i
Roberta Galetti
Evaluation of mechanical properties of dental
tissue of patients who undergone radiotherapy
Análise das propriedades mecânicas dos tecidos dentários de
pacientes submetidos à radioterapia
PIRACICABA
2015
iii
Universidade Estadual de Campinas
Faculdade de Odontologia de Piracicaba
Roberta Galetti
Evaluation of mechanic behavior of dental tissue of
patients who undergone radiotherapy
Análise das propriedades mecânicas dos tecidos dentários de
pacientes submetidos à radioterapia
Doctorate thesis presented to the Dental Materials Post Graduation Program of the Piracicaba Dental School, State University of Campinas, to obtain the PhD grade in Dental Materials
Tese apresentada à Faculdade de Odontologia de Piracicaba, da Universidade Estadual de Campinas, para obtenção do título de Doutora em Materiais Dentários.
Orientador: Prof. Dr. Alan Roger dos Santos Silva
Coorientador: Prof. Dr. Mario Fernando de Goes
Este exemplar corresponde à versão final da tese
defendida por Roberta Galetti e orientada pelo
Prof. Dr. Alan Roger dos Santos Silva.
______________________
Assinatura do Orientador
PIRACICABA
2015
vii
ResumoEste estudo avaliou o comportamento mecânico de tecidos dentários de pacientes com câncer de cabeça e pescoço submetidos à radioterapia. No capítulo I, o ensaio mecânico da nanoindentação foi utilizado para determinar a dureza e módulo de elasticidade do esmalte, dentina e da região de união restauradora em dentina (adesivo, camada híbrida e dentina subjacente). Foram utilizados seis dentes incisivos inferiores irradiados in vivo e não irradiados (grupos controle). A dureza e o módulo de elasticidade e foram obtidos após a realização da nanoindentação com pico de força de 1000 µN em dentina intertubular e região de união restauradora e 1500 µN em esmalte (centro do prisma) usando o microscópio de força atômica equipado com nanoidentador com tempo 5-2-5 seg para carregamento, aplicação e descarregamento da carga. A análise de variância a um fator foi aplicada com nível de significância de 0.05%. O valor da nanodureza e módulo de elasticidade não foram estatisticamente diferentes entre os tecidos avaliados em ambos os grupos irradiados e controle. Desta foma, pode-se concluir que tanto a dureza como o módulo de elasticidade de dentes submetidos à radioterapia in vivo não apresentam alterações das propriedades mecânicas no esmalte, dentina e região de união adesivo/dentina devido á ação direta da radioterapia. No capítulo II, foram avaliadas as propriedades viscoelásticas (storage e loss modulus) de três regiões diferentes: esmalte, junção amelo-dentinária (JAD) e dentina de dentes irradiados in vivo. Cinco dentes não irradiados (grupo de controle, n = 5) e cinco dentes irradiados in vivo (grupo irradiado, n = 5) foram utilizados para produzir cinco fatias de cada para avaliar a três áreas distintas: o esmalte, o JAD , e a dentina. A análise por mapeamento (Modulus Mapping Analysis) foi escolhida para avaliar a perda e armazenamento de energia mediante uma carga aplicada. Três regiões de dados foram coletados de cada área de tecido de cada fatia, totalizando quinze mapeamentos por tecido por grupo. Os valores do módulo foram calculados pelo software Hysitron® e a análise da variância (ANOVA Plot Split) e teste de Tukey a 5% de significância foram utilizados para comparar os grupos e tecidos. As três áreas avaliadas de ambos os grupos controle e irradiado revelaram diferença estatística no módulo de perda e armazenamento. Ambos os valores de perda e de armazenamento apresentaram-se maiores no grupo irradiado para esmalte (164,44 ± 36,60 GPa; 177,59 ± 58,84 GPa), JAD (50,85 ± 35,78 GPa; 83,33 ± 38,59 GPa) e dentina (21,18 ± 18,61 GPa; 52,44 ± 26,56 GPa) do que no grupo controle para o esmalte (127,15 ± 74,45 GPa; 162,85 ± 74,63 GPa), JAD (25,72 ± 9,64 GPa; 21,93 ± 52,78 GPa) e dentina (10,39 ± 8,65 GPa; 32,10 ± 20,39 GPa), respectivamente. Foi possível concluir neste estudo, que as propriedades viscoelásticas dos dentes irradiados in vivo apresentam-se diferentes das do grupo controle. Estes resultados sugerem que, após a radioterapia, os tecidos dentais estariam mais suscetíveis a fraturas.
Palavras chave: Nanoindentação, radioterapia, esmalte, dentina, camada híbrida, viscoelasticidade.
ix
AbstractThis study evaluated the mechanic properties of enamel, dentin, and dentin bond interface of patients who undergone head and neck cancer treatment. On I chapter, the nanoindentation technique was used to determine the hardness (H) and reduced modulus of elasticity (Er) of the control group on enamel, dentin, and dentin bond interface (adhesive layer, hybrid layer and underlyer dentin). The Er and H were obtained after completion of nanoindentation with peak force of 1000 μN on intertubular dentin and restorative dentin interfaces and 1500 μN on enamel (prism center) using the atomic force microscope with nanoindenter accopled with test time 5-2-5 seconds for loading, holding and unloading. The one-way analysis of variance (p≤0.05) was applied and the valus for H and Er for both groups and tissues were no statistical different. As conclusion, the nanohardeness and elastic modulus behavior of the enamel, dentin and dentin bond interface was not impacted by the radiotherapy treatment of head and neck cancer. On II chapter, the viscoelastic properties were assessed (storage and loss modulus) of three different regions: enamel, dentin-enamel junction (DEJ) and dentin irradiated teeth in vivo. Five non irradiated teeth (control group, n=5) and five in vivo irradiated teeth (irradiated group, n=5) were used to produce five beams that were used to evaluate three different areas: the enamel, the DEJ, and the dentin. Perpendicular sections to the long axis of the teeth were made at middle region of the crown to produce the beams. The Modulus Mapping Analysis was chosen to evaluate the loss and storage moduli of each area. Three data regions were collected of each tissue area of each beam, summing a total of fifteen data per tissue per group. The modulus values were calculated by the Hysitron® software and an Analysis of Variance (ANOVA Split Plot) and Tukey test at 5% of significance was used to compare groups and tissues. All the three areas evaluated of control and irradiated group revealed statistical difference on the Loss and Storage Moduli. Both the loss and storage values are higer on the irradiated group for enamel (164.44±36.60 GPa; 177.59±58.84 GPa), DEJ (50.85±35.78 GPa; 83,33±38,59 GPa) and dentin (21.18±18.61 GPa; 52.44±26.56 GPa) than control group values for enamel (127.15±74.45 GPa; 162.85±74.63 GPa), DEJ (25.72±9.64 GPa; 21.93±52.78 GPa) and dentin (10.39±8.65 GPa;32,10±20,39 GPa), respectivally. The viscoelastic properties of in vivo irradiated teeth are different from control group. The enamel, DEJ and dentin presented the higer values on the in vivo irradiated group. These finds suggest that after radiotherapy, the dental tissues are more susceptible to fractures.
Keywords
xi
SUMÁRIO
DEDICATÓRIA ………... xiii
AGRADECIMENTOS ESPECIAIS ………. xv
AGRADECIMENTOS ... xvii
INTRODUÇÃO ...1
CAPÍTULO 1: The quasi static nanoindentation technique as a tool to the
investigation on in vivo irradiated teeth ...6
CAPÍTULO 2: The dentino-enamel junction of the in vivo irradiated teeth by
nanoDMA analisys………...………...24
CONCLUSÃO ...37
REFERÊNCIAS ...38
APÊNDICE 1 ...39
APÊNDICE 2 ...40
ANEXO I ...41
xiii
DEDICATÓRIA
Dedico este trabalho,
Ao meu marido Andre Corsetti e meu filho Alvaro Corsetti Neto, meus
amores e companheiros de vida, que me tornam cada dia mais feliz e realizada.
Aos meus pais Marco Antonio Galetti e Angela Montaninini Galetti, irmãos
Renata Galetti e Rafael Galetti, pelo incentivo e apoio, pelo carinho e amor
dedicados, por sempre me darem forças para que pudesse alcançar este objetivo.
xv
AGRADECIMENTOS ESPECIAIS
À Deus, por estar sempre comigo, guiando e iluminando meus passos,
dando saúde e força para concluir mais um projeto em minha vida.
Ao Professor Doutor Alan Roger dos Santos Silva, por coordenar e orientar
esta pesquisa.
Ao Professor Doutor Mário Fernando de Goes, pela colaboração e
co-orientação das atividades desta pesquisa. Pelo incentivo aos estudos e novas
experiências profissionais e pessoais fora do país.
A Professora Doutora Ana Karina Bedran-Russo, por aceitar minha
permanência em seu laboratório, oferecendo a oportunidade de crescimento
pessoal e profissional fora do país, na Universidade de Illinois em Chicago.
Aos meus amigos, por fazer dos meus objetivos parte de suas vidas, pelo
companheirismo e incentivo, onde dispuseram seu tempo e atenção em meu favor
sempre com muito carinho e amor.
xvii
AGRADECIMENTOS
À Faculdade de Odontologia de Piracicaba-UNICAMP em nome do Prof. Dr.
Guilherme Elias Pessanha Henriques.
Aos professores da Área de Materiais Dentários da Faculdade de
Odontologia de Piracicaba–UNICAMP, pelo empenho, respeito e ensino.
À Thais Bianca Brandão, coordenadora do setor de odontologia oncológica
do Instituto do Cancer do Estado de São Paulo, por ter cedido, gentilmente, o
espaço físico e o material biológico que viabilizou a execução desta dissertação.
Aos profissionais do setor de odontologia oncológica do Instituto do Cancer
do Estado de São Paulo, pela colaboração.
Ao Engenheiro Mecânico Marcos Blanco Cangiani, técnico especializado da
Área Materiais Dentários, a funcionária Selma Aparecida Barbosa Segalla e ao
biólogo Adriano Luis Martins pelo carinho e disposição em ajudar sempre.
À CAPES pela concessão da bolsa de estudo que me deu a oportunidade
para a realização deste curso de pós-graduação.
1
INTRODUÇÃO
A radioterapia, junto da cirurgia, é a modalidade terapêutica mais
frequentemente aplicada no controle do câncer de cabeça e pescoço (Kielbassa et
al., 2006). Entretanto, a eficiência da radioterapia no controle local e regional da
doença é realizada às custas de diversas injurias patológicas agudas e crônicas
nos tecidos normais localizados no interior ou nas adjacências do campo irradiado
(Galetti et al., 2014).
Efeitos colaterais comuns em pacientes irradiados durante o tratamento do
câncer de cabeça e pescoço incluem mucosite, hipossalivação, radiodermite,
infecções oportunistas de origem fúngica e viral, disgeusia (perda do paladar), dor
crônica, osteorradionecrose, trismo e cárie relacionada à radioterapia (CRR), entre
outros (Kielbassa et al., 2006).
Do ponto de vista odontológico, uma das complicações mais importantes e
complexas é a “cárie relacionada a radiação”. O risco para o desenvolvimento da
cárie relacionada a radiação (CRR) estará presente por toda a vida dos pacientes
após a radioterapia em cabeça e pescoço. Notavelmente, esta doença possui
início e progressão rápidos, caráter recorrente e tratamento desafiador; já que
materiais restauradores odontológicos possuem baixa longevidade nos dentes
destes pacientes (Silva et al.,2009). Sabe-se que o desenvolvimento da cárie em
pacientes tratados com radioterapia é maior em relação a pacientes que não foram
submetidos ao tratamento (Hong et al., 2010).
No que diz respeito à incidência da CRR em pacientes brasileiros com
câncer de cabeça e pescoço, um estudo realizado de modo prospectivo
acompanhou pacientes irradiados antes, durante e depois do tratamento, por mais
de 2 anos, e demonstrou incidência de 11% (Bonan et al., 2006). Embora pareça
pequena, a incidência de cárie após o tratamento radioterapêutico nestes
pacientes revelou-se alta, uma vez que 30% dos pacientes eram edentulos e 70%
dos pacientes dentados, continham em média 10 dentes presentes. Além disso,
2
antes do tratamento, os pacientes passaram por avaliação bucal e em grande
parte destes, foram feitas em média 8,6 extrações.
Na forma clínica mais comum, a lesão localiza-se na região cervical dos
dentes, onde as superfícies dentárias adquirem coloração marrom escura (Silva et
al., 2009). Além disso, a sua rápida progressão pode determinar a perda das
coroas dentárias. A formação das lesões tem sido atribuída tanto ao dano
radiogênico direto no tecido dentário quanto aos efeitos indiretos relacionados à
radioxerostomia (Grötz et al., 1997). Por isso, pesquisas vêm sendo feitas para
elucidar questões referentes aos efeitos bucais nocivos causados pela
radioterapia. O uso de dentes irradiados in vivo foi utilizado por Silva et al. (2009),
onde constataram não haver diferença estrutural nos tecidos cariados de dentes
irradiados e não irradiados. Por isso, o tratamento restaurador indicado antes,
durante e após o tratamento radioterapêutico poderia não ser diferente daquele
realizado em pacientes não irradiados.
Entretanto esta suposição não é uma realidade, na maioria dos casos o
profissional tem dúvidas no planejamento do tratamento restaurador em pacientes
submetidos à radioterapia. Isso acontece, pois não há indicação consistente na
literatura sobre o material restaurador a ser aplicado nestes casos, e sim a
generalização sobre a recomendação de restauração direta (Jansma 1992). Em
alguns casos, sugere-se o uso de ionômero de vidro como material restaurador
em pacientes pediatricos com câncer de cabeça e pescoço (Albuquerque et al.,
2007). Porém, Hu et al. (2005) acompanharam durante dois anos o desempenho
clínico de restaurações feitas com cimentos de ionômeros de vidro em dentes que
apresentavam cárie de radiação. O estudo mostrou que este tipo de material
apresentou 82% de falhas em função ao deslocamento do material da cavidade.
Além disso, correlações entre metodologias in vitro e in vivo mostram que a
capacidade de liberação de flúor do material não pode ser diretamente relacionada
à biodisponibilidade e consequente formação de fluoreto de cálcio, como à inibição
de cárie secundária. (Randal et al., 1999, Papagiannoulis et al., 2002)
3
Em contraste a estes materiais, os compósitos resinosos quando unidos a
dentina de dentes irradiados (in vivo) por agentes monoméricos específicos
(adesivos) têm apresentado resultados de resistência mecânica por tração
similares entre dentes irradiados e não irradiados (Genhardt et al., 2001, Genhardt
et al., 2003, Galetti et. al., 2014). Estes estudos sugerem a possibilidade de
realizar o procedimento restaurador com sistemas adesivos associados às resinas
compostas como meio terapêutico restaurador de dentes em pacientes que foram
submetidos à radioterapia.
Contrariamente, a análise micromorfométrica e micromorfológica in vitro, in
situ e in vivo da superfície do esmalte desmineralizado irradiado e não irradiado foi
estudada por Grötz et al. em 1998. Neste estudo, houve diferença estatística
significante entre os grupos, sendo que nos grupos na qual foi aplicada irradiação
ocorreu perda total da estrutura prismática da subsuperfície do esmalte. Este
comportamento levou os pesquisadores a acreditar que o esmalte é um tecido
menos resistente ao ataque ácido após a radioterapia. A fragilidade do esmalte
seria a primeira evidência do desenvolvimento acelerado da cárie em pacientes
submetidos à radioterapia de cabeça e pescoço.
Seguindo este conceito, a iniciação e a progressão da cárie da radiação
foram estudadas por Jansma et al. (1993) usando um modelo in situ. A morfologia
de lesões de cárie natural foi comparada com da cárie de radiação. As cáries de
radiação induzida e natural mostraram os mesmos padrões de degradação. Em
pacientes irradiados grande parte do esmalte foi severamente desmineralizado
dentro de 6 semanas, visto que no grupo controle o esmalte não mostrou
desmineralização significativa após 12 semanas. Por outro lado, Silva et al,
demonstraram não haver alterações morfológicas na dentina dos dentes irradiados
in vivo quando comparados com dentes não irradiados. Isto fica mais evidente
quando os resultados de resistência mecância por tração obtidos em restaurações
em dentina de dentes irradiados também não apresentaram diferenças
significativas em relação aos tecidos dentinários não irradiados. Entretanto, a
4
propriedade mecânica dos tecidos (esmalte e dentina), além da região da união do
adesivo com a dentina ainda não foi avaliada na condição irradiada in vivo.
Dentre os tipos de ensaios mecânicos que avaliam os tecidos e suas
estruturas, o ensaio laboratorial de nanodureza, mostrou-se eficiente para
caracterizar o perfil mecânico das estruturas dentais (Lewis G. & Nyman J. S.,
2008). A dureza e o módulo de elasticidade são propriedades que podem predizer
a capacidade de resposta de uma estrutura a diferentes condições. Este método
tem sido usado para quantificar a dureza e o módulo de elasticidade da região de
união dente/adesivo/compósito resinoso e é conhecido como Quasi-Static
analysis. Esta região é considerada capaz de aliviar tensões de contração durante
e após a restauração, assim como reforçar biomecânicamente as estruturas dos
dentes unidos à restauração polimérica (Van Meerbeek et al., 1993). Além deste
tipo de ensaio de nanoindentação, existem também as avaliações de diferentes
propriedades e em diferentes condições durante as endentações de forma
dinâmica, conhecido como análises mecânicas nano-dinâmicas (nanoDMA).
Dentro dos diferentes tipos de ensaios dinâmicos, está a análise por mapeamento
da imagem (Modulus Mapping analysis). Este tipo de avaliação vem sendo cada
vez mais aplicada em tecidos dentários, por permitir uma análise mais detalhada
sobre as respostas viscolasticas de materiais mediante cargas e frequências
diferentes. Sendo o dente, um órgão composto por diferentes tecidos e com
propriedades intrincicas dependentes da composição e meio externo, o uso do
nanoDMA para avaliar possíveis alterações nos tecidos tem aumentado.
Diante dos estudos existentes sobre os efeitos da radioterapia nos tecidos
dentários, as contradições e a falta de caracterização das condições clínicas
envolvidas no processo de tratamento radioterápico (mucosite, disfagia, disgeusia,
trismo, xerostomia), mantém aberta a questão a cerca dos efeitos diretos da
radiação.
Dessa forma este estudo tem por objetivo avaliar através de ensaios na
escala nanométrica (nanoindentação), o comportamento mecânico dos tecidos
(esmalte e dentina) de dentes irradiados in vivo e compará-lo ao comportamento
5
mecânico de dentes que não foram submetidos a qualquer tipo de tratamento
oncológico (controle). Além disso, avaliar também a zona de união produzida por
um monômero (adesivo) e o tecido dentinário, juntamente com a dentina e o
adesivo ajacentes à região da união.
A hipótese do estudo é de que não haverá diferença nos valores de
nanodureza, módulo de elasticidade, storage e loss modulus, obtidos através do
ensaio de nanoindentação quasi estática e dinâmica nos tecidos de dentes
irradiados in vivo e não irradiados.
6
Capítulo 1: The Quasi-static nanoindentation technique as a tool to the
investigation on in vivo irradiated teeth.
Galetti R1, Silva A R S2, De Goes M F1, Bedran-Russo A K3, Lopes M A.2
1 Department of Restorative Dentistry, Piracicaba Dental School - University of Campinas, 13414-903, Campinas – São Paulo, Brazil.
2 Department of Oral Pathology, Piracicaba Dental School - University of Campinas, 13414-903, Campinas - São Paulo, Brazil.
3 Department of Restorative Dentistry, University of Illinois at Chicago, 60612, Chicago – Il, USA.
Abstract
This study evaluated the hardeness (H) and elastic modulus (Er) of enamel (EIG), dentin (DIG), and dentin bond interface (RIG) of patients who undergone head and neck cancer treatment (n=6). The non-irradiated group (n=6) was used as control (enamel= ECG, dentin= DCG, and dentin bond interface= RCG). It was used the quasi-static nanoindentation technique with 1000 μN of peak force on intertubular dentin and restorative dentin interfaces and 1500 μN on enamel (prism center). The ANOVA one-way analysis (p≤0.05) showed no difference in the H values and Er (GPa). The H and Er values of control and irradiated group were, repectivaly: ECG=4.5±0.3 and EIG=4.7±0.4; DCG= 0.9±0.1 and DIG=0.9±0.1 and ECG=105.2±13.7 and EIG=106.3±8.7; DCG=20.4±5.2 and DIG: 20.7±1.4. For the dentin-restored interfaces the H and Er values were: adhesive layer (RCG=0.3±0.1 and RIG=0.5±0.3), hybrid layer (RCG=1.1±0.57 and RIG=1.5±0.5) and underlying dentin (RCG=1.3±0.4 and RIG=1.2±0.2) and adhesive layer (RCH=7.4±4.2 and RIC=9.2±4.7), hybrid layer (RCG=24.1±11.8 and RIG=32.9±11) and underlying layer (RCG=26.7±8.3 and RIG=25.4±4.9) showed no statistical difference between groups. The study concludes that the mechanical proppeties behavior of the enamel, dentin and dentin bond interface was not impacted by the radiotherapy treatment of head and neck cancer.
Keywords
Nanoindentation, radiation therapy, viscoelasticity, enamel, dentin, hybrid layer.
7
IntroductionThe discussion about dental treatment of head and neck cancer patients who underwent radiation therapy has increased. Although the treatment is very effective for tumor control, the radiation also causes a series of toxicity issues to the normal tissues surrounding the tumor and within the radiation field. [1-4].
The common information of most of the recent studies about these changes in dental structures is the clinical appearance of the caries in these patients. The cervical and incisal lesions with brown discoloration are a common post radiation effect seen by clinicians. The progression of this dental carie is fast and the fragility of the structure of these tissues seems to be greater than regular teeth [5,7].
Many studies search for the reason of the effects of radiation on dental hard tissues, as in the enamel, dentin, and the dentin bond interface [8-18]. But, understanding how exactly the structures of the dental tissues are affected by radiation therapy is also important and stills an open question.
The fragility of the enamel it was been related as the first evidence of accelerated development of caries in patients undergoing radiotherapy for head and neck. Grotz et al., (1998) evaluated in vitro, in situ and in vivo, the demineralized enamel surface irradiated and non-irradiated by the micromorphometric and micromorphological analysis. On the non-irradiated enamel, there was a total loss of prismatic structure. This behavior led researchers to believe that the enamel is less resistant to acid attack tissue after radiotherapy [19].
Already when the tissue evaluated is the dentin, the resin composites bonded to irradiated dentin by specific monomeric agents (adhesives) have presented similar bond strength results of the non-irradiated dentin [10, 17, 14]. These studies suggest the possibility of performing the restorative procedure with adhesive systems associated with composites as therapeutic means of restoring teeth in patients who underwent radiotherapy.
However, this assumption is not a reality. In most of cases, the professional has doubts about the restorative treatment planning of patients undergoing radiotherapy. This happens
8
because there is unconsistent indication in the literature that indicates the restorative material to be applied in these cases, but the generalization on the recommendation of direct restoration [1]. In some cases, it is suggested that the use of glass ionomer as a restorative material in pediatric patients with head and neck cancer [20]. However, Hu et al. (2005) followed two years clinical performance of restorations made with glass ionomer cement in teeth that had decay radiation. The study showed that this type of material showed 82% of failures due to displacement of material from the cavity [21]. Because of the failure of these materials, it can be suggest the possibility of performing the restorative procedure with adhesive systems associated to the composite restorative resins such therapeutic means of teeth in patients who underwent radiotherapy.
The morphology of natural caries lesions was compared with the radiation caries and both conditions showed the same patterns of degradation. Curiously, most of part of the irradiated patients was severely demineralized enamel within 6 weeks, whereas the control group showed no significant enamel demineralization after 12 weeks. On the other hand, no morphological changes of in vivo irradiated teeth was observed when compared with non-irradiated teeth [5, 6]. This becomes more evident when the results of tensile strength obtained in irradiated dentin restorations also showed no significant differences when compared to non-irradiated dentin.
Since the enamel and dentin are considered viscoelastic materials, the nanoindentation technique is indicated to gather information, allowing prediction of alterations on those structures after radiation therapy. The knowledge of the mechanical properties of the hard tissues are essential for predicting the effects of microstructural alterations due to aging, development of the carie, chemical and physical treatments on tooth strength [22].
One of the first to apply nanoindentation to the study of dentin and dental materials was Van Meerbeek et al., who assessed the hardness and elasticity of the resin-dentin bonding area. This region is considered able to relieving tensions of contraction during and after restoration, as well as biomechanically strengthening of dental structures bonded to the restoration [23].
A nanometer probe tip approaching the surface of the materials that are under test has largely been used to measure the reduced modulus of elasticity and hardness of those types of
9
materials. The modern theory of indentation, where the force and displacement are used to measure these mechanical properties, has been applied to mineralized tissues [24]. The technique enables the use of a minimal sample size, is a nondestructive technique, and can predict the responsiveness of a structure in different conditions.
One issue of the treatment for head and neck cancer, as with radiation therapy, is the side effects in dental teeth. The effects can be analyzed by the nanoindentation as a way to know if the structure of the enamel and dentin and the dentin restoration interface were direct affected by the radiation.
On this way, the propose of this study was evaluate at the nanoscale (nanoindentation), the mechanical behavior of in vivo irradiated teeth (enamel and dentin) and compare it to the mechanical behavior of nonirradiated teeth. Besides, we also evaluate the dentin bond restorative area produced by a monomer (adhesive) and resin.
The null hypothesis tested in this study was that there is no difference in the mechanical behavior between non-irradiated and in vivo irradiated teeth.
Materials and methods
The study was approved by the Ethics Committee of Piracicaba Dental School (protocol 003/2012). There were two groups in the study, the first was comprised by six adult non-irradiated lower incisors (control group), and the second was comprised by six human lower incisors extracted from five patients who underwent head and neck radiotherapy (in vivo irradiated group). The extractions and storage of the samples followed the protocols of Silva et al [5]. All patients of the second group underwent conventional bi-dimensional radiotherapy with parallel-opposed lateral fields with the entire mandible included in the radiation field. Radiotherapy plans from all patients were assessed for the field of radiation and total tumor dose estimation values (Table 1).
10
Table 1. Clinical pathologic aspects of patients and specimens used.
Patient Age (Years)
Anatomic site
(SCC) Treatment Oral side effects
Radiation dose (Gy) 1 67 Base of the Tongue Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70 2 60 Base of the Tongue Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70 3 60 Tonsil Surgery + Radiotherapy Radiation-related caries + Xerostomia 60
4 66 Pyriform Sinus Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70 5 60 Base of the Tongue Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70
SCC = squamous cell carcinoma Specimen preparation
A total of twelve lower incisor teeth were used in this study, six from the control group and six from the in vivo irradiated group, and it was evaluated three dental regions: enamel control group/enamel in vivo irradiated group (ECG/EIG), dentin control group/dentin in vivo irradiated group (DCG/DIG), and restoration interface control group/restoration in vivo irradiated group (RCG/RIG).
At preparation, each tooth was divided into three parts producing eighteen specimens per group. The crowns of each tooth were secctioned into three parts: incisal, that was used to evaluate the enamel; the midlle part, that was used to evaluate the dentin; and cervical, that was used to evaluate the restoration interface. The specimens were allocated to evaluate one of the three dental regions, resulting in six specimens per region for each group.
For the enamel specimens, the bucal surface was polished following the curve of the crown until it became a plane surface. For the dentin specimen, the bucal surface was polished following
11
the curve of the crown until it completely exposed the dentin. For the restoration group, the bucal surface was polished as the dentin group, and were restored using the Adper Single Bond Plus (3M ESPE, St. Paul, MN, USA). The bonding procedures were performed following the respective manufacturer’s instructions. After 24 hours of storage, the restoration was secctioned in a half to obtain the dentin bond interface area.
All specimens were embedded in epoxy resin, and when the resin cured, the embedded sample’s surface were further polished through silicon carbide paper of decreasing granulation sizes, from 400 to 1200 (Buehler Inc., Lake Bluff, IL, USA), and polished using colloidal diamond paste of 9, 6, 3, 1 and 0,25 µm (Buehler Inc., Lake Bluff, IL, USA), been cleaned using deionized water in ultrasonic cleaning for 5 minutes after each paste. The root-mean-square roughness of the finished surface was about 30 nm, measured by scanning the surface.
Quasi-static nanoindentation test
The technique consists in the measurement of the indenter load as a function of depth of penetration by the contact stiffness (Sc). These measures were obtained from the derivative of the unloading curve evaluated at the peak force. To maintain the reproducibility of the study, care was taken to remove any excess machine compliance. The indentation modulus E, sometimes referred to as the reduced modulus, is determined from the corrected contact stiffness, and the contact areas for each indentation. Hardness and reduced elastic modulus were calculated using the TriboScan 8.1.1 software (Hysitron Incorporated, Minneapolis, MN) based in a model of mechanical properties and load–displacement [23].
The nanoindenter used for all testing is a Hysitron Ubi 1 Triboindenter (Hysitron Incorporated, Minneapolis, MN). The manufacturer has specified a load noise floor of 100 nN with a load resolution of 1 nN. The maximum force is 10 mN or 30 mN. The displacement resolution is 0.04 nm with a noise floor of 0.2 nm in the z direction and a displacement resolution is 4.0 nm with a noise floor of 10 nm in the x-y direction. The thermal drift wasw less than 0.05 nm/sec.
The indentations were made using a Berkovich probe. The tip radius curvature is approximately 150nm, and the standard of the contact depth in dentin were about 150nm and 50
12
nm in enamel, that were calibrated using fused silica. A fluid cell Berkovich tip was used at force 1000 µN in dentin and RIG. When the indentation was done in enamel, the maximum load applied was 1500 µN. A standard trapezoidal load function of 5–2–5 seconds for load, hold, unload, was used respectively. The measurements were performed under hydrated conditions where the samples were kept immersed in deionized water.
An optical system (either Top-Down or Tip-View) integrated into the Ubi® system (Hysitron Incorporated, Minneapolis, MN) allowed positioning the probe over the desired testing location. So, the indentations were made in three different areas spaced at 200 μm of each. Five individual indentations were made in each area with spaced approximately 10 μm each. In enamel, the indentations were done in the prism location and the sheath indentations were discarded. Individual indentations were made on the intertubular dentin, where at least 5 μm of spacing was set between adjacent indentations. For the RIG, the indentations were made in the adhesive layer, hybrid layer and underlying dentin following the same parameter of the dentin group. The locations were carefully chosen to centralize the indents within each region of evaluation.
Statistical analysis
The data of the Quasi-static indentation test was listed by the TriboScan 8.1.1 Analisys Software (Hysitron Incorporated, Minneapolis, MN) and analyzed by one-way analysis of variance (ANOVA). Statistical tests run at 5% level of significance.
Results
From the optical system, in the enamel image captured (Figure 1), it can be seen the prism and sheath structural location, showing that the prisms are perpendicularly to the sample surface. The results for Er in the ECG (105.2 ± 13.7 GPa) and EIG (106.3 ± 8.7 GPa), and H in the ECG (4.5 ± 0.3 GPa) and EIG (4.7 ± 0.4 GPa) showed no statistical difference (p>0.05). Even in the enamel data, in the intertubular dentin (Figure 2), the Er for DCG (20.4 ± 5.2 GPa) and DIG (20.7 ± 1.4 GPa), and H in DCG (0.9 ± 0.1 GPa) and DIG (0.9 ± 0.1 GPa) failed to demonstrate statistical
13
difference (p>0.05). The Figure 3, present the restoration interface of material and dentin. All these three areas of evaluation (adhesive layer, hybrid layer and underlying dentin) have no statistical difference among control and in vivo irradiated group (Table 2).
Figure 1. Image from the surface of enamel specimen. The P letter shows the prism center, where the three series of five indents (*) were made.
Figure 2. Dentin surface image. The I letter show the intertubular dentin, where the three series of five indents (*) were made.
Figure 3. Dentin-Restoration interface image. The R letter show the composite resin layer. The A letter represent the adhesive layer. The HL letters show the hybrid layer and the D letter represent the underlying dentin. The three series of five indents (*) were made in the adhesive layer, hybrid layer and underlying dentin.
14
Table 2. Results from nanoindentations in restoration interface of dentin.
Area of evaluation Er H Control group Irradiated group Control group Irradiated group Adhesive layer 7.4 ±4.2 9.2±4.7 0.3±0.1 0.5±0.3 Hybrid layer 24.1±11.8 32.9±11 1.1±0.5 1.5±0.5 Underlying Dentin 26.7±8.3 25.4±4.9 1.3±0.4 1.2±0.2 Discussion
The null hypothesis of the present study was accepted, since the H and Er did not differ between the groups of irradiated and non-irradiated teeth.
The studies related with effects of radiotherapy on dental tissues are contradictory, and encompass a variety of methodology. Specially, the head and neck radiation therapy is a physical treatment that involve different tissues of oral cavity, divers effects can be seen and act as adjunt in a dental effects after the therapy. While the treatment is done, the trismus, mucositis, and decrease of buccal pH, are unforeseeable effects that can limit the oral hygiene capacity [1, 2, 25, 26]. When the in vitro studies are conducted, these oral side effects are not presented and in the most of these studies, the properties evaluated showed bad results when compared with non-irradiated teeth [8, 9, 13]. On the other hand, if the patient has transient effects, good oral hygiene, and receives care of a specialist dentist during the treatment, the development of caries is minor [3, 4].
The involvement of these oral changes on the caries development it seems be the reason by which the in vivo radiation is more applicable to research of the real effects of radiation therapy in teeth, than in vitro studies. Possibly, this difficulty of reproducing all the variables involved in the initiation and progression of caries in cancer patients, post-RDT process, is the base to explain the contradictory results described above and existing in the relevant in vitro scientific literature.
15
Therefore, it is imperative to encourage the development of more trustworthy models of the complex oral environment of patients with CCP and the RDT protocols aimed to these patients, allowing a better understanding of the biological processes involved in the etiology of CRR.
Remarkably, rather than analysing samples that were irradiated in vitro, this study analysed enamel, dentin and dentin bond interface from teeth that were irradiated in vivo and received high doses of radiation because they were included in the primary field of radiation. Additionally, such samples were exposed to the hostile oral environment of post-head and neck radiation patients and
suffered the effects of a highly cariogenic environment due to demineralization attributed to
hyposalivation and diet, among others.
All patients in the current study underwent conventional bi-dimensional radiotherapy with parallel-opposed lateral fields. Unlike intensity-modulated radiotherapy, conventional bi-dimensional radiotherapy does not allow the study of detailed dose distributions to the teeth of patients receiving head and neck radiotherapy. When the tolerance dose is above the 25–40 Gy, the damage to the salivary glands is usually irreversible [25-28]. However, during sample selection, the radiotherapy plans for all patients were assessed for field of radiation and total tumour dose estimation and it was possible to confirm that all samples included in the irradiated group were obtained from patients with late stage disease where the entire mandible was included in the radiation field as well as all mandibular anterior teeth.
These considerations are essential to the results of this study, since the mechanical behavior of both in vivo irradiated and non irradiated group did not differ in all tissues and areas evaluated. What may be happening was that after the radiation therapy treatment, the effects of buccal environment affected oral tissues, but not as a direct radiation effect in enamel, dentin, and restoration interface of dentin. A recent dosimetric study revealed, in accordance of the diagnosis and total dose of treatment, that when the teeth are in the radiation’s field, the dose absorbed by the teeth is close to the total dose applied by the radiation machine [29]. Therefore, it is possible to accept in this study, that all in vivo irradiated samples were subjected to a total radiation dose similar to the final dose delivered to the tumor.
16
It can be seem in the results from this study, that the H of the enamel and dentin of both control and in vivo irradiated group revealed a consistent value with others studies [30]. These findings show that the mechanical behavior of the analyzed tissues has the same behavior of the tissues from people that did not received the therapy. Also it can predict, that the behavior of these structures that compose the teeth is the same before and after radiation therapy, as showed by this and others studies by the normal pulp response, prospective clinical evaluations, and mechanical tests [10, 17, 31-35]. The current study exhibit normal mechanical properties response compared as non-irradiated teeth and the radiation therapy is conducted in vivo situations, what is goal of the results of this study.
The assumption that after radiation therapy the structure of enamel prism is altered and this condition causes reduced physical properties [36], is also declined by the results of this study. As the indentation is in a nanometric scale, if some alteration or degradation of the prism is present, the result between in vivo irradiated and non-irradiated shoud be observed, what is not the case of results of this study. Also, in dentin, the same assumption can be seen. In the dentin the hardness and reduced modulus of elasticity is compatible with in vivo irradiated teeth and non-irradiated teeth. Because of the nanosize of indentations, the alteration reported by the effect of radiation in water content of dentin was rejected. The free radicals, result of radiolysis, could denature the collagen and reduce the mechanical properties [37]. So, once the free radicals are released in dentin, the metallo-proteinases (MMPs) would activated, and the dentin bonding could be affected by degradation of the collagen resulting in decay of restorations or second caries [38, 39]. But, if these considerations are valid for head and neck radiation therapy, in the case of this study, our results should be different, since the Er and H are the mechanical behavior that can predict if the tissues are healthy or have some alteration.
The values of these measurements are the response of the mineral and organic component of the tissues. In enamel, that is a high mineral content structure, the hardness is greater than in dentin [40]. That is expected because the organic content in dentin make the tissue softer, what implies that the depth contact of tip indenter can be greater. As the technique measures the
17
indenter load as a function of depth of penetration from which the contact stiffness was obtained, these affirmations become true. Therefore, in this way, if these structures had any problem, the contact and unload curve would present an unusual behavior.
The bond procedures on dentin by acid etching increase the permeability of resins to the tissue [41]. In dentin, this is a form of tissue engineering, since the phosphoric acid demineralize the surface 5–8 µm to create nanometersized porosities within the underlying collagen fibrillar matrix. When the nanohardness and reduced modulus of elasticity values for hybrid layer and underlayer dentin were similar in this study, is possible to confirm that the interaction of dentin-adhesive-resin create a effective structure with properties comparable to dentin. This revealed the good tissue engineering as on the in vivo irradiated teeth as on nonirradiated teeth.
The relevance of the nanoindentation in hybrid layer concerns the restorative practice. The new evaluation is the use of this methodology to validate the difference of tissues irradiated by oncology treatments. Since them, the mechanical behavior of the hybrid layer has been used to show the difference of materials applied, as dentin bond agents, and the surface treatment of dentin with chemical agents and physical methods alterations [42]. The quality of the interaction between the collagen fibrils and bond agents result in a good performance of restoration. Changes in the structure of the collagen would come to make a poor hybrid layer construction, as well as the collapse of the fibrils make no permeation of the bond agent into the tubules.
When analyzed the results of restoration interface of this study, there were no changes in the layers of restoration. Both in adhesive layer, hybrid as an underlying dentin, the values are similar for the control and in vivo radiation group. It known that, if the resin is poorly infiltrated or if the resin slowly hydrolyzes and leaches from the hybrid layer, the intrinsic collagenolytic and gelatinolytic activity of the dentin matrix can be expressed and attack the collagen, causing it to solubilize [43]. The dissolution of the mineral content of dentin as result of the exposure of organic matrix degradated by bacterial enzymes and collagen degrading proteases occurs during caries progression. This weakens the hybrid layer and shifts more functional stress to the remaining fibrils,
18
causing them to defibrillate and enlarging the porosities within the hybrid layer, resulting in a low mechanic properties values [44].
However, as showed in this current study by the relative results of the H and Er on hybrid layer and underlayer dentin, the adhesives resins can seal the acid etched dentin from water, and can preserve their mechanical/chemical properties against the hydrolytic attack by MMPs. The findings of this study showed that probably the radiation therapy did not affect directly the dentin structures, once the all in vivo irradiated specimen’s indentations exhibit the same performance of the control specimens.
Conclusions
This work asserts that for these samples, the H and Er did not present variation between the respective values of both in vivo irradiated and non-irradiated groups. By the way, the radiation therapy was not able to impair the structure of enamel, dentin, as the hybrid layer formation during restorative procedures.
Acknowledgments
The authors would like to thank the financial support of CAPES (Coordination of Improvement of Higher Level).
References
1- Jansma J, Vissin kA, Spijkervet FKL, Roodenburg JLN, Panders AK, Vermey A, Szabó BG, Gravenmade EJ. Protocol for the prevention and treatment of oral sequelae resulting from head and neck radiation therapy. Cancer 1992;70:2171–2180.
2- Shaw MJ, Kumar NDK, Duggal M, Fiske J, Lewis DA, Kinsella T, Nisbet T. Oral management of patients following oncology treatment: literature review. Br J Oral Maxillofac Surg 2000;38:519–24.
19
3- Hancock PJ, Epstein JB, Sadler GR. Oral and dental management related to radiation therapy for head and neck cancer. J Can Dent Assoc 2003;69(9):585-90.
4- Andrews N, Griffiths C. Dental complications of head and neck radiotherapy: Part 1. Aust Dent J 2001;46:88–94.
5- Silva ARS, Alves FA, Antunes A, De Goes MF, Lopes MA. Patterns of demineralization and dentin reactions in radiation-related caries. Caries Res 2009;43:43–49.
6- Silva ARS, Alves FA, Berger SB, Gianini M, De Goes MF, Lopes MA. Radiation-related caries and early restoration failure in head and neck cancer patients. A polarized light microscopy and scanning electron microscopy study. Support Care Cancer 2010; 18:83– 87.
7- Al-Nawas B, Grotz KA, Rose E, Duschner H, Kann P, Wagner W. Using ultrasound transmission velocity to analyse the mechanical properties of the teeth after in vitro, in situ, and in vivo irradiation. Clin Oral Invest 2000;4:168-72.
8- Kielbassa AM. In situ induced demineralization in irradiated and non-irradiated human dentin. Eur J Oral Sci 2000;108:214–221.
9- Kielbassa AM, Schendera A, Schulte-Mönting J. Microradiographic and microscopic studies on in situ induced initial caries in irradiated and nonirradiated dental enamel. Caries Res 2000;34:41–4.
10- Gernhardt CR, Kielbassa AM, Hahn P, Schaller HG. Tensile bond strengths of four different dentin adhesives on irradiated and non-irradiated human dentin in vitro. J Oral Rehabil 2001;28:814–820.
11- Mcomb D, Erickson RL, Maxymiw WG, Wood RE. A clinical comparison of glass ionomer, resin modified glass ionomer and resin composites restorations in the treatment of cervical caries in xerostomic head and neck radiation patients. Oper Dent 2002;27:430–437.
20
12- Haveman CW, Summit JB, Burgess JO, Carlson K. Three restorative materials and topical fluoride gel used in xerostomic patients: A clinical comparison. J Am Dent Assoc 2003;134:177-84.
13- Naves LZ, Novais VR, Armstrong SR, Correr-Sobrinho L, Soares CJ. Effect of gamma radiation on bonding to human enamel and dentin. Support Care Cancer 2012;20:2873–78.
14- Galetti R, Santos-Silva AR, Nogueira da Gama Antunes A, de Abreu Alves F, Lopes MA, de Goes MF. Radiotherapy does not impair dentin adhesive properties in head and neck câncer patients. Clin Oral Invest 2014;18(7):1771-8.
15- Yadav S, Yadav H. Ionizing irradiation affects the microtensile resin dentin bond strength under simulated clinical conditions. J Conserv Dent 2013;16: 148-51.
16- Van Meerbeek B, Willems G, Celis JP, Roos JR, Braem M, Lambrechts P, Vanherle G. Assessment by nano-indentation of the Hardness and Elasticity of the Resin-Dentin Bonding Area. J Dent Res 1993;72: 1434-1442.
17- Gernhardt CR, Koravu T, Gerlach R, Schaller HG. The influence of dentin adhesives on the demineralization of irradiated and non-irradiated human root dentin. Oper Dent 2004;29:454–461.
18- Gonçalves LM, Palma-Dibb RG, Paula-Silva FW, Oliveira HF, Nelson-Filho P, Silva LA, Queiroz AM. Radiation therapy alters microhardness and microstructure of enamel and dentin of permanent human teeth. J Dent. 2014;42(8):986-92
19- Grötz KA, Duschner H, Kutzner J, Thelen M, Wagner W. Histotomography studies of direct radiogenic dental enamel changes. Mund Kiefer Gesichtschir 1998;2(2):85-90.
20- Albuquerque RA, Morais VLL, Sobral APV. Odontologic protocol of attendance the pediatric oncology patients: review of literature. Rev Odontol UNESP 2007;36(3):275-280
.
21
21- Hu J Y, Chen X C, Li Y Q, Smales R J, Yip K H. Radiation- induced root surface caries restored with glass-ionomer cement placed in conventional an ART cavity preparations: Result at two years. Aust Dent J. 2005; 50 (3): 183-190
22- Angker L, Swain MV. Nanoindentation: application to dental hard tissue investigations. J Mater Res 2006;21 (8):1893-1905
23- Van Meerbeek B, Willems G, Celis JP, Roos JR, Braem M, Lambrechts P, Vanherle G. Assessment by nano-indentation of the Hardness and Elasticity of the Resin-Dentin Bonding Area. J Dent Res 1993;72:1434-1442.
24- Oliver WC, Pharr GM. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 1992;7:6.
25- Seikaly H,Jha N,Harris R,Barnaby P,Liu R,Williams D,McGraw T, Rieger J, Wolfaardt J, Hanson J. Long-term outcomes of submandibular gland transfer for prevention of postradiation xerostomia. Arch Otolaryngol head Neck Surg 2004;130:956–961.
26- Eisbruch A, ten Haken RK, Kim HM, Marsh LH, Ship JA. Dose, volume and function relationships in parotid salivary glands following conformal and intensity-modulated irradiation of head and neck cancer. Int J Radiat Oncol Biol Phys 1999;45:577–587.
27- Walker MP, Wichman B, Cheng AL, Coster J, Williams KB. Impact of radiotherapy dose on dentition breakdown in head and neck cancer patients, author manuscript. Pract Radiat Oncol 2001;1(3):142–148.
28- Roesink JM, Moerland MA, Battermann JJ, Hordijk GJ, Terhaard CH. Quantitative dose-volume response analysis of changes in parotid gland function after radiotherapy in the head-and-neck region. Int J Radiat Oncol Biol Phys 2001;51:938–946.
22
29- Walker MP, Wichman B, Cheng AL, Coster J, Williams KB. Impact of Radiotherapy Dose on Dentition Breakdown in Head and Neck Cancer Patients. Pract Radiat Oncol 2011;1(3): 142–148.
30- Lewis G, Nyman JS. The Use of nanoindentation for characterizing the Properties of Mineralized Hard Tissues: State-of-the Art review. J Biomed Mater Res B Appl Biomater 2008;87(1):286-301.
31- Pioch T, Staehle HJ. Experimental investigation of the shear strengths of teeth in the region of the dentinoenamel junction. Quintessence Int 1996;7:711–714 28.
32- Bulucu B, Avsar A, Demiryürek EO, Yesilyurt C. Effect of radiotherapy on the microleakage of adhesive systems. J AdhesDent 2009;11:305–309.
33- Hey J,Seidel J,Schweyen R,Paelecke Habermann Y, Vordemark D, Gernhardt C, Kuhnt T. The influence of parotid gland sparing on radiation damages of dental hard tissue. Clin Oral Investig 2013;17:1619–1625 30.
34- Gomez DR, Estilo CL, Wolden SL, Zelefsky MJ, Kraus DH, Wong RJ, Shaha AR, Shah JP, Mechalakos JG, Lee NY. Correlation of osteoradionecrosis and dental events with dosimetric parameters in intensity-modulation radiation therapy for head and neck cancer. Int J Radiat Oncol Biol Phys 2011;81:207–213.
35- Bodrumlu E, Avsar A, Meydan AD, Tuloglu N. Can radio- therapy affect the apical sealing ability of resin-based root canal sealers. J Am Dent Assoc 2009;140:326–330.
36- Franzel W, Gerlach R, Hein HJ, Schaller HG. Effect of tumor therapeutic irradiation on the mechanical properties of teeth tissue. Z Med Phys 2006;16:148–154.
37- Pioch TD, Golfels D, Staehle HJ. An experimental study of the stability of irradiated teeth in the region of the dentinoenamel junction. Endod Dent Traumatol 1992;8:241–244.
38- Al-Ammar A1, Drummond JL, Bedran-Russo AK. The use of collagen cross-linking agents
to enhance dentin bond strength. J Biomed Mater Res B Appl Biomater 2009;91(1):419-24.
23
39- De Munck J, Mine A, Van den Steen PE, Van Landuyt KL, Poitevin A, Opdenakker G, Van Meerbeek B. Enzymatic degradation of adhesive-dentin interfaces produced by mild self- etch adhesives. Eur J Oral Sci 2010;118:494–501.
40- He LH, Swain MV. Undestanding the mechanical behavior of humen enamel from its structural and compositional characteristics. J Mech Behav Biomed Mater 2008;1(1):18-29.
41- Pashley DH, Tay FR, Breschi L, Tjäderhanee L, Carvalho RM, Carrilho M, Tezvergil-Mutluay A. State of the art etch-and-rinse adhesives. dental materials 2011; 27:1-16.
42- Higashi C, Michel MD, Reis A, Loguercio AD, Gomes OMM, Gomes JC. Impact of adhesive application and moisture on the mechanical properties of the adhesive interface Determined by the Nano-indentation Technique. Oper Dent 2009;34:51-57.
43- Sano H. Microtensile testing, nanoleakage and biodegradation of resin-dentin bonds Journal of Dental Research 2006; 85(1):11-14.
44- Komori PCP, Pashley DH, Tjaderhane L, Breschi L, Mazzoni A, De Goes MF, Wang L, Carrilho MR. Effect of 2% Chlorexidine Digluconate on the bond strength to normal versus caries-affected dentin. Oper Dent 2009;34-2, 157-165.
24
Capítulo 2: The dentino-enamel junction of the in vivo irradiated teeth by
nanoDMA analisys.
Galetti R1, Silva A R S2, De Goes M F1, Bedran-Russo A K3, Lopes M A2.
1 Department of Restorative Dentistry, Piracicaba Dental School - University of Campinas, 13414-903, Campinas – São Paulo, Brazil.
2 Department of Oral Pathology, Piracicaba Dental School - University of Campinas, 13414-903, Campinas - São Paulo, Brazil.
3 Department of Restorative Dentistry, University of Illinois at Chicago, 60612, Chicago – Il, USA.
Abstract
Aim: To evaluate the viscoelastic properties of the enamel, dentino-enamel junction (DEJ) and dentin of in vivo irradiated teeth.
Material and method: Five non irradiated teeth (control group, n=5) and five in vivo irradiated teeth (irradiated group, n=5) were used to produce five beams that were used to evaluate three different areas: the enamel, the DEJ, and the dentin. Perpendicular sections to the long axis of the teeth were made at middle region of the crown to produce the beams. The Modulus Mapping Analysis was chosen to evaluate the loss and storage moduli of each area. Three data regions were collected of each tissue area of each beam, summing a total of fifteen data per tissue per group. The modulus values were calculated by the Hysitron® software and an Analysis of Variance (ANOVA Split Plot) and Tukey test at 5% of significance was used to compare groups and tissues. Results: All the three areas evaluated of control and irradiated group revealed statistical difference on the Loss and Storage Moduli. Both the loss and storage values are higer on the irradiated group for enamel (164.44±36.60;177.59±58.84), DEJ (50.85±35.78; 83,33±38,59) and dentin (21.18±18.61; 52.44±26.56) than control group values for enamel (127.15 ±74.45; 162.85±74.63), DEJ (25.72±9.64; 21.93±52.78) and dentin (10.39±8.65;32,10±20,39), respectivally.
Conclusions: The viscoelastic properties of in vivo irradiated teeth are different from control group. The enamel, DEJ and dentin presented the higer values on the in vivo irradiated group. These found suggest that after radiotherapy, the dental tissues are more susceptible to fractures.
25
IntroductionThe mechanical behavior of teeth are evaluated with the purpose of characterizing the dental materials, and to use this information to try to mimic or improve the composition of tooth in restorative procediments [1]. Nanoindentation is an attractive evaluation method because enamel and dentin are complex in structure and morphology, and the transition from the high mineralization area (enamel) to the low mineralization area (dentin) is very thin. Over the last years, the literature on the nanomechanical properties of bone and teeth tissues has burgeoned [2]. Furthermore, the methods to evaluate the behavior of the dental materials have been developed and improved to create a dynamic analysis in small areas resulting in a nondestructive evaluation method.
The Nanoscopic Dynamic Mechanical Analysis (nanoDMA®), made by an atomic force microscope with a nanoindenter coupled, is a good example of means to characterize the properties of isolated components of the microstructure, and/or property gradients exhibited from, anatomical structures of interest. NanoDMA® enables the characterization of spatial variations in both the storage and loss moduli (components describing the elastic and viscous behavior), as well as the distinction of the junctions between discrete constituents [1] like dentino-enamel junction (DEJ). The dental tissues are human body’s parts composed of high mineralized, organic and vascularized tissues. The enamel is the external coverage of the teeth composed in weight of 92-96% of inorganic matter, 1-2% of organic matrix and 3-4% of water, which makes it the hardest tissue on the human body. The dentin is a more resilient tissue located under the enamel, composed in weight of 70% inorganic matter, 18% organic matrix and 12% of water. The joint of these different tissues are known as junctions, and it supports the integrity of these tissues dissipating the stress during an applied force, that is responsible for preventing fractures [3,4].
All the differences of those tissues make the intrinsic mechanical behavior of teeth. The structure of the DEJ is described in the literature as having 25–100 µm, scallops shaped with convexities directed toward dentin. Each scallop includes finer structures down to nanometer-scale features. In addition to these topographic features that enhance the surface area of junctions, gradients in phase are regulated by a precise control of the protein expression and
26
biomineralization near the junction. These gradients in phase change mechanical analysis results in monotonic variations of mechanical properties [5,6,7].
In the case of head and neck malignancy-related radiotherapy, due to all the vital structures in the oral region, one of the most severe secondary consequences of this therapy is the destructive result to teeth and therefore to bucal function [8]. Radiation-related caries, also known as “radiation caries”, is considered a chronic and late side effect of head and neck radiotherapy [9] and generally affects the cervical region of teeth, with tooth surfaces acquiring a dark or brownish colour [10,11]. It is still unclear which factors cause this destruction of teeth. It could be the alteration of the dental hard tissues or the combination of hyposalivation, change in oral hygiene and a more soft and carbohydrate-rich diet [8]. The better understanding of this pathology can be used to make a more adequate preventive and restorative treatment plan for an individual patient before and after radiotherapy in the head and neck region.
Because of this lack of information about the radiation effects in the dental junctions and the distrust of these areas modified by the direct radiation, the aim of this study was try to understand if the enamel, dentin and the junction of those tissues have different viscoelastic behavior due to radiation treatment using the nanoDMA® methodology.
The null hypothesis tested in this study was that there is no difference in the viscoelastic behavior between non-irradiated and in vivo irradiated teeth.
Materials and methods
All samples of this study have the approval of the Ethics Committee of Piracicaba Dental School (protocol 003/2012). The experiment protocol used followed Galetti et al. [12], and the samples of the irradiated group were extracted and stored according to Silva et al [11].
Five adults non-irradiated lower incisors extracted by clinical indication from patients unknown (control group, CG), and five adults lower incisors extracted due to advanced periodontal disease from five patients who underwent head and neck radiotherapy (irradiated group, IG) were included in this study. All patients underwent conventional bi-dimensional radiotherapy with
27
opposed lateral fields. Radiotherapy plans from all patients were assessed for field of radiation and total tumor dose estimation (Table 1).
Table 1. Clinical pathologic aspects of patients of specimens used.
Patient Age (Years)
Anatomic site
(SCC) Treatment Oral side effects
Radiation dose (Gy) 1 67 Base of the Tongue Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70 2 60 Base of the Tongue Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70 3 60 Tonsil Surgery + Radiotherapy Radiation-related caries + Xerostomia 60
4 66 Pyriform Sinus Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70 5 60 Base of the Tongue Chemotherapy + Radiotherapy Radiation-related caries + Xerostomia 70
SCC = squamous cell carcinoma Specimen preparation
For each samples, one long axis central flat slice was produced using a low speed diamond saw cut machine (Isomet 1000; Buehler, Lake Bluff, USA) under water-cooling, and embedded in epoxy resin. The surfaces were then sanded using silicon carbide paper of decreasing size from 400 to 1200 grit, and polished using colloidal diamond paste of 9, 6, 3,1 and 0,025 mm and cleaned using deionized water by ultrasonic cleaning for 10 minutes in each paste. The root-mean-square (RMS) roughness of the finishing surface obtained by scanning the surface was about 30 nm.
28
NanoDMA® – Modulus Mapping testThe Modulus Mapping combines the in-situ imaging capabilities with the ability to perform nanoDMA® tests (Hysitron, Inc, Minneapolis, USA). During the imaging process, the machine system continuously monitores the stiffness of the specimen, and plot this stiffness as a function of the position on the specimen. At each pixel in the image, the stiffness is presented and the modulus is calculated. After acquiring the values, the software calculates storage (E’) and loss (E”) moduli.
The nanoDMA® test was performed in five specimens of each group in the DEJ (CG: n=5; IG: n=5) region. Fifteen scans were performed per group, three per specimen at different locations: one in enamel; one in dentin; and one in the junction.
The scan size window of 30 µm was used throughout. The frequency of scan was 0.2 Hz, with a set point of 1.5µN. A dynamic load of 50% of the static component was applied with a frequency of 100 Hz. The lock-in control settings used was found after pilot studies, where the results showed a dynamic amplitude between 0.4 nm. The sensitivity of 50 mV, time constant of 3 ms, and dynamic load of 1.5 µN were used to obtain the images.
Statistical analysis
The data were processed by the SPM Software (Hysitron, Inc, Minneapolis,USA) and the statistical analysis was made using the Analysis of Variance (ANOVA Split Plot) and Tukey test at 5% of significance.
Results
The nanoDMA® test was performed on the polished specimens to evaluate whether there were differences in properties between CG and IG, with indents maintained within the elastic range (including elastic and viscoelastic deformation). The results of the loss and storage moduli of the enamel, dentin, and junction areas of both groups are in the table 2 and 3 respectivelly.
The loss and storage moduli values presented different statatistical values between control and treatment teeth, table 2 and 3. Also, there was statistical difference between tissues, which was expected.
