UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
Jessica Montenegro Fonseca
ALTERAÇÕES DO COMPLEXO DENTINA-POLPA EM CÁRIE CONVENCIONAL E CÁRIE RELACIONADA À RADIAÇÃO: UM ESTUDO COMPARATIVO.
DENTIN-PULP COMPLEX REACTIONS IN CONVENTIONAL AND RADIATION-RELATED CARIES: A COMPARATIVE STUDY.
Piracicaba 2017
Jessica Montenegro Fonseca
ALTERAÇÕES DO COMPLEXO DENTINA- POLPA EM CÁRIE CONVENCIONAL E CÁRIE RELACIONADA À RADIAÇÃO: UM ESTUDO COMPARATIVO.
DENTIN-PULP COMPLEX REACTIONS IN CONVENTIONAL AND RADIATION-RELATED CARIES: A COMPARATIVE STUDY.
Dissertação apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Estomatopatologia, na Área de Estomatologia.
Dissertation presented to the Piracicaba Dental School of the University of Campinas in partial fulfilment of the requirements for the degree of Master in Pathology, in Stomatology area.
Orientador: Prof. Dr. Mario Fernando De Goes Coorientador: Prof. Dr. Alan Roger Dos Santos Silva
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA JESSICA
MONTENEGRO FONSECA E
ORIENTADA PELO PROFº. DRº MARIO FERNANDO DE GOES.
Piracicaba 2017
Agência(s) de fomento e nº(s) de processo(s): CAPES, 758/2012 ORCID: http://orcid.org/0000-0002-1217-7298
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159
Fonseca, Jessica Montenegro, 1991-
F733a Alterações do complexo dentina-polpa em cárie convencional e cárie relacionada à radiação : um estudo comparativo / Jessica Montenegro Fonseca. – Piracicaba, SP : [s.n.], 2017.
Orientador: Mario Fernando de Goes. Coorientador: Alan Roger dos Santos Silva.
Dissertação (mestrado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
1. Câncer. 2. Radioterapia. 3. Cárie dentária. 4. Dentes. 5. Polpa dentária. I. Goes, Mario Fernando de,1954-. II. Santos-Silva, Alan Roger,1981-. III. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. IV. Título.
Informações para Biblioteca Digital
Título em outro idioma: Dentin-pulp complex reactions in conventional and radiation- related caries : a comparative study
Palavras-chave em inglês: Cancer Radiotherapy Dental caries Teeth Dental pulp
Área de concentração: Estomatologia Titulação: Mestra em Estomatopatologia Banca examinadora:
Alan Roger dos Santos Silva [Coorientador] José Flávio Affonso de Almeida
Karina Morais Faria
Data de defesa: 17-07-2017
UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE ODONTOLOGIA DE
PIRACICABA
A Comissão Julgadora dos trabalhos de Defesa de Dissertação de Mestrado, em sessão pública realizada em 17 de Julho de 2017, considerou a candidata JESSICA MONTENEGRO FONSECA aprovada.
PROF. DR. ALAN ROGER DOS SANTOS SILVA
PROFª. DRª. KARINA MORAIS FARIA
PROF. DR. JOSÉ FLÁVIO AFFONSO DE ALMEIDA
A Ata da defesa com as respectivas assinaturas dos membros encontra-se no processo de vida acadêmica do aluno.
DEDICATÓRIA
Dedico este trabalho aos meus pais Jane Montenegro Fonsêca e Ruy Leal Fonsêca, que por amor e altruísmo abdicaram dos próprios caminhos, para acompanhar-me nos meus;
À minhas irmãs, Taís Montenegro Fonsêca e Elaine Montenegro Fonsêca por igual apoio.
AGRADECIMENTOS
À Deus por sua presença em mim, todos os dias, na forma de fé, dando- me saúde e força para superar as dificuldades;
À Universidade Estadual de Campinas, na pessoa do Magnífico Reitor, Prof. Dr. Marcelo Knobel;
À Faculdade de Odontologia de Piracicaba, na pessoa do seu Diretor Prof. Dr. Guilherme Elias Pessanha Henriques, e seu Diretor Associado, Prof. Dr. Francisco Haiter Neto;
Ao Professor Doutor Márcio Ajudarte Lopes, coordenador do Programa de Pós Graduação em Estomatopatologia, pela atenção com cada um dos alunos do programa e por todas as oportunidades cedidas;
Ao Professor Doutor Alan Roger dos Santos Silva, pela orientação acadêmica e profissional e por todo o cuidado e atenção do início ao fim do meu mestrado, sempre incentivando a criar novos conhecimentos e sendo exemplo de ética e profissionalismo;
Aos Professores Doutores, Oslei Paes de Almeida, Pablo Agustin Vargas, Jacks Jorge Júnior, Edgard Graner e Ricardo Della Coletta, da Área de Patologia da Faculdade de Odontologia de Piracicaba, pelo apoio, dedicação e oportunidades de aprendizado;
Aos profissionais do OROCENTRO, em especial ao Rogério de Andrade Elias, Elisabete, Maria Aparecida Campion, Daniele Cristina Castelli Morelli, pelo apoio, bom convívio e aprendizado profissional;
Aos profissionais do Laboratório de Patologia Oral, da Faculdade de Odontologia de Piracicaba, em especial ao Adriano Luís e Fabiana Casarotti, por todo suporte, atenção e também pelo aprendizado;
À Thaís Bianca Brandão, chefe do serviço de Odontologia Oncológica do Instituto do Câncer do Estado de São Paulo (ICESP) por ter cedido o material biológico que viabilizou a execução desta Dissertação e por todo o suporte que tem oferecido ao Programa de Pós Graduação em Estomatopatologia da FOP - UNICAMP;
À Fundação Brasileira de Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo apoio financeiro, através da concessão da bolsa de mestrado no período de Agosto de 2015 à Julho de 2017;
À professora Doutora Águida Cristina Gomes Henriques Leitão, minha orientadora de iniciação científica, pelo apoio e incentivo contínuo desde a minha graduação até hoje;
Aos colegas Wagner Gomes, Mariana Paglioni e Karina Morais pela colaboração na minha pesquisa e nos experimentos;
À minha turma de mestrado, Gleyson Amaral, Natália Palmier, Rodrigo Soares, Lígia Miyahara e Camila Weissheimer pela amizade, união, cumplicidade e por todo o apoio profissional e pessoal;
Aos meus companheiros de moradia Daniel Chan, Lara Caponi, Lucas Del Vigna, Thaís Tavares, Jordana Carvalho, Rebeca Barros e Bruno Mariz pela irmandade e carinho durante toda a minha estadia em Piracicaba, facilitando a minha jornada longe de casa;
Por fim a todos os meus colegas e amigos da Faculdade de Odontologia, pelo bom convívio, troca de experiências e apoio durante o período do meu mestrado.
EPÍGRAFE
“Sempre que houver alternativas, tenha cuidado. Não opte pelo conveniente, pelo confortável, pelo respeitável, pelo socialmente aceitável, pelo honroso. Opte pelo que faz o seu coração vibrar. Opte pelo que gostaria de fazer, apesar de todas as consequências.”
RESUMO
Os carcinomas espinocelulares de cabeça e pescoço são diagnosticados predominantemente em estadios clínicos avançados da doença e, consequentemente, tratados por meio de protocolos multimodalidade, incluindo quimiorradioterapia (QRDT). Apesar dos avanços tecnológicos, protocolos contemporâneos de radioterapia (RDT) atingem, além do tumor primário, tecidos sadios adjacentes ao tumor, gerando toxicidades agudas e crônicas em pele, mucosas, glândulas salivares, ossos e dentes, entre outros. A cárie relacionada à radioterapia (CRR) é uma toxidade crônica que afeta aproximadamente 25% dos pacientes submetidos à RDT na região de cabeça e pescoço, podendo gerar destruição generalizada dos dentes, perda de eficiência mastigatória, infecção crônica e risco aumentado para o desenvolvimento da osteorradionecrose. A inclusão dos dentes no campo de radiação dos tumores de cabeça e pescoço (CCPs) sugere que a CRR resulta da destruição radiogênica do esmalte, da junção amelodentinária e do complexo dentina-polpa. Desta forma, é imperioso compreender se sua patogênese é consequência direta ou indireta da RDT. O objetivo deste estudo foi comparar padrões microscópicos de resposta do complexo dentina-polpa à progressão da cárie convencional e da CRR a fim de testar a hipótese de que a RDT é capaz de modificar diretamente as respostas micromorfológicas do complexo dentina-polpa à progressão da CRR. Foram utilizados 22 dentes extraídos de pacientes com CCPs, divididos entre grupo controle (cárie convencional) e grupo estudo (CRR). Os espécimes dos dois grupos foram pareados por grupo anatômico dental, padrões clínicos de progressão da cárie [“Post-Radiation Dental Index” (PRDI)] e profundidade de invasão microscópica das lesões de cárie. As amostras foram desmineralizadas e analisadas por meio da microscopia de luz óptica e da histomorfometria a fim de investigar a hierarquia tecidual da polpa e padrões microscópicos da resposta do complexo dentina-polpa à progressão da cárie. Todos os eventos avaliados neste estudo foram semelhantes entre os grupos, incluindo PRDI (CRR=3.8 vs. controle=3.2), profundidade de invasão microscópica (CRR=1,056.89 μm vs. controle=1,158.58 μm; p= 0.79), evidência de fibrose pulpar (p=0.9), calcificação pulpar (p=0.34), necrose pulpar (p=1.0), inflamação pulpar (p= 0.28) e deposição de dentina reacional (p=0.28). Em conclusão, o presente estudo identificou preservação da hierarquia tecidual da polpa em ambos os grupos, rejeitando a hipótese de que a RDT é capaz de modificar diretamente as respostas micromorfológicas do complexo dentina-polpa à progressão da CRR.
ABSTRACT
Head and neck squamous cell carcinomas are mainly diagnosed in late stages, which consequently increases the necessity for multimodality treatment protocols, such as chemoradiotherapy (CRT). Although the technological improvement in radiotherapy (RT) treatment protocols, radiation still damage not only the tumour site but also adjacent healthy tissues leading to the development of acute and chronic toxicities on the skin and mucosal tissues, salivary glands, bones, teeth, among others. Radiation-related caries (RRC) is a chronic toxicity that affects about 25% of the patients who have undergone head and neck RT (HNRT), and it can lead to generalized tooth breakdown, loss of masticatory efficiency, recurrent infections and increased risk for the development of osteoradionecrosis. The inclusion of the teeth inside the radiation field of head and neck tumours (HNC) suggests that RRC results of a radiogenic destruction of enamel, enamel junction (DEJ) and dentin-pulp complex. Therefore, it`s of great importance to further understand if it’s the direct or indirect influence of RT on the pathogenesis of RRC. The aim of the present study was to compare microscopic patterns of the dentin-pulp complex response to the progression of conventional caries lesions and RRC, testing the null hypothesis that the RT is able to directly modify the micromorphological response of the dentin-pulp complex facing RRC progression. Twenty-two teeth extracted from HNC patients were divided into control (conventional caries) and study (RRC) groups. The samples from both groups were paired matched by anatomic dental group, clinical patterns of caries progression [“Post-Radiation Dental Index” (PRDI)] and depth of microscopic invasion of caries lesions. Samples were demineralized and analysed by means of light microscopy and histomorphometry in order to investigate the pulp tissue hierarchy and microscopic patterns of the dentin-pulp complex response to caries. The results found in the present study were similar between the groups, including PRDI (Control= 3.2 vs RRC= 3.8), depth of microscopic invasion (Control= 1,158.58 μm vs RRC= 1,056.89 μm; p=0.79), pulp fibrosis evidence (p=0.9), pulp calcification (p=0.34), pulp necrosis (p=1.0), pulp inflammation (p=0.28) and tertiary dentin deposition (p=0.28). In conclusion, the present study observed preservation of pulp tissue hierarchy in both groups rejecting the null hypothesis that the RT is able to directly modify the micromorphological response of the dentin-pulp complex facing RRC progression.
SUMÁRIO
1 INTRODUÇÃO 12
2 ARTIGO: Dentin-Pulp Complex Reactions In Conventional And Radiation-Related Caries: A Comparative Study
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3 CONCLUSÃO 35
REFERÊNCIAS 36
ANEXOS
Anexo 1: Parecer Consubstanciado do CEP 40
Anexo 2: Certificado de submissão ao periódico: Clinical Oral Investigations
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1 INTRODUÇÃO
A incidência do câncer aumentou de modo exponencial nos últimos anos em todo o mundo, e passou a apresentar um grande desafio no cenário de políticas em saúde pública. Estima-se que, até 2030, 27 milhões de casos novos serão diagnosticados no mundo. No Brasil, para 2017, foram estimados 596 mil novos casos de câncer (INCA, 2016). Neste contexto, o câncer de cabeça e pescoço (CCP) representa 6% de todas as neoplasias malignas, afetando frequentemente os tecidos moles e duros da boca, lábios, faringe, laringe, glândulas salivares menores e maiores, cavidade nasal e seios paranasais (Sloan et al., 2017). Entre todos os casos de câncer de cabeça e pescoço, 90% correspondem a carcinomas espinocelulares (CECs) de boca orofaringe (Rodrigues et al., 2014; Sloan et al., 2017).
Indivíduos do gênero masculino, idosos, tabagistas e etilistas de longa data e grande intensidade representam o principal perfil clínico para o câncer de boca e orofaringe (Pelucchi et al., 2008; Neville et al., 2016). Recentemente, outro grupo composto por pacientes mais jovens (menos de 40 anos de idade) e sem fatores de risco comumente associados ao câncer de boca foi identificado (Ribeiro et al., 2009). A infecção pelo papiloma vírus humano (HPV), principalmente o genótipo 16 e 18, também é atualmente considerada fator de risco para o CEC de orofaringe e, ao que tudo indica, está associada ao comportamento sexual (sexo oral) (Sloan et al., 2017) .
Em adição à problemática da alta frequência dos CECs bucais e de orofaringe, é importante esclarecer que a maior parte destes tumores é diagnosticada tardiamente, em estadiamentos clínicos avançados da doença, o que torna seu tratamento desafiador e associado a baixas taxas de sobrevida em cinco anos. Os protocolos de tratamento contemporâneos do CEC de boca e orofaringe costumam envolver a cirurgia, a quimioterapia (QT) e a radioterapia (RDT) associadas ou, mais frequentemente, combinadas (Huang e O´sullivan, 2013; Marta et al., 2015).
A radiação ionizante atua diretamente sobre as células tumorais durante o ciclo celular, produzindo espécies reativas de oxigênio que interagem, por sua vez, com o DNA, o RNA e as enzimas celulares de forma a desorganizar seus nucleotídeos e danificar seu material genético de modo que não consegue ser reparado pelos mecanismos regulatórios de células malignas, gerando apoptose, morte celular e diminuição da capacidade proliferativa do tumor (Huber e Terezhalmy, 2003).
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A dose de RDT é expressa pela quantidade de energia absorvida pelo tecido irradiado. A unidade que padroniza a dose absorvida pelo tecido é conhecida como Gray (Gy = 1J/Kg) (Huber; Terezhalmy, 2003; Rolim; Costa; Ramalho, 2011). Com o objetivo de minimizar toxicidades ao paciente, a RDT em cabeça e pescoço costuma ser administrada ao longo de 5 a 7 semanas com frações diárias de 2 Gy, 5 vezes por semana com intervalos de dois dias (aos finais de semana) a fim de que os tecidos sadios adjacentes ao tumor possam se recuperar (Vissink et al., 2003; Kielbassa et al., 2006).
A QT também cumpre papel importante no tratamento dos CECs bucais e de orofaringe e tem sido associada à RDT (quimiorradioterapia - QRDT) de forma a elevar as chances de sucesso terapêutico, podendo ser aplicada antes (indução), simultaneamente (neoadjuvante) ou após o tratamento radioterápico (adjuvante). Desta forma, a QRDT atua reduzindo o tamanho do tumor primário, potencializando a atividade citotóxica do tratamento e reduzindo o risco de metástases (Bucheler et al., 2012). No entanto, esta modalidade pode intensificar as toxicidades bucais que comumente geram dor intensa, necessidade de interrupção dos protocolos de tratamento oncológico, redução nas taxas de sucesso do tratamento e aumento dos custos globais de tratamento do câncer (Seiwert et al., 2007).
O tratamento radioterápico associado ou não à QT para os CCPs promove reações aos tecidos sadios adjacentes ao campo irradiado, incluindo os dentes. De acordo com Cooper et al. (1995), a região de cabeça e pescoço é complexa do ponto de vista anatômico, onde estruturas muito distintas do ponto de vista biológico reagem de uma maneira muito variável à radiação. Neste cenário, as principais toxicidades da RDT incluem mucosite, disgeusia, disfagia, hipossalivação, infecções oportunistas, osteorradionecrose, CRR e trismo, entre outros (Vissink et al., 2003; Lobo; Martins, 2009). Estas toxicidades podem se manifestar logo no início (agudas) ou após meses da conclusão da RDT (crônicas), sendo que as últimas podem apresentar duração permanente aos pacientes que sobreviverem (Vissink et al., 2003).
Nesta problemática, a CRR é uma toxicidade crônica que surge após aproximadamente 12 meses da conclusão do tratamento radioterápico e afeta cerca de 25% dos pacientes tratados por RDT em cabeça e pescoço. Interessantemente, os padrões clínicos de início, desenvolvimento e progressão da CRR são distintos aos da cárie convencional (Hong et al., 2010). O início da CRR geralmente afeta as áreas cervical e incisal dos dentes anteriores (Kielbassa et al., 2006), gerando primariamente perda do brilho e translucidez do esmalte da coroa dental seguida por alteração da coloração da coroa que passa a assumir tom esbranquiçado que, se não tratado, progride para marrom escuro em associação a trincas e fraturas nas superfícies livres de esmalte. Posteriormente, o esmalte sofre o fenômeno de
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“delaminação” caracterizado pelo desprendimento de grandes áreas de esmalte que acaba expondo a dentina (tecido mais frágil) ao ambiente bucal dos pacientes irradiados, marcado por alto potencial cariogênico e que permitirá rápida deterioração das coroas dentárias que acabam amputadas da raiz, expondo o canal radicular ao meio bucal, tornando-se fácil acesso para entrada de microrganismos nos tecidos periapicais. (Frank et al., 1965; Schweyen et al., 2012). Caso não seja instituído um protocolo de prevenção ou tratamento à CRR, pode ocorrer destruição generalizada dos dentes de pacientes oncológicos, representando um grande risco para o desenvolvimento de focos de infecção crônica e, consequentemente, de osteorradionecrose (Vissink et al., 2003; Silva et al., 2009). Geralmente, a deterioração dos tecidos dentais relacionada à CRR não está associada a queixa de dor espontânea, ou estimulada, por parte dos pacientes (Schweyen et al., 2012).
O conjunto de sinais e eventos clínicos “atípicos” descritos acima, no contexto da CRR, acaba por estimular clínicos e pesquisadores a atribuir o início e a progressão da CRR a danos radiogênicos diretos sobre a microestrutura dentária. Esta eventual destruição radiogênica seria responsável pela perda gradual da vitalidade pulpar e consequente retração dos processos odontoblásticos, gerando fragilidade na junção amelodentinária (JAD) (Dahllöf et al., 1994; Grötz et al., 2001). Outros autores sugeriram que alterações radiogênicas diretas alterariam o fluxo vascular da polpa e reduziriam a condução nervosa dos nociceptores pulpares, permitindo que a CRR progredisse de modo mais rápido na ausência dos mecanismos protetores de dor nos pacientes oncológicos irradiados (Knowles et al., 1986; Schweyen et al., 2012). Ainda no campo da vascularização pulpar, estudos clínicos demonstraram que os níveis de oxigenação da polpa diminuem durante a radioterapia para tumores de cabeça e pescoço. Contudo, o mesmo grupo de autores, em outro estudo mais recente, demonstrou níveis normais de oxigenação da polpa após 4 a 6 anos da conclusão do tratamento radioterápico, sugerindo que a radioterapia provavelmente não tem influência a longo prazo na vitalidade da polpa (Kataoka et al., 2011; Kataoka et al., 2016).
Os estudos supramencionados validam evidências sobre o fato de que a patogênese da CRR permanece controversa e incerta, especialmente no que se refere à capacidade da RDT em gerar efeitos diretos na estrutura dentária. Uma série de pesquisadores e especialistas acreditam que a CRR não deve ser atribuída aos efeitos diretos da RDT sobre os dentes; ao contrário, seria resultado de um “agrupamento de sintomas bucais” representados por efeitos indiretos da radiação que se desenvolvem em pacientes com CCPs submetidos à RDT, como as alterações quantitativas (hipossalivação) e qualitativas (microbiota bucal) da saliva que são
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notoriamente atribuídas ao aumento do risco de cárie (Kielbassa et al., 2006; Sciubba et al., 2006). Tais alterações na quantidade e na composição salivar alteram a microbiota oral, estimulando o desenvolvimento de microrganismos acidófilos com patogenicidade aumentada como Streptococcus mutans, Lactobacillus e espécies de Cândida (Al-Nawas et al., 2006; Kielbassa et al., 2006).
O “agrupamento de sintomas orais” é ainda composto por alterações na dieta do paciente com CPP submetido à RDT. O déficit nutricional típico deste perfil de pacientes oncológicos é compensado por meio da ingestão de alimentos mais calóricos, de consistência pastosa ou líquida, ricos em carboidratos, em porções pequenas e frequentes, o que favorece a adesão à superfície dental de um ambiente sem saliva e aumenta a predisposição à cárie rampante (Deng et al., 2015). Além disso, esta população enfrenta um desafio no controle da placa dental devido a um curto intervalo de tempo entre as refeições e também à dor relacionada à mucosite (induzida pela QRDT) que afeta diretamente o estímulo à higiene oral e a capacidade de remoção mecânica do biofilme dental (Vissink et al., 2003; Kielbassa et al., 2006; McGuire et al., 2014; Brennan et al., 2010).
Adicionalmente a esta teoria multifatorial acerca da patogênese da CRR, a hipótese do dano radiogênico direto para os tecidos dentários permanece latente na literatura científica. Estudos anteriores utilizando modelos experimentais animais ou técnicas de RDT já em desuso defendem a ideia de alterações pulpares em dentes submetidos a altas doses de radiação (Meyer et al., 1962; Hutton et al., 1974; Vier –Pelisser et al., 2007). Do mesmo modo, pesquisas in vitro, sustentam a existência de alterações na estrutura mineralizada de dentes irradiados (Pioch et al., 1992; Springer et al., 2005; De Barros da Cunha et al., 2017). Springer et al., (2005), concluiu em um estudo que a radiação é capaz de causar dano direto ao colágeno presente na polpa dental, não ocorrendo em tecido mineralizado devido a concentração relativamente baixa desta proteína na dentina e esmalte.
Contrariamente a estes resultados, estudos recentes de nossa equipe de pesquisadores -baseados em dentes irradiados in vivo- indicam que os efeitos diretos da radiação não são capazes de causar alterações micromorfológicas nos tecidos dentários. Em outras palavras, é pouco provável que a radiação ionizante dos protocolos oncológicos de RDT em cabeça e pescoço represente um fator de risco independente para o início e a progressão da CRR (Silva et al., 2009; Faria et al., 2014; Gomes-Silva et al., 2017).
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A despeito dos resultados mencionados, tornou-se imperioso aprimorar o conhecimento do impacto da RDT sobre o complexo dentina-polpa e sua habilidade de responder à progressão da CRR, tendo em vista os resultados de pesquisas sugerindo que os efeitos diretos da radiação limitariam a resposta do complexo dentina-polpa e justificariam a rápida progressão clínica da CRR (Springer et al., 2005; El-Faramawy et al., 2013; McGuire et al., 2014).
Portanto, o presente estudo se propôs a comparar os padrões microscópicos de resposta do complexo dentina-polpa à progressão da cárie convencional e da CRR, com a finalidade de testar a hipótese de que a RDT é capaz de modificar diretamente as respostas micromorfológicas do complexo dentina-polpa à progressão da CRR.
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2 ARTIGO
Este trabalho foi realizado no formato alternativo, conforme Informação CCPG/001/2015, da Comissão Central de Pós-Graduação (CCPG) da Universidade Estadual de Campinas.
Dentin-pulp complex reactions in conventional and radiation-related caries: A comparative study.
Jéssica Montenegro Fonsêcaa Wagner Gomes Silvaa,b Natalia Rangel Palmiera Pablo Agustin Vargasa Mario Fernando De Goesc Marcio Ajudarte Lopesa João Victor Salvajolid
Ana Carolina Prado Ribeiroa,b Thais Bianca Brandãoa,b Alan Roger Santos-Silvaa,b
a
Oral Diagnosis Department, Semiology and Oral Pathology Areas, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba, São Paulo, Brazil.
b
Dental Oncology Service, Instituto do Câncer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
c
Department of Dental Materials, Piracicaba Dental School, Av. Limeira, 901, Piracicaba, SP 13414-903, Brazil.
d
Radiotherapy Service, Instituto do Câncer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
Corresponding Author
Alan Roger Santos-Silva DDS, MSc, PhD Department of Oral Diagnosis, Semiology Area
Piracicaba Dental School, University of Campinas (UNICAMP)
Av. Limeira, 901, Bairro Areão, Piracicaba-SP, Brasil. CEP 13414-903 alanroger@fop.unicamp.br Telephone: +55 19 21065320
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ACKNOWLEDGMENTS
The authors would like to gratefully acknowledge the financial support of the São Paulo Research Foundation (FAPESP) processes numbers 2013/18402-8 and 2012/06138-1 as well as The Coordination for the Improvement of Higher Education Personnel
(CAPES/PROEX process number 758/2012). The authors also thank Fabiana Facco
Cassarotti and Adriano Luis Martins who provided technical support and contributed to the experimental development of the study.
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ABSTRACT
Introduction: radiation-related caries (RRC) is one of the most significant oral toxicities of head and neck radiotherapy (HNRT); however, the potential of radiation to directly cause harmful dentin and pulpal effects and impair response to caries progression is controversial. Therefore, the aim of this study was to characterize the reactions of the dentin-pulp complex in teeth affected by RRC. Methods: twenty-two carious teeth extracted from 22 head and neck cancer patients were divided into control (conventional caries; n=11) and irradiated (RRC; n=11) groups and matched by dental homology, clinical patterns of caries progression
following the Post-Radiation Dental Index (PRDI) and microscopic depth of carious invasion (1,158.58µm control vs. 1,056.89µm irradiated; p=0.79). Optical light microscopy and histomorphometry descriptively investigated histopathological characteristics based on morphological hierarchy and cell populations of dental pulp, including blood vessels, neural elements, extracellular matrix components, presence of inflammation, patterns of carious invasion and reactionary dentin presence. Results: pulp histopathological changes and dentin reaction patterns were similar between groups and varied according to the PRDI scores and carious lesions depth. Conclusions: dentin and pulp reactions to RRC are highly preserved. Clinical relevance: direct effects of radiation therapy may not be directly injurious to the pulp to the extent of impairing the response to caries progression.
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INTRODUCTION
Head and neck cancer (HNC) represent 6% of all human malignancies and
approximately 650,000 new cases are diagnosed annually worldwide. Treatment protocols often involve the combination of surgery, chemotherapy, and head and neck radiotherapy (HNRT). Although considered highly effective in the locoregional control of cancer, HNRT results in a myriad of acute and chronic toxicities to non-targeted tissues, including oral mucositis, hyposalivation, oral opportunistic infections, trismus, radiation-related caries (RRC) and osteoradionecrosis, among others [1, 2].
RRC, also known as “radiation caries”, is a chronic side effect of HNRT that affects up to 25% of patients who underwent HNRT. Its hallmark is a high potential for generalized dentition destruction and clinical patterns of progression that differ from conventional caries, being characterized by widespread cervical demineralization, incisal edges and cusp tips lesions and diffuse brownish to black discoloration of the smooth surface of enamel. RRC rapidly progresses causing enamel cracks, delamination and amputation of teeth crowns, leading to teeth destruction. In addition, it can increase the risk for the development of
osteoradionecrosis and negatively impact the overall oral function as well as the quality of life of cancer survivors [3, 4].
One of the most controversial topics in the scenario of HNRT side effects is the ability of ionizing radiation to cause direct radiogenic destruction to the teeth. Although many studies have suggested direct radiogenic damage to the dentin and pulp that would lead to RRC [5, 6, 7], others have linked the increased risk of caries in post-HNRT patients with the indirect effects of radiation therapy. These would include hyposalivation, oral microbiota alterations, impaired self-cleaning properties, poor oral health status prior to and after
treatment, increased dietary intake of carbohydrates, and insufficient fluoride exposure, which form a cluster of oral symptoms that predisposes patients to rampant caries regardless of the direct effect of radiation on teeth [8, 9, 10].
Hence, considering that HNRT is routinely used in more than 90% of all HNC patients [2], it is paramount to precisely understand its impact on the reactions of dentin and pulp to caries progression. To date, no study has been conducted to investigate biological evidence that radiation therapy could be directly injurious to the dentin-pulp complex and impair response to caries progression. Therefore, this study aimed to evaluate if in vivo irradiated
21
human teeth affected by RRC have microscopically discernible effect on dentin and pulp responses when compared to conventional caries teeth samples.
PATIENTS AND METHODS
Patients and specimen collection
This study was approved by the local Ethics Committee (protocol number 023/2015) and was conducted in accordance with the Declaration of Helsinki. Carious teeth (n=22) from HNC patients were collected independently of the particulars of the study. Dental extractions were performed due to advanced caries or periodontal disease in both teeth groups (control and irradiated). Immediately after the extractions, teeth were identified, placed in plastic containers with 10% buffered formalin solution and fixed for at least 72 h at 4 °C [3].
Eleven teeth affected by RRC were extracted from 11 patients with head and neck squamous cell carcinomas (SCC) who were subjected to clinical radiation protocols with tridimensional conformal HNRT in 6-mV linear accelerators on the Synergy Platform (Elekta AB, Stockholm, Sweden) with cumulative doses that ranged from60 to 70Gy (2 Gy/day, five days per week). The tridimensional HNRT plan of the patients was retrieved from the CMS system XiO version 4.60 (Elekta CMS software, St. Louis, MS, USA) to study the radiation field and the total dose directed to the teeth [11]. Eleven carious non-irradiated teeth
specimens were extracted from 11 head and neck SCC patients before radiation treatment during mouth conditioning protocols.
For clinical characterization of the patients, the electronic medical record system was consulted and the following data were collected: age, gender, tumor topography, alcohol consumption and smoking habit, tumor histological type, clinical cancer stage (according the American Joint Committee on Cancer), total radiation dose prescribed to tumor treatment (Gy), anatomic origin of extracted teeth, and time between the end of HNRT and teeth extraction.
Macroscopic analysis
All twenty-two carious teeth samples were divided into two groups: control (conventional caries; n=11) and irradiated (RRC; n=11), cataloged and subjected to
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dental homology (anatomic group), clinical patterns of caries progression established by the Post-Radiation Dental Index (PRDI) [12] and microscopic depth of carious invasion (refer to microscopy analysis).
Demineralization and histological preparation
All specimens were cleaned up with manual periodontal curettes to remove residual soft tissues and decalcified in Ana Morse’s solution (equal volumes of 20% sodium citrate and 50% formic acid) at 4 °C for three weeks, with changes every two days. The
decalcification was monitored and confirmed by weekly periapical radiographs. Specimens were sectioned along the longitudinal teeth axis through the center of the deepest carious lesions with the aid of a histologic disposable razor. The samples were embedded in Paraplast Plus® (Leica Biosystems Richmond, Inc., Richmond, IL, USA) to produce 5-µm-thick sections on a microtome (Leica, Nussloch, Germany) in silanized slides for hematoxylin and eosin (H&E) morphological evaluation.
Optical light microscopy analysis
An optical light microscope (OLM) (DM4000 B Leica, Wetzlar, Germany) was used for the micromorphological study of the cell populations of dental pulp, including blood vessels, neural elements, extracellular matrix components, inflammation, depth of carious invasion and reactionary dentin presence. Three demineralized histological sections of each specimen were analyzed and illustrative microscopic images were captured.
Two previously calibrated oral pathologists analyzed the slides. A descriptive analysis was performed for the morphologic criteria [13,14] regarding microscopic dentin and pulp reactions to caries progression, which were evaluated in a semi-quantitative way and compared between groups: presence and preservation of hierarchy of the dental pulp; presence and preservation of blood vessels; pulp extracellular matrix components (fibrosis, calcification, necrosis or inflammation) and the presence of reactionary dentin. Examiners were instructed to come to a consensus in discordant cases. Results were analyzed by using descriptive statistics, absolute values, and percentages.
Mean microscopic depth of caries invasion was quantitatively determined by measuring the distance from the surface of the demineralized dentin to the deepest point of
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caries-affected dentin in three demineralized histological sections of each specimen, in both groups, which was obtained in microns (µm) by using the software LAS version 4.2.0 (Leica Microsystems, Switzerland).
Statistical analysis
Morphological outcomes were descriptively analyzed and the results generated were analyzed by using descriptive statistics (Fisher's exact test), absolute values, and percentages. Mean values of microscopic depth of caries invasion for each specimen were compared between both groups using the Student’s t-test for independent samples. The software IBM SPSS Statistics for Windows version 22.0 (Armonk, NY, USA) was used with the
significance level set at α=0.05.
RESULTS
Demographic features and clinicopathological data obtained from the 22 patients are described in Table 1. The control and irradiated groups consisted of 6 molars (54.54%), 3 pre-molars (27.27%), 1 incisor (9.09%) and 1 canine (9.09%) each. The mean time for teeth extraction following HNRT was 33 months, ranging from 4 to 62 months. The mean dose received by each tooth sample was 53.47 Gy, ranging from 38.79 to 69.33 Gy (Table 1).
Mean PRDI scores were 3.2 for the control group and 3.8 for the irradiated group, with values ranging from 2 to 5 in both groups. The dentin demineralization patterns were similar between the groups and marked by triangular demineralization with the base at the tooth surfaces and the apex pointing towards the pulp. Demineralization depth due to caries progression was controlled during specimens’ selection, leading to a mean depth of 1,158.58 μm (ranging from 471,57 to 2,498.48 μm) in the control group and 1,056.89 μm (ranging from 750,75 to 1,407.54 μm) in the irradiated group (p=0.79).
All pulp specimens presented well-preserved polarized odontoblastic layers arranged in palisade, subodontoblastic cell-poor layers of Weil and central zones with prominent normal blood vessels, fibroblasts, and neural bundles. The microscopic analysis revealed the presence and the preservation of the pulp cellular layers hierarchy in 8 (72.7%) cases for the control group and 8 (72.7%) cases of the irradiated group (p=0.28) (Figures 1A and 1B). In the other cases of both groups, the presence and the preservation of the pulp cellular layers
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hierarchy was changed because of calcification, diffuse chronic inflammation and necrosis associated to bacterial invasion. Blood vessels presence and vascular architectural
preservation was observed in all (100%) samples of both groups.
All samples from both groups (100%) presented well-preserved subodontoblastic layers, which were closely related to well-preserve odontoblasts layers. Odontoblasts from all samples were characterized by tall columnar cells arranged in palisade and located at the periphery of the dental pulp. Cell processes arising from the odontoblasts' cell body could be observed penetrating into the dentin and in close contact between fibroblasts of all studied samples (Figures 2A and 3A).
Superficial caries-infected dentin composed of disorganized dentin and bacterial colonies, as well as an inner demineralized layer with affected, but not disrupted, dentin was consistently observed in all studied specimens of both groups (Figures 2B and 3B). Pulp extracellular matrix components were similarly detected in both groups and characterized by focal areas of fibrosis: 8 (72.7%) control cases vs. 7 (63.6%) irradiated cases (p=0.9);
calcification: 5 (45,5%) cases in each studied group (p = 0.34); necrosis: 4 (36.4%) control cases vs. 3 (27.3%) irradiated cases(p=1.00); and chronic inflammation: 7 (63.6%) control cases vs. 6 (55,5%) irradiated cases (p=0.28) (Figures 2C and 3C). Reactionary dentin formation was detected underlying caries demineralization fronts. The presence of reactionary dentin was observed in 6 (55,5%) cases of the control group and 5 (45,5%) cases of the
irradiated group (p=0.28) (Figures 2D and 3D).
No significant difference was encountered between irradiated and non-irradiated groups in any of the analyzed parameters. No evidence of abnormal cellular components or architecture could be detected.
DISCUSSION
The potential of HNRT to cause direct harmful dentin and pulpal effects that could impair the response of the dentin-pulp complex to caries progression is controversial. In this context, Kataoka et al. [15] showed that the levels of pulp oxygenation are decreased during radiotherapy for malignant intraoral and oropharyngeal tumors. More recently, the same group of authors [16] demonstrated normal pulp oxygenation levels after 4 to 6 years of the conclusion of HNRT, suggesting that radiation therapy may not have a long-term influence on
25
pulp vitality. Influenced by their results, we decided to perform a study to investigate
microscopic evidence that radiation therapy could be directly injurious to the pulp and impair response to caries progression by using teeth extracted after a mean time of 33 months following the conclusion of HNRT (ranging from 4 to 62 months).
The clinicopathological profile of patients enrolled in this study is in accordance with the traditional features of oral and oropharyngeal SCC patients observed worldwide [17, 18], which is marked by elderly male individuals, smokers and drinkers with poor oral health status who were diagnosed at late stages of tumor progression [19, 20, 21].
The clinical aggressiveness and the potential for generalized dental destruction have been well documented in RRC patients and predominately linked to post-radiation
hyposalivation [9, 22, 23]. However, observations based on in vitro studies proposed that the direct effects of HNRT on tooth-mineralized structure might also be a significant causal factor for RRC [5, 7, 12, 24].
Although the potential benefits in terms of reducing treatment-associated toxicities, the intensity-modulated radiotherapy (IMRT) technology is not available in many centers of the world, where tridimensional conformal radiotherapy (3DRT) is still routinely used [25, 26, 27]. In this context, 3DRT was the technology used in all of the patients investigated in the present study and its use is still widely accepted because no overall survival benefits have been observed in HNC patients treated with IMRT [28]. Most importantly, high doses of radiation were directed to the irradiated group samples of the current study (mean dose of 53.47Gy) and the influence of the radiation technique should not be considered an issue in terms of radiogenic effects, especially because a collaborative study recently demonstrated that IMRT and 3DRT deliver similar doses of radiation to the teeth of HNC patients [29].
One of the limitations of the current study was the small number of teeth specimens analyzed; wherein larger sample sizes would probably lead to more robust results. However, it is important to clarify that this limitation was a consequence of the strict methodological design used, which was based on a paired-matched sampling approach by teeth anatomic origin, clinical stage of RRC progression and microscopic depths of caries invasion. In addition, this study relied on human in vivo irradiated teeth (which are seldom collected because of the risk of osteoradionecrosis) rather than in samples irradiated in vitro.
Comparisons between studies that evaluated in vivo and in vitro effects of radiotherapy on dental pulp should be carefully established because in vitro simulated radiotherapy does not
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represent real clinical conditions concerning the cariogenic microenvironment or the dosimetric standards for HNRT [11].
From a microscopic point of view, patterns of dentin demineralization presented normal architecture [13,14] and varied according to the PRDI in both groups. The
microscopic analysis found homogeneous results between the groups, showing no statistical difference to the criteria of presence and preservation of hierarchy of the dental pulp; presence and preservation of blood vessels; pulp extracellular matrix components (fibrosis,
calcification, necrosis or inflammation) and the presence of reactionary dentin. Apparently, this was the first study to compare RRC and conventional caries specimens by using matched-paired teeth groups.
Previous studies based on animal experimental models or with an obsolete technique of radiotherapy, also investigated pulpal reactions related to radiation, but not to caries progression, and concluded that only teeth subjected to high doses of radiation (more than 50 Gy) showed alterations in the dental pulp tissue, including fibrotic and inflammatory
degenerations [30, 31]. Conversely, a recent study failed to detect morphological changes or altered patterns of immunohistochemical expression of proteins related to vascularization (CD34 and smooth muscle actin), innervation (S-100, NCAM/CD56, and neurofilament), and extracellular matrix (vimentin) in dental pulp after in vivo HNRT, showing broad preservation of the microvascular and neural pulp components following high doses of radiation [32].
In conclusion, the present study rejected the hypothesis that HNRT is able to impair the micromorphological pulp reactions to RRC progression. Therefore, direct effects of radiation may not be regarded as an independent factor to explain the rapid onset and aggressive clinical patterns of RRC progression.
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COMPLIANCE WITH ETHICAL STANDARDS Conflict of Interest
The authors declare that they have no conflict of interest.
Funding
- Research Foundation (FAPESP) processes numbers 2013/18402-8 and 2012/06138-
- Coordination for the Improvement of Higher Education Personnel (CAPES/PROEX) process number 758/2012.
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent
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TABLES
Table 1. Clinicopathological profile of studied patients
Ir
rad
iate
d
Group
Patient Age Gender Tobacco Alcohol Site T N M CH RDT Dose Dental Dose
1 58 M Yes Yes Tongue 3 0 0 Yes 3D 70 Gy 67.83 Gy
2 65 M Yes Yes Oropharynx 4 3 0 Yes 3D 70 Gy 38.79 Gy
3 52 F Yes Yes Tongue 4 2 0 No 3D 70 Gy 59.35 Gy
4 54 M Yes Yes Tongue 2 3 0 Yes 3D 70 Gy 46.71 Gy
5 56 F No No Nasopharynx 4 1 0 Yes 3D 70 Gy 63.83 Gy
6 65 F Yes Yes Tongue 3 0 0 No 3D 60 Gy 40.04 Gy
7 65 F Yes Yes Tongue 4 0 0 No 3D 70 Gy 69.33 Gy
8 66 M Yes Yes Tongue 4 2 0 Yes 3D 70 Gy 46.71 Gy
9 62 M Yes Yes Oropharynx 2 2 0 Yes 3D 70 Gy 53.11 Gy
10 47 M Yes Yes Oropharynx 1 2 0 Yes 3D 70 Gy 51.81 Gy
11 54 M Yes Yes Oropharynx 4 1 0 Yes 3D 70 Gy 51.81 Gy
Contr
ol G
rou
p
1 52 M Yes Yes Nasopharynx 3 3 0 - - - -
2 68 M Yes No Larynx 4 3 0 - - - -
3 76 M Yes Yes Submandibular gland 3 2 0 - - - -
4 55 M Yes Yes Hypopharynx 1 2 0 - - - -
5 60 M Yes Yes Larynx 4 3 0 - - - -
6 53 M Yes Yes Oral Cavity 4 2 0 - - - -
7 55 M Yes Yes Tongue 2 0 0 - - - -
8 61 M Yes Yes Soft Palate 4 0 0 - - - -
9 51 F Yes Yes Tongue 4 1 0 - - - -
10 48 M Yes Yes Oral Cavity 1 2 0 - - - -
11 61 M Yes Yes Soft Palate 1 2 0 - - - -
M: Male; F: Female; CH: Chemotherapy; RDT: Radiotherapy; 3D: Tridimensional conformal radiotherapy; Dental Dose: mean radiation dose delivered to each tooth.
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FIGURES
Figure 1. Microscopic overview of irradiated teeth crowns showing preservation of the dental pulp layers hierarchy (Hematoxylin and Eosin-stained sections, 2X magnification). A. Irradiated molar sample. B. Irradiated premolar sample.
Figure 2. Control group samples exhibiting preservation of the dental pulp layers hierarchy (Hematoxylin and Eosin-stained sections). A. Dentin (D), predentin (PD), odontoblasts (O) and pulp central region (C). B. Patterns of bacterial invasion of the dentin. C. Chronic
34
inflammation affecting irradiated pulp tissue. D. Preservation of the dental pulp micromorphology and dentin-pulp complex reactions to caries. Dentin (D), reactionary dentin (RD), predentin (PD), odontoblastic layer (O), cell-poor zone (CP), cell-rich zone (CR) and central region (C) with preserved fibroblasts, and vascular bundles.
Figure 3. Irradiated group samples exhibiting preservation of the dental pulp layers hierarchy (Hematoxylin and Eosin-stained sections). A. Presence of preserved neural vascular bundles, dystrophic calcification, and hyperemia in irradiated pulp tissue. Dentin (D), predentin (PD), odontoblasts (O) and pulp central region (C). B. Superficial caries-infected dentin composed of bacterial colonies and disorganized dentin. Inner demineralized layer with affected dentin showing normal patterns of bacterial invasion of the irradiated dentin. C. Chronic inflammation affecting irradiated pulp tissue. D. Preservation of the dental pulp layers hierarchy and dentin-pulp complex reactions to caries. Reactionary dentin (RD), cell-poor zone (CP), cell-rich zone (CR), central region (C), odontoblasts (O), predentin (PD), and dentin (D). Note the presence of preserved fibroblasts and neural vascular bundles.
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3 CONCLUSÃO
Não foram identificadas diferenças microscópicas significativas entre os padrões de resposta do complexo dentina-polpa entre dentes afetados por cárie convencional e CRR.
Este estudo rejeitou a hipótese de que a RDT é capaz de modificar diretamente as respostas micromorfológicas do complexo dentina-polpa à progressão da CRR.
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ANEXOS
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Anexo 2 - Certificado de submissão ao periódico
