UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
LENITA MARANGONI LOPES
EFEITOS DA RADIAÇÃO GAMA E DO FLUORETO SOBRE
DENTES DECÍDUOS – ESTUDOS IN VITRO.
Effects of gamma radiation and fluoride on primary teeth - in vitro studies.
Piracicaba 2018
LENITA MARANGONI LOPES
EFEITOS DA RADIAÇÃO GAMA E DO FLUORETO SOBRE DENTES DECÍDUOS – ESTUDOS IN VITRO.
Effects of gamma radiation and fluoride on primary teeth - in vitro studies.
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Odontologia, na Área de Odontopediatria.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dentistry, in Pediatric Dentistry area.
Orientador: Profª. Drª. Marinês Nobre dos Santos Uchôa
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA LENITA MARANGONI LOPES, E ORIENTADA PELA PROFA. DRA.
MARINÊS NOBRE DOS SANTOS UCHÔA.
Piracicaba 2018
DEDICATÓRIA
AGRADECIMENTOS
À Universidade Estadual de Campinas, na pessoa do seu Magnífico Reitor Prof.
Dr. Marcelo Knobel; à Faculdade de Odontologia de Piracicaba, na pessoa do seu diretor Prof. Dr. Guilherme Elias Pessanha Henriques; à coordenadora da Pós-Graduação da
FOP-UNICAMP Profª. Drª. Cínthia Pereira Machado Tabchoury; ao coordenador do programa de Pós-Graduação em Odontologia FOP- UNICAMP Prof. Dr. Marcelo de
Castro Meneghim agradeço a oportunidade de poder fazer parte como aluna de
pós-graduação desta conceituada universidade.
Ao Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela concessão da bolsa de Doutorado.
À minha orientadora, Profª. Drª. Marinês Nobre dos Santos Uchôa, por toda a orientação, paciência, exemplo, incentivo e conhecimentos transmitidos.
As professoras do departamento de Odontologia Infantil da FOP-UNICAMP
Profª. Drª. Carolina Steiner Oliveira Alarcon, Profª. Drª. Fernanda Miori Pascon, Profª. Drª. Maria Beatriz Duarte, Profª. Drª. Marinês Nobre dos Santos Uchôa e Profª. Drª. Regina Puppin Rontani por todo o conhecimento transmitido a mim ao longo
da minha pós-graduação.
Ao técnico do laboratório de Odontopediatria Sr. Marcelo Corrêa Maistro pela ajuda nas análises bioquímicas.
À secretária do departamento de Odontologia Infantil, Sra. Shirley Rosana
Sbravatti Moreto pelo apoio recebido.
Aos professores da banca do exame de qualificação, Profª. Drª. Débora Alves
Nunes Leite Lima, Prof. Dr. Matheus Lima De Oliveira e Prof. Dr. Rafael Nobrega Stipp e aos professores da banca de Defesa desta Tese, Profª. Drª. Cínthia Pereira Machado Tabchoury, Profª. Drª. Fernanda Miori Pascon, Profª. Drª. Lidiany Karla Rodrigues Azevedo, Profª. Drª. Marinês Nobre dos Santos Uchôa, e Profª. Drª. Thaís Manzano Parisotto, por todas as contribuições dadas a esta Tese.
À Accecil, ao Departamento de Radiologia da FOP - Unicamp, ao Hospital
- Unicamp, e à Universidade Vale do Paraíba e por possibilitarem a execução de
algumas etapas desta pesquisa.
Agradeço a Deus, a quem deposito minha fé e me apoio em todos os momentos, entregando minha vida e meus caminhos.
À minha mãe Marcia e minha irmã Amanda, pelo amor incondicional, apoio e compreensão. Ao meu pai Aloisio, por todas as lições que deixou.
Ao Jair, por todo o carinho, apoio e pela compreensão nos momentos de ausência. À toda minha família, por estarem sempre ao meu lado. Aos amigos da pós-graduação, em especial a Andréia Cardoso, Bruna Zancopé, Camila Freitas, Daniele
Picco, Emerson Tavares, Darlle Araújo, Gabriela Borghi, Lívia Pagotto e Micaela Cardoso pela ajuda e pelos momentos de descontração.
RESUMO
OBJETIVO: Avaliar se a radioterapia por radiação gama causa mudanças na composição mineral e orgânica, microdureza e morfologia do esmalte e da dentina dos dentes decíduos; na susceptibilidade dos dentes decíduos irradiados ao desafio cariogênico; e se a associação de dentifrício fluoretado e aplicação tópica de flúor fosfato acidulado (FFA) inibe a perda mineral do esmalte decíduo irradiado. MATERIAL E MÉTODOS: No artigo I, os espécimes de esmalte e a dentina de dentes decíduos (n=30) foram submetidos à microdureza superficial, composição (Raman) e microscopia eletrônica de varredura (MEV) antes, e ao atingir as doses de 1080, 2160 e 3060 cGy de radiação. No artigo II, foi utilizado um modelo biológico para avaliar a susceptibilidade do esmalte decíduo irradiado ao desafio cariogênico (n=18). A profundidade, área e volume da lesão da cárie foram avaliados por microtomografia computadorizada e MEV. No artigo III, espécimes foram randomizados em oito grupos (n=13): Não irradiado + Dentifrício não fluoretado (DNF) (G1); Não irradiado + Dentifrício fluoretado (DF – 1100 ppm) (G2); Não irradiado + DNF + FFA (G3), Não irradiado + DF + FFA (G4); Irradiado + DNF (G5); Irradiado + DF (G6); Irradiado + DNF + FFA (G7); Irradiado + DF + FFA (G8). Todos os grupos foram submetidos a ciclagem de pH e ao respectivo dentifrício duas vezes ao dia. Foi realizada microdureza transversal e microscopia de luz polarizada. RESULTADOS: Os resultados do artigo I mostraram que a microdureza da superfície do esmalte diminuiu ao atingir a dose de 2160 cGy. Para a dentina, a microdureza da superfície diminuiu ao atingir 1080 cGy e 2160 cGy. O conteúdo mineral de fosfato, carbonato, amida e hidrocarbonetos do esmalte diminuiu ao atingir 3060 cGy. Para a dentina, observamos um aumento crescente do conteúdo de fosfato v1, amida e hidrocarbonetos ao atingir as doses de 1080 e 2160 cGy e uma redução ao atingir 3060 cGy. As imagens por MEV mostraram trincas no esmalte e a degradação da dentina peri-tubular. No artigo II, observou-se que a profundidade e a área da lesão no grupo irradiado foram significativamente maiores do que no grupo não irradiado. No entanto, o volume da lesão não diferiu entre os grupos. As imagens por MEV mostram perda de estrutura do esmalte no grupo irradiado. No artigo III, os grupos G1 e G3 apresentaram perda mineral estatisticamente maior do que G2 e G4. Não foi encontrada diferença estatística entre os grupos irradiados. As imagens por microscopia de luz polarizada mostraram maior birrefringência positiva nos grupos G5, G6, G7 e G8, e presença da linha de remineralização nos grupos G2, G3, G4 e G6. CONCLUSÃO: A radioterapia usando radiação gama causou redução da microdureza da superfície, alteração da composição
mineral e orgânica e alterações morfológicas no esmalte e na dentina dos dentes decíduos. Essas mudanças tornaram os dentes decíduos irradiados mais susceptíveis ao desafio cariogênico e o uso de dentifrício fluoretado e FFA não teve sua efetividade detectada na redução da perda mineral de dentes decíduos irradiados.
ABSTRACT
AIM: To evaluate whether the radiotherapy with gamma radiation causes changes on the mineral and organic composition, microhardness, and morphology of enamel and dentin of primary teeth; the susceptibility of primary teeth to a cariogenic challenge; and if the association of fluoride dentifrice and topical application of fluoride reduces the mineral loss of irradiated primary enamel. MATERIAL AND METHODS: In Article I, specimens of primary enamel and dentin (n=30) were subjected to surface microhardness, composition, and scanning electron microscopy (SEM) analyses before, at 1080, 2160, and 3060 cGy of gamma radiation. In Article II, a biological model was used to evaluate the susceptibility of the irradiated primary enamel(n=18) to the cariogenic challenge. The depth, area, and volume of the caries lesion were evaluated using microcomputer tomography, and a qualitative analysis was performed using SEM analysis. In Article III, 104 specimens of primary enamel were randomized into eight treatment groups (n=13): Non-irradiated + Non-fluoride dentifrice (NFD) (G1); Non-irradiated + Fluoride dentifrice (FD – 1100 ppm) (G2); Non-irradiated + Acidulated phosphate fluoride (APF-gel) + NFD (G3), Non-irradiated + APF-gel + FD(G4); Irradiated + NFD (G5); Irradiated + FD (G6); Irradiated + APF-gel + NFD (G7); Irradiated + APF-gel + FD (G8). All groups were submitted to a pH cycling with the respective dentifrice twice daily. Specimens were submitted to transverse microhardness analysis and polarized light microscopy (PLM) analysis. RESULTS: The results of Article I showed that microhardness of the enamel surface decreased at 2160 cGy of radiation. For dentin, the surface microhardness decreased at 1080 cGy and 2160 cGy doses. The phosphate, carbonate, amide III, and hydrocarbons contents of enamel, decreased at 3060 cGy. For dentin, we noticed a growing increase in phosphate v2, amide III and hydrocarbons content at 1080 and 2160 cGy and a reduction at 3060 cGy. SEM images showed a cracked aspect on enamel surface, and degradation of peri-tubular dentin. In Article II, it was observed that the lesion depth and area of caries lesion in the irradiated group were significantly higher than in the non-irradiated group. However, the lesion volume did not differ among the groups. The SEM images showed enamel breakdown in irradiated group. In Article III, G1 and G3 groups showed statistically higher mineral loss than G2 and G4 groups. The reduction of mineral loss provided by fluoride dentifrice, APF application as well as by their combination, did not differ from control group. The PLM images show a higher positive birefringence in the groups G5, G6, G7, and G8, and the presence of a surface layer of remineralization in groups G2, G3, G4 and G6. CONCLUSION: Radiotherapy
with gamma radiation reduced surface microhardness, changed mineral and organic composition, and promoted morphological changes on the enamel and dentin surface of primary teeth. These changes rendered irradiated primary teeth more susceptible to a cariogenic challenge. In addition, the effectiveness of fluoride dentifrice and APF-gel in reducing the mineral loss of gamma irradiated primary teeth could not be demonstrated. Key words: Deciduous Tooth. Dental Caries. Radiotherapy.
SUMÁRIO
1 INTRODUÇÃO 13
2 ARTIGOS
2.1 Artigo: Radiotherapy changes microhardness,
composition, and morphology of primary teeth - In vitro study. 15
2.2 Artigo: Susceptibility of gamma irradiated primary teeth
to a cariogenic challenge - In vitro study. 34
2.3 Artigo: Effect of association of fluoride dentifrice and
APF-gel on the susceptibility of gamma irradiated primary teeth
to the mineral loss - In vitro study. 46
3 DISCUSSÃO 58
4 CONCLUSÃO 61
REFERÊNCIAS 62
ANEXO 1 – Certificado do Comitê de Ética e Pesquisa 65
1 INTRODUÇÃO
Como consequência da radioterapia, dependendo da área de abrangência, do tipo de tecido irradiado, da dose de radiação e do protocolo de tratamento, podem ocorrer efeitos colaterais nas células normais de tecidos adjacentes que se encontram na direção do feixe de radiação (Peterson e D'Ambrosio, 1992). Quando a radioterapia incide na região supraclavicular, podem ocorrer então, alterações nas glândulas salivares presentes na direção do feixe de radiação, o que pode afetar a produção e composição de saliva (Marangoni-Lopes et al., 2016), e/ou promover modificações nos tecidos dentários. Essas modificações poderiam tornar o esmalte/dentina mais susceptíveis ao desenvolvimento de cárie.
De fato, crianças submetidas à radioterapia na região de cabeça e pescoço, apresentam maior prevalência de cárie dental (Pajari et al., 1995; Gawade et al., 2014). Neste contexto, o estudo realizado pelo nosso grupo de pesquisa (Marangoni-Lopes et al., 2016) mostrou que crianças com Linfoma de Hodgkin, irradiadas na região cervical, tiveram redução do fluxo salivar e da capacidade tampão da saliva o que pode significar menor capacidade de autolimpeza da cavidade bucal e neutralização dos ácidos. Quanto às alterações nos tecidos dentários devido à radioterapia, os resultados de uma revisão sistemática da literatura mostram grande quantidade de estudos revelando alterações e danos aos tecidos dentários permanentes (Lieshout e Bots, 2014).
Os efeitos da radiação sobre as estruturas dentárias dependem da dose de radiação, da composição e conteúdo dessas estruturas. O esmalte dentário é formado por 92-96% em peso de matéria inorgânica, o que o torna suscetível a danos em sua porção mineral (Gwinnett, 1992). Pesquisas realizadas com dentes permanentes submetidos à radiação mostraram destruição da estrutura prismática do esmalte dentário, observada por microscopia confocal (Grötz et al., 1998), diminuição das propriedades mecânicas, como resistência à tração (Soares et al., 2010), e ao ataque ácido (Grötz et al., 1998). No entanto, alterações em dentina decorrentes da radioterapia resultam numa redução significativa da microdureza desse substrato (Fränzel e Gerlach, 2009, Gonçalves et al., 2014), da resistência à tração (Soares et al., 2010), e da estabilidade da junção amelo-dentinária (Pioch et al., 1992; Madrid et al., 2017) com redução da birrefringência à luz polarizada e aumento dos espaços interprismáticos do esmalte cervical.
Com relação aos dentes decíduos, a literatura demonstra que estes, apresentam características morfológicas e físico-químicas diferentes daquelas observadas para os dentes permanentes, substrato utilizado nos estudos já descritos na literatura. O dente
decíduo apresenta menor espessura de esmalte (de Menezes Oliveira et al., 2010), maior porosidade (Zamudio-Ortega et al., 2014) e menor grau de mineralização com um maior conteúdo de carbonato (Sonju Clasen, 1997). Sendo os efeitos da radiação dependentes da composição mineral e orgânico, estas características orgânicas e inorgânicas diferentes do dente permanente podem resultar também em um comportamento diferente frente à exposição radioterápica.
Um estudo recente mostrou alterações estruturais observadas por microscopia eletrônica de varredura no esmalte e na dentina, bem como na microdureza transversal de dentes decíduos irradiados com até 6000 cGy de radiação (de Siqueira Mellara et al., 2014). Outro estudo mostrou redução no conteúdo de orgânico da dentina em dentes decíduos irradiados com 5400 cGy (de Sá Ferreira et al., 2015). Essas alterações, na presença de um alto desafio cariogênico, poderiam tornar o esmalte mais susceptível à desmineralização. De fato, neste estudo, a ciclagem de pH reduziu o conteúdo de fósforo e a proporção Ca/P no esmalte e o conteúdo de carbonato na dentina de dentes decíduos irradiados (de Sá Ferreira et al., 2015). Além disso, alterações na junção amelo-dentinária e na dentina, poderiam promover uma rápida progressão de cárie. Sendo assim, o primeiro objetivo da presente tese foi avaliar se a radiação gama causa mudanças na composição mineral e orgânica, microdureza e morfologia do esmalte e da dentina dos dentes decíduos e o segundo objetivo desta tese foi investigar a susceptibilidade dos dentes decíduos irradiados por radiação gama ao desafio cariogênico.
Considerando-se a maior susceptibilidade dos dentes decíduos ao desafio cariogênico, a utilização de fluoreto torna-se imprescindível na prevenção da cárie dentária (dos Santos et al., 2013). Dentre os métodos tópicos de uso de fluoreto, o meio mais racional é o dentifrício fluoretado, por manter constantes os níveis de fluoreto no meio bucal (Pessan et al., 2015). Em pacientes considerados de alto risco à cárie, como aqueles em tratamento radioterápico, a associação de outros métodos de uso de fluoreto, por exemplo, a aplicação tópica de flúor fosfato acidulado (FFA) combinada com o uso de dentifrício fluoretado tem sido indicada (Adair, 2006). Dessa forma, o terceiro objetivo desta pesquisa foi avaliar se a associação de dentifrício fluoretado com a aplicação tópica de FFA, reduz a perda mineral do esmalte decíduo irradiado por radiação gama. Com os três objetivos, esta tese abrangeu as perguntas de pesquisa sequenciais: se a radiação gama interfere nas propriedades químicas, físicas e morfológicas do esmalte e da dentina de dentes decíduos, se o esmalte irradiado se comporta de maneira diferente quando submetido a um desafio cariogênico e se a associação de dentifrício fluoretado e aplicação tópica de FFA reduz a perda mineral do esmalte decíduo.
2 ARTIGOS
2.1. Radiotherapy changes microhardness, composition, and morphology of primary teeth - In vitro study.
Lenita Marangoni-Lopes, Gabriela Rovai Pavan, Carolina Steiner-Oliveira, Marinês Nobre-dos-Santos.
Artigo submetido ao periódico Caries Research (Anexo I).
Abstract
Objective: We aimed to evaluate whether radiotherapy causes changes in the mineral composition, hardness, and morphology of enamel and dentin of primary teeth. Materials and Methods: Thirty specimens of primary teeth were subjected to radiotherapy. At baseline and after 1080, 2160, and 3060 cGy, the specimens were subjected to microhardness, FT-Raman spectroscopy, and scanning electron microscopy (SEM) analysis. The pH of artificial saliva was determined, as were the calcium and phosphate concentrations. The data were subjected to the Shapiro-Wilks normality test, showed a non-normal distribution, and were compared by the Kruskal-Wallis test. Results: The results showed that the microhardness of the enamel surface decreased after 2160 cGy (281.5±58) (p=0.045). For dentin, the surface hardness decreased after 1080 cGy (34.9±11.4) and 2160 cGy (26±3.5) (p<0.0001). The mineral and organic contents of phosphate (p<0.0001), carbonate (p<0.0001), amide (p=0.0002), and hydrocarbons (p=0.0031) of enamel decreased after 3060 cGy (5178±1082, 3868±524, 999±180, and 959±168, respectively). For dentin, we noticed a growing increase in phosphate v2, amide, and hydrocarbon content after 1080 (8210±2599, 5730±1818, and 6118±1807, respectively) and 2160 cGy (10071±2547, 7746±1916, and 8280±2079, respectively) and a reduction after 3060 cGy (6782±2175, 3558±1884, and 3565±1867, respectively) (p<0.0001). SEM images showed cracks on enamel and degradation of peri-tubular dentin. Conclusion: We concluded that radiotherapy caused a reduction in surface hardness, changed mineral composition, and promoted morphological changes on the enamel and dentin of primary teeth.
Introduction
Children subjected to head and neck radiotherapy have a higher prevalence of dental caries [Tulunoglu et al., 2006], related to changes in their diet and/or oral microbiota and difficulty in performing adequate oral hygiene. In addition, direct factors such as changes in the salivary glands can also affect salivary production and composition. In this regard, a recent study conducted by our research group showed that children with Hodgkin's lymphoma and receiving radiation in the cervical region of the body had reduced salivary flow and salivary buffering capacity [Marangoni-Lopes et al., 2016]. These changes in salivary properties may contribute to less self-cleaning capacity of the oral cavity and to a reduced acid neutralization capacity of saliva. In this context, the susceptibility of irradiated enamel to dental erosion also seems to be increased in oncology patients, since they suffer from low salivary flow rates [Järvinen et al., 1991]. In addition, according to a literature review, the higher caries prevalence observed in these patients is not only a result of a reduced salivary flow rate but also a consequence of the direct effects of radiation on the dental hard tissue [Lieshout and Bots, 2014].
The effects of radiotherapy on teeth are dependent on the mineral and organic composition of the structures of enamel or dentin [Gwinnett, 1992]. Studies concerning permanent teeth undergoing radiotherapy showed destruction of the prismatic structure of dental enamel [Grötz et al., 1998], reduction of mechanical properties such as tensile strength [Soares et al., 2010], wear resistance [Jervoe, 1970], and acid attack resistance [Grötz et al., 1998]. However, changes in dentin caused by radiotherapy are due to damage to collagen fibrils [Cheung et al., 1990], resulting in a significant reduction of the hardness and elasticity of this substrate [Fränzel et al., 2009], tensile strength, and stability of the amelo-dentin junction [Pioch et al., 1992].
The features of the primary teeth differ from those of the permanent teeth and include thinner thickness and higher numerical density of rods [Zamudio-Ortega et al., 2014], abundant microporosities, exposed prisms, and major carbonate incorporation [de
Menezes Oliveira et al., 2010]. These differing features are responsible for making primary teeth more susceptible to the development and progression of caries. In addition, these peculiarities may result in different effects of radiotherapy on the dental structures of primary teeth. In this way, it would be relevant to investigate how the enamel and dentin of primary teeth behave under radiotherapy. A recent study [de Siqueira Mellara et al., 2014] showed that the transverse microhardness of enamel decreased at a dose of 6000 cGy, whereas the values of dentin microhardness did not change. When one considers that the lower the microhardness, the lower the mineral content, another important aspect to be investigated is the mineral composition of irradiated enamel and dentin. In this regard, an early study [de Sá Ferreira et al., 2015] evaluated the effects of radiotherapy on the composition of primary enamel and concluded that radiotherapy reduced the organic content of the primary enamel. However, in this study, the authors evaluated only the changes occurring in enamel after the final 5400 cGy dose. In this way, their findings may not represent what really occurs in enamel, if we consider that there are treatment protocols with lower radiation doses for children with cancer. Thus, it would be relevant to evaluate the effects of a lower irradiation dose on primary teeth.
Therefore, the aim of this study was to investigate whether irradiation of enamel and dentin with 1080, 2160, and 3060 cGy causes changes in the mineral composition, hardness, and morphology of the enamel and dentin of primary teeth.
Materials and methods Experimental Design
For this study, we used 30 specimens of primary teeth measuring 3x3x2 mm. These specimens were kept in artificial saliva and subjected to 17 daily fractions of 180 cGy radiation, resulting in a total dose of 3060 cGy. This protocol simulates the German Society of Pediatric Oncology and Hematology–Hodgkin's Disease (GPOH-HD95) protocol, used in the treatment of Hodgkin’s lymphoma. The specimens were subjected to surface microhardness and mineral composition (RAMAN) analyses in four
experimental phases: prior to radiotherapy (baseline) and after the completion of 1080, 2160, and 3060 cGy of radiation therapy. In addition, pH and concentrations of calcium and phosphate of the artificial saliva were determined. At each experimental phase, two specimens were set apart for scanning electron microscopy (SEM) analysis. The results were compared among the experimental phases.
Ethical Considerations
This study was approved by the Committee of Research Ethics of Piracicaba Dental School (Protocol Number 024/2014), and the Certificate of Presentation for Ethical Appreciation number is 27478814.0.0000.5418. This study was conducted in compliance with the Helsinki Declaration.
Sample Preparation
To perform this study, we used the same sample size as that used by Soares et al. [2010] (n = 20), increased by 20% to prevent possible losses (n = 24) and increased 2 extra specimens per phase for SEM analysis (n = 30).
The donated caries-free primary teeth were stored in thymol (0.1 M, pH 7.0). After
inspection, teeth with developmental defects were excluded. The selected teeth were cut
in the neck portion to separate the dental crown from the root, by means of a cutting machine (Isomet 1000, Buehler®, Lake Bluff, IL, USA) with a double-sided diamond disc (Buehler®). The crowns were then sectioned, yielding specimens with dimensions of 3x3x2 mm on the buccal and lingual surfaces. For specimen grinding and polishing, an
APL-4 polisher (AROTEC, Cotia, SP, Brazil) was used. For enamel polishing, sandpaper grains of 600 and 1200 and felt discs with diamond solution (Buehler®) were used for 1 min each. For dentin, we used sandpaper grains of 800 and 1200 and felt discs with a diamond solution for 1 min each. Specimens were then washed in ultrasound USC 1400
(Unique, São Paulo, SP, Brazil) for 2 min, with distilled and deionized water after each sandpaper and detergent solution use (Buehler®).
The specimens were numbered, and the initial surface microhardness of the enamel and dentin surfaces were measured. Three indentations were created near the central region by means of the microhardness tester FM-ARS Tech Future (Future-Tech Corp., Tokyo, Honshu, Japan) with a Knoop indenter, with loads of 50 kgf for enamel and 25 kgf for dentin, for 5 s. The mean and standard deviation of Knoop hardness number were calculated for each specimen and each surface (enamel and dentin). Samples were excluded from the experiment if the mean Knoop hardness was greater or lower than the general mean (300.76 for enamel and 58.16 for dentin) plus two times the standard deviation (46.70 for enamel and 7.67 for dentin). This procedure was performed to standardize the enamel and dentin microhardness, and outliers were excluded.
Enamel and Dentin Irradiation
Daily fractions of 180 cGy were applied every 5 days, with a 2-day interval that corresponded to the weekend. This approach aimed to simulate the radiotherapy protocol used in children with cancer like Hodgkin's lymphoma. In total, the specimens received 17 daily fractions of 180 cGy of gamma radiation, resulting in a total dose of 3060 cGy. This phase of the study was performed at the Oncology Center (Ceon) of Cane Suppliers Hospital (Piracicaba, São Paulo, Brazil). The specimens were kept in individual wells in a 24-well plaque and immersed in artificial saliva containing 1.5 mM Ca, 0.9 mM PO4,
and 150 mM KCl in 20 mM Tris buffer, pH 7.0. Considering 0.5 cm of artificial saliva, 1 cm of air, and a bolus of 1.5 cm height, we used the Varian - Clinac 6EX (Varian, Palo Alto, CA, USA) with a linear accelerator and beam photons of 6 MeV to irradiate the specimens with a 99.9% margin of the total dose, using the following parameters: focus
of 100 cm in a campus of 15x15 cm. After each phase of the study, the enamel and dentin samples were placed in new artificial saliva.
Enamel and Dentin Microhardness Analysis
To evaluate microhardness, we created three indentations near the central area of each enamel and dentin specimen, using a microhardness tester, FM-ARS Tech Future (Future-Tech Corp., Tokyo, Honshu, Japan) with a Knoop indenter with a 50 kgf load for enamel and 25 kgf load for dentin, for 5 sec. The average of the three indentations was calculated for each specimen of enamel and dentin, and the final median microhardness of enamel and dentin were calculated and expressed as the Knoop hardness number (KHN).
FT-Raman Spectroscopy Analysis
The spectrum was obtained by means of an FT-Raman spectroscope RFS 100/S (Bruker Inc., Karlsruhe, Baden-Württemberg, Germany) with a Ge diode detector cooled with liquid nitrogen. To promote spectrum excitation, the FT-Raman spectroscope used a Nd:YAG ƛ = 1064.1 nm air-cooled laser operating in the focus mode. The maximum energy of the incident laser on the specimen’s surface was 77 mW with a spectrum resolution of 4 cm-1. The specimens were placed in a sample-holder, and the lens collected the radiation transmitted within an angle of 180°. The FT-Raman spectra were obtained from 700 scans with explored frequencies ranging from 400 to 1800 cm-1. From each enamel and dentin specimen, three spectra were obtained at three different points in a circular area of 2 mm diameter delimited by a black pencil on the surface, with Microcal Origin5.0 software (Microcal Software, Northampton, MA, USA). This analysis was performed twice, once in the enamel face and another on the dentin face. The peak intensities of 430 and 961 (phosphate v2 and v1), 1071 (carbonate), 1245 (amide), and
1450 cm-1 (hydrocarbons) bands of the three spectra for enamel and for dentin were isolated. The medians of the three spectra of each peak intensity of each band were calculated and expressed as arbitrary units (AU) for statistical analyses
Determination of Calcium/Phosphate and pH of Artificial Saliva
After each experimental phase, the artificial saliva in which the specimens were kept during enamel and dentin irradiation was subjected to pH measurement and determination of calcium and phosphate concentrations. These analyses were performed with the aim of observing if possible changes occurring in enamel and dentin after irradiation would have any effect on the saturation of artificial saliva. The pH was determined by means of the Accumet® microelectrode (Cole-Parmer International, Vernon Hills, IL, USA) coupled to a pre-calibrated Orion® pH meter (Thermo Scientific, Waltham, MA, USA). The calcium and phosphate concentrations were determined with Bioclin Kits (Bioclin/Quibasa, Belo Horizonte, MG, Brazil) with the spectrophotometer previously calibrated with a blank and a standard containing 10 mg/dL of calcium and 5.0 mg/dL of
phosphate. The results were expressed as mg/mL.
Scanning Electron Microscopy Analysis
At each experimental phase, two specimens were dehydrated in silica for at least 48 h while fixed in stubs with double-sided carbon tape (Electron Microscopy Sciences, Washington, USA), and covered with gold-palladium (Balzers sputter coater SCD 050, Leica Microsystems, São Paulo, Brazil). Then, the specimens were examined under a scanning electron microscope (JEOL, JSM – 5600 LV, Tokyo, Japan) at 15 kV acceleration voltage. Standardized images were acquired at a 2500 magnification for the enamel surfaces and a 1500 magnification for the dentin surfaces.
Statistical Analysis
The data for Knoop hardness number, mineral and organic content, pH, and calcium/phosphate concentrations in artificial saliva were subjected to the Shapiro-Wilks normality test and were shown to follow a non-normal distribution. All data were compared longitudinally along the experimental phases (paired data) by the Kruskal-Wallis test. For these analyses, a 5% significance level was established. The tests were performed with Bioestat software (Ayres, Belem, PA, Brazil).
Results
Enamel and Dentin Microhardness
For the enamel, the surface hardness after 2160 cGy radiation (281.5 ± 58.0) was significantly lower (p =0.045) than that observed at baseline (323.6 ± 59.5). After 1080 (301.6 ± 62.7) and 3060 cGy (312.9 ± 38.2), the values were intermediate and did not differ from those of the other phases. The dentin surface hardness decreased after 1080 cGy (34.9 ± 11.4) and decreased further after 2160 cGy (26.0 ± 3.5) (p< 0.0001) when compared with previous phases. However, after 3060 cGy (26 ± 6.8), the surface hardness showed intermediate values, differing only from baseline values (56.5 ± 7.7) (Figure 1).
Figure 1. Medians and interquartile ranges of enamel surface microhardness along
the experimental phases (n=24). Distinct letters indicate statistically significant differences (p <0.05). Bars represent the interquartile deviations.
A AB B AB 0 50 100 150 200 250 300 350 400
Baseline 1080 cGy 2160 cGy 3060 cGy
Kn o o p H ard ne ss Numbe r (KH N)
Figure 2. Medians and interquartile ranges of dentin surface microhardness along
the experimental phases (n=24). Distinct letters indicate statistically significant differences (p <0.05). Bars represent the interquartile deviations.
Enamel and Dentin Mineral Composition
The contents of phosphate v2 (p < 0.0001), carbonate (p < 0.0001), amide (p = 0.0002), and hydrocarbons (p = 0.02) in enamel decreased significantly after 3060 cGy as compared with baseline values as well as with lower doses. After 2160 cGy, phosphate
v1 content was lower than that observed after 1080 cGy (p = 0.01), but did not differ from
baseline (p > 0.05). In addition, a significant reduction (p = 0.0002) in phosphate v1 content was noted after 3060 cGy (Table 1). For dentin, a significant and progressive increase in phosphate v2, amide, and hydrocarbon content was observed after 1080 (p = 0.01, p = 0.004, and p = 0.04, respectively) and 2160 cGy radiation (p < 0.0001). In addition, a significant reduction in phosphate v2, amide, and hydrocarbon content was found after 3060 cGy radiation (p < 0.0001, p = 0.002, and p = 0.01, respectively). The carbonate content increased only after 2160 cGy and decreased after 3060 cGy (p < 0.0001). Concerning phosphate v1 content, a significant increase was observed only after 3060 cGy (p < 0.0001) (Table 2). A B C BC 0 10 20 30 40 50 60 70
Baseline 1080 cGy 2160 cGy 3060 cGy
Kn o o p H ard ne ss Numbe r (KH N)
Table 1. Medians and interquartile deviations of phosphate, carbonate, amide III, and
hydrocarbons content (AU) in enamel along the experimental phases (n=24).
Baseline 1080 cGy 2160 cGy 3060 cGy
Phosphate v1 51630 ± 496 B 57936 ± 6705 A 54280 ± 3915 B 44805 ± 6822 C Phosphate v2 6265 ± 1331 A 6297 ± 783 A 6198 ± 1931 A 5178 ± 1082 B
Carbonate 4775 ± 1526 A 4664 ± 1251 A 4557 ± 732 A 3868 ± 524 B
Amide III 1338 ± 343 A 1102 ± 222 A 1259 ± 293 A 999 ± 180 B
Hydrocarbons 1269 ± 344 A 1045 ± 205 A 1151 ± 293 A 959 ± 168 B
* Distinct letters in the same line indicate statistically significant difference among
experimental phases (p < 0.05).
Table 2. Medians and interquartile deviations of phosphate, carbonate, amide III, and
hydrocarbons content (AU) in dentin along the experimental phases (n=24).
Baseline 1080 cGy 2160 cGy 3060 cGy
Phosphate v1 28353 ± 17026 A 30485 ± 9916 A 32389 ± 7261 A 4315 ± 2163 B Phosphate v2 7250 ± 3317 C 8210 ± 2599 B 10071 ± 2547 A 6782 ± 2175 C
Carbonate 7293 ± 3443 B 7992 ± 3371 B 9849 ± 2862 A 3824 ± 1912 C
Amide III 4602 ± 1635 C 5730 ± 1818 B 7746 ± 1916 A 3558 ± 1884 D
Hydrocarbons 4913 ± 1584 C 6118 ± 1807 B 8280 ± 2079 A 3565 ± 1867 D * Distinct letters in the same line indicate statistically significant difference among experimental phases (p < 0.05).
Inorganic Composition and pH of Artificial Saliva
The pH of artificial saliva did not vary during the experimental phases (Table 3). The phosphate concentration in the artificial saliva increased progressively during the experimental phases (p < 0.0001). After 1080, 2160, and 3060 cGy, the calcium concentrations were significantly higher (p < 0.0001) than that observed at baseline, but no difference among these phases was noted (p > 0.05). At baseline, the values of pH and phosphate and calcium concentrations did not show interquartile deviations, because a single analysis was performed (n = 1).
Table 3. Medians and interquartile deviations of pH, phosphate and calcium
concentration (mg/mL) in artificial saliva along the experimental phases (n=24).
Baseline 1080 cGy 2160 cGy 3060 cGy
pH 7.00 A 6.42 ± 0.34 A 6.58 ± 0.17 A 6.44 ± 0.51 A
Phosphate 0.27 D 0.30 ± 0.07 C 0.37 ± 0.05 B 0.45 ± 0.05 A Calcium 0.45 B 1.04 ± 0.40 A 1.28 ± 0.39 A 1.21 ± 0.35 A
* Distinct letters in the same line indicate statistically significant difference among
experimental phases (p <0.05).
Morphological Changes on Enamel and Dentin Surfaces
Images obtained by SEM analysis showed progressive morphological alterations on enamel as well as on dentin. Surface cracks could be visualized on the enamel surface after 2160 cGy. For dentin, after 1080 cGy, a progressive degradation of peri-tubular structure was observed, resulting in progressive obliteration of the tubules over the experimental phases (Figure 3).
Figure 3. Representative scanning electron micrographs images of enamel (left)
and dentin (right) at baseline (T0), at 1080 cGy (T1), 2160 cGy (T2) and 3060 cGy (T3) radiation doses showing enamel cracks (yellow arrows), degradation of peri-tubular
Discussion
We performed an in vitro evaluation of the effects of radiotherapy on primary teeth. Despite the fact that this was an in vitro study, we performed a simulation of a radiotherapy regime to which sound primary teeth could be subjected. In this regard, a previous follow-up study demonstrated that radiotherapy changed salivary properties and reduced the salivary flow and buffering capacity of individuals with Hodgkin’s disease [Marangoni-Lopes et al., 2016]. Although the present study does not represent a real clinical condition of post-radiation in the oral environment, we used a representative sample size and were able to demonstrate changes occurring in primary enamel and dentin, even at doses lower than those used previously [de Siqueira Mellara et al., 2014; de Sá Ferreira et al., 2015].
The results of the present study demonstrated that radiotherapy significantly reduced the surface microhardness of primary enamel after 2160 cGy radiation as compared with baseline. However, no change in surface hardness was found among the phases (1080 and 3060 cGy). In contrast, Siqueira Mellara et al. [2014] observed an increase in the surface hardness only after 4000 cGy of radiation. This different result may be explained because these authors performed a transverse microhardness analysis, while in the present study a surface microhardness measurement was carried out. In the current study, the radiation
protocol was calculated to deliver a total dose to the enamel surface specimen. For this
purpose, the specimens were irradiated without being sectioned, a procedure that
corresponds to the real situation, instead of being transversely sectioned, which would
expose the inner enamel for evaluation. In this regard, it is known that the hydroxyapatite
crystals and the extracellular enamel matrix are different on the enamel surface as compared with the inner enamel. The inorganic content of enamel is more evident on the enamel surface, while the organic content is usually found in higher volumes in the inner enamel [de Menezes Oliveira et al., 2010].
For dentin, our results showed that irradiation significantly reduced the dentin microhardness of primary teeth after 1080, 2160, and 3060 cGy. Our results agree with those of the study by Siqueira Mellara et al. [2014], who also observed lower dentin microhardness after 1000, 2000, and 3000 cGy radiation when compared with baseline values. In this regard, radiation may cause a reduction in the water content of tissues, causing tissue dehydration in the organic matrix [Fajardo, 2005]. In summary, these results suggest that radiotherapy promotes changes in the dentin matrix and its physical conformation, which could make the tissue more friable. In addition, the reduction in dentin microhardness was progressive after 1080 and 2160 cGy. Considering that radiotherapy has a cumulative dose of radiation fractions, a progressive effect, suggesting a dose-dependent response, was expected in a sensitive organic tissue like dentin.
Regarding the enamel composition after radiotherapy, the Raman spectroscopy results of the present study showed a reduction in phosphate, carbonate, amide, and hydrocarbon contents in the enamel after 3060 cGy, as well as an increase in phosphate
v2, amide, and hydrocarbon contents in dentin after 1080 and 2160 cGy of radiation and
a reduction after 3060 cGy radiation. In this regard, the work of Sa Ferreira et al. [2015] reported that a 5400 cGy radiation dose reduced the organic content of primary enamel. According to these authors, the initial damage from irradiation occurred in the organic portion of enamel. Gamma radiation promotes oxidation of water molecules in hydroxyl and hydrogen ions. These hydrogen free-radicals could cause organic degradation. However, in their analysis, those authors did not differentiate enamel from dentin, nor did they use a follow-up design with intermediate experimental stages.
Another result of the present study was that significant reductions in phosphate and carbonate contents were found after 3060 cGy. These results can be partially explained if we consider that the calcium and phosphate concentrations were significantly increased in the artificial saliva in which the specimens were kept during enamel and dentin
irradiation. Taking into account that this solution was used to keep the specimens only during radiotherapy, and that no pH change could be detected, the increase in calcium and phosphate ions in saliva could be supposed to originate from the specimens. Thus, it is suggested that oxidation of water molecules inside the enamel specimen could affect not only its organic content but also its mineral content. This calcium and phosphate loss from enamel surfaces may render this substrate undersaturated in relation to saliva.
The scanning electron microscopy images showed a melting and fusing aspect on the enamel surface and the obliteration of dentin tubules after 2160 and 3060 cGy. Regarding the enamel surface, Madrid et al. [2017] found irregularities and a loss of enamel organic matrix in SEM images of transverse sections of irradiated permanent teeth after radiation doses from 5000 up to 7000 cGy. Despite the fact that these authors used doses from 1.6 to 2.3 times greater than the one we investigated, we also found changes in primary enamel, although this substrate had mineral and organic contents lower than those of permanent teeth [Zamudio-Ortega et al., 2014].
Regarding dentin, an early study by Gonçalves et al. [2014] reported fragmentation of collagen fibers in permanent teeth after 3000 and 6000 cGy of radiation. This finding may be related to the reduction in the intensity of the collagen Raman band after 3060 cGy as observed in our study, which could have occurred as a consequence of the water oxidation and collagen proteolysis as previously explained. In this context, a previous study demonstrated a higher expression of metalloproteinase in the dentin-enamel junction of in vitro-irradiated teeth. The proteolytic activity of metalloproteinase could degrade the protein structure, contributing to the etiology of radiation-related caries [McGuire et al., 2014]. With respect to collagen, McGuire et al. [2014] showed the importance of type IV collagen in the dentin-enamel junction (DEJ). Reduction of this component may represent biochemical changes and instability of the DEJ of irradiated teeth.
According to Lieshout and Bots [2014], the formation of recurrent and atypical patterns of dental caries, namely radiation caries, in irradiated teeth is due not only to loss of saliva but also to a combination of both hyposalivation and the direct effects of radiation on permanent teeth. We believe that the reduction in surface hardness and changes in the mineral composition promoted by radiotherapy, associated with hyposalivation and diet changes, could render the primary teeth more susceptible to dental erosion and demineralization in the presence of a high cariogenic challenge. The cracks observed in the SEM images could favor acid penetration in dentin during a cariogenic challenge. Moreover, these morphological changes could represent the result of reduced ultimate tensile strength in the dentin-enamel junction, as previously reported [Soares et al., 2010], and could be responsible for enamel breakdown. In this regard, cracked and broken down enamel can expose the dentin, which has a lower critical pH, thus favoring the progression of caries lesions. In addition, reduced salivary flow rate is a risk factor for dental erosion [Järvinen et al., 1991]. In this way, oncology patients who undergo head and neck radiotherapy could be at high risk for the development of dental erosion, since they suffer from hyposalivation.
We conclude that radiotherapy caused a reduction in surface hardness, changed the mineral composition, and promoted morphologic changes in the enamel and dentin of primary teeth. The changes caused by radiotherapy in primary teeth found in this research may occur in pediatric patients with other malignancies in the region of the head and neck. Despite the fact that the dose delivered to the tooth structure in patients subjected to radiotherapy does not correspond to the total dose administered, and that recent technologies, such as Intensity Modulated Radiotherapy, allow for lower and more targeted doses, reducing side-effects, we found important changes occurring in enamel and dentin even with low doses. Results of the current study could contribute to future investigations testing the susceptibility of gamma-irradiated primary teeth to an erosive
and/or cariogenic challenge. In addition, the evidence provided by this research may be useful in future studies that investigate the effectiveness of chemical compounds such as chlorhexidine and fluoride dentifrices, varnish, mouthwash, and gel in preventing dental caries and/or erosion. Furthermore, effective mechanical biofilm control and advice regarding the frequency of fermentable carbohydrate consumption are recommended strategies to promote caries prevention, and to improve the quality of life for this
caries-risk group undergoing radiotherapy of the head and neck region.
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2.2. Susceptibility of gamma irradiated primary teeth to a cariogenic challenge - In vitro study.
Lenita Marangoni-Lopes, Gabriela Rovai Pavan, Carolina Steiner-Oliveira, Marinês Nobre-dos-Santos.
Abstract
AIM: This study aimed to evaluate whether radiotherapy with gamma radiation changes the susceptibility of primary teeth to a cariogenic challenge. MATERIAL AND METHODS: Specimens of primary teeth (n = 18) were subjected to gamma radiation simulating the radiotherapy treatment and 18 specimens were used as control group. A microbiological model with S. mutans of caries induction was used to evaluate the susceptibility of these irradiated specimens to the caries development. The depth, area and volume of caries lesion formed were evaluated using microcomputer tomography analysis (μ-CT) and changes occurring on the enamel surface were evaluated using scanning electron microscopy (SEM). Data of the lesion depth, lesion area and volume were submitted to independent T-Test with a 5% of significance limit. RESULTS: It was observed that the lesion depth and area of caries in the irradiated group (196.61 ± 60.41 µm and 564.52 ± 343.37 µm2 respectively) were significantly higher (p = 0.02 and p = 0.03 respectively) than in the non-irradiated group (151.70 ± 32.16 µm and 386.64 ± 169.75 µm2 respectively). However, the lesion volume did not differ (p = 0.15) between the groups (7797.89 ± 3675.69 µm3 for irradiated and 6509.03 ± 2902.65 µm3 for non-irradiated group). The SEM images show greater enamel breakdown in the group of irradiated specimens. CONCLUSION: Gamma radiation protocol used for cancer treatment turns primary teeth more susceptible to a cariogenic challenge.
Introduction
Cancer survivor´s children who underwent radiotherapy in head and neck have an impaired dental health and high caries prevalence (Pajari et al., 1995; Gawade et al., 2014). According to a literature review, the high caries prevalence observed in patients submitted to head and neck radiotherapy to cancer treatment, is not only a result of the reduced salivary flow rate. In fact, a combination of this effect with the direct effects of radiation on the enamel composition, play a significant role on the dental hard tissue (Lieshout & Bouts, 2014).
Radiotherapy used cancer treatment, delivers radiation doses close to the tooth structure. A previous study showed a dose-volume histogram of children treated with 4600 to 6600 cGy of gamma radiation for a variety of tumors including rhabdomyosarcoma, sarcoma, teratoma and carcinoma. The authors found that a mean dose of 3700 cGy per tooth was delivered (Thompson et al., 2013).
Regarding the radiation effects on primary teeth, recent studies showed changes in primary teeth submitted to radiation simulation. In this regard, de Siqueira Mellara et al. (2014), showed increased enamel transversal microhardness with 4000 and 6000 cGy. Another study showed a reduction in the organic content of dentin in primary teeth irradiated with 5400 cGy (de Sá Ferreira et al., 2015). In addition, a recent study performed by our group showed a decrease in the surface microhardness, mineral and organic composition, and morphological changes on the enamel and dentin of primary teeth after irradiation with lower doses (1080, 2160 and 3060 cGy) (Marangoni-Lopes et al., 2017).
With respect to the primary teeth, features as thinner thickness (de Menezes Oliveira at el., 2010), abundant microporosities, exposed prisms and high carbonate incorporation (Zamudio-Ortega et al., 2014) are responsible for the higher susceptibility of these teeth to mineral loss as compared with permanent teeth (Sonju Clasen et al., 1997). To intensify these features, the lower enamel microhardness and mineral content, promoted by gamma radiation (Marangoni-Lopes et al., 2017) may turn the primary teeth even more susceptible to a cariogenic challenge and to caries development and progression. In fact, de Sá Ferreira et al. (2015) showed that after a pH cycling, the irradiated primary teeth showed a reduced phosphate content, a reduced Ca / P ratio in the enamel, and a lower carbonate content in the dentin.
Despite the higher caries risk of children submitted to radiotherapy, the early studies showing biochemical and morphological alterations promoted by gamma radiation on irradiated primary enamel, we were not able to find any study evaluating the susceptibility
of irradiated primary teeth to demineralization. Thus, the aim of the present study was to evaluate whether gamma radiation changes the susceptibility of primary teeth to a cariogenic challenge.
Materials and methods Experimental design
A biological model of caries induction was used to evaluate the susceptibility of the irradiated primary enamel (n=18) to the development of caries in parallel, to a control group of non-irradiated specimens (n=18). After 14 days at the end of the experiment, the specimens were submitted to microcomputer tomography (μ-CT) to calculate the caries lesion depth, area and volume and to qualitative analysis using scanning electron microscopy analysis (SEM). The results were compared between the irradiated and non-irradiated groups.
Ethical considerations
This study was approved by the local Committee of Research Ethics (Protocol Number 024/2014) and Certificate of Presentation for Ethical Appreciation number was 27478814.0.0000.5418. This study was performed in accordance with the Helsinki Declaration.
Sample size
The sample size of this study was calculated using the Bioestat® software (Ayres, Belem, PA, Brazil) based on the values of means and standard deviations of microhardness obtained in a previous study (Marangoni-Lopes et al., 2017), considering 85% as test power and an alpha level of 0.05. The result (n=14) was increased by 30% to prevent possible losses (n=18).
Sample preparation
The donated primary molars were stored in thymol (0.1M, pH 7.0). After a previously inspection teeth with developmental defects were excluded. The selected teeth were cut using a cutting machine Isomet 1000 (Buehler®, Lake Bluff, IL, USA) with a double-sided diamond disc (Buehler®, Lake Bluff, IL, USA) to obtain a specimen of the buccal and lingual surfaces with 3 x 3 x 2 mm dimensions. For specimens grinding and polishing, an APL-4 polisher (Arotec, Cotia, SP, Brazil) with sandpaper grains 1200 for 40 s and 2400 for 1 min, and felt discs with diamond solution (Buehler®, Lake Bluff, IL,
USA) were used for 1 min. At the end, specimens were washed in ultrasound USC 1400 (Unique, Sao Paulo, SP, Brazil) for 10 min, using distilled and deionized water, and a detergent solution (Buehler®, Lake Bluff, IL, USA). The specimens were numbered, and the initial surface microhardness was measured. Three indentations were performed near the central region, using the microhardness tester FM-ARS Tech Future (Future-Tech Corp., Tokyo, Honshu, Japan) with a Knoop indenter, load of 50 kgf, for 5 seconds. The mean and standard deviation of the Knoop hardness number was calculated for each specimen. Samples were excluded from the experiment if the mean Knoop hardness number was greater than or lower than the general mean (300.76) plus two times the standard deviation (46.70). This procedure was performed to standardize the surface enamel microhardness. The selected specimens were randomized between the two groups using the Excel.
Irradiation of enamel specimens
This approach aimed to simulate the radiotherapy protocol used for children under cancer treatment. Specimens of the irradiated group were submitted to daily fractions of 180 cGy, for 17 weekdays, resulting in a total dose of 3060 cGy. This protocol simulates the German Society of Pediatric Oncology and Hematology–Hodgkin's Disease (GPOH-HD95) protocol, used in the treatment of the Hodgkin lymphoma. The following parameters were used: 0.5 cm of artificial saliva, 1 cm of air, a wax bolus of 1.5 cm height, 99.9% margin of the total dose, focus of 100 cm, and a campus of 15 x15 cm. The equipment used was the Varian - Clinac 6EX (Varian, Pallo Alto, CA, USA) with a linear accelerator and beam photons of 6 MeV. The specimens were kept in individual wells in a 24-wells-plaque and immersed in artificial saliva containing 1.5mM Ca, 0.9 mM PO4,
150 mM KCl in buffer 20 mM Tris, pH 7.0 (Thaveesangpanich et al., 2005). After each week, the specimens were placed in new artificial saliva.
Biological model for caries lesion formation
The specimens were protected with acid-resistant varnish except for the enamel face, fixed individually in a 24-wells-plaque with an orthodontic wire, kept in relative humidity, and sterilized using ethylene oxide. After sterilization, specimens were removed from the distilled water and immersed in a sterile artificial saliva medium comprised of meat extract, 1 g/l; yeast extract, 2 g/l; proteose peptone, 5 g/l; type III hog gastric mucin, 2 g/l; sodium chloride, 0.2 g/l; calcium chloride, 0.3 g/l; potassium chloride, 0.2 g/l; 1.25 ml/l of a 0.2 µm filter-sterilized solution of 40% urea, added after
autoclaving, and pH adjusted to 6.61. Each artificial saliva well (2.0 ml) was inoculated with 0.5 ml (1-2´108 colony-forming units / ml) of an overnight culture of Streptococcus mutans UA 159. The specimens were incubated at 37o C and a partial 10% CO2 pressure.
After 24 hours, we initiate the sucrose baths. Sucrose baths were performed 3 times a day for 5 min, using a 40% sucrose solution. After the last sucrose bath of the day, specimens were transferred to a fresh saliva medium. This procedure was repeated for 14 days (Steiner-Oliveira et al., 2011).
Determination of depth, area and volume of caries lesion
After caries production using the microbiological model, the specimens were submitted to μ-CT analysis for determination of the depth, area and volume of caries lesion. Initially, the acid resistant varnish was removed. The specimens were fixed with cyanoacrylate glue (Superbonder Loctite, São Paulo, Brazil) forming a vertical column composed of 9 blocks, with the carious surfaces arranged in the same direction in a way that they remain free. This procedure was performed to avoid the inference of adjacent structures. These totems were exposed to 50 kV and 800 μA μ-CT SkyScan 1174 scanner (SkyScan, Kontich, Belgium) using a 1 mm aluminum filter to eliminate the low-energy X-rays.
For the images reconstruction, the NRecon software (NRecon, SkyScan, Kontich, Belgium) was used. During reconstruction, the necessary image corrections were made, such as misalignment compensation, smoothing level 3, reduction of the beam hardening artifact at 35%, and the ring artifact level 11, keeping the same scanning parameters.
After reconstruction, the datasets of each specimen were open in a Data viewer program (Dataviewer, SkyCan, Kontich, Belgium) to save the longitudinal view, which could be observed the transversal section of enamel and dentin. Then, the central dataset of each specimens was open in the CTAn program (SkyScan, Kontich, Belgium) at 300 magnifications, and the region of interest (enamel) for the measurements was selected. The threshold was established at 106 pixels, and the depth, area and volume of the lesion were evaluated. The pixels values were converted to micrometers, and the lesion depth was expressed in µm, the lesion area was expressed in µm², and the lesion volume in µm³ (Delbem et al., 2009).
Scanning electron microscopy analysis
At the end of the experiment, 2 specimens of each group were dehydrated in silica for at least 48 h, fixed in stubs with carbon double-sided tape (Electron Microscopy
Sciences, Washington, USA) and covered with gold-palladium (Balzers SCD 050 sputter coater, Liechtenstein, Germany). Then, the specimens were examined under a scanning electron microscope (JEOL, JSM – 5600 LV, Tokyo, Japan) at 15 kV acceleration voltage. Standardized images of the enamel surface were acquired at 1000 magnification.
Statistical analysis
To test the hypothesis that lesion depth, area and lesion volume of irradiated and non-irradiated groups were different, the data were submitted to the Lilliefors normality test and showed to follow normal distribution. Then non-irradiated (control) and irradiated (test) groups were compared using the independent T-Test. For these analyses, a 5% significance one-tailed alpha level was established. The tests were performed using the Bioestat software (Ayres, Belem, PA, Brazil).
Results
Table 1 shows the depth, the area and the volume of caries lesion. It was observed that in irradiated group, the lesion depth was around 30% statistically higher than that of the non-irradiated group (p = 0.02). Moreover, the lesion area in the irradiated group was around 46% significantly higher than that of the non-irradiated group (p = 0.03). In addition, the lesion volume in the irradiated group was numerically 20% higher, however, no difference between irradiated and non-irradiated group was detected (p = 0.15).
Figure 1 shows representative scanning electron micrographs of irradiated and non-irradiated specimens. The changes on enamel surface have major aggressiveness in the irradiated group than in non-irradiated group. It is possible to observe cracks (yellow arrows) and enamel breakdown (red arrows) in the irradiated group.
Table 1. Means and standard deviations of lesion depth, lesion area, and lesion volume of Irradiated and non-irradiated primary teeth (n=18). Depth area (µm)* p t Power Effect Lesion area (µm2)* p t Power Effect Lesion volume (µm3) p t Power Effect Irradiated 196.61 (60.41) 0.02 2.46 0.79 0.44 564.52 (343.37) 0.03 2.24 0.72 0.40 7797.89 (3675.69) 0.15 1.47 0.43 0.24 Non-irradiated 151.70 (32.16) 386.64 (169.75) 6509.03 (2902.65)
Figure 1. Representative scanning electron micrographs of irradiated and non-
irradiated specimens. Surface morphology of irradiated (I - upper images) and non-irradiated (N - lower images) primary teeth showing cracks (yellow arrows) and enamel
breakdown (red arrows).
Discussion
In the present study we performed a simulation of low radiation doses of radiotherapy treatment to which children having cancer is submitted (Marangoni-Lopes et al., 2016). In fact, radiotherapy used for cancer treatment, delivers gamma radiation to teeth structure (Thompson et al., 2013) and with an adequate methodology, we demonstrated that radiation turns primary teeth more susceptible to a cariogenic challenge.
The association of sucrose and S. mutans is consolidated in literature as a simulation of cariogenic challenge considering the acidogenicity and aciduricity of this microorganism and the formation of extra-cellular polysaccharides with this carbohydrate (Arthur et al., 2011). The saliva medium with three sucrose baths per day, which could simulate the saliva remineralization period and cariogenic challenge, seems to be suitable to study dental caries formation (Steiner-Oliveira et al., 2011). The µ-CT analysis used to
determine the caries lesion depth, area and volume seems to be adequate in high cariogenic challenge and lesions with loss of enamel integrity.In this regard, a previous study showed that μ-CT and microhardness give similar results in areas with a high demineralization pattern (Delbem et al., 2009).
Using this methodology, we observed that the depth and area of caries lesion of the irradiated teeth were respectively around 30 and 46 % higher than the depth and area in the non-irradiated teeth. These results show that the irradiated primary teeth are more susceptible to the cariogenic challenge used in the present study and to the caries development. We were not able to find any study evaluating the susceptibility of irradiated either primary or permanent teeth to demineralization. However, early studies showed physical, chemical and morphological alterations in irradiated enamel (Marangoni-Lopes et al., 2017; de Sá Ferreira et al., 2015; de Siqueira Mellara et al., 2014), which could at least partially, explain the higher susceptibility of irradiated primary enamel to the cariogenic challenge used in the present study.
Considering dental caries as a dynamic process of alternating periods of demineralization and remineralization, the mineral content of the teeth is an important issue. The reduced microhardness was an evidence of reduced mineral content (Marangoni-Lopes et al., 2017). These conditions also turn enamel more susceptible to mineral loss in other situations, like white spot lesions (Wierichs et al., 2016) and pre-erupted teeth (Kotsanos and Darling, 1991, Palti et al., 2008), since a less mineralized surface is more susceptible to the mineral dissolution and to acids diffusion into deeper parts of the lesion.
Regarding the morphological changes occurring on the surface of primary enamel surface after gamma irradiation, the scanning electron micrographs of irradiated specimens showed cracks and enamel breakdown. These findings are in line with a previous investigation (Marangoni-Lopes et al., 2017) which also found cracks on enamel surface of irradiated primary teeth due to the dehydration promoted by gamma radiation. These cracks could be explained by a reduced ultimate tensile strength (Soares et al., 2010) and could turn enamel more friable and susceptible to breakdown, as observed in the irradiated group. In this regard, the cracked enamel can tear off teeth and expose the dentin, which has a lower critical pH.
The lesion volume of irradiated teeth was numerically 20 % higher than that of the non-irradiated teeth. However, no statistically significant difference between these two groups could be detected. Despite the fact that the sample size calculation was carried out with an 85% power test, and it was a representative sample size, the test power of lesion
