FACULDADE DE ODONTOLOGIA
CAROLINE CORRÊA DA SILVA RAMOS MENDONÇA
ANÁLISE MOLECULAR DO EFEITO ANTIMICROBIANO DA
TERAPIA FOTODINÂMICA COMO COADJUVANTE NO
TRATAMENTO ENDODÔNTICO: UM ESTUDO CLÍNICO
NOVA FRIBURGO
2016
CAROLINE CORRÊA DA SILVA RAMOS MENDONÇA
ANÁLISE MOLECULAR DO EFEITO ANTIMICROBIANO DA
TERAPIA FOTODINÂMICA COMO COADJUVANTE NO
TRATAMENTO ENDODÔNTICO: UM ESTUDO CLÍNICO
Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense / Campus Universitário de Nova Friburgo, como parte dos requisitos exigidos para a obtenção do título de Mestre em Odontologia, na Área de concentração Biologia e Patologia Buco- dental.
Orientadora: Profa. Dra. Natalia Iorio Lopes Pontes Póvoa
Orientador: Prof. Dr. Leonardo dos Santos Antunes
ANÁLISE MOLECULAR DO EFEITO ANTIMICROBIANO DA
TERAPIA FOTODINÂMICA COMO COADJUVANTE NO
TRATAMENTO ENDODÔNTICO: UM ESTUDO CLÍNICO
Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense / Campus Universitário de Nova Friburgo, como parte dos requisitos exigidos para a obtenção do título de Mestre em Odontologia, na Área de concentração em Biologia e Patologia Buco-dental.
Aprovado em: 15 de dezembro de 2016
BANCA EXAMINADORA
Profa. Dra. Lívia Azeredo Alves Antunes
Profa. Dra. Natalia Iorio Lopes Pontes Póvoa
Profa. Dra. Paula Alvarez Abreu NOVA FRIBURGO
Agradeço a Deus pela permissão de concretizar mais uma etapa em minha vida e ter-me dado coragem para persistir no meu objetivo, apesar das adversidades encontradas no caminho.
Agradeço à minha família e amigos pelo apoio incondicional.
Agradeço a todos os professores envolvidos no projeto, em especial aos meus orientadores Natalia Iorio Lopes Pontes Póvoa e Leonardo dos Santos Antunes, assim como seus respectivos cônjuges, Helvécio Cardoso Corrêa Póvoa e Lívia Azeredo Alves Antunes. Agradeço por terem me recebido e contribuído para minha evolução e amadurecimento profissional. Muito obrigada por compreenderem e exercerem perfeitamente o real propósito da orientação, que por muitas vezes é esquecido pelos docentes. Acredito que o desenvolvimento de uma pesquisa só é possível quando somos orientados por bons mestres e a competência de vocês é indiscutível.
Agradeço aos alunos de graduação e pós-graduação participantes do projeto.
Agradeço a todos os alunos do Laboratório de Microbiologia Experimental pela ajuda e pelo senso de trabalho em equipe.
Agradeço às meninas do laboratório multidisciplinar I pela receptividade. Agradeço aos pacientes pelo consentimento na participação da pesquisa.
Meu sincero agradecimento a todos. Sem a contribuição de cada um, a conclusão deste projeto não seria possível.
Microrganismos desempenham um papel crítico na etiologia e patogênese da periodontite apical. Enterococcus faecalis e Candida sp. são freqüentemente associados com infecções persistentes. O objetivo deste estudo foi avaliar a eficácia do uso da terapia fotodinâmica antimicrobiana (TFDa) como terapia coadjuvante no tratamento endodôntico contra E. faecalis, Candida sp. e domínio Bacteria, por biologia molecular. Foram analisadas 10 dentes unirradiculares [grupo controle (GC) = 4 e grupo teste (GT) = 6] com polpa necrótica e periodontite apical primária. As amostras foram coletadas antes e após preparo químico-mecânico (PQM), após TFDa (para GT) e na segunda sessão após a remoção da restauração temporária para verificar a possível ação residual da TFDa. No GT a TFDa foi realizada com laser diodo de baixa intensidade (1 J/cm; λ = 660 nm; 100 mW; 40 s) após a sensibilização com o azul de metileno por 5 min. Em seguida, outra iluminação foi realizada em um ponto gengival, próximo ao forame apical (3 J/cm; λ = 660 nm; 100 mW; 30 s). Os genes específicos para E. faecalis, Candida sp. e domínio Bacteria, foram detectados por PCR (“polymerase chain reaction”). No GT, o dente que apresentou amostra positiva para Candida sp. antes do PQM não foi detectada nas coletas subsequentes, além disso, não foi detectado E. faecalis na segunda sessão, mesmo a espécie sendo detectada nas amostras coletadas de dois dentes após o PQM e uma após a TFDa. A TFDa apresentou-se como uma terapia coadjuvante eficaz no tratamento endodontico primário de dentes permanentes, apresentando significante redução da incidência de E. faecalis antes da obturação dos canais radiculares.
Palavras-chave: Fotoquimioterapia, Necrose da Polpa Dentária, Reação em Cadeia da Polimerase, Microbiologia, Enterococcus faecalis, Candida.
ABSTRACT
Microorganisms play a critical role in the etiology and pathogenesis of apical periodontitis. Enterococcus faecalis and Candida are frequently associated with persistent infections. The aim of this clinical study was evaluated the antimicrobial effects of primary endodontic conventional treatment, followed by an adjunct therapy with the antimicrobial photodynamic therapy (aPDT). Microbial detection was performed with polymerase chain reaction (PCR) assays. Were analyzed 10 uniradicular teeth [control group (CG) = 4 and test group (TG) = 6] with necrotic pulp and apical periodontitis. Samples were taken before and after chemical-mechanical instrumentation (CMI), after the aPDT (for the TG) and after removed the temporary obturation at the second session, in order to obseve the residual effect. In TG the aPDT was performed with low intensity laser diode (1J/cm; λ = 660 nm; 100 mW; 40 s), after 5 min of the photosensitizer methylene blue. Another illumination of 30 sec and energy of 3 J was performed, in a punctual and contact mode, in an extra root point, in the attached gingival close to the apical foramen. The genes specific for E. faecalis, Candida sp. and Bacteria domain, were detected by PCR. For TG, the tooth that was positive for Candida before the CMI was not detected in subsequent samples, additionally E. faecalis was not detected at the second session, but the species was present in two teeth after CMI and one after aPDT. The aPDT may be used as an effective adjunct in the endodontic treatment of permanent teeth, resulting in a significant reduction of the incidence of E. faecalis before final obturation at the second session in teeth with primary endodontic infections.
Keywords: Photochemoterapy, Dental Pulp Necrosis, Polymerase chain reaction, Microbiology, Enterococcus faecalis, Candida.
SUMÁRIO 1.INTRODUÇÃO... 1 2. OBJETIVOS ... 5 3. CAPÍTULOS ... 6 3.1. Artigo ... 6 3.1.1. Resumo ... 7 3.1.2. Introdução ... 8 3.1.3. Material e Métodos ... 9 3.1.4. Resultados ... 14 3.1.5. Discussão e Conclusão ... 16 3..1.6. Agradecimentos ... 19 3.1.8. Referências ... 19 4. CONSIDERAÇÕES FINAIS ... 25 5. REFERÊNCIAS ... 26 6. ANEXOS ...
6.1 Anexo 1. Aprovação do CEP ... 6.2 Anexo 2. Termo De Consentimento Livre Esclarecido ...
6.3 Anexo 3. Normas para publicação no periódico Journal of Biomedical Optics. 32
32 34 35
1. INTRODUÇÃO
Todas as superfícies do corpo humano são colonizadas por microrganimos, que constituem a microbiota do hospedeiro. Entretanto, em situações de desequilíbrio, constituintes da microbiota bucal podem tornar-se patógenos oportunistas, causando doença se tiverem acesso as áreas normalmente estéreis do corpo, como a polpa dental e tecidos perirradiculares (Baumgartner et al., 2006).
A via de acesso microbiano mais comum para o sistema de canais radiculares é a cárie dentária. A cárie é uma infecção localizada, destrutiva e progressiva da dentina, que quando não detectada e removida, pode resultar em necrose pulpar e possível perda do dente. Produtos e subprodutos microbianos provenientes da dissolução de matéria orgânica e inorgânica da dentina promovida pela cárie, modulam os efeitos da infecção na polpa, assim como a permeabilidade dentinária (Fouad e Levin, 2006).
A permeabilidade da dentina está relacionada a presença dos túbulos dentinários. Sendo a quantidade e o diâmetro dos túbulos aumentados a medida em que se aproximam da polpa. Quando são expostos, servem de via para a invasão microbiana ao espaço pulpar. As bactérias e seus subprodutos podem ter um efeito sobre a polpa mesmo antes de uma exposição direta (Pashley e Liewehr, 2006). Conforme a lesão cariosa progride, há uma reação pulpar igualmente progressiva. Na presença de inflamação pulpar grave, há formação de micro-abcessos e alterações circulatórias. A polpa coronal e subsequentemente a polpa radicular podem tornar-se não irrigadas e, então, necróticas (Fouad e Levin, 2006).
Desta forma, a profundidade da lesão cariosa está diretamente relacionada ao desenvolvimento da infecção endodôntica (Rôças et al., 2015), pois os microrganismos presentes nesta lesão podem alcançar de forma direta ou indireta o sistema de canais radiculares (Pashley e Liewehr, 2006). Uma vez estabelecida a infecção dentro do canal radicular, o ambiente endodôntico oferece um habitat seletivo para o estabelecimento de uma microbiota diversa, incluindo àqueles não-cultiváveis (Paula, 2013).
A comunidade polimicrobina presente nos canais radiculares possui diversas propriedades biológicas e patogênicas, como antigenicidade, atividade mitogênica, quimiotaxia, histólise enzimática (Nair, 2004). Esses microrganismos têm um papel crítico na etiologia e patogênese da periodontite apical (Aw, 2016) e seus produtos podem avançar até a região do periápice (Nair, 2004), como consequência ocorre a resposta do hospedeiro, composta por: células de defesa, sinalizadores intercelulares,
anticorpos e moléculas efetoras. Os microrganismos e o sistema imune do hospedeiro confrontam-se e promovem a destruição do tecido periapical, resultando na formação de várias categorias de lesões de periodontite apical (Nair, 2004).
O objetivo do tratamento endodôntico consiste na antissepsia dos canais radiculares, proporcionada pelo preparo químico-mecânico (PQM) (Peters e Peters, 2006). Através da instrumentação mecânica e do uso de soluções irrigadoras antissépticas, há a remoção do tecido pulpar necrótico e da dentina infectada, promovendo a redução dos microrganismos presentes. Porém, a complexidade dos espaços que necessitam ser acessados, modelados, limpos e preenchidos é ampla (Peters e Peters, 2006).
A complexidade anatômica do sistema de canais radiculares é ampla, sendo encontradas foraminas múltiplas (Elnour et al., 2016), canais acessórios (Leoni et al., 2014), deltas apicais (Gao et al., 2016) e canais laterais adjacentes ao canal principal (Nair, 2006). Adicionalmente, ainda é possível observar canais em forma da letra “C” (Martins et al., 2013).
Irrigantes e medicação intracanal complementam a limpeza mecânica, e auxiliam a antissepsia das regiões que não são tocadas mecanicamente. Entretanto, os microrganismos presentes nas áreas intocadas bem como nos túbulos dentinários podem sobreviver (Al-Nazhan et al., 2014). A solução irrigadora comumente utilizada no PQM é o hipoclorito de sódio (Waltimo et al., 2005; Sakamoto, 2007). A inserção da medicação intracanal (hidróxido de cálcio, paramonoclorofenolcanforado e glicerina), utilizada entre as sessões da terapia endodôntica, suplementa o protocolo de antissepsia do sistema de canais radiculares (Rôças e Siqueira, 2011).
Juntos, e realizados corretamente, todos os procedimentos referentes ao protocolo para tratamento endodôntico são a base para o sucesso biológico (Peters e Peters, 2006). A falha no tratamento do canal radicular pode estar relacionada a fatores como a complexidade anatômica dos canais radiculares e a persistência microbiana (Siqueira e Rôças, 2008). Esforços devem ser realizados para eliminar a maior carga bacteriana antes da obturação (Siqueira e Rôças, 2008). Estudos recentes indicam a correlação entre infecções endodônticas persistentes e a presença de Enterococcus faecalis e/ou Candida (Kumar et al., 2015, Tennert et al., 2014, Murad et al., 2014, Dumani et al., 2012, Poptani et al., 2013).
urinário, endocárdio, abdome e nas vias biliares (Michaud et al., 2000), além de estar relacionada a infecções endodônticas (Siqueira e Rôças, 2004) São capazes de viver e persistir em ambientes extremos e com nutrientes escassos (Richards et al, 2010). Sobrevivem a ação de vários irrigantes e na presença de medicação intracanal (Rodríguez-Niklitschek, 2015). Têm a capacidade de formar biofilmes, se aderindo ao colágeno mediados por adesinas na superfície celular (Kayaoglu et al., 2005). Invade os túbulos dentinários, onde pode sobreviver por um período prolongado sob condições adversas (Al- Nazhan et al., 2014). É capaz de tolerar pH extremamente alcalino, sendo resistente a medicação intracanal com hidróxido de cálcio, além de resistir ao hipoclorito de sódio (Evans et al., 2002).
Candida albicans está associada à infecções persistentes (Poptani et al., 2013), bem como a periodontites periapicais primárias (Dumani et al., 2012). Apresenta vários fatores de virulência importantes como a aderência, que a permite iniciar a colonização de superfícies sólidas como esmalte e dentina, e formação de hifas, que possibilitam a penetração nos túbulos dentinários (Al-Nazhan et al., 2014). Sobrevivem em ambientes pobres em nutrientes (Richards et al.,2010) e são resistentes a ação da medicação antimicrobiana com hidróxido de cálcio, uma vez que suportam pH extremamente alcalinos (Mohammadi et al., 2012).
Novos protocolos têm sido avaliados para melhorar a antissepsia, em estudos in vitro ou ex vivo, especialmente para E. faecalis e/ou Candida (Vatkar et al., 2016, Valera et al., 2016, Sabino et al., 2015), como: laser Nd-YAG, acrônimo do inglês Neodymium-doped Yttrium Aluminium Garnet (Vatkar et al., 2016), laser diodo (Vatkar et al., 2016; Sabino et al., 2015), óleo de rícino (Valera et al., 2016), aloe vera e extrato de gengibre glicólicos (Valera et al., 2016).
A terapia fotodinâmica antimicrobiana (TFDa) é uma estratégia antimicrobiana que envolve a combinação de um fotossensibilizador não-tóxico e uma fonte de luz. O fotossensibilizador excitado reage com oxigênio molecular e produz oxigênio altamente reativo, que induz lesão e morte de microrganismos, sem promover efeitos citotóxicos ao hospedeiro (Xu, 2009; Singh, 2014).
Dessa forma, a terapia fotodinâmica é um processo fotoquímico em que a excitação eletrônica do fotossensibilizador provoca dois mecanismos: tipo I – transferência de elétrons - e tipo II – transferência de energia. No mecanismo tipo I, há a formação de produtos oxidados e ocorre em cerca de 5% da reação. Nos outros 95%,
diferentemente da necrose, não provoca injúria aos tecidos adjacentes. Na apoptose, a célula tem morte programada com encolhimento dessa e formação de vesículas (corpos apoptóticos) que são fagocitadas por macrófagos posteriormente. Como não há lise celular, não há extravasamento do conteúdo citoplasmático, evitando assim, lesão tecidual. Isso torna a TFDa uma terapia eficaz e segura (Kubler et al., 1998; Konig et al., 2000).
O fotossensibilizador deve possuir uma banda de absorção ressonante com o comprimento de onda da fonte de luz a ser utilizada; deve possuir estabilidade biológica, eficiência fotoquímica, seletividade pela célula-alvo e mínimo efeito tóxico às células do hospedeiro (Seal et al., 2002), é importante que a fonte de luz seja absorvida pelo corante para que a TFDa seja efetiva na inviabilização de células (Ackroyd et al., 2001).
A utilização de um agente de absorção óptica, não tóxico, que se fixe à parede celular bacteriana atraindo para si a luz é necessária para que ocorra a ação antimicrobiana sobre os microrganismos (Karu, 1999). Postula-se que o efeito microbicida da laserterapia de baixa potência ocorra em virtude da absorção de fótons por cromóforos endógenos (4-aminoantipirina e hemoglobina), com concomitante produção de moléculas altamente reativas e citotóxicas, que provocam a ruptura da membrana e morte microbiana (Xhevdet et al., 2014).
Estudos recentes demonstram diferentes aplicações da terapia fotodinâmica na Medicina e na Odontologia (Garcez 2015; Araújo, 2015; Ornellas et al., 2016; Lin, 2016). A terapia endodôntica convencional associada à TFDa tem se mostrado um método eficaz na redução de microrganismos in vitro (Arneiro et al., 2014) e ex vivo (Xhevdet et al., 2014, Ng et al., 2011). Os estudos clínicos que associam a TFDa ao tratamento endodôntico convencional demonstram eficácia (Bonsor et al., 2006a; Bonsor et al., 2006b; Garcez et al., 2008; Garcez et al., 2010; Garcez et al., 2015). Entretanto, não há estudos moleculares que avaliem os efeitos da TFDa frente à E. faecalis, Candida sp. e domínio Bacteria.
2. OBJETIVOS 2.1 Objetivo geral
Avaliar qualitativamente, por PCR, a eficácia da terapia fotodinâmica antimicrobiana como coadjuvante ao tratamento endodôntico.
2.2 Objetivos específicos
1) Detectar a presença de E. faecalis, Candida e microrganismos do domínio Bactéria em um grupo controle utilizando antissepsia com hipoclorito de sódio a 2,5%;
2) Detectar a presença de de E. faecalis, Candida e microrganismos do domínio Bactéria em um grupo teste utilizando antissepsia com hipoclorito de sódio a 2,5% + TFDa;
3) Avaliar o efeito residual da TFDa através de coletas feitas na segunda sessão, uma semana após a primeira sessão, para detecção da presença de E. faecalis, Candida e microrganismos do domínio Bactéria;
4) Comparar os resultados obtidos entre o grupo teste e o grupo controle nas coletas realizadas na segunda sessão, uma semana após a primeira sessão, para detecção da presença de E. faecalis, Candida e microrganismos do domínio Bactéria.
3. CAPÍTULOS
3.1 Artigo: Trabalho a ser submetido ao periódico “Journal of Biomedical Optics”
NOVEL ANTIMICROBIAL THERAPY ASSOCIATED WITH THE CONVENTIONAL ENDODONTIC TREATMENT: A CLINICAL AND
MOLECULAR MICROBIOLOGICAL STUDY
Caroline Corrêa da Silva Ramos Mendonça, DDS, MSc student Sérgio Pinto Chaves Júnior, DDS, MSc
Gabriela Lorena Dias Pereira, DDS student Lívia Azeredo Alves Antunes, DDS, MSc, PhD
Karla Bianca Fernandes da Costa Fontes, DDS, MSc, PhD Helvécio Cardoso Corrêa Póvoa, BSc, MSc, PhD
Leonardo Santos Antunes, DDS, MSc, PhD
Natalia Lopes Pontes Póvoa Iorio, BSc, MSc, PhD
Correspondence to Dr. Natalia Lopes Pontes Póvoa Iorio, Universidade Federal Fluminense, Nova Friburgo, RJ, Brazil
Rua Doutor Silvio Henrique Braune, 22 – Centro, Nova Friburgo, Rio de Janeiro Brazil, CEP- 28625-650
E-mail address: iorionlp@yahoo.com.br Fax: +55-22-25287168
ABSTRACT
Microorganisms play a critical role in the etiology and pathogenesis of apical periodontitis. Enterococcus faecalis and Candida are frequently associated with persistent infections. The aim of this study was to evaluate the antimicrobial effects of primary endodontic conventional treatment, followed by an adjunct therapy with the antimicrobial photodynamic therapy (aPDT). Microbial detection was performed by polymerase chain reaction (PCR). Ten uniradicular teeth [control group (CG)= 4 and test group (TG)= 6] with endodontic infections were analyzed. Samples were collected before and after chemical-mechanical instrumentation (CMI), after the aPDT (for the TG) and after removed the temporary obturation at the second session. In TG the aPDT was performed with low intensity laser diode (methylene blue/ 5 min; 1J/cm; λ = 660 nm; 100 mW; 40 s). The genes for E. faecalis, Candida sp. and Bacteria domain were detected. For TG, the tooth that was positive for Candida before CMI was not detected in subsequent samples, additionally E. faecalis was not detected at the second session, but the species was present in two teeth after CMI and one after aPDT. The aPDT may be used as an effective adjunct, resulting in a significant reduction of the incidence of E. faecalis before final obturation.
Keywords: Photochemoterapy, Dental Pulp Necrosis, Polymerase chain reaction, Microbiology, Enterococcus faecalis, Candida.
INTRODUCTION
Apical periodontitis is generally a sequel of endodontic infections1 and the
microorganisms have a critical role in its etiology and pathogenesis2 .The microorganisms associated with primary endodontic infection are diverse, including the uncultivable ones3. Therefore,the reduction of microorganisms in infected root canal systems is a fundamental component of successful endodontic treatment4.
The failure of root canal treatment can be related to several factors such as the anatomical complexities of root canals and bacterial persistence4. Efforts should be expended to eliminate the maximal bacterial from the root canals before filling4. Enterococcus faecalis and/or Candida are frequently associated with endodontic failures5-9. These microorganisms can easily penetrate dentinal tubules of root canals10.
With this regard, novel protocols to enhance disinfection have been evaluated in vitro or ex vivo, especially for E. faecalis and/or Candida, as: Neodymium-doped yttrium aluminum garnet laser11, diode laser11,12,13, castor oil14, glycolic Aloe vera and glycolic ginger extracts14, and passive ultrasonic irrigation13. The antimicrobial photodynamic therapy (aPDT) is an antimicrobial strategy that involves the chemical interaction of a topical nontoxic photosensitizer and a light source at a specific wavelength. The excited photosensitizer reacts with the molecular oxygen from the environment in order to restricts the action of highly reactive oxygen species and causes the bacterial cell wall to rupture killing the bacteria15. In addition, unlike antibiotics, which have unique microbial targets, reactive oxygen generated from a photodynamic reaction has multifunctional characteristics and can damage several cellular structures, thus decreasing possible development of PDT-resistant bacteria16, showing any cytotoxic effects to the host cell17.
The combination of conventional endodontic therapy and aPDT has been shown as an effective on microorganisms in vitro18, ex vivo19,13 and clinical20-23 studies.
Therefore, the aim of this clinical study was evaluated the antimicrobial effects of endodontic conventional treatment, followed by an adjunct therapy with the aPDT. Microbial detection was performed with polymerase chain reaction (PCR) assays.
MATERIAL AND METHODS Patient selection
This study was approved by Research Ethics Committee of Federal Fluminense University (protocol number: 707.933), and informed written consent was obtained from all participants. Nine patients attended, during april 2015 and june 2015, at the Dental Clinical at Fluminense Federal University, from Nova Friburgo city in the Rio de Janeiro state, Brazil were included in this study. Each patient contributed with one or two teeth, totalizing 10 teeth with apical periodontitis. The individuals were from both genders with ages ranging between 17 and 65 years old. The selection criteria used were patients that have endodontic infections with only single root and a single-canal teeth to permit better aseptic control during sample collection and to increase the chances of obtaining a significant sample from the main root canal. Selected teeth should presented intact pulp chamber walls, necrotic pulp confirmed by sensitivity pulp tests, and clinical and radiographic evidence of asymptomatic apical periodontitis. Exclusion criteria were teeth with: gross carious lesions, root or crown fracture, previous endodontic treatment, gingival recession and periodontal pockets deeper than 4 mm.
The patients pregnant, lactating, with systemic diseases that could compromise the immune system, individuals who received antibiotic therapy within the previous 3 months or immuno-suppressive therapy were also excluded. The indivuduals included
in this study were attended, and randomly assigned into two groups: control group (CG) and test group (TG), according to the procedures.
Treatment procedures
In both groups, after anesthesia with lidocaine 2% 1:50.000 (Dentsply, Catanduva, SP, Brazil), the supragingival calculus and biofilm were removed from each tooth by scaling and cleaning with pumice. Than, rubber dam (Madeitex, São José dos Campos, SP, Brazil) was applied, the carious tissues were removed with a spherical rotary bur no. 4 (KG Sorensen, São Paulo, SP, Brazil) at high rotation and cooling with sterile saline drip. After that, the teeth and the operating field was cleaned and disinfected by the sequential use of 3% hydrogen peroxide (Rioquímica, São José do Rio Preto, SP, Brazil), 2% iodine (Rioquímica), 3% hydrogen peroxide (Rioquímica) once again, 2.5% NaOCl (Rioquímica), and then 5% sodium thiosulfate (Química Moderna, Barueri, SP, Brazil) was used to inactivate residues of both iodine and 2.5% NaOCl (Rioquímica). The access preparation was completed with another sterile bur under sterile saline irrigation, and the operating field, now including the pulp chamber, was once again cleaned and disinfected as above protocol.
Root canal instrumentation was done at the same appointment in all cases. The crown-down technique24, was performed using Gates Glidden (Dentsply Maillefer, Ballaigues, Switzerland) and Kerr files (Dentsply Maillefer) with anatomic diameter compatible to the radicular canal, and the irrigation was performed with 5 ml of 2.5% NaOCl between each endodontic file. The working length was established 1 mm short of the radiographic apex. Smear layer was removed by rinsing the canal with 17% ethylene diamine tetra-acetic acid (Biodinâmica Química e Farmacêutica LTDA, Ibiporã, PR, Brazil), which was left in the canal for 5 minutes, followed by a final irrigation with 15 mL of 2.5% NaOCl. The canal was dried with sterile paper points
(Endopoints, Paraíba do Sul, RJ, Brazil) and then flushed with 5 mL of 5% sodium thiosulfate to inactivate any residual NaOCl.
The calcium hydroxide paste, calcium hydroxide P.A. (Biodinâmica Química e Farmacêutica LTDA) and paramonoclorophenol (Biodinâmica Química e Farmacêutica LTDA) diluted in sterile 0.9% saline, was inserted with spirals Lentulo caliber 35 (Dentsply Maillefer), at low speed, filling the root canals as temporary medication between the sessions25. The coronal sealing was performed using Coltosol (Coltène,
Altstatten, Switzerland), followed by glass ionomer Maxxion R (FGM, Joinville, SC, Brazil).
At the second session, after 7 days, the teeth and rubber dam were cleaned and disinfected as previously reported. The temporary obturation was removed, and the teeth and operating field were once again cleaned and disinfected. After that, the root canal was filled by the hybrid Tagger technique, with a condenser Mc Spadden (Dentsply Maillefer).
Antimicrobial photodynamic therapy - aPDT
In the test group, after the conclusion of the instrumentation at the first day, 0.01% methylene blue aqueous solution (Fórmula & Ação, São Paulo, SP, Brazil) was inserted into the canal and kept in place for 5 min, as an incubation time. After this time, the excess of the photosensitizer was removed with a paper point and the tooth was irradiated with an InGaAIP diode laser (MMOptics, São Paulo, SP, Brazil). The illumination was performed in the root canal with a disposable 55 mm plastic optical fiber-coupled diode laser (MMOptics) previously disinfected with NaOCl. The laser delivered 660 nm light at a total power of 100 mW and 1 J of energy out of the fiber for a period of 40 sec. The fiber was placed in the apical portion 1 mm short of the radiographic root apex. Then, spiral movements, from apical to cervical, were
performed to ensure a diffusion of the light inside the root canal. Another illumination of 30 sec and energy of 3 J was performed, in a punctual and contact mode, in an extra root point, in the attached gingival close to the apical foramen. In each day of illumination, the output power of the laser equipment was measured using a power meter (Laser Check, MMOptics). Then, the root canals were filled with temporary medication, calcium hydroxide paste, between the sessions, as described above.
Sample collection
Two sterility control samples were taken from the cavo surface angle of the access cavity by scrubbing with sterile paper points no. 15 (Endopoints) after the protocol of clean and disinfection in both sessions, to verify the efficacy of this protocol.
The microbiological samples were taken in three moments to CG and four to TG: i) before chemical-mechanical instrumentation (CMI) (samples A); ii) after CMI (samples B); iii) after aPDT, exclusively to TG (samples C); iv) before final obturation (samples D). The samples were taken with two sterile paper points no. 15 (Endopoints) to each moment, the paper point were left in the canal, previously filled with sterile saline solution, for 30 sec to a level approximately 1 mm short of the radiographic root apex.
Culture analysis
The paper points from sterility control samples were immediately placed in flasks containing Fluid Thioglycollate Medium (Becton, Dickinson and Company, MD, Sparks, USA) and incubated for 14 days at 36oC to determined the presence/absence of cultivable microorganisms. For inclusion of the tooth in this study, these control samples had to be negative for culture.
The microbiological samples (A, B, C and D) were placed in cryotubes containing 0.5 mL of Tris-EDTA buffer solution pH 8.0 (Sigma-Aldrich, Buchs, Switzerland) and immediately frozen at -20oC. Deep frozen samples were thawed and dispersed by constant stirring for 30 min. Paper points were removed and samples were centrifuged at 5000 g for 10 min. After centrifugation the supernatant was discarded and the pellet containing microbial cells were resuspended in 180µL of ATL buffer (Qiagen, Valencia, CA, USA), and the DNA was extracted using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s instructions. The DNA extracts were stored at -20oC until required for further analysis.
Amplification of whole-genome and quantification of DNA from microbiological samples
The DNA extracts were subjected to whole-genome amplification using the Illustra GenomiPhi V2 DNA Amplification kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions, in order to increase the DNA amount. The DNA was quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), with the Qubit dsDNA HS Assay kit (Life Technologies).
PCR for E. faecalis, Candida sp. and domain Bacteria
Presence/absence E. faecalis, Candida sp. and domain Bacteria in microbiological samples was determined by using end-point PCR. Aliquots of 10ng of the extracted DNA were used in PCR protocols for E. faecalis26, Candida genus27 and members of the domains Bacteria28. Positive controls consisted of DNA extracted from E. faecalis (ATCC 29212) for detection of E. faecalis and domain Bacteria and C. albicans (ATCC 10321) for Candida genus. PCR amplifications were performed in a DNA thermocycler (Life Technologies). Amplified products were analyzed by 1% of
agarose gel electrophoresis with GelRed 1X (Biotium, Hayward, CA, USA) and visualized on UV transilluminator (Kasvi, Curitiba, PR, Brazil).
Statistical Analysis
The data were analyzed using the statistical program Epi Info version 7 (Centers for Disease Control and Prevention, Atlanta, GA, USA). Fisher's exact test was applied to evaluate and compare the incidence of positive PCR results in the teeth in different stages of treatment, differences were considered significantly when values of p<0.05 were obtained.
RESULTS
Ten permanent teeth with necrotic pulp, from nine patients were included in this study, being four for CG and six for TG. All paper points of sterility control that placed in flasks containing Fluid Thioglycollate Medium (Becton, Dickinson and Company) determined the absence of cultivable microorganisms.
The results of presence/absence of DNA from E. faecalis species, fungal genus Candida and domain Bacteria in the samples before CMI, after CMI, after aPDT and at the beginning of the second session are shown in Table 1.
Table 1. Detection of domain Bacteria, E. faecalis and Candida sp. in microbiological samples by PCR.
Before CMI (A) After CMI (B) After aPDT (C) Second session (D) Sample
16s E.f C 16s E.f C 16s E.f. C 16s E.f. C CG (without aPDT) 1 + - - + - - na na na + - - 2 + - + - - - na na na + - - 3 + - + - - - na na na + - + 4 + - - - na na na + - - CG % 100 0 50 25 0 0 na na na 100 0 25 TG (with aPDT) 5 + - - + - - + - - + - - 6 + + - + - - + - - + - - 7 + - - + - - + - - + - - 8 + + - + + - + + - + - - 9 + + + + - - - + - - 10 + + - + + - + - - + - - TG % 100 66.7 16.7 100 33.3 0 83.3 16.7 0 100 0 0 TOTAL % 100 40 30 70 20 83.3 16.7 0 100 0 10 CMI: chemical-mechanical instrumentation; aPDT: antimicrobial photodynamic therapy; 16s: domain Bacteria; E.f.: E. faecalis; C: Candida sp.; CG: Control group; TG: Test group; +: presence; -: absence; na: no applicable.
For both groups the analysis of the first microbiological samples (A) showed the presence of bacteria in all teeth. E. faecalis were detected in four (40%) and Candida in three (30%) samples before CMI (A) (Table 1). After the CMI (samples B), the presence of bacteria was detectable in seven teeth. E. faecalis were present in two samples and Candida was not detectable after the CMI. No significant difference was found for the incidence of bacteria (7/10; p=0.2105), E. faecalis (2/4; p=0.4286); and Candida (0/3; p=0.1) after and before CMI.
In relation to the CG, the presence of bacteria was detectable in all samples of the second session (samples D). One of the two teeth that were showed a positive result for Candida in samples before the CMI (A) continues with this profile in the second
session (D). E. faecalis were not present in samples before the CMI (A) in the CG. No significant difference was found for the incidence of Candida (1/2; p=1) in samples D and samples A.
For the TG, five of the six teeth that carried bacteria in the samples after CMI (B) continue with this profile in the samples after aPDT (C). Two teeth showed presence of E. faecalis in the samples after CMI (B), but after aPDT (samples C) only were detected in sample of one tooth. At the second session, the samples D for all teeth were positive for bacterial detection. However, E. faecalis that were present in samples before CMI (A) of four teeth, were not detectable in samples collected in the second session (D), as well as the Candida detected before the CMI (sample A) for only one tooth. Significant difference was found for the incidence of E. faecalis (0/4; p=0.0286) in samples collected in second session (D) and before the CMI (samples A) in the TG.
DISCUSSION AND CONCLUSION
A better disinfection of root canal have been desired along the years. In our study, we performed a clinical study to evaluate the effective of conventional endodontic therapy in combination aPDT against E. faecalis, Candida sp. and domain Bacteria. We observed that the combination of these therapies could be responsible to the absence of E. faecalis and Candida sp. before final obturation at the second session in permanent teeth with primary endodontic infections.
Recent studies have shown the different applicability of the photodynamic therapy in Medicine and Dentistry29-32. Previous studies showed that combination of
conventional endodontic therapy followed by aPDT was effective on microorganisms in clinical studies20-23. There is no molecular evaluation in clinical studies about the effect of aPDT against Candida and E. faecalis previously reported. To the best of our
knowledge, this is the first report that evaluated the detection of E. faecalis, Candida sp. and domain Bacteria, by PCR, after the conventional endodontic therapy followed by antimicrobial aPDT in the permanent teeth with apical periodontitis.
The incidence of positive cultures after CMI is lower for culture methods, ranged from 17% to 55%20,33,34; in comparison to PCR results, between 54% to 67% 34-36. It can be explicated by the high sensitiveness of PCR in comparison to culture method, and also allow the detection of difficult-to-grow and uncultivable microorganisms37. In the present study the incidence of positive PCR after CMI was of 70%, this rate was high compared with previously report that used PCR35,36, but in the present study the DNA extracts were subjected to whole-genome amplification and increased of the DNA amount (data not show) and consequently the sensibility of the method.
E. faecalis and C. albicans have been associated with endodontic failures5-9,38. The incidence of positive cultures in primary endodontic infections ranged from 9 to 20%39,40 and 12.5 to 30%39,41, for Candida and E. faecalis, respectively. We detected the species E. faecalis in four (40%) and Candida in three (30%) teeth before CMI in this study, by PCR, probably due the high sensitiveness of the method.
Although we not deteted Candida after the CMI, one tooth of CG carried this microorganism at the second session. This fact can be justified due the anatomical complexities of root canals and formation of biofilms on the surface of root apex. E. faecalis and C. albicans tend to be resistant to many environmental factors and antimicrobial agents39 , and can easily penetrate dentinal tubules of root canals10.
Xhevdet and coworkers (2014), showed that the aPDT is an effective approach in reducing of these microorganisms ex vivo13. In case of TG, the tooth that was present a positive result for Candida before the CMI (A) showed negative results in subsequent
samples (B, C and D), additionally E. faecalis was not detected at the second session, but the species was present in two teeth after CMI and one after aPDT. Similar results were found in a recently ex vivo study that showed, by confocal laser scanning microscopy, that the both laser methods evaluated were effective in eliminating the vitality of E. faecalis in contrast to conventional irrigants11. In contrast, an ex vivo study that used culture methods did not observe presence of C. albicans or E. faecalis after treatment with conventional irrigants14.
Although E. faecalis were not found in CG before CMI, it is now that the viable E. faecalis entombed at the time of root canal filling may provide a long-term nidus for subsequent infection4. The search for the ideal materials, instruments and techniques should continue in order to further enhance the success of endodontic treatment2.
The absence of E. faecalis and Candida sp. at the second session in the TG may be related with the extra illumination of 30 sec (3J), in punctual and contact mode, in an extra root point, in attached gingival close to the apical foramen in order to achieve the microorganisms present in the accessory canals and apical foramen and/or with the benefits from the inside effects of methylene blue that was not remove at the end of first session. An ex vivo study that used 2% chlorhexidine gel as irrigant and 14 days of calcium hydroxide intra-canal dressing observed, by culture, presence of E. faecalis in 20% (5/15) of the samples at the second session42. Williams and coworkers (2007) found, by qPCR, the presence of E. faecalis at the second session in all single-canalled teeth with primary endodontic infections that were positive for this bacteria before the CMI, but when these authors used the culture method the presence of E. faecalis at second session was not observed38.
Studies using culture methods report a high incidence of negative cultures after CMI20,34. However, because of the low sensitivity of culture methods, a negative result
does not necessary imply in a sterile channel, but only means that bacterial numbers is undetectable per culture33,43. The complete bacterial eradication is still impossible and
not all treatment results in resolution of apical periodontitis2. In this study we detected bacteria in all samples at the second session. In contrast with only one positive of eleven cultures found in a clinical study33, but our results can be attributed to the high sensitiveness of the molecular method used. According to the complex anatomy of the root system, the complete microorganism eradication is utopic for most cases4.
Therefore, the significantly reduction of microorganisms from the root canal system is a goal of endodontic treatment4,39. Further clinical studies are necessary to evaluate the microorganism reduction after aPDT as well as the residual effect of PDT at the second session comparing with a conventional endodontic therapy.
The aPDT may be used as an effective adjunct in the endodontic treatment of permanent teeth, resulting in a significant reduction of the incidence of E. faecalis before final obturation at the second session in teeth with primary endodontic infections.
ACKNOWLEDGMENTS
This study was supported by grants from: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Pró-Reitoria de Pesquisa, Pós-Graduação e Inovação / Plano de Desenvolvimento Institucional / Universidade Federal Fluminense (PROPPI/PDI/UFF). The authors deny any conflicts of interest related to this study.
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4. CONSIDERAÇÕES FINAIS
Este é o primeiro trabalho que avalia a ação da TFDa, por biologia molecular, frente à E. faecalis, Candida sp. e domínio Bacteria, comparando grupos submetidos e não submetidos a TFDa, através de coletas antes e após o preparo químico mecânico, após a TFDa e antes da obturação dos canais radiculares (segunda sessão).
A TFDa apresentou-se como uma terapia coadjuvante eficaz no tratamento endodontico primário de dentes permanentes, apresentando significante redução da incidencia de E. faecalis antes da obturação dos canais radiculares.
São necessários estudos futuros que avaliem a redução de microrganismos depois da TFDa, bem como seu efeito residual na segunda sessão, comparando à terapia endodôntica convencional.
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6. ANEXOS
! !
TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
Título do Projeto: TERAPIA FOTODINÂMICA ANTIMICROBIANA ASSOCIADA AO
TRATAMENTO ENDODÔNTICO CONVENCIONAL EM DENTES DECÍDUOS E PERMAMENTES: EFICÁCIA E UTILIZAÇÃO CLÍNICA
Pesquisador Responsável: Lívia Azeredo Alves Antunes
Instituição: Faculdade de Odontologia ! Universidade Federal Fluminense ! NF Telefones de contato: (22) 25287168
O(a) Sr.(a) está sendo convidado(a) a participar do projeto de pesquisa "#$TERAPIA
FOTODINÂMICA ANTIMICROBIANA ASSOCIADA AO TRATAMENTO ENDODÔNTICO CONVENCIONAL EM DENTES DECÍDUOS E PERMAMENTES: EFICÁCIA E UTILIZAÇÃO CLÍNICA% de responsabilidade da Professora Lívia Azeredo A. Antunes.
Objetivo do estudo: avaliar uma terapia complementar para o tratamento de canal. Justificativa do estudo: a condição bucal e saúde geral podem influenciar a vida do
indivíduo sob aspecto psicossocial gerando impactos relacionados a qualidade de vida dessas pessoas. A terapia fotodinâmica antimicrobiana (TFDa) têm sido amplamente empregada nas diversas especialidades médicas e, mais atualmente, na odontologia para com intuito de uma melhor qualidade de tratamento. Como será feito: Crianças e adultos que já se submeteriam a tratamento de canal poderão complementar o tratamento utilizando a TFDa coma o uma forma auxiliar de eliminação de bactérias de dentro do canal.
Desconfortos e risco: são mínimos, uma vez que será realizado um procedimento de
rotina. É dado o direito de procurar obter indenização por danos eventuais. Caso necessário poderá ser oferecido, pelo pesquisador responsável, auxílio transporte e alimentação para o participante da pesquisa Benefícios: O paciente será tratado e, além disso, irá contribuir para melhorar a evidência científica a respeito da terapia utilizada. Participação voluntária: A sua participação é voluntária. Você terá plena liberdade de recusar-se a participar ou retirar seu consentimento, em qualquer fase da pesquisa, sem penalização alguma. Sigilo: É garantida de manutenção do sigilo e sua privacidade durante todas as fases da pesquisa
Confidência: As informações fornecidas pelo senhor(a) serão confidenciais, assim como os
resultados obtidos pela pesquisa. Você receberá uma cópia do documento de consentimento.
Atenciosamente,
___________________________________ Lívia Azeredo Alves Antunes Prof. Adjunto II - FO/UFF/PUNF
Eu, _______________________________________________________________,RG nº _______________________, declaro ter sido informado e concordo com a minha participação, como voluntário, no projeto de pesquisa acima descrito
_______________,______ de ______________ de _________ ________________________
Assinatura do paciente
__________________________________ ___________________________________
Testemunha Testemunha
6.2. TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
6.3 NORMAS PARA PUBLICAÇÃO NO PERIÓDICO “Journal of Biomedical Optics”
General Guidelines and Policies for Journal Authors Preparing a Manuscript
Overview
Manuscripts should be submitted in English, and the presentation should be as succinct as comprehension will permit. Manuscripts are reviewed and refereed. Those accepted for publication are edited for conformance to the journal's style.
For peer review, manuscripts should be submitted with the figures/tables and their captions incorporated into the same file as the manuscript text. However, upon first revision or acceptance, authors will be asked to submit individual figure files and a properly formatted manuscript for use in production.
SPIE journals typically allow only one round of major revision. Authors should carefully address all reviewer comments when submitting a revised manuscript.
Claims of "new" or "novel" work or of being the "first" to report on a topic should be avoided unless they can be fully substantiated.
Cover Letter
Authors are required to include a separate cover letter with their submission explaining the significance and novelty of the work, the problem that is being addressed, and why the manuscript belongs in this journal.
English Language Editing
If you are not a native English speaker, SPIE recommends that your manuscript be professionally edited prior to submission. SPIE authors will receive a 10% discount off the language editing services provided by Editage, a recommended independent editorial service. More detailed information can be found at spie.org/EnglishEditing.
