Loiane Massunari 

 

 

 

 

 

Efeito de flavonoides sobre microrganismos de 

interesse endodôntico e sua influência na expressão 

de marcadores de mineralização 

 

 

 

 

 

 

 

Araçatuba - SP 

2018



 
 

Loiane Massunari 

 

 

 

Efeito de flavonoides sobre microrganismos de 

interesse endodôntico e sua influência na expressão 

de marcadores de mineralização 

 

Tese de Doutorado apresentada à 

Faculdade de Odontologia da 

Universidade Estadual Paulista “Júlio de 

Mesquita Filho”, Campus de Araçatuba, 

como parte dos requisitos para a 

obtenção de título de Doutor em Ciência 

Odontológica - Área de Concentração: 

Endodontia. 

 

Orientadora: Profa. Dra. Cristiane 

Duque        

 

 

 

Araçatuba - SP 

2018



 
 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Catalogação na Publicação (CIP) 

Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP 

 
 Massunari, Loiane. 
M422e Efeito de flavonoides sobre microrganismos de interesse 
 endodôntico e sua influência na expressão de marcadores 
 de mineralização / Loiane Massunari. - Araçatuba, 2018 
 152f. : il. ; tab. 
 
 Tese (Doutorado) – Universidade Estadual Paulista,  
 Faculdade de Odontologia de Araçatuba 
 Orientadora: Profa. Cristiane Duque 
 
 1. Flavonoides 2. Testes de sensibilidade microbiana 
 3. Dentinogênese 4. Osteogênese 5. Técnicas de cultura de 
 células I. T. 
 
    Black D24 
    CDD 617.67 
 

Responsável: Claudio Hideo Matsumoto – CRB-8/5550



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Dedicatória 



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À Deus 

Meu criador 

Sempre em primeiro lugar 

À minha família 

Pai, Mãe, Ni, Glau, Jhone, Mia e Márcio 

Nada teria sentido sem vocês!!! 



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Agradecimentos Especiais  



5 
 

À Deus 

Por estar sempre comigo, guiando os meus passos e pensamentos; 

Pelo seu amor e misericórdia perante os meus erros; 

Por seus ensinamentos que nunca me faltaram. 

“Ao único Deus, nosso Salvador, mediante Jesus Cristo, Senhor nosso, glória, magestade, 
império e soberania, antes de todas as eras, e agora, e por todos os séculos. Amém” Judas 1:25 

Aos meus pais Jorge e Elenice 

Pelo amor incondicional; 

Por toda a dedicação e cuidados; 

Pelas correções e conselhos sempre oportunos; 

Por sempre me proporcionarem muito mais do que precisei; 

Por me aguardarem aos finais de semana sempre com muito amor e carinho. 

Pessoas essenciais na minha formação, os responsáveis por cada conquista minha, vocês 
sempre serão as minhas melhores referências de integridade, honestidade e dedicação. Amo 
vocês!! 

Aos meus irmãos Nicole e Glauber 

Pelo amor e cuidados; 

Por serem tão presentes; 

Pelos momentos únicos;  

Por me ouvirem e me aconselharem. 

Os maiores presentes da minha vida, juntos nós passamos por muitos momentos, aprendemos 
e crescemos. Nosso amor é incomparável, inexplicável e eterno. Vocês realmente me 
completam! Amo vocês!! 

 

 

 



6 
 

Ao meu noivo Márcio 

Pelo amor e carinho; 

Pelos conselhos e cuidados; 

Pelos momentos únicos que tivemos e ainda teremos. 

Você realmente entrou na minha vida para acrescentar. Sempre me encorajando e apoiando 
nos momentos difíceis. Quero você sempre ao meu lado! 

Às minhas avós Mineco e Rosa 

(in memoriam) 

Pela companhia e cuidados; 

Pelos ensinamentos transmitidos, 

Por tudo que fizeram pela nossa família. 

Vocês sempre serão grandes exemplos para mim, donas dos corações mais puros e bondosos 
que Deus me permitiu amar. Guardarei cada ensinamento por toda a minha vida, buscando 
sempre ser uma pessoa melhor. 

À minha cunhada Jaqueline 

Pela amizade; 

Pelos momentos de alegria; 

Por ser tão presente e cuidadosa. 

Você já faz parte da nossa família, sempre muito atenciosa e disposta á ajudar. 

À Faculdade de Odontologia de Araçatuba - Unesp 

Nas pessoas dos professores, Dr. Wilson Roberto Poi digníssimo Diretor e, Dr. João Eduardo 
Gomes Filho digníssimo Vice-Diretor. 

 

 

 



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À minha orientadora Prof. Dra. Cristiane Duque 

Por toda ajuda durante a elaboração desse trabalho, pelas sugestões e auxílio sempre que 
precisei. A senhora sempre esteve disponível para me ajudar, me orientando e buscando 
extrair o meu melhor. O aprendizado não foi apenas técnico, terá sempre a minha admiração 
e gratidão. 

Aos meus colegas de Pós-Graduação da Endodontia, Farmacologia 
ePediatria 

Pela amizade, convívio, ajuda e experiências trocadas. 

À minha professora Dra Ana Cláudia Okamoto 

Pela orientação durante a graduação e no trabalho de conclusão de curso. A senhora me 
inspirou á entrar na pós-graduação; agradeço cada ensinamento transmitido e conselho dado. 
Lembro como se fosse hoje, a senhora me incentivando á participar de congressos e fazer 
aulas de inglês. Será sempre um exemplo para mim. 

À minha professora Dra Daniela Micheline dos Santos 

Pela orientação durante a iniciação científica, sempre orientando com muito amor e carinho, 
muito obrigada por toda a paciência e ensinamentos. 

À minha amiga Índia 

Por ter me acompanhado e ensinado os primeiros passos na cultura de células, sempre muito 
centrada e disposta á ajudar. Agradeço também as conversas e momentos de descontração 
durante todo esse período de pós-graduação. Sua amizade foi um presente para mim. 

À minha amiga Paula 

Por todas as conversas, conselhos e carinho. Quantas vezes você me lembrou que o maior 
significado disso tudo é Deus, me acalmando em momentos difíceis sempre com palavras 
sábias e de muito carinho. Mesmo distante, sempre se fez presente quando precisei, agradeço 
à Deus por ter te colocado em meu caminho durante a pós-graduação. 

Às minhas amigas Gaby, Juliana, Annelise e Lívia 

Pela amizade, carinho, conversas e conselhos. Estarei sempre torcendo por cada uma de vocês 
e espero tê-las sempre por perto. 



8 
 

Aos docentes da disciplina de Endodontia da FOA - Unesp  

Prof. Dr. Roberto Holland, Prof. Dr. Mauro Juvenal Nery, Prof. Dr. José Arlindo Otoboni 
Filho, Prof. Dr. Elói Dezan Júnior, Prof. Dr. João Eduardo Gomes Filho; Prof. Dr. Luciano 
Tavares Ângelo Cintra, Prof. Dr. Rogério Castilho Jacinto e Prof. Dr. Gustavo Sivieri Araújo. 

Pelos ensinamentos transmitidos e contribuição na minha formação profissional. 

À Prof Drª Sandra Helena Penha de Oliveira 

Por ter aberto as portas do seu laboratório, me permitindo realizar parte desse trabalho. 
Agradeço o carinho, disponibilidade e sugestões. 

Ao Prof Dr Carlos Alberto de Souza Costa 

Por ter me recebido em seu laboratório, me permitindo realizar parte desse trabalho. Sou 
muito grata por todos os ensinamentos e carinho transmitidos durante esse período. O senhor 
realmente inspira todos à sua volta, não somente pelo saber, mas também pelo lado humano e 
íntegro que possui. 

Às meninas do Laboratório de Patologia Experimental e 
Biomateriais 

Pelo ótimo convívio, ensinamentos e ajuda sempre que precisei. 

À querida Malu 

Por ter me recebido em sua casa durante todo o período que precisei, sempre com muito 
carinho e disponibilidade para me ajudar. Muito obrigada pelos ensinamentos no laboratório, 
ajuda durante os experimentos, conversas e conselhos que tornou a minha estadia em 
Araraquara muito melhor do que eu poderia imaginar. Você sempre estará guardada no meu 
coração. 

À Giovana 

Por ter me ensinado á executar todos os protocolos que realizei em Araraquara, sempre com 
muito carinho e atenção. Você foi essencial para a finalização desse trabalho. 

Ao curso de pós-graduação em Ciência Odontológica da FOA  

Na pessoa do coordenador Prof. Dr. Luciano Tavares Ângelo Cintra. 



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Aos funcionários do Departamento de Odontopediatria  

Ricardo, Mário e Luisinho. 

Pela amizade e ajuda sempre que precisei; 

Por manterem a organização no laboratório. 

Aos funcionários do Departamento de Endodontia 

Nelci, Elaine e Peterson. 

Pela amizade e ajuda sempre que precisei.  

Aos funcionários da biblioteca da FOA  

Ana Cláudia, Luzia, Ivone, Cláudio, Maria Cláudia, Luiz, Denise e Izamar. 

Pela atenção e disponibilidade. 

Aos funcionários da sessão de Pós Graduação da FOA  

Valéria, Cristiane e Lilian. 

Pelo profissionalismo e disponibilidade. 

À Fundação de Amparo à Pesquisa do Estado de São Paulo   

Pela concessão da bolsa de estudo (Processo 2015/00812-0). 

 

Muito Obrigado!!! 

 



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Resumo 



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Massunari, L. Efeito de flavonoides sobre microrganismos de interesse endodôntico e 

sua influência na expressão de marcadores de mineralização. [Tese] Universidade 

Estadual Paulista (Unesp), Faculdade de Odontologia de Araçatuba. Araçatuba, 2018. 

Resumo 

O tratamento endodôntico de dentes permanentes jovens representa um grande 

desafio clínico devido a presença de paredes dentinárias finas, divergentes ou 

paralelas e ápice aberto, o que dificulta a desinfecção e a execução dos procedimentos 

convencionais. Terapias de regeneração endodôntica envolvem o uso de materiais 

capazes de promover uma desinfecção eficaz sem causar citotoxicidade, além de 

induzir a diferenciação de células-tronco ou bioestimular células remanescentes do 

tecido pulpar mesmo após a injúria. Nesse contexto, os flavonoides, polifenóis 

presentes em frutas e vegetais, poderiam ser agentes interessantes para o tratamento 

endodôntico de dentes imaturos devido a sua amplitude terapêutica. Dessa forma, o 

objetivo do presente trabalho foi avaliar o efeito antimicrobiano, citotóxico e indutor 

de mineralização de flavonoides com finalidade de aplicação endodôntica. Este 

trabalho de tese foi dividido em três capítulos. O capítulo 1 avaliou a toxicidade dos 

flavonoides taxifolina, crisina, pinocembrina e galangina sobre fibroblastos pelo ensaio 

de MTT, a atividade antimicrobiana pela determinação da concentração inibitória e 

bactericida mínima, e a ação antibiofilme do flavonoide com melhor efeito 

antimicrobiano, por meio de ensaios em placas de poliestireno e em dentina radicular 

bovina por meio da análise por microscopia confocal. Os resultados mostraram que o 

flavonoide taxifolina não foi tóxico para os fibroblastos em nenhuma das 

concentrações analisadas, enquanto que os flavonoides crisina, pinocembrina e 

galangina apresentaram efeitos citotóxicos. Crisina, pinocembrina e galangina não 

apresentaram efeito antimicrobiano frente E. faecalis and S. mutans nas 

concentrações testadas. A taxifolina foi capaz de inibir todas as bactérias testadas, 

eliminar biofilmes de E. faecalis e S. mutans em placas de poliestireno e reduzir 

significantemente o biofilme de E. faecalis em túbulos dentinários. O capítulo 2 avaliou 

a citotoxicidade do flavonoide taxifolina e o seu potencial sobre a indução de 

marcadores de mineralização dentinária (produção de fosfatase alcalina - ALP, nódulos 

de mineralização – NM e expressão dos genes DSPP – sialofosfoproteína dentinária e 



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DMP-1 – proteína da matriz dentinária - 1) em células semelhantes a odontoblastos 

MDPC-23, após tratamentos de 24, 72h e contínuo. A taxifolina não apresentou 

citotoxicidade em nenhum dos três tipos de tratamento analisados. Todas as 

concentrações do tratamento de 24h e as concentrações de 10 e 5µM do tratamento 

de 72h aumentaram a atividade de ALP. A formação de NM aumentou com os 

tratamentos de taxifolina à 10µM em ambos os tratamentos de 24 e 72h, e à 5µM no 

tratamento de 24h. A expressão de DMP-1 aumentou com o tratamento de taxifolina 

em ambos os tratamentos de 24 e 72h, enquanto que a de DSPP aumentou apenas 

com o tratamento de 72h na concentração de 5µM. O capítulo 3 avaliou a 

citotoxicidade da taxifolina, e o seu potencial sobre a indução de marcadores de 

mineralização óssea (ALP, NM e expressão dos genes ALP e colágeno 1 - Col-1) em 

células semelhantes a osteoblastos Saos-2, após tratamentos de 24, 72h e contínuo. Os 

resultados mostraram que os tratamentos com taxifolina nas concentrações de 10, 5 e 

1µM não foram citotóxicos em nenhum dos períodos analisados. O tratamento de 72h 

da taxifolina à 10µM foi capaz de aumentar a atividade de ALP e a formação de NM, 

além de aumentar a expressão de Col-1 após 13 dias. O tratamento de 24h da 

taxifolina na concentração de 10µM aumentou a expressão de ALP após 6 dias. 

Conclui-se que a taxifolina é um flavonoide com potencial uso para o tratamento 

endodôntico de dentes permanentes jovens, devido à sua ação 

antimicrobiana/antibiofilme, baixa citotoxicidade e capacidade de estimular a 

mineralização em odontoblastos e osteoblastos. 

 

Palavras-chave: Flavonoides, testes de sensibilidade microbiana, dentinogênese, 

osteogênese, técnicas de cultura de células. 

 

 



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Abstract 



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Massunari, L. Effect of flavonoids on endodontic microorganisms and their influence 

on the expression of mineralization markers. [Thesis] São Paulo State University 

(Unesp), School of Dentistry, Araçatuba, 2018. 

Abstract 

The endodontic treatment of young permanent teeth represents a great clinical 

challenge due to the presence of thin, divergent or parallel dentin walls and the open 

apex that makes it difficult to disinfect and perform conventional endodontic 

procedures. Endodontic regeneration therapies involve the use of materials capable of 

promoting effective disinfection without causing cytotoxicity, in addition to inducing 

differentiation of stem cells or biostimulating remaining pulp tissue cells even after 

injury. In this context, the flavonoids, polyphenols present in fruits and vegetables, 

could be interesting agents for the endodontic treatment of immature teeth due to the 

wide therapeutic use. Thus, the objective of the present study was to evaluate the 

antimicrobial, cytotoxic effects and capacity of mineralization induction of flavonoids 

for endodontic application. This thesis was divided into three chapters. The chapter 1 

evaluated the toxicity of taxifolin, chrysin, pinocembrin and galangin flavonoids on 

fibroblasts by the MTT method, antimicrobial activity by determining the minimum 

inhibitory and bactericidal concentrations and analyzed the antibiofilm action of the 

flavonoid with the best antimicrobial effect, by means of the biofilm assays in 

polystyrene plates and in bovine root dentin and confocal microscopy analysis. The 

results showed that the flavonoid taxifolin was not toxic on fibroblasts in any tested 

concentration, while chrysin, pinocembrin and galangin flavonoids showed cytotoxic 

effects. Chrysin, pinocembrin and galangin showed no antimicrobial effect against E. 

faecalis and S. mutans in any tested concentrations. Taxifolin was able to inhibit all 

tested bacteria, to eliminate E. faecalis and S. mutans biofilms on polystyrene plates 

and significantly reduce E. faecalis biofilms from the dentin tubules. The chapter 2 

evaluated the cytotoxicity of taxifolin and its potential on the induction of dentin 

mineralization markers (alkaline phosphatase production - ALP, mineralization nodules 

- MN and expression of genes DSPP - dentin sialophosphoprotein and DMP-1 - dentin 

matrix protein - 1) on odontoblast-like cells MDPC-23, after treatments of 24, 72h and 

continuous. Taxifolin did not present cytotoxicity at any of the three types of 



15 
 

treatments analyzed. All concentrations of 24h-treatment and 10 and 5μM of 72h-

treatment increased ALP activity. NM formation increased with taxifolin treatments at 

10μM in both 24 e 72h treatments, and at 5μM in the 24h-treatment. Expression of 

DMP-1 increased with taxifolin in both 24 e 72h-treatments, whereas DSPP expression 

increased only with 72h-treatment at 5μM. The chapter 3 evaluated the cytotoxicity of 

taxifolin and its potential on the induction of bone mineralization markers (ALP, NM 

and expression of ALP and collagen 1 -Col-1 genes) on Saos-2 osteoblast-like cells, after 

treatments of 24, 72h and continuous. The results showed that taxifolin treatments at 

10, 5 and 1μM were not cytotoxic in any of the periods analyzed. The 72h-treatment of 

taxifolin at 10μM was able to increase ALP activity and NM formation, in addition to 

increasing Col-1 expression after 13 days. The 24h-treatment of taxifolin at 10μM 

increased ALP expression after 6 days. It is concluded that taxifolin is a flavonoid with 

potential use for endodontic treatment of young permanent teeth due to its 

antimicrobial/antibiofilm action, low cytotoxicity and ability to stimulate 

mineralization in odontoblasts and osteoblasts. 

 

Keywords: Flavonoids, microbial sensitivity tests, dentinogenesis, osteogenesis, cell 

culture techniques. 

 

 

 

 

 



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Lista de Abreviaturas 

 



17 
 

Lista de abreviaturas 

A. israelii – Actinomyces israelii 

ALP – Fosfatase alcalina (alkaline phosphatase) 

ANOVA – Análise de variância (analysis of variance) 

BHI – Infusão cérebro e coração (brain heart infusion) 

BSA – Albumina do soro bovino (bovine serum albumin) 

C. albicans – Candida albicans 

cDNA – Ácido desoxirribonucleico complementar (complementary deoxyribonucleic 
acid)  

CFU – Unidades formadoras de colônia (colony-forming unit) 

CH – Hidróxido de cálcio (calcium hydroxide) 

CHX – Digluconato de clorexidina (chlorhexidine digluconate) 

CLSI – Instituto de padrões clínicos e laboratoriais (Clinical and Laboratory Standard 
Institute) 

CLSM – Microscopia confocal de varredura a laser (confocal laser scanning microscopy) 

CO2 – Gás carbônico (carbon dioxide) 

Col-1 – Colágeno 1 (collagen 1) 

Ct – Ciclo limiar (Threshold cycle) 

CT – Tratamento contínuo (continuous treatment) 

d – Dias (days) 

DAP – Pasta biantibiótica (double antibiotic paste) 

ΔΔCt – Delta delta ciclo limiar (delta delta threshold cycle) 

DMEM – Meio de Eagle modificado por Dulbecco (Dulbecco Modified Eagle’s Medium) 

DMP-1 – Proteína da matriz dentinária (dentin matrix protein – 1) 

DMSO – Dimetilsulfóxido (dimethyl sulfoxide) 

DPSCs – Células-tronco da polpa dentária (Dental pulp stem cells)  

DSPP – Sialofosfoproteína dentinária (dentin sialophosphoprotein) 

E. coli – Escherichia coli 

E. faecalis – Enterococcus faecalis 



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EGCG – Epigalocatequina galato (Epigallocatechin gallate) 

FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (São Paulo Research 
Foundation) 

FBS – Soro fetal bovino (Fetal bovine serum) 

FIOCRUZ – Fundação Oswaldo Cruz (Oswaldo Cruz Foundation) 

FOA – Faculdade de Odontologia de Araçatuba (School of Dentistry, Araçatuba) 

g – Aceleração da gravidade (Gravitational acceleration) 

g/mL – Gramas por mililitros (Grams per milliliters) 

GAPDH – Gliceraldeído-3-fosfato desidrogenase (Glyceraldehyde-3-phosphate 
dehydrogenase) 

h – Horas (Hours) 

HDPCs – Células da polpa dentária humana (Human dental pulp cells) 

HGF – Fibroblastos gengivais humanos (Human gingival fibroblasts) 

IL- 1 – Interleucina 1 (Interleukin 1) 

IL-17 – Interleucina 17 (Interleukin 17) 

IL-1β – Interleucina 1 beta (Interleukin 1 beta) 

IL-6 – Interleucina 6 (Interleukin 6) 

ISO – Organização internacional de normalização (International Organization for 
Standardization) 

L929 – Linhagem celular de fibroblastos de camundongo (Murine fibroblast cell line) 

LDH – Lactato desidrogenase (Lactate dehydrogenase) 

Log – Logaritmo (Logarithm) 

MBC – Concentração bactericida mínima (Minimum bactericidal concentration) 

MDPC-23 – Células da papila dentária de camundongo (Mouse dental papilla cells) 

mg/mL – Miligramas por mililitros (Milligrams per milliliters) 

MIC – Concentração inibitória mínima (Minimum inhibitory concentration) 

min – Minutos (Minutes) 

mm – Milímetros (Millimeters) 

mM – Milimolar (Millimolar) 

mmol/L – Milimol por litro (Milimol per liter) 



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mRNA – Ácido ribonocleico mensageiro (Messenger ribonucleic acid) 

MSA – Ágar mitis salivarius (Mitis salivarius agar) 

MTA – Agregado de trióxido mineral (Mineral trioxide aggregate) 

MTT – Brometo de 3- (4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) 

n – Tamanho da amostra (Sample size) 

NF-Kβ - Fator nuclear Kappa beta (Nuclear factor kappa beta) 

nm – Nanômetros (Nanometers) 

NM – Nódulos de mineralização (Mineralization nodule) 

0C - Grau Celsius (Degrees Celsius) 

OD – Densidade óptica (Optical density) 

OH – Radical hidroxila (Hydroxyl radical)  

PDLSCs – Células-tronco do ligamento periodontal (Human periodontal ligament stem 
cells) 

qPCR – Reação da polimerase em cadeia (Quantitative Polymerase Chain Reaction) 

RANK - Receptor ativador do fator nuclear Kappa beta (Receptor activator of nuclear 
factor-kappa beta) 

RANKL - Ligante do receptor ativador do fator nuclear Kappa beta (Receptor activator 
of nuclear factor-kappa beta ligand) 

RE – Regeneração endodôntica (Regenerative Endodontics) 

RET – Terapias de regeneração endodôntica (Regenerative endodontic therapies) 

RNA – Ácido ribonucleic (Ribonucleic acid) 

S. aureus – Staphylococcus aureus 

S. mutans – Streptococcus mutans  

Saos-2- Linhagem de células semelhantes a osteoblastos humanos (Human osteoblast-
like cell line) 

SD = Desvio padrão (Standard deviation) 

SHED – Células-tronco de dentes decíduos exfoliados humanos (Stem cells from human 
exfoliated deciduous teeth) 

TAP – Pasta triantibiótica (Triple antibiotic paste) 

TNF-α – Fator de necrose tumoral alfa (Tumor necrosis factor alpha) 



20 
 

U/L – Unidades por litro (Units per liter) 

U/mL – Unidades por mililitros (Units per milliliters) 

UNESP – Universidade Estadual Paulista (São Paulo State University) 

VPT – Terapia de vitalidade pulpar (Vital pulp therapy) 

µg/mL – Microgramas por mililitros (Micrograms per milliliters) 

µL – Microlitros (Microliters) 

µm – Micrometros (Micrometers)  

µM – Micromolar (Micromolar) 

 

 

 

 

 

 

 

 

 

 

 



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Lista de Figuras 

 



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Lista de Figuras 

Capítulo 1 

Figure 1. Mean (bars) of the percentage of cell viability of fibroblasts from L929 line 

after exposure to different concentrations of flavonoids, using MTT 

assays…………………………………………………………………………………………………………………………58 

Figure 2. Bacterial recovery (Log CFU+1) after 24h of taxifolin and chlorhexidine 

digluconate (CHX) treatments (10x MBC) on 48h-growth biofilm……………………………….59 

Figure 3. Representative CLSM images (63x) of E. faecalis biofilms inside dentin tubules 

after 48h of the treatments …………….…………………………………………………………………………59 

Figure 4. Means (bars-standard deviation) of the percentage of dead cells obtained 

after CLSM analysis of E. faecalis biofilm……………………………………………………………………60 

Capítulo 2 

Figure 1. Cell viability immediately after 24 and 72 h of treatments with different 

concentrations of taxifolin, using MTT assays.  ………………………………….………………………79 

Figure 2. Cell viability of cells after 6 and 13 days of the three forms of treatment (24h, 

72h, and continuous treatment) with taxifolin, using MTT assays………………………………80 

Figure 3.  Alkaline phosphatase (ALP) activity of cells after 6 days of the three forms of 

treatment with taxifolin……………………………………………………………………………………………..81 

Figure 4.  Mineralization ability of cells after 13 days of the three forms of treatment 

with taxifolin, using alizarin red staining…………………………………………………………………….81 

Figure 5. Representative images of alizarin red staining showing mineralization ability 

of MDPC-23 cells after 13 days of taxifolin treatment………………………………………………..82 

  



23 
 

Figure 6. Expression of dentin matrix protein - 1 (A) and dentin sialophosphoprotein 

(B) from MDPC-23 cells after 6 and 13 days of treatment with taxifolin…………………….83 

Capítulo 3 

Figure 1. Cell viability immediately after 24 and 72h of treatments with different 

concentrations of taxifolin, using MTT assays ……………………………………………….…………101 

Figure 2. Cell viability of cells after 6 and 13 days of the three forms of treatment (24h, 

72h and continuous treatment) with taxifolin, using MTT assays  ……………………………101 

Figure 3.  Alkaline phosphatase (ALP) activity of cells after 6 days of the three forms of 

treatment with taxifolin……………………………………………………………………………………………102 

Figure 4.  Mineralization ability of cells after 13 days of the three forms of treatment 

with taxifolin, using alizarin red staining………………………………………………………………….102 

Figure 5. Representative images of alizarin red staining showing mineralization ability 

of Saos-2 cells after 13 days of taxifolin treatments…………………………………………………103 

Figure 6. Expression of alkaline phosphatase (A) and collagen-1 (B) from Saos-2 cells 

after 6 and 13 days of treatment with taxifolin…………………………………..……………………104 

  

 

 

 



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Lista de Tabelas 

 



25 
 

Lista de Tabelas 

Capítulo 1 

Table 1. Minimum inhibitory concentration (MIC) and minimal bactericidal 

concentration (MBC) of flavonoids…………………………………………………………………………….58 

Capítulo 2 

Table 1. Description of groups chosen for the present study………………..……………………79 

Capítulo 3 

Table 1.  Description of groups chosen for this study…………………………………….…………100 

Table 2. Nucleotides sequence of primers used for each selected gene………………….100 

 

 

 

 

 

 

 



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Sumário 

 



27 
 

Sumário 

 

Introdução geral ......................................................................................................................... 29 

Proposição .................................................................................................................................. 36 

Capítulo 1 .................................................................................................................................... 38 

Anti-biofilm and cytotoxic effect of flavonoids intended for endodontic purposes .................... 38 

Capítulo 2 .................................................................................................................................... 62 

Cytotoxicity and potential of taxifolin, a catechol-type flavonoid, to induct mineralization 

markers in odontoblast-like cells ................................................................................................. 62 

Capítulo 3 .................................................................................................................................... 85 

Effect of Taxifolin on viability and mineralization markers of osteoblast-like cells .................... 85 

Conclusão .................................................................................................................................. 106 

Referências Bibliográficas ........................................................................................................ 108 

Anexo A ..................................................................................................................................... 117 

Guidelines for Publishing Papers in the Biofouling .................................................................... 117 

Anexo B ..................................................................................................................................... 121 

Guidelines for Publishing Papers in the International Endodontic Journal ............................... 121 

Anexo C ..................................................................................................................................... 135 

Guidelines for Publishing Papers in the Archives of Oral Biology .............................................. 135 

Anexo D ..................................................................................................................................... 143 

Comitê de Ética no Uso de Animais .......................................................................................... 143 

Anexo E ..................................................................................................................................... 144 

Protocolos Experimentais .......................................................................................................... 144 

 

 

 

 

 



28 
 

 

 

 

 

 

 

 

 

 

 

 

 

Introdução Geral 

 



29 
 

Introdução geral  

 

 Após a erupção do dente permanente na cavidade bucal ainda são necessários 

aproximadamente 3 anos para o desenvolvimento completo das raizes e o fechamento 

dos respectivos ápices radiculares (Bhasker 1991). Desta forma, durante esta fase, a 

ocorrência de traumatismos dentários ou lesões de cárie que comprometam a 

vitalidade do tecido pulpar, podem interromper a formação da raiz, resultando na 

presença de paredes dentinárias finas, divergentes ou paralelas e ápice aberto, o que 

dificultará a execução dos procedimentos endodônticos convencionais, vindo a 

comprometer o resultado do tratamento a longo prazo (Rafter 2005, Iglesias-Linares et 

al. 2013). Além disso, nesses casos de dentes permanentes jovens, além da 

necessidade de um tratamento que promova a descontaminação do sistema de canais 

radiculares, é desejável que esse permita a continuação do desenvolvimento radicular, 

bem como a recuperação dos tecidos periapicais quando já estiverem comprometidos 

(Hargreaves et al. 2013).   

 Durante muitos anos, o tratamento de dentes permanentes com rizogênese 

incompleta baseou-se na vitalidade do tecido pulpar afetado. Assim, para dentes que 

apresentam vitalidade pulpar tem se indicado procedimentos, como a pulpotomia, que 

consiste na remoção da polpa infectada e manutenção da polpa radicular, estimulando 

o desenvolvimento fisiológico contínuo e o término da formação radicular pelo 

processo de apicogênese. Os casos de dentes que apresentam vitalidade pulpar, 

porém o tecido pulpar encontra-se inflamado irreversivelmente, bem como os casos 

de dentes que apresentam necrose pulpar indica-se a técnica de apicificação, que se 

baseia na indução do fechamento do forame apical, por meio da deposição de uma 

barreira de tecido duro, na região apical ou ainda pela indução do desenvolvimento 

apical, o que se encontra diretamente relacionado à manutenção da bainha epitelial 

de Hertwig (American Association of Endodontics 2003; Rafter 2005; Soares et al. 

2008).    

 Na técnica de apicificação, o hidróxido de cálcio (HC) ainda é o material mais 

empregado, permitindo que ocorra o selamento biológico pela formação de um tecido 

duro, semelhante ao cemento. Entretanto, a capacidade indutora de mineralização do 



30 
 

HC é dependente da manutenção da alcalinidade do mesmo, durante todo o período 

de tratamento, que varia de 8 – 12 meses (Soares et al. 2008). Sendo assim, são 

necessárias trocas periódicas do material, visando renovar a sua propriedade alcalina. 

Além da necessidade de múltiplas sessões, o que aumenta o risco de contaminação; a 

apicificação resulta na formação de paredes dentinárias delgadas e desenvolvimento 

radicular incompleto, tornando o dente suscetível à fratura (Hargreaves et al. 2013).  

 A técnica de apicificação também pode ser empregada por meio da colocação 

de uma barreira apical artificial - constituída por materiais à base de agregado de 

trióxido mineral (MTA) - que permite a obturação do canal radicular logo após o 

protocolo de descontaminação (American Association of Endodontists, 2003). O MTA 

tem sido indicado principalmente em casos onde há a impossibilidade de 

acompanhamento a longo-prazo, e tem se mostrado capaz de induzir a formação da 

barreira apical, semelhantemente ao HC (Shabahang et al. 1999). Além da redução no 

número de sessões durante o tratamento (Damle et al. 2012, Marí-Beffa et al. 2017), o 

emprego do MTA também tem sido associado a um menor número de fraturas 

(Jeeruphan et al. 2012). Entretanto, a técnica de apicificação, seja com HC ou plug de 

MTA, não permite o desenvolvimento radicular completo, tanto em espessura quanto 

em comprimento (Jeeruphan et al. 2012), além de não restabelecer funcionalmente o 

tecido pulpar (Hargreaves et al. 2013). 

 Recentemente, terapias biológicas denominadas de regeneração endodôntica 

(RE), que visam recuperar as funções fisiológicas de estruturas do complexo dentino-

pulpar e permitir a formação radicular completa estão sendo estudadas (Hargreaves et 

al. 2013, Marí-Beffa et al. 2017). Banchs e Trope (2004) trouxeram o conceito de RE 

pela técnica de revascularização, por meio da indução do sangramento apical após o 

emprego de uma pasta triantibiótica, observando então o desenvolvimento contínuo 

da raiz, evidenciado não só pelo aumento do seu comprimento, mas também pelo 

espessamento das paredes do canal radicular. Embora a técnica de revascularização 

tenha se mostrado eficaz na continuação da formação radicular (Petrino et al. 2010) 

alguns estudos avaliaram o tecido neoformado no espaço do canal radicular e 

observaram que o mesmo corresponde a um tecido conjuntivo semelhante ao 

ligamento periodontal, e a um tecido semelhante ao cemento ou cemento - ósseo 



31 
 

(Becerra et al. 2014, Torabinejad et al. 2015). Dessa forma, do ponto de vista clínico, a 

regeneração do tecido perdido não pode ser alcançada, visto que não há o 

restabelecimento da vascularização, inervação e deposição de dentina como no tecido 

pulpar normal. Técnicas da bioengenharia têm sido propostas visando a formação de 

um tecido semelhante à polpa, bem como a diferenciação odontoblástica, por meio do 

uso de “scaffolds” - estruturas tridimensionais que permitem a adesão, proliferação e 

diferenciação de células tronco mesenquimais - associado ao emprego de biomateriais 

ou moléculas de sinalização que estimulem esses processos (Hargreaves et al. 2008), 

como o fator de crescimento fibroblástico básico (bFGF) (Chang et al. 2017), fator de 

crescimento derivado de plaquetas (PDGF) (Cai et al. 2016), plasma rico em plaquetas 

(PRP) (Torabinejad & Faras 2012, Torabinejad et al. 2015), fibrina rica em plaquetas 

(PRF) (Subash et al. 2016), entre outros. 

 A RE em dentes permanentes jovens apresenta um prognóstico favorável 

devido à ampla comunicação entre o canal radicular e os tecidos periapicais. Essa 

vascularização tem se mostrado capaz de manter a viabilidade celular mesmo após 

danos aos tecidos pulpar e/ou periapicais (Tsukiboshi et al. 2017). Células-tronco são 

células progenitoras que apresentam ampla capacidade de auto-renovação e 

diferenciação (Gronthos et al. 2002, Huang et al. 2006, Limjeerajarus et al. 2014, 

Maglione et al. 2017), podendo ser classificadas em totipotentes - apresentam a 

capacidade de originar qualquer tipo celular, inclusive tecidos extraembrionários como 

a placenta; pluripotentes – originam todas as células de um organismo, exceto de 

tecidos extraembrionários; e multipotentes – originam linhagens específicas de células 

(Rodriguez-Lozano et al. 2011). Células-tronco mesenquimais apresentam grande 

potencial em procedimentos de RE, uma vez que estão presentes na própria polpa 

dentária - DPSCs (Gronthos et al. 2002), dentes decíduos – SHED (Miura et al. 2003), 

ligamento periodontal - PDLSCs (Jo et al. 2007) e papila apical - SCAP (Huang et al. 

2008). Mesmo na presença de processo inflamatório, células da polpa dentária e da 

região periapical, apresentaram alta expressão de marcadores de células-tronco 

mesenquimais (Alongi et al. 2010, Liao et al. 2011). 

 A busca por tratamentos capazes de induzir a diferenciação de células (Asgary 

et al. 2014, Chen et al. 2015, Huang et al. 2016), bioestimular células remanescentes 



32 
 

em concentrações não tóxicas (Washington et al. 2011, Kuang et al. 2016, Chen et al. 

2015), promover uma desinfecção eficaz sem citotoxicidade (Bottino et al. 2013, 

Palasuk et al. 2014, Kamocki et al. 2015), ou ainda criar um scaffold que permita a 

proliferação e diferenciação celular, além de oferecer suporte adequado para 

biomoléculas (Bottino et al. 2015, Kuang et al. 2016) tem sido alvo de diversos estudos 

no campo da RE. A análise de marcadores de diferenciação osteogênica após 

determinado tratamento proposto em células indiferenciadas (d'Aquino et al. 2007, 

Wang et al. 2017), ou ainda em células diferenciadas que poderiam ser remanescentes 

à determinada injúria (Satué et al. 2013, Zeng et al. 2013, Liu et al. 2017) tem sido 

amplamente realizada com o intuito de analisar a capacidade bioestimulatória de 

diferentes materiais, visando induzir o processo de reparo. Dentre esses marcadores 

de mineralização estão a fosfatase alcalina (ALP – enzima responsável pela 

mineralização da matriz dentinária ou óssea); cuja expressão ocorre em células 

envolvidas no processo de mineralização, como os odontoblastos e osteoblastos, a 

proteína da matriz dentinária (DMP-1) e a sialofosfoproteína dentinária (DSPP) que são 

proteínas não-colagenosas que participam da mineralização da dentina e maturação 

das fibras colágenas durante o processo de dentinogênese. Essas proteínas 

permanecem no interior do substrato dentinário, sendo liberadas em resposta às 

injúrias teciduais, para estimular odontoblastos primários a produzirem dentina 

terciária reacional, ou ainda para estimular a diferenciação de células pulpares em 

odontoblastos que irão produzir dentina terciária reparadora (Ferracane et al. 2010, de 

Souza Costa et al. 2014). 

 A desinfecção do sistema de canais radiculares também representa um desafio 

dentro das terapias de RE, uma vez que esses dentes permanentes jovens apresentam 

canal radicular e ápice amplos, além de paredes dentinárias delgadas, inviabilizando o 

preparo biomecânico convencional. A pasta triantibiótica (metronidazol, ciprofloxacina 

e minociclina) tem se mostrado eficaz em tratamentos de RE para dentes imaturos 

(Windley et al. 2005, Petrino et al. 2010); entretanto, foi observada uma significativa 

redução na viabilidade de células-tronco da papila apical (Ruparel et al. 2012) e 

fibroblastos do ligamento periodontal (Yadlapati et al. 2014) após a sua aplicação. Esse 

efeito citotóxico também foi demostrado em células da polpa humana, onde a pasta 



33 
 

na concentração de 2,5mg/mL, ou seja bem menor do que aquelas empregadas 

durante as terapias de RE (aproximadamente 1g/mL), foi capaz de reduzir a 

proliferação celular (Labban et al. 2014). Devido à algumas desvantagens, como a 

descoloração dentinária (Kim et al. 2010), a minociclina – antibiótico semisintético 

derivado da tetraciclina – foi removida da pasta triantibiótica. A pasta biantibiótica 

(metronidazol e ciproflofaxacina) tem apresentado bons resultados frente à infecção 

endodôntica (Iwaya et al. 2001), porém com maior efeito citotóxico, reduzindo a 

viabilidade de células pulpares à partir da concentração de 0,5mg/mL (Labban et al. 

2014). De maneira semelhante, o hidróxido de cálcio também não demonstrou 

toxicidade em baixas concentrações (até 2,5mg/mL),  entretanto a concentração 

utilizada clinicamente é de aproximadamente 0,7g/mL  (Labban et al. 2014). 

 Compostos biativos derivados de plantas como os flavonoides, têm recebido 

destaque na literatura devido à sua amplitude terapêutica. Os flavonoides são 

moléculas polifenólicas presentes em frutas e vegetais (Panche et al. 2016) que 

apresentam propriedades antimicrobiana, antioxidante, osteogênica e 

antiosteoclastogênica (Pietta 2000, Tripoli et al. 2007, Sharan et al. 2009, Pilsakova et 

al. 2010, Domitrovic 2011). Dentre eles, a taxifolina, um flavonoide tipo catecol, 

isolado de diversas plantas (da Costa et al. 2014, Wang et al. 2015, Park et al. 2016 ), e 

mais recentemente do chá verde (Wang et al. 2017), tem apresentado ampla ação 

terapêutica, entre as quais, atividade antimicrobiana contra Enterococcus faecalis 

resistente a vancomicina (Jeong et al. 2009), Acinetobacter  hemolyticus (Chatzopoulou 

et al. 2010); atividade antifúngica (Kanwal et al. 2010); capacidade de estimular o 

aumento da expressão gênica de osteocalcina, osteoprotegerina e sialoproteína óssea, 

além de diminuir a expressão de RANKL (marcador de reabsorção óssea) em 

osteoblastos (Satué et al. 2013) e estimular a diferenciação de células-tronco 

mesenquimais em osteoblastos (Córdoba et al. 2015, Wang et al. 2017). 

 Própolis, substância resinosa produzida por abelhas por meio da mistura de 

suas secreções com produtos obtidos de diversas plantas, tem sido amplamente 

estudada devido as suas propriedades terapêuticas (Yokoyama et al. 2014, Aral et al. 

2015, Asawahame et al, 2015). Dentre essas, a sua atividade antimicrobiana tem sido 

relatada por alguns autores (Uzel et al. 2005, de Luca et al. 2014), inclusive frente à 



34 
 

microrganismos de interesse endodôntico, como Enterococcus faecalis, Candida 

albicans, Streptococcus mutans e Pseudomonas aeruginosa (Uzel et al. 2005). Dentre 

os principais compostos isolados da própolis estão os flavonoides galangina, crisina e 

pinocembrina. 

 Huh et al. (2013) observou que a galangina reduziu as citocinas inflamatórias IL-

1β, TNF-α e IL-17; inibiu a formação de osteoclastos e fatores osteoclastogênicos; além 

de aumentar a expressão gênica de osteoprotegerina em osteoblastos. Liu et al. (2017) 

também observaram um aumento na expressão de marcadores de diferenciação 

osteogênica, como colágeno, fosfatase alcalina (ALP), osteocalcina e osteopontina em 

células derivadas de osteosarcoma após o tratamento com galangina. Esse flavonoide 

mostrou ainda atividade inibitória contra S. aureus e Enterococcus (Li et al. 2012), 

apresentando como possível mecanismo de ação um dano direto à membrana 

citoplasmática ou indireto à parede celular, por provocar a perda de íons potássio 

(Cushnie e Lamb 2005). O flavonoide crisina também demonstrou atividade contra 

bactérias Gram positivas e Gram negativas, incluindo S. aureus e E. coli com 

concentrações inibitórias mínimas (CIM) que variaram de 50 à 6,25 µg/mL (Liu et al. 

2010) e promoveu aumento da expressão de marcadores osteogênicos, como 

colágeno, osteocalcina e osteopontina em osteoblastos (Zeng et al. 2013). A 

pinocembrina apresentou atividade antimicrobiana contra C. albicans e S. aureus, com 

CIMs de 6.25 e 12.5 μg/ml respectivamente (Katerere et al. 2012), além de atenuar a 

resposta inflamatória induzida por lipopolissacarídeos (Giri et al. 2016). Assim, os 

flavonoides apresentam diversas propriedades desejáveis nas terapias de regeneração 

endodôntica devido às suas múltiplas funções biológicas.  

 

 

 

 

 

 



35 
 

 

 

 

 

 

 

 
 

Proposição 
 



36 
 

Proposição 

 

 O objetivo geral do presente trabalho foi avaliar o efeito antimicrobiano, 

citotóxico e indutor de mineralização de flavonoides com finalidade de aplicação 

endodôntica, tanto em procedimentos de vitalidade pulpar quanto na regeneração 

endodôntica. 

 Os objetivos específicos foram: 

 - Avaliar o efeito citotóxico dos flavonoides taxifolina, crisina, galangina e 

pinocembrina em cultura de fibroblastos (L-929); 

 - Avaliar a atividade antimicrobiana dos flavonoides frente Streptococcus 

mutans, Enterococcus faecalis e Actinomyces israelii; 

 - Avaliar o efeito antibiofilme do flavonoide taxifolina em biofilme formado em 

placas de poliestireno e túbulos dentinários; 

 - Avaliar o efeito citotóxico de diferentes tratamentos com a taxifolina em 

células semelhantes a odontoblastos (MDPC-23) e a osteoblastos (Saos-2); 

 - Avaliar a atividade da fosfatase alcalina e produção de nódulos de 

mineralização após os tratamentos com a taxifolina nas células MDPC-23 e Saos-2; 

 - Avaliar a expressão gênica de DSPP e DMP-1 nas células MDPC-23 após 24 e 

72h de tratamento com a taxifolina; 

 - Avaliar a expressão gênica de ALP e Col-1 nas células Saos-2 após 24 e 72h de 

tratamento com a taxifolina. 



37 
 

 

 

 

 

 

 

 

 

 
 

 

Capítulo 1 
 



38 
 

Cytotoxic, antibacterial and anti-biofilm activities of flavonoids intended for 

endodontic purposes* 

Abstract 

The rhizogenesis process can be interrupted after dental pulp injury. The aim of this 

study was to evaluate the toxicity on fibroblasts, antibacterial and anti-biofilm of 

flavonoids. MTT assays were conducted to analyzed taxifolin, chrysin, pinocembrin, 

and galangin treatments on fibroblasts viability. Minimal inhibitory and bactericidal 

concentrations were determined for the same flavonoids against Enterococcus 

faecalis, Actinomyces israelii, and Streptococcus mutans. Biofilm assays were 

performed only with taxifolin in polystyrene plates and inside dentin tubules. The 

results showed that taxifolin had no toxic effect on fibroblasts, while the other 

flavonoids showed cytotoxic effects. Taxifolin was the unique flavonoid able to inhibit 

all tested bacteria growth through the microdilution broth method, besides to 

eliminate E. faecalis and S. mutans biofilms in polysterene plates, and reduce E. 

faecalis biofilm on dentin tubules. Taxifolin presents potential use in permanent teeth 

for endodontic purposes, due to its antimicrobial/anti-biofilm effects and low toxicity 

on fibroblasts. 

 

Keywords: Flavonoids, Cell Culture Techniques, Microbial Sensitivity Tests, Biofilms. 

 

 

 

 

 

 

*The manuscript is according to the guide for authors of Biofouling (Anexo A). 



39 
 

Introduction 

Dental caries or trauma in immature permanent teeth can interrupt the normal 

apical closure process due to pulp tissue injury. The exposed pulp allows root canal 

contamination by bacteria from the oral cavity or those found in deep dentinal caries, 

such as Streptococcus mutans and Actinomyces israelii (van Houte et al. 1996; Munson 

et al. 2004; Rôças et al. 2015), which are able to invade the pulp via dentinal tubules 

and trigger an endodontic infection. Primary endodontic infection is polymicrobial in 

nature, probably due to oxygen variation and nutrient availability in root canal thirds. 

Studies have reported a prevalence of facultative anaerobic bacteria in primary 

endodontic infections (Gomes et al. 2006; Sedgley et al. 2006; Ruviére et al. 2007; 

Ledezma-Rasillo et al. 2010) and similarity in the microbial composition between 

permanent young teeth and completely formed teeth (Baumotte et al. 2011). Due to a 

wide root canal, open apex, and thin dentinal walls, chemo-mechanical root canal 

preparation is limited and therefore disinfection occurs mainly by intracanal 

medicament.  

For a long time the treatment of choice for young permanent teeth was 

apexification with calcium hydroxide (CH). Despite the success attributed to this 

technique in virtue of formation of an apical hard tissue barrier (Rafter et al. 2005), the 

multiple sessions and long-term treatment frequently increases the potential for 

recontamination, the formation of fragile dentin walls, and subsequent susceptibility 

to fractures (Hargreaves et al. 2013). Another technique that has been used is artificial 

apical barrier through the application of mineral trioxide aggregate (MTA) as a plug in 

the tooth apical third. One advantage of the MTA technique is the reduction in clinical 

appointments (Marí-Beffa et al. 2017). Inevitably, both these techniques lead to loss of 

vital pulp tissues including odontoblasts, fibroblasts, stem cells, and the Hertwig 

epithelial root sheath need for complete root development (Chueh et al. 2006). 

Studies have appointed higher levels of mesenchymal stem cell markers in dental pulp 

stem cells even in inflamed pulps or in periapical progenitor cells from inflamed 

periapical tissue collected during endodontic surgical procedures (Alongi et al. 2010; 

Liao et al. 2011).  



40 
 

Regenerative endodontic therapies (RET) have been considered as 

bioengineering therapies as part of the search for new materials that allow the 

synthesis of a new pulp-like tissue able to restore lost functions (Mohammad et al. 

2015; Marí-Beffa et al. 2017). RET are possible in any permanent teeth, however 

immature permanent teeth present apical enlargement that provides communication 

between root canal and periapical tissues providing a large blood supply, carrying 

cellular and molecular components of the immune system (Tsukiboshi et al. 2017). 

Immature teeth also have been presented a large number of undifferentiated 

mesenchymal cells and odontoblasts than adult teeth (de Souza Costa et al. 2014), 

likely due the recruitment of these cells during physiological tooth wear, carious 

process and cavity preparation in adult teeth. 

 The material most commonly used based on these techniques is the triple 

antibiotic paste (TAP - metronidazole, minocycline, and ciprofloxacin) for microbial 

decontamination followed by the induction of revascularization by blood clot 

formation (Petrino et al. 2010). However, the use of TAP has generated some 

discussion regarding bacterial resistance, crown discoloration, and possible allergic 

reaction (Cohenca et al. 2010; Kim et al. 2010; Akcay et al. 2014). Others antimicrobials 

commonly used in RET, such as CH and double antibiotic paste (DAP – metronidazole 

and ciprofloxacin) also had show drawbacks related to their cytotoxic effect. Safest 

non-toxic concentrations of TAP, DAP and CH were lower than the intracanal 

concentrations that have been advocated in RET (Labban et al. 2014). 

Plants are a wide source of bioactive compounds, which present several 

biological activities (Dimech et al. 2013; Coronado-Aceves et al. 2016; Wang et al. 

2017). Among these natural compounds, flavonoids are an important class known for 

their therapeutic use. They are plant secondary metabolites, mainly pigments that 

color flowers, fruits, and seeds, constituted by a polyphenolic structure (Panche et al. 

2016). Taxifolin is a catechol-type flavonoid isolated from Hymenaea courbaril (da 

Costa et al. 2014) Morus laevigata (Wang et al. 2015), Hovenia dulcis (Park et al. 2016), 

and green tea (Wang et al. 2017) among others. Studies have reported the 

antimicrobial potential (Chatzopoulou et al. 2010; Kanwal et al. 2010; Jeong et al. 

2009) of taxifolin and its ability to stimulate osteogenic differentiation (Satué et al. 



41 
 

2013; Wang et al. 2017). Other flavonoids frequently isolated from propolis, such as 

pinocembrin, galangin, and chrysin have been related to antimicrobial activity of this 

resinous substance produced by bees (Uzel et al. 2005; Suleman et al. 2015). Among 

them, galangin has also inhibited osteoclastogenic factors (Hu et al. 2013) and both 

galangin and chrysin enhanced the expression of osteoblast differentiation markers 

and mineralization (Liu et al. 2017; Zeng et al. 2013). 

Based on the fact that the commonly used antimicrobial agents have presented 

cell toxicity in the concentrations advocated for endodontic regeneration (Yadlapati et 

al. 2014; Labban et al. 2014), the aim of this study was to evaluate the antimicrobial 

activity and toxicity of taxifolin, chrysin, pinocembrin, and galangin on fibroblasts, 

besides to analyze the anti-biofilm effect of the flavonoid with the best antimicrobial 

effect, intended to be used for endodontic purposes. The null hypotheses tested were: 

(1) flavonoids present cytotoxic effect on L929 fibroblasts, (2) flavonoids do not have 

antibacterial activity, and (3) taxifolin does not have anti-biofilm effect. 

Materials and Methods 

Cell viability assays 

Flavonoids  

Taxifolin (#78666), chrysin (#C80105), pinocembrin (#P5239), and galangin 

(#282200) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A stock solution 

was prepared in Dimethyl Sulfoxide (DMSO - Sigma-Aldrich, St. Louis, MO, USA) and 

stored at -20°C for each flavonoid. Solution with the final concentration of 0.5% DMSO 

was included as the control group in the experiments. Chlorhexidine digluconate (CHX, 

Sigma Aldrich, St. Louis, MO, USA) was used as a positive control. 

Cell Lines 

Mouse fibroblasts (L-929) were grown in Dulbecco Modified Eagle’s Medium 

(DMEM) supplemented with 10% fetal bovine serum (FBS, GIBCO BRL, Gaithersburg, 

MD) streptomycin (50 g/mL), and 1% antibiotic/antimycotic cocktail (300 U/mL 

penicillin, 300 mg/mL streptomycin, 5 mg/mL amphotericin B, and L-glutamine 0.3 g/L) 



42 
 

(GIBCO BRL, Gaithersburg, MD) under standard cell culture conditions (37ºC, 100% 

humidity, 95% air, and 5% CO2). 

MTT assay 

The cells were plated at a density of 1x10⁵ cells/well in 96-well plates and 

incubated for 24 hours in a humidified air atmosphere of 5%  at 37ºC to allow cell 

attachment. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 

assay was used to determine the cell viability according to Takamiya et al. (2016). 

Briefly, after the cell attachment, the flavonoid groups (600, 300, 150, 75, 37.5, 18.75, 

and 9.37µg/mL) were added to the cells. The controls were cultured in medium 

without flavonoid treatment.  At 24h after addition, the treatments were removed and 

the MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells and the 

plates were incubated at 37°C for 4h protected from light. This tetrazolium salt is 

metabolically reduced by viable cells to yield a purple insoluble formazan crystal. Next, 

the MTT solution was discarded and 200µL of isopropyl alcohol was added to each well 

to dissolve the crystals under continuous agitation for 30 min. The solution was 

transferred to a 96-well plate to measure the optical density (OD) at 570nm in a 

spectrophotometer (Shimadzu MultSpec-1501; Shimadzu Corporation, Tokyo, Japan). 

The experiments were performed in triplicate. 

Antimicrobial assay 

Microbial conditions 

The following microbial strains used in the present study were kindly provided 

by the Oswaldo Cruz Foundation (FIOCRUZ - Rio de Janeiro, RJ, BR): Streptococcus 

mutans (ATCC 25175), Enterococcus faecalis (ATCC 51299), and Actinomyces israelii 

(ATCC 12102). Stock microbial suspensions were inoculated in Brain Heart Infusion 

Agar (BHI, Difco Laboratories, Detroit, MI, USA) for E. faecalis and A. israelii or Mitis 

Salivarius Agar base (MSA, Difco Laboratories) with 0.2 U mg ml−1 bacitracin (Sigma-

Aldrich) for S. mutans. Representative colonies of these strains were grown overnight 

in BHI broth (Difco Laboratories) and incubated at 37°C for 24 hours in 5% CO₂ 

(Incubator Ultra Safe, HF212-UV). All microorganisms were incubated in these 



43 
 

atmospheric conditions to simulate the low oxygen concentration inside root canals. 

Growth curve assays were performed for each microorganism in order to determine 

the optical density at the mid-log phase [approximately 0.5 (1-5x10⁸ CFU/mL)] to be 

used in the following experiments. The absorbance was measured using a microplate 

reader (Eon Microplate Spectrophotometer, BioTek Instruments, Winooski, Vermont, 

USA) to assess the cell growth.  

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal 

concentration (MBC) 

MIC and MBC were determined through the microdilution broth method, in 96-

well microtiter plates, following the criteria previously described by Clinical and 

Laboratory Standard Institute M7-A9 (CLSI, 2012). The final concentration of bacterial 

suspension in the wells was 1-5x10⁵CFU/mL. Initially, the flavonoid stock solutions 

were serially diluted in water and after that, correctly adjusted microbial suspensions 

were inoculated in each well. The flavonoid concentrations tested ranged from 2 – 

0.0002mg/mL. The plates were incubated at 37°C for 24 hours in a 5% CO₂ 

atmosphere. Afterwards, 15µL of 0.01% resazurin (R7017 Sigma-Aldrich – St. Louis, 

Missouri, USA) was applied in each well and incubated for 4 hours to promote 

oxidation-reduction and determine the cell viability through visual detection of the 

color change. Posteriorly the wells corresponding to MIC (the last blue well) and at 

least three previous wells were homogenized, six times diluted and plated on Mueller-

Hinton Agar (Difco Laboratories) for E. faecalis and A. israelii or Mitis Salivarius Agar 

base (Difco Laboratories) with 0.2 U mg ml−1 bacitracin (Sigma-Aldrich) for S. mutans 

to determine the MBC. The plates were incubated at 37°C for 24 hours in a 5% CO₂ 

atmosphere. The colonies were counted and the number of viable bacteria determined 

in CFU/mL. The MBC was considered when the flavonoid treatment killed more than 

99% of the tested microbial culture. Chlorhexidine digluconate (CHX) was used as a 

positive control. The negative control was the culture without antimicrobial agents. 

Assays were repeated three times for each microorganism, in three independent 

experiments. 

Anti-biofilm assays 



44 
 

Biofilm formation in polystyrene microplates 

Taxifolin was selected for biofilm assays as it presented the best results in the 

previous assays: MTT, MIC, and MBC. Biofilm assays were conducting according to 

Massunari et al. (2017) with E. faecalis, S. mutans, and A. israelii. Twenty microliters of 

each microorganism suspension (approximately 1-5x10⁶CFU/mL) were inoculated in 

sterile U-shaped bottom polystyrene 96-well microplates containing 180µL of BHI 

supplemented with 0.5% glucose. The plates were incubated at 37°C in a 5% CO₂ 

atmosphere. After 48 hours, the culture medium was removed and the wells were 

washed with sterile saline for subsequent addition of 200µL of previously diluted 

taxifolin per well. The concentration of taxifolin was 10 times higher than the MBC 

concentration (10x MBC) obtained for each microorganism. The microplates were 

incubated again in the same conditions for 24 hours. All cultures in the wells were 

diluted six times and plated in brain heart infusion agar and incubated for 24 hours. 

After this period, the colony forming units (CFU)/mL were determined. Chlorhexidine 

digluconate (10x MBC) was used as a positive control as well as biofilm in medium 

without antimicrobial agents as a negative control. Assays were repeated three times 

for each microorganism, in three independent experiments. 

Biofilm formation in dentin tubules of bovine roots  

 Biofilm assays for confocal laser scanning microscopy (CLSM) analysis were 

conducted with E. faecalis, using taxifolin and CHX, according to the method proposed 

by Ma et al. (2011) with some modifications. Briefly, dentin blocks (Ethics Committee 

on the Use of Animals – FOA/UNESP, Protocol 01194-2017) with a length of 4 mm 

were obtained from bovine roots (n = 3/group), sectioned horizontally 1 mm below the 

cement-enamel junction. After enlargement of the root canals with Gates Glidden drill 

#6 (1.5 mm diameter) (Dentsply, Tulsa, USA), each cylindrical dentin block was 

fractured into two semi cylindrical halves and ground using 600-grit silicon carbide 

paper until reaching the final size of 3 × 3 × 2 mm. The blocks were cleaned in an 

ultrasonic bath initially using 17% EDTA solution for 3 minutes and then distilled water 

for 5 minutes. After autoclaved, the blocks were fixed in a microtube with a resin 

composite (3M ESPE, USA) and light cured for 40 seconds. Five hundred microliters of 



45 
 

E. faecalis suspension at 107 CFU/mL in BHI broth were added to each microtube and 

sequentially harvested at 1400, 2000, 3600, and 5600 g, twice each, for 5 minutes. A 

fresh suspension of bacteria was inserted between every centrifugation and the final 

solution was discarded. Dentin blocks were incubated individually in 48-well plates in 

BHI broth for 15 days, replacing the culture medium every 72h. After this period, the 

blocks were washed twice with sterile saline and transferred, under aseptic conditions, 

to a new plate and exposed to taxifolin (at 10x MBC) and CHX (100 and 1000x MBC) for 

2h under agitation and a further 46h in static conditions. After that, the dentin blocks 

were washed again twice, cut into transverse slices of 1mm thickness, and polished 

with 1200 grit sandpaper disks.  

 After new washing with sterile water, the dentin blocks were stained with 

100µL of fluorescent LIVE/DEAD BacLight Bacterial Viability stain (L13152, Molecular 

Probes, Eugene, OR) containing SYTO9 and propidium iodide, according to the 

manufacturer’s instructions. The excitation/emission wavelengths were 480/500 nm 

for SYTO 9 and 490/635nm for propidium iodide. Two additional untreated specimens 

were stained using the same protocol as the negative controls. The mounted 

specimens were observed using a 63x NA 1.4 oil immersion lens. CLSM images were 

acquired using the software LAS AF (Leica Mic-systems). The Z stack was obtained from 

the top until the bottom of the biofilms. Using a 0.5µm interval stack between each 

frame, 4 randomly selected areas of each dentin specimen were made for each 

sample. Each 2D (two-dimension) image was obtained by the max projection of the Z 

stack. The ratio of red fluorescence to green-and-red fluorescence indicated the 

proportion of dead cells for each antimicrobial agent, measured using Image J software 

(Rasband, W.S., Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA, 

https://imagej.nih.gov/ij/, 1997-2016). 

Statistical analysis 

The results were presented as means ± standard deviation (SD) and analyzed 

using SPSS version 17.0 software (SPSS, IL, USA). Data from bacterial recovery from 

biofilm assays were converted in logarithmic scale log10 (CFU+1) and calculated the 

percentage (%) of bacterial growth compared to control groups. Values were normally 

distributed as verified by Kolmogorov-Smirnov test. Analysis of variance (ANOVA) 



46 
 

followed by Bonferroni tests were performed for all results, considering p<0.05 as 

significant.  

Results 

Cell viability  

Taxifolin showed no toxic effect on L-929 fibroblast cells at any tested 

concentration. Chrysin demonstrated low cytotoxic effect over 150 µg mL-1. 

Pinocembrin was cytotoxic from 600 to 150 µg mL-1. Galangin was the most cytotoxic 

flavonoid reducing its toxicity only below 37.5 µg mL-1. Chlorhexidine digluconate was 

cytotoxic from 600 to 18.75 µg mL-1 (Figure 1). 

Antimicrobial activity 

 MIC and MBC values for flavonoids ranged between 0.03 and 1 mg mL-1 (Table 

1). All flavonoids tested demonstrated antibacterial activity against A. israelii, except 

chrysin that had no effect against any of the bacteria on tested concentrations. Only 

taxifolin was able to inhibit all tested bacteria growth through the microdilution broth 

method. S. mutans and E. faecalis growth was not affected by pinocembrin, galangin, 

or chrysin (Table 1). The control chlorhexidine presented the lowest values of MIC and 

MBC for all tested bacteria (Table 1). 

Anti-biofilm effect in polystyrene plates 

 Taxifolin at 10x MBC concentration was able to completely eliminate E. faecalis 

and S. mutans biofilms, and reduce A. israelii biofilm. Chlorhexidine digluconate in the 

same conditions (10x MBC) was able to eliminate only S. mutans biofilm and reduce A. 

israelii biofilm, but did not present an effect against E. faecalis biofilm (Figure 2). 

Anti-biofilm effect in dentin tubules of bovine roots 

 Representative CLSM images of E. faecalis biofilms formed in bovine root 

dentin specimens are presented in Figure 3A-D. Both taxifolin and CHX showed higher 

quantification of dead cells (red points) when compared to the control (culture 

medium). Taxifolin at 10x MBC strongly reduced E. faecalis (99.74% dead cells). Similar 



47 
 

reduction (93.73% dead cells) was observed for CHX at 1000x MBC and a lower 

reduction (64.61% dead cells) for CHX at 100x MBC (Figure 4).  

Discussion 

 The aims of this study were to evaluate the cytotoxicity and antibacterial 

activity of four flavonoids, as well to analyze the anti-biofilm effect of the flavonoid 

which was highlighted in previous assays. The null hypothesis 1 was partially accepted, 

since taxifolin was the unique tested flavonoid without cytotoxicity on L929. The 

hypothesis 2 also was partially accepted, once only chrysin did not have antibacterial 

activity; and the hypothesis 3 was rejected because taxifolin showed anti-biofilm 

effect. 

  Among the flavonoids tested, taxifolin did not affect viability of fibroblast cells 

up to 600 µg mL-1. Although taxifolin caused 15% of reduction on L929 cell viability at 

600 µg mL-1, this effect is considered low, since ISO 10993-5:2009 recommendations 

characterized a reduction of from 30% in cell viability as a cytotoxic effect. Gómez-

Florit et al. (2014) also evaluated the effect of chrysin, taxifolin and galangin on human 

primary gingival fibroblasts (HGF cells) and observed that 100-200 µM chrysin, 500 µM 

galangin and doses higher than 10µM taxifolin produced a significant release of lactate 

dehydrogenase (LDH) activity compared to controls. In this same study, the highest 

concentration of taxifolin (500 µM – around 150 µg mL-1) caused low toxicity (20%) on 

fibroblasts; different from the present study, which showed minimal toxic effect of 

taxifolin at 600 µg mL-1. Likely this difference is related to different cell types used in 

the studies. Primary cells gradually reduce their proliferation rate in vitro, shortening 

of telomeres, coming in cellular senescence (Milyavsky et al. 2003); while immortalized 

cells, such as L929, presents the elongation of telomeres increasing the stability of 

chromosomes (Maqsood et al. 2013).  

Chlorhexidine is a broad-spectrum antiseptic, used as endodontic irrigant or 

root canal dressing (Gomes et al. 2006, Rôças et al. 2016), however have been 

presented cytotoxicity against eukaryotic cells (Hidalgo & Domingues 2001; Faria et al. 

2007). The present study showed cytotoxic effect of the CLX up to 20 µg mL-1 on L929. 

Similarly to ours, Faria et al. (2007) observed CHX at 0,002% (20 µg mL-1) caused a 



48 
 

significant increase in the percentage of necrotic cell (34%). Faria et al. (2009) showed 

CHX causes endoplasmic reticulum overexpression in L929 due accumulation of 

proteins, resulting in cell death by necrosis or apoptosis, depending on the CHX 

concentration. According the authors CLX at high concentration (from 0,002%) might 

retard the periapical healing, due cell necrosis, a passive form of cell death that results 

from disruption of cell membranes and release of cell components to the extracellular 

matrix, triggering an inflammatory reaction.  

Another important characteristic of a biomaterial indicated for endodontic 

purposes is the antimicrobial activity, mainly against deep caries-related bacteria, such 

like S. mutans and A. israelii, and persistent endodontic infection such as E. faecalis. 

These bacteria were selected in the present study due their close relationship with the 

endodontic polymicrobial infection. Tsukiboshi et al. (2017) showed the presence of 

apical periodontitis in radicular vital pulp, characterizing a partial pulp necrosis in 

immature permanent teeth. According the authors this clinical condition can be due 

large foraminal opening which carries cellular and molecular components of the 

immune system, delaying the necrosis procces. Therefore, early colonizers such as S. 

mutans could play a role at the beginning of apical periodontitis formation. A. israelii is 

a common facultative anaerobe isolated from the root canal and periapical tissues due 

its capacity to form biofilm and break into soft tissue (Xia & Baumgartner 2003). The 

high production of extracellular polymers can result in a well-organized biofilm with 

difficult elimination, even after both taxifolin and chlorhexidine treatments at high 

concentrations, as showed in the Figure 2. E. faecalis was included in this study due its 

presence in persistent endodontic infections, surviving at alkaline pH by proton pump 

(Evans et al. 2002), and invading dentin tubules forming biofilm into them, as observed 

in Figure 3. 

Taxifolin was able to inhibit all tested bacteria, while other flavonoids 

demonstrated activity against A. israelii, excepted for chrysin that had no effect against 

any of the tested bacteria. Jeong et al. (2009) also found important taxifolin activity 

against E. faecalis and vancomycin-resistant E. faecalis showing MIC values ranging 

from 128 to 512 µg mL-1. In the current study, other flavonoids did not inhibit E. 

faecalis and S. mutans growth at the tested concentrations. Studies evaluating the 



49 
 

isolated effect of these flavonoids on oral bacteria were not found. On the other hand, 

Suleman et al. (2015) observed a noteworthy antimicrobial activity of propolis samples 

from Brazil and South Africa against important pathogens such as E. faecalis, S. aureus, 

and C. albicans and attributed this property to flavonoid content composed mainly of 

pinocembrin, galangin, and chrysin, possibly through the synergic effect between 

them. Antimicrobial activity of flavonoids has been related to their structure, mainly 

their B ring. The hydroxy groups of taxifolin were able to bind to site of β-Ketoacyl 

carrier protein synthase (KAS III) of E. faecalis, by hydrogen-bonding networks, 

showing as a candidate inhibitor of this protein. KAS lll is an enzyme responsible for 

catalyze condensation reaction in type II fatty acid synthesis pathway, that is essential 

for bacterial survival (Jeong et al. 2009). 

In the present study, taxifolin was selected for biofilm assays based on its 

previous antibacterial and non-cytotoxic effects. This is the first study to evaluate the 

anti-biofilm effect of taxifolin. Biofilm assays were performed at a 10x MBC 

concentration for taxifolin as the architecture of biofilms provides advantages to 

bacteria, such as more resistance regarding environmental stresses, including 

antimicrobial agent effects. In the present study, taxifolin and CHX at 10x MBC were 

able to eliminate S. mutans biofilm. Lee et al. (2016) also showed that CHX treatment 

reduced bacterial viability in mature S. mutans biofilms. CHX antimicrobial activity is 

related to its cationic nature, which allows it to bond strongly to anionic sites on cell 

membranes and walls (Jensen, 1977). A. israelli biofilm presented a remarkable 

reduction after CHX and taxifolin treatment, but elimination was not observed. 

Barnard et al. (1996) suggested that this difficulty in A. israelii elimination is due to the 

genotypic tolerance of the species. Only taxifolin at 10x MBC eliminated E. faecalis in 

both biofilm assays. E. faecalis has demonstrated high resistance to endodontic 

medicaments (Eswar et al. 2013), and the ability to form biofilms both in treated and 

untreated root canals (Gomes et al. 2006, Vidana et al. 2016). The absence of a CHX 

effect on E. faecalis biofilm is associated with the concentration chosen for the biofilm 

assays in polystyrene plates (10x MBC). In dentin tubules, CHX at 100x MBC was 

effective in reducing E. faecalis biofilm and practically eliminated biofilm at 1000x 



50 
 

MBC. In other studies, CHX was also effective against E. faecalis biofilm at high 

concentrations (0.2-2% or 2-20 mg mL-1) (Eswar et al. 2013, Komiyama et al. 2016). 

This study was performed with fibroblast cells due its wide application, being 

present in both the pulp and periapical tissues. Primary these cells are known as 

structural elements, constructing an important structure to support tissue integrity 

and repair. Moreover, fibroblasts can behave as immunoregulatory cell, producing 

chemokines that initiate the recruitment of bone-marrow-derived cells, after activated 

by several bacterial products, such as LPS (Smith et al 1997). Further studies are need 

to test cytotoxic effect of taxifolin on undifferentiated cells, besides their capacity to 

promote pulp regeneration and stimulate the formation of mineralized tissue when 

proposed as pulp-capping agent. 

Chrysin, pinocembrin and galangin showed cytotoxic effects at any tested 

concentrations besides not displaying antibacterial activity against S. mutans and E. 

faecalis. Taxifolin promotes a potential antimicrobial/anti-biofilm effect against all 

tested bacteria, without toxicity on fibroblasts, could be indicated for endodontic 

purposes, such as vital pulp and regenerative endodontic therapies.   

Acknowledgements 

The authors would like to thank Oswaldo Cruz Foundation (FIOCRUZ) by 

supporting the microbial strains.  

Disclosure statement 

The authors reported no potential conflict of interest. 

Funding 

This work was supported by the São Paulo Research Foundation (FAPESP) under 

grants #2015/00812-0 and 2017/10940-1. 

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58 
 

Figures and Tables 

 

Figure 1. Mean (bars) of the percentage of cell viability of fibroblasts from L-929 line after 

exposure to different concentrations of flavonoids, using MTT assays.  

aDifferent lower case letters show statistical differences between the groups, considering each 
concentration separately, according to ANOVA and Bonferroni tests, considering p<0.05.  
 

Table 1. Minimum inhibitory concentration (MIC) and minimal bactericidal concentration 

(MBC) of flavonoids. 

 

Flavonoid Bacteria 
MIC 

(mg/mL) 

MBC 

(mg/mL) 

Taxifolin 

S. mutans 0.5 1 

E.faecalis 0.25 1 

A.israelii 0.25 0.5 

Chrysin 

S. mutans >2 >2 

E.faecalis >2 >2 

A.israelii >2 >2 

Pinocembrin 

S. mutans >2 >2 

E.faecalis >2 >2 

A.israelii 0.03 0.03 

Galangin 

S. mutans >2 >2 

E.faecalis >2 >2 

A.israelii 0.03 0,03 

Chlorhexidine  

Digluconate 

S. mutans 0.0002 0.002 

E.faecalis 0.004 0.004 

A.israelii 0.002 0.002 

    

 
   
   
   

 
   
   
   

  



59 
 

 

Figure 2. Bacterial recovery (Log CFU+1) after 24h of taxifolin and chlorhexidine 

digluconate (CHX) treatments (10x MBC) on 48h-growth biofilm.  

aDifferent lower case letters show statistical differences between the groups, considering each 
strain separately, according to ANOVA e Bonferroni tests, considering p<0.05. 
 
 

 

Figure 3. Representative CLSM images (63x) of E. faecalis biofilms inside dentin tubules 

after 48h of the treatments. (A) control – culture medium; (B) taxifolin 10x MBC; (C) 

CHX 100x MBC; (D) CHX 1000x MBC. 



60 
 

 

 

 

 

 

 

 

 

Figure 4. Means (bars-standard deviation) of the percentage of dead cells obtained 

after CLSM analysis of E. faecalis biofilm.  

Different lower case letters show statistical differences between the groups, considering each 

strain separately, according to ANOVA e Bonferroni tests, considering p<0.05.  

 



61 
 

  

 

 

 

 

 

Capítulo 2 



 

62 
 

Cytotoxicity and potential of taxifolin, a catechol-type flavonoid, to induct 

mineralization markers in odontoblast-like cells* 

 

Abstract 

 

The aim of this study was to evaluate the effects of treatment with taxifolin flavonoid 

on the viability of odontoblast-like cells and expression of mineralization markers. 

MDPC-23 was exposed to different concentrations of taxifolin treatment (10, 5, and 

1µM) for different periods (24h, 72h, and continuous treatment). Cell viability, alkaline 

phosphatase (ALP) activity, mineralization nodule formation, and expression of DMP-1 

and DSPP were determined. For all periods, taxifolin treatment was not cytotoxic to 

cells in the concentrations tested, according to ANOVA and Tukey tests. Taxifolin at the 

concentrations 10 to 1µM for 24h and 10 to 5µM for 72h stimulated ALP activity of 

cells. The percentage of mineralization nodule formation at 13d increased after 24h of 

treatment with taxifolin at 10 and 5µM, and 72h of treatment at 10µM. Continuous 

treatment did not stimulate ALP activity or mineralization by cells. The most elevated 

DMP-1 mRNA levels were observed on day 13 after 72h of treatment with 10µM of 

taxifolin. Groups exposed to taxifolin at 5µM for 72h presented elevated DSPP mRNA 

levels on days 6 and 13 without statistical difference between them, according to 

Kruskal-Wallis and Mann-Whitney tests. In conclusion, taxifolin treatments of 24 and 

72h were more effective than continuous treatment, demonstrating that a lower dose 

of taxifolin over short periods of time has a biostimulatory effect on MDPC-23 cells. 

Our results suggest that taxifolin could be used as a biomaterial to stimulate remaining 

primary odontoblasts and odontoblast-like cells to produce a mineralized tissue barrier 

in vital pulp procedures. 

 

Running title: Taxifolin biostimulate MDPC-23 

Keywords: Cell Culture Techniques, Flavonoids, Odontoblasts, Pulpotomy. 

 

*The manuscript is according to the guide for authors of International Endodontic 

Journal (Anexo B). 



 

63 
 

Introduction 

Immature permanent teeth with pulp exposed by caries, trauma, or restorative 

procedures require different treatments depending on the pulpal status, size of 

exposure, and microbial contamination (Bortolluzi et al. 2008). Vital pulp therapy (VPT) 

procedures have been extensively studied aiming at maintaining the vitality of 

radicular pulp and thus allowing dentin formation and completed root development 

(Lima et al. 2011, Keswani et al. 2014, Tsukiboshi et al. 2017). VPT includes pulp 

capping and pulpotomy procedures and their success depends mainly on the capacity 

of healing of the pulp tissue. 

Pulpotomy is a procedure based on amputation of the infected and inflamed 

coronal pulp and treatment of remaining vital radicular pulp tissue with medicaments 

(American Academy of Pediatric Dentistry 2014). When there is little pulp exposed, 

direct pulp capping is the treatment of choice (Keswani et al. 2014, Taha & Khazali 

2017). Different from pulpotomy, pulp-capping material is placed in contact with the 

exposed pulp tissue, without amputation, however, both procedures allow the 

formation of mineralized tissue on the exposed area and apexogenesis - the continued 

physiological development and formation of the root apex. VPT procedures have been 

indicated for young permanent teeth without clinical or radiographic signs of 

irreversible pulpitis or necrosis (American Academy of Pediatric Dentistry 2014).  

Odontoblasts are the main link between the dentin and pulp, whose primordial 

functions are synthesis and deposition of dentin matrix. These specialized pulp cells 

are the first line of defense of the dentin-pulp complex, and are also therefore the first 

cells injured by harmful effects (de Souza Costa et al. 2014). A complicating factor in 

VPT is the difficulty of predicting the degree of pulpal damage. Generally, low intensity 

injuries induce primary odontoblasts to produce reactionary dentin; however the 

repair of severe injuries involves the recruitment of undifferentiated mesenchymal 

cells from pulp due to the death of the primary odontoblasts. These stem cells 

differentiate into odontoblast-like cells and start to produce reparative dentin (de 

Souza Costa et al. 2014). 

Calcium hydroxide (CH) and mineral trioxide aggregate (MTA) are conventional 

endodontic materials frequently used in VPT (Keswani et al. 2014, Taha & Khazali 2017, 



 

64 
 

Tsukiboshi et al. 2017). In partial pulpotomy, MTA has presented a higher success rate 

than CH (Taha & Khazali 2017). However, both materials demonstrate drawbacks. 

Tunnel defects in dentin bridges and microleakage have been observed in pulpotomy 

with CH which, over time, can lead to infection/necrosis of pulp (Schuurs et al. 2000). 

Studies have pointed out difficulties with the handling and insertion of MTA due to its 

grainy consistency and the possibility of break down due to a long setting time and 

prolonged maturation phase (Kogan et al. 2006), allied to high cost and tooth 

discoloration (Belobrov & Parasho 2011). 

Bioactive molecules have been suggested as VPT materials in several studies 

(Bortolluzi et al.2008, Parolia et al. 2010, Li et al. 2011, Lima et al. 2011, Kim et al. 

2013, Keswani et al. 2014, Daltoé et al. 2016, Balata et al. 2017). According to Tziafas 

et al. (2000), an ideal pulp-capping agent should present effective adhesion to dentin, 

antimicrobial effect, and promote dentinogenesis, stimulating the formation of dentin 

bridge, indicative of a favorable prognosis after VPT (Kim et al. 2013). Alkaline 

phosphatase (ALP), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 

(DMP1) are mineralization markers and have been used to assess the biostimulating 

action of materials (Li et al. 2011, Kim et al. 2013, Daltoé et al. 2016, Wang et al. 2017) 

Flavonoids are secondary plant metabolites constituted by a polyphenolic 

structure (Panche et al. 2016). Taxifolin is a catechol-type flavonoid isolated from 

green tea (Wang et al. 2017), which has presented antimicrobial activity (Jeong et al. 

2009) as well as stimulating osteoblast differentiation in the mouse osteoblastic cell 

line (Satué et al. 2013) and in human bone marrow mesenchymal stem cells (Wang et 

al. 2017). The biostimulatory effect of taxifolin on odontoblast cells has not yet been 

studied. Therefore, the present study aimed to evaluate the effects of taxifolin 

treatment on the viability of odontoblast-like cells and expression of mineralization 

markers. The null hypotheses tested were: (1) taxifolin present cytotoxic effect on 

MDPC-23 cells, and (2) taxifolin treatments would not stimulate odontoblast-like cells 

to increase the expression of mineralization markers. 

 

 



 

65 
 

Materials and Methods  

Materials  

Culture medium, antibiotics, and reagents were purchased from Thermo Fisher 

Scientific (Pittsburgh, PA, USA). Taxifolin (#78666, Sigma-Aldrich, Saint Louis, MO, USA) 

was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO, USA) and 

the stock solution stored at -20o C.  

Cell Culture and study design  

Immortalized odontoblast-like (MDPC-23) cell line was cultured in Dulbecco’s 

modified Eagle’s medium (DMEM, GIBCO, Grand Island, NY, USA) with 10% fetal 

bovine serum (FBS; GIBCO, Grand Island, NY, USA), penicillin (100 IU/ml), streptomycin 

(100 μg/ml), and glutamine (2 mmol/L) (GIBCO, Grand Island, NY, USA). Cells were 

seeded (2.5x103 cells/well) in 96-well plates and incubated at 37oC under a 5% CO2 and 

95% air atmosphere (Thermo Plate, Fisher Scientific, Pittsburgh, PA, USA) for 24 h. 

After incubation, taxifolin (T) treatments were performed as follows: 24h treatment – 

cells were exposed once to the flavonoid treatment; 72h treatment – cells were 

exposed three times; continuous treatment – cells were exposed daily up to 13 days. 

All treatments were assayed with three taxifolin concentrations: 10, 5, and 1µM (T10, 

T5, and T1). After 24 or 72h of taxifolin treatment, the DMEM was replaced every 24h 

until completion of the experimental period (6 or 13 days). The negative control was 

DMEM without flavonoid, and the control group was DMSO 10µM (Table 1). 

Cell Viability Analysis 

 Methylthiazol tetrazolium assay was performed to determine cell viability 24h, 

72h, 6d, and 13d after beginning treatments. MTT assay is based on the succinate 

dehydrogenase enzyme produced by mitochondria, which reduces the MTT salt 

metabolically, converting it into formazan crystals. The treatments or DMEM were 

aspirated, then MTT 5mg/mL (Sigma-Aldrich, Saint Louis, MO, USA) was applied into 

wells and the plate was incubated at 37o C under a 5% CO2 and 95% air atmosphere for 

4 h. Thereafter, the MTT solution was aspirated and replaced by acidified isopropanol 

solution to dissolve the formazan crystals. Cell viability was determined by absorbance 

in a spectrophotometer (Synergy H1 Hybrid multi-Mode Microplate Reader; Biotek 

Instruments, Winooski, VT, USA) at 570 nm. This assay was performed in duplicate. 



 

66 
 

Alkaline Phosphatase Assays 

Total Protein Production 

 The quantification of total protein was performed according to Huck et al. 

(2017) with some modifications. On the sixth day, the treatments were aspirated and 

200µL of sodium lauryl sulfate 0.1% (Sodium dodecyl sulfate, Sigma-Aldrich, Saint 

Louis, MO, USA) previously dissolved in deionized water were added to each well to 

lyse the cells. After 40 min at room temperature, the solution was homogenized and 

100µL were separated for ALP activity assay. Next, 100µL of Lowry reagent (Sigma-

Aldrich, Saint Louis, MO, USA) were added to the lysed cells and incubated for 20 min 

at room temperature. Posteriorly, 50µL of Folin (Folin-Ciocalteu’s phenol reagent, 

Sigma-Aldrich, Saint Louis, MO, USA), previously diluted in deionized water at a ratio of 

1:3, were applied to each well and incubated for 30 min. After this period, all the 

samples were read in a spectrophotometer (Synergy H1 Hybrid multi-Mode Microplate 

Reader; Biotek Instruments, Winooski, VT, USA) to determine absorbance at 655 nm. A 

standard curve containing 32, 64, 96, 128, and 160 µg mL-1 of bovine albumin was 

determined to measure total protein of each sample (BSA, Sigma-Aldrich, Saint Louis, 

MO, USA). 

Alkaline Phosphatase Activity 

 Alkaline phosphatase (ALP) activity was evaluated fo