UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Camila Galletti Espir Passador Emprego da microtomografia computadorizada na análise de preparo, limpeza e obturação de canais radiculares ovalados Araraquara 2018 UNESP - Universidade Estadual Paulista Faculdade de Odontologia de Araraquara Camila Galletti Espir Passador Emprego da microtomografia computadorizada na análise de preparo, limpeza e obturação de canais radiculares ovalados Tese apresentada à Universidade Estadual Paulista (Unesp), Faculdade de Odontologia, Araraquara para obtenção do título de Doutor em Odontologia, na Área de Endodontia Orientador: Prof. Dr. Mario Tanomaru Filho Araraquara 2018 Passador, Camila Galletti Espir Emprego da microtomografia computadorizada na análise de preparo, limpeza e obturação de canais radiculares ovalados / Camila Galletti Espir Passador. -- Araraquara: [s.n.], 2018 113 f. ; 30 cm. Tese (Doutorado em Odontologia) – Universidade Estadual Paulista, Faculdade de Odontologia Orientador: Prof. Dr. Mario Tanomaru Filho 1. Endodontia 2. Obturação do canal radicular 3. Preparo de canal radiocular I. Título Ficha catalográfica elaborada pela Bibliotecária Ana Cristina Jorge, CRB-8/5036 Universidade Estadual Paulista (Unesp), Faculdade de Odontologia, Araraquara Serviço Técnico de Biblioteca e Documentação Camila Galletti Espir Passador Emprego da microtomografia computadorizada na análise de preparo, limpeza e obturação de canais radiculares ovalados Comissão julgadora Tese para obtenção do grau de doutor em Odontologia (Endodontia) Presidente e orientador Prof. Dr. Mario Tanomaru Filho 2º Examinador Prof. Dr. Joni Augusto Cirelli 3º Examinador Prof. Guilherme Ferreira da Silva 4º Examinador Prof. Dr. Marco Antonio Hungaro Duarte 5º Examinador Profa. Dra. Laila Gonzales Freire Araraquara, 19 de fevereiro de 2018. DADOS CURRICULARES Camila Galletti Espir Passador NASCIMENTO: 08/04/1988 – Jales – São Paulo FILIAÇÃO: Estela Maria S. Galletti Abdulmassih Espir e Frederico Abdulmassih Espir Março 2007 - Dez 2011: Graduação em Odontologia pela Faculdade de Odontologia de Araraquara – UNESP Agosto 2010 - Junho 2011: Extensão em Endodontia (100 horas) - Fundação Araraquarense de ensino e pesquisa em Odontologia – FAEPO Março 2012 - Março 2014: Mestrado do programa de Pós-Graduação em Odontologia - Endodontia pela Faculdade de Odontologia de Araraquara – UNESP Março 2012 - Março 2014: Especialização em Endodontia pela Fundação Araraquarense de ensino e pesquisa em Odontologia – FAEPO Abril 2014 – atual - Doutoranda do programa de Pós-Graduação em Odontologia - Endodontia pela Faculdade de Odontologia de Araraquara - UNESP Quando iniciei meu Mestrado, tive a honra de ter em minha turma uma pessoa que não imaginava o quanto se tornaria especial em minha vida. Costumo dizer que ele foi um anjo! Nossa convivência infelizmente foi muito rápida mas tenho certeza que representou uma das amizades mais especiais que já tive! Dedico este trabalho a você, Adinael! Onde quer que você esteja hoje, tenho a certeza de que em nenhum momento estive sozinha. Por nenhum segundo se quer nos momentos mais difíceis que fossem, me senti desamparada por você. E nessas horas, era apenas pensar no seu rosto doce ou lembrar da sua risada única que tudo parecia ficar bem. Obrigada por ter sido uma das melhores pessoas da minha vida e por ter me dado a honra de, mesmo que rápido, ter tido sua convivência e poder te chamar de AMIGO! AGRADECIMENTOS Durante toda minha trajetória, o que não me faltaram foram motivos para agradecer. Costumo dizer que tive muita sorte! Estar sempre rodeada de amigos e pessoas que querem te ver bem é uma honra! E por isso, agradeço primeiramente a Deus, sendo realmente muito grata pela minha vida. E esta gratidão começa desde que nasci. Tive a honra de ser filha de Frederico e Estela. E bota honra nisso! Agradeço eternamente os ensinamentos, o amor e o carinho e tudo que fizeram e ainda fazem por mim sem medir esforços! Tenho certeza que sem vocês eu não seria nada! Minha eterna gratidão a vocês. Como se não bastasse, esta família me dá de presente o meu maior presente: uma irmã! Costumamos dizer que somos metades e nossa maior saudade diária. Não tenho dúvidas disso! Acredito que irmãos deveriam ficar para sempre na mesma casa. Mas, como isso não é possível, ainda bem que tenho você sempre comigo. Não posso deixar de agradecer também a toda minha família. Pessoas que mesmo de longe sempre se fizeram muito presentes em toda minha vida. Uma energia boa e sentimentos tão puros que impulsionam e nos fazem sempre seguir em frente. Em se tratando de família, hoje posso dizer que tenho a minha. Um outro presente de Deus, conheci o Felipe ainda na época da graduação e por uma alegria do destino, estamos juntos até hoje, casados e muito felizes! Em alguns momentos, confesso que implorei para que ele tivesse feito pós-graduação para me entender melhor rs. Tenho certeza que sem ele ao meu lado nada teria sido tão leve. Obrigada por me fazer sempre melhor e por estar ao meu lado todos os dias! Neste caminho que decidi trilhar, acabei ainda ganhando uma outra família. E que sorte a minha! Por todos estes anos, o Departamento de Endodontia da Faculdade de Odontologia de Araraquara foi minha segunda casa e ali, a cada ano que passava, conquistava novos amigos que se tornavam parte de mim. E quando digo que tive sorte, tive mesmo! Encontrei ali um orientador que hoje posso dizer que é um amigo e uma das melhores pessoas que já conheci. Desde a graduação, no meu mestrado e no doutorado, tive a honra de ter suas orientações. E além delas, pude também crescer muito com seus ensinamentos de vida. Nossas conversas em sua sala muitas vezes não eram apenas de trabalho. Falamos de viagens, família, acontecimentos. Como foi bom pra mim trabalhar todos esses anos com o senhor. Sou eternamente grata a você, professor Mario. Ou melhor dizendo, meu pai da pesquisa! Agradeço também à professora Juliane, ou apenas Ju! Sua risada única vou levar comigo para sempre e a forma leve e alegre de levar a vida também! Obrigada por tudo que me ensinou, e acima de tudo, pela sua amizade. Aos professores do Departamento, mais um motivo de sorte. Conviver com o professor Idomeo e todo seu conhecimento clínico; professor Fabio e sua incansável busca por algo novo; professor Renato com seu coração que não cabe no peito; professora Gisele e seu conhecimento impecável, além de sua amizade; e professor Kuga e seu conhecimento único, foi realmente uma honra! Jamais vou esquecer tudo que aprendi com cada um de vocês. Meu muito obrigada! Mais que ensinamentos, uma questão de amizade. E não uma amizade qualquer, mas aquela que te impulsiona, que te acompanha nos momentos mais difíceis e que aparece em sua vida da forma mais leve e também turbulenta. Amigos de pós-graduação, amigos de pesquisa, amigos de laboratório, amigos para a vida! De uma forma cíclica, pessoas que vem e em seguida se vão, mas que deixam sua marca especial. Agradeço a todos, sem exceção. Mas alguns não teria como não fazer um agradecimento especial. Gisselle, minha peruana favorita! O que teria sido de mim sem você? Além de uma amiga, ganhei uma mãe! Que presente mais especial foi ter te conhecido. Agradeço simplesmente tudo! Seu carinho por mim, sua paciência, sua competência e disciplina motivantes, e até mesmo suas lamentações! Rs Muito obrigada por tudo! Roberta, minha querida amiga. Comigo desde a graduação, não teria palavras para te agradecer! Você hoje é uma saudade diária, mas saber que está feliz já me deixa completa. Obrigada pela nossa parceria sempre! Fernanda, a irmã que a pós me deu. Dividir meus dias ao seu lado foi algo único! Nossos dias bons e ruins não teriam sido tão especiais sem você ali. Obrigada pela sua amizade, por sermos um time e por ter sido tudo exatamente da maneira que foi. Muito obrigada! Ana Lívia, Leticia e Raqueli, amigas que também não poderia esquecer. Comigo também desde o começo, foram pessoas muito importantes e que levarei comigo para sempre! Ao Jader e à Mariana, meu agradecimento é por um motivo especial. Além da nossa amizade, agradeço muito pela confiança e pela oportunidade que me deram. Foi para mim uma honra passar um pouco daquilo que sei e ajudar no trabalho de vocês. Muito obrigada de coração! Gostaria de deixar também um agradecimento especial a algumas pessoas que fizeram parte em especial de minha formação em pesquisa. Minha amiga Camila, que se tornou uma parceira fundamental em todas as etapas da nossa pesquisa. Professor Joni, agradeço sua atenção e todo empenho em me ajudar com o micro-CT. Professores Laila e Bruno, minha eterna gratidão! Terem me passado todo conhecimento e tirado tantas dúvidas é algo que jamais vou me esquecer. Tenho certeza que esta pesquisa não teria sido metade sem vocês. Muito obrigada! Professor Guilherme, muito obrigada! Me lembro de você desde a minha graduação, nos laboratórios, nos corredores e na copinha do Departamento. Obrigada por todos os ensinamentos, amizade e conselhos. Agradeço também à Luana, minha técnica de laboratório favorita e que compartilhou comigo os momentos mais inusitados desta trajetória! Por fim, agradeço à Faculdade de Odontologia de Araraquara – UNESP, na pessoa da sua Diretora Profa. Dra. Elaine Maria Sgavioli Massucato e vice-diretor Prof. Dr. Edson Alves Campos. Agradeço também às agências de fomento Capes pela concessão da bolsa de estudo para realização do doutorado e FAPESP pela concessão do auxílio para financiamento da pesquisa (Processo nº 2015/03437-6). Espir CG. Emprego da microtomografia computadorizada na análise de preparo, limpeza e obturação de canais radiculares ovalados [tese]. Araraquara: Faculdade de Odontologia da UNESP; 2018. RESUMO Este estudo avaliou morfologia, preparo e obturação com diferentes técnicas e materiais em canais radiculares ovais. Quinhentos e vinte incisivos inferiores foram avaliados por meio de radiografias digitais nos sentidos vestíbulo lingual (VL) e mesio distal (MD), sendo obtidos a partir da relação de diâmetro VL/MD: 23.3% canais achatados; 41.3% ovais; 27.3% arredondados; 4.5% redondos e 3.6% com achatamento VL (Publicação 1). Os incisivos classificados como ovais foram preparados: Reciproc 40 (R40) reciprocante anti-horário; MTwo 40, taper .06 (M 40.06) reciprocante horário (150° horário e 30° anti-horário); e MTwo 20, taper .06 seguido de MTwo 40, taper .06 (M 20/40.06) em reciprocante horário (Publicação 2); R40, Unicone 20.06 seguido de 40.06 reciprocante (Uni20/40.06) e sistema Mtwo até 40.06 rotatório (Mtwo seq) (Publicação 3); e R40, M40.06 e Mtwo seq (Publicação 4). A obturação foi realizada pelas técnicas de cone único (CU) ou condensação lateral (CL) e cimento AH Plus (AHP) (Publicação 4); CU e AHP ou Neo MTA Plus (NMTAP) (Publicação 5); e CL e MTA Fillapex (MTAF) ou AHP (Publicação 6). Escaneamentos foram realizados antes e após cada etapa experimental, utilizando o microtomófrafo SkyScan 1176. Volume, porcentagem de debris, de superfície não instrumentada e de falhas na obturação foram obtidos. Dados foram submetidos aos testes estatísticos ANOVA e Tukey, Kruskal-Wallis e Dunn, T Pareado ou Mann-Whitney (α=0.05). Reciproc e MTwo usando diferentes cinemáticas apresentaram aumento similar de volume do canal, e de superfície não instrumentada. M20/40.06 apresentou o menor percentual de debris no terço médio. O movimento reciprocante para R40 e M40.06 apresentaram preparos similares. Melhor limpeza de debris no terço médio foi obtida utilizando dois instrumentos com diâmetros diferentes (Publicação 2). O preparo Unicone apresentou maior aumento de volume, e maior percentual de debris e de superfície não instrumentada no canal e no terço médio. Maior limpeza e menor percentual de debris foi obtido para preparo reciprocante com R40 e MTwo seq (Publicação 3). MTwo seq apresentou maior percentual de debris e superfície não instrumentada no terço cervical, além de maior percentual de falha para técnica de CU na extensão total e terços cervical e apical após este preparo. O preparo de canais ovais com R40 e M40.06 proporcionaram maiores percentuais de limpeza, e obturação semelhante para CU e CL (Publicação 4). Não foram observadas diferenças significantes nos diferentes terços para obturação com cimentos AHP ou NMTAP. O cimento NMTAP apresenta capacidade de preenchimento de canais ovais pela técnica de CU similar ao AHP (Publicação 5). Obturação com MTAF apresentou maior percentual de falhas no terço cervical, sem diferença nos demais terços. A análise da alteração volumétrica demonstrou maior perda volumétrica para MTAF. MTAF proporciona maior porcentagem de falhas no preenchimento de canais ovais, e apresenta maior perda volumétrica que o AHP. (Publicação 6). Palavras chave: Endodontia. Obturação do canal radicular. Preparo de canal radicular. Espir CG. Use of computerized microtomography in the analysis of preparation, cleaning and obturation of oval root canals [tese]. Araraquara: Faculdade de Odontologia da UNESP; 2018. ABSTRACT This study evaluated the morphology, preparation and filling using different techniques and materials in oval-shaped canals. Five hundred and twenty mandibular incisors were evaluated using digital radiography in the buccolingual (BL) and mesio distal (MD) directions, being obtained from the VL / MD diameter ratio: 23.3% flattened canals; 41.3% ovals; 27.3% rounded; 4.5% round e 3.6% with flattening BL (Publication 1). The oval-shaped incisors were prepared: Reciproc 40 (R40) counterclockwise reciprocating; MTwo 40, taper .06 (M 40.06) clockwise reciprocating (150° clockwise and 30° counterclockwise); and MTwo 20, taper .06 and MTwo 40, taper .06 (M 20/40.06) clockwise reciprocating (Publication 2); R40, Unicone 20.06 and 40.06 reciprocanting (Uni20/40.06) and Mtwo rotary system until 40.06 (Mtwo seq) (Publication 3); and R40, M40.06 e Mtwo seq (Publication 4). Filling was performed using single-cone technique (SC) or lateral condensation (LC) and AH Plus sealer (AHP) (Publication 4); SC and AHP or Neo MTA Plus (NMTAP) sealer (Publication 5); and LC using MTA Fillapex (MTAF) or AHP sealer (Publication 6). Scans were performed before and after each experimental stage, using the SkyScan 1176 micro-computed tomography. Volume, percentage of debris, percentage of uninstrumented surface and voids in obturation were obtained. Data were submetted to ANOVA and Tukey’s tests or Kruskal–Wallis and Dunn tests, non- paired T test or Mann-Whitney (α=0.05). Reciproc and Mtwo using different kinematics were associated with a similar increase in root canal volume. M20/40.06 was associated with significantly lower debris in the middle third. The reciprocation motion for R40 and the clockwise reciprocation motion for Mtwo resulted in similar root canal preparations. Less remaining debris was present in the middle third when two instruments with diferente diameters were used (Publication 2). Unicone preparation was associated with higher debris, increase in root canal volume and uninstrumented surface in entire root canal and in the middle third. Better cleaning and less remaining debris was observed when R40 and Mtwo seq were used (Publication 3). MTwo seq was associated with higher debris and uninstrumented surface in the cervical third, and higher percentage of failure for the SC in the entire root canal and in cervical and apical thirds after this prepare. R40 and M40.06 were associated with less remaining debris and a similarity between SC and LC (Publication 4). No significant differences were observed in the different thirds for AHP or NMTAP sealer. NMTAP sealer presents the ability to fill oval canals by SC similar to AHP (Publication 5). Filling using MTAF showed higher percentual of voids in cervical third, with no difference in the other thirds. The volumetric change analysis revealed higher volumetric loss to MTAF. MTAF provides a greater percentage of voids in the filling of oval canals, and presents greater volumetric loss than the AHP. (Publication 6). Keywords: Endodontics. Root canal obturation. Root canal preparation. SUMÁRIO 1 INTRODUÇÃO ................................................................................................ 12 2 PROPOSIÇÃO ..................................................................................................15 2.1 Proposição Geral ..........................................................................................15 2.2 Proposições Específicas ............................................................................. 15 3 PUBLICAÇÕES ................................................................................................ 16 3.1 Publicação 1 ................................................................................................. 16 3.2 Publicação 2 ................................................................................................. 28 3.3 Publicação 3 .................................................................................................. 41 3.4 Publicação 4 .................................................................................................. 55 3.5 Publicação 5 .................................................................................................. 67 3.6 Publicação 6 .................................................................................................. 75 4 DISCUSSÃO ......................................................................................................85 5 CONCLUSÃO .....................................................................................................90 REFERÊNCIAS ................................................................................................. 91 APÊNDICE A – METODOLOGIA DETALHADA ................................................ 98 ANEXO A - AUTORIZAÇÃO PARA UTILIZAÇÃO DA PUBLICAÇÃO 1 ..........108 ANEXO B – AUTORIZAÇÃO PARA UTILIZAÇÃO DA PUBLICAÇÃO 2 .........109 ANEXO C – APROVAÇÃO COMITÊ DE ÉTICA EM PESQUISA ..................... 110 12 1 INTRODUÇÃO A morfologia do canal radicular exerce influência na qualidade do preparo1-4. Dentre as diversas anatomias, canais radiculares denominados ovais apresentam diâmetro mesiodistal (MD) menor que o vestíbulo-lingual (VL), são mais susceptíveis à fratura radicular5,6, e apresentam áreas de mais difícil acesso durante o preparo. Versiani et al.2 avaliaram por meio de análise microtomográfica o preparo de canais ovais utilizando diferentes sistemas (SAF, Reciproc, WaveOne e ProTaper Universal) e concluíram que nenhum sistema foi capaz de preparar completamente o canal radicular. Incisivos inferiores apresentam na maioria das vezes um único canal7,8,9 além de elevada incidência de canais com morfologia oval, em prevalência que pode exceder um percentual de 50%9,10. Esta morfologia é empregada para estudos de preparo e limpeza do canal radicular por apresentar áreas de difícil acesso11. A cinemática dos instrumentos também pode influenciar o preparo do canal radicular. O movimento reciprocante consiste em movimento rotatório não contínuo em direções opostas (direção de corte do instrumento e direção de liberação do instrumento). Esta cinemática previne o travamento do instrumento no canal radicular, permite o preparo com uso de um único instrumento e promove redução de fadiga cíclica12-15. Os sistemas Reciproc e WaveOne são usados com movimento reciprocante16-19, apresentando um maior movimento no sentido anti-horário que corresponde ao corte do instrumento, seguido de menor rotação no sentido inverso (horário), proporcionando a progressão do instrumento para o ápice20. Reciproc consiste em sistema de lima única fabricado com liga de Niquel titânio M-Wire, disponível em três diferentes tamanhos (R25, R40 e R50). O sistema Unicone (UnicOne, Medin, Nové Město Moravě, Czech Republic) apresenta ponta inativa e secção triangular, disponível nos tamanhos 20.06, 25,06 e 40.06 sendo preconizado uso em movimento reciprocante anti-horário, como o Sistema Reciproc. Em análise comparativa utilizando este sistema, um menor transporte foraminal para o Sistema Unicone foi observado quando comparado aos sistemas Reciproc e ProTaper21. Considerando-se a capacidade de corte no sentido horário dos instrumentos rotatórios, o uso dos mesmos em movimento reciprocante no sentido horário vem sendo avaliado17,22. O sistema rotatório Mtwo (VDW, Munich, Germany) consiste em uma sequência de limas de NiTi, que apresenta efetividade no preparo23, incluindo 13 limpeza de debris no terço apical24. MTwo e Reciproc são instrumentos com secção transversal semelhante (em forma de “S”), porém direção de corte inversa. Desta forma, o uso da cinemática reciprocante horária para instrumentos com a capacidade de corte para direita vem sendo avaliada13,17,25,26, utilizando rotação horária maior que a anti-horária. Com base nestas considerações, a utilização da cinemática reciprocante em diferentes sistemas mecanizados pode reduzir a fadiga cíclica e torsional dos instrumentos13,25,26, podendo permitir a redução do número de instrumentos para o preparo. O preparo biomecânico dos canais radiculares visa, além de limpeza, proporcionar conformação cônica, adequada para a obturação do canal27. Assim, a qualidade da obturação pode ser influenciada pelo preparo com diferentes sistemas de instrumentação28, utilização de diferentes técnicas de obturação29, e materiais obturadores30-32. Dentre as técnicas preconizadas, a compactação lateral é um método bastante usado devido ao baixo custo e habilidade seladora33, além de apresentar menores taxas de sobre-obturação34. Quando comparada à técnica de cone único, apresenta resultados similares quanto a quantidade de guta-percha35,36, habilidade seladora37,38 e qualidade da obturação39. A técnica de cone de guta- percha único tem sido bastante utilizada atualmente em associação aos preparos mecanizados40. No entanto, a falta de padronização nas medidas de fabricação dos cones dentro de um mesmo sistema e as diferentes anatomias do canal radicular podem promover falhas na obturação41-43. Além da técnica utilizada, o cimento obturador é um fator importante para o sucesso do tratamento. Associado aos cones de guta-percha, os diferentes cimentos visam o selamento do sistema de canais radiculares. Porém, o material obturador pode permitir infiltração bacteriana30-32, conduzindo ao insucesso do tratamento endodôntico. AH Plus é um material endodôntico à base de resina epóxica que apresenta habilidade seladora além de baixa solubilidade, o que garante uma maior integridade do cimento com menor perda de estrutura para o ambiente oral44. MTA Fillapex tem sido proposto como material obturador endodôntico, apresentando algumas propriedades físicas adequadas45, como escoamento e radiopacidade conforme recomendações ISO. No entanto, em análise de propriedades físico- químicas e mecânicas, altos valores de solubilidade foram observados46,47. Neo MTA Plus consiste em um material a base de silicato de cálcio que apresenta adequada 14 radiopacidade e hidratação, não promove descoloração, radiopacidade satisfatória e liberação de íons cálcio e hidroxila significativamente maior que MTA Plus, além de apresentar biocompatibilidade48-50. As finas partículas dos componentes do material podem favorecer o escoamento do cimento51, além de ter sido demonstrada penetrabilidade do Neo MTA Plus nos túbulos dentinários52. A maior parte dos estudos existentes para análise de materiais, em especial para Neo MTA Plus, consiste em avaliar suas propriedades físico-químicas e biológicas. A comparação deste material com relação à atuação em canais radiculares ovais avaliando falhas na obturação, sendo ainda comparados a um cimento padrão ouro como AH Plus ainda se encontra escassa na literatura. Para análise comparativa das etapas do tratamento endodôntico realizadas com diferentes variáveis, dentre os métodos atuais destaca-se a microtomografia computadorizada (micro CT). A possibilidade de executar análises não destrutivas permite que um mesmo espécime seja utilizado na análise dessas diferentes etapas do tratamento, como preparo e obturação. A análise do preparo avaliando parâmetros de volume, superfície não instrumentada, aumento de volume, debris, entre outros53,54 e da obturação de canais radiculares, em especial analisando falhas na obturação55,56 é realizada empregando esta ferramenta que possibilita alta resolução e qualidade de imagens. Suas características de análise permitem a avaliação de detalhes tridimensionais, especialmente falhas da obturação. Sendo assim, a análise do preparo de canais radiculares ovais empregando diferentes sistemas, cinemáticas e instrumentos, além da obturação com diferentes técnicas e materiais são propostas deste estudo. 15 2 PROPOSIÇÃO Os objetivos do presente estudo foram: 2.1 Proposição Geral Classificar incisivos inferiores e avaliar por meio de micro-CT o preparo e obturação de canais radiculares ovais. 2.2 Proposições específicas Publicação 1: Avaliar e classificar a morfologia do canal radicular e espessura dentinária, comparando análises radiográficas e em micro-CT. Publicação 2: Avaliar por micro-CT o preparo de canais radiculares ovais empregando movimento reciprocante com diferente ciemática utilizando os sistemas Reciproc e MTwo. Publicação 3: Avaliar por meio de micro-CT o preparo de canais radiculares ovais empregando movimento reciprocante e rotatório utilizando os sistemas Reciproc, Unicone e MTwo. Publicação 4: Avaliar por meio de micro-CT a qualidade de obturação de canais ovais preparados com sistemas Reciproc R40, Mtwo 40.06 e sequência Mtwo e obturados com cone único e compactação lateral. Publicação 5: Avaliar a qualidade de obturação de canais radiculares ovais com diferentes cimentos (Neo MTA Plus e AH Plus) após preparo com sistema Unicone e obturados pela técnica de cone único. Publicação 6: Avaliar a qualidade de obturação de canais radiculares ovais com diferentes materiais (MTA Fillapex e AH Plus) obturados pela técnica de cone único, e a alteração volumétrica dos cimentos endodônticos. 16 3 PUBLICAÇÕES 3.1 Publicação 1 Radiographic and Micro-CT classification of root canal morphology and dentin thickness of mandibular incisors ABSTRACT Context: Root canal anatomy is evaluated using different methodologies. Aims: To evaluate and classify root canal morphology and dentin thicknesses (DT), comparing radiographic and Micro-CT analysis. Methods and Material: Canal diameter and DT of mandibular incisors (n=520) were evaluated using digital radiographs in buccolingual (BL) and mesiodistal (MD) directions. The diameter ratio (DR) BL/MD was classified: flattened (FL, DR>4); oval (OV, 2≤DR≥4); rounded (RN, 1.12); round (RO, 0.9≤DR≥1.1); and with BL flatness (BL, DR<0.9). OV (n=110) were subjected to Micro-CT. DT and DR were evaluated at 3, 6 and 9 mm. ANOVA, Tukey and paired Wilcoxon tests (P<0.05) were used. Results: Radiographic classification was 23.3% FL; 41.3% OV; 27.3% RN; 4.5% RO and 3.6% BL. DT was similar. Radiographic DT at 3 and 9 mm was greater than Micro-CT (P<0.05), and were similar at 6 mm (P>0.05). DR differed between the analyses. Oval canals were predominant at all levels radiographically and at 9 and 6 mm in Micro-CT analysis, with greater variation at 3mm. Conclusion: Oval root canals are predominant in mandibular incisors at 9 mm. Radiographic DT is larger than observed in Micro-CT at 3 and 9 mm, and the classification differed in each root level. The classification at 9 mm is indicated. Key Words: Endodontics, radiography, root canal anatomy, x-Ray Microtomography. Key Messages: The oval shape is predominant in mandibular incisors with a single canal, at 9 mm from the apex. When comparing the radiographic and Micro-CT analyses, the radiographic mesial and distal thickness is greater at 3 and 9 mm. The classification at 9 mm from the apex must be indicated for root canal classification.  Artigo publicado e descrito segundo as normas do periódico Journal of Conservative Dentistry (Confira autorização da editora para publicação no Anexo A) . 17 INTRODUCTION Root canal morphology is evaluated using different methodologies, such as staining and clearing techniques, radiographic, cone beam computed tomography1 or microtomographic analysis2,3,4. Mandibular incisors are commonly used for different analysis, especially considering the number of main and accessory canals5. Mandibular incisors are also widely used in Endodontics studies because they have flattened and oval canals. The cross section morphology of the root canal is classified according to the radiographic measurement of the mesiodistal (MD) and buccolingual (BL) diameter ratio (DR)6,7: round (similar DR), oval (DR two times larger), long oval (DR up to 4 times larger), flattened (DR 4 or more) and irregulars (undefined values)7. It has been defined based on different root levels7 or sections at 1, 2, 3, 4 and 5 mm from the apex (DR≥2)6. Mandibular incisors commonly have a single canal8 and high prevalence of oval root canal2,3, been extensively used to evaluation of root canal cleaning and preparation using different instruments9,10. Microtomographic analysis has also been widely used to evaluate this morphology, such as root canal preparation, root canal filling and retreatment protocols11,12. Dentin thickness of root walls at the different levels are important to the planning of root canal preparation maintaining the root strength13. Roots with a MD dimension smaller than the BL are more susceptible to fracture, as observed in maxillary premolars, mesial roots of mandibular molars, and mandibular incisors14. Moreover, planning for intracanal posts15 depends on evaluating the remaining dentin thickness16. This thickness may be evaluated radiographically6, with possible occurrence of distortions due to superimposition of root wall structures. Evaluation by using microtomographic analysis obtain precise measurements of this thickness2,17. Considering the importance of root canal morphology and dentin thickness of root walls for research protocols and clinical procedures, the aims of this study is to evaluate and classify the root canal shape and dentin wall thicknesses of mandibular incisors, and compare radiographic and Micro-CT analyses. 18 MATERIAL AND METHODS Five hundred and twenty mandibular incisors approved by Ethics Committee (#699.061) were radiographed (Kodak RVG 6100 Atlanta, GA, USA) in the buccolingual (BL) and mesio distal (MD) directions to select teeth with single canal, absence of endodontic treatment and without extensive restorations. Radiographic Analysis The images obtained were analyzed by CTan (V1.11.8; SkyScan, Belgium) software, measuring the root canal diameter in the BL and MD direction (n=520). These measurements were made at 9 mm from the apex (middle third) to classify the root canal shape. According to the BL/MD diameter ratio (DR), were classified: flattened canals (FL, DR>4); oval canals (OV, 2≤DR≥4); and an additional classification, with rounded canals (RN, 1.12); round (RO, 0.9≤DR≥1.1) and with flattening BL (BL, DR<0.9) (Figure 1A). The dentin thickness (DT) of buccal, lingual, mesial and distal walls was also evaluated in each sample, by using the same tool. The measurements were made at 3 and 9 mm from the apex. The distance between the root canal and external wall was considered as the value corresponding to the dentin thickness of the walls (Figure 1B). Fig. 1 1A: Buccolingual (BL) and mesiodistal (MD) radiographic images (I). Analysis of mesiodistal (MD) and buccolingual (BL) diameter (D) of root canal at 9mm from the apex (II). Classification according to the observed diameter ratio (DR) BL/MD (III). 1B: Radiographic images (I) used to analysis the dentin thickness (DT) of mesial (M) and distal (D) (II) dentin walls and e buccal (B) and lingual (L) (III) at 3 and at 9 mm from the apex. 19 Microtomographic and Radiographic Analysis At this stage, a hundred and ten mandibular incisors radiographically classified as oval were subjected to microtomographic scannings using the Micro-CT scanner (SkyScan 1176, Bruker-microCT, Kontich, Belgium). The scanning parameters were based on a pilot study and were performed at 70 kV, 353 mA and 360° rotation, and a 0.5° rotation step, resulting in an image with a 17.42-µm voxel size, filter made of aluminum and duration time of 23 minutes 14 seconds. Thereafter, the images were reconstructed using NRecon (V1.6.4,7; SkyScan, Belgium) software. The adjustment of reconstruction parameters were made in order to suppress noises. Fine-tuning function enabled set artifact correction values, such as Gaussian filter, beam hardening correction, post alignment and ring artifact correction. The resulting images were processed in Data Viewer (V1.5.1.2; Sky Scan, Belgium) software, to obtain sagittal sections and were analyzed with CTan (V1.11.8; SkyScan, Belgium) (Figure 2A). The BL/MD DR, and dentin thickness of mesial and distal walls of the samples was measured at 3, 6 and 9 mm from the apex (Figure 2B), as previously described. These 110 samples were also evaluated radiographically again, with the DR and dentin thickness being measured at 3, 6, and 9 mm from the apex. Fig. 2 2A - Diameter mesiodistal (MD – a1, a3, a5) and buccolingual (BL – a2, a4, a6) evaluated in radiographic images (red lines). Diameter ratio (DR) (b1, b2, b3) observed (blue lines) in micro CT images at 3 (I), 6 (II) e 9 (III) mm from the apex. 2B - Images obtained using CTAn software and measurements of dentin thickness (white lines) of mesial (M) e distal (D) dentin walls at 3 (IV), 6 (V) and 9 (VI) mm from the apex. 20 Statistical Analysis The data obtained were submitted to the ANOVA and Tukey tests and Wilcoxon paired correlation test, at a 5% level of significance. RESULTS Radiographic Analysis The 520 mandibular incisors evaluated at 9 mm from the apex were classified: 121 (23.3%) flattened; 215 (41.3%) oval; 142 (27.3%) rounded; 23 (4.5%) round; and 19 (3.6%) with BL flatness. As regards the dentin wall measurements, the thicknesses at 3 and 9 mm from the apex were similar between the different classifications, except for the buccal wall that was greater for flattened canals compared with those considered rounded, both at 3 and at 9 mm (Table 1). Microtomographic and Radiographic Analysis A hundred and ten samples radiographically considered oval were evaluated. A comparison between the values obtained radiographically and by Micro-CT analysis was performed. The radiographic dentin thickness was greater than that obtained by Micro-CT for the mesial and distal walls at 3 and 9 mm (P<0.05), and were similar at 6mm (P>0.05) (Table 2). Based on the values for the DR at the 3 root levels, the authors reclassified the samples considered oval by the radiographic exam at 9 mm level. Therefore, using Micro CT at 9mm level, the classification was: 94 (85.4%) oval; 14 (12.7%) flattened; and 2 (1.8%) rounded. By the Micro-CT analysis at 6mm: 58 (52.7%) were oval; 9 (8.1%) flattened; 28 (25.4%) rounded; 11 (10%) round; and 4 (3.6%) with BL flatness. At 3 mm, 35 (31.8%) were considered oval; 1 (0.9%) flattened; 48 (43.6%) rounded; 17 (15.4%) round; and 9 (8.1%) with BL flatness. Radiographically at 9 mm, the 110 samples were classified as oval. However, at 6mm, 69 (62.7%) were considered oval; 27 (24.5%) flattened; 12 (10.9%) rounded; and 2 (1.8%) round and at 3 mm, 62 (56.3%) were considered oval; 8 (7.27%) flattened; 25 (22.7%) rounded; 13 (11.8%) round; and 2 (1.8%) with BL flatness (Table 3). 21 Table 1 – Distribution of mandibular incisors classified at 9 mm from apex by diameter ratio (DR); buccolingual (BL) and mesio distal (MD) ratio. Mean and standard deviation of dentin thickness (in mm) of buccal (B); lingual (L); mesial (M); and distal (D) walls after radiographic analysis Radiographic evaluation of mandibular incisors with single canal (n-520) Classification Total 520 % Dentin thickness (DT) 3mm 9mm B L M D B L M D *Flattened (DR>4) 212 23.3 1.55a (±0.2) 1.56a (±0.2) 0.83a (±0.2) 0.87a (±0.2) 1.98a (±0.3) 2.00a (±0.2) 1.25a (±0.2) 1.29a (±0.2) *Oval (2≤DR≥4) 215 41.3 1.48a,b (±0.2) 1.54a (±0.2) 0.83a (±0.19) 0.88a (±0.19) 1.91a,b (±0.2) 1.96a (±0.2) 1.25a (±0.2) 1.27a (±0.2) **Rounded (1.12) 142 27.3 1.43b (± 0.2) 1.49a (±0.2) 0.83a (±0.19) 0.87a (±0.18) 1.85b (± 0.2) 1.95a (±0.2) 1.29a (±0.2) 1.27a (±0.2) **Round (0.9≤DR≥1.1) 23 4.5 1.39a,b (±0.2) 1.44a (±0.2) 0.80a (±0.16) 1.21a (±1.4) 1.99ª,b (±0.2) 1.97a (±0.2) 1.25a (±0.17) 1.31a (±0.2) ** BL flatness (DR<0.9) 19 3.6 1.43a,b (±0.2) 1.50a (±0.2) 0.78a (±0.17) 0.83a (±0.18) 1.78ª,b (±0.2) 1.95a (±0.2) 1.20a (±0.2) 1.21a (±0.15) Different letters in each column indicate statistically significant differences (P<0.05). *Classification according to Jou et al. (20) **Additional Classification proposed Table 2 - Means and standard deviation (±) for mesial (M) and distal (D) dentin thickness values (in mm). Buccolingual (BL) and mesio distal (MD) diameter values (in mm) of the radiographic and microtomographic analyses. Mandibular incisors with root canal radiographically classified at 3, 6 and 9 mm from Apex 22 Radiographic and microtomographic analyses of the dentin thickness and canal diameters Analysis Dentin thickness Canal Diameters 3mm 6mm 9mm 3mm 6mm 9mm M D M D M D BL MD BL MD BL MD Rx 0.77a ±0.19 0.85a ±0.18 0.97a ±0.19 1.06a ±0.2 1.21a ±0.2 1.26a ±0.2 0.5a ±0.24 0.22a ±0.08 0.97a ±0.33 0.31a ±0.12 1.37a ±0.37 0.51a ±0.14 Micro CT 0.71b ±0.13 0.74b ± 0.16 0.92a ±0.13 0.98a ±0.7 1.10b ± 0.16 1.09b± 0.15 0.52a ±0.18 0.33b ± 0.1 0.78b ±0.27 0.37b ±0.11 1.30b ± 0.3 0.39b ± 0.1 Different letters in each column indicate statistically significant differences (P<0.05). Table 3 - Reclassification according to the buccolingual/mesio distal diameter ratio by radiographic and microtomographic analyses at 3, 6 and 9 mm of mandibular incisors samples with root canals radiographically classified as oval at 9 mm from the Apex Radiographic and microtomographic reclassification of oval root canals Classification DR (VL/MD) Radiographic Analysis Microtomographic Analysis 3mm 6mm 9mm 3mm 6mm 9mm *Flattened DR>4 8 (7.27%) 27 (24.5%) 0 1 (0.9%) 9 (8.1%) 14 (12.7%) *Oval 2≤DR≥4 62 (56.3%) 69 (62.7%) 110 (100%) 35 (31.8%) 58 (52.7%) 94 (85.4%) **Rounded 1.12 25 (22.7%) 12 (10.9%) 0 48 (43.6%) 28 (25.4%) 2 (1.8%) **Round 0.9≤DR≥1.1 13 (11.8%) 2 (1.8%) 0 17 (15.4%) 11 (10%) 0 ** BL flatness DR<0.9 2 (1.8%) 0 0 9 (8.1%) 4 (3.6%) 0 *Classification according to Jou et al.20 **Additional Classification proposed 23 DISCUSSION In the present study, high variability of the DR BL/MD was observed by using radiographic measurements at 9 mm from the apex. This DR showed values ranging from 0.9 to values higher than 4. The root canal morphology is classified by means of various criteria, and it is normally considered oval when the DR values are over 29. Therefore, considering this variability and the need for standardizing the planning of different studies in Endodontics, it was considered: flattened (FL, DR>4) and oval (OV, 2≤DR≥4) canals, as previously described7, as well as an additional classification: rounded (RN, 1.12), round (RO, 0.9≤DR≥1.1) and BL flatness (BL, DR<0.9). The root level used to determine the classification of cross section morphology of the root canal is variable and frequently not specifically described14,18,19. The authors observed high variability in the values for DR used in teeth selection12,20, as well as divergence between the classification of oval or flattened canals10,13. This study used specific values to determine the points of measurement for the analysis (3, 6 and 9 mm) in order to achieve more standardized definition for the different types of studies. Cross section morphology of the root canal considered oval after radiographic classification at 9 mm from the apex presented other classifications at 3 and 6 mm from the apex, corroborating with previous studies17. Moreover, radiographic and microtomographic analyses showed no correlation, demonstrating different classifications between them. Many studies make this measurement in the apical third,4,13 since an oval morphology is commonly found at 3 mm from the apex. However, at this distance, the authors found higher percentages of samples considered rounded (43.6%) in Micro-CT, corroborating with previous study2. At 9 mm, a higher correlation to the DR was obtained, with lower variability and a higher prevalence of oval morphology at this point (Table 3). This higher prevalence of oval classification for mandibular incisors was also reported, varying from 76.2% at 3mm2; 56% at 5mm6; or even 88% from 3-5 mm11. In the present study, the MD canal diameter at 3mm was 0.22mm, corroborating with previous study a constant MD distance in the 3 mm apical measurements, with values between 0.20 and 0.25mm2. In the remaining levels of measurement (6 and 9 mm), the MD and BL diameters showed values increasing progressively in the coronal direction (Table 2). These variations in diameter 24 significantly interfere in the BL/MD diameter ratios of root canals, influencing the distribution of classifications (Table 3). The mesial and distal dentin thickness showed values between 0.9 and 1.0 at 6mm level, without difference between the radiographic and microtomographic analyses. However, the measurements at 3 and 9 mm showed values between 0.71 and 1.26, with difference between them (Table 2). The analyses by using Micro-CT show values closer to real21, since they eliminate the superimposition of structures. Moreover, it is possible to observe the flattening dentin wall, specially evaluating the sagittal section (Figure 2). In spite of being a simpler method, radiographic evaluation may show a larger thickness than that obtained by Micro-CT, due this superimposition of structures and distortions of the image. No flattened areas (apical or middle third) were shown in the radiographs, and this may influence planning of apical instrumentation or preparation for intracanal posts. Although radiographic is clinically the most widely used method, microtomography is suggested as a tool for the selection of samples to be used in different studies, especially when specific morphologies are selected, standardizing results with high clinical relevance. A poorer image details was observed, for example, to cone beam computed tomography in comparison to microcomputed tomography, with difference when used in the different root thirds17. The root wall morphology and structure has a direct influence on planning of root canal preparation13 and intracanal posts15. An incorrect planning may favor micro-cracks or diminish the strength of the root. Smaller dentin thickness may increase susceptibility root fracture22. Although fractures predominantly occur in the BL direction23, a considerable number of fractures in the MD direction was observed24, with different types of cracks and DT values (DT≤0.3 or DT≥0.3). Therefore, the proximal DT observed (values ≥0.3 – as demonstrated in Table 2) may be used for planning studies about the influence of it on apical instrumentation of the root canal, or the use of intracanal posts. CONCLUSION Oval root canals is predominant in mandibular incisors with a single canal, after radiographic and Micro-CT analysis at 9 mm from the apex. In radiographic analysis, dentin thickness is similar between the different classifications (i.e. oval, 25 round, flattened). However, when comparing the radiographic and Micro-CT analysis of oval canals, the radiographic mesial and distal thickness is greater at 3 and 9 mm, and the diameter ratio values vary according to the root level. The classification at 9 mm from the apex showed greater proximity between the analyses, and must be indicated for root canal classification. The authors declare that they have no conflict of interest. REFERENCES 1- Lin Z, Hu Q, Wang T, Ge J, Liu S, Zhu M, Wen S. Use of CBCT to investigate the root canal morphology of mandibular incisors. Surg Radiol Anat 2014; 36:877-82. 2- Milanezi de Almeida M, Bernardineli N, Ordinola-Zapata R, Villas-Bôas MH, Amoroso-Silva PA, Brandão CG, Guimarães BM, Gomes de Moraes I, Húngaro- Duarte MA. Micro-computed tomography analysis of the root canal anatomy and prevalence of oval canals in mandibular incisors. J Endod 2013; 39:1529-33. 3- Leoni GB, Versiani MA, Pécora JD, Damião de Sousa-Neto M. Micro- computed tomographic analysis of the root canal morphology of mandibular incisors. J Endod 2014; 40:710-6. 4- Freire LG, Gavini G, Cunha RS, Santos Md. Assessing apical transportation in curved canals: comparison between cross-sections and micro-computed tomography. Braz Oral Res 2012; 26:222-7. 5- Vertucci FJ. Root canal anatomy of the mandibular anterior teeth. J Am Dent Assoc 1974; 89:369–71. 6- Wu MK, R’Oris A, Barkis D, Wesselink PR. Prevalence and extent of long oval canals in the apical third. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000; 89:739–43. 7- Jou YT, Karabucak B, Levin J, Liu D. Endodontic working width: current concepts and techniques. Dent Clin North Am 2004; 48:323-35. 8- Boruah LC, Bhuyan AC. Morphologic characteristics of root canal of mandibular incisors in North-East Indian population: An in vitro study. J Conserv Dent 2011; 14:346-50. 26 9- Siqueira JF Jr, Alves FR, Almeida BM, de Oliveira JC, Rôças IN. Ability of chemomechanical preparation with either rotary instruments or self-adjusting file to disinfect oval-shaped root canals. J Endod 2010; 36:1860-5. 10- De Melo Ribeiro MV, Silva-Sousa YT, Versiani MA, Lamira A, Steier L, Pécora JD, de Sousa-Neto MD. Comparison of the cleaning efficacy of self-adjusting file and rotary systems in the apical third of oval-shaped canals. J Endod 2013; 39:398-401. 11- Paes da Silva Ramos Fernandes LM, Rice D, Ordinola-Zapata R, Alvares Capelozza AL, Bramante CM, Jaramillo D, Christensen H. Detection of various anatomic patterns of root canals in mandibular incisors using digital periapical radiography, 3 cone-beam computed tomographic scanners, and micro-computed tomographic imaging. J Endod 2014; 40:42-5. 12- Crozeta BM, Silva-Sousa YT, Leoni GB, Mazzi-Chaves JF, Fantinato T, Baratto-Filho F, Sousa-Neto MD. Micro-Computed Tomography Study of Filling Material Removal from Oval-shaped Canals by Using Rotary, Reciprocating, and Adaptive Motion Systems. J Endod 2016; 42:793-7. 13- Abou El Nasr HM, Abd El Kader KG. Dentinal damage and fracture resistance of oval roots prepared with single-file systems using different kinematics. J Endod 2014; 40:849-51. 14- Rivera ER, Walton RE. Longitudinal tooth fractures: findings that contribute to complex endodontic diagnoses. Endod Top 2009; 16:82–111. 15- Cheung W. A review of the management of endodontically treated teeth post, core and the final restoration. J Am Dent Assoc 2005; 136:611–9. 16- Alomari QD, Barrieshi KM, Al-Awadhi SA. Effect of post length and diameter on remaining dentine thickness in maxillary central and lateral incisors. Int Endod J 2011; 44:956-66. 17- Marca C, Dummer PM, Bryant S, Vier-Pelisser FV, Só MV, Fontanella V, Dutra VD, de Figueiredo JÁ. Three-rooted premolar analyzed by high-resolution and cone beam CT. Clin Oral Investig 2013; 17:1535-40. 18- Arruda MP, Carvalho Junior JR, Miranda CE, Paschoalato C, Silva SR. Cleaning of Flattened Root Canals with Different Irrigating Solutions and Nickel- Titanium Rotary Instrumentation. Braz Dent J 2009; 20:284-9. 19- 2Versiani MA, Pécora JD, Sousa-Neto MD. Microcomputed tomography analysis of the root canal morphology of single-rooted mandibular canines. Int Endod J 2013; 46:800-7. 27 20- Rechenberg DK, Paqué F. Impact of cross-sectional root canal shape on filled canal volume and remaining root filling material after retreatment. Int Endod 2009; 46:547-55. 21- Balto K, Muller R, Carrington DC, Dobeck J, Stashenko P. Quantification of periapical bone destruction in mice by micro-computed tomography. J Dent Res 79:35–40. 22- Wilcox LR, Roskelley C, Sutton T. The relationship of root canal enlargement to finger-spreader induced vertical root fracture. J Endod 1997; 23:533– 4. 23- Bellucci C, Perrini N. A study on the thickness of radicular dentine and cementum in anterior and premolar teeth. Int Endod J 2002; 35:594–606. 24- Sathorn C, Palamara JEA, Palamara D, Messer HH. Effect of root canal size and external root surface morphology on fracture susceptibility and pattern: a finite element analysis. J Endod 2005; 31:288–92. 28 3.2 Publicação 2 Counterclockwise or clockwise reciprocating motion for oval root canal preparation: a micro-CT analysis ABSTRACT Aim To evaluate oval root canal preparation using one or two instruments in counterclockwise or clockwise reciprocating motion. Methodology The radiographic diameter of mandibular human incisors was evaluated, and oval canals were selected (2 ≤ Diameter Ratio ≤ 4). Fiftyseven teeth were assigned to root canal preparation (n = 19): Reciproc 40 (R40) in a counterclockwise reciprocating motion; Mtwo size 40, .06 taper (M 40.06) in a clockwise reciprocating motion or Mtwo size 20, .06 taper and size 40, .06 taper (M 20/ 40.06) in a clockwise reciprocating motion. Mtwo instruments were coupled to an ENDO DUAL motor, turning 150° clockwise and 30° counterclockwise. Scanning was performed before and after root canal preparation using a SkyScan 1176 micro- computed tomography. Volume, percentage of debris and percentage of uninstrumented surface were analysed in the entire root canal and in each third of the canal. Data were compared using ANOVA and Tukey’s tests or Kruskal–Wallis and Dunn tests. Results The Reciproc and Mtwo systems using diferente kinematics were associated with a similar increase in root canal volume. Additionally, both system had similar percentage of uninstrumented surface (P > 0.05). Mtwo size 20, .06 taper and size 40, .06 taper was associated with significantly lower debris (P < 0.05) in the middle third (0.56%) when compared to R40 (1.31%) and M size 40, .06 taper (1.54%). Conclusions The conventional reciprocation motion for R40 and the clockwise reciprocation motion for Mtwo resulted in similar root canal preparations. Less remaining debris was present in the middle third when two instruments with diferente diameters were used. Keywords: micro-computed tomography, reciprocating motion, root canal preparation.  Artigo publicado e descrito segundo as normas do periódico International Endodontics Journal (Confira autorização da editora para publicação no Anexo B) 29 INTRODUCTION Reciprocating motion is a noncontinuous rotation, originally with a movement towards the cutting direction of the instrument (counterclockwise), followed by a minor rotation in the release direction (clockwise). Rotary instruments are produced to cut in clockwise rotation and the use of these instruments in reciprocating motion has been evaluated (Yared 2008, De-Deus et al. 2010a, Gavini et al. 2012, Giuliani et al. 2014), using clockwise rotation greater than the counterclockwise rotation. Single-file root canal preparation has some potential advantages, promoting simple and adequate shaping ability (Burklein et al. 2012, Stern et al. 2012, Versiani et al. 2013). However, in complex root canals and isthmus areas, the use of more than one instrument may be preferred, promoting better root canal cleaning with less debris (Robinson et al. 2013). Preparation with an additional instrument can also decrease the area of uninstrumented canal wall (Amoroso-Silva et al. 2016). Mtwo and Reciproc (VDW, Munich, Germany) are systems with S-shaped cross-sectional design. However, Mtwo is a rotary NiTi file system used in clockwise rotation, whilst Reciproc is a single-file system manufactured with NiTi M-wire alloy, used with reciprocating counterclockwise motion. Cross-sectional root canal morphology directly influences many endodontic procedures, such a root canal preparation (Versiani et al. 2013) and filling (Rechemberg & Paqué 2013). Oval root canals are often studied (Oliveira et al. 2015, Farmakis et al. 2016, de Siqueira et al. 2016), especially as they are more difficult to clean effectively (Versiani et al. 2013). Thus, the aim of this study was to assess the preparation of oval canals with one or two instruments using different kinematics. The null hypothesis was that there are no differences in the shaping ability and cleaning capacity between the clockwise or counterclockwise reciprocating motion and the use of one or two instruments. MATERIAL AND METHODS Selection of teeth After Institutional Ethics Committee approval (CEP #31725014.7.0000.5416), extracted mandibular incisors were selected. They were submitted to digital radiography (Kodak RVG 6100), and the diameter ratio (DR) was obtained by 30 measuring the mesiodistal and buccolingual length of the root canal. Oval-shaped root canals were selected when the buccolingual distance was 2–4 times larger than the mesiodistal diameter (2 ≤ DR ≤ 4). Fifty-seven teeth with single roots and fully formed apices were selected and stored in 0.1% thymol solution at 5 °C. All teeth were mounted on a custom attachment and scanned using a high-definition micro-CT scanner SkyScan 1176 (SkyScan 1176, Bruker-microCT, Kontich, Belgium). The parameters used were 70 kV, 353uA, 360° rotations, a 0.5-mm-thick aluminium filter and a 0.5° rotation step, resulting in an image with a 17.42-lm voxel size. Images of all specimens were reconstructed using NRecon software (NRecon v.1.6.3, Bruker- microCT). A 3D evaluation of the initial volume and surface area of the root canal was performed using CTAn (CTAn v.1.14.4, BrukermicroCT), and based on morphological parameters of the root canals (length, diameter ratio, volume and surface area), teeth were divided into three experimental groups (n = 19). After washing in water for 48 h, access to the root canals was created using high-speed diamond burs (n.2, KG Sorensen, Sao Paulo, SP, Brazil) and the root canals were explored using a size 10 K-file (Dentsply Sirona, Ballaigues, Switzerland). When the tip of the instrument became visible through the apical foramen, the work length (WL) was established 1 mm shorter. Then, a glide path was established using a size 10 K-file (Dentsply Sirona) and a single operator with clinical experience with Reciproc and Mtwo systems prepared all samples. Root canal preparation with Reciproc 40 R40 (size 40, .06 taper) instruments were driven with an electric motor (VDW.SILVER, VDW GmbH, Munich, Germany) in a reciprocating motion, according to manufacturer’s instructions. The instrument was gradually inserted into root canal in a slow in and- out motion, at the three levels (cervical, middle and apical), with a brushing motion against the walls of the root canals. A new R40 instrument was used for each root canal preparation. Root canal preparation with Mtwo size 40, .06 taper Mtwo 40 (size 40, .06 taper) instrument was used in an alternating rotation (150° in clockwise [CW] 150° and counterclockwise [CCW] 30°) driven with the ENDO DUAL electric motor (Satelec, By Dental srl, Pistoia, Italy). The recommended in-and-out motion was the same as described for the R40. A new M size 40, .06 taper instrument was used for each root canal preparation. 31 Root canal preparation with Mtwo size 20, .06 taper and size 40, .06 taper In this group, before the use of Mtwo size 40, .06 taper instruments as previously described, an Mtwo 20 (size 20, .06 taper) instrument was used. Both instruments prepared each canal with the same kinematic described for M size 40, .06 taper. A new M size 20, .06 taper and M size 40, .06 taper instruments were used for each root canal preparation. Root canal irrigation was performed with 2 mL 2.5% NaOCl after preparation of each third. After preparation, the root canals were irrigated with 5 mL 2.5% NaOCl followed by a final irrigation with 2 mL 17% EDTA. The time of preparation was also recorded. Micro-CT analysis The scans before and after root canal preparation were reconstructed using NRecon software. A geometric alignment was performed using the 3D registration function in Data Viewer software (Data Viewer v.1.5.1, Bruker-microCT). After Data Viewer analysis, images were quantitatively analysed using CTAn software (CTAn v.1.14.4, Bruker-microCT). A density histogram was used in a global threshold method. This segmentation determined the dentine before and after instrumentation, and the values ranged from 65 to 73. Task lists were applied and arithmetic and logical operations between the superimposed cross-sectional images were performed. The initial volume (IV) and the final volume (FV) – after preparation – were obtained. Based on these values, the increase in volume (FV - IV) and the percentage of increase [(FV 9 100/IV) - 100] was calculated. The percentage of debris (material with a density similar to dentine previously occupied by air) and percentage of uninstrumented surface were obtained, using specific formulas as follows: %debris = final debris vol x 100 final volume %of uninstrumented areas = uninstrumented area x 100 initial área Hard-tissue debris is formed inside the root canal as confirmed in a previous study using high-resolution micro-CT imaging (Paque et al. 2009). The threedimensional (3D) study also enables the evaluation of the previous state of dentine surface before the root canal preparation, allowing the evaluation of the free 32 debris area or an unprepared area (De-Deus et al. 2011). Voxels representing the dentine wall in the main root canal that were removed during instrumentation and then occupied by hard-tissue debris could not be measured because of the impossibility to discern between accumulated hard-tissue debris and dentine on the scans (Paque et al. 2009, Freire et al. 2015). Matched images of the surface areas of the root canals before and after preparation were examined to evaluate the percentage of uninstrumented canal wall surface (number of static voxel surface) (da Silva Limoeiro et al. 2016). All values were calculated by subtracting the scores for the treated canals from those recorded for their untreated counterparts and then converted into percentages (Amoroso-Silva et al. 2016). All analyses were conducted in the entire canal and in each third of the root canal (apical, middle and cervical). Statistical evaluation Data obtained for the initial volume (IV) and the final volume (FV) were submitted to one-way ANOVA and Tukey’s tests (a<0.05). The data obtained for the percentage relation between the volume measures (%V), the percentage of debris (%D) and the percentage of uninstrumented surface (%S) were submitted to Kruskal–Wallis test, complemented by Dunn’s test for multiple comparisons (a < 0.05). RESULTS Volume Table 1 shows the results of initial volume (mm3), final volume (mm3) and percentage of increase in the volume of the entire root canal and of each third. For the entire canal and each third, there was no significant difference amongst the groups (P > 0.5). The entire preand postoperative volumes (mm3) of the root canals were not different amongst the R40 (3.37 and 6.05 mm3, respectively), M size 40, .06 taper (3.53 and 6.59 mm3, respectively) and M size 20, .06 taper/size 40, .06 taper (3.64 and 6.33 mm3, respectively) (P > 0.05). The preparation with regard to volume using different instruments was similar, as demonstrated by the increase of volume (%), with no significant difference for the entire canal and in its thirds (P > 0.05). The 3D reconstructions for the groups with regard to canal volume are shown in Fig. 1a,b. Debris 33 The results for debris are shown in Table 2 for the entire root canal and its thirds. The R40 and M size 40, .06 taper preparation produced in the middle third more debris than M size 20, .06 taper/size 40, .06 taper (P < 0.05). Throughout the canal and in the other thirds, there were no significant diferences (P > 0.05). The Fig. 1d,e shows representative images, in which the black colour indicates the total accumulated hard-tissue debris plus instrumented areas. Uninstrumented surfaces The results of uninstrumented surfaces are also shown in Table 2 for the entire canal and its thirds. No differences were observed amongst the groups (P > 0.05). Changes in the canal shape are demonstrated by the superimposition of preoperative root canal (green) and post-instrumentation (red), showing untouched areas (Fig. 1c). Preparation time The time of preparation using R40, M size 40, .06 taper and M size 20, .06 taper/size 40, .06 taper were not significantly different (P > 0.05), with a mean of 2.658, 2.827 and 3.207 min, respectively. Table 1 - Initial, final and increase in the volume (%) of root canal volume (means and standard deviations). Experimental groups R40 M40.06 M20/40.06 Volume (mm3) (initial) All* 3.37±0.79 3.53±1.84 3.64±1.12 Cervical 2.27±0.57 2.38±1.42 2.50±0.79 Middle 0.9±0.32 0.99±0.41 0.85±0.3 Apical 0.31±0.15 0.33±0.15 0.26±0.08 Volume (mm3) (final) All* 6.05±1.01 6.59±1.15 6.33±0.77 Cervical 3.71±0.61 3.98±0.8 3.98±0.49 Middle 1.73±0.3 1.94±0.29 1.8±0.24 Apical 0.59±0.19 0.64±0.15 0.57±0.11 Increase in volume (%) All* 79.52±34.37 86.68±51.35 73.9±23.61 Cervical 63.43±24.07 67.22±41.67 59.2±33.93 Middle 92.2±43.38 95.95±51.33 111.76±47.91 Apical 90.32±60.15 93.93±70.21 119.23±56.64 No statistically significant difference was observed between groups in any variable measured throughout the canal and in each region (P > 0.05), according to ANOVA and Tukey tests. *All: entire root canal. 34 Table 2 – Median percentage and minimum and maximum values (interquartile range) of different parameters measured after root canal preparation Experimental groups R40 M40.06 M20/40.06 Debris (%) All* 1.87 (0.36 – 5.85) 2.14 (0.08 – 6.61) 1.43 (0.16 – 5.69) Cervical 0.16 (0.01 – 2.26) 0.39 (0.01 – 2.65) 0.11 (0.01 – 1.11) Middle 1.21 (0.05 – 4.08) a 1.47 (0.01 – 5.11) a 0.41 (0.01 – 2.37) b Apical 0.78 (0.01 – 3.25) 0.69 (0.01 – 2.98) 0.49 (0.04 – 2.35) Uninstrumented surface (%) All* 17.81 (3.93 - 36.75) 16.99 (1.15 – 34.83) 10.27 (2.02 – 37.82) Cervical 2.99 (0.27 – 19.08) 4.77 (0.03 – 13.35) 1.75 (0.19 – 14.59) Middle 6.32 (1.48 – 18.75) 6.89 (0.06 – 17.83) 4.82 (0.14 – 21.76) Apical 4.41 (0.01 – 18.65) 4.1 (0.19 – 15.5) 3.13 (0.64 – 26.21) Different superscript bold letters in the same line indicate statistical significant difference between groups (Kruskal–Wallis test, P < 0.05). No statistically significant difference was observed between groups in any variable measured throughout the canal and in each region (P > 0.05), except for the analysis of accumulated debris after preparation measured in the middle third (P< 0.05). *All: entire root canal. 35 Figure 1 Three-dimensional micro-CT scans reconstruction of the external and internal anatomy of oval canals of mandibular incisors of the R40, M40.06 and M20/40.06 groups. (a) The root canal before (green) and (b) after (red) preparation. (c) Superimposition of preoperative root canal (a) and post-instrumentation (b). (d) Superimposition of accumulated hard-tissue debris plus instrumented areas (black areas) over the postoperative anatomy (green). (e) Total accumulated hard-tissue debris plus instrumented areas (black areas). DISCUSSION The effectiveness of root canal preparation depends on many factors, including root canal morphology, techniques and instruments. Several studies have investigated the unprepared surfaces on root canal walls (Paque et al. 2010, Ruckman et al. 2013, Busquim et al. 2015) or accumulated hard-tissue debris (Paque et al. 2012, De-Deus et al. 2014, Freire et al. 2015, Versiani et al. 2016). Reciprocating motion using a clockwise rotation greater than the counterclockwise motion allows the use of a greater number of instruments available. Furthermore, reciprocating motion as an alternative to continuous rotation may reduce the risk of instrument fracture and root canal aberrations (Yared 2008, Varela-Patino et al. 2010). A side effect of root canal preparation is the production of debris retained on dentine or on instrumented surfaces. Smear layer and accumulated hard-tissue debris are collect specifically in isthmuses, irregularities and ramifications of the complex root canal network (Paque et al. 2009, De-Deus et al. 2014). Accumulated hard-tissue debris may interfere with disinfection of the root canal (Paque et al. 2012) or with root filling (Metzger et al. 2010, Freire et al. 2015). The use of nondestructive micro-computed tomography allows the scanning of teeth before and after preparation, making possible the measurement of the debris using software (De- Deus et al. 2014, Freire et al. 2015, Versiani et al. 2016). According to Robinson et al. (2013), ‘pixels that were occupied by air and then became dentine must be debris’. In the present study, this tool was used to quantify the volume of debris and its percentage in the root canal. It was calculated using specific tasks lists and arithmetic and logical operations between the superimposed cross-sectional images (Freire et al. 2015). According to the present study, more debris was observed in the middle third of the root canal. Mandibular incisors commonly have a single canal (Vertucci 1974, 36 Miyashita et al. 1997, Mauger et al. 1998) with variable morphology and high prevalence of oval root canal (Wu et al. 2000, Milanezi de Almeida et al. 2013), classified according to radiographic measurement of diameter ratio (Wu et al. 2000, Jou et al. 2004). Root canals are normally considered oval when the cross-sectional long-short diameter ratio is ≥2 (Wu et al. 2000, Milanezi de Almeida et al. 2013) or when the buccolingual diameter is 2.5 or more times larger than the mesiodistal diameter (Versiani et al. 2013). The straightening in the mesiodistal direction in this anatomy makes irregularities more prevalent in the middle thirds, contributing to unprepared surfaces and accumulating microorganisms and debris in the canal system (Metzger et al. 2010, Moura-Netto et al. 2013). The use of an instrument with smaller diameter may improve disinfection in areas with difficult access. In the presente study, the use of Mtwo size 20, .06 taper before Mtwo size 40, .06 taper was associated with less debris accumulation in the middle third, without increasing preparation time. Probably, the smallest diameter of the instrument allowed the cleaning of irregularities and areas difficult to access. The majority of debris is detected in uninstrumented regions of the teeth including fins or recesses in the canal wall (Robinson et al. 2013), and it is concentrated in the middle third (De-Deus et al. 2014). M size 20, .06 taper/size 40, .06 taper had the lowest percentage of uninstrumented areas [10.27 (2.02–37.82) %] compared with the R40 [17.81 (3.93–36.75) %] and M size 40, .06 taper [16.99 (1.15–34.83) %]. It can explain the presence of less debris in the middle third of oval canals when prepared using M size 20, .06 taper/size 40, .06 taper. The preparation using this technique promotes more effective cleaning of the canals in the middle region. The use of more than one instrument has been considered previously (Busquim et al. 2015, Marceliano-Alves et al. 2015, Amoroso-Silva et al. 2016). Oval- shaped root canals prepared by the conventional ProTaper full sequence was associated with a significant improvement in debridement than the single-file F2 ProTaper technique (De-Deus et al. 2010b). The use of a sequence of instruments or the association of more than one instrument may be preferred, specially related to debris accumulation and regions difficult to access and clean (Robinson et al. 2013, Busquim et al. 2015, Amoroso-Silva et al. 2016). CONCLUSIONS 37 Reciprocating motion in the counterclockwise direction for R40 and in the clockwise direction for Mtwo instruments resulted in similar root canal preparations. Less debris in the middle third of oval root canals was obtained when two instruments with different diameters were used. Acknowledgements This work is supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) grant (2015/03437-6). Conflict of interest The authors have stated explicitly that there are no conflict of interests in connection with this article. REFERENCES Amoroso-Silva P, Alcalde MP, Duarte MA et al. (2016) Effect of finishing instrumentation using niti hand files on volume, surface area and uninstrumented surfaces in C-shaped root canal systems. International Endodontics Journal 2016. doi: 10.1111/iej.12660. [Epub ahead of print]. Bürklein S, Hinschitza K, Dammaschke T, Schäfer E (2012) Shaping ability and cleaning effectiveness of two single-file systems in severely curved root canals of extracted teeth: Reciproc and WaveOne versus Mtwo and ProTaper. Intetnational Endodontics Journal 45, 449-61. Busquim S, Cunha RS, Freire L, Gavini G, Machado ME, Santos M (2015) A micro- computed tomography evaluation of long-oval canal preparation using reciprocating or rotary systems. International Endodontics Journal 48, 1001-6. da Silva Limoeiro AG, Dos Santos AH, De Martin AS et al. (2016) Micro-Computed Tomographic Evaluation of 2 Nickel-Titanium Instrument Systems in Shaping Root Canals. Journal of Endodontics 42, 496-9. de Siqueira Zuolo A, Zuolo ML, da Silveira Bueno CE, Chu R, Cunha RS (2016) Evaluation of the Efficacy of TRUShape and Reciproc File Systems in the Removal of Root Filling Material: An Ex Vivo Micro-Computed Tomographic Study. Journal of Endodontics 42, 315-9. 38 De-Deus G, Moreira EJ, Lopes HP, Elias CN (2010a) Extended cyclic fatigue life of F2 ProTaper instruments used in reciprocating movement. International Endodontics Journal 43, 1063-8. De-Deus G, Barino B, Zamolyi RQ et al. (2010b) Suboptimal debridement quality produced by the single-file F2 ProTaper technique in oval-shaped canals. Journal of Endodontics 36, 1897-1900. De-Deus G, Reis C, Paciornik S (2011) Critical appraisal of published smear layer- removal studies: methodological issues. Oral Surgery, Oral Medicine, Oral Pathololy and Endodontics 112,531–43. De-Deus G, Marins J, Neves Ade A et al. (2014) Assessing accumulated hard-tissue debris using micro-computed tomography and free software for image processing and analysis. Journal of Endodontics 40, 271-6. Farmakis ET, Sotiropoulos GG, Abràmovitz I, Solomonov M (2016) Apical debris extrusion associated with oval shaped canals: a comparative study of WaveOne vs Self-Adjusting File. Clinical Oral Investigation 20, 2131-8. Freire LG, Iglecias EF, Cunha RS, Dos Santos M, Gavini G (2015) Micro-Computed Tomographic Evaluation of Hard Tissue Debris Removal after Different Irrigation Methods and Its Influence on the Filling of Curved Canals. Journal of Endodontics 41, 1660-6. Gavini G, Caldeira CL, Akisue E, Candeiro GT, Kawakami DA (2012) Resistance to flexural fatigue of Reciproc R25 files under continuous rotation and reciprocating movement. Journal of Endodontics 38, 684-7. Giuliani V, Di Nasso L, Pace R, Pagavino G (2014) Shaping Ability of WaveOne Primary Reciprocating Files and ProTaper System Used in Continuous and Reciprocating Motion. Journal of Endodontics 40, 1468-71. Jou YT, Karabucak B, Levin J, Liu D (2004) Endodontic working width: current concepts and techniques. Dental clinics of North America 48, 323-35. Marceliano-Alves MF, Sousa-Neto MD, Fidel SR et al. (2015) Shaping ability of single-file reciprocating and heat-treated multifile rotary systems: a micro-CT study. International Endodontics Journal 48, 1129-36. Mauger MJ, Schindler WG, Walker WA 3rd (1998) An evaluation of canal morphology at different levels of root resection in mandibular incisors. Journal of Endodontics 24, 607–9. 39 Metzger Z, Zary R, Cohen R, Teperovich E, Paqué F (2010) The quality of root canal preparation and root canal obturation in canals treated with rotary versus self- adjusting files: a threedimensional micro-computed tomographic study. Journal of Endodontics 36, 1569-73. Milanezi de Almeida M, Bernardineli N, Ordinola-Zapata R et al. (2013) Micro- computed tomography analysis of the root canal anatomy and prevalence of oval canals in mandibular incisors. Journal of Endodontics 39, 1529-33. Miyashita M, Kasahara E, Yasuda E, Yamamoto A, Sekizawa T (1997) Root canal system of the mandibular incisor. Journal of Endodontics 23, 479–84. Moura-Netto C, Palo RM, Camargo CH, Pameijer CH, Bardauil MR (2013) Micro-CT assessment of two different endodontic preparation systems. Brazilian Oral Research 27, 26-30. Oliveira MA, Alves LD, Pereira AG, Raposo LH, Biffi JC (2015) Influence of flexion angle of files on the decentralization of oval canals during instrumentation. Brazilian Oral Research doi: 10.1590/1807-3107BOR-2015.vol29.0078. [Epub 2015 Jun 16]. Paqué F, Laib A, Gautschi H, Zehnder M (2009) Hard-tissue debris accumulation analysis by high-resolution computed tomography scans. Journal of Endodontics 35, 1044-7. Paqué F, Balmer M, Attin T, Peters OA (2010) Preparation of oval-shaped canals in mandibular molars using nickeltitanium rotary instruments: a micro-computed tomography study. Journal of Endodontics 36, 703–7. Paqué F, Al-Jadaa A, Kfir A (2012) Hard-tissue debris accumulation created by conventional rotary versus selfadjusting file instrumentation in mesial root canal systems of mandibular molars. International Endodontic Journal 45, 413–8. Rechemberg DK, Paqué F (2013) Impact of cross sectional root canal shape on filled canal volume and remaining root filling material after retreatment. International Endodontics Journal 46, 547-55. Robinson JP, Lumley PJ, Cooper PR, Grover LM, Walmsley AD (2013) Reciprocating root canal technique induces greater debris accumulation than a continuous rotary technique as assessed by 3-dimensional micro-computed tomography. Journal of Endodontics 39, 1067-70. Ruckman JE, Whitten B, Sedgley CM, Svec T (2013) Comparison of the Self- Adjusting File with rotary and hand instrumentation in long-oval-shaped root canals. Journal of 40 Endodontics 39, 92–5. Stern S, Patel S, Foschi F, Sherriff M, Mannocci F (2012) Changes in centring and shaping ability using three nickel-titanium instrumentation techniques analysed by micro-computed tomography (μCT). International Endodontics Journal 45, 514-23. Varela-Patiño P, Ibanez-Parraga A, Rivas-Mundina B, Cantatore G, Otero XL, Martin- Biedma B (2010) Alternating versus continuous rotation: a comparative study of the effect on instrument life. Journal of Endodontics 36, 157–9. Versiani MA, Leoni GB, Steier L et al. (2013) Micro-computed tomography study of oval-shaped canals prepared with the self-adjusting file, Reciproc, WaveOne, and ProTaper universal systems. Journal of Endodontics 39, 1060-6. Vertucci FJ (1974) Root canal anatomy of the mandibular anterior teeth. Journal of American Dental Association 89, 369–71. Versiani MA, Alves FR, Andrade-Junior CV et al. (2016) Micro-CT evaluation of the efficacy of hard-tissue removal from the root canal and isthmus area by positive and negative pressure irrigation systems. International Endodontics Journal 49, 1079-87. Wu MK, R’Oris A, Barkis D, Wesselink PR (2000) Prevalence and extent of long oval canals in the apical third. Oral surgery, oral medicine, oral pathology, oral radiololy and endodontics 89, 739–4. Yared G (2008) Canal preparation using only one Ni-Ti Rotary instrument: preliminary observations. International Endodontics Journal 41, 339–44. 41 3.3 Publicação 3 Shaping ability of rotary or reciprocating systems for oval root canal preparation: a micro-computed tomography study ABSTRACT Objectives To evaluate the shaping ability and cleaning after oval root canal preparation using one or more instruments in reciprocating or rotary motion. Materials and methods Oval-shaped mandibular incisors were selected, based on the radiographic diameter (2 ≤ Diameter Ratio ≤ 4), and assigned according root canal preparation (n=18): single-file (Reciproc R40); two reciprocating files (Unicone size 20 and 40, .06 taper) or Mtwo rotary files until size 40, .06 taper file. Scanning was performed before and after preparation using SkyScan 1176 with a voxel size of 17.42µm. Volume, percentage of debris and percentage of uninstrumented surface were analyzed in the entire root canal and in each root canal third. Data were compared using ANOVA and Tukey or Kruskal-Wallis and Dunn tests (α = 5%). Results: The initial volume were similar among the groups (p>.05). Unicone preparation was associated with higher debris, increase in root canal volume and uninstrumented surface in entire root canal and in the middle third (p<.05). Mtwo was associated with lower uninstrumented surface in the entire root canal and in the cervical third. The apical third were similar for the 3 preparations. Conclusions: Unicone system using two instruments in reciprocating motion resulted in higher increase in volume. However, less remaining debris was observed when Reciproc single-file and Mtwo rotary systems were used. Clinical relevance: A preparation that volumetrically increases the root canal is not necessarily associated with better cleaning. Shaping and hard tissue debris removal depends on root canal anatomy, kinematics, number of instruments and instrument design. Artigo em revisão e descrito segundo as normas do periódico Clinical Oral Investigation 42 INTRODUCTION Root canal preparation may be influenced by the design [1], kinematics [2-4], diameter and taper [5], and the number of instruments used [6, 7]. Reciprocating motion allows preparation with a lower number of instruments [8]. However, the importance of using more than one instrument to promote better root canal cleaning with less debris and decrease in the area of uninstrumented canal wall, including different diameters, has been demonstrated [6, 7, 9]. Oval-shaped and flattened root canals may make it difficult to root canal prepration, especially because they present areas that are difficult to access, favoring the accumulation of debris and microorganisms [10, 11]. The oval-shaped anatomy has been studied [3, 4, 12, 13] and self-adjusting instruments [14] or different additional disinfection procedures [15, 16] have been proposed for cleaning these kind of canals. Nevertheless, some regions of the canal may remain unprepared [17- 19]. The Unicone System (Medin, Nove Mesto Morave, Czech Republic) is composed of 3 instruments for reciprocating motion, with diameters #20, #25 and #40 and taper 6%. When used as a single-file (size 25, .06 taper), this system was associated with a more conservative preparation, and lower foramen transportation when compared with the Reciproc (R25) and ProTaper (sequence until F2) systems [20]. However, it had less cyclic fatigue resistance when compared with the Reciproc R25 and WaveOne Primary instruments [21]. No studies have been found to analyze the preparation characteristics using this system. Reciproc is a single-file instrument made of a special NiTi alloy (M-Wire), used with reciprocating counterclockwise motion and available in 3 different sizes (R25, R40 and R50) to be used in different canal diameter. This instrument produces a similar preparation with regard to the amount of touched dentin walls to Self- Adjusting File, WaveOne and Protaper systems when used in oval-shaped root canals [3], and greater increase in the root canal volume when compared with the BioRace rotary system until size 40, .04 taper file in long-oval root canals [22]. MTwo (VDW, Munich, Germany) is a system of NiTi files with S-shaped cross-sectional design, manufactured to operate in a rotary clockwise motion. In comparison to Reciproc, it demonstrated similar apical preparation in mandibular molars [23]. 43 The aim of this study was to evaluate by means of micro-computed tomography (micro-CT) the shaping ability and cleaning effectiveness of different root canal preparation systems (Reciproc, Unicone and MTwo) in oval root canals. MATERIAL AND METHODS Selection of teeth and preparation Extracted mandibular incisors (CEP #31725014.7.0000.5416) were selected by using digital radiography (Kodak RVG 6100). Mesiodistal and buccolingual length measurements were used to define the diameter ratio (DR) at 9 mm from anatomic apex of each specimen. Only single rooted teeth, with long oval canals (2≤ DR ≤4), completely formed apices and a root length of 20±2 mm were initially selected. Sixty- six teeth were selected and stored in a glass bottle containing 0.1% thymol solution at 5°C. The selected specimens were scanned using a high-definition micro-CT scanner SkyScan 1176 (SkyScan 1176, Bruker-microCT, Kontich, Belgium) at 70 kV, 353uA, 360° rotations, a 0.5-mm-thick aluminium filter and 0.5° rotation step, resulting in an image with 17.42 µm voxel size. An initial reconstruction using NRecon software (NRecon v.1.6.3, Bruker-microCT) and analysis using CTAn software (CTAn v.1.14.4, Bruker-microCT) with regard morphological parameters of the root canals (length, volume and surface area) ensured another selection of specimens and fifty-four root canals (n=54) were randomly assigned to one of the three different instrumentation groups. After washing in water for 48 h, a conventional access to the root canals was created using high-speed diamond burs (n2, KG Sorensen, São Paulo, Brazil) and the patency of the root canals was confirmed when a #10 K-file (Dentsply Sirona, Ballaigues, Switzerland) was visible at the apical foramen. The working length was then set at 1.0 mm shorter and a single operator with clinical experience prepared all samples. Root canal preparation with Reciproc 40 (n=18): R40 (size 40, .06 taper) instruments were acted in reciprocating motion (VDW.SILVER, VDW GmbH), according to manufacturer’s instructions. The instrument was gradually inserted into root canal in a slow in-and-out motion in the three levels (cervical, middle and apical), with a brushing motion against the walls. After prepare of each third, the instrument was 44 cleaned in a gauze and the canal was irrigated. A new R40 instrument was used for each root canal preparation. Root canal preparation with UniCone 20.06 and 40.06 (n=18): a Unicone 20 (size 20, .06 taper) instrument was used before the Unicone 40 (size 40, .06 taper) instrument in reciprocating motion (VDW.SILVER, VDW GmbH). The recommended in-and-out motion was the same as described above for the R40 instrument. Preparation with Mtwo files (n=18): Mtwo files were used in sequence and in rotary motion and 300 rpm (VDW.SILVER, VDW GmbH). For cervical, middle and apical preparation, size 25, .07 taper, size 25, .06 taper and size 20, .06 taper instruments, respectively, were used. After this, a sequence of Mtwo files from size 25, .06 taper instrument up to size 40, .06 taper instrument were used. After the use of each instrument, it was cleaned in a gauze and the canal was irrigated. Root canal irrigation during prepare was performed with a total of 6 mL 2.5% NaOCl (2 mL for each third to Reciproc, 1 mL for each third after both instruments to Unicone and 1 mL for each instrument change to Mtwo rotary system). A 30-G NaviTip needle (NaviTip, Ultradent Products, South Jordan, UT, USA) placed to 1 mm short of working length in a 5-mL syringe (Ultradent Products, South Jordan, UT, USA) was used with a flow rate of 2 mL /min and simultaneous suction by using a 0.014-in tip (Capillary tips, Ultradent, USA). The continuous flow of the irrigant was associated with an in-and-out movement. A final irrigation with 5 mL 2,5% NaOCl followed by 2 mL 17% EDTA was performed in each sample. Root canals were aspirated with a capillary tip and dried with absorbent paper points (Dentsply Sirona), and the specimens submitted to postoperative scanning and reconstruction, applying the aforementioned parameters. Micro-CT analysis After reconstruction of the scans before and after root canal preparation by using NRecon software, the datasets were geometrically aligned by using the 3D registration function of the Data Viewer software (Data Viewer v.1.5.1, Bruker microCT). Images were quantitatively analyzed using CTan software (CTAn v.1.14.4, Bruker microCT) and defined parameters of increase in volume, percentage of debris and percentage of uninstrumented surface were obtained as previously described [7, 24-26). The analysis was performed in the total extension of the samples (from the coronal limit that enamel became invisible to the apex of the root) and in thirds (cervical, middle and apical). 45 Statistical evaluation Data were compared using one-way ANOVA and Tukey to initial volume; to increase in volume (%) in the cervical, middle and apical thirds; to debris (%) in the total and apical third; and to uninstrumented surface (%) in the total and middle and apical thirds (α = 5%). Kruskal-Wallis and Dunn tests were used to increase in volume (%) in the total; to debris (%) in the cervical and middle thirds; and to uninstrumented surface (%) in the cervical third (α = 5%). RESULTS The results obtained showed that there was no difference among the systems evaluated with regard the initial volume, confirming the degree of homogeneity (baseline) of the groups (P>.05). Unicone group was associated with the highest increase in volume values (P<.05), with no difference among groups in the cervical and apical thirds (P>.05), as shown in Table 1. For this system, a higher percentage of debris was also observed in the entire root canal and in the middle third (P<.05). With regard to the percentage of uninstrumented surface, there was no significant difference among the systems in the middle and apical thirds (P>.05). However, a higher percentage of uninstrumented surface in the entire root canal and in the cervical third was observed for Unicone Group when compared with Reciproc group (P<.05) (Table 1). Figure 1 shows representative 3D reconstructions of mandibular incisors before and after canal preparation with the tested systems. Table 1 – Means and standard deviations (±)* or median percentage (maximum and minimum values)** of the parameters analyzed in the different experimental groups 46 Experimental groups R40 UniCone 20/40.06 MTwo files Initial volume Total* Cervical* Médio* Apical* 3.23±0.89a 3.48±0.96a 3.91±1.03a 2.15±0.63a 2.32±0.87a 2.49±0.73a 0.81±0.25a 0.98±0.3a 0.99±0.32a 0.26±0.08a 0.27±0.11a 0.31±0.1a Increase in volume (%) Total** 84.32 (41.7-186.9)b 111.9(83.53-203.2)a 82.08(58.17-117.1)b Cervical* 74.22±28.92a 81.82±27.54a 78.81±25.47a Médio* 121.9±62.99a 83.49±27.98b 108.0±34.99a,b Apical* 147.9±95.23a 203.4±101.5a 147.4±66.1a Debris (%) Total* 2.33±1.95b 4.45±2.05a 2.87±1.69b Cervical** 0.18(0.005-2.26)b 0.8(0.01-2.32)a 1.22(0.09-3.17)a Médio** 1.36(0.05-4.08)b 4.38(2.92-9.5)a 0.82(0.13-4.8)b Apical* 1.04±0.97a 1.05±0.69a 0.95±0.72a Uninstrumented surface (%) Total* 17.3±10.41b 30.0±7.64a 23.15±12.17a,b Cervical** 3.3(0.16-19.08)b 8.25(2.88-16.65)a 6.64(0.46-23.43)a,b Médio* 6.59±4.82a 10.04±3.45a 8.43±5.69a Apical* 6.3±5.22a 6.28±2.06a 6.21±2.52a * Different superscript letters in the same line indicate statistical significant difference between groups (ANOVA and Tukey* or Kruskal–Wallis and Dunn** tests, P < 0.05). 47 Figure 1 – Tridimensional micro-CT scan reconstructions of the external and internal anatomy of oval canals of mandibular incisors, in the Reciproc, Unicone and Mtwo groups. (A) The root canal before (green) and (B) after (red) preparation. (C) Superimposition of preoperative root canal (A) and post- instrumentation (B). (D) Superimposition of accumulated hard-tissue debris plus instrumented areas (black areas) on the postoperative anatomy (green). (E) Total accumulated hard-tissue debris plus instrumented areas (black areas). (F) Axial view of superimposed root canals before (green) and after (red) preparation at coronal (c), middle (m), and apical (a) thirds. DISCUSSION The volumetric increase of the root canal, percentage of debris and uninstrumented surface have been evaluated by means of computed microtomography. Oval canal preparation represents a clinical challenge [18, 19] and the use of different instruments [14] or even auxiliary means of cleaning [15,16] did not promote complete preparation and cleaning of oval canals, corroborating the findings of the present study. The teeth were first selected by means of radiographic analysis, as other studies [3, 19, 27]. Selected teeth were then analyzed using micro- CT with regard morphological parameters of the root canals and could be randomly 48 assigned to one of the experimental groups. The similar initial volume to all groups proved the corrected distribution (Table 1). In the present study, the higher increase in the root canal volume throughout the extension of the canal and in the middle third was observed for the Unicone reciprocating system, using two instruments. However, in spite of the increase in volumetric values, a higher percentage of untouched areas were also observed for this system. In previous studies, WaveOne, for example, promoted a higher increase in the volume value of the canal, with a similar percentage of uninstrumented surfaces of oval canals to Easy ProDesign Logic and OneShape systems [28]. It was also observed that although the SAF system touched the canal walls to a larger extent, it removed less dentin in the preparation of oval root canals [19]. Corroborating with our results, these data demonstrated that root canal preparation was influenced by diverse factors, such as instrument design, kinematics and number of instruments. An increase in canal volume indicates that the instrument provided greater wear on the dentin walls, but does not mean that this wear accompanied the anatomy. What probably occurred in this study is that in the Unicone group there was more prepare in the mesiodistal direction and in the middle and cervical thirds. Therefore, areas of flattening of the canal in the buccal-lingual direction were not touched by the instrument and still housed debris. The accumulation of debris may also have been favored by the desing of the instrument. In spite of standardizing the size (instruments with tip #40) and taper (taper .06), the taper may be influenced the preparation. The Unicone instruments present a constant taper throughout the entire active part, while Reciproc presents taper .06 in the apical 3 mm and a reduction to 0.04 mm up to the end of the active part. This reduction may have favored the grater action in the middle of the oval canal, which narrows significantly [10,11], thereby promoting a larger area of instrumented surface, and a reduction in the percentage of debris. It means that the reduction of taper to Reciproc probably improve its capacity of prepare because it could reach regions of more difficult access. In the apical region, where the tip and taper were similar among the systems, no difference in the uninstrumented surface value or accumulation of debris was observed. The size of the apical enlargement of root canal preparation can be associated to a significant effect on apical crack initiation [29]. However, with the purpose of performing a correct cleaning and disinfection of the root canals, studies have shown 49 that the use of larger instruments is significantly more effective in eliminating bacteria [30-32]. The enlargement of the canal to 3 sizes larger than the first apical binding file is considered adequate [33]. With respect to mandibular incisors, besides presenting greater straightening in the mesiodistal direction with irregularities more prevalent in the middle thirds, contributing to unprepared surfaces and accumulating microorganisms and debris in the canal system [10,11], this anatomy presents the prevalence of oval canals at the apical third [12,34,35], which presents difficulty to achieve efficient cleaning [17-19]. With basis on these concepts, regarding the anatomy and the systems used in our study with their availability of instruments, we standardized the preparation up to file #40, .06 taper. The use of a file #25, for example, could lead to doubts about a cleaning preparation that falls short of what is expected when disinfecting and removing debris are evaluated. The highest percentage of uninstrumented surface in the preparation with Unicone, and the highest percentage of debris may also be related to its metalic nucleus. Reciproc and Mtwo instruments have a cylindrical nucleus, which gives them greater flexibility and escape area [36]. The Unicone instrument has a conical nucleus, which makes the instrument more rigid and less resistant, difficulting its performance in all the walls of the root canal. These characteristics corroborate previous studies, which have demonstrated low flexibility and a short lifetime during cyclic fatigue test [21, 37]. Furthermore, the Unicone instruments presented smaller escape areas [38] and interlaminar distance [39] than the Reciproc instruments. Areas that remained untouched during preparation may be colonized by biofilm, capable of compromising the endodontic treatment [16, 41]. In relation to cleaning (considered by the percentage of debris), the preparation of oval canals were shown to be cleaner (lower percentage of debris) with the reciprocating R40, and rotary system with the Mtwo sequence of instruments. A lower percentage of debris in the middle third of oval canals prepared with MTwo or R40 instruments was observed when compared with the preparation performed with a single Mtwo 40.06 instrument [7]. Favorable results were also obtained with the use of the Mtwo sequence of instruments, in the analysis of preparation in mandibular molars with two separate mesial canals and severe curvature [40]. The production of debris during preparation usually involves their accumulation in specific regions, such as isthmus, irregularities and ramifications [26]. Oval canals present mostly straightening in the middle region of the root canal 50 [11], making this region critical to cleaning and prepare. The smaller volumetric increase of this region observed for the Unicone system may be related to a reduced capacity of hard tissue debris removal, maintaining its accumulation in this third. CONCLUSION The Unicone system using two instruments in reciprocating motion resulted in higher increase in volume values. However, less remaining debris was present when Reciproc single-file and Mtwo rotary system were used. A preparation that volumetrically increases the root canal is not necessarily associated with better cleaning. Shaping and cleaning depend on root canal anatomy, kinematics, number of instruments and instrument design. Acknowledgements This work is supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant (2015/03437-6). REFERENCES 1- Peters OA (2009) Current challenges and concepts in the preparation of root canal systems: a review. J Endod 30:559-567. 2- De-Deus G, Barino B, Zamolyi R, et al (2010) Suboptimal debridement quality produced by the single-file F2 Protaper technique in oval-shaped canals. J Endod 36: 1897–1900. doi: 10.1016/j.joen.2010.08.009 3- Versiani MA, Leoni GB, Steier L et al (2013) Micro-computed tomography study of oval-shaped canals prepar