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UNESP - Universidade Estadual Paulista  

“Júlio de Mesquita Filho”

Faculdade de Odontologia de Araraquara

Lívia Jacovassi Tavares

ARARAQUARA

2017

Eficácia da terapia fotodinâmica 
antimicrobiana associada ao metronidazol em 

biofilmes de Fusobacterium nucleatum e
Porphyromonas gingivalis

 

 

 

 

 

 



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UNESP - Universidade Estadual Paulista

“Júlio de Mesquita Filho”

Faculdade de Odontologia de Araraquara

Lívia Jacovassi Tavares 

ARARAQUARA

2017

Tese apresentada ao Programa de Pós-Graduação 
em Reabilitação Oral – Área de Prótese da
Faculdade de Odontologia de Araraquara, 
Universidade Estadual Paulista para o título de 
Doutor em Reabilitação Oral.

Orientador: Prof ª Dr ª Ana Cláudia Pavarina

 

Eficácia da terapia fotodinâmica antimicrobiana 
associada ao metronidazol em biofilmes de 

Fusobacterium nucleatum e Porphyromonas gingivalis

 

 
 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                    Tavares, Lívia Jacovassi   

            Eficácia da terapia fotodinâmica antimicrobiana associada ao 

metronidazol em biofilmes de Fusobacterium nucleatum e 

Porphyromonas gingivalis / Lívia Jacovassi Tavares.-- Araraquara: 

[s.n.], 2017 

               117f. ; 30 cm. 

 

               Tese (Doutorado em Prótese) – Universidade Estadual Paulista, 

Faculdade de Odontologia 

               Orientadora: Profa. Dra. Ana Cláudia Pavarina 

                                                                                            

    1. Fotoquimioterapia  2. Metronidazol  3. Fusobacterium nucleatum 

4. Porphyromonas gingivalis I. Título 

 
     Ficha   catalográfica  elaborada   pela  Bibliotecária Ana Cristina Jorge, CRB-8/5036  

   Serviço Técnico de Biblioteca e Documentação da Faculdade de Odontologia de Araraquara / UNESP 



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Lívia Jacovassi Tavares

Eficácia da terapia fotodinâmica antimicrobiana associada ao 

metronidazol em biofilmes de Fusobacterium nucleatum e 

Porphyromonas gingivalis.

TESE PARA OBTENÇÃO DO GRAU DE DOUTOR

COMISSÃO JULGADORA

Presidente e Orientadora: Profa. Dra. Ana Cláudia Pavarina

2º Examinador: Prof. Dr. Ana Carolina Pero Vizoto

3º Examinador: Prof. Dr. Daniela Leal Zandim-Barcelos 

4º Examinador: Prof. Dr. Natalia Mayumi Inada

5º Examinador: Profa. Dra. Karin Hermana Neppelenbroek

Araraquara, 16 de março de 2017.

 

 



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DADOS CURRICULARES

Lívia Jacovassi Tavares

Nascimento: 29/05/1986 – Juiz de Fora - MG

Filiação: Antônio Celso Tavares e Cleusa Aparecida Jacovassi Tavares

2006-2010 Graduação em Odontologia. 

Faculdade de Odontologia de Araraquara - UNESP

2011-2013 Pós-graduação em Implantodontia – Nível Especialização

Fundação Araraquarense de Ensino e Pesquisa- FAEPO

2013-2017 Pós-graduação em Reabilitação Oral– Nível Doutorado

Faculdade de Odontologia de Araraquara - UNESP

 

 



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Aos meus pais, Antônio Celso e Cleusa, por serem os melhores do 

mundo. Vocês são as pessoas mais importantes da minha vida. Obrigada por acreditar 

nos meus sonhos. Esta conquista também é de vocês.

Ao Luiz Guilherme Freitas de Paula por ser meu parceiro em todos os 

momentos. Obrigada por acreditar no meu potencial. A vida é muito melhor com você 

ao meu lado.

Á Ana Cláudia Pavarina, pela disposição e paciência com que orientou 

meus passos até aqui. Muito obrigada por esta oportunidade.

À Denise Madalena Spolidório, por disponibilizar o uso do laboratório 

para a execução deste projeto e por ser sempre solícita nos momentos em que precisei.

Á Érica Dorigatti de Avila - Não tenho palavras para agradecer tudo o 

que fez por mim. O que aprendi com você vai muito além do laboratório. Espero um dia 

ser uma pequena parte desta profissional que você é. Obrigada pela amizade, pela 

paciência e por estar sempre presente nos momentos bons e ruins.

À Beatriz Helena Dias Panariello pela amizade desde a época da 

graduação. Obrigada pelo apoio durante esta trajetória.

 



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À Kahena Rodrigues Soldati pela convivência durante todo este tempo.

Obrigada pela paciência e parceria. Que a nossa amizade seja sempre assim. 

Aos amigos e colegas do Laboratório de Microbiologia que fizeram com que 

dias e noites de trabalho fossem divertidos e agradáveis. Desejo muito sucesso a todos.

Aos meus amigos de coração que Araraquara me proporcionou: Vinícius, 

Suelen, Cibele, Gabi, Ju, Fer, Jeff, Geisi.... sentirei saudades!

Às alunas de pós-graduação Carol Tonon e Patty Maquera pelo apoio na 

execução deste projeto. Torço muito por vocês, meninas. Sucesso!

Às professoras Marlise, Janaína, Daniela, Ana Carolina, Karin e 

Natália pela contribuição neste trabalho.

À Faculdade de Odontologia de Araraquara (FOAr-UNESP) por toda a 

minha formação.

Á Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), 

pela concessão da bolsa de estudos.

 

 



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Tavares LJ. Eficácia da terapia fotodinâmica antimicrobiana associada ao metronidazol 

em biofilmes de Fusobacterium nucleatum e Porphyromonas gingivalis [Tese de 

Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2017.

Resumo

O objetivo deste estudo foi avaliar a eficácia da terapia fotodinâmica antimicrobiana 

associada (aPDT) ao metronidazol (MTZ) em biofilmes periodontopatogênicos. Para tal 

finalidade, foram realizadas as seguintes etapas: (1) determinação do tempo de adesão 

(24 e 48 horas) e formação de biofilme mono e duo-espécie (3, 5 e 7 dias) de 

Fusobacterium nucleatum (NCTC 11326) e Porphyromonas gingivalis (ATCC 33277); 

(2) aplicação da aPDT mediada por PDZ associada ao MTZ em biofilmes mono-espécie 

de F. nucleatum e P. gingivalis. Foram avaliadas diferentes concentrações do PDZ (50, 

75 e 100 mg/L) e dose de luz de 50 J/cm2 (660nm). Após a aplicação da aPDT, os 

biofilmes foram incubados com diferentes concentrações do MTZ (MIC, 50x MIC e 

100x MIC) por 24 horas. Os grupos controles positivos (L-F-) não receberam 

fotossensibilizador e não foram iluminados. A viabilidade dos microrganismos após os 

tratamentos foi avaliada por meio da contagem de UFC/ml. Os resultados demonstraram 

que o período de adesão de 24 horas, seguido de 5 dias de formação de biofilme foi 

satisfatório para a obtenção de biofilmes maduros mono-espécie. Para F. nucleatum, os 

resultados demonstraram que aPDT 75 mg/mL associado com MTZ 100x MIC e aPDT 

100 mg/L associado com MTZ nas concentrações de 50x MIC e 100x MIC reduziu 

significativamente o número de UFC/mL, 2,99; 2,9 e 3,94 Log10 respectivamente. Para 

P. gingivalis, a redução mais significativa de UFC/mL foi obtida quando a associação 

de aPDT 100 mg/L e MTZ 100x MIC foi realizada, resultando em 5 Log10 de redução. 

Adicionalmente, houve redução significativa nos grupos que foram expostos apenas à 

luz ou à maior concentração de antibiótico, 1,71 e 3,07 Log10, em comparação com o 

 

  

 



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grupo sem tratamento. O efeito do tratamento da aPDT associada à MTZ foi potenciado 

quando comparado aos tratamentos isolados.

Palavras chave: Fotoquimioterapia. Metronidazol. Fusobacterium nucleatum.

Porphyromonas gingivalis. 

 



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Tavares LJ. Efficacy of antimicrobial photodynamic therapy associated with 

metronidazole on biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis

[Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2017. 

Abstract

The aim of this study was to evaluate the efficacy of metronidazole (MTZ) associated 

antimicrobial photodynamic therapy (aPDT) on periodontopathogenic biofilms. For this 

purpose, the following steps were performed: (1) determination of adhesion period (24 

and 48 hours) and single and duo species biofilm formation (3, 5 and 7 days) of 

Fusobacterium nucleatum (NCTC 11326) and Porphyromonas gingivalis (ATCC 

33277); (2) Photodithazine ® (PDZ)- mediated aPDT in association with MTZ in 

single-specie biofilms of F. nucleatum and P. gingivalis. Different concentrations of 

PDZ (50, 75 e 100 mg/L) and light dose of 50 J / cm2 (660nm) were evaluated. After 

application of aPDT, the biofilms were incubated with different concentrations of MTZ 

(MIC, 50x MIC and 100x MIC) for 24 hours. Positive control groups (L-F-) received no 

photosensitizer and were also not illuminated. The viability of the microorganisms after 

the treatments was evaluated by counting CFU/ml. The results demonstrated that the 24 

hours adhesion period followed by 5 days of biofilm formation was satisfactory for 

obtaining a mature biofilm in single-specie. For F. nucleatum, the results demonstrated 

that 75 mg/L aPDT associated with MTZ 100x and 100 mg/mL aPDT associated with 

MTZ at 50x MIC and 100x MIC concentrations significantly reduced the number of 

CFU/mL, 2.99; 2.9 and 3.94 Log10 respectively. For P. gingivalis, the greatest reduction 

of CFU/mL was obtained when the association of aPDT 100 mg/L and MTZ 100x MIC 

was performed, resulting in 5 Log10 reduction. Additionally, there was a significant 

reduction in the groups that were exposed only to the light or the highest concentration 

of antibiotic, 1.71 and 3.07 Log10, compared to the group without treatment. The 

 

   



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treatment effect of MTZ-associated aPDT was potentiated when compared to the 

isolated treatments.

Key words:  Photochemotherapy. Metronidazole. Fusobacterium nucleatum. 

Porphyromonas gingivalis.

 



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SUMÁRIO

1 INTRODUÇÃO .........................................................................12

2 PROPOSIÇÃO ..........................................................................18

3 PUBLICAÇÃO 1 .......................................................................19

4 PUBLICAÇÃO 2 .......................................................................58

5 PUBLICAÇÃO 3 ........................................................................78

6 CONSIDERAÇÕES FINAIS ....................................................107

7 CONCLUSÃO ...........................................................................109

REFERÊNCIAS ........................................................................110

ANEXO A ..................................................................................115

ANEXO B ...................................................................................117  

 



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1 INTRODUÇÃO

De acordo com o “Instituto Nacional da Saúde” (NIH), infecções causadas por 

microrganismos organizados em biofilmes, são consideradas um problema crônico de 

saúde pública, uma vez que as doenças decorrentes desta comunidade microbiana estão 

associadas a 80% de todas as infecções em seres humanos 

(http://grants.nih.gov/bolsas/guia/pa-files/PA-07-288.html). Biofilmes são estruturas 

biológicas constituídas por microrganismos envoltos por uma matriz extracelular de 

polissacarídeos , que assegura a sobrevivência dos mesmos (Lamfon et al.35, 2005; 

Ramage et al.53, 2006), agindo como uma barreira protetora a agentes físicos e químicos 

externos, o que pode limitar a penetração de agentes antimicrobianos (Evans et al.13, 

1990). Particularmente na cavidade oral, o biofilme é responsável pelo desenvolvimento 

de diversas patologias, incluindo a doenças periodontal e peri-implantar.

Peri-implantite, assim como a periodontite, é caracterizada pela perda de 

inserção e destruição do osso alveolar adjacente. Embora estas patologias sejam 

mediadas e reguladas por processos inflamatórios advindos do próprio hospedeiro, as 

bactérias são responsáveis pelo seu desenvolvimento (Rosen et al.56, 2013). De acordo 

com Socransky, Haffajee62, 2002, as bactérias envolvidas nestas patologias agrupam-se 

em complexos, cujo o complexo conhecido como laranja, representado pelas espécies 

Peptostreptococcus micros, Prevotella intermedia, Prevotella nigrescens, 

Fusobacterium periodonticum e Fusobacterium nucleatum têm a capacidade de 

interagir e favorecer a implantação do complexo vermelho, representado pelas espécies 

Porphyromonas gingivalis, Treponema denticola  e Tannerella forsythia (Socransky et 

al.61, 1998; Socransky, Haffajee62, 2002).

P. gingivalis e F. nucleatum, são bactérias anaeróbias Gram-negativas 

associadas com a periodontite crônica, periodontite agressiva localizada e doença peri-



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implantar. P. gingivalis tem sido considerada uma das principais espécies que 

desenvolvem a doença periodontal, devido aos seus numerosos fatores de virulência 

(Hajishengallis, Lamont26, 2012). F. nucleatum é considerado fundamental para a 

maturação dado biofilme dental, devido à grande capacidade de co-agregação com 

outros microrganismos, tais como P. gingivalis (Kolenbrander, Andersen32, 1989; 

Bradshaw et al.7, 1998). Estudos relatam aumento no grau de patogenicidade, 

determinado pelos fatores de virulência expressos durante a interação entre essas 

espécies (Sundqvist et al.65, 1979; Baumgartner et al.4, 1992; Feuille et al.23, 1996; 

Ebersole et al.17, 1997). Estas interações são definidas como um sinergismo patogênico 

que conduzem a uma relação de cooperação que contribui para a sobrevivência e a 

persistência de ambas em diversos nichos orais (Metzger et al.41, 2009).

Indivíduos portadores de doença periodontal apresentam grande quantidade de 

microrganismos patogênicos na cavidade oral. Em caso da perda de dentes 

comprometidos, estas bactérias permanecem sobre os dentes remanescentes e podem 

influenciar a microbiota peri-implantar (Metzger et al.41, 2009). Estudos anteriores 

identificaram alta prevalência de bactérias anaeróbias Gram-negativas ao redor de 

implantes com sinais clínicos de peri-implantite (Mombelli et al.44, 1987; Mombelli, 

Mericske‐ster43, 1990; Shibli et al.60, 2008; Tabanella et al.66, 2009). As bactérias 

encontradas nestas regiões foram similares as espécies envolvidas na periodontite, 

incluindo as bactérias do complexos vermelho (P. gingivalis, Treponema dentícola e 

Tannerella forsythia) e laranja (Fusobacterium sp. and Prevotella intermedia) 

(Socransky et al.61, 1998). Espécies bacterianas como P. gingivalis and P. intermedia

mostraram ter alta afinidade pelo titânio. Esta capacidade de se aderirem diretamente ao 

titânio poderia causar infecções na região peri-implantar (Kuula et al.34, 2004). A 

inflamação em consequência da presença do biofilme bacteriano na região subgingival, 



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é considerada um dos principais responsáveis pela perda dos implantes após o processo 

de osseointegração (Hayek et al.27, 2005; Elter et al.20, 2008).

O tratamento da peri-implantite deve ser focada na descontaminação da 

superfície do implante e na regeneração dos tecidos perdidos (Schou et al.59, 2004; 

Bautista, Huynh-Ba5, 2013). No entanto, o desenho e os tratamentos de superfície do 

titânio podem facilitar a adesão de bactérias e desenvolvimento de biofilme bacteriano 

(Schou et al.59, 2004; Bautista, Huynh-Ba5, 2013). O tratamentos convencional, tal 

como o debridamento mecânico, é insuficiente para a remoção completa do biofilme 

(Karring et al.29, 2005). Então, o uso de antibiótico pode ser indicado para potencializar 

a redução bacteriana desde exista o reconhecimento da etiologia infecciosa da doença. 

O metronidazol foi considerado o antibiótico de escolha, dada a sua capacidade de 

causar dano ao DNA bacteriano, especialmente em anaeróbios como P. gingivalis

(Müller47, 1983). Embora os antibióticos tenham seus benefícios, a utilização de grandes 

quantidades por longos períodos é indesejável na prática clínica devido aos efeitos 

adversos que causam na microbiota e ao aumento do potencial para induzir resistência.

Recentemente, a terapia fotodinâmica antimicrobiana (aPDT) foi introduzida 

como uma nova abordagem na descontaminação de superfície de implantes (Marotti et 

al.40, 2008; Lima et al.36, 2009). No processo fotodinâmico, a célula-alvo deve ser 

tratada com o fotossensibilizador (FS) de absorção máxima de luz específica, em um 

processo conhecido como fotossensibilização. Em seguida, a interação da luz com 

comprimento de onda adequado, com o FS e na presença de oxigênio, resulta em 

espécies reativas de oxigênio capazes de induzir a morte celular. Esse mecanismo 

envolve a absorção de fótons da fonte de luz pelo FS, o que leva os elétrons a um estado 

excitado. Na presença de oxigênio, o FS excitado pela luz pode reagir com moléculas 

vizinhas, por meio da transferência de elétrons ou hidrogênio (reação do tipo I) ou pela 



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transferência de energia ao oxigênio (reação do tipo II), levando à produção de espécies 

reativas (Bonnett, Martınez6, 2001) e, consequentemente, à  morte celular. Estudos têm 

demonstrado que aPDT é mais eficaz na inativação de bactérias Gram-positivas que 

Gram-negativas, possivelmente pela estrutura química da parede celular das mesmas 

(Malik et al.38, 1992). O efeito bactericida da aPDT associada ao azul de metileno (MB) 

foi avaliado em culturas planctônicas de Aggregatibacter actinomycetemcomitans, F. 

nucleatum, P. gingivalis, Prevotela Intermedia e Streptococcus. sanguis. Os dados 

mostraram que as espécies Gram-negativas foram mais resistentes e S. sanguis foi a 

espécie mais suscetível (Chan, Lai9, 2003), sugerindo uma relação direta da composição 

química da parede celular com o fotossensibilizador utilizado. Estudos anteriores 

relataram que as suspensões de P. gingivalis e F. nucleatum são suscetíveis a aPDT 

(Chan, Lai9, 2003; Habiboallah et al.25, 2014). Por outro lado, não foi observada a 

inativação completa desses microrganismos, quando organizados em biofilmes (Street et 

al.64, 2010).  Estudos anteriores sugeriram que a aplicação prévia de aPDT associada a 

um antibiótico poderia potencializar a redução bacteriana (Di Poto et al.14, 2009; Barra 

et al.3, 2015; Ronqui et al.55, 2016). 

Atualmente, uma nova classe de FS vem sendo empregada em aPDT, os 

fotossensibilizadores de segunda geração. Dentre estes compostos estão as clorinas, 

porfirinas hidrofílicas reduzidas que apresentam forte banda de absorção na região 

vermelha do espectro fotomagnético. O Photodithazine ® (PDZ) é uma clorina e6 que 

mostrou ter efeito significativo em células tumorais (Corrêa11, 2006). Em estudos 

preliminares (Dovigo et al.16, 2013; Quishida et al.52, 2015; Carmello et al.8, 2016) foi 

avaliado a eficácia da PDZ na fotoinativação de cepas de Candida albicans, Candida 

glabrata e Candida tropicalis isoladas de pacientes com estomatite protética. As 

suspensões foram tratadas com 25, 50 e 75mg/L de PDZ e expostas a luz LED a 37,5; 



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25,5 e 18,0J/cm2 e os biofilmes foram tratados com maiores concentrações de PDZ (100 

e 125mg/L). Os resultados demonstraram que a aPDT promoveu redução significativa 

na viabilidade da C. tropicalis e da C. glabrata enquanto cinco cepas de C. albicans

foram completamente inativadas após a aPDT. A maior redução do biofilme foi 

observada com a utilização de 125 mg/L de PDZ. Para C. albicans, houve uma redução 

de 62,1% enquanto para a C. tropicalis e C. glabrata foi observada uma redução de 76 e 

76,9%, respectivamente (Dovigo et al.16, 2013). Quando um biofilme misto formado por 

C albicans, C glabrata, e S mutans foi submetido a aPDT com o PDZ foi observada 

redução significativa na viabilidade das colônias das três espécies avaliadas, e redução 

significativa na atividade metabólica dos biofilmes submetidos a aPDT (Quishida et 

al.52, 2015). No tratamento da candidíase oral em um modelo murino, aPDT mediada 

por PDZ foi tão eficaz quanto a nistatina na inativação de C. albicans e apresentou 

regressão completa das lesões orais após seis aplicações (Carmello et al.8, 2016)

Apesar dos resultados promissores, seria interessante o desenvolvimento de 

estratégias que aumentassem a suscetibilidade dos microrganismos aos métodos 

antibacterianos já conhecidos (Di Poto et al.14, 2009; Ronqui et al.55, 2016). A 

avaliação do pré-tratamento do biofilme maduro de Streptococcus aureus com aPDT 

seguida pela aplicação de vancomicina reduziu significantemente a concentração 

bacteriana, sugerindo que aPDT poderia provocar a desintegração da matriz extracelular 

e, consequentemente, aumentaria a suscetibilidade ao antibiótico (Di Poto et al.14, 

2009). Em biofilmes de S. aureus e Escherichia coli resultados significativos também 

foram observados na combinação entre aPDT e o ciprofloxaxino, com redução de 5.4 

Log10 para S. aureus e aproximadamente 7 Log10 para E. coli (Ronqui et al.55, 2016).

Até o presente momento, os autores não localizaram informações de aPDT 

mediada pela PDZ sobre bactérias periodontopatogênicas, tão pouco sobre a influência 



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no tratamento com antibióticos. Dessa forma, o objetivo do presente estudo será avaliar 

se a aplicação da aPDT poderia atuar no biofilme bacteriano formado pela P. gingivalis

e F. nucleatum e favorecer a suscetibilidade destas bactérias ao antibiótico MTZ. 



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2 PROPOSIÇÃO

O presente trabalho tem como objetivo geral avaliar in vitro a eficácia da aPDT 

associada ao MTZ em biofilmes mono-espécie. Para isso, este projeto foi subdividido 

em 3 publicações, com os seguintes objetivos:

Objetivos Específicos

Publicação 1- Revisão de literatura para melhor entendimento dos 

mecanismos que envolvem aPDT na peri-implantite

Publicação 2 - Avaliar a adesão e o período satisfatório para a formação de 

biofilme maduro mono e duo-espécie de P. gingivalis e F. nucleatum

Publicação 3– Avaliar a eficácia da aPDT associada ao MTZ em biofilme

mono-espécie de P. gingivalis e F. nucleatum



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3 PUBLICAÇÃO 1*

* ANEXO B



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The impact of antimicrobial photodynamic therapy on peri-implant disease: What 

mechanisms are involved in this novel treatment?

Lívia Jacovassi Tavares, DDS, PhD Studenta, Ana Claudia Pavarina, DDS, MSc, PhD, 

Adjunct Professora, Carlos Eduardo Vergani, DDS, MSc, PhD, Full Professora, Erica 

Dorigatti de Avila, DDS, PhD, Postdoctoral Research Fellowa

aDepartment of Dental Materials and Prosthodontics, School of Dentistry at Araraquara, 

Univ Estadual Paulista - UNESP, Rua Humaitá, 1680, 14801-903 Araraquara, SP, 

Brazil. 



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Abstract

According to the American Academy of Implant Dentistry, 3 million Americans have 

dental implants, and this number is growing by 500,000 each year. Proportionally, the 

number of biological complications is also increasing. Among them, peri-implant 

disease is considered the most common cause of implant loss after osseointegration. In 

this context, microorganisms residing on the surfaces of implants and their prosthetic 

components are considered to be the primary etiologic factor for peri-implantitis. Some 

research groups have proposed combining surgical and non-surgical therapies with 

systemic antibiotics. The major problem associated with the use of antibiotics to treat 

peri-implantitis is that microorganisms replicate very quickly. Moreover, inappropriate 

prescription of antibiotics is not only associated with potential resistance but also and 

most importantly with the development of superinfections that are difficult to eradicate. 

Although antimicrobial photodynamic therapy (aPDT) was discovered several years 

ago, aPDT has only recently emerged as a possible alternative therapy against different 

oral pathogens causing peri-implantitis. The mechanism of action of aPDT is based on a 

combination of a photosensitizer drug and light of a specific wavelength in the presence 

of oxygen. The reaction between light and oxygen produces toxic forms of oxygen 

species that can kill microbial cells. This mechanism is crucial to the efficacy of aPDT. 

To help us understand conflicting data, it is necessary to know all the particularities of 

the etiology of peri-implantitis and the aPDT compounds. We believe that this review 

will draw attention to new insights regarding the impact of aPDT on peri-implant 

disease. 

Keywords: Photodynamic therapy; peri-implantitis; photosensitizer; microorganisms



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1. Introduction

According to the National Institutes of Health (NIH), infections caused by 

microorganismal biofilms are considered to be a public health problem, as biofilm-

associated diseases might be responsible for 80% of all infections in humans 

(http://grants.nih.gov/grants/guide/pa-files/PA-07-288.html). A biofilm is a complex 

interaction between a surface and microbial cells that are protected by an extracellular 

matrix of polymeric substances [1, 2], which confers resistance to antibiotic treatment 

[3]. In addition, these microbial networks are responsible for the most common oral 

diseases: dental caries, periodontitis, and peri-implantitis [4-6]. 

With the growing number of dental implant procedures, the prospective number 

of sites with implant-associated diseases has also increased [7]. Specifically, given the 

common incidence of peri-implantitis [8-10] and considering that the etiopathogenesis 

of peri-implantitis is not well delineated, the most effective treatment for peri-

implantitis has not been conclusively established. Similarly to periodontal disease, peri-

implantitis is a destructive inflammatory process that leads to pocket formation and loss 

of supporting bone; in peri-implantitis in particular, the disease site surrounds an 

osseointegrated implant. Peri-implantitis has been estimated to occur in 10.7–47.2% of 

dental implant patients within 10 years of post-treatment observation, and these data are 

considered alarming [11]. According to NHANES 2009-2010, the prevalence of 

periodontitis in the United States among adults aged 30 years and older was 47.2%. This 

percentage is even higher at 70.1% for adults older than 65 years [12]. The cost 

associated with the treatment and prevention of this disease reached 14.3 billion dollars 

in 1999 [13]. In an attempt to reduce these numbers, antibiotic therapy is often 

recommended for patients receiving periodontitis and peri-implantitis treatment 

procedures [14]. According to some authors, the advantage of antibiotic use is the short 



23 

 

course of administration, which may contribute to patient compliance [15]. Despite the 

clinical relevance and the effective use of systemic antibiotics to treat numerous 

infectious diseases, the currently available scientific information on the use of these 

agents in the treatment of periodontal and peri-implant diseases is insufficient to support 

any official recommendations on the use of these medicines [16]. It is important to 

emphasize that antibiotics are antimicrobial substances that can lead to side effects of 

varying intensities, and their unselective use can increase selection for bacteria that are

resistant to antibiotics. In 2014, a new report by the World Health Organization (WHO) 

revealed that antimicrobial resistance is currently a serious threat and is no longer 

simply a future problem. This phenomenon is occurring across many different regions 

of the world and can affect anyone, independent of age or country. 

Although dental implants are a successful treatment modality [17], peri-

implantitis is the most common cause of late failure and can occur years after 

osseointegration [18]. To address this issue, increased attention has been paid to non-

surgical alternatives for treatment of localized infections [19]. Recently, antimicrobial

photodynamic therapy (aPDT) has been considered as an adjunct treatment approach to 

the bacterial decontamination of teeth and implants affected by periodontal and peri-

implant disease. aPDT involves exposure to a combination of a photosensitizer [20] and 

an appropriate wavelength of laser light, resulting in the destruction of different oral 

pathogens in planktonic and biofilm forms [21, 22]. In vitro and in vivo studies 

confirmed that a major periodontopathogenic bacterium, Porphyromonas gingivalis, is 

susceptible to aPDT [22-24]. Despite promising results, several factors should be 

considered in order to obtain good treatment outcomes in patients, such as the type of 

PS, total exposure time, wavelength, intensity of laser irradiation, and the combination 



24 

 

of another treatment with aPDT. Thus, we address the impact of aPDT on peri-implant 

disease and discuss all of the factors related to this novel therapy.

1.1 Bacterial adherence to implant surfaces – a key factor in peri-implantitis 

Peri-implantitis is a complex and interesting disease in which alterations in bone and 

connective tissue homeostasis involve intricate interactions between bacteria and the 

inflammatory immune response of the host [25]. Bacteria are considered to play a 

principal role in initiating the host inflammatory process [14]. Increased understanding 

of the various factors contributing to peri-implantitis has revealed that the clinical 

phenotype is not simply the translation of microbial challenge into a standard host 

response. Strong evidence has suggested that smoking, diabetes, and susceptibility to 

periodontitis are powerful determinants of peri-implantitis development as well as 

disease severity [26, 27]. To create a strategy for treating peri-implantitis, it is crucial to 

understand all the factors involved in the development of the disease and its 

mechanisms of action. 

Regarding the bacteria that are responsible for initiating host inflammatory 

processes and bone loss, two points should be considered: the bacterial species involved 

and the host immune response to the bacteria. Molecular analysis of oral 

microorganisms has identified approximately 700 species of bacteria inside the mouth 

of any individual [28-30]. Due to high diversity, it is therefore necessary that oral 

bacteria adhere to solid surfaces for the development of oral disease. This specificity 

occurs via mechanisms of adherence, i.e., several cell surface structures (especially 

those proteinaceous and carbohydrate molecules) of different bacterial species can 

identify receptors in the salivary pellicle, and these structures coat enamel and/or dental 

implant materials and their prosthetic components. Importantly, the chemical 



25 

 

composition of different materials can have a significant impact on biofilm formation 

[31-33], initiating gene expression and determining the bacterial profile of the species 

adhering to the biofilm. Recently, an in vitro study evaluated the effect of several 

implant materials in comparison to enamel on bacterial adhesion. A preference of 

Streptococcus mutans and P. gingivalis for the chemical composition of enamel surfaces 

was suggested [34], as it was not possible to detect bacteria on titanium or zirconia 

materials. In general, streptococci and actinomyces initially dominate the bacterial 

composition of the tooth surface and can recognize receptors in the salivary pellicle [35-

37]. In the case of dental implant surfaces, while some findings have reported 

similarities in the microbiota composition between the surfaces of both healthy and 

infected implants and teeth [38-41], other findings have indicated that peri-implantitis 

may be more complex and diverse than periodontitis [40, 42]. Overall, black-pigmented 

Prevotella species, Aggregatibacter actinomycetemcomitans, and P. gingivalis are 

found in higher quantities in peri-implantitis lesions than in healthy control tissue and at 

comparable levels in periodontitis samples; however, enterobacteria and staphylococci 

have been identified around implants [43]. Another important factor that regulates 

bacterial colonization profiles and should be considered before planning treatment is the 

type of edentulism: either full or partial. Some findings demonstrated that 1 month after 

total dental extraction in individuals with periodontal disease, A. 

actinomycetemcomitans and P. gingivalis were undetectable in the oral cavity [44]. 

Similarly, Streptococcus sanguinis, S. mutans, and lactobacilli were visibly reduced in 

edentulous adults with or without standard removable dentures compared with dentate 

patients [45]. Therefore, the environment can be considered as the main factor that 

influences the microbial colonization profile. 



26 

 

1.2 Biofilm complexity and bacterial invasion

The persistence of dental plaque changes the dental ecosystem, and new bacterial 

composition appears to affect the environment, thus resulting in clinical disease. The 

cell-to-cell interactions involved in coaggregation are responsible for dynamic biofilm 

construction, which is categorized as either cooperative or competitive [46]. It has been 

known that bacteria of the genus Fusobacterium exhibit partnerships with initial, early, 

and late colonizers and thus serve as a bridge in the succession of genera in naturally 

developing dental plaque [47, 48]. The ability of F. nucleatum to adhere to biofilm at 

different stages can be explained by its two distinct types of adherence, classified based 

on their inhibition by either D-galactose or L-arginine. While the adherence of F. 

nucleatum to Gram-negative bacteria is galactose sensitive, its adherence to Gram-

positive bacteria is mediated by arginine-inhibitable adhesins [49]. Below the gum line, 

the environment changes and becomes anaerobic. In this context, subgingival anaerobic 

bacteria dominate the environment, which has a higher overall species diversity than 

that of supragingival biofilms [28]. Among the anaerobic bacteria considered to be 

periodontopathogens, P. gingivalis is known to misdirect the host defense and increase 

tissue-destructive inflammation [50], thus influencing disease initiation and progression. 

Scientific evidence has shown that P. gingivalis is commonly found in patients with 

periodontitis [51, 52] and is associated with peri-implantitis [53, 54]. Additionally, 

interaction with early microbial colonizers, such as Streptococcus species, can also 

promote the migration of P. gingivalis in subgingival biofilms [55].

In addition to their interactions with other bacteria, some pathogenic species 

adhere to oral epithelial cells and induce interleukin production [56]. A small number of 

microorganisms are able to bind to and invade different types of host cells, thereby 

eliciting proinflammatory responses and periodontal destruction [56, 57]. P. gingivalis, 



27 

 

for example, is capable of producing a number of virulence factors such as fimbriae, 

lipopolysaccharide (LPS), capsules, and proteases, which can bind to and activate 

human epithelial cells, thus resulting in cytokine release [58, 59]. Another bacterial 

species involved in the stimulation of the innate immune response is F. nucleatum.

Recently, a novel type of adhesion was identified as being involved in bacterial 

attachment to host epithelial cells; this type of adhesion is unique to the oral microbiota 

and may play an important role in Fusobacterium colonization in the host [60]. It has 

been postulated that these bacteria not only induce peptide production against 

periodontopathogens but also influence the immune response through the induction of 

cytokines and chemokines [61]. The invasion of epithelial cells [62] was also 

demonstrated by the Gram-negative anaerobic bacteria Treponema denticola, 

characterized as the “red complex” by Socransky et al. [63]. T. denticola possesses 

several virulence factors responsible for adherence, tissue penetration, cytotoxicity, and 

immunomodulation and is involved in inhibiting the complement system [64].

Gingival epithelial cells are the first human cells with which bacteria of the 

biofilm interact. Once bacterial proteins binds to their receptors, gingival epithelial cells 

produce a wide array of responses, thus increasing the abundance of proinflammatory 

cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukins (ILs), which 

indirectly attempt to eliminate the infection [65]. Different cytokine response profiles 

are induced by distinct bacterial species; pathogenic species, for example, can provoke 

an inflammatory response, while those considered to be commensal produce an 

insignificant inflammatory response. In an in vitro study, primary human gingival 

epithelial cells (HGECs) were incubated with several species of dental plaque bacteria 

to determine the levels of specific interleukins. The results showed that the cells 

stimulated with live P. gingivalis produced high levels of IL-1b but that the same cells 



28 

 

stimulated with live A. actinomycetemcomitans produced high levels of IL-8. In contrast 

to pathogenic bacteria, the commensal Streptococcus gordonii induced low levels of 

pro-inflammatory cytokines [65]. 

Peri-implant tissue surrounding the implants provides a barrier resisting 

frictional forces and protecting the soft tissue against microorganisms. Thus, the 

penetration and injury of the epithelial layer are important steps in the pathogenesis of 

peri-implantitis.

1.3 Stages of peri-implant disease

When individuals lose their teeth due to periodontal disease, the pathogenic 

microorganism remains inside the mouth. Within almost 30 minutes of transmucosal 

implant placement, bacteria initiate colonization on the implant surfaces [66]. The 

progression of the adherent biofilm on the dental implant seems to be the driving force 

in the commencement and development of peri-implant disease. When signs of 

inflammation without loss of connective tissue are identified following initial bone 

remodeling around the implant during healing, it is believed that peri-implant mucositis 

has been established. For this stage of the disease, mechanical therapy (with or without 

adjunctive use of antiseptic rinses) is commonly the initial treatment of choice [67, 68]. 

However, during disease progression, inflammatory mediators produced by the soft 

tissue activate osteoclastogenesis and the subsequent loss of the marginal, supporting 

bone around the functioning implant [25]. At this stage, peri-implantitis becomes 

established. The presence of increased levels of pathogens in peri-implantitis is a 

serious treatment issue, as discussed below. Treatment difficulties at this point are 

directly related to the complexity of the biofilm, the probing depth, and the 

inflammatory immune response of the host. 



29 

 

Overall, evidence from in vivo studies points to a questionable theory of 

microbial similarity between teeth and implants [39, 69, 70]. This information has 

guided the treatment of peri-implantitis to be similar to that of periodontitis. Efforts to 

control peri-implantitis have been made with different methods of open or closed 

debridement, systemic or local delivery of antibiotics, aPDT, and combinations of these 

therapies.

1.4 Treatment options

Although peri-implantitis is modulated and mediated by the host, supportive peri-

implant therapy is a critical procedure for preventing the incidence and/or for treating 

the disease [71-73]. One of the main challenges in the treatment of peri-implantitis is 

the disinfection process of dental implant surfaces to reduce inflammation and stimulate 

re-osseointegration. Although periodontitis and peri-implantitis share similar etiological 

factors, in peri-implantitis, the irregular structure of the dental implant can promote 

plaque accumulation when exposed to the oral cavity [74] and can interfere with the 

quantity and quality of the biofilm that adheres to the implants. Conventional treatment 

for periodontal disease involves debridement of the root surfaces with mechanical 

instruments. Considering that decontamination of the implant surface is much more 

problematic than decontamination of natural root surfaces, mechanical therapy alone 

could be insufficient for biofilm elimination in peri-implantitis [75]. Furthermore, 

titanium curettes could severely damage the implant surface, thus increasing its 

roughness and bacterial adherence. Consequently, plastic curettes were introduced in an 

attempt to reduce the damage caused by metal instruments, but plastic curettes cannot 

reach the macro- or micro-pores of these dental implant substrates. The ineffectiveness 

of these instruments results in large residual plaque areas after treatment [76]. Another 



30 

 

treatment option is the use of an air-abrasive device. This procedure is effective for the 

removal of biofilm from implant surfaces [77], but one disadvantage is the risk of 

emphysema after treatment [78]. Thus, to further facilitate bacterial reduction, 

additional approaches have been used, such as the use of systemically or locally 

administered antibiotics that act directly on active subgingival species in the dental 

plaque or in adjacent epithelial tissues lining the peri-implant pocket. It is believed that 

local or systemic antibiotics eliminate periodontopathogenic bacteria to a greater extent 

than conventional therapy. This phenomenon is explained by several findings that the 

short-term clinical benefits achieved with conventional methods (scaling and root 

planing) are frequently not sustained in the long term, especially in more progressive 

cases [79] and in cases associated with risk factors such as smoking [80] and diabetes 

[81]. However, it is imperative to highlight that antibiotics are biologically active 

substances that can lead to side effects of differing severity. Additionally, the WHO has 

questioned the current practice of indiscriminate antibiotic use, which is progressively 

leading to antibiotic resistance, the persistence of infections, and treatment failure 

(http://www.who.int/mediacentre/facsheets/fs194/en/). In an attempt to diminish the 

inflammatory process and reduce the potential for pathogen resistance, alternative 

treatment methods have been introduced. One of the most promising methods for 

treating peri-implant mucositis and peri-implantitis is aPDT. 

1.5 aPDT - definition, application, and mechanism of action

The use of light with a sensitizing agent was first described in the medical literature 

more than 100 years ago [82]. Interestingly, the discovery occurred incidentally after a 

medical student observed that paramecia, unicellular protozoa, were killed only when a 

dye was exposed to strong daylight. Since then, various studies have investigated the 



31 

 

efficacy and efficiency of this approach, mainly as a cancer therapy. The applicability of 

this therapy is a consequence of its mechanism of action. aPDT involves the activation 

of a drug using light, and at the trigger time, exposure of the drug to excitation light 

leads to cell death via apoptosis or necrosis. The mechanism of action, although not 

completely understood, involves the production of reactive oxygen species (ROS), 

which can damage the target cell. Regarding its effects on microorganisms, the literature 

has shown that aPDT is more effective in inactivating Gram-positive bacteria than 

Gram-negative bacteria due to the chemical structure of the cell walls [83]. The driving 

force of aPDT is photosensitization. For this therapy to work, the PS molecule must 

penetrate the cell walls of the microorganisms until it reaches its final destination and 

binds to the plasma membrane of the microbial cell. However, besides a pronounced 

antimicrobial efficacy, PS should not be toxic toward mammalian cells. Since PS play a 

pivotal role in aPDT therapy this substance should be effective in the selectivity for 

microbial cells over host mammalian cells [84]. In this context, the cytotoxicity to 

normal tissue are minimized due to high selective affinity of the PS to the diseased 

tissue and microbial cells, and by delivering the light in a spatially confined and focused 

manner. Increasing the selective accumulation of the PS into target cells can be 

explained by the strong interaction between PS with low-density lipoprotein (LDL) 

overexpressed on cancer cells [85]. In fact, which factors in the chemical structures of 

the PS are involved for maximizing the selectivity for the tumor over normal tissue and 

microbial cells are still not completely understood [86]. However, studies have been 

performed to investigate if a desired therapeutic dosage might kill microbes effectively 

without damaging the adjacent cells. The data found in the literature have demonstrated 

low toxicity against mammalian cells when PS is applied to a specific area [87, 88]. 



32 

 

The membrane affinity of a PS is directed by its amphiphilic properties, and this 

is dependent on the chemical organization of hydrophobic and hydrophilic regions in its 

structure [89, 90]. However, the type of membrane barriers of the bacterial cell, for 

example, can limit the simple dissemination of a PS into the bacterial cytosol. The 

composition of Gram-positive bacteria differs in several key ways from their Gram-

negative counterparts. Overall, the outer membrane surrounding Gram-positive bacteria 

becomes the cell wall of this bacterial class, and their outer membrane is more 

permeable to hydrophobic small molecules. This structure plays a key role in protecting 

Gram-negative bacteria from the environment by eliminating toxic molecules and 

offering an additional stabilizing layer around the cell. However, a thick layer of 

peptidoglycans around Gram-positive microorganisms could limit the diffusion of the 

PS into the bacteria. Threading through these layers of peptidoglycans are teichoic 

acids, which are long anionic polymers whose negative charge can attract cationic 

molecules [91]. The outer membrane is composed of glycolipids, principally LPS, a 

well-known molecule responsible for much of the toxicity of Gram-negative organisms. 

LPS induces the production of different mediators associated with septicemia [92]. The 

human innate immune system is sensitized to LPS, which is an unquestionable indicator 

of infection. Therefore, aPDT-mediated killing of Gram-positive bacteria is definitely 

much easier to accomplish than that of Gram-negative bacteria. Thus, it is more 

challenging to obtain a highly potent PS for mediating aPDT against Gram-negative 

bacteria, as their cell wall prevents the uptake of anionic and neutral PSs. This theory is 

corroborated by previous results presented in the scientific literature. 

In this review, we discuss aPDT as an alternative method for eradicating bacteria 

from peri-implant pockets; however, we should be cautious considering that 

antimicrobial/antibacterial treatment results have revealed a CFU reduction rate of 



33 

 

greater than 3 log10, as stated by the American Society of Microbiology (ASM) in 2010 

[93]. The bactericidal effect of aPDT using methylene blue (MB) was studied in 

planktonic cultures of A. actinomycetemcomitans, F. nucleatum, P. gingivalis, P. 

intermedia, and S. sanguinis. Consistent with the theory described above, the data 

showed that Gram-negative species were more resistant to aPDT-mediated killing than 

Gram-positive species. S. sanguinis was the most susceptible strain [94]. MB belongs to 

the phenothiazinium family of positively charged sanguinis dyes, and the cationic 

molecules present in this PS may interact with anionic regions from S. sanguinis cell 

walls. The results of this study also demonstrated that the bactericidal effect of aPDT is 

wavelength-dependent, dose-dependent, and bacterial species-dependent. Another 

example for phenothiazinium dye, which has been tested for inactivation of planktonic 

cells and biofilm, is toluidine blue (TB). The interesting and promisors previous data 

obtained from in vitro studies [95, 96] instigated the continued use of this PS in current 

reports. Recently, the effectiveness of the TB on multispecies biofilm grown on bovine 

enamel slabs was evaluated within the oral cavity. For initially adherent oral anaerobic 

microorganisms, the results showed significant CFU reduction from a native in situ

biofilm. The effect was sustained during the subsequent biofilm formation and the 

number of cultivable microorganisms within mature oral biofilms declined by 2.21 

log10. However, more important than the capacity of reducing the number of bacteria is 

the regular oral microflora disturbed by this therapy [97]. Remarkably, the data revealed 

that F. nucleaum, for example, could not be detected in the biofilm after the application 

of aPDT using TB [97]. Since this bacterium plays an important role in the 

establishment of anaerobes species in the periodontal pocket, F. nucleatum reduction 

could affect the survival of periodontopathogens [98]. Similar to MB, TB was initially 

used by the dye industry due to its affinity for nucleic acids, and therefore binds to 



34 

 

nuclear material of tissues with a high DNA and RNA content [99]. Those properties 

also conferred it negative aspects of the clinical use related to its capacity to stain hard 

tissues of the tooth. However, it has been reported that residual staining of teeth and 

gingival tissue with TB is not visible after the aPDT application, and therefore, caused 

no esthetic problems for the patients [100].

Other hydrophobic compounds often used in aPDT include porphyrins, chlorins, 

and phthalocyanines, which are structurally comparable heterocyclic macrocycles. 

Porphyrins, for example, are endogenous substances and Gram-positive cell wall 

constituents; moreover, they act as PSs and induce a lethal auto-photosensitization 

process that kills bacteria via an oxidative burst similar to the photodynamic 

inactivation of bacteria. Furthermore, the membrane affinity for PS molecules facilitates 

the penetration of porphyrins [101]. However, varying results were observed even when 

different Gram-positive bacterial species were examined. In an interesting report, 

researchers tested the effect of the porphyrin 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-

21H,23H-porphine tetra-p-tosylate salt (TMPyP) against Enterococcus faecalis

monospecies biofilm and verified the inefficacy of the treatment. One explanation for 

this outcome is the large molecular structure of TMPyP, which may delay the 

penetration of this PS through the extracellular polymeric substances [99]. Additionally, 

electrostatic interactions between the positively charged TMPyP and negatively charged 

EPS could delay PS diffusion [102]. In the same report, the authors suggested that the 

emission of the LED light-curing unit was not ideal for excitation of TMPyP, which is 

another important point to consider. The wavelength of the light source excites the PS to 

produce free radicals and/or ROS. If the PS compound is unable to absorb laser energy, 

the therapy will not be efficient [94]. 



35 

 

Limited data were obtained when aPDT was applied with a cationic chlorin-e6 

derivative, commercially marketed as Photodithazine®, on fungal biofilms from 

Candida albicans and Candida glabrata. The results showed a CFU reduction of 

approximately 1 log10 [21, 103]. Similar to Gram-positive bacteria, fungi have a thick 

cell wall and no outer membrane, but fungi have a unique chemical composition. The C. 

albicans cell wall, for example, is primarily composed of glucan and chitin, very 

hydrophobic carbohydrates responsible for the mechanical strength of the cell wall, as 

well as mannoproteins [104]. Quite divergent data were acquired in recent and 

innovator investigations. A succession of in situ studies has shown high antimicrobial

effects of aPDT with chlorine e6 (Ce6) against initial and mature in oral biofilm, 

reducing significantly the numbers of viable anaerobic microorganisms. The differences 

in the susceptibility for microorganisms presented in these studies clearly underline the 

wavelength-dependence, since the authors combined visible-light [96] and water-

filtered infrared A (wIRA) with this cationic PS [105, 106]. Noticeably, we must 

consider the particularities and limitations of both studies that could have interfered 

with the final results, such as the time of biofilm formation, type of PS used, PS 

concentration, wavelength, intensity of laser irradiation, and light source. The chemical 

properties of the fungal cell wall reflect the limited interactions among chlorin, 

carbohydrates, and proteins. In another study, the investigators demonstrated that both 

XF-73 and TMPyP, porphyrin molecules, exposed to blue light effectively 

photodynamically killed C. albicans in suspension [107]. The positive interaction 

between porphyrin molecules and chitosan, a chitin derivative, can be explained by the 

fact that chitosan promotes greater adsorption of porphyrins on phospholipid 

monolayers and allows the porphyrin to stay in its monomeric form [108]. Closely 

associated with PS interaction, the fact of the tests have been performed against 



36 

 

planktonic microorganisms can also have contributed to the experimental success. The 

conflicting results found in the scientific literature prompt us to consider and further 

explore the possible reasons underlying this discrepancy. In the case of PS, for example, 

it was previously shown that TMPyP attaches to and has a high affinity for C. albicans 

cells. XF-73 compounds show a similar property. Although porphyrins and chlorin 

share similar chemical properties, the fact that Photodithazine® does not have an 

antimicrobial effect inspires new investigation into the particularities of each 

microorganismal species. 

Another important and intriguing class of PSs recently introduced in aPDT is 

curcumin [109]. This compound has been isolated from the plant Curcuma longa, and 

because this product is natural and confers antimicrobial properties, accumulating 

studies have investigated its therapeutic efficacy in various inflammatory diseases [110, 

111]. Among these studies, the antifungal effect of curcumin-mediated aPDT against 

oral candida infections caused by Candida spp has been evaluated. Previous findings

have indicated this PS as an effective photosensitizing agent for the inactivation of C. 

albicans in both its planktonic and biofilm forms [87]. In addition to its antifungal 

properties, curcumin has been noted for its beneficial treatment outcomes for dentine 

carious lesions. Impressive results were obtained when mature, multispecies biofilms of 

S. mutans and Lactobacillus acidophilus were exposed to a curcumin solution for 5 

minutes and were irradiated for 5 minutes with blue light, leading to a CFU reduction of 

more than 3 log10. However, a different outcome was observed when dentin carious 

lesions were exposed to this compound under the same concentration, time, and light 

conditions [112]. The depth of dentin could have reduced curcumin penetration due to 

their unique physicochemical properties. Dentin is a highly hydrophilic connective 

tissue, whereas curcumin is a hydrophobically derived polyphenol, and this difference 



37 

 

can explain the discrepant results observed in distinct experimental designs. As 

curcumin is a natural product, its mechanism of action is also attractive with regard to 

human healthcare. It has been demonstrated that curcumin is a potent inhibitor of the 

generation of ROS, which are mediators of inflammation. The photodynamic effect of 

curcumin involves hydrogen peroxide production without the generation of singlet 

oxygen [113], which in turn potently enhances heme oxygenase-1 (HO-1) expression. 

However, it was shown that the activity of HO-1 in angiogenesis upregulates the 

synthesis of vascular endothelial growth factor (VEGF) under both physiological and 

pathological conditions [114]. Thus, the benefits of this compound depend on both its 

dose and the chemical environment.

1.6 A new insight into light source for aPDT success

Antimicrobial PDT requires a set of procedures to work. The evidences presented above 

display the impact of the light source to improve the interaction between PS and cell 

compositions from different microorganisms. Most PS is activated by specific 

wavelength. In spite of the PS excitation is required, the degree of penetration can 

compromise the tissue health and cause injuries [115].

Depth of light penetration in human tissue is wavelength-dependent. Up to date, 

a variety of light sources have been employed for aPDT protocols, such as: nonlaser 

light generators (halogen or light-emitting diode [LED] lamps). The main issue of 

halogens lamps is the gas contained inside the tube that makes the light much brighter 

and can induce tissue overheating [116]. On the other hand, the intensity of light emitted 

by LEDs on the skin is lower, since its cells maintain a good interaction with the light. 

However, LEDs produce relatively limited bands of green, yellow, orange or red light 

and this restricted emission wavelength spectrum [117]has not provided antimicrobial 



38 

 

effects, so far. Thus, alternative strategies have been introduced in an attempt to 

combine the PS with the appropriate light source and improve the effect of aPDT to 

treat oral diseases. 

Recent investigations, combining visible light with water-filtered infrared-A 

(VIS+ wIRA), have described a significant reduction of the total oral bacterial for the 

chronic wound treatments [118]. This potential effect has directed the use of the VIS + 

wIRA device to improve the efficacy of aPDT. The combination of both light and 

radiation is based on a natural process in which mankind has developed, i.e., the heat 

radiation of the sun, in moderate climatic zones, is filtered by water vapor in the 

atmosphere of the earth. Similar to sun heat radiation, the water-filtering allows to high 

penetration properties with a low thermal load to the surface of the skin, (within 780-

1400 nm) [119]. The mechanism of action involves the cells and cellular structures 

stimulation by direct radiation effect. Some reports have shown that wavelengths within 

wIRA influence interactions between cells and extra- cellular matrices, increasing the 

amount of ATP available [120, 121], participating in wound repair processes and 

modulating the immune system and/or to induce necrosis/apoptosis of damaged cells 

and of bacteria [122]. Thus, it seems probable that VIS+wIRA could increase the 

desired PDT outcomes, in a number of dental procedures.

Since the concept of the aPDT involves the production of ROS, responsible to 

damage the target cell, the rising production of ROS and singlet oxygen would improve 

its antimicrobial effects. As discussed in the previous topic, effectiveness of aPDT 

approach using VIS+wIRA in combination with PS has been tested on in situ

experiments [97, 105, 106]. Besides a successful outcome demonstrated by CFU 

reduction, viability assay enabled understanding the relevant contribution of the light 

source in the eradication of biofilm bacteria. Interestingly, when the authors exposed the 



39 

 

PS onto the oral biofilms in the absence of VIS+wIRA, the cells preserved their 

viability, indicating a VIS+wIRA-dependence to destroy a vast amount of 

microorganisms. This new insight about the impact of the light sources on aPDT 

efficacy could be tested onto a pathogenic environment and be directed, in future, to 

treat peri-implantitis.

1.7 Could the inflammatory response activated by aPDT modulate bone 

resorption?

Initially, aPDT was discovered because of its antimicrobial properties. However, as 

researchers began to understand part of its mechanism of action, this therapy was 

directed towards cancer treatment. Accordingly, activation of the immune response is 

necessary for effective tumor control [123]. aPDT activates several cell-signaling 

cascades and the release of cell fragments, cytokines, and inflammatory mediators, 

which stimulate the recruitment of neutrophils [124]. In an interesting report, the 

authors investigated two distinct mechanisms of neutrophil migration induced by aPDT, 

and they found that the early phase reaction may be regulated by TNF-α, neutrophil 

chemo-attractants, or IL-6. Recently, a research group evaluated inflammatory cytokine 

expression after aPDT application in the treatment of oral candidiasis in a murine model 

[125]. Consistent with the mechanism of action of this therapy [126], the results 

revealed high TNF-α expression; however, the expression levels of IL-1 and IL-6 in the 

aPDT group were lower than and similar to those in the untreated group, respectively 

[125]. During the delayed phase reaction, neutrophil chemo-attractants and IL-1b are 

the factors regulating neutrophil migration [127]. With regard to peri-implantitis, this 

disease involves the destruction of alveolar bone, which leads to implant loss. During 

the bone resorption process, different types of cells, such as neutrophils, macrophages, 



40 

 

dendritic cells (DCs), and T cells, participate in the immune response [128]. 

Furthermore, similar cytokines produced by immune cells after aPDT irradiation are 

released during the disease process [129]. The association among IL-1/6, TNF-α, and 

peri-implantitis has already been well documented [128, 130]. The main question in this 

field is if aPDT stimulates an inflammatory response in tumor cells, could this therapy 

also treat peri-implantitis and exacerbate bone loss? Clearly, we must consider several 

issues such as the short aPDT irradiation time and the levels of cytokines produced 

during treatment. We believe that the present review provides new insights into the 

possible connections of the immune response triggered by aPDT with peri-implantitis to 

ensure the safety of this therapeutic approach. 

2. Final considerations

Could PDT be considered as a novel modality for treating peri-implant disease?

In this review, we have summarized the most important factors related to aPDT for peri-

implantitis treatment and have focused on the outcomes of previous in vitro and in vivo

studies. The selected bacteria and/or fungi used in the in vitro experiments demonstrate 

the mechanism of action of PSs within microorganisms of different classes. Although 

the effects of aPDT on peri-implant disease have previously been investigated, the exact 

mechanism of action of aPDT against peri-implantitis remains largely unknown. The 

insufficient results found in the scientific literature with regard to using aPDT against 

pathogenic biofilms have not discouraged new investigations due to the advantage of 

this therapy in avoiding antibiotic resistance. Accordingly, we made significant effort to 

describe and discuss all the contradictory results found in the literature. We have 

demonstrated that the microorganism selected, PS properties, wavelength, and light 

source play critical roles in the clinical efficacy of aPDT. Focusing on peri-implant 



41 

 

disease and considering that peri-implantitis is dominated by Gram-negative anaerobic 

bacteria, the PS composition ultimately determines the affinity and specificity of the PS 

between different species. LPS is a highly anionic and important pathogenic factor 

present in Gram-negative bacteria that extends beyond outer membrane proteins. 

Additionally, all carbon atoms that are not bound to nitrogen or oxygen atoms from the 

thin peptidoglycan layer confer a hydrophobic property. Thus, new studies should be 

directed towards the development of specific PSs. Indeed, a detailed understanding of 

the mechanisms of action of aPDT could position this therapy as the treatment of choice 

in selected cases and as an important adjunct to other therapies. 

Conflicts of Interest: The authors declare no conflicts of interest. 



42 

 

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4 PUBLICAÇÃO 2*

An in vitro model for periodontopathogenic biofilm 

Lívia Jacovassi Tavares, DDS,a Marlise Inêz Klein, DDS, MSc, PhD,b Beatriz Helena 

Dias Panariello, DDS, MSc,a Erica Dorigatti de Avila, DDS, PhD,c Ana Cláudia 

Pavarina, DDS, MSc, PhDd

aPhD Student, Department of Dental Materials and Prosthodontics, Araraquara Dental 

School, Univ Estadual Paulista - UNESP, Araraquara, SP, Brazil. 

bResearcher, Department of Dental Materials and Prosthodontics, Araraquara Dental 

School, Univ Estadual Paulista - UNESP, Araraquara, SP, Brazil. 

cPostdoctoral Research Fellow, Department of Dental Materials and Prosthodontics, 

Araraquara Dental School, Univ Estadual Paulista - UNESP, Araraquara, SP, Brazil.

dAdjunct Professor, Department of Dental Materials and Prosthodontics, Araraquara 

Dental School, Univ Estadual Paulista - UNESP, Araraquara, SP, Brazil.

Corresponding author:

Dr Ana Cláudia Pavarina.

Present address: Department of Dental Materials and Prosthodontics, Araraquara Dental 

School, Univ Estadual Paulista - UNESP. Rua Humaitá, 1680, Araraquara, São Paulo, 

14801-903 Brasil, Tel: +55-16-3301-6424 / PABX: +55-16-3301-6406. E-mail:

pavarina@foar.unesp.br 

*De acordo com “Journal of Prosthetic Dentistry”



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ACKNOWLEDGMENTS 

This work was supported by Coordination for the Improvement of Higher Level -or 

Education- Personnel (CAPES) and CEPID/CEPOF - Research, Innovation and 

Diffusion Centers/ Research Center for Optics and Photonics under Grant 2013/07276-

1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



60 

 

ABSTRACT 

Statement of problem. Consistent in vitro biofilm models are required to elucidate the 

understanding about the interactions among species and to assess the antimicrobial 

effect of specific materials as well as to investigate the efficiency of different 

treatments. 

Purpose. The goal of this study was to design a standard in vitro periodontopathogenic 

biofilm model to lead therapeutic approaches in future studies.

Material and Methods. Fusobacterium nucleatum and Porphyromonas gingivalis 

strains were growth under anaerobic conditions in single and dual bacteria species. First, 

bacterial biomass was evaluated at 24 and 48 hours to determine adhesion phase onto 

saliva coated polystyrene surfaces. Thereafter, the biofilm development was assessed 

overtime by crystal violet staining and the biofilm maturity was confirmed by scanning 

electron microscopy (SEM). An unpaired t test, one tailed, was applied to define the 

best time point to adhesion period. In case of biofilm formation, one way analysis of 

variance (ANOVA), with a Tukey's posthoc test, was employed to indicate the 

difference among the periods previously established. 

Results. The data showed a significant difference in total biomass of bacteria adhered 

after 48 hours for P. gingivalis in single and dual species. For biofilm development 

approaches, P. gingivalis in single and dual species, the biomasses accumulated were 

substantially higher after 7 days than after 3 days of incubation; but no significant 

difference was obtained between 5 and 7 days growth. On the other hand, the biomass 

of F. nucleatum biofilm was higher at earlier time point and the results did not show any 

difference among 3, 5 and 7 days of incubation. 

Conclusion. The assessment of this research were efficient in revealing the pathogenic 

bacterial growth periods and the establishment of mature biofilm, describing an 



61 

 

important sequence in the development an in vitro model of periodontopathogenic 

biofilm in single and dual species. 

CLINICAL IMPLICATIONS

An in vitro periodontopathogenic biofilm model construction is crucial to lead a deeper 

understanding about the efficiency of new antimicrobial materials surfaces as well as 

chemical agents development to treat oral disease.  



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INTRODUCTION

Periodontal and periimplant diseases are triggered infections associated to complex 

biofilms structure, which induce an inflammatory response causing the destruction of 

the connective tissues.1, 2 The prevalence of periodontitis in adults is about 47%,3 being 

the 6th most prevalent oral disease,4 while periimplantitis is present in 28% of subjects 

examined.5 Porphyromonas gingivalis (P. gingivalis) is a red complex anaerobic Gram 

negative bacteria, strongly associated with the advancement of both oral infections.6-8

The mechanisms involved in bacteria colonization on natural and artificial surfaces as 

well as the surround periodontal tissues consist of straight attaching themselves to saliva 

proteins, to epithelial and connective cells receptors and/or interacting with others 

intermediate and/or early bacterial colonizers.9-12 F. nucleatum is also a Gram negative 

bacteria, regarded as a central organism for dental biofilm maturation, due to its wide 

ability of coaggregation to other microorganisms, as P. gingivalis.13-16 These 

coaggregation, known as mutually beneficial, promotes a high number of virulence 

factors expressed by both species.17 Consequently, those molecules may contributes to 

survival, presence and pathogenicity of these microorganisms in various oral niches the 

bacterial pathogenicity.13, 18 Once bacteria are attached to a surface, the dynamic 

interactions between the host and the bacteria evolve into an organized and complex 

microbial community, protected from mechanical and chemical damage.19 The 

development of promising strategies for fighting biofilm related infections requires in 

vitro models of mature biofilm, which are useful in obtaining a better understanding 

about the action mechanism of some drugs, for example. Such models are essential for 

evaluating the efficiency of therapies that aim to control and prevent oral diseases 

caused by periodontopathogenic biofilm. 



63 

 

Scientific literature has reported different in vitro biofilm representations to 

assess the effects of specific materials, as well as to investigate the efficiency of 

treatments.20-23 However, there is limited knowledge on the adequate periods necessary 

for establishing a mature biofilm. Therefore, in this investigation, P. gingivalis and F. 

nucleatum were grown onto saliva coated surfaces with the goal of developing in vitro 

models of periodontopathogenic single and dual species biofilm to evaluate therapeutic 

approaches in future studies. Additionally, since the bacterial growth pattern24-26

understanding is important to know the ideal concentration to initiate the biofilm 

development, the growth curve of both bacteria species was described.  

MATERIAL AND METHODS

Human saliva samples from three healthy adult male volunteers were collected under 

the approval of the Ethics Committee for Research in Humans (CAAE 

26142014.0.0000.5416) (ANEXO A) and after informed consent obtaining. None of the 

participants had been treated for oral diseases or had taken any prescription medication 

during the three months previous to the study.27 The saliva preparation was performed 

as described in the previous studies.28 Before its use, the supernatant obtained after 

centrifugation was purified with a 0.22 μm membrane filter (Millipore), and stored at -

80°C.29, 30 

The pathogenic bacteria strains selected to this study were P. gingivalis ATCC 

32277 and F. nucleatum NCTC 11326. The microorganisms stored at -80ºC wer