) from the initial readings. The enamel
surface microhardness was calculated using the
equation: % SMH (Δ) = 100 * (initial reading -
nal reading) / initial reading.
Statistical analysis
Data were analyzed using SigmaPlot
12.0 software (Systat Software Inc., San Jose, USA).
The ANOVA and Tukey’s multiple comparisons
post hoc tests were used to assess total biolm
biomass and microhardness. The presence of
culturable cells in the biofilms was compared
among groups by ANOVA with Tukey’s post-test
post hoc paired comparisons. Data from microbial
biolm composition were transformed in log 10,
and the signicance level was set at 5%.
RESULTS
Total biolm biomass
The results of quantication of total biomass by
dry weight of biolm AMP D1-23, in both studied
concentrations, reduced biolm biomass. However,
these results were not statistically different from the
positive control group (p > 0.05).
Following are the dry weight and standard
deviation data: DMM (15.5+1.8), AMP
D1-23 0,2mM (13.0+3.12), AMP D1-23 1mM
(9.83+3.55), Chlorhexidine (20.8+3.01).
Quantication and identication of biolm
culturable cells
Figure 1 shows that AMP D1-23 at a con-
centration of 1 mM showed an antimicrobial
activity similar to chlorhexidine against
S. mutans
(p > 0.05), while it did not present antimicrobial
activity against
Streptococci
or
Candida sp
in the
investigated concentrations (p > 0.05).
Different letters represent statistical difference.
(p <0.05, ANOVA with Tukey’s post-test).
Effect of AMP D1-23 on enamel demineral-
ization
The SMH results are shown in Table I. Loss
of enamel surface hardness was showed in all
groups after 7 days of biolm growth and 24 hours
of treatment. When the cariogenic biolm was
treated with 1 mM AMP D1-23, it was observerd
the lowest enamel demineralization (p <0.05).
DISCUSSION
AMP D1-23, at the investigated concentrations,
did not play an important role in controlling the
cariogenic biolm. However, it positively affected
the control of enamel demineralization following
cariogenic biolm growth.
The microcosm biolm model, used in this
study, was applied in preclinical evaluations to
evaluate the potential of antimicrobials in the
development of biologically active materials [11,14].
Table I - Analysis of Knoop surface microhardness of dental enamel according to the treatment performed
Treatment Surface microhardness
(baseline)* Mean ± SD
Surface microhardness
After treatment Mean± SD**
Percentage Enamel
demineralization after treatment
Defined Medium Mucine (DMM) 327.7±35.2 28.4±2.5 91.26a#
D1-23 0.2 mM 271.6±33.6 58.3±6.4 78.47a
D1-23 1 mM 286.6±63.5 112.1±19.0 58.04b
Chlorhexidine 2% (0.03mM) 284.7±81.1 72.9±12.8 73.55a
* Surface microhardness of sound tooth enamel (baseline). ** Surface microhardness after 7 days of biofilm growth and 24h-treatment. #
Different letters represent statistically different values (p<0.05. ANOVA with Tukey test).
Figure 1 - Graphical representation of antibiofilm activity through
quantitative analysis of Colony Forming Units (CFU) of complex
biofilm of
Candida sp
,
S. mutas
and
Streotococci sp
, treated with
AMP D1-23 0.2mM and AMP D1-23 1mM.
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Silva PV et al.
Role of antimicrobial peptide on cariogenic biofilm and dental enamel demineralization
Silva PV et al. Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
It is important to mention that the mature biolm,
rich in extracellular polysaccharide matrix, is
characterized by an organized structure that makes
its mechanical removal [15] and the action of
antimicrobials difcult [16]. Thus, approaches to
control the formation of pathogenic biolms can be
a strategy for the prevention of dental caries [15]
Bovine tooth discs were used as substrate
to develop the microcosm biolm model, which
has advantages such as its easy obtention and
handling. Furthermore, it is known that the
structure of bovine enamel and dentin is similar
to that of human enamel and dentin in terms of
quantity, size, shape, diameter, and density of
dentin tubules [17].
Antibiofilm activity was evaluated by
quantifying the total biomass and culturable
cariogenic microorganisms present in the biolm
after antimicrobial treatment. Although no
studies were found with AMP D1-23 in complex
biolms [9], it was showed the effect of AMP
D1-23 on planktonic cells and simple
S. mutans
biolm. Greater reduction in total biolm biomass
was observed by confocal microscopy after
biofilm treatment with AMP D1-23 than with
chlorhexidine [9]. The effect of antimicrobial
agents on biomass depends on treatment
depletion in the wet phase, antibiotic penetration,
and biome physiology [16]. However, only
antimicrobial penetration and biolm formation
and maturation can be associated with biomass
tolerance in the
in vitro
microcosm biolm model.
When the number of viable cells in the
dental biofilm was evaluated, AMP D1-23, at
both concentrations, showed effects similar to
those of the negative control group, that is, low
efcacy against the studied strains. Contrary to
our results, Kreking et al. [9] observed a more
signicant effect of 0.2 mM AMP D1-23 compared
to 0.03 mM (2%) chlorhexidine against
S. mutans
.
However, it was shown that more mature and
thicker biolms are invariably less susceptible
to antimicrobial agents than younger and less
dense biolms due to the more complex structural
nature of the extracellular polysaccharide
matrix [16], which could explain the difference
between the two studies. In our study, 4-day
complex biolms, for the rst time investigated
under the effect of AMP D1-23, were obtained
from human saliva, contrary to the previous
investigation, where the effect of AMP D1-23 was
evaluated on a 48-hour
S. mutans
biolm [9].
Biolm tolerance to antimicrobials is known to
comprise the large number of challenges that can
inuence susceptibility in a specic biolm, such
as microbial composition, substrate material, cell
density or biolm thickness, and biolm age [11].
S. mutans
is considered one of the initial
colonizers of the cariogenic biofilm, being a
potent acid producer, which helps in modulating
the presence of other microorganisms and
makes the biofilm more pathogenic [17].
Therefore, a reduction in
S. mutans
levels
may contribute to a lower acidogenicity of
the biolm by reducing sugar metabolism and
biofilm complexity [15].
Streptococci
, which
were also evaluated in this study, are related to
the constant maintenance of the acidic biolm
pH and the progression of caries lesions. In this
study, AMP D1-23 had the same performance as
the negative control against
Streptococci
, which
differed from Kreling et al. [9], who observed
higher bactericidal activity of AMP D1-23 against
the same species in planktonic state.
Candida
sp
contributes to the formation of multispecies
biolms, increasing the chances of adhesion of
other microorganisms in the oral environment.
In our study, the count of
Candida
species in the
donor was low, corroborating previous studies
that showed that
Candida
constitutes less than
1% of the total of microorganisms found in the
non-biolm composition of saliva [18]. However,
the same donor, at different times of repeated
collections, has a signicantly different microora
composition, suggesting that the observed
transition in dental biolm composition between
health and disease is driven by a microbiota
response to environmental changes [18].
The antimicrobial effect of AMP D1-23 in
complex biolm is thought-provoking, since it is
known that the effect of an antimicrobial is different
and limited in complex biolms when compared
to microorganisms in planktonic state [19].
This considers the fact that microorganisms in
biolms are incorporated into the extracellular
polysaccharide matrix, which offers physical
protection against mechanical and chemical
challenges [20].
There is no doubt that chlorhexidine
has a potent antimicrobial effect. However,
although the broad-spectrum antimicrobial
action of chlorhexidine is well established in the
literature [21], the suppression of oral microbiota
by chlorhexidine represents a disadvantage [22],
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Silva PV et al.
Role of antimicrobial peptide on cariogenic biofilm and dental enamel demineralization
Silva PV et al. Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
since an effective antimicrobial must be associated
with the re-establishment of oral microbiome
balance and not its total elimination.
A reduction in enamel demineralization after
treatment with 1 mM of AMP D1-23 was observed in
our study. Although no previous studies on the effect
of AMP D1-23 on tooth enamel have been found, the
AMP TVH19 (TKRQQVVGLLWHLLHHLLH-NH2),
synthesized with bifunctional activity
(antimicrobial and remineralizing action), showed
a remineralizing effect on tooth enamel.
The control of dental enamel demineralization
by AMP D1-23 at the two investigated concentrations
observed in this study could be explained by the
reduction of total biolm biomass, since the initial
demineralization process occurs by complex
biolm formation [12] and the interruption of
this process could contribute to the reduction of
biolm acidogenicity. However, more studies are
necessaries to explain AMP D1-23 activity.
Positive results regarding the action of
AMP fragments on complex biofilms open up
interesting possibilities for their use in the
literature, taking into account that the cost
of synthesizing this material decreases when
studying its fragments [4]. Despite the promising
results of the present study, some limitations
prevent our ndings from being extrapolated to
clinical practice. Although a microcosm biolm
resembling the dynamics of the dental biolm
was used, it still represents an
in vitro
model,
which does not fully portray the reality of
biolm formation. This process depends on the
availability of nutrients, as well as the substrate
made available by each individual and the biolm
pH, which changes according to the substrate
being metabolized by the microorganisms [23].
The search for agents with anti-cariogenic
properties such as antimicrobial capacity [24],
antibiofilm properties [4], and the ability
to inhibit demineralization and to stimulate
remineralization [24] still represents a challenge
in cariology.
Even with the limitations of our results, the
possibilities of studies with peptide fragments
become more viable every day [9]. Among the
advantages of investigating these molecules, we
described the fact that dentistry is no longer looking
for materials that eliminate microorganisms of the
oral cavity, but those that provide the rebalancing
of the oral microbiome [20]. In this context, AMP
D1-23 may represent a promising strategy against
dental caries, as it has antibiolm action and an
ability to control demineralization. However,
investigations on the effect of AMP D1-23 in
the production of extracellular and intracellular
polysaccharides are still needed for controlling
biolm acidity, as well as on the effectiveness
of its incorporation into uoride varnishes and
restorative materials.
CONCLUSIONS
Based on the results of this study, the
following conclusions can be made:
1. AMP D1-23 regardless of concentration
reduced cariogenic biofilm biomass,
which is an important factor in controlling
demineralization.
2. 1mM AMP D1-23 decreased mineral loss,
controlling the progression of caries lesions.
3. AMP D1-23 may be a potential treatment
of non-cavitated initial carious lesions. The
findings of cariogenic biofilm control and
prevention of demineralization are relevant
in the control and treatment of dental caries,
making the results promising. However, further
studies are needed to assess the mechanism of
action and efcacy in clinical treatment.
Acknowledgements
The authors are grateful to the Graduate
Program in Dentistry (PPGCOI -UNIC) for
partially funding the execution of this research.
Author’s Contributions
PVS: Data Curation, Writing- Original, Draft
Preparation; TPC: Methodology, Resources,
Writing- Review & Editing; JLSU: Data Curation;
ARSJ: Formal Analysis, Writing- Review & Editing;
MSC: Methodology, Validation, Writing- Review
& Editing; AMFA: Conceptualization, Funding
Acquisition, Project Administration, Supervision,
Validation, Writing- Review & Editing.
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.
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Silva PV et al.
Role of antimicrobial peptide on cariogenic biofilm and dental enamel demineralization
Silva PV et al. Role of antimicrobial peptide on cariogenic biofilm and dental
enamel demineralization
Funding
This study was partially or totally supported by
CAPES-PROSUP and the Graduate Program in Inte-
grated Dental Sciences [88882.365929/2019-01].
Regulatory Statement
This study was conducted in accordance
with all the provisions of the local human subjects
oversight committee guidelines and policies of the
Research.
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Andreza Maria Fábio Aranha
(Corresponding address)
Universidade de Cuiabá, Faculdade de Oodntologia, DEpartamento de
Ciências Oodntológicas Integradas, Cuiabá, MT, Brazil.
Email: andreza.aranha@gmail.com
Date submitted: 2024 Feb 02
Accept submission: 2024 July 25
