alteraÇÕes do complexo dentina-polpa em cÁrie...

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

Programa de Pós-Graduação: Estomatopatologia

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.”

(Osho)

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.

Palavras-chave: Câncer; Radioterapia, Cárie Relacionada à Radiação, Dentes, Polpa

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, dentin-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.

Key Words: Cancer; Radiotherapy, Radiation-Related Caries, Teeth, Pulp.

SUMÁRIO

1 INTRODUÇÃO 12

2 ARTIGO: Dentin-Pulp Complex Reactions In Conventional And

Radiation-Related Caries: A Comparative Study

17

3 CONCLUSÃO 35

REFERÊNCIAS 36

ANEXOS

Anexo 1: Parecer Consubstanciado do CEP 40

Anexo 2: Certificado de submissão ao periódico: Clinical Oral

Investigations

41

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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

15

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).

16

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.

17

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

[email protected] Telephone: +55 19 21065320

mailto:[email protected]

18

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.

19

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.

Key Words: Cancer; Caries; Radiotherapy, Radiation-Related Caries, Teeth, Pulp.

20

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 from 60 to 70 Gy (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

photographic documentation. Teeth samples from both groups were classified and matched by

22

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

23

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

24

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

26

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.

27

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

Informed consent was obtained from all individual participants included in the study.

28

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32

TABLES

Table 1. Clinicopathological profile of studied patients

Irra

dia

ted

Gro

up

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


5 56 F No No Nasopharynx 4 1 0 Yes 3D 70 Gy 63.83 Gy







Con

trol

Gro

up

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.

33

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.

35

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.

36

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ANEXOS

Anexo 1 – Parecer Consubstanciado do CEP

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Anexo 2 - Certificado de submissão ao periódico

alteraÇÕes do complexo dentina-polpa em cÁrie...

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