UNIVERSIDADE ESTADUAL DE CAMPINAS

FACULDADE DE ODONTOLOGIA DE PIRACICABA

BEATRIZ OMETTO SAHADI

EFEITO DE SOLUÇÕES DE PRIMERS EXPERIMENTAIS À BASE

DE FLAVONÓIDES NA RESISTÊNCIA DE UNIÃO À DENTINA E
NA MORFOLOGIA DA ÁREA DE UNIÃO DENTINA-ADESIVO

EFFECT OF FLAVONOID-BASED EXPERIMENTAL PRIMER

SOLUTIONS ON DENTIN BOND STRENGTH AND DENTIN-
ADHESIVE INTERFACE MORPHOLOGY

PIRACICABA
2021
 BEATRIZ OMETTO SAHADI

EFEITO DE SOLUÇÕES DE PRIMERS EXPERIMENTAIS À BASE

DE FLAVONÓIDES NA RESISTÊNCIA DE UNIÃO À DENTINA E
NA MORFOLOGIA DA ÁREA DE UNIÃO DENTINA-ADESIVO

EFFECT OF FLAVONOID-BASED EXPERIMENTAL PRIMER

SOLUTIONS ON DENTIN BOND STRENGTH AND DENTIN-
ADHESIVE BONDING INTERFACE MORPHOLOGY

Dissertação apresentada à Faculdade de

Odontologia de Piracicaba da Universidade
Estadual de Campinas como parte dos
requisitos exigidos para obtenção do título
de Mestra em Clínica Odontológica, na Área
de Dentística.
Dissertation presented to the Piracicaba
Dental School of the University of Campinas
in partial fulfillment of the requirements for
the degree of Master in Clinical Dentistry, in
Operative Dentistry area.

Orientador: Prof. Dr. Marcelo Giannini

ESTE EXEMPLAR CORRESPONDE A VERSÃO

FINAL DA DISSERTAÇÃO DEFENDIDA PELA
ALUNA BEATRIZ OMETTO SAHADI, E
ORIENTADA PELO PROF. DR. MARCELO
GIANNINI.

PIRACICABA
2021
Agência(s) de fomento e nº(s) de processo(s): CNPq, nº 131656/2019-8;
FAPESP, nº 2019/14973-7.
 UNIVERSIDADE ESTADUAL DE CAMPINAS

Faculdade de Odontologia de Piracicaba

A Comissão Julgadora dos trabalhos de Defesa de Dissertação de Mestrado, em sessão

pública realizada em 15 de julho de 2021, considerou a candidata BEATRIZ OMETTO
SAHADI aprovada.

PROF. DR. MARCELO GIANNINI

PROF. DR. VICTOR PINHEIRO FEITOSA

PROF. DR. MÁRIO ALEXANDRE COELHO SINHORETI

A Ata da defesa, assinada pelos membros da Comissão Examinadora, consta no

SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da Unidade.
 DEDICATÓRIA

Dedico este trabalho à minha mãe, Maria Lúcia Ometto Sahadi, meu
exemplo de vida, que sempre me apoiou, incondicionalmente, nessa jornada e não
poupou esforços para que eu realizasse o sonho de formação educacional e
profissional. Sou grata por tudo o que fez e ainda faz por mim!
 AGRADECIMENTOS

Primeiramente à Deus, que iluminou meu caminho durante esta jornada.

Ao meu orientador, Prof. Dr. Marcelo Giannini, um exemplo de profissional dedicado

à pesquisa com honestidade e seriedade. Obrigada por ter me dado oportunidade de
trabalhar e aprender com profissionalismo e competência, pela paciência na
orientação desta pesquisa, pelos conhecimentos transmitidos e oportunidades
concedidas.

À minha coorientadora, Profa. Dra. Carolina Bosso André, um exemplo de

profissional. Agradeço a paciência, dedicação e disposição constante em me ajudar
desde o delineamento desse trabalho até a sua conclusão. Obrigada pela amizade,
conselhos sinceros e momentos de descontração.

À Universidade Estadual de Campinas – UNICAMP, na pessoa do magnífico Reitor

Prof. Dr. Antônio José de Almeida Meirelles e à Faculdade de Odontologia de
Piracicaba (FOP), por meio do diretor Prof. Dr. Francisco Haiter Neto.

À coordenadora geral dos cursos de Pós-graduação da FOP-UNICAMP Profa. Dra.

Karina Gonzales Silvério Ruiz.

Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq pela

bolsa concedida no primeiro ano do mestrado (processo 131656/2019-8).

À Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP, pela bolsa

de estudo concedida (processo 2019/14973-7).
Ao Funcionário do centro de Microscopia Eletrônica de varredura da FOP-UNICAMP,
Adriano L. Martins, pelo apoio, ensinamentos transferidos, pela atenção prestada e
por ser sempre muito solícito a ajudar nos momentos de dificuldade.

Aos professores da banca de qualificação, Profa. Dra. Vanessa Cavalli Gobbo,

Profa. Dra. Letícia Cidreira Boaro e Profa. Dra. Ana Paula Ayres pela
disponibilidade e valiosas contribuições ao trabalho.

Aos professores da banca de defesa, Prof. Dr. Marcelo Giannini, Prof. Dr. Mário
Alexandre Coelho Sinhoreti e Prof. Dr. Victor Pinheiro Feitosa pela disponibilidade
e valiosas contribuições ao trabalho.

Ao meu amigo e fiel escudeiro, Jorge Rodrigo Montero-Soto, agradeço a amizade e

por ter se tornado um irmão para mim. Obrigada pelos conselhos na pesquisa e,
principalmente, na vida. Um exemplo de pesquisador, pai, marido, amigo e
companheiro. Vou levar essa amizade para o resto da minha vida. Com certeza as
nossas conversas e os momentos de descontração fizeram diferença na minha
caminhada.

Aos amigos de orientação, Mayara dos Santos Noronha, Maicon Sebold, Vitaliano
Gomes de Araújo Neto e Eduardo Fernandes de Castro pela amizade, momentos
de descontração e conselhos. Com certeza os meus dias no laboratório foram mais
leves e felizes com vocês. Vocês foram os melhores amigos de orientação que eu
poderia ter nessa caminhada.

Aos amigos, Iana Maria Gonçalves, Danielle Ferreira, Gleyson do Amaral, Rodrigo
Lins e Juliana Pucci pela amizade e pelos bons momentos vividos durante essa
caminhada. Vocês tornaram os meus dias mais leves. A amizade e companheirismo
de vocês fizeram grande diferença para que eu não desanimasse nos dias mais
difíceis.

À Marina Rodrigues Santi que esteve comigo desde o início da minha caminhada,
quem se tornou meu ponto de apoio em tantos momentos. Obrigada pelo
companheirismo e parceria nos trabalhos e na vida.

À minha mãe, Maria Lúcia Ometto Sahadi, pelo amor, carinho e apoio incondicionais,
sempre me incentivando a seguir em frente e nunca me deixar abater pelos obstáculos
encontrados.

A todos que diretamente ou indiretamente contribuíram para o desenvolvimento

desse trabalho, meus sinceros agradecimentos.
RESUMO
A degradação das fibrilas colágenas é uma das causas de falhas da adesão.
Alguns compostos têm sido sugeridos para serem aplicados previamente aos
adesivos ou incorporados a eles. O objetivo desse estudo in vitro foi avaliar o efeito
de quatro soluções de primers experimentais na resistência de união à dentina por
microtração, após 24 horas e um ano de armazenamento em saliva artificial, e na
morfologia da área de união dentina-adesivo. Dois diferentes flavonóides foram
diluídos em álcool absoluto: Kaempferol (Ka) e Naringina (Na) em diferentes
concentrações (10 mM e 20 mM), obtendo quatro soluções de primers. Os primers
foram aplicados por 30 ou 60 segundos em dentina previamente condicionada com
ácido fosfórico a 35%, seguido à aplicação de um sistema adesivo convencional de
dois passos (Optibond S, Kerr). No grupo Controle Negativo (CN) não houve aplicação
de nenhum primer previamente à aplicação do adesivo e no grupo Controle Positivo
(CP) foi usado solução de Digluconato de Clorexidina 0,2% por 30 ou 60 segundos.
Oitenta e oito terceiros molares humanos foram distribuídos, aleatoriamente, em onze
grupos (n=8), de acordo com o tipo, concentração e tempo de aplicação dos primers
para o ensaio de resistência à união (RU). Os dentes foram restaurados de acordo
com cada grupo e seccionados nas direções mesial-distal e vestíbulo-lingual, obtendo-
se espécimes no formato de “paralelepípedos”. Metade dos espécimes foram
submetidos ao ensaio de RU após 24 horas, enquanto a outra metade foi armazenada
a 37º em solução de saliva artificial por um tempo de um ano e testada. Para a análise
da morfologia da área de união dentina-adesivo, quarenta e quatro dentes (n=4) foram
restaurados seguindo a mesma metodologia do ensaio de RU, porém, no adesivo foi
adicionado o corante Rodamina B na concentração 0,1% em peso. Após 24 horas, os
dentes foram seccionados em fatias de 1 mm de espessura. As fatias foram polidas e
analisadas em Microscopia Confocal de Varredura a Laser (MCVL). Os dados de RU
foram avaliados quanto à distribuição e homoscedasticidade, seguidas por análises
estatísticas apropriadas (α=0,05). Em relação aos resultados do ensaio de RU, não
foi encontrada diferença estatística entre os grupos experimentais e o CN em 24
horas. No entanto, os primers experimentais contendo 20 mM Na, 10 e 20 mM Ka,
aplicados por 60 segundos na dentina, apresentaram maiores valores de RU em
relação ao CN e CP após um ano. Formação de camada híbrida e penetração do
adesivo nos túbulos dentinários foram observadas em todos os grupos. A aplicação
de todos os primers experimentais à base de flavonóides em dentina por 60 segundos
foi capaz de aumentar a RU em comparação ao CN após um ano de envelhecimento
artificial.

Palavras-Chave: Flavonoides, Flavanonas, Adesivos Dentinários, Dentina,

Condicionamento ácido do dente.
ABSTRACT

The degradation of collagen fibrils is one of the causes of adhesion failures.

Somo compounds have been suggested to be applied before adhesives or
incorporated into them. The objective of this in vitro study was to evaluate the effect of
four experimental primer solutions on dentin bond strength (µTBS), after 24 hours and
one year of storage in artificial saliva, and the dentin-resin bonding interface
morphology. Two different flavonoids were diluted in absolute alcohol: Kaempferol (Ka)
e Naringin (Na) at different concentrations (10 mM and 20 mM), obtaining four primer
solutions. Primers were applied for 30 or 60 seconds in dentin previously conditioned
with 35% phosphoric acid, followed by the application of a two-steps etch-and-rinse
adhesive system (Optibond S, Kerr). The Negative Control (NC) consisted of no primer
application prior to adhesive, while the Positive Control (PC) comprised 0.2%
Chlorhexidine Digluconate solution used for 30 seconds or 60 seconds. Eighty-eight
third human molars were randomly assigned into eleven groups (n=8), according to
the type, concentration and time of application of the primer for the µTBS test. The
teeth were restored according to each group and were cut into lingual-buccal and
mesial-distal directions sections, obtaining stick-shaped specimens. Half of the
specimens were submitted to the µTBS test after 24 hours, while the other half was
stored at 37ºC in an artificial saliva solution for one year and tested. For the analysis
of the morphology of the bonding interface morphology, forty-four teeth (n=4) were
restored following the same methodology of the µTBS methodology. However, the
Rhodamine B dye was added to the adhesive at 0.1% weight concentration. After 24
hours, the teeth were sectioned into 1 mm thick slices. The slices were polished and
analyzed in Confocal Laser Scanning Microscopy (CLSM). All methodological analyses
were evaluated for distribution and homoscedasticity, followed by appropriate
statistical analyses (α=0.05). Regarding µTBS, no statistical difference was found
among the experimental groups and NC at 24 hours. However, experimental primers
containing 20 mM Na, 10 and 20 mM Ka applied for 60 s presented higher means than
that obtained for NC and PC at one year. Hybrid layer formation and adhesive
penetration into dentinal tubules were observed in all groups. The application of all
flavonoid-based experimental primers for 60 s was able to produce higher µTBS than
those obtained with NC after one-year of artificial aging.
Key words: Flavonoids, Flavanones, Dentin-bonding agents, Dental acid etching,
Dentin.
 SUMÁRIO

1 INTRODUÇÃO 14

2 ARTIGO: Effect of flavonoid-based experimental primers on dentin

18
microtensile bond strength and interface morphology

3 CONCLUSÃO 48

REFERÊNCIAS 49

ANEXOS 52

Anexo 1 - Comprovação da submissão do artigo “Effect of flavonoid- 52

based experimental primers on dentin microtensile bond strength and
interface morphology”

Anexo 2 – Certificado do Comitê de Ética em Pesquisa 53

Anexo 3 – Verificação de Originalidade e Prevenção de Plágio 55

 14

1 INTRODUÇÃO

Embora os avanços nas técnicas restauradoras adesivas, na química dos

materiais adesivos e no conhecimento sobre os mecanismos de união à dentina
(Bedran-Russo AK. et al., 2013) ainda há problemas associados à estabilidade da
união dentina-resina ao longo do tempo, com relatos de diminuição significativa na
resistência de união dos adesivos à dentina (Breschi et Al., 2008; Pashley et al., 2011;
Tjaderhane L, 2015). Os adesivos sofrem sorção dos fluidos orais, degradação
polimérica e, consequentemente, a lixiviação de monômeros e oligômeros (De Munck
et al., 2003; Tay FR et al., 2003).
A longevidade de uma restauração adesiva também está intimamente ligada à
formação de retenções micromecânicas de um infiltrado resinoso no esmalte e na
dentina (Leme-Kraus AA. et al., 2020). O condicionamento ácido dentinário tem a
função de remover a smear layer e a porção inorgânica superficial da dentina, para
facilitar a penetração dos monômeros adesivos entre as fibrilas colágenas
(Nakabayashi et al., 1982). Este processo é conhecido como hibridização
(Nakabayashi & Pashley, 1998), e resulta na formação de uma camada ácido-
resistente de dentina “reforçada” por resina adesiva fluida (camada híbrida), a qual é
responsável pela retenção do material restaurador ao substrato dentinário (Martins et
al., 2008).
Entretanto, como as fibrilas de colágeno não são completamente encapsuladas
pelos monômeros resinosos, principalmente na base da camada híbrida (Wang Y. et
al., 2003), as metaloproteinases da matriz dentinária (MMPs) podem degradar esse
colágeno, o qual perdeu o conteúdo mineral intra e extra-fibrilar (Perdigão J. et al.,
2013). A perda mineral torna as fibrilas desprotegidas e limita a longevidade da união
dentina-adesivo. A difusão prejudicada da resina adesiva na parte inferior da camada
híbrida também pode ser causada por alterações de fase da solução do adesivo,
devido ao seu contato com água residual deixada entre as fibras colágenas (Sebold
M. et al., 2019). Desta forma, com a deficiente infiltração da resina adesiva,
principalmente na base da camada híbrida, as fibrilas de colágeno desprotegidas são
propensas à degradação enzimática (Tay et al., 2002).
O colágeno é uma unidade elemental estrutural da matriz extracelular. As
fibrilas colágenas apresentam estabilidade química pelas ligações cruzadas
enzimáticas inter- e intra-moleculares (Bedran-Russo AK. et al., 2014). Assim, a
 15

estrutura e composição da matriz extracelular dentinária desempenham um papel

fundamental nos procedimentos restauradores adesivos e na longevidade das
restaurações adesivas (Bertassoni LE. et al., 2017). Aumentar a resistência do
colágeno contra à hidrólise por meio da biomodificação de proteínas é uma alternativa
para melhorar a estabilidade da área de união dentina-adesivo. Substâncias naturais
e sintéticas são capazes de aumentar o número de ligações peptídicas intra e
intermoleculares, além das microfibrilares do colágeno (Bedran-Russo AK. et al.,
2013). O resultado é a melhoria das propriedades mecânicas da matriz de colágeno
desmineralizada (Scheffel et al., 2014).
O digluconato de clorexidina (CHX) é um agente sintético capaz de prevenir a
degradação da camada híbrida (Breschi et al., 2009; Tjaderhane 2013). O CHX pode
inativar as proteases endógenas da área de união dentina-adesivo (Tjaderhane L. et
al., 2013, 2015) e ainda, pode se ligar a várias proteínas por um mecanismo de
quelação (Negrelo Newton AP. et al., 2004). Assim, o CHX pode evitar ligações de
íons como Zn²+ e Ca²+ às estruturas das MMPs, inibindo as suas atividades catalíticas
(Sousa ABS et al., 2016). O CHX também apresenta propriedades catiônicas, com
alta afinidade por estruturas orgânicas (colágeno) e inorgânicas (hidroxiapatita) da
dentina, especialmente quando aplicada após o condicionamento com ácido fosfórico
(Iskander M. et al., 2015). Alguns estudos relatam efeito detergente do CHX, que pode
aumentar a impregnação dos monômeros resinosos na dentina desmineralizada
(Breschi L. et al., 2010), enquanto outros mostram que a aplicação de CHX em dentina
desmineralizada aumenta a formação de tags resinosos, que ocluem os túbulos
dentinários e favorecem a retenção micromecânica (Gendron R. et al., 1999).
O uso de compostos naturais derivados de plantas para preservar a área de
união também é uma alternativa atraente aos agentes sintéticos (Tjaderhane L. et al.,
2013). Na literatura, há estudos relatando o uso da proantocianidina (PA), a qual é
classificada como um flavonóide e sub-classificada como um flavonol. Este composto
é um agente antioxidante e de reticulação de colágeno, além de possuir atividades
biológicas e funcionais (Bedran-Russo AK. et al., 2014). A principal fonte de PA está
no extrato de semente de uva (Joshi SS. Et al., 2001), que demonstra melhorar as
propriedades mecânicas e capaz de reduzir as taxas de biodegradação da dentina
desmineralizada (Phansalkar RS. et al., 2015) por multi-interação com componentes
da matriz dentinária, incluindo colágeno tipo I, proteoglicanos e proteases endógenas
(Bedran-Russo AK. et al., 2014). Sugere-se que o mecanismo de ação de ligação
 16

cruzada entre o colágeno e a PA pode ocorrer por meio de ligações covalentes e

pontes de hidrogênio. O isolamento de compostos altamente bioativos, a partir do
extrato de semente de uva mostrou recentemente resultados promissores para o
planejamento futuro de um material de intervenção clínica (Phansalkar RS. et al.,
2015).
O uso de um primer elaborado com agentes de ligação cruzada na dentina
desmineralizada, como um pré-tratamento e antes da aplicação da resina adesiva
parece ser uma abordagem promissora para estender a longevidade das restaurações
de compósito (Tjaderhane L. et al., 2013, 2015), uma vez que a esses primers
experimentais podem aumentar a estabilidade funcional da matriz orgânica da dentina
(Hicci HA. et al., 2010). Sabendo-se da afinidade entre flavonóides e proteínas ricas
em prolina e da capacidade do grupo fenil-hidroxila em formar pontes de hidrogênio
com os grupos carbonila presentes nas fibrilas de colágeno (Hagerman AE. et al.,
1981), os compostos Kaempferol, subclassificado como flavonol, e Naringina,
subclassificado como flavonona, podem ser utilizados na tentativa de reduzir as taxas
de biodegradação da dentina desmineralizada.
Os flavonóides são compostos naturais e possuem propriedades anti-
oxidativas, anti-inflamatórias, anti-mutagência e anti-carcinogênica, além disso
possuem capacidade de modular a chave da função celular enzimática. Eles também
são conhecidos por serem potentes inibidores de várias enzimas, como a xantina
oxidase (XO), ciclo-oxigenase (COX), lipo-oxigenase e fosfoinositídeo 3-quinase.
Esses flavonóides são uma classe de metabólitos secundários que contêm estruturas
fenólicas e a sua subclassificação se dá pela posição da ligação do anel benzênico B
no C, e o grau de instauração e oxidação do anel C (Figura 1). Os flavonols como o
Kaempferol possuem um grupo cetônico. Já a flavonona (Naringina), possui uma
ligação saturada no anel benzênico C (Panche AN et al, 2016).
 17

Figura 1. Flavonóides. (A) Esqueleto químico de um flavonóide. (B) Molécula do

Kaempferol. (C) Molécula da Naringina. *Adaptado de Panche AN et al, 2016.

Apesar da sua aplicação na Odontologia ainda não ter sido relatada,

espera-se que estes compostos tenham ação similar à PA por serem da mesma
classe. Porém, a PA, por ser uma molécula oligomérica, apresenta um tamanho
molecular maior (592,5 g/mol), podendo apresentar limitada penetração na dentina
desmineralizada, além de pigmentar a dentina, provocando problemas clínicos
(Epasinghe DJ. et al., 2016). Os flavonóides sugeridos apresentam como vantagem
menor tamanho molecular: Kaempferol (286,24 g/mol) e Naringina (580,53 g/mol), que
pode facilitar a penetração na dentina desmineralizada e, além disso, podem causar
menor ou nenhuma pigmentação à dentina.
Portanto, este trabalho tem como objetivo avaliar o efeito de primers
experimentais à base de flavonóides, aplicados em dentina desmineralizada
previamente ao adesivo, na resistência à união por microtração e na morfologia da
área de união dentina-adesivo.
 18

2 ARTIGO: Effect of flavonoid-based experimental primers on dentin microtensile bond

strength and interface morphology

Artigo submetido ao periódico Journal of Dentistry (Anexo 1).

Beatriz Ometto Sahadia, Carolina Bosso Andréb, Maicon Seboldc, Marcelo Gianninid.

a DDS, MSc Student, Department of Restorative Dentistry, Operative Dentistry

Division, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil.

b DDS, MSc, PhD, Adjunct Professor, Department of Restorative Dentistry, Operative

Dentistry Division, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

c DDS, MSc, PhD Student, Department of Restorative Dentistry, Operative Dentistry

Division, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil.

d DDS, MSc, PhD, Associate Professor, Department of Restorative Dentistry,

Operative Dentistry Division, Piracicaba Dental School, University of Campinas,
Piracicaba, SP, Brazil.

Corresponding author:
Beatriz Ometto Sahadi
Department of Restorative Dentistry – Operative Dentistry Division
Piracicaba Dental School – State University of Campinas
Av. Limeira, 901 – Bairro Areião
Piracicaba – SP – Brazil Zip Code: 13414-903
Phone: 55 19 21065340 Fax: 55 19 21065218
e-mail: [email protected]
 19

ABSTRACT

Objectives: This study evaluated the effect of four flavonoid-based experimental

primers on the dentin bond strength (µTBS) and the bonding interface morphology of
an adhesive. Methods: Four experimental primer solutions containing two different
flavonoids (Kaempferol - Ka and Naringin - Na) in two concentrations (10 mM and 20
mM) were applied for 30 s or 60 s on dentin after phosphoric acid etching, followed by
an etch-and-rinse adhesive system (Optibond S, Kerr). A Negative Control (NC)
consisting of dentin not treated with experimental primer solutions and Positive
Controls (PC) that used 0.2% chlorhexidine solution for 30 and 60 s were also tested.
Eighty-eight teeth were selected for dentin µTBS (n=8). Dentin-adhesive interface
morphology and adhesive resin infiltration (n=4) were analyzed by confocal laser
scanning microscopy (CLSM). Data were analyzed by mixed three-way ANOVA
(µTBS) and two-way ANOVA (CLSM) (α=0.05). Dentin µTBS was analyzed at 24h and
one-year, while the bonding interface morphology and adhesive resin infiltration were
evaluated at 24 hrs. Results: Regarding µTBS, no statistical difference was found
between the experimental groups and NC at 24 h. However, experimental primers
containing 20 mM Na, 10 and 20 mM Ka applied for 60 s presented higher values
compared to NC at one-year. Hybrid layer formation and adhesive infiltration was
observed in all groups by CLSM. Conclusions: The application of flavonoid-based
experimental primers for 60 s was able to produce higher µTBS than those obtained
with NC and PC after one-year of artificial aging, without interfering with interface
morphology.

Clinical significance: Flavonoid-based primers were able to preserve the bond

strength of acid-etched dentin after artificial aging. These formulations have the
potential to improve the longevity of adhesive restorations.

Keywords: flavonoids; kaempferol; naringin; dental bonding; microtensile bond

strength; adhesive.
 20

1. INTRODUCTION

In past decades, dental research has pushed forward remarkable evolution and
advancement in the field of adhesive restorative techniques, as well as in the chemistry
of adhesive resin-based materials, resulting in broader knowledge about dentin
bonding mechanisms [1]. However, there are still concerns regarding the stability of
dentin-resin bonding over time, with reports of a significant decrease in dentin bond
strength associated with acid etching [2,3]. It is believed the degradation of collagen
fibrils due to the activation of dentin matrix metalloproteinases (MMPs), and the
hydrolytic degradation of adhesive resin polymer [4] contribute significantly to the
failure of composite restorations [5,6].

Within this context, the development of experimental dentin primer solutions

might be a promising clinical strategy, with a few protocols already under investigation
[7]. For example, increasing collagen resistance to hydrolysis through protein
biomodification could be an alternative to improve the stability of dentin bonding.
Natural and synthetic substances can increase the number of intra- and intermolecular
peptide bonds within collagen fibrils [1], improving the mechanical properties of the
collagen matrix that lost the minerals around it [8]. In addition, these substances have
been shown to inhibit and reduce MMP activity [8], which could contribute to the
longevity of dentin-resin bonding.

Chlorhexidine digluconate (CHX) is one of the synthetic agents capable of

inactivating endogenous proteases within the dentin-resin bonding interface [9,10],
which might increase the longevity of the hybrid layer [11,12]. CHX binds to several
proteins by a chelation mechanism [13], and consequently it avoids ions such as Zn²+
and Ca²+ from binding to MMPs, inhibiting their proteolytic activity [7]. Also, CHX has
cationic properties, presenting strong affinity for organic (collagen) and inorganic
(hydroxyapatite) dentin structures, especially when applied after acid etching [14].

Apart from synthetic agents, the use of natural, plant-derived compounds,

such as flavonoids, has been demonstrated to reduce the degradation of collagen
fibrils [15]. Previous studies have reported on the application of proanthocyanidin,
which is an antioxidant and collagen crosslinker provided with biological and functional
activities [16] that can preserve the integrity of the hybrid layer over time [17].
Flavonoids have anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-
 21

carcinogenic properties, while also being able to modulate key processes of cell
enzymatic function [18]. They are also known to be potent inhibitors of various
enzymes, namely xanthine oxidase, cyclooxygenase, lipooxygenase and
phosphonoside 3-kinase [18]. In fact, some flavonoids have shown better performance
in reducing the degradation of the hybrid layer when compared to proanthocyanidins
[19,20].
Flavonols such as Kaempferol have a ketone group, while Naringin has a
saturated bond in the benzene ring C [18], similar to the chemical structure of other
flavonoids. Although their application in dentistry has not yet been reported, these
compounds are expected to have a similar action mechanism compared to
proanthocyanidin, because they come from the same class of biomodifiers, which
induce irreversible changes in the catalytic domain or allosteric inhibition of other
modular domains of proteases (MMPs) involved in collagen degradation [21].
Considering proanthocyanidin is an oligomeric molecule with a larger molecular size
(592.5 g/mol), it might present limited penetration into demineralized dentin.
Furthermore, it causes dentin staining, leading to clinical problems [22]. Conversely,
flavonoids have the smallest molecular size advantage: Kaempferol (286.24 g/mol)
and Naringin (580.53 g/mol). This suggests they would better penetrate demineralized
dentin, presenting promising results in the preservation of the dentin-resin bond [23].
Therefore, the aim of this study was to evaluate the effect of the application of
flavonoid-based (Kaempferol and Naringin) experimental primers on dentin bonding at
different concentrations (10 mM or 20 mM) and application times (30 s or 60 s) after
phosphoric acid etching. The null hypotheses of this study were: (1) flavonoid-based
primers would not influence the results of dentin microtensile bond strength (µTBS)
after 24 h or one-year of storage in artificial saliva when compared to untreated dentin
(Negative Control); (2) flavonoid-based primers would not influence the results of µTBS
after 24 h or one-year of storage in artificial saliva when compared to dentin treated
with CHX (Positive Control); (3) different concentrations and application times would
not influence the µTBS results; and (4) flavonoid-based primers would not interfere
with the morphology of the bonding interface.

2. MATERIALS AND METHODS

 22

2.1. Teeth Selection and Preparation

One hundred and thirty-two sound human third molars were obtained after the
approval by the Ethics Committee in Research of the Piracicaba Dental School (CAAE
#19855119.1.0000.5418). All teeth were cleaned by hand-scaling with a periodontal
curette (SS White Duflex, Juiz de Fora, MG, Brazil), and polished with a paste of
pumice and water. Afterwards, they were stored in aqueous solution of 0.5%
Chloramine-T (Merck KGaA, Darmstadt, Germany) at 4 °C for no longer than three
months.

2.2. Experimental Primer Solutions

Naringin (Merck KGaA, Darmstadt, Germany) and Kaempferol (Merck KGaA,

Darmstadt, Germany) were diluted at concentrations of 10 mM and 20 mM in absolute
ethanol. The physical and chemical properties of these flavonoids are displayed in
Table 1. The compounds were diluted and homogenized with the aid of a magnetic
stirrer and kept in light-proof containers, with their lids closed to prevent solvent
evaporation. The pH of these solutions was determined using a digital pH meter (MS
Tecnopon Equipment’s, Piracicaba, SP, Brazil) (Table 2). Chlorhexidine 0.2% in
aqueous solution was used as Positive Control.

Table 1. Physical and Chemical characteristics of flavonoids used for the experimental
primers.

Substanc Empiric 2D Chemical Number of Molecul Solubili Solubili Colo

e al structure* hydroxyfe ar Mass ty in ty in r
formula nyl water ethanol
radicals
Naringin C27H32O 2 580.53 1mg/mL 0.1 Whit
≥95% 14 g/mol at 40º C g/mL e to
HPLC faint
(Merck beige
KGaA,
Darmsta
dt,
Germany
)
 23

Kaempfe C15H10O 4 286.24 440mg/ 0.02 Yello

rol ≥97% 6 g/mol L at 25º g/mL w
HPCL very
(Merck dark
KGaA, yello
Darmsta w
dt,
Germany
)

Information provided by the Manufacturer. (*) According to PubChem.

Table 2. Characteristics of experimental primer solutions.

Experimental Primers pH Color of the solution

Naringin 10 mM 7.2 Colourless

Naringin 20 mM 7.7 Colourless
Kaempferol 10 mM 7.3 Yellow
Kaempferol 20 mM 7.1 Yellow

2.3 Dentin Microtensile Bond Strength Test (µTBS)

This study followed the methodology described in the guidelines of the Academy
of Dental Materials which were published in Dental Materials [24]. The occlusal enamel
of each tooth was removed with a diamond saw (Buehler Ltd., Lake Bluff, IL, USA)
using a precision cutting machine, and dentin was exposed at medium depth in relation
to the pulp. Dentin surfaces were polished with silicon carbide paper (600-grit) for 5 s
under water-cooling to create a flat surface and standardize the smear layer.

Eighty-eight teeth were randomly assigned to eleven groups (n=8), according to

type of primer, concentration of primer main ingredient, and application time (Table 3).
Dentin was etched with 35% phosphoric acid (Ultra-Etch, Ultradent Products, Inc.,
South Jordan, UT, USA) for 15 s and rinsed with water for 15 s by an oil-free air/water
spray. Dentin was kept moist before primer application. An amount of 15 μL of each
primer was applied passively to the dentin surfaces, according to the application time
 24

of each group. Excess primer solution was removed with absorbent paper. Then, a 2-
step, etch-and-rinse adhesive (Optibond S, Kerr Dental, Orange, CA, USA) was
applied for 15 s using light brushing motion, followed by air thinning for 3 s, and light-
cured according to the manufacturer's instructions. The Negative Control consisted in
the application of adhesive resin without a previous priming step.

Table 3. Treatments, type of flavonoid-based primer, concentrations and primer

application time for experimental groups and controls.

Treatment Primer/Concentration Application time (in

(abbreviation) seconds)

Chlorhexidine 0.2% (CHX/30) 30

Positive Controls
Chlorhexidine 0.2% (CHX/60) 60

Naringin 10 mM (Na10/30) 30

Naringin 10mM (Na10/60) 60

Naringin 20 mM (Na20/30) 30

Flavonoid-based Naringin 20 mM (Na20/60) 60

goups Kaempferol 10 mM (Ka10/30) 30

Kaempferol 10mM (Ka10/60) 60

Kaempferol 20 mM (Ka20/30) 30

Kaempferol 20 mM (Ka20/60) 60

Negative Control No primer application -

A resin composite (Spectra Smart, Dentsply Sirona, Pirassununga, SP, Brazil)

build-up of approximately 4-mm height was placed over bonded dentin, using 2
consecutive layers of composite with 2-mm thickness. Each composite layer was light-
cured for 20 s (Valo, Ultradent Products Inc., South Jordan, UT, USA). The light-curing
unit was verified by a spectroradiometer (MARC-PS, BlueLight Analytics Inc., Halifax,
NS, Canada) to ensure the delivery of a radiant exposure of at least 16.8 J/cm 2 (50).
All restorative materials used in this study are described in Table 4.
 25

Table 4. Commercial name, composition and batch number of the materials used in
the study.

Material Composition Batch

Number
Ultra-Etch Phosphoric acid, dimethicone. BFDJ8
Optibond S Alkyl dimethacrylate resins, barium 6840312
aluminoborosilicate glass, fumed silica (silicon
dioxide), sodium hexafluorosilicate and ethyl alcohol.
Spectra Smart Glass powder, colloidal silica hydrophobic, 356416K
dimethacrylate, benzophenone-3, ethylamine
benzoate, camphorquinone, butylated
hydroxytoluene, yellow and red iron oxide, black iron
oxide and titanium dioxide.
SureFil SDR Modified urethane dimethacrylate resin, triethylene 02419
Flow glycol dimethacrylate, polymerizable dimethacrylate
resin, polymerizable trimethacrylate resin,
camphorquinone, ethyl-4(dimethylamino)benzoate,
butylated hydroxy toluene, fluorescent agent, UV
stabilizer, barium-alumino-fluoro-borosilicate glass,
silanated strontium alumino-fluoro-silicate glass,
surface treated fume silicas, ytterbium fluoride,
synthetic inorganic iron oxide pigments and titanium
dioxide (70.5 wt% / 47.4 vol% filler).

Restored teeth were kept at relative humidity, at 37 °C, for 24 h. Thereafter,

teeth were sectioned in lingual-buccal and mesial-distal directions to obtain at least 8
to 16 stick-shaped specimens per tooth with a cross-section area of approximately 1.0
mm2. Half of the specimens per teeth were tested after 24 h of storage in artificial saliva
(12.9 mM KCl, 1.9 mM KSCN, 2.4 mM Na2SO4 10 H2O, 3.3 mM NH4Cl, 1.5 mM CaCl2
2H2O, 7.5 mM NaHCO3, 0.02 mM ZnCl2 and 5 mM HEPES buffer pH 7.4) [25] at 37°C,
while the other half was tested after 12 months of storage under the same conditions.
The artificial saliva was replaced monthly.

Specimens were tested on a microtensile device attached to a universal testing

machine (EZ Test, Shimadzu, Kyoto, Japan). Each specimen was fixed to the device
with cyanoacrylate-based glue (Super Bonder Gel, Henkel/Loctite, Diadema, SP,
Brazil) and tested at a constant speed of 1.0 mm/min until failure. The cross-section
area of the specimens was measured by means of a digital caliper (Mitutoyo Co.,
Kanagawa, Japan), and bond strength means were calculated from the average of the
specimens per tooth, according to the evaluation time. Data were submitted to normal
 26

distribution and homoscedasticity tests (Shapiro-Wilk and Levene tests) and were
analyzed by three-way mixed ANOVA, followed by Bonferroni’s test (between-subject
factors: “type of primer” and “primer application time”; within-subject factor: “evaluation
time”). The Negative Control was analyzed by paired T test and compared to other
groups by one-way ANOVA, followed by Dunnett’s test [43].

2.4 Failure Mode Analysis by Scanning Electron Microscopy (SEM)

The surfaces involved in the fracture of each specimen after the microtensile
bond strength test were analyzed by SEM regarding failure pattern classification. The
fractured specimens were fixed on metallic stubs with carbon tape, keeping the areas
involved in the fractures facing upwards. Then, these fragments were sputter-coated
with gold (SDC 050 Sputter coater, Baltec, Balzers, Liechtenstein) and observed under
SEM (JSM 5600LV, Jeol, Tokyo, Japan), using 100x and 400x magnifications. Failure
modes were classified according to the structures involved [26]: Type I - cohesive
failure within the resin composite; Type II - adhesive failure between resin composite
and the bonding agent; Type III - adhesive failure between dentin and the bonding
agent; Type IV - mixed failure (dentin, bonding agent, and resin composite can be
observed in the same fractured surface); Type V - cohesive failure within the bonding
agent layer; Type VI - cohesive failure within the hybrid layer; and Type VII - cohesive
failure within dentin. In order for a specimen not to be considered mixed, 70% or more
of a single specific failure mode should be present on the evaluated surface.
Descriptive analysis was used to report failure modes with their respective percentages
of occurrence.

2.5 Adhesive-Dentin Bonding Interface Morphology Analysis by Confocal Laser

Scanning Microscopy (CLSM)

Forty-four human third molars (n = 4) were restored following the same

methodology described for the microtensile bond strength test. However, Rhodamine
B dye (Sigma-Aldrich, St. Louis, MO, USA), which presents a pink-reddish color, was
previously added to the adhesive system at a concentration of 0.1 wt% [27]. After acid
etching and the application of the tested primers, the adhesive resin containing
 27

Rhodamine B was applied and light-cured. A flowable bulk-fill resin (SureFil SDR Flow
(Dentsply Sirona, Konstanz, Germany) was used to cover the adhesive-bonded
surfaces and the samples were stored in vegetable oil (Soya, Bunge Brasil, São Paulo,
SP, Brazil) to avoid water loss and/or dye dissolution, at 37 °C for 24 h. Then, teeth
were sectioned into 1-mm thick slices. These slices were manually polished with 2000-
grit silicon carbide paper for 30 s.
Polished samples were analyzed under CLSM (TCS SP5, Leica Microsystems,
Mannheim, Baden-Württemberg, Germany). The excitation energy provided by the
argon (488 nm) and He-Ne (543 nm) lasers, and the photomultipliers amplification
were constant throughout the whole investigation. A layer approximately 10 μm below
the surface of the sample was observed and CLSM micrographs were obtained in
fluorescent and reflectance modes with oil immersion objective lens (63x
magnification, 3x zoom, numeric aperture of 1.3, pinhole of 5.5 μm). At least three sets
of micrographs were obtained for each sample, which comprised (1) an image of dye
detection in fluorescent mode, (2) an image formed by overlapping the micrographs of
fluorescent and reflectance modes, and (3) a gray-scale image of the sample surface
in transmission mode. The hybrid layer thickness was measured using the Image J
software (Fiji, Version 1.0). Data were submitted to normal distribution and
homoscedasticity tests (Shapiro-Wilk and Levene tests) and analyzed by two-way
ANOVA and one-way ANOVA (comparing negative controls).

3. RESULTS

3.1. Dentin Microtensile Bond Strength Test

Mean (± standard deviation) µTBS values are presented in Table 5. Three-way

mixed ANOVA analysis showed that “type of primer” (p < 0.001), “primer application
time” (p = 0.009), “evaluation time” (p < 0.001), and all interactions between each two
factors significantly influenced µTBS results.
 28

Table 5. Means (± standard deviation) dentin µTBS (in MPa), comparing the
experimental groups with the Negative Control at different evaluation time and primer
application time on dentin.

24 hours One year

Treatments 30s 60s 30s 60s

Chlorhexidine 0.2% 69.4 (10.7) Aa 65.1 (11.1) Ab 84.6 (9.5) Aa 76.2 (11.1) Ac

Naringin 10 mM 76.2 (9.4) Aa 80.3 (6.9) Aa 83.9 (10.0) Aa& 88.3 (4.8) Abc&*

Naringin 20 mM 71.4 (9.3) Aa 81.2 (11.6) Aa 86.0 (7.8) Ba 97.7 (9.3) Aab*

Kaempferol 10 mM 73.0 (12.4) Aa 67.2 (8.2) Aab 91.3 (9.9) Aa* 96.9 (3.5) Aab*

Kaempferol 20 mM 73.2 (5.4) Aa 75.4 (11.6) Aab 94.6 (4.8) Ba* 107.1 (8.6) Aa*

Negative Control 71.1 (10.1) 75.9 (4.4) &

Means followed by different letters indicate significant difference (p<0.05). Lowercase

letters compare treatments within the same application and evaluation times.
Uppercase letters compare application times within the same treatment and evaluation
time. (&) Indicate no significant difference compared to the 24-hour results for the same
treatment and the application time. (*) Indicate significant difference from the Negative
Control within the same evaluation time.

When applied for 30 or 60 s, none of the flavonoid-based experimental primers

showed higher µTBS than Negative Control (no primer application) at 24 hours.
Differences among treatments were observed when experimental primers were
applied for 60 s, as naringin-based primers presented higher µTBS means at 24 hours
compared to CHX/60, for both concentrations tested (Na10/60 and Na20/60).

After one year of storage, most groups of the tested flavonoid-based

experimental primers had higher µTBS means compared to CHX/60. Also, the µTBS
of most experimental primer groups increased after one year, while Na10/30 and
Na10/60 presented stable µTBS results over time. In general, flavonoids-based
primers presented higher µTBS means compared to the Negative Control group at one
year, except for Na10/30 and Na20/30. For CHX, the application time did not influence
the results, regardless of the evaluation time.
 29

3.2 Failure Mode Analysis

The percentages of occurrence of failure modes for each tested group are
presented in Figure 1. Cohesive failures within resin composite (Type I) were
predominant (27% or more) for all groups, regardless of evaluation time (Figure 2.1).
Cohesive failures within dentin (Type VII) were also observed for all experimental and
control groups (10% or more) (Figure 2.7). The sum of both cohesive failures (within
resin composite and dentin) was greater than 50%. Adhesive failures between resin
composite and bonding agent (Type II) were the most predominant adhesive fracture
(Figure 2.2), while adhesive failures between dentin and bonding agent (Type III -
Figure 2.3) were not always observed (at 24 hours: Na20/30 and at one year: Na20/30,
Na20/60, and Ka10/30) or had a low incidence. Failure modes Type IV (Figure 2.4)
and V (Figure 2.5) showed intermediate frequency of occurrence, and Type VI failures
were very uncommon (Figure 2.6). Failure modes for the experimental and control
groups did not seem to change after one year of storage.
 30

Figure 1. Failure pattern percentage for each group. (A) at 24 hours and (B) at one
year. Type I: cohesive failure within resin composite; Type II: adhesive failure between
resin composite and bonding agent; Type III: adhesive failure between dentin surface
and bonding agent; Type IV: mixed failure; Type V: cohesive failure within adhesive
layer; Type VI: cohesive failure in the hybrid layer; Type VII: cohesive failure within
dentin.
 31

D
RC
AL
 32

Figure 2. Representative SEM images of each failure pattern at 400x magnification.

2.1: (Type I) cohesive failure within resin composite; 2.2: (Type II) adhesive failure
between resin composite and bonding agent; 2.3: (Type III) adhesive failure between
dentin surface and bonding agent; 2.4: (Type IV) mixed failure; 2.5: (Type V) cohesive
failure within adhesive layer; 2.6: (Type VI) cohesive failure in the hybrid layer; 2.7:
(Type VII) cohesive failure within dentin. RC: resin composite; AL: adhesive layer; D:
dentin.

3.3 Adhesive-Dentin Bonding Interface Morphology Analysis

Representative CLSM images of the dentin bonding interface morphology and

measurements of hybrid layer thickness are depicted in Figure 3 and Table 6,
respectively. The average hybrid layer thickness of the tested groups ranged from 2.6
to 4.3 μm. Resin tags formation was observed in all groups, despite the application of
a dentin primer. Slight differences were observed among groups regarding the number
and length of resin tags. However, this feature depends on the dentin cutting direction
and on the Z depth of CLSM. The concentration of CHX and flavonoids, as well as the
application time did not influence the hybrid layer thickness means (Table 6).
33
34
35
 36

Figure 3. CLSM images of each group tested. (I) Overlapping of the micrographs on
fluorescent mode and reflectance mode. (II) Micrographs on reflectance mode. The
area between dotted lines indicates the hybridization zone. Arrows indicate resin tags
formation. RC: resin composite; AL: Adhesive layer; D: dentin.
 37

Table 6. Hybrid layer thickness means (in µm) for each tested group, according to the
primer application time.

Group 30s 60s

CHX 2.7 (0.4) 2.9 (0.3)

Na10 3.2 (0.8) 3.2 (0.6)

Na20 3.6 (0.1) 2.8 (1.1)

Ka10 3.4 (0.6) 2.9 (0.3)

Ka20 3.4 (0.5) 3.9 (0.3)

NC 3.6 (0.8)

No significant difference was observed among groups.

4. DISCUSSION

Many researchers have investigated and discussed alternative clinical

approaches to increase the stability and longevity of dentin-resin bonds [2,18,23]. The
use of experimental primers based on flavonoids has been shown to increase collagen
resistance against hydrolysis through the biomodification of proteins, which
consequently improves the stability of the adhesive interface [7]. Although several
flavonoids have already been extensively investigated, the search for different
subclasses of these compounds for the development of new techniques and materials
remains extremely important in the current scenario of restorative dentistry, especially
regarding the longevity of adhesive restorations after dentin acid etching.

This study showed flavonoids used as experimental primer solutions could

influence μTBS values when compared with the Negative Control (untreated dentin, i.
e. no primer application). Thus, the first null hypothesis was rejected. As observed in
Table 5, the tested dentin primers had a positive effect on μTBS results only after one
year of aging compared to the Negative Control, unlike what occurred for the 24-hour
 38

evaluation time, when results did not differ statistically from the Negative Control.
Although other studies corroborate the data herein presented [28], additional research
approaches are needed in order to fully understand the mechanism of action of these
compounds, and their long-term effects on dentin bonding.

The present study deals with the use of flavonoids, which are a group of natural
substances presenting varied phenolic structures. They can be classified into different
subclasses, depending on the position of the carbon element in their molecular ring,
the position in which their B ring is attached, and the degree of oxidation of their C ring
[18]. Despite the similarity between flavonoids, considerable variations in biological
properties can be caused by even minor changes in their molecular structures, such
as the number and specific positions of hydroxyl groups and their substitutions [29].
Naringin belongs to the group of flavones, which have a saturated C ring in their
structure, while Kaempferol belongs to the subgroup of flavonols, which are
characterized by presenting a ketone group and compared to flavones, flavonols have
a hydroxyl group at position 3 of the C ring (Table 1). Both subclasses are diverse in
methylation and hydroxylation patterns [18].

In addition to the structural difference, the interaction between natural

compounds and dentin will depend on the type of existing chemical bonding, following
four proposed types: covalent [16], hydrogen [9], ionic [30] or hydrophobic [31] bonds.
Thus, a potential positive effect of flavonoids on dentin bonding could be related to the
number of hydrogen bonds between their reactive phenolic groups which may react
with the carboxyl, amine, hydroxyl, or amide groups in collagen fibrils [32], suggesting
these compounds might play an important role in stabilizing these fibrils. Due to its
triple-helix structure, collagen allows the binding of hydrogen molecules present in the
flavonoids to carbonyl oxygen in the peptide of collagen fibrils [33].

There are also a few factors that might contribute to the cross-linker effect of
flavonoids on demineralized dentin: I) the smaller molecular size (weight) of the
flavonoids that facilitates their diffusion ; II) the number of molecules available in the
experimental primer solution; III) the molecular solubility index and its influence on the
miscibility of the vehicle used for dilution of the flavonoids; and IV) the amount and type
of reactive sites of the flavonoid molecules (Table 1) [23]. Kaempferol presents a higher
amount of hydroxyphenyl sites, lower molecular weight, and better solubility in alcohol
 39

compared to Naringin. These features of Kaempferol might have reflected on the

results obtained by this type of flavonoid applied for 60 s, even after artificial aging of
the samples for one year.

Although Naringin has similar characteristics to Kaempferol, they differ

regarding molecular weight. Naringin has double the molecular weight of Kaempferol,
and it has two hydroxyphenyl radicals less than Kaempferol. These differences may
have hindered infiltration and interaction with demineralized dentin for Naringin.
Increasing the concentration of Naringin to 20 mM and the application time of its
derived primer to 60 s improved the bond strength to dentin when compared to
CHX/60. Therefore, the amount of Naringin present in the primer may influence the
interaction with demineralized dentin. In fact, the highest results found of μTBS at one
year were Ka10/60, Ka20/60, and Na20/60 compared to CHX and NC.

The primer application time on dentin surface was also a factor that influenced
the results of μTBS. When studies about the application of natural compounds on
dentin were initiated, the recommended application time was of at least 10 min [34].
However, this time would be clinically unfeasible. More recently, studies have shown
that application times between 60 and 120 s could be considered acceptable clinical
times [35]. These short application times were sufficient to increase the degree of
crosslinking and the μTBS results in demineralized dentin [36]. The present study
proposed different application times, including a period of time even shorter than those
reported in literature so far (30 s).

At 24 hours, the primer application time did not cause any change in μTBS
results, which was not observed at one year. After artificial aging, the Na/20 and Ka/20
showed better results when the primers were applied to dentin for 60 s compared to
30 s, rejecting the third null hypothesis. Although the application for 30 s was not
comparable with the results of 60 s, it did not demonstrate a poor performance in μTBS
after one year of storage. In fact, most of the tested groups were able to increase the
μTBS values, except for Na10/30 and Na10/60. Also, the concentration of 10 mM or
20 mM did not affect μTBS comparing within the same flavonoid and application time,
for both evaluation times. However, Na10/60 had no statistical difference compared to
CHX, while Na20/60 had a statistically significant difference compared to CHX.
 40

A solution of chlorhexidine digluconate at 0.2% was used as a positive control

in this study, because it has shown positive effects on bonding interface stability in
other studies [11,12]. However, the duration of these effects has been questioned.
Albeit CHX has an inhibitory effect on dentin MMPs in the adhesive interface [37], the
longevity of CHX-induced effects is a limitation to solve the problem of hybrid layer
degradation. Therefore, the second null hypothesis of this study should also be
rejected, since some experimental primers based on flavonoids differed from CHX
(Na10/6, Na20/6 for 24-hour evaluation; Na20/60, Ka10/60, and Ka20/60 after one-
year aging).

Another factor that might be related to the better μTBS performance of the
experimental primers after aging compared to CHX is the vehicle that was used to
dilute the tested flavonoids. Alcohol, in addition to exerting positive effects on dentin
bonding [38], can have a synergistic effect with flavonoids, affecting polyphenolic
collagen interactions and the amount of hydrogen bonds formed [39]. These
interactions occur due to a decrease in the dielectric constant, which can reduce the
interfibrillar spaces filled with water, thus preventing collagen degradation [40].
Moreover, flavonoids may be more reactive when diluted in alcohol, as in the case of
Naringin [41]. Despite the described differences in μTBS, no great changes are
observed in failure modes among groups. The high prevalence of cohesive failures
within resin composite or within dentin demonstrates the bonding area, which was able
to withstand tensile testing, might be more resistant that the cohesive strength of the
composite or dentin. Additionally, Type III failures (between dentin and adhesive) were
very uncommon or not present at all, even for Negative Control, showing the Optibond
S adhesive interacted properly with etched dentin.

The analysis of interface morphology by CLSM shows all tested groups had
hybrid layer formation and adhesive infiltration into dentin coupled with the formation
of resin tags, though none or little influence on hybrid layer thickness was seen. Hence,
the fourth null hypothesis was accepted. The thickness of the hybrid layer is not always
related to higher μTBS values [42], which can be observed comparing the average
thickness of Na20/60 (2.8 μm) and Ka10/60 (2.9 μm) and their respective 24-hour
μTBS results with other groups.
 41

Although the μTBS values presented in this study seem promising, especially
after artificial aging, the application of the experimental primer solutions on etched
dentin is an additional step during the bonding procedure, which can increase chair-
side time and technique sensitivity. Moreover, further studies are necessary to
investigate the interaction of the tested flavonoids with etched dentin and their effects
on MMP activity. However, the beneficial effects produced by flavonoids isolated from
natural compounds should be considered for the development of future techniques or
adhesive restorative materials, focusing on the reduction of bonding interface
degradation.

CONCLUSION

Within the limitations of the present study, the following conclusions can be drawn:

- All the tested flavonoid-based primers seemed to improve dentin bond strength after
one year compared to untreated dentin when applied for 60 s, while only Kaempferol
improved dentin bond strength after one year when applied for 30 s;

- Kaempferol at both tested concentrations and Naringin at 20 mM concentration

presented better results after one year compared to chlorhexidine when applied for 60
s;

- The investigated flavonoid-based primers did not seem to interfere with adhesive
interface morphology and failure mode.

- The effectiveness of the experimental primers might depend on the type of flavonoid
used and the time of application on dentin;

- Better results were obtained when the flavonoid-based primers were applied for 60
s.
 42

ACKNOWLEDGEMENTS
This research was supported by Brazilian Financial Agencies: CNPq: 131656/2019-8

and FAPESP: 2019/14973-7.

CrediT authorship contribution statement

Beatriz Ometto Sahadi: Methodology, Investigation, Writing - Original Draft,

Carolina Bosso André: Conceptualization, Formal analysis, Writing - Review &
Editing. Maicon Sebold: Methodology, Writing – Review and Editing. Marcelo
Giannini: Conceptualization, Writing - Review & Editing, Project administration.

FIGURE LEGENDS

FIGURE 1. Failure mode percentages of occurrence for each group at 24 hours (A)
and at one year (B). Type I: cohesive failure within resin composite; Type II: adhesive
failure between resin composite and bonding agent; Type III: adhesive failure between
dentin surface and bonding agent; Type IV: mixed failure; Type V: cohesive failure
within adhesive layer; Type VI: cohesive failure within the hybrid layer; Type VII:
cohesive failure within dentin.

FIGURE 2. Representative SEM images of each failure pattern at 400x magnification.

2.1: (Type I) cohesive failure within resin composite; 2.2: (Type II) adhesive failure
between resin composite and bonding agent; 2.3: (Type III) adhesive failure between
dentin surface and bonding agent; 2.4: (Type IV) mixed failure; 2.5: (Type V) cohesive
failure within adhesive layer; 2.6: (Type VI) cohesive failure in the hybrid layer; 2.7:
(Type VII) cohesive failure within dentin. RC: resin composite; AL: adhesive layer; D:
dentin.
 43

FIGURE 3. CLSM images of each group tested. (I) Overlapping of the micrographs in
fluorescent and reflectance modes. (II) Micrographs in reflectance mode. The area
between dotted lines indicates the hybridization zone. Arrows indicate resin tags. RC:
resin composite; AL: adhesive layer; D: dentin.

REFERENCES

1. Bedran-Russo AK, Karol S, Pashley DH, Viana G, Site specific properties of

carious dentin matrices biomodified with collagen cross-linkers, Am J Dent. 26
(2013) 244-248.
2. Pashley DH, Tay FR, Imazato S, How to increase the durability of resin-dentin
bonds, Compend Contin Educ Dent. 32 (2011) 60-66.
3. Tjäderhane L, Dentin Bonding: Can We Make it Last? Oper Dent. 40 (2015) 4-
18, https://doi.org/10.2341/14-095-BL.
4. Reis AF, Carrilho MR, Ghaname E, Pereira PN, Giannini M, Nikaido T, Tagami
J, Effects of water-storage on the physical and ultramorphological features of
adhesives and primer/adhesive mixtures, Dent Mater J. 29 (2010) 697-705,
https://doi.org/10.4012/dmj.2009-091.
5. Mazzoni A, Carrilho M, Papa V, Tjaderhane L, Gobbi P, Nucci C, Di Lenarda R,
Mazzotti G, Tay FR, Pashley DH, Breschi L, MMP-2 assay within the hybrid
layer created by a two-step etch-abd-rinse adhesive: biochemical and
immunohistochemical analysis, J Dent. 39 (2011) 470-477,
https://doi.org/10.1016/j.jdent.2011.04.004.
6. Mazzoni A, Scaffa P, Carrilho M, Tjaderhane L, Di Lenarda R, Polimeni A,
Tezvergil-Mutluay A, Tay FR, Pashley DH, Breschi L, Effects of etch-and-rinse
and self-etch adhesives on dentin MMP-2 and MMP-9, J Dent Res. 92 (2013)
82-86, https://doi.org/10.1177/0022034512467034.
7. Silva Sousa AB, Vidal CMP, Leme-Krauss AA, Pires-de-Souza FCP, Bedran-
Russo AK, Experimental primers containing synthetic and natural compounds
reduce enzymatic activity at the dentin-adhesive interface under cyclic loading,
Dent Mater. 32 (2016) 1248-1255, https://doi.org/10.1016/j.dental.2016.07.012.
 44

8. Scheffel DL, Hebling J, Scheffel RH, Agee K, Turco G, de Souza Costa CA,
Pashley D, Inactivation of matrix-bound matrix metalloproteinases by cross-
linking agents in acid-etched dentin, Oper Dent. 39 (2014) 152-158,
https://doi.org/10.2341/12-425-L.
9. Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S,
Tezvergil-Mutluay A, Carrilho M, Carvalho RM, Tay FR, Pashley DH, Strategies
to prevent hydrolytic degradation of the hybrid layer—a review, Dent Mater. 29
(2013) 999–1011, https://doi.org/10.1016/j.dental.2013.07.016.
10. Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S,
Tezvergil-Mutluay A, Carrilho MR, Carvalho RM, Tay FR, Pashley DH,
Optimizing dentin bond durability: control of collagen degradation by matrix
metalloproteinases and cysteine cathepsins, Dent Mater. 29 (2013) 116-135,
https://doi.org/10.1016/j.dental.2012.08.004.
11. Breschi L, Mazzoni A, Nato F, Carrilho M, Visintini E, Tjäderhane L, Ruggeri A
Jr, Tay FR, Dorigo Ede S, Pashley DH, Chlorhexidine stabilizes the adhesive
interface: a 2-year in vitro study, Dent Mater. 26 (2010) 320-325,
https://doi.org/10.1016/j.dental.2009.11.153.
12. Carrilho MR, Geraldeli S, Tay F, de Goes MF, Carvalho RM, Tjäderhane L, Reis
AF, Hebling J, Mazzoni A, Breschi L, Pashley D, In vivo preservation of the
hybrid layer by chlorhexidine, J Dent Res. 86 (2007) 529-533,
https://doi.org/10.1177/154405910708600608.
13. Negrelo Newton AP, Cadena SM, Merlin Rocha ME, Skare Carnieri EG,
Martinelli de Oliveira MB, New data on biological effects of chlorexidine: Fe2+
induced lipid peroxidation and mitochondrial permeability transition, Toxicol
Lett. 151 (2004) 407-416, https://doi.org/10.1016/j.toxlet.2004.02.013.
14. Iskander M, Elkassas D, Mohsen MA, Effect of two matrix metalloproteinase
inhibitors on the color stability of a nanofilled resin composite, Oper Dent. 40
(2015) E11-20, https://doi.org/10.2341/12-428-L.
15. Hiraishi N, Sono R, Sofiqul I, Yiu C, Nakamura H, Otsuki M, Takasuka J, In vitro
evaluation of plant-derived to preserv dentin collagen, Dent Mater. 29 (2013)
1048-1054, https://doi.org/10.1016/j.dental.2013.07.015.
16. Bedran-Russo AK, Pauli GF, Chen SN, McAlpine J, Castellan CS, Phansalkar
RS, Aguiar TR, Vidal CM, Napotilano JG, Nam JW, Leme AA, Dentin
 45

biomodification: strategies, renewable resources and clinical applications, Dent

Mater. 30 (2014) 62–76, https://doi.org/10.1016/j.dental.2013.10.012.
17. Leme-Kraus AA, Aydin B, Vidal CM, Phansalkar RM, Nam JW, McAlpine J,
Pauli GF, Chen S, Bedran-Russo AK, Biostability of the proanthocyanidins-
dentin complex adhesion studies, J Dent Res. 96 (2017) 406-412,
https://doi.org/10.1177/0022034516680586.
18. Panche AN, Diwan AD, Chandra SR, Flavonoids: an overview, J Nutr Sci. 5
(2016) e47, https://doi.org/10.1017/jns.2016.41.
19. Islam S, Hiraishi N, Nassar M, Yiu C, Otsuki M, Tagami J, Effect of natural cross-
linkers incorporation in a self-etching primer on dentine bond strength, J Dent.
40 (2012) 1052-1059, https://doi.org/10.1016/j.jdent.2012.08.015.
20. Islam MS, Hiraishi N, Nassar M, Yiu C, Otsuki M, Tagami J, Effect of hesperidin
incorporation into a self-etching primer on durability of dentin bond, Dent Mater.
30 (2014) 1205-1212, https://doi.org/10.1016/j.dental.2014.08.371.
21. Sela-Passwell N, Rosenblum G, Shoham T, Sagi I, Structural and functional
bases for allosteric control of MMP activities: can it pave the path for selective
inhibition? Biochim Biophys Acta. 1803 (2010) 29-38,
https://doi.org/10.1016/j.bbamcr.2009.04.010.
22. Epasinghe DJ, Yiu C, Burrow MF, Effect of flavonoids on remineralization
remineralization of artificial root caries, Aust Dent J. 61 (2016) 196-202,
https://doi.org/10.1111/adj.12367.
23. Davila-Sanchez A, Gutierrez MF, Bermudez JP, Mendez-Bauer ML, Hilgemberg
B, Sauro S, Loguercio AD, Arrais CAG, Influence of flavonoids on long-term
bonding stability on caries-affected dentin, Dent Mater. 36 (2020) 1151-1160,
https://doi.org/10.1016/j.dental.2020.05.007.
24. Armstrong S, Breschi L, Ozcan M, Pfefferkorn F, Ferrari M, Van Meerbeek B,
Academy of Dental Materials guidance on in vitro testing of dental composite
bonding effectiveness to dentin/enamel using micro-tensile bond strength
(muTBS) approach, Dent Mater. 33 (2017) 133-143,
https://doi.org/10.1016/j.dental.2016.11.015.
25. Sabatini C, Ortiz PA, Pashley DH, Preservation of resin-dentin interfaces
treated with benzalkonium chloride adhesive blends, Eur J Oral Sci. 123 (2015)
108-15, https://doi.org/10.1111/eos.12176.
 46

26. Sebold M, André CB, Ambrosano GMB, Nascimento FD, Giannini M, Bond
strength and adhesive interface analysis using EDTA as a dentin conditioner,
Int J Adhes Adhes. 77 (2017) 157-163.
27. Langer A, Ilie N, Dentin infiltration ability of different classes of adhesive
systems, Clin Oral Investig. 17 (2013) 205-216, https://doi.org/10.1007/s00784-
012-0694-4.
28. Baldion PA, Cortes CC, Castellanos JE, Betancourt DE, Effect of myricetin on
odontoblast-like cells and its potential to preserve resin-dentin bonds, J Mech
Behav Biomed Mater. 117 (2021) 104392,
https://doi.org/10.1016/j.jmbbm.2021.104392.
29. Sim GS, Lee BC, Cho HS, Lee JW, Kim JH, Lee DH, Kim JH, Pyo HB, Moon
DC, Oh KW, Yun YP, Hong JT, Structure activity relationship of antioxidative
property of flavonoids and inhibitory effect on matrix metalloproteinase activity
in UVA-irradiated human dermal fibroblast, Arch Pharm Res. 30 (2007) 290-
298, https://doi.org/10.1007/BF02977608.
30. Shahidi F, Varatharajan V, Oh WY, & Peng H, Phenolic compounds in agri-food
by-products, their bioavailability and health effects, Journal of Food
Bioactives, 5 (2019) 57-119, https://doi.org/10.31665/JFB.2019.5178.
31. Pandey KB, Rizvi SI, Plant polyphenols as dietary antioxidants in human health
and disease, Oxid Med Cell Longev. 5 (2009) 270-278,
https://doi.org/10.4161/oxim.2.5.9498.
32. Aguiar TR, Vidal CM, Phansalkar RS, Todorova I, Napolitano JG, McAlpine JB,
Chen SN, Pauli GF, Bedran-Russo AK, Dentin biomodification potential
depends on polyphenol source, J Dent Res. 93 (2014) 417-422,
https://doi.org/10.1177/0022034514523783.
33. Ku CS, Sathishkumar M, Mun SP, Binding affinity of proanthocyanidin from
waste Pinus radiata bark onto proline-rich bovine achilles tendon collagen type
I, Chemosphere. 67 (2007) 1618-1627,
https://doi.org/10.1016/j.chemosphere.2006.11.037.
34. Bedran-Russo AK, Pereira PN, Duarte WR, Drummond JL, Yamauchi M,
Application of crosslinkers to dentin collagen enhances the ultimate tensile
strength, J Biomed Mater Res B Appl Biomater. 80 (2007) 268-272,
https://doi.org/10.1002/jbm.b.30593.
 47

35. Fang M, Liu R, Xiao Y, Li F, Wang D, Hou R, Chen J, Biomodification to dentin

by a natural crosslinker improved the resin-dentin bonds, J Dent. 40 (2012) 458-
466, https://doi.org/10.1016/j.jdent.2012.02.008.
36. Liu R, Fang M, Xiao Y, Li F, Yu L, Zhao S, Shen L, Chen J, The effect of transient
proanthocyanidins preconditioning on the cross-linking and mechanical
properties of demineralized dentin, J Mater Sci Mater Med. 22 (2011) 2403-
2411, https://doi.org/10.1007/s10856-011-4430-4.
37. Breschi L, Maravic T, Comba A, Cunha SR, Loguercio AD, Reis A, Hass V,
Cadenaro M, Mancuso E, Mayer-Santos E, Niu L, Pashley DH, Tay FR, Mazzoni
A, Chlorhexidine preserves the hybrid layer in vitro after 10-years aging. Dent
Mater. 36 (2020) 672-68, https://doi.org/10.1016/j.dental.2020.03.009.
38. Tay FR, Pashley DH, Kapur RR, Carrilho MR, Hur YB, Garrett LV, Tay KC,
Bonding BisGMA to dentin--a proof of concept for hydrophobic dentin bonding,
J Dent Res. 86 (2007) 1034-1039,
https://doi.org/10.1177/154405910708601103.
39. Nalla RK, Balooch M, Ager JW 3rd, Kruzic JJ, Kinney JH, Ritchie RO, Effects of
polar solvents on the fracture resistance of dentin: role of water hydration, Acta
Biomater. 1 (2005) 31-43, https://doi.org/10.1016/j.actbio.2004.08.002.
40. García-Estévez I, Ramos-Pineda AM, Escribano-Bailón MT, Interactions
between wine phenolic compounds and human saliva in astringency perception,
Food Funct. 9 (2018) 1294-1309, https://doi.org/10.1039/c7fo02030a.
41. Cui L, Zhang ZH, Sun E, Jia XB, Effect of β-cyclodextrin complexation on
solubility and enzymatic conversion of naringin, Int J Mol Sci. 13 (2012) 14251-
14261, https://doi.org/10.3390/ijms131114251.
42. Yoshiyama M, Matsuo T, Ebisu S, Pashley D, Regional bond strengths of self-
etching/self-priming adhesive systems, J Dent. 26 (1998) 609-616,
https://doi.org/10.1016/s0300-5712(97)00046-8.
43. Tichy A, Brabec M, Bradna P, Hosaka K, Tagami J, A competing risk model for
bond strength data analysis, Dent Mater. 36 (2020) 1508-1515,
https://doi.org/10.1016/j.dental.2020.09.004.
 48

3 CONCLUSÃO

Dentro das limitações desse estudo in vitro, pode-se concluir que todos os
primers testados à base de flavonóides pareceram melhorar a resistência à união à
dentina após um ano, quando aplicados por 60 s, em comparação com a dentina não
tratada. Enquanto, apenas o Kaempferol melhorou a resistência à união à dentina
após um ano quando aplicado por 30 s. O Kaempferol nas suas concentrações
testadas e a Naringina na concentração de 20 mM apresentaram melhores resultados
após um ano em relação à Clorexidina quando aplicado por 60 s. Além disso, os
primers à base de flavonóides pareceram não interferir na morfologia da interface
adesiva e no modo de falha. E, a eficácia dos primers experimentais pode depender
do tipo de flavonóide utilizado e do tempo de aplicação na dentina e os melhores
resultados foram obtidos quando os primers à base de flavonóides foram aplicados
por 60 s.
 49

REFERÊNCIAS *

Bedran-Russo AK, Karol S, Pashley DH, Viana G. Site specific properties of carious
dentin matrices biomodified with collagen cross-linkers. Am J Dent. 2013 Oct;26(5):
244-8.

Bedran-Russo AK, Pauli GF, Chen SN, McAlpine J, Castellan CS, Phansalkar RS, et
al. Dentin biomodification: strategies, renewable resources and clinical applications.
Dent Mater. 2014 Jan;30(1):62-76. doi: 10.1016/j.dental.2013.10.012.

Bertassoni LE, Swain MV. Removal of dentin non-collagenous structures results in the
unraveling of microfibril bundles in collagen type I. Connect Tissue Res. 2017 Sep;
58(5):414-423. doi: 10.1080/03008207.2016.1235566.

Breschi L, Cammelli F, Visintini E, Mazzoni A, Vita F, Carrilho M, et al. Influence of

chlorhexidine concentration on the durability of etch-and-rinse dentin bonds: a 12-
month in vitro study. J Adhes Dent. 2009 Jun;11(3):191-8.

Breschi L, Mazzoni A, Nato F, Carrilho M, Visintini E, Tjäderhane L, et al. Chlorhexidine

stabilizes the adhesive interface: a 2-year in vitro study. Dent Mater. 2010 Apr;26(4):
320-5. doi: 10.1016/j.dental.2009.11.153.

Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R, De Stefano Dorigo E.

Dental adhesion review: aging and stability of the bonded interface. Dent Mater. 2008
Jan;24(1):90-101. doi: 10.1016/j.dental.2007.02.009.

De Munck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K, et al. Four-

year water degradation of total-etch adhesives bonded to dentin. J Dent Res. 2003
Feb;82(2):136-40. doi: 10.1177/154405910308200212.

Epasinghe DJ, Yiu C, Burrow MF. Effect of flavonoids on remineralization of artificial

root caries. Aust Dent J. 2016 Jun;61(2):196-202. doi: 10.1111/adj.12367.

Gendron R, Grenier D, Sorsa T, Mayrand D. Inhibition of the activities of matrix

metalloproteinases 2, 8, and 9 by chlorhexidine. Clin Diagn Lab Immunol. 1999 May;
6(3):437-9. doi: 10.1128/CDLI.6.3.437-439.1999.

Hagerman AE, Butler LG. The specificity of proanthocyanidin-protein interactions. J

Biol Chem. 1981 May;256(9):4494-7.

Iskander M, Elkassas D, Mohsen MA. Effect of two matrix metalloproteinase inhibitors

on the color stability of a nanofilled resin composite. Oper Dent. 2015 Jan-Feb;40(1):
E11-20. doi: 10.2341/12-428-L.

* De acordo com as normas da UNICAMP/FOP, baseadas na padronização do

International Committee of Medical Journal Editors – Vancouver Group. Abreviatura
dos periódicos em corformidade com o Pubmed.
 50

Joshi SS, Kuszynski CA, Bagchi D. The cellular and molecular basis of health benefits
of grape seed proanthocyanidin extract. Curr Pharm Biotechnol. 2001 Jun;2(2):187-
200. doi: 10.2174/1389201013378725.

Leme-Kraus AA, Phansalkar RS, Dos Reis MC, Aydin B, Sousa ABS, Alania Y, et al.
Dimeric Proanthocyanidins on the Stability of Dentin and Adhesive Biointerfaces. J
Dent Res. 2020 Feb;99(2):175-181. doi: 10.1177/0022034519892959.

Martins GC, Franco APGO, Godoy EP, Maluf DR, Gomes JC, Gomes OMM. Adesivos
Dentinários. Rev. Gaúcha Odontol. 2008 out-dez; v. 56, n. 4, p. 429-436.

Nakabayashi N, Kojima K, Masuhara E. The promotion of adhesion by the infiltration

of monomers into tooth substrates. J Biomed Mater Res. 1982 May;16(3):265-73. doi:
10.1002/jbm.820160307.

Nakabayashi N, Pashley DH. Evolution of dentin-resin bonding In: Hybridization of

Dental Hard Tissues. Quintessence Publishing. 1998;1-20.

Negrelo Newton AP, Cadena SM, Merlin Rocha ME, Skäre Carnieri EG, Martinelli de
Oliveira MB. New data on biological effects of chlorhexidine: Fe2+ induced lipid
peroxidation and mitochondrial permeability transition. Toxicol Lett. 2004 Aug;151(3):
407-16. doi: 10.1016/j.toxlet.2004.02.013.

Panche AN, Diwan AD, Chandra SR. Flavonoids: an overview. J Nutr Sci. 2016
Dec;29;5:e47. doi: 10.1017/jns.2016.41.

Pashley DH, Tay FR, Imazato S. How to increase the durability of resin-dentin bonds.
Compend Contin Educ Dent. 2011 Sep;32(7):60-4,66.

Phansalkar RS, Nam JW, Chen SN, McAlpine JB, Napolitano JG, Leme A, et al. A
galloylated dimeric proanthocyanidin from grape seed exhibits dentin biomodification
potential. Fitoterapia. 2015 Mar; 101:169-78. doi: 10.1016/j.fitote.2014.12.006.

Perdigão J, Reis A, Loguercio AD. Dentin adhesion and MMPs: a comprehensive

review. J Esthet Restor Dent. 2013 Aug; 25(4):219-41. doi: 10.1111/jerd.12016.

Ricci HA, Sanabe ME, Costa CA, Hebling J. Effect of chlorhexidine on bond strength
of two-step etch-and-rinse adhesive systems to dentin of primary and permanent teeth.
Am J Dent. 2010 Jun; 23(3):128-32.

Scheffel DL, Hebling J, Scheffel RH, Agee K, Turco G, de Souza Costa CA, et al.
Inactivation of matrix-bound matrix metalloproteinases by cross-linking agents in acid-
etched dentin. Oper Dent. 2014 Mar-Apr; 39(2): 152-8. doi: 10.2341/12-425-L.

Sebold M, André CB, Carvalho RM, Giannini M. Dry-bonding to dentin using alternative
conditioners based on iron-containing solutions or nitric acid. J Mech Behav Biomed
Mater. 2019 Jun; 94:238-248. doi: 10.1016/j.jmbbm.2019.03.015.
 51

Silva Sousa AB, Vidal CMP, Leme-Kraus AA, Pires-de-Souza FCP, Bedran-Russo AK.
Experimental primers containing synthetic and natural compounds reduce enzymatic
activity at the dentin-adhesive interface under cyclic loading. Dent Mater. 2016 Oct;
32(10):1248-1255. doi: 10.1016/j.dental.2016.07.012.

Tay FR, Hashimoto M, Pashley DH, Peters MC, Lai SC, Yiu CK, et al. Aging affects
two modes of nanoleakage expression in bonded dentin. J Dent Res. 2003 Jul;82(7):
537-41. doi: 10.1177/154405910308200710.

Tay FR, Pashley DH, Suh BI, Carvalho RM, Itthagarun A. Single-step adhesives are
permeable membranes. J Dent. 2002 Sep-Nov;30(7-8):371-82.

Tjäderhane L. Dentin bonding: can we make it last? Oper Dent. 2015 Jan-Feb;40(1):
4-18. doi: 10.2341/14-095-BL.

Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S, et al.

Strategies to prevent hydrolytic degradation of the hybrid layer-A review. Dent Mater.
2013 Oct;29(10):999-1011. doi: 10.1016/j.dental.2013.07.016.

Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S, et al.

Optimizing dentin bond durability: control of collagen degradation by matrix
metalloproteinases and cysteine cathepsins. Dent Mater. 2013 Jan;29(1):116-35. doi:
10.1016/j.dental.2012.08.004.

Wang Y, Spencer P. Hybridization efficiency of the adhesive/dentin interface with wet

bonding. J Dent Res 2003 Feb; 82:141-5. doi: 10.1177/154405910308200213.
 52

ANEXO 1 – Comprovação da submissão do artigo

 53

ANEXO 2 - Certificado do Comitê de Ética em Pesquisa

54
 55

Anexo 3 - Verificação de Originalidade e Prevenção de Plágio