Pelotas, 2011

Marcos Antonio Pacce

UNIVERSIDADE FEDERAL DE PELOTAS

Programa de Pós-graduação em Odontologia

Tese

Influência de bráquetes e tipos de ligadura no acúmulo

microbiano e na desmineralização do esmalte adjacente a

dispositivos ortodônticos

2

MARCOS ANTONIO PACCE

INFLUÊNCIA DE BRÁQUETES E TIPOS DE LIGADURA NO ACÚMULO

MICROBIANO E NA DESMINERALIZAÇÃO DO ESMALTE ADJACENTE A

DISPOSITIVOS ORTODÔNTICOS

Tese apresentada ao Programa de Pós-

Graduação em Odontologia da Universidade

Federal de Pelotas, como requisito parcial

para obtenção do título de Doutor em

Odontologia, (área de concentração:

Odontopediatria).

Orientador: Prof. Dr. Maximiliano Sérgio Cenci

Co-Orientadores: Profª. Drª. Sandra B. Chaves Tarquínio

Prof. Dr. Douver Michelon

Pelotas, 2011

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Dados de Catalogação da Publicação

P114i Pacce, Marcos Antonio

Influência de bráquetes e tipos de ligadura no acúmulo microbiano e na desmineralização do esmalte adjacente a dispositivos ortodônticos / Marcos Antonio Pacce ; orientador: Maximiliano Sérgio Cenci ; co-orientadores: Sandra Beatriz Chaves Tarquinio, Douver Michelon . – Pelotas: UFPel, 2011.

79 f. : tab. ; fig.

Tese (Doutorado) Odontopediatria. Faculdade de Odontologia. Universidade Federal de Pelotas. Pelotas.

1. Bráquetes. 2. Cárie dentária. 3. Biofilme. 4. Placa Dentária. 5. Tratamento ortodôntico. I. Cenci, Maximiliano Sérgio (orient.) II. Tarquinio, Sandra Beatriz Chaves (co-orient.) III. Michelon, Douver (co-orient.) IV. Título.

D602

Bibliotecário: Fabiano Domingues Malheiro CRB -10/1955

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Banca examinadora: Prof. Dr. Maximiliano Sérgio Cenci

Prof. Dr. Gustavo Hauber Gameiro

Prof. Dr. Tiago Aurélio Donassollo

Profa. Dra. Dione Dias Torriani

Profa. Dra. Elenara Ferreira de Oliveira

Profa. Dra. Ana Regina Romano (suplente)

Prof. Dr. Miguel Roberto Simões Regio (suplente)

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Dedicatória

- À minha querida mãe Maria que mesmo distante, com muito amor torna minha vida

mais alegre e prazerosa.

- Ao meu pai Duilio, obrigado pelo seu imenso coração e bondade. Sua proteção e

incentivo são grandes responsáveis por mais esta conquista.

- À minha filha Violetta um exemplo de humildade e simplicidade.

- Ao meu filho Benjamin que me ensinou o significado de lutar pela vida.

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Agradecimentos

- Ao meu orientador, Prof. Maximiliano Cenci pela incansável dedicação. Muito

obrigado por me ensinar e permitir que trabalhássemos mesmo nos finais de

semana. Sua ajuda tornou simples até as fases mais complicadas. Por isso, foi

possível a realização deste trabalho. Muito obrigado também a Profª. Tatiana pela

participação na etapa mais importante e por disponibilizar sua casa para que

pudéssemos desenvolver várias atividades.

- Ao meu co-orientador, Prof. Douver Michelon, pela oportunidade de aprender

contigo. Muito obrigado pelas discussões que enriqueceram a elaboração teórica e

pela ajuda nas tarefas práticas deste trabalho.

- À minha co-orientadora, Profª. Sandra B. Chaves Tarquínio, obrigado por ter

sido fundamental na definição das diretrizes e características desta pesquisa.

- À coordenadora de área de Odontopediatria, Profª. Dione Dias Torriani, que

muitas vezes sacrifica horas de lazer com ânimo para manter a qualidade do

Programa de Pós-Graduação em Odontologia, obrigado pelo exemplo e amizade.

- Às minhas colegas professoras de Clínica Infantil, Ana Regina Romano,

Lizandrea Shardosim e Maria Laura Menezes Bonow, obrigado pela convivência

agradável e incentivo.

- Ao coordenador do PPGO Prof. Flávio Fernando Demarco, responsável pela

estruturação deste programa que tem incentivado a formação docente e elevado o

nome de nossa faculdade. Obrigado pela oportunidade.

- Aos professores do PPGO, pelos conhecimentos divididos.

- Ao Dr. Fábio Garcia Lima e aos alunos de graduação Júlia da Rosa de Almeida,

Marcos Rodolfo Bolfani e Murilo Souza Luz pela contribuição com logística dos

estudos e leitura dos dados.

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- Aos colegas do PPGO, pela convivência, especialmente à Marina Azevedo e à

Françoise Van de Sande pela ajuda no laboratório e na revisão dos artigos.

- À secretária do PPGO, Josiane Silva, pela paciência e simpatia.

- Aos alunos de pós-graduação Carolina Camporese e Vanessa Pereira, da

graduação Camila Nascimento, Fabiane, Francine, Ana Paula Gonçalves, Cacá

(Carolina Ramalho), Laura Baes das Neves, Nathália Motta Martins, Silene Barbieri

e Wagner da Silva. Aos Cirurgiões-Dentistas Fabiano Botelho e Flávia Wendt.

Muito obrigado, esse trabalho não seria possível sem a colaboração de vocês.

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Resumo PACCE, Marcos Antonio. Influência de bráquetes e tipos de ligadura no acúmulo microbiano e na desmineralização do esmalte adjacente a dispositivos ortodônticos. 2011. 80f. Tese (Doutorado) – Programa de Pós Graduação em Odontologia. Universidade Federal de Pelotas, Pelotas. O tratamento ortodôntico é relacionado com o desenvolvimento de lesões de cárie, uma vez que o aparelho constitui-se em fator de retenção do biofilme. Este trabalho de tese teve como objetivo avaliar o efeito do tipo de ligadura e tipo de bráquete ortodôntico na perda mineral e no acúmulo de biofilme adjacente a estes dispositivos. Para cumprir esse objetivo, três estudos foram realizados. O primeiro estudo consistiu em numa revisão sistemática da literatura, onde bancos de dados eletrônicos (PubMed, Embase, Cochrane Central Register of Controlled Trials, ISI Web of Knowledge TRIP, Scopus e SciELO) foram pesquisados até julho de 2011. Ensaios clínicos ou estudos in situ que avaliaram o efeito de tipos de bráquetes ou ligaduras no acúmulo de biofilme e/ou a desmineralização do esmalte foram selecionados. Estudos não controlados, in vitro, ou que não apresentaram os desfechos procurados foram excluídos. O segundo estudo avaliou a desmineralização do esmalte em torno de seis tipos de combinações de bráquetes / tipos de ligadura. Biofilmes foram formados a partir de inóculos de saliva (microcosmos) e cultivados em microplacas de 24 poços sobre discos de esmalte por 14 dias em saliva artificial, sob desafio. Os grupos (n = 10 por grupo) foram: bráquetes autoligantes (AL); bráquetes convencionais ligados com amarrilhos de aço inoxidável (CA); bráquetes convencionais (CE), bráquetes com ganchos (GE), bráquetes de cerâmica (KE), e bráquetes de compósito (RE), todos os quatro ligados com anéis elastoméricos. O biofilme formado em torno dos bráquetes foi coletado e teve seu peso seco determinado (mg). A perdam mineral adjacente aos bráquetes foi avaliada por microdureza de secção longitudinal. O terceiro estudo avaliou in situ o efeito combinado de tipos de bráquetes e tipos de ligadura sobre a desmineralização do esmalte, o acúmulo de biofilme e a sua composição microbiana. O estudo teve um desenho experimental randomizado, duplo-cego e de boca dividida. Voluntários (n = 17) usaram placas palatais removíveis contendo discos de esmalte com bráquetes ortodônticos durante 14 dias. Para fornecer desafio cariogênico, solução de sacarose 20% foi gotejada 8x/dia em cada disco. As quatro condições em estudo foram: CE; CA; GE e AL. Os biofilmes formados em torno dos bráquetes foram coletados para análises microbiológicas e a perda mineral foi determinada pelo método descrito acima. Os resultados da revisão sistemática mostraram divergência entre os estudos, com muita variabilidade metodológica entre eles. Os artigos revisados não permitem chegar a uma conclusão sobre o papél dos tipos de bráquetes ou ligadura no desenvolvimento de lesões de cárie ou no acúmulo de biofilme. Não foram observadas diferenças in vitro ou in situ quanto a biomassa do biofilme (P> 0,05), exceto para o grupo GE, o qual apresentou menor acúmulo de biofilme in vitro (P<0,05). Menor desmineralização foi observada associada à bráquetes autoligantes (P< 0,05). Bráquetes com um desenho mais complexo promovem maior desmineralização do que os autoligantes (P< 0,05). Os métodos de ligadura também afetaram a desmineralização, e anéis elastoméricos promovem lesões de cárie do que amarrilhos de aço (P< 0,05). Palavras-chave: Bráquetes. Placa Dentária. Cárie dentária. Biofilme. Tratamento ortodôntico.

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Abstract

PACCE, Marcos Antonio. Influência de bráquetes e tipos de ligadura no acúmulo microbiano e na desmineralização do esmalte adjacente a dispositivos ortodônticos. 2011. 79f. Tese (Doutorado) – Programa de Pós Graduação em Odontologia. Universidade Federal de Pelotas, Pelotas.

The orthodontic treatment has been related to caries lesions development, since the devices used are biofilm retentive. This thesis aimed to evaluate the effect of ligation type and type of orthodontic bracket in the mineral loss and biofilm accumulation to these devices. To accomplish this goal, three studies were conducted. The first study consisted in a systematic literature review, where electronic databases (PubMed, Embase, Cochrane Central Register of Controlled Trials, ISI Web of Knowledge, TRIP, Scopus, and SciELO) were searched up to July 2011. Clinical trials or in situ studies that assessed the effect of types of brackets or ligatures on biofilm accumulation and/or enamel demineralization were selected. Non-controlled studies, in vitro studies, or studies not reporting on the established outcomes were excluded. The second study assessed the enamel demineralization around six types of brackets/archwire ligation combinations. In that study, microcosm plaque biofilms were grown in 24-well microplates on enamel discs for 14 days in artificial saliva. Growth condition comprised cariogenic challenge. The groups (n=10 per group) were: self-ligating brackets (SL); conventional brackets ligated with stainless steel wires (CW); conventional brackets (CE), brackets with hooks (HE), ceramic brackets (KE), and composite brackets (RE), all the four ligated with elastomeric rings. The biofilm formed around the brackets was collected and dry-weighted and the mineral loss around the brackets was determined by cross-sectional microhardness. The third study evaluated in situ the combinated effect of types of brackets and archwire ligation on enamel demineralization, on the accumulation and on microbiological composition of dental plaque. The study design was a modified in situ model, randomized, double-blind and split-mouth. Volunteers (n=17) wore palatal removable appliances containing enamel discs with bonded orthodontic brackets during 14 days. To provide cariogenic challenge, 20% sucrose solution was dripped 8x/day onto each disc. The four conditions under study were: CE; CW; HE; and SL. The biofilm formed around the brackets was collected for microbiological analyses and the mineral loss was determined by cross-sectional microhardness measurement. The systematic review results showed contradictions among studies, and a great methodology variation among them. The reviewed papers did not allow any conclusion about the effect of types of brackets and ligatures on caries lesions and biofilm accumulation. No differences were observed in vitro or in situ regarding biofilm biomass (P>0.05), except for the HE group wich presented lower biofilm accumulation in vitro (P<0.05). Lower demineralization was observed associated to self-ligated brackets, and brackets with more complex design promote higher demineralization than self-ligated brackets. The ligature methods also affected demineralization, and elastomeric rings are more prone to promote caries lesions then steel wires. Key-words: Brackets, Dental Caries, Dental Plaque, Biofilm, Orthodontic treatment

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Sumário

1 Projeto de Pesquisa ...................................................................................... 11

2 Relatório do trabalho de campo ................................................................... 23

3 Artigo 1............................................................................................................... 4 Artigo 2.............................................................................................................

5 Artigo 3 .....................................................................................................

26

45

57

6 Conclusões .......................................................................................................

73

7 Referências Complementares......................................................................... 74

8 Apêndices ......................................................................................................... 76

9 Anexos .............................................................................................................. 79

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Projeto de pesquisa

1. Antecedentes e Justificativas

A desmineralização do esmalte adjacente aos bráquetes ortodônticos é

um grande problema clínico, aumentando a prevalência e gravidade das lesões de

cárie durante e após o tratamento ortodôntico fixo (MIZRAHI, 1982; BOERSMA et

al., 2005). A prevalência de lesões de mancha branca em pacientes ortodônticos

varia entre 12,6% e 50% (ÅRTUN; BROBAKKEN, 1986). Os aparelhos ortodônticos

quando fixados em todos os dentes, são os responsáveis pela criação de novas

áreas de retenção, as quais favorecem a formação e o acúmulo de biofilme. Diante

do consumo aumentado de carboidratos fermentáveis, essa condição pode

favorecer a colonização e o aumento de espécies cariogênicas como os

estreptococos do grupo mutans e lactobacilos. Ainda, sem a limpeza completa dos

dentes, podem ocorrer danos ao esmalte e ao periodonto. Sendo assim, cuidados

mais intensivos são necessários durante o tratamento ortodôntico (ZACHRISSON;

ZACHRISSON, 1971).

Uma das formas de prevenção destes problemas, a qual tem sido bastante

explorada na literatura, diz respeito ao aprimoramento das técnicas de colagem e

sobretudo ao uso de materiais para cimentação que teriam potencial antimicrobiano

para diminuir o acúmulo de biofilme durante o tratamento ortodôntico fixo,

especialmente em pacientes com má higiene bucal (BISHARA et al.,1998;

ROSENBLOOM; TINANOFF, 1991). Adicionalmente aos fatores relacionados à

colagem, aspectos relacionados ao tipo de dispositivo ortodôntico a ser fixado

devem ser explorados, considerando seu potencial de promover retenção

microbiana e consequentemente de facilitar o desenvolvimento de lesões de cárie.

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Embora o efeito dos aparelhos ortodônticos fixos sobre a flora microbiana e o

estado periodontal tenham sido avaliados (CORBETT et al.,1981, FORSBERG et al.,

1991) poucos estudos têm incluído os métodos de ligadura e/ou o tipo de bráquetes

como fatores adicionais na retenção de biofilme (SUKONTAPATIPARK et al., 2001;

TURKKAHRAMAN et al., 2005). Alguns estudos atribuem a formação do biofilme

durante o tratamento com aparelhos fixos principalmente à complexidade do

desenho do bráquete (LEE et al., 2001; TABAK; BOWEN, 1989).

Recentemente surgiram no mercado bráquetes autoligáveis, que dispensam

qualquer tipo de amarração, os quais teriam por finalidade reduzir o tempo de

atendimento clínico e de tratamento, proporcionando maior conforto e maior

facilidade de higienização ao paciente (HENAO; KUSY, 2004; REDLICH et al.,

2003). Um estudo in vivo, através da bioluminescência, concluiu que o desenho dos

bráquetes auto-ligantes quando comparados com os de ligadura elástica reduzem

significativamente o acúmulo de bactérias ao redor dos mesmos (PELLEGRINI et al.,

2009).

O potencial de acúmulo microbiano e a consequente facilitação de

desmineralização adjacente aos dispositivos ortodônticos, especificamente em

função da arquitetura dos bráquetes com ligaduras e auto-ligantes permanece

inexplorado em condições controladas. Recentemente um modelo de estudo

controlado in situ foi desenvolvido, o qual permite verificar a perda mineral adjacente

a bráquetes ortodônticos unidos por segmento de arco (GAMEIRO et al., 2009). A

utilização deste modelo permite avaliar o efeito do desenho dos diversos tipos de

bráquetes e ligaduras disponíveis comercialmente sobre a perda mineral em

esmalte.

O efeito do acúmulo microbiano e do desafio cariogênico ao redor das

diferentes superfícies dos bráquetes ortodônticos e da forma de ligadura não foi

estudado em condições simulando a realidade clínica, como a condição in situ.

As lesões de cárie que se desenvolvem durante o tratamento ortodôntico fixo

podem regredir após a remoção do aparelho. No entanto, lesões extensas podem

resultar em sequelas estéticas importantes, com necessidade de intervenção

restauradora (van der VEEN et al., 2007). Assim, a completa prevenção do

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desenvolvimento de lesões de cárie deveria ser uma preocupação importante

durante o tratamento ortodôntico. Por essa razão, justifica-se a necessidade de

estudar os fatores relacionados aos dispositivos ortodônticos fixos frente à condição

clínica de desafio cariogênico, tornando possível aprimorar estratégias preventivas a

serem usadas durante o tratamento ortodôntico.

2. Hipótese

A hipótese nula a ser testada é:

O tipo de bráquete ortodôntico e o tipo de ligadura não modificam a aderência

microbiana e a quantidade de desmineralização do esmalte adjacente a esses

dispositivos.

3. Objetivos

3.1 Geral Avaliar o efeito dos bráquetes ortodônticos na perda mineral e no acúmulo de

biofilme adjacente aos mesmos.

3.2 Específicos

Analisar in situ o efeito do tipo de bráquete e tipo de ligadura sobre o acúmulo

microbiano.

Verificar in situ o efeito do tipo de bráquete e tipo de ligadura sobre perda

mineral adjacente aos bráquetes ortodônticos.

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4. Metodologia

4.1 Estudo in situ

4.1.2 Delineamento experimental

Este estudo in situ envolverá será do tipo duplo-cego e boca dividida para

indução de cárie ao redor de bráquetes ortodônticos por acúmulo de biofilme e

exposição à sacarose. Dezesseis voluntários serão convidados a participar do

estudo, que será conduzido em uma fase de 14. O fator em avaliação será tipo de

bráquete ortodôntico+ligadura em 04 níveis: bráquete convencional + ligadura

metálica; bráquete convencional + ligadura elástica; bráquete convencional com

gancho + ligadura elástica; e bráquete auto-ligante. Desta forma serão obtidos 4

grupos que serão aleatoriamente alocados nas placa palatinas de cada um dos

voluntários. Durante a fase experimental, os voluntários usarão uma placa palatina

contendo 04 cavidades (17 × 7 × 4 mm3), onde 03 discos de esmalte bovino (05 mm

de diâmetro e 02 mm de altura) serão colocados em cada cavidade. Os discos serão

montados de forma que as superfícies do esmalte fiquem 01 mm abaixo da

superfície da placa palatina para permitir o acúmulo de biofilme. Cada 3 discos de

esmalte alojados cavidades da placa palatina receberão uma combinação de

bráquete/tipo de ligadura, caracterizando um modelo boca-dividida. Aos voluntários

serão fornecidas escovas dentárias tamanho médio padronizadas e dentifrício

fluoretado (1.100 mg F / g - NaF). Eles serão instruídos a escovar os dentes e as

placa palatinas, exceto os discos de esmalte e bráquetes, durante 01 minuto, 03

vezes/dia e deverão abster-se de quaisquer outros procedimentos de higiene oral,

ou do uso de antimicrobianos. Ao final da fase experimental, o biofilme acumulado

sobre os bráquetes será coletado para quantificação microbiana (porcentagens de

estreptococos do grupo mutans e porcentagens de lactobacilos em relação aos

microrganismos totais cultiváveis). Adicionalemente, a perda mineral adjacente aos

dispositivos ortodônticos será verificada por microdureza de secção tansversa. Os

dados obtidos serão submetidos à análise exploratória para seleção de modelo

estatístico adequado. Para fins de análise, os voluntários serão considerados como

blocos estatísticos e como unidades experimentais.

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4.2 Cálculo da amostra

O cálculo amostral foi feito no programa Sigmastat® (Versão 3.5, Systat

Software Inc.) presumindo-se que seria feita posteriormente uma Análise de

Variância, considerando 04 grupos experimentais e adotando os seguintes

parâmetros: poder do estudo de 80%, erro tipo alfa de 5%, valores da diferença

entre as médias de perda mineral de 10 % em volume, e desvio padrão médio

presumido de 8 %. Para as médias e desvios esperados foram utilizados os dados

de microdureza Knoop convertidos em % de volume mineral perdido em esmalte do

trabalho de Gameiro et al. (2009). Com esses parâmetros, obteve-se um n amotral

calculado de 14 indivíduos. Considerando que a perda estimada de voluntários em

estudos in situ é de 10%, 16 voluntários serão convidados a participar deste estudo.

4.3 Seleção da amostra

Os responsáveis pelo estudo convidarão a participar da pesquisa alunos da

graduação e pós-graduação de odontologia da Universidade Federal de Pelotas

(RS) através da fixação de folhetos e chamadas periódicas até a obtenção do

número amostral, perfazendo um total de 16 voluntários.

Os critérios de inclusão serão:

1. Adultos com idade entre 18 e 50 anos, que aceitem as condições do

estudo

2. Pacientes com boa condição de saúde bucal (sem lesões cariosas

ativas ou doença periodontal moderada ou severa).

3. Pacientes com boa condição de saúde geral.

4. Pacientes com fluxo salivar normal.

Os critérios de exclusão serão:

1. Grávidas ou lactantes

2. Fumantes

3. Pacientes sob tratamento ortodôntico

4. Pacientes que estiverem utilizando ou que tenham utilizado antibióticos

e/ou antimicrobianos ou medicamentos que interferem no fluxo salivar no último

mês.

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5. Pacientes com lesões cariosas cavitadas ou ativas ou doença

periodontal.

4.4 Obtenção e preparo dos discos de esmalte e das placas palatinas

Após aprovação pelo Comitê de Ética em Pesquisa, amostras de esmalte

serão obtidas de incisivos bovinos livres de falhas, obtidos em um frigorífico local. Os

dentes serão raspados, limpos e armazenados em água destilada (-20ºC). Para

obtenção de discos de esmalte de 5 mm de diâmetro e 2 mm de espessura, o terço

médio vestibular será seccionado em furadeira industrial com broca de núcleo de

diamante (tipo trefina) em velocidade de 400 RPM. A porção em dentina dos discos

de esmalte será planificada em politriz com lixas 80, sob irrigação com água, e os

discos de esmalte serão autoclavadas e armazenadas em solução estéril a 5ºC até

utilização. A esterilização em autoclave será realizada conforme protocolo padrão, à

121ºC por 30 minutos, a 15 libras de pressão. O controle de eficácia será realizado

com indicador biológico (bacillus stearothermophilus).

Os discos de esmalte serão posicionados em cada placa palatina removível

onde serão construidos 4 cavidades com dimensões de 17 X 7 X 4 mm3,

compatíveis com o alojamento de 3 discos de esmalte bovino (5 mm de diâmetro e 2

mm de altura) serão colocados com cera pegajosa em cada cavidade. Sobre esses

discos de esmalte serão fixados os bráquetes, sendo que os diferentes tratamentos

(combinações de bráquetes e ligaduras) serão alocados aleatoriamente as

condições experimentais descritas acima.

Para confecção das placas palatinas (uma para cada voluntário), será feita a

moldagem com alginato da arcada superior de todos os voluntários. As placa

palatinas serão confeccionados em resina acrílica autopolimerizável sobre os

modelos vazados em gesso pedra.

4.5 Montagem dos dispositivos ortodônticos

Bráquetes convencionais de base metálica (Vector- ADITEK, Cravinhos, São

Paulo, Brasil), a serem ligados com amarrilho metálico, ou anéis elastoméricos; ou

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bráquetes com clipes automáticos (Sistema Easy Clip- ADITEK, Cravinhos São

Paulo, Brasil), de dimensões 2,90 × 3,33 mm2 serão colados no centro dos discos

de esmalte com adesivo ortodôntico Transbond (3M do Brasil Ltda, São Paulo,

Brasil), de acordo com as instruções do fabricante. Um fio ortodôntico de aço

inoxidável (0,016 polegadas) será inserido nos três bráquetes em cada cavidade.

4.6 Fase Clínica

Os voluntários usarão por 14 dias as placas móveis palatinas contendo os

dispositivos ortodônticos fixados sobre os discos de esmalte, de acordo com as

combinações experimetnais em estudo. Os voluntários receberão instruções de

não utilizar produtos fluoretados ou com propriedades antimicrobianas na semana

antecedente ao estudo e durante todo o período experimental.

Durante a fase clínica, os voluntários utilizarão dentifrício fluoretado

(contendo 1100 µgF/g, como NaF). Os voluntários não saberão qual o tratamento

proposto (cegos) e as condições experimentais serão aleatoriamente alocadas

nas placas palatinas para evitar viés. Durante o período clínico, os voluntários

deverão utilizar a placa palatina em tempo integral, removendo-a apenas durante

as refeições, e para higienização (3 vezes por dia), a qual deverá ser realizada da

seguinte forma: as faces em contato com a mucosa bucal da placa palatina

deverão ser escovadas com o dentifrício fornecido, bem como todas as regiões

que não contenam os dispositivos e os discos de esmalte. No entanto, a região

que contém os espécimes deverá entrar em contato com a suspensão formada

pelo dentifrício fluoretado, a qual deverá ser levada para a região sem atrito.

Todos os voluntários receberão treinamento e recomendações por escrito

(APÊNDICE B), juntamente com as caixas contendo as placas palatinas, escovas

dentais e dentifrício.

Para estimular o desenvolvimento de lesões de cárie adjacente aos

bráquetes, os voluntários aplicarão extra-oralmente uma solução de sacarose a

20% por cinco minutos, 8 vezes ao dia, pingando uma gota sobre cada espécime

alojado na placa palatina em horários pré-determinados. No 14º dia os biofilmes

formados serão coletados e avaliados quanto à composição microbiana.

19

4.7 Avaliação da Desmineralização

a) Análise da Microdureza do esmalte

Serão realizadas três fileiras de quatro endentações, localizadas ao centro do

bráquete (controle), a 50 e a 100 m da borda do bráquete. As endentações em

cada fileira serão realizadas em profundidade a distâncias de 10, 20, 30, 50, 70 e 90

m da superfície de esmalte.

b) Análise da perda mineral por microscopia eletrônica de varredura (MEV)

Após a realização da avaliação da perda mineral por microdureza, os

espécimes embutidos em resina acrílica e sequencialmente polidos (lixas de

granulação 1000, 1200, 1500, e 2000, seguido por polimento com discos de feltro e

suspensão de diamante. Após serão secos e recobertos com ouro para avaliação

em MEV (SSX-550, Shimadzu, Japão). Serão aleatoriamente selecionados para

análise em MEV 5 espécimes de cada grupo, e estes também serão avaliados por

espectroscopia dispersiva de raio X (EDS). A avaliação por EDS será realizada em

10 linhas onde será avaliada a concentração de Ca e P da superfície para a porção

mais interna do esmalte submetido ao desafio cariogênico. O tempo de aquisição

para o espectro de EDS será de 100 segundos, a uma voltagem de aceleração de

15 kV e com corrente de 0,1 nA. Essas análises serão realizadas de acordo com o

método descrito por Nakata et al., (2009).

c) Análise microbiológica do biofilme dental

Amostras (1 mg) do biofilme formado adjacente aos dispositivos ortodônticos

serão coletadas com espátulas plásticas estéries. Essas amostras serão colocadas

em tubos contendo 1mL RTF (meio de transporte reduzido), e sonicadas (Sonicador

Vibra Cell - Sonics and Materials, Danbury,CT, USA) com potência de 40W,

amplitude de 5%, usando 6 pulsos de 9,9s cada (BOWEN; PRUCHNO; BELLONE,

1986) para obtenção do biofilme em suspensão homogênea. Será então realizada

diluição seriada das suspensões de biofilme para contagem de microrganismos

totais, estreptococos do grupo mutans, lactobacilos (TENUTA et al., 2006), e

microrganismos acidúricos totais. As suspensões serão diluídas em RTF em séries

até 1:107 e imediatamente inoculadas em duplicata nos seguintes meios de cultura:

20

Ágar sangue para microrganismos totais; Ágar mitis salivarius com 0,2 unidades de

bacitracina/mL (MSB), para quantificação de estreptococos do grupo mutans (GOLD;

JORDAN; VAN HOUTE, 1973); Ágar Rogosa SL para lactobacilos; e BHI com pH

ajustado a 4,7 para quantificação de microorganismos totais acidúricos. As placas

serão incubadas em condição de anaerobiose (80% N2, 10% CO2 e 10% H2), a

37ºC por 96h. As unidades formadoras de colônia serão contadas e os resultados

expressos em UFC/mg de espécime de biofilme (peso úmido) e em porcentagem de

estreptococos do grupo mutans, de lactobacilos, em relação aos microrganismos

totais cultiváveis.

4.8 Tratamento estatístico

De posse dos resultados experimentais deste projeto, o método estatístico

será escolhido com base na aderência ao modelo de distribuição normal e igualdade

de variância. Para todos os testes será considerado o valor p < 0,05 como

estatisticamente significativo. Será utilizadada ANOVA seguida do teste de Tukey

para comparações entre grupos. Os voluntários serão considerados como unidades

experimentais e blocos estatísticos, a fim de diminuir a variabilidade do experimento.

Será empregado o pacote SigmaStat (Versão 3.51, Systat Software Inc.).

4.9 Aspectos éticos

O projeto foi encaminhado ao Comitê de Ética em Pesquisa da Faculdade de

Odontologia da Universidade Federal de Pelotas (FO-UFPel - RS). Os voluntários

receberão uma carta de informação sobre o estudo (APÊNDICE B) e deverão

assinar um termo de consentimento livre e esclarecido, a fim de autorizar sua

participação no estudo (APÊNDICE A).

21

5. Cronograma

ANO

2009 2010 2011

MÊS J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D

Pesquisa

bibliográfica

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

Submissão ao comitê de ética

x

Qualificação x

Aquisição dos

materiais

x x x

Preparo dos dentes (in

situ)

x x x x

Seleção dos

pacientes (in

situ)

x x

Confecção dos

aparelhos

x x

Fase in situ x x

Avaliação dos

espécimes

x x x x x

Análise do biofilme

x x x x x

Descrição dos

resultados e análise

estatística

x x x x x

Redação dos artigos

x x x x x x

Redação

final da tese

x x x x x

Defesa da tese

x

22

7. Orçamento

Material Valor unitário Valor total

Espátula de resina 55,00 110,00 Brocas 10,00 40,00 Escovas dentais 7,00 112,00 Creme dental 3,00 48,00 Alginato 16,00 32,00 Resina acrílica 60,00 60,00 Gesso pedra 6,00 12,00 Lixas para desgaste 1,00 30,00 Luvas para procedimento 18,00 18,00 Mascaras 10,00 10,00 Pincel descartável 10,00 10,00 Pasta para polimento 16,60 16,60 Adesivo Concise 160,00 160,00 Bráquetes metálicos 3,00 450,00 Bráquetes autoligantes 20,00 1000,00 Ligaduras elásticas 8,50 17,00 Ligaduras metálicas 20,00 20,00 Fio ortodôntico 2,00 40,00 Condicionador ácido a 35% 12,00 24,00 Caixa de aparelho removível 8,00 112,00 Açúcar refinado 2,00 2,00 Frascos plásticos 0,90 12,60 Ependorfs 28,00 28,00 Preparo dos espécimes 10,60 (hora) 159,00 Uso do Durômetro 11,50 (hora) 805,00 Serviço de revisão do inglês 500,00 500,00 Xerox 100,00 100,00 Despesas com impressão 400,00 400,00 Gastos com apresentação em congressos 2000,00 2000,00 Gastos com publicação em periódico 300,00 300,00 Investimento total ------- 6628,20

FONTES DE FINANCIMENTO: Este estudo será financiado com recursos do PPGO-UFPEL (PROAP), PRODOC-CAPES (R$ 12.000,00 / ano) e com recursos próprios dos pesquisadores envolvidos.

23

Relatório do trabalho de campo

1. Introdução

Este estudo foi realizado com voluntários estudantes do programa de Pós-

Graduação e de graduação da Faculdade de Odontologia da Universidade Federal

de Pelotas.

2. Confecção dos aparelhos

Após seleção e consentimento (APÊNDICE A) dos voluntários procederam-se

as moldagens para confecção dos 17 aparelhos, tipo placa palatina removível, para

suportar os dispositivos necessários para este estudo “in situ”. Após prontos e

testados em cada voluntário foram inseridos os discos e os dispositivos ortodônticos.

2.1 Entrega dos aparelhos

Nas dependências da Clinica Infantil da Faculdade foi realizado um

treinamento teórico-prático de aproximadamente uma hora no dia da entrega dos

aparelhos. Na primeira etapa foram distribuídas e lidas as instruções de uso e de

cuidados com os aparelhos (APÊNDICE B). Em seguida foram instalados os

aparelhos e distribuídos os kits-estojos contendo escova de dente, creme dental,

compressas de gaze estéril, porta aparelho e um frasco de sacarose. Por último,

foram agendados os retornos, no mesmo local, para as trocas periódicas dos frascos

24

de sacarose. Todas as atividades foram supervisionadas pelo pesquisador

responsável. Estas ocorreram no dia 19 de julho de 2010.

3. Período de uso do aparelho

O pesquisador responsável monitorou, via telefone, as rotinas dos voluntários

durante o período de uso do aparelho (19 de julho a 02 de agosto de 2010) na

ocasião não foram constatados nenhum problema com os participantes.

4. Coleta dos aparelhos

O agendamento para as entregas realizou-se com intervalos de 10 minutos

entre cada participante. A partir do momento da entrega do aparelho, um grupo de

apoio com experiência laboratorial efetuava o processamento das amostras. A

equipe se dividiu para as seguintes etapas: recebimento e remoção dos dispositivos

da placa; aplicação do sonicador, coleta e processamento do biofilme e

armazenamento dos discos para posterior analise no microdurômetro. Esta etapa

ocorreu nas primeiras horas do dia 02 de agosto de 2010.

5. Análise da microdureza

Os exames de microdureza foram precedidos pela preparação dos

espécimes. Cada disco com dispositivo colado foi seccionado longitudinalmente

resultando em duas metades iguais. Estas foram embutidas em grupos de oito em

resina acrílica e levadas ao microdurômentro. Esta etapa foi executada, por um

examinador experiente, de agosto a dezembro de 2010.

25

6. Microscopia Eletrônica de Varredura (MEV)

No projeto inicial estava prevista a avaliação qualitativa das lesões de cárie

formadas in situ adjacentes aos dispositivos ortodônticos através de MEV. No

entanto, após realização de estudo piloto, observou-se que essas imagens não

agregariam dados relevantes ao trabalho, e como alternativa outros estudos foram

propostos, conforme descrição abaixo.

7. Estudo in vitro e revisão sistemática

Durante a execução do estudo in situ, adventou-se a possibilidade de realizar

um estudo in vitro com modelo de biofilme de microcosmos para avaliação do

acúmulo microbiano e da perda mineral adjacente a diferentes tipos de bráquetes e

de sistemas de ligadura, o que la foi desenvolvido de acordo com o planejamento.

Paralelamente, o grupo de trabalho decidiu realizar uma revisão sistemática da

literatura sobre o tema geral do projeto a fim de melhor fundamentar o trabalho de

tese.

26

Artigo 1#

Effects of types of brackets and archwire ligation on biofilm accumulation and on enamel demineralization: A systematic review

Marcos Antonio Paccea, Marina Sousa Azevedoa, Sandra Beatriz Chaves Tarquiniob, Douver Michelonb, Maximiliano Sérgio Cencib

a DDS, Msc, PhD student, Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil b DDS, MSc, PhD, Associate Professor, Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil

Reprint requests to: M.S. Cenci – Rua Gonçalves Chaves 457, 5th floor, 96015-560 Pelotas, RS (Brazil) Tel./Fax +55 53 3225 6741 – 134 - E-Mail m.s.cenci@pq.cnpq.br.

#Formatado segundo normas do periódico American Journal of Orthodontics and Dentofacial Orthopedics

27

ABSTRACT

Introduction: Considering that caries lesions are still a matter of concern for

patients under orthodontic treatment, this systematic review aimed to identify and

assess the evidence on the effects of types of brackets and ligature methods on

biofilm accumulation and enamel demineralization adjacent to fixed orthodontic

devices. Methods: Electronic databases (PubMed, Embase, Cochrane Central

Register of Controlled Trials, ISI Web of Knowledge, TRIP, Scopus, and SciELO)

were searched up to July 2011. Clinical trials or in situ studies that assessed the

effect of types of brackets or ligatures on biofilm accumulation and/or enamel

demineralization were selected. Non-controlled studies, in vitro studies, or studies

not reporting on the established outcomes were excluded. Results: Twelve relevant

articles were identified, 2 were in situ studies, 9 were randomized clinical trials and 1

was a controlled trial. Demineralization/caries was assessed by 4 studies,

periodontal status by 2 studies and microbiological analysis by 9 studies.

Conclusions: From this systematic review no definite conclusions can be drawn

regarding which type of ligation or bracket design has a beneficial influence to

orthodontic patients. Besides that, it was impossible to make any current

recommendations on the usage of fluoride releasing elastomers to prevent caries

and/or cariogenic bacteria.

28

INTRODUCTION

The close relationship between the development of carious lesions and the

biofilm accumulation in areas adjacent to the bonded orthodontic appliances is still a

matter of concern in orthodontics.1 The reported prevalence of white spots after fixed

appliance treatment varies between 2 and 96 per cent, considering different studies.2-

4 White spot formation during orthodontic treatment has been attributed to the effect

of prolonged accumulation and retention of bacterial biofilm promoted by the newly

installed orthodontic devices, which represent retentive sites. Fixed appliances make

conventional oral hygiene more difficult, and adjacent to the brackets the clearance

of plaque by saliva and cheeks is also reduced.5 The carious lesions developed

during orthodontic treatment can regress after removal of the device; however

extensive lesions may have need for restorative intervention.6

Several treatment alternatives have been proposed to control caries in

patients under fixed orthodontic treatment. In this context, resin sealers have been

applied on the facial surfaces of bracketed teeth to prevent enamel demineralization.7

Also, remineralizating substances such as amorphous calcium phosphate-containing

products were tested for improving remineralization and inhibition of caries lesion

development around brackets.8,9 Other studies testing the benefits of topical fluoride

treatment10 and chlorhexidine varnish application in order to prevent the

development of white spot lesion during orthodontic treatment have been

accomplished.11 Also, the effect of fluoride incorporation in the elastomeric ligature

rings was investigated. Despite that, white spot lesions continues to be one of the

most common side effects of use of fixed appliances in orthodontic treatment.12

However, the most directly related factor to the biofilm accumulation and

possible demineralization associated to orthodontic devices that is the bracket design

per se has been scarcely investigated. Also, the effect of the type of ligature method

on biofilm accumulation and enamel demineralization has been evaluated by some

studies, but the results are controversial.13 Since orthodontic devices per se create

extra retention sites, studies that evaluated the effect of different devices’ designs in

dental plaque accumulation and in caries development should be revised in order to

establish evidence-based clinical protocols for patients’ treatment.

29

Therefore, the aim of this systematic review was to evaluate the effect of types

of brackets and archwire ligation on biofilm accumulation and on enamel

demineralization in the area adjacent to these devices.

MATERIAL AND METHODS

This systematic review was conducted in accordance with the guidelines of

Transparent Reporting of Systematic Reviews and Meta-Analyses [PRISMA

statement30]. The question being focused on was as follows: what is the effect of

types of brackets and types of ligatures on enamel caries and biofilm accumulation,

in a patient undergoing orthodontic treatment?

Search Strategy

Internet sources were used to search for appropriate papers that satisfied the

study purpose. These included the NationalLibrary of Medicine, Washington, D.C.

(MEDLINE-PubMed), the Cochrane Central Register of Controlled Trials (CENTRAL),

EMBASE (Excerpta Medical Database by Elsevier), ISI Web of Knowledge, TRIP

Database, Scopus, and SciELO (Scientific Electronic Library Online). The databases

were searched for studies conducted in the period up to and including July 17, 2011.

The structured search strategy was designed to include any published paper that

evaluated the effect types of brackets and types of ligatures on enamel caries and

biofilm accumulation. The search strategy included the following terms and their

combinations: orthodontic, caries, biofilm, dental plaque, in situ, clinical trial,

The following eligibility criteria were used:

• randomized controlled clinical trials (RCTs), or controlled clinical trials, or in situ

controlled studies;

• conducted in humans;

• subjects: adolescents or adults, no other limit age;

• intervention: types of brackets or ligature methods;

• control: standard devices;

• clinical parameters: enamel demineralization and biofilm accumulation.

Screening and Selection

Two reviewers (M.A.P and M.S.C.) independently screened titles and

abstracts for eligible papers. If information relevant to the eligibility criteria was not

available in the abstract, or if the title was relevant but the abstract was not available,

the paper was selected for a full reading of the text. Next, full-text papers that fulfilled

30

the eligibility criteria were identified and included into this study. The two reviewers

hand-searched the reference lists of all of the selected studies for additional

published papers that could possibly meet the eligibility criteria of this study. Papers

that fulfilled all of the selection criteria were processed for data extraction.

Types of comparisons:

Comparison between brackets: Metal (stainless steel) brackets compared with

other materials such as acrylic, composite, ceramics, sapphire. Or conventional metal

brackets compared with modified design brackts (such as presence of hooks).

Comparison between types of ligatures: elastomeric rings or stainless still wieres

compared to self-ligated bracket systems.

Outcomes:

• Primary Outcome:

Caries lesions (demineralization, white spot lesions or cavities) developed

adjacent to the orthodontic devices.

• Secondary Outcome:

Biofilm accumulation, assessed by Oral Hygienne Index, Visible Plaque

Indexes, or other clinical parameters.

Biofilm accumulation evaluated by biomass quantification, or biofilm microbial

composition

Periodontal status: presence of gengivitis, evaluated by gingival bleeding

index.

Secondary outcomes also included differences before and after orthodontic

appliance placement on plaque/gingivitis, calculus and/or pocket depth.

Data Extraction

Data from the papers that met the selection criteria were processed for

analysis. Data were extracted with regard to the effect types of brackets and types of

ligatures on enamel caries and biofilm accumulation.

For studies that presented intermediate assessments, the baseline and final

evaluations were used for this review. Data were extracted by M.A.P and M.S.A.

31

RESULTS

A total of 354 titles and abstracts were identified in the electronic databases

used (Fig. 1). Application of the inclusion and exclusion criteria on the referred

studies allowed identification of 12 relevant publications (Fig. 1 –Table 1). From the

total publications included 2 were in situ studies, 9 were randomized clinical trials and

1 was a controlled trial. Seven of these studies used split-mouth design.

The type of brackets was assessed only by 3 studies.1,14,15 Demling et al,

201014 analyzed the biofilm formation on brackets coated with polytetrafluoroethylene

(PTFE) compared to uncoated brackets. Two compared self-ligating with

conventional brackets.1,15

Studies investigating the archwire ligation were most commonly found.13,16-23

Six studies compared fluoridated elastomeric ligatures with non-fluoridated

elastomeric ligatures.16-18,20,21,23 Three compared elastomeric rings with steel ligature

wires.13,19,22

Four studies13,16,18,20 assessed enamel demineralization, but none of them used the

same methodology for this outcome assessment. Only 2 studies reported statistically

significant differences in demineralization between the control and the experimental

group, both were randomized clinical trials16,20 Banks et al.,16 verified enamel

demineralization by direct clinical observation using the Enamel Decalcification

Index.24 The enamel demineralization was subjectively measured in the Mattick’s

study using a semi-quantitative index based on the Enamel Defect Score.2 Gameiro

et al. and Doherty et al13,18 using in situ caries model did not found differences

between tested groups, enamel demineralization around the brackets was evaluated

by cross-sectional microhardness in the Gameiro13 study and by transverse

microradiography by Doherty et al.18

Two studies assessed periodontal status.15,22 Periodontal pocket depth and bleeding

on probing were the two periodontal measures used in common between the studies.

In 9 studies microbiological issue was assessed.1,13-15,17,19,21-23 Most of the studies

used plaque/biofilm as microbiological samples1,13,14,17,19,21. In the study of Miura et

al21, plaque and saliva samples were collected for microbiologic analysis and

Wilson23 collected only saliva sample for microbiological purpose. Total bacteria, total

32

aerobic bacteria, total anaerobic bacteria, total streptococci, mutans streptococci,

and lactobacilli counts were used by the studies as microbiological composition

parameters.

All studies provided patient numbers. The age of the subjects ranged from 11 years

to 35 years. Mattick et al20 did not report the age of the subjects. Gameiro et al.13 and

Banks et al16 gave only the mean age of the subjects.

DISCUSSION

The placement of fixed orthodontic appliances creates new retention areas resulting

in plaque accumulation. The design of different orthodontic brackets, as well as the

method of ligation can contribute to plaque adhesion and therefore influence caries

around bracket and periodontal status of the patients.

Our review identified three studies that compared the method of ligation13,19,22 and

only 2 studies that compared the design of the brackets.1,15 The other studies

included not compared the design of the bracket or the ligature method, but tested a

treatment/coating applied on bracket14 or ligatures containing fluoride.17,18,20,21,23,24

The single feature common to included studies which compared the method of

ligation or the design of the brackets was the microbiological assessment. While Van

Gastel et al15 and Turkk22 also verified the periodontal status and Gameiro13 the

enamel demineralization.

Although the 3 studies of the ligature methods compared elastomeric rings and steel

wire, the microbiological outcomes were different.13,19,22 The 2 studies that did not

found statistical difference investigated cariogenic bacteria, as S. mutans and

lactobacilli.13,22 Only one found statistical significant differences showing an increase

in the total number of bacteria in the plaque in ligation with elastomeric ring than

ligation with steel wires.19 The cariogenic bacteria were assessed by this study only

in the saliva sample, which is not adequate to analyze since a split-mouth design was

used. According to this author, in orthodontic patients whose oral hygiene is not

optimal, the use of elastomeric rings for ligation cannot, therefore, be recommended,

as they may significantly increase the microbial accumulation on tooth surfaces

adjacent to the brackets, leading to a predisposition for the development of dental

caries and gingivitis. The predominant species from diseased sites are different from

33

those found in healthy sites25, thus the knowledge of the plaque composition can be

more relevant than the total number of bacteria per se.

The two studies comparing the design of the brackets found disparities among

microbiological outcome.1,15 Both of them compared self-ligating with conventional

brackets ligated with elastomeric rings. Pellegrini1 showed fewer total bacteria and

oral streptococci in teeth bonded with sel-ligating brackets. On the other hand, Van

Gastel15 found that self-ligating bracket sites in general allowed more plaque

formation than conventional bracket sites. The increased CFU counts in the self

ligating bracket sites were not expected because of the limited dimensions of the

self-ligating bracket and the presence of a smooth clip instead of an elastomeric

ligature. In order to clarify this issue Van Gastel15 used scanning electron

microscopic (SEM) to find differences in the surface between the two brackets. These

qualitative SEM images revealed remarkable irregularities on the interfaces between

the different parts of the self-ligating attachments used (both of the bracket and the

tube). These parts seem to be welded together causing an irregular surface, which

might have lead to the increased plaque adhesion in the self-ligating brackets.

Five randomized clinical trials used a split-mouth design1,15,17,19,20, in which each

subject receives greater than or equal to 2 treatments, each to a separate section of

the mouth.26 The main purpose of the split-mouth design is to remove all components

related to differences between subjects from the treatment comparisons. By making

within-patient comparisons, rather than between-patient comparisons, thus the error

variance (noise) of the experiment can be reduced.27 Two of these studies used split-

mouth design to compare fluoride releasing modules and non-fluoride releasing

modules.17,20 However, when considering products containing fluoride, it is possible

that the fluoride released will affect all quadrants, not just those using the

experimental material; the possibility of some crossover effect to the control side

must be considered.28 Although it is possible that a crossover effect of fluoride

occurred via saliva and it can reduce the differences between the groups, the studies

did not mention the possible bias of this type of design.

Three studies investigated the effect of fluoridated elastomeric ligatures on the

bacterial count of dental plaque and/or saliva.17,21,23 The main idea of these studies is

that if fluoridated elastomers can affect local cariogenic bacteria, this will be

important in reducing enamel demineralization around orthodontic brackets.

However, it is known that fluoride can only affect bacteria in high concentrations, and

34

therefore the effect of fluoride released from elastomers would be more physic-

chemical on the demineralization/remineralization process. The fluoridated

elastomers were supposed to be an effective method since a constant supply of

fluoride over orthodontic treatment can be released and because can be placed in

close proximity to the bracket. However, the reduction of cariogenic bacteria was not

relevant in the studies included. A reason pointed by Benson17 was that fluoridated

elastomers release high levels of fluoride initially; this release rapidly drops to a point

that will not affect bacterial growth and metabolism.29 Thus, these fluoridated

elastomeric ligatures can be not relevant to current orthodontic practice because the

time between adjustment visits during orthodontic treatment will be longer than the

time of fluoride release. In spite of that, other 2 studies included tested fluoride

elastomers and investigated caries outcome showing a reduction in the incidence of

decalcification around brackets during a complete course of orthodontic

treatment.16,20 It is worth to mention that in both studies the use of this ligature

significantly reduced, but did not eliminate the incidence of decalcification following

orthodontic treatment.

Our systematic review identified 4 studies investigating caries outcome.13,16,18,20 The

main problems were differences in the studies design and differences in the outcome

measures used. For example, one study used in situ model to evaluate the effect of

ligatures on enamel demineralization on blocks of bovine enamel, the enamel

demineralization was evaluated by cross-sectional microhardness.13 Other one, for

example, was a randomized clinical trial and enamel decalcification incidence and

distribution were recorded for all teeth individually using an index by direct clinical

observation, the Enamel Decalcification, by one operator. Although all study types

tested the type of ligature and were included, they are clearly not directly

comparable.

Periodontal outcome was assessed by two studies reported.15,22 Türkkahraman et

al22 reported that the same examiner evaluated the periodontal status and was

calibrated before, however a kappa index was not mentioned. The other study

reported that the examiner was blinded, to ensure blind evaluation the

measurements were carried out after removal of the brackets and the researcher did

not know which bracket was bonded on which tooth15, the calibration was not

35

reported by this study. Bleeding on probing and pocket depth was used as

periodontal measurements common to both studies. However, one compared the

ligation method22 and the other the bracket design15, thus the results were not

comparable.

Deficiencies in the way data were reported, the differences in sample size, sample

teeth, registration times and study design were some problems encountered among

the studies in this systematic review.

CONCLUSION

Studies exhibited conflicting data as to whether or not type of brackets and archwire

ligation significantly prevent or inhibit caries and biofilm accumulation in fixed

orthodontic patients. Thus, from this systematic review no definitive conclusions can

be drawn regarding which type of ligation or bracket design has a beneficial influence

to orthodontic patients. Besides that, it was impossible to make any current

recommendations on the usage of fluoride releasing elastomers to prevent caries

and/or cariogenic bacteria.

Further randomized and blinded controlled studies are needed to determine whether

or not the design of the brackets, the type of archwire ligation and treatment applied

on/in these appliances can contribute to prevent caries and plaque accumulation.

36

REFERENCES

1. Pellegrini P, Sauerwein R, Finlayson T, McLeod J, Covell DA, Jr., Maier T et al.

Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative

comparison of oral bacteria and detection with adenosine triphosphate-driven

bioluminescence. Am J Orthod Dentofacial Orthop 2009;135:426 e421-429;

discussion 426-427.

2. Artun J, Brobakken BO. Prevalence of carious white spots after orthodontic

treatment with multibonded appliances. Eur J Orthod 1986;8:229-234.

3. Gorelick L, Geiger AM, Gwinnett AJ. Incidence of white spot formation after

bonding and banding. Am J Orthod 1982;81:93-98.

4. Mitchell L. Decalcification during orthodontic treatment with fixed appliances--an

overview. Br J Orthod 1992;19:199-205.

5. Mattousch TJ, van der Veen MH, Zentner A. Caries lesions after orthodontic

treatment followed by quantitative light-induced fluorescence: a 2-year follow-up.

Eur J Orthod 2007;29:294-298.

6. van der Veen MH, Mattousch T, Boersma JG. Longitudinal development of caries

lesions after orthodontic treatment evaluated by quantitative light-induced

fluorescence. Am J Orthod Dentofacial Orthop 2007;131:223-228.

7. Hu W, Featherstone JD. Prevention of enamel demineralization: an in-vitro study

using light-cured filled sealant. Am J Orthod Dentofacial Orthop 2005;128:592-

600; quiz 670.

8. Beerens MW, van der Veen MH, van Beek H, ten Cate JM. Effects of casein

phosphopeptide amorphous calcium fluoride phosphate paste on white spot

lesions and dental plaque after orthodontic treatment: a 3-month follow-up. Eur J

Oral Sci 2010;118:610-617.

9. Behnan SM, Arruda AO, Gonzalez-Cabezas C, Sohn W, Peters MC. In-vitro

evaluation of various treatments to prevent demineralization next to orthodontic

brackets. Am J Orthod Dentofacial Orthop 2010;138:712 e711-717; discussion

712-713.

10. Suri L, Huang G, English JD, Jr., Owen S, Nah HD, Riolo ML et al. Ask us.

Topical fluoride treatment. Am J Orthod Dentofacial Orthop 2009;135:561-563.

11. Derks A, Frencken J, Bronkhorst E, Kuijpers-Jagtman AM, Katsaros C. Effect of

chlorhexidine varnish application on mutans streptococci counts in orthodontic

patients. Am J Orthod Dentofacial Orthop 2008;133:435-439.

37

12. Ogaard B, Rolla G, Arends J. Orthodontic appliances and enamel

demineralization. Part 1. Lesion development. Am J Orthod Dentofacial Orthop

1988;94:68-73.

13. Gameiro GH, Nouer DF, Cenci MS, Cury JA. Enamel demineralization with two

forms of archwire ligation investigated using an in situ caries model--a pilot study.

Eur J Orthod 2009;31:542-546.

14. Demling A, Elter C, Heidenblut T, Bach FW, Hahn A, Schwestka-Polly R et al.

Reduction of biofilm on orthodontic brackets with the use of a

polytetrafluoroethylene coating. Eur J Orthod 2010;32:414-418.

15. van Gastel J, Quirynen M, Teughels W, Coucke W, Carels C. Influence of bracket

design on microbial and periodontal parameters in vivo. J Clin Periodontol

2007;34:423-431.

16. Banks PA, Chadwick SM, Asher-McDade C, Wright JL. Fluoride-releasing

elastomerics--a prospective controlled clinical trial. Eur J Orthod 2000;22:401-

407.

17. Benson PE, Shah AA, Campbell IF. Fluoridated elastomers: effect on disclosed

plaque. J Orthod 2004;31:41-46; discussion 16.

18. Doherty UB, Benson PE, Higham SM. Fluoride-releasing elastomeric ligatures

assessed with the in situ caries model. Eur J Orthod 2002;24:371-378.

19. Forsberg CM, Brattstrom V, Malmberg E, Nord CE. Ligature wires and

elastomeric rings: two methods of ligation, and their association with microbial

colonization of Streptococcus mutans and lactobacilli. Eur J Orthod 1991;13:416-

420.

20. Mattick CR, Mitchell L, Chadwick SM, Wright J. Fluoride-releasing elastomeric

modules reduce decalcification: a randomized controlled trial. J Orthod

2001;28:217-219.

21. Miura KK, Ito IY, Enoki C, Elias AM, Matsumoto MA. Anticariogenic effect of

fluoride-releasing elastomers in orthodontic patients. Braz Oral Res 2007;21:228-

233.

22. Turkkahraman H, Sayin MO, Bozkurt FY, Yetkin Z, Kaya S, Onal S. Archwire

ligation techniques, microbial colonization, and periodontal status in

orthodontically treated patients. Angle Orthod 2005;75:231-236.

38

23. Wilson TG, Gregory RL. Clinical effectiveness of fluoride-releasing elastomers. I:

Salivary Streptococcus mutans numbers. Am J Orthod Dentofacial Orthop

1995;107:293-297.

24. Banks PA, Richmond S. Enamel sealants: a clinical evaluation of their value

during fixed appliance therapy. Eur J Orthod 1994;16:19-25.

25. Marsh PD. Dental plaque as a biofilm and a microbial community - implications

for health and disease. BMC Oral Health 2006;6 Suppl 1:S14.

26. Antczak-Bouckoms AA, Tulloch JF, Berkey CS. Split-mouth and cross-over

designs in dental research. J Clin Periodontol 1990;17:446-453.

27. Hujoel PP, DeRouen TA. Validity issues in split-mouth trials. J Clin Periodontol

1992;19:625-627.

28. Rogers S, Chadwick B, Treasure E. Fluoride-containing orthodontic adhesives

and decalcification in patients with fixed appliances: a systematic review. Am J

Orthod Dentofacial Orthop 2010;138:390 e391-398; discussion 390-391.

29. Wiltshire WA. Determination of fluoride from fluoride-releasing elastomeric

ligature ties. Am J Orthod Dentofacial Orthop 1996;110:383-387.

30. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JP, Clarke

M, Devereaux PJ, Kleijnen J, Moher D. The PRISMA statement for reporting

systematic reviews and meta-analyses of studies that evaluate health care

interventions: explanation and elaboration. J Clin Epidemiol 2009;62:e1-34

39

PRISMA Flow Diagram30

IDE

NT

IFIC

AD

OS

S

EL

EC

ION

AD

O

S

ES

CO

LH

IDO

S

INC

LU

ÍDO

S

Artigos após remoção das

duplicatas (n=354)

Textos completos selecionados

(n=66)

Artigos excluídos pelos seguintes motivos: fora do desfecho, anais, casos clínicos e estudos in vitro. (n=288)

28

Artigos - textos completos avaliados (n=66) Artigos – textos completos

excluídos por não haver grupo controle. (n=52)

Artigos – textos completos escolhidos (n=14)

Incluídos (n=12)

ISI WEB OF

KNOWLEDGE

(n=78)

SCOPUS

(n=274)

COCHRANE

BVS

(n=230)

Artigos – textos completos excluídos por deficiência metodológica. (n=2)

PUBMED

(n=611)

TRIP +

EMBASE

(n=59)

SCIELO

(n=292)

Table 1 . Studies included for review that fulfilled selection criteria Study Study

Design

Sam

ple

size

Mean

age

(range)

Type of

bracket

Type of

archwire

ligation

Caries outcome Periodontal

outcome

Microbiological

outcome

Conclusion/Findings

Miura et al.

2007

RCT 40 12-20

years

ND Fluoride-

releasing

elastomeric

rings X

Convention

al

elastomeric

rings

NT NT Seven, 14 and

28 days after

placement of the

ligature ties

saliva and

plaque were

collected to

determine the

number of CFU

of

Streptococcus

mutans.

There was no significant reduction in S.

mutans in saliva or plaque around

fluoride-releasing elastomeric ligature

ties.

Türkkahram

an et al.

2005

Split-

mouth,

CT

21 15.37

(11.6-

25.7)

Convention

al

brackets

Elastomeric

rings X steel

ligature

wires

NT The gingival

index

(GI), bonded

bracket plaque

index (BBPI),

bleeding

on probing

(BOP) values,

and pocket

depth (PD)

values

were recorded

before bonding,

1 week later,

and 5 weeks

after bonding.

The number of

total bacteria, S.

mutans and

lactobacilli were

recorded before

bonding,

1 week later,

and 5 weeks

after bonding.

Although teeth ligated with elastomeric

rings exhibited a slightly greater

number of microorganisms than teeth

ligated with steel ligature wires, the

differences were not statistically

significant and could be ignored. No

significant effect of archwire ligation

technique was

determined in the GI, BBPI, and PDs of

bonded teeth.

However, the teeth ligated with

elastomeric rings were more prone to

bleeding. Therefore, the use of

elastomeric rings is not recommended

in patients with poor oral hygiene.

Gameiro et

al.

2009

Split-

mouth,

In situ

4 27 years Convention

al

brackets

Elastomeric

rings X

stainless

steel

ligatures

Enamel blocks

were removed

from the

appliances and

enamel

demineralizatio

NT The total

biofilm formed

on the enamel

blocks, under

the

ligatures and

The ligatures evaluated did not differ

significantly from each other regarding

biofilm weight, total

bacteria, total streptococci, mutans

streptococci, or lactobacilli counts

(P>0.05).

41

n around the

brackets was

evaluated by

cross-sectional

microhardness.

around the

brackets, was

collected,

weighed,

and assessed

microbiologicall

y (total bacteria,

total

streptococci,

mutans

streptococci,

and lactobacilli

counts)

Enamel demineralization was also not

different around the brackets for the

different ligation methods

(P>0.05). However, a statistical power

analysis based on the data showed a

trend to higher demineralization around

brackets ligated with elastomeric rings.

Benson et

al. 2004

splith-

mouth

RCT

30 14.2

years

(11.8-

20.6)

ND Fluoridated

elastomeric

rings X

nonfluoridat

ed

elastomeric

rings

NT NT Each

elastomeric

sample were

assessed for

total aerobic,

total anaerobic,

and total Mitis

Salivarius

colony forming

units.

Fluoridated elastomers are not effective

at reducing

streptococcal or anaerobic bacterial

growth in

local plaque surrounding an orthodontic

bracket after

a mean time of 40 days in the mouth.

Doherty et

al.

2002

In situ,

RCT

14 14.8

years

(13.3-

17.7)

ND Fluoridated

elastomeric

rings X non-

fluoridated

elastomeric

rings

Enamel lesions

were prepared

on extracted

pre-molar teeth.

After removal

from the mouth,

the specimens

were sectioned

and transverse

microradiograph

y was carried

out (mineral

loss (delta Z),

lesion depth

(ld), lesion

width (lw), and

ratio (delta

NT NT Fluoride-releasing ligatures do not

provide a significant anti-cariogenic

benefit in patients undergoing

orthodontic treatment using the in situ

caries model

42

Z/ld).

van Gastel

et al.

2007

Split-

mouth,

RCT

16 17-27

years

Convention

al

brackets -G

and self-

ligation-S

Convention

al

elastomeric

ring and

self-ligation

NT Periodontal

pocket depth

(PPD), the

crevicular fluid

flow and

bleeding on

probing (BOP)

were recorded at

baseline, on

days 3 and 7.

Microbiological

samples were

taken from

the brackets and

the teeth on

days 3 and 7.

Total

number of

anaerobic and

aerobic colony-

forming units

(CFU) were

counted. From

these data, the

CFU ratio

[CFUaerobe/

CFUanaerobe

(CFUae and

CFUanae)]

was also

calculated.

Bracket design can have a significant

impact on bacterial load and on

periodontal parameters Both anaerobe

and aerobe colony-forming units (CFU)

were significantly

higher in S-sites than in G-sites

(p50.0002, p50.02). The

aerobe/anaerobe

CFU ratio was significantly lower in S-

sites than in G-sites (p50.01). On day 3,

the

crevicular fluid flow was significantly

higher in S-sites than in control sites

(p50.01).

More hypertrophy was seen in S- than

in G- and control sites

(p50.05). No significant differences for

bleeding on probing were observed.

.

Pellegrini et

al.

2009

Split-

mouth,

RCT

14 11-17

years

Self ligating

brackets X

conventiona

l brackets

Convention

al

elastomeric

ring X self-

ligation

NT NT Standard

microbiology

techniques were

used to confirm

the quantity

and to identify

the bacteria

(total bacteria

and oral

streptococci)

At 1 and 5 weeks after bonding,

the means for SL vs E brackets were

statistically lower

for total bacteria and oral streptococci

(P\0.05). The findings indicate that self-

ligating appliances

might promote reduced retention of

bacteria, including

streptococci, than appliances with

elastomeric ligation.

Demling et

al.

2010

RCT 13 11.2

years (8-

16)

polytetraflu

oroethylene

Elastomeric

ring

NT NT Analysis of

quantitative

biofilm

formation was

Uncoated orthodontic brackets are

highly susceptible to biofilm formation

that endangers the integrity of oral hard

and soft tissues by means of

43

coated

conventiona

l bracket X

uncoated

conventiona

l bracket

performed with

the Rutherford

backscattering

detection

(RBSD)

method, a

scanning

electron

microscopy

(SEM)

technique

decalcification and periodontal disease.

A PTFE coating on brackets reduced

the biofilm formation to a minimum.

Forsberg et

al.

1991

Split-

mouth,

RCT

12 12-14

years

Convention

al

brackets

Elastomeric

rings X steel

ligatures

NT NT Number of

microorganisms

in samples of

plaque after 4,

10, 19, 34 and

61 weeks.

The mean number of bacteria was

higher in ligation with elastomeric ring

than ligation with steel wires on all

occasions (P<0.001). The method of

ligation of the arch wire influence the

degree of plaque development.

Wilson and

Gregory,

1995

RCT 24 13-35

years

Convention

al

brackets

Fluoride

releasing

elastomeric

rings X

conventiona

l

elastomeric

rings

NT NT A

total of 7 saliva

samples were

collected from

each subject

over a 13-week

period to

quantify the

number of oral

streptococci and

S. mutans.

Results

showed that the control group

demonstrated no significant changes (/3

> 0.05) in the percentage

of S. mutans over the 13-week study

period. However, after the fluoride-

releasing elastomers were

placed, the percent of salivary S.

mutans decreased significantly (p <

0.01) in the experimental

group. There was no significant effect

after the fluoride-releasing elastomers

were in place for 2 or

more weeks.

Banks et al.

2000

RCT 100 Control

group

(16.5

years)

Experim

ental

group

Convention

al brackets

Standard

elastomeric

rings X

fluoride

releasing

elastomeric

rings

Enamel

decalcification

incidence and

distribution

were recorded

using an index

by direct

NT NT The reduction in the incidence of

decalcification in the experimental teeth

was highly significant (P<0.001).

Fluoride releasing elastomerics appear

to provide a clinically worthwhile

reduction in enamel decalcification

during fixed appliance therapy when

44

(15.5yea

rs)

clinical

observation, the

Enamel

Decalcification

Index.

they are changed at each treatment visit.

Mattick et

al. 2001

Spli-

mouth,

RCT

21 ND ND Fluoride

releasing

elastomeric

rings X non-

fluoride-

releasing

elastomeric

ringss

The degree of

decalcification

was assessed in

each tooth

quadrant, using

a modification

of the Enamel

Defect Score.

NT NT Decalcification was found to occur in

both treatment groups, though to a

significantly greater degree on the

control side (p = 0.002). The fluoride

module side showed significantly fewer

serious decalcified lesions than the

control (p = 0.013). It would appear that

the use of fluoride releasing elastomeric

modules reduces the degree of

decalcification experienced during

orthodontic treatment

NT=not tested ND=not described

RCT= randomized clinical trial

CT= controlled trial

Artigo 2#

Effect of types of brackets and archwire ligation on enamel demineralization assessed in a microcosm biofilm model

Marcos Antonio Paccea, Júlia Rosa de Almeidab, Marina Sousa Azevedoa, Marcos Rodolfo Bolfonib, Françoise Hèléne van de Sandea, Sandra Beatriz Chaves

Tarquinioc, Douver Michelonc, Maximiliano Sérgio Cencic

a DDS, Msc, PhD student, Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil b Undergraduate student, School of Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil c DDS, MSc, PhD, Associate Professor, Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil

Reprint requests to: M.S. Cenci – Rua Gonçalves Chaves 457, 5th floor, 96015-560 Pelotas, RS (Brazil) Tel./Fax +55 53 3225 6741 – 134 - E-Mail m.s.cenci@pq.cnpq.br.

#Formatado segundo normas do periódico American Journal of Orthodontics and Dentofacial Orthopedics

46

ABSTRACT

Introduction: Brackets’ designs and/or the type archwire ligations could affect biofilm

accumulation and enamel demineralization during fixed orthodontic treatment. The

aim of this study was to assess enamel demineralization around six types of

brackets/archwire ligation combinations. Methods: Microcosm plaque biofilms were

grown in 24-well microplates on enamel discs for 14 days in artificial saliva. Growth

condition comprised cariogenic challenge (artificial saliva supplemented with 1%

sucrose 6 h/day). The groups under study (n=10) were: self-ligating brackets;

conventional brackets ligated with stainless steel wires; conventional brackets,

brackets with hooks, ceramic brackets, and composite brackets, all the four ligated

with elastomeric rings. The biofilm formed around the brackets was collected and dry-

weighted and the mineral loss around the brackets was determined by cross-

sectional microhardness measurement. Results: Lower biofilm biomass was

observed adjacent to brackets with hooks (P<0.05). Lower demineralization was

observed associated to self-ligated brackets, composite brackets, and steel wire

ligated brackets than adjacent to brackets with hooks (P<0.05). Ceramic brackets

and conventional brackets ligated with elastomeric rings presented intermediate

results (P>0.05). Conclusion: The design of the bracket and/or type of archwire

ligation affects demineralization adjacent to the devices, and clinicians should be

aware of this factor to better control their patients.

Key Words: Biofilm, Dental caries, Dental plaque, Orthodontic brackets

47

INTRODUCTION

Patients with fixed orthodontic appliances have an increased risk for development of

enamel caries, since these appliances increase dental plaque retention and make

tooth-brushing more difficult1. Even after the removal of fixed appliances and

restoration of oral hygiene, most of the white spot lesions developed during treatment

does not regress, even leading to restorative needs in some cases2, 3.

Several treatment alternatives have been proposed to control caries in patients under

fixed orthodontic treatment. In this context, resin sealers have been applied on the

facial surfaces of bracketed teeth to prevent enamel demineralization4. Also,

remineralizating substances such as amorphous calcium phosphate-containing

products were tested for improving remineralization and inhibition of caries lesion

development around brackets5, 6. Others studies testing the benefits of topical

fluoride treatment7 and chlorhexidine varnish application in order to prevent the

development of white spot lesion during orthodontic treatment have been

accomplished8. Also, the effect of fluoride incorporation in the elastomeric ligature

rings was investigated9-11. Despite that, white spot lesions continues to be one of the

most common side effects of use of fixed appliances in orthodontic treatment12.

The complexity of the oral environment and ethical problems associated with in vivo

studies of oral diseases in humans have inevitably led to the development of

laboratory models, which simulate the oral environment in vitro 13, 14. Aiming to

answer specific questions related to caries, community cultures of biofilm

microcosms derived from the natural oral microflora have been used and appear to

reflect the complexity, diversity and heterogeneity of in vivo plaques15-19. Studies

using these models showed that the responses of the plaque microbiota, formation of

biomass and pH of supplemental sucrose are different among people20, but the

overall caries lesion development is similar under the same cariogenic conditions14.

The effect of ligature on enamel demineralization has been evaluated previously21, as

well as the changes on periodontal status and microbial flora related to archwire

ligation techniques22. However, the combined effect of fixed orthodontic appliances

and the method of ligation in the plaque accumulation and on enamel

demineralization have not been sufficiently investigated, especially under controlled

laboratory conditions. Since orthodontic devices per se create extra retention sites,

the effect of different devices’ designs in dental plaque accumulation and in caries

development should be further investigated.

48

Preclinical research is essential to develop and test new treatments, and to achieve

knowledge on the factors affecting caries development around orthodontic devices.

Thus, this study was designed to investigate the combination effect of types of

brackets and archwire ligation on biofilm accumulation and enamel demineralization,

under a cariogenic microcosm biofilm model. The hypothesis tested is that brackets

and ligation methods with less complex designs promote lower biofilm accumulation

and lower demineralization on the adjacent enamel.

METHODS

Experimental Design

A completely randomized, evaluator-blinded, in vitro design for caries development

under sucrose exposure and biofilm accumulation originated from saliva of one donor

was grown around orthodontic brackets, using a previously described method14.

Biofilm was grown in a chemically defined saliva analogue with mucin (DMM)23

supplemented with sucrose alternated with pure DMM. Bovine enamel discs

measuring 6 mm diameter and 2 mm thickness were obtained and used for

simulating the clinical use of orthodontic brackets, ligatures and archwire. The factors

under evaluation were bracket type and ligatures/attachment at 6 levels: conventional

bracket (Aditek, Cravinhos, SP, Brazil) + elastomeric rings; conventional bracket +

stainless steel ligatures; conventional bracket with hooks + elastomeric rings;

ceramic bracket (Trancend Unitek, 3M, Sumaré, SP, Brazil) + elastomeric rings;

composite bracket (Morelli, São Paulo, SP Brazil) + elastomeric rings; and self-

ligating bracket. Brackets, 3 × 4 mm were bonded to the centre of the enamel blocks

with Transbond XT Light Cure Adhesive System (3M ESPE, St Paul, MN, USA),

following the manufacturer’s instructions. For each experimental unit, a 0.016 inch

stainless steel archwire was inserted in bracket slots in all enamel discs (n= 10

samples per group). At the end of the experimental phase, biofilms were collected

and assessed for total biomass (dry weight, in mg). The enamel discs were assessed

regarding integrated mineral loss (ΔS) adjacent to the brackets by cross-sectional

microhardness. The research protocol of this study was approved by the Local Ethics

Committee (Protocol number 168/2010) and written informed consent was obtained

from the saliva donor.

49

Biofilm growth conditions and cariogenic challenge

All samples were sterilized by gamma radiation (Theratronics - Eldorado 78,

radioisotope cobalt 60, radiation energy of 1.25 MeV, 1161 KGy dose and exposure

time of 21min51s, at a distance of 2 cm from the irradiating source) before the

experiment. Saliva was used as inoculum to provide a multispecies microcosm

biofilm. Approximately 40 mL of stimulated saliva (Parafilm ―M"®, American National

CanTM, Chicago, IL, USA) was collected from a healthy donor in the morning (21

years old), 2 h after the last meal and the volunteer abstained from oral hygiene 24 h

prior to collection. An aliquot of 0.6 mL of fresh and homogenized saliva was

inoculated on each specimen, in 24-well plates. After 1 h the saliva was gently

aspirated from each well and growth media supplemented with 1% sucrose (1.8 mL)

was added to each sample. Every day, each individual biofilm (n=10 per group)

received DMM with 1% of sucrose (DMM+s) for 6 h, and after the sugar challenge

the discs were dip washed for 10 s in sterile saline solution and transferred to a new

plate with pure DMM for 18 h. These alternate cariogenic challenge / non-cariogenic

challenge was repeated until the final of the experiment, on the day 14th. Details of

the biofilm method were previously published14.

Dental biofilm analysis

On the 14th day, the dental biofilm from each set comprising enamel disc, bracket,

ligature and archwire was collected. Each set was inserted in sterile tube containing

10 mL of 0.9% NaCl solution and sonicated. An aliquot of the homogenized

suspension was used for dry weight determination. Three volumes of cold ethanol (–

20°C) were added to 0.2 mL of the cell suspension, and the resulting precipitate

collected (10 000g for 10 min, 4°C). The supernatant was discarded, and the cell

pellet was washed twice with cold ethanol, and then lyophilized and weighed24.

Enamel mineral loss assessment

All discs from each set were sectioned longitudinally through the centre for enamel

cross-sectional microhardness (CSMH) determination. The enamel demineralization

was evaluated using a microhardness tester (Future-Tech FM, Tokyo, Japan) with a

50

Knoop diamond indenter under a 25 g load for 5 s. Two lines of indentations were

made: one corresponding to the 50 µm next to the bracket margin and other 100 µm

from the bracket margin 21. Indentations were made at 10, 20, 30, 50, 70, 100 and

150 μm from the outer enamel surface. The CSMH determination of all enamel discs

was carried out by one blind and trained examiner, and the results per sample

considering each distance from the bracket margin were used to calculate the

integrated mineral loss (Δ S)25.

Statistical analyses

Data of enamel demineralization and biofilm weight were analyzed by ANOVA

followed by the Holm-Sidak method for multiple comparisons. In order to attend the

assumptions of normality of distribution and equality of variances, data of biofilm

weight were transformed by log (10) and data of ΔS at 50 µm from the bracket edge

were transformed by square root. The SigmaStat software (version 3.5; Systat Inc,

USA) was used and the significance level set at P < 0.05.

RESULTS

Lower biomass was recovered from the conventional bracket with hook ligated with

elastomeric rings (P=0.04), except in the comparison with the brackets ligated with

steel wires (P>0.05). No differences among the other types of brackets or ligature

methods were found (P>0.05) (Fig. 1). The type of bracket / ligature method affected

demineralization (P<0.0001). Overall, lower demineralization was observed around

self-ligating brackets, regardless of the distance from the bracket margin (P<0.05).

The differences among groups are showed in the Fig. 2.

DISCUSSION

The present study, was the first to evaluate several combinations of types of

brackets and archwire ligation methods and their effect on enamel demineralization

under a controlled microcosm biofilm conditions. The model used has been shown to

replicate the variability and heterogeneity of plaques in vivo, simulating the

complexity of the oral environment, and thus overcoming the limitations of in vivo

experiments. This model has been shown to provide reproducible plaque biofilms

that are established from each salivary donor and are able to replicate the population

51

dynamics of natural plaque development20, and promote reproducible caries lesions

allowing comparison of different experimental conditions14, 19.

The overall results showed that the type of bracket/ligature can affect the

amount of demineralization adjacent to these devices, which is important in a clinical

perspective, since clinicians should be aware of these differences to better prevent

caries in patients undergoing orthodontic treatment. Previous studies have shown

that even after removal of fixed orthodontic devices, the developed caries lesions are

not completely solved by the removal of retentive sites and restoration of regular oral

hygiene2, 3. Therefore the study hypothesis was partially accepted.

Lower biofilm accumulation was observed for the conventional bracket with

hook ligated with elastomeric rings. This observation was not expected, since this

bracket design is the one with higher potential for biofilm accumulation, which was

confirmed by the mineral loss data, where these brackets exhibited significantly

higher demineralization adjacent to than the other groups. These results could be

explained by a faster turnover which could be expected in this experimental group,

since the presence of a complex design with hook could favor faster biofilm

development, and consequently a higher rate of maturation, establishment of climax

communities, and the consequent detachment of the biofilm surface layer26. Other

studies showed controversial results about biofilm composition and biomass formed

adjacent different types of brackets or ligatures. A in situ study have not found

differences in biofilms formed around conventional brackets ligated with stainless

steel wires or elastomeric rings21. Pellegrini27 showed fewer total bacteria and oral

streptococci in teeth bonded with self-ligating brackets compared to conventional

brackets. On opposite, Van Gastel28 found that self-ligating bracket sites in general

allowed more plaque formation than conventional bracket sites, and the authors

attributed their finds to the irregularities observed under scanning electron

microscopic (SEM). Only one study showed differences comparing elastomeric rings

or steel wires as methods of ligation, where an increase in the total number of

bacteria in the plaque was evidenced under elastomeric rings use29.

Few studies have assessed the mineral loss and caries lesions development

adjacent to orthodontic devices. Gameiro et al.21 (2009) evaluated the effect of two

different archwire ligation techniques, elastomeric rings and steel ligature wires, on

52

caries adjacent to brackets and showed that there was a trend to higher

demineralization of enamel around brackets tied with elastomers than steel ligatures.

Our results corroborate that finds, and also showed that reduced demineralization

was found adjacent to self-ligating brackets. Also, our results showed that more

complex and retentive brackets and ligatures can promote increased

demineralization, and therefore more attention should be given to the patients using

these devices during orthodontic treatment.

CONCLUSION

The results of this study show that the type of bracket and ligature system used to

attach the bracket to the orthodontic archwire segment affects the mineral loss during

treatment, and brackets with complex design provide higher risk for caries lesions

development.

53

REFERENCES

1. Ousehal L, Lazrak L, Es-Said R, Hamdoune H, Elquars F, Khadija A.

Evaluation of dental plaque control in patients wearing fixed orthodontic appliances: A clinical study. Int Orthod. 2011; 9:140-155

2. van der Veen MH, Mattousch T, Boersma JG. Longitudinal development of caries lesions after orthodontic treatment evaluated by quantitative light-induced fluorescence. Am J Orthod Dentofacial Orthop. 2007;131:223-8

3. Mattousch TJ, van der Veen MH, Zentner A. Caries lesions after orthodontic treatment followed by quantitative light-induced fluorescence: A 2-year follow-up. Eur J Orthod. 2007;29:294-8

4. Hu W, Featherstone JD. Prevention of enamel demineralization: An in-vitro study using light-cured filled sealant. Am J Orthod Dentofacial Orthop. 2005;128:592-600; quiz 670

5. Behnan SM, Arruda AO, Gonzalez-Cabezas C, Sohn W, Peters MC. In-vitro evaluation of various treatments to prevent demineralization next to orthodontic brackets. Am J Orthod Dentofacial Orthop. 2010;138:712 e711-717; discussion 712-3

6. Beerens MW, van der Veen MH, van Beek H, ten Cate JM. Effects of casein phosphopeptide amorphous calcium fluoride phosphate paste on white spot lesions and dental plaque after orthodontic treatment: A 3-month follow-up. Eur J Oral Sci. 2010;118:610-7

7. Suri L, Huang G, English JD, Jr., Owen S, Nah HD, Riolo ML, Shroff B, Southard TE, Turpin DL. Ask us. Topical fluoride treatment. Am J Orthod Dentofacial Orthop. 2009;135:561-3

8. Derks A, Frencken J, Bronkhorst E, Kuijpers-Jagtman AM, Katsaros C. Effect of chlorhexidine varnish application on mutans streptococci counts in orthodontic patients. Am J Orthod Dentofacial Orthop. 2008;133:435-9

9. Mattick CR, Mitchell L, Chadwick SM, Wright J. Fluoride-releasing elastomeric modules reduce decalcification: A randomized controlled trial. J Orthod. 2001;28:217-9

10. Benson PE, Douglas CW, Martin MV. Fluoridated elastomers: Effect on the microbiology of plaque. Am J Orthod Dentofacial Orthop. 2004;126:325-30

11. Doherty UB, Benson PE, Higham SM. Fluoride-releasing elastomeric ligatures assessed with the in situ caries model. Eur J Orthod. 2002;24:371-8

12. Ogaard B, Rolla G, Arends J. Orthodontic appliances and enamel demineralization. Part 1. Lesion development. Am J Orthod Dentofacial Orthop. 1988;94:68-73

13. Tang G, Yip HK, Cutress TW, Samaranayake LP. Artificial mouth model systems and their contribution to caries research: A review. J Dent. 2003;31:161-71

14. Azevedo MS, van de Sande FH, Romano AR, Cenci MS. Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Res. 2011;45:510-7

15. Sissons CH. Artificial dental plaque biofilm model systems. Adv Dent Res. 1997;11:110-26

16. Filoche SK, Soma KJ, Sissons CH. Caries-related plaque microcosm biofilms developed in microplates. Oral Microbiol Immunol. 2007;22:73-9

54

17. Sissons CH, Anderson SA, Wong L, Coleman MJ, White DC. Microbiota of plaque microcosm biofilms: Effect of three times daily sucrose pulses in different simulated oral environments. Caries Res. 2007;41:413-22

18. Cenci MS, Pereira-Cenci T, Cury JA, Ten Cate JM. Relationship between gap size and dentine secondary caries formation assessed in a microcosm biofilm model. Caries Res. 2009;43:97-102

19. van de Sande FH, Azevedo MS, Lund RG, Huysmans MC, Cenci MS. An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies. Biofouling. 2011;27:1057-63

20. Filoche SK, Soma D, van Bekkum M, Sissons CH. Plaques from different individuals yield different microbiota responses to oral-antiseptic treatment. FEMS Immunol Med Microbiol. 2008;54:27-36

21. Gameiro GH, Nouer DF, Cenci MS, Cury JA. Enamel demineralization with two forms of archwire ligation investigated using an in situ caries model--a pilot study. Eur J Orthod. 2009;31:542-46

22. Turkkahraman H, Sayin MO, Bozkurt FY, Yetkin Z, Kaya S, Onal S. Archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients. Angle Orthod. 2005;75:231-6

23. Wong L, Sissons C. A comparison of human dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva. Arch Oral Biol. 2001;46:477-86

24. Koo H, Hayacibara MF, Schobel BD, Cury JA, Rosalen PL, Park YK, Vacca-Smith AM, Bowen WH. Inhibition of streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol. J Antimicrob Chemother. 2003;52:782-9

25. Sousa RP, Zanin IC, Lima JP, Vasconcelos SM, Melo MA, Beltrao HC, Rodrigues LK. In situ effects of restorative materials on dental biofilm and enamel demineralisation. J Dent. 2009;37:44-51

26. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche. J Bacteriol. 1994;176:2137-42

27. Pellegrini P, Sauerwein R, Finlayson T, McLeod J, Covell DA, Jr., Maier T, Machida CA. Plaque retention by self-ligating vs elastomeric orthodontic brackets: Quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence. Am J Orthod Dentofacial Orthop. 2009;135:426 e421-429; discussion 426-7

28. van Gastel J, Quirynen M, Teughels W, Coucke W, Carels C. Influence of bracket design on microbial and periodontal parameters in vivo. J Clin Periodontol. 2007;34:423-31

29. Forsberg CM, Brattstrom V, Malmberg E, Nord CE. Ligature wires and elastomeric rings: Two methods of ligation, and their association with microbial colonization of streptococcus mutans and lactobacilli. Europ J Orthod. 1991;13:416-20

55

56

Figure 2. Mean (± SE) of the integrated mineral loss (ΔS) adjacent to orthodontic

brackets used, at the distances of 50 µm and 100 µm from the edge of the bracket. Different letters indicate statistically significant differences between groups, within each distance from the edge of the bracket (p <0.05).

57

Artigo 3#

Effect of types of brackets and archwire ligation on enamel demineralization in situ

Marcos Antonio Paccea, Marina Sousa Azevedoa, Fábio Garcia Limab, Wagner Missio da Silvac, Sandra Beatriz Chaves Tarquiniod, Douver Michelond, Maximiliano

Sérgio Cencid

a DDS, Msc, PhD student, Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil b DDS, MSc, PhD, Post-Doctoral Fellow, Graduate Program in, Federal University of Pelotas, Pelotas, RS, Brazil c Undergraduate student, School of Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil d DDS, MSc, PhD, Associate Professor, Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil

Reprint requests to: M.S. Cenci – Rua Gonçalves Chaves 457, 5th floor, 96015-560 Pelotas, RS (Brazil) Tel./Fax +55 53 3225 6741 – 134 - E-Mail m.s.cenci@pq.cnpq.br.

#Formatado segundo normas do periódico American Journal of Orthodontics and Dentofacial Orthopedics

58

ABSTRACT Introduction: Biofilm accumulation and enamel demineralization are facilitated by

orthodontic devices, and this in situ study was performed to investigate the

combination effect of types of brackets and archwire ligation on enamel

demineralization, on the accumulation and on microbiological composition of dental

plaque. Methods: The study design was a modified in situ model, randomized,

double-blind and split-mouth. Volunteers (n=17) wore palatal removable appliances

containing enamel discs with bonded orthodontic brackets during 14 days. To provide

cariogenic challenge, 20% sucrose solution was dripped 8x/day onto each disc. The

four conditions under study were: conventional brackets ligated with elastomeric

rings (CE); conventional brackets ligated with stainless steel wires (CS); brackets

with hooks ligated with elastomeric rings (HE); and self-ligating brackets (SL). The

biofilm formed around the brackets was collected for microbiological analyses and

the mineral loss was determined by cross-sectional microhardness measurement.

Results: There were no statistically significant differences regarding biofilm weight,

or percentages of mutans streptococci and lactobacilli in relation to total

microrganisms (P>0.05, Friedman’s test). The demineralization according to the

experimental groups was as follows: CE≥HE≥CS>SL (P<0.05, ANOVA and Tukey’s

test). Conclusions: Bracket design and/or type of archwire ligation affects

demineralization adjacent to the devices, and clinicians should select less biofilm-

retentive orthodontic devices such as the self-ligating brackets.

59

INTRODUCTION

Patients with fixed orthodontic appliances have an increased risk for development of

enamel caries/white spot lesions, since fixed orthodontic appliances increase dental

plaque retention and make tooth-brushing more difficult1. Moreover, even after the

removal of fixed appliances and restoration of oral hygiene, most of the white spot

lesions developed during treatment does not regress or improve, even leading to

restorative needs in some cases2, 3.

Several treatment alternatives have been proposed to control caries in patients under

fixed orthodontic treatment. In this context, resin sealers have been applied on the

facial surfaces of bracketed teeth to prevent enamel demineralization4. Also,

remineralizating substances such as amorphous calcium phosphate-containing

products were tested for improving remineralization and inhibition of caries lesion

development around brackets5, 6. Others studies testing the benefits of topical

fluoride treatment7 and chlorhexidine varnish application in order to prevent the

development of white spot lesion during orthodontic treatment have been

accomplished8. Also, the effect of fluoride incorporation in the elastomeric ligature

rings was investigated9-11. Despite that, white spot lesions continues to be one of the

most common side effects of use of fixed appliances in orthodontic treatment12.

The effect of ligature on enamel demineralization has been evaluated previously13, as

well as the changes on periodontal status and microbial flora related to archwire

ligation techniques14. However, the combined effect of fixed orthodontic appliances

and the method of ligation in the plaque accumulation, on microbial microflora and on

enamel demineralization have not been sufficiently investigated. Since orthodontic

devices per se create extra retention sites, the effect of different devices’ designs in

dental plaque accumulation and in caries development should be investigated.

Thus, this in situ study was performed to investigate the combination effect of types

of brackets and archwire ligation on enamel demineralization, on the accumulation

and on microbiological composition of dental plaque. The hypothesis tested is that

brackets and ligation methods with less complex designs promote lower biofilm

accumulation and lower demineralization on the adjacent enamel.

60

METHODS

Experimental Design

A randomized, double-blind, split-mouth in situ design for caries development around

orthodontic brackets was conducted in one phase of 14 days. A custom-made acrylic

resin intraoral palatal device containing two cavities on each side (17 × 7 × 4 mm3)

was made for each volunteer. In each cavity three bovine enamel discs measuring 6

mm diameter and 2 mm thickness were placed and fixed with stick wax (Fig 1). The

discs were mounted 1 mm below of the device surface to allow biofilm accumulation.

The factors under evaluation were bracket type and ligatures/attachment at 4 levels:

conventional bracket + elastomeric rings; conventional bracket + stainless steel

ligatures; conventional bracket with hooks + elastomeric rings and self-ligating

bracket. Brackets, 3 × 4 mm (Aditek, Cravinhos, SP, Brazil) were bonded to the

centre of the enamel discs with Transbond XT Light Cure Adhesive System (3M

ESPE, St Paul, MN, USA), following the manufacturer’s instructions. A 0.016 inch

stainless steel archwire was inserted in the three bracket slots in all sets of

brackets/enamel discs. The position of placement for each set was randomly decided

for each volunteer, using a computer program for randomization, following the split-

mouth design. During the experimental phase volunteers brushed their teeth and the

appliance 3 times/day with fluoride dentifrice (1100 mg F / g – NaF), except the

region containing the dental discs. At the end of the experimental phase, biofilms

were collected and assessed for microbiological composition. The enamel discs were

assessed regarding mineral loss adjacent to the brackets by cross-sectional

microhardness.

Subjects and Ethical aspects

The research protocol of this study was approved by the Local Ethics Committee

(Protocol number 168/2010) and written informed consent was obtained from all

subjects. Seventeen healthy adults, aged 20-45 years (average 28.2 years), with

normal salivary flow rates, able to comply with the experimental protocol, participated

in this study. The exclusion criteria for the subjects were: active caries lesions,

periodontal disease, smokers, pregnant and lactating, use of fixed orthodontic

61

devices, use of any antibiotics and/or antimicrobial within the 1 month prior to study

initiation.

Sample size calculation

The sample size was carried out with the software Sigmastat 3.5 (Systat Software

Inc.), assuming that data would be analyzed by ANOVA under the following

parameters: power test of 80%, and type I error of 5%. Data from a previously

published paper 15 were used as reference. From these parameters, a calculated

sample size of 14 volunteers was obtained and considering the possibility of drop-

outs, 17 volunteers were invited to participate in the study. These volunteers were

considered as experimental units in the study, and as statistical blocks for data

analysis.

Intra-oral phase

During the experimental period, the volunteers received standardized fluoridated

dentifrice (1100 µg F/g, silica-based) (Sorriso Fresh Plus, Colgate, Brazil) and

received a medium-sized, soft bristled toothbrush (Colgate 360, Colgate, Brazil).

They were instructed to brush their teeth and the palatal device, except brackets and

enamel discs, for one minute three times per day and to refrain from any other oral

hygiene procedures, such as the use of anti-microbial mouth-rinses.

In order to provide a cariogenic challenge, the volunteers were instructed to remove

the appliances from the mouth and drip one drop of 20% sucrose solution onto each

enamel disc eight times per day at predetermined times13, 16. After 5 minutes, the

appliances were replaced in the mouth. The subjects were instructed to wear the

appliances at all times, removing them only during mealtimes13.

Dental biofilm analysis

On the 14th day, after the experimental phase the dental biofilm from the

discs+brackets was collected. For the microbiological analyses, the three discs with

brackets of each group/set were inserted in sterile microcentrifuge tubes containing

10 mL of 0.9% of NaCl solution and sonicated. The suspensions were diluted in 0.9%

NaCl in series up to 1:107 and immediately inoculated in duplicate in the following

62

culture media: blood agar, for total cultivable microbiota; mitis salivarius agar plus 0·2

units of bacitracin/ ml (MSB), for mutans streptococci group and Rogosa SL agar

(Difco 248020; Becton Dickinson, Sparks, MD, USA), for lactobacillus. All plates were

incubated anaerobically at 37°C for 4 days and the numbers of colony-forming units

(CFUs) were then determined by one trained operator based on colony morphology

and cell morphology using a microscope and stereoscopic microscope. Data were

expressed as percentages of mutans streptococci in relation to total cultivable

microorganisms; and percentages of lactobacilli in relation to total cultivable

microorganisms.

For the dry weight determination, an aliquot of the homogenized suspension was

used, three volumes of cold ethanol (–20°C) were added to 0.2 mL of the cell

suspension, and the resulting precipitate collected (10 000g for 10 min, 4°C). The

supernatant was discarded, and the cell pellet was washed twice with cold ethanol,

and then lyophilized and weighed17.

Enamel mineral loss assessment

All discs from each set were sectioned longitudinally through the centre for enamel

cross-sectional microhardness (CSMH) determination. The enamel demineralization

was evaluated using a microhardness tester (Future-Tech FM, Tokyo, Japan) with a

Knoop diamond indenter under a 25 g load for 5 s. Two lines of indentations were

made: one corresponding to the 20 µm next to the bracket margin and other 100 µm

from the bracket margin 13. Indentations were made at 10, 20, 30, 40, 50, 70 and 100

μm from the outer enamel surface (Fig 1). The CSMH determination of all enamel

discs was carried out by one blind and trained examiner.

Statistical analyses

Volunteers were considered as statistical blocks. Biofilm weight and microbiological

counts were analyzed using the Friedman paired test. Data of enamel

demineralization were analyzed by split-plot ANOVA followed by Tukey’s test. In

order to attend the assumptions of normality of distribution and equality of variances,

data of enamel demineralization were rank-transformed. The SAS software (version

63

9.1; SAS Institute Inc., Cary, North Carolina, USA) was used and the significance

level set at P < 0.05.

RESULTS

The type of bracket/ligature method did not affected the amount of biofilm

accumulated around the devices (P=0.083; Table 1) or the percentages of mutans

streptococci (P = 0.754) and percentages of lactobacilli (P = 0.241) in the collected

biofilm (Figure 2). The type of bracket/ligature method affected demineralization

(P<0.0001). Lower demineralization was observed around self-ligating brackets,

regardless of the distance from the bracket margin (P<0.05). The demineralization

according to the experimental groups was as follows: conventional brackets ligated

with elastomeric rings ≥ conventional brackets with hooks ligated with elastomeric

rings ≥ conventional brackets ligated with stainless steel wires > self-ligating brackets

(P<0.05; Figures 3 and 4). There was no statistically significant differences for

demineralization according to the distances (20 or 100 µm) from the bracket margin

(P=0.09). Demineralization was higher next to the outer enamel surface, and

decreased according to the depth (P<0.05, Figures 3 and 4).

DISCUSSION

The present study, based on the literature assessed, was the first to evaluate

several combinations of types of brackets and archwire ligation methods and their

effect on enamel demineralization. The overall results showed that the type of

bracket/ligature can affect the amount of demineralization adjacent to these devices,

which is important in a clinical perspective, since clinicians should be aware of these

differences to better prevent caries in patients undergoing orthodontic treatment.

Previous studies have shown that even after removal of fixed orthodontic devices,

the developed caries lesions are not completely solved by the removal of retentive

sites and restoration of regular oral hygiene2, 3.

The hypothesis tested was partially rejected since the different brackets/

ligature methods did not affected the biofilm amount or composition associated with

the tested treatments. Previous studies with similar experimental design also have

not found differences in biofilms formed around conventional brackets ligated with

stainless steel wires or elastomeric rings13, 14. This lack of difference could be

explained considering the split-mouth design of both studies, and the fact that the in

situ model is somewhat biofilm-retentive per se. Moreover, all sites with brackets

64

were exposed to a high-cariogenic challenge by dripping sucrose 8 times a day, and

the material composition of all tested brackets was very similar, and the only factor in

study was bracket design and ligature method. During the 14-day clinical phase, it is

expected some biofilm detachment under in situ conditions, and this fact also could

explain why we have found higher mineral loss adjacent to the theoretically more

biofilm retentive brackets/ ligatures, while no differences in biofim dry weight or

composition were detected.

Other studies found contradictory results regarding biofilm accumulation and

composition considering types of brackets and ligature methods. Pellegrini18 showed

fewer total bacteria and oral streptococci in teeth bonded with sel-ligating brackets

compared to conventional brackets. On opposite, Van Gastel19 found that self-ligating

bracket sites in general allowed more plaque formation than conventional bracket

sites. Only one study showed differences comparing elastomeric rings or steel wires

as methods of ligation, where an increase in the total number of bacteria in the

plaque was evidenced under elastomeric rings use20.

The present in situ model is suitable for studying dental caries13, 21 and it was

used to assess caries around orthodontic appliances9, 13, 22. Also, bovine enamel is

considered an acceptable alternative to human enamel21. Some limitations of the

present study are the relatively short-term enrolled in the clinical phase, and the

complete lack of brushing over the region with brackets and ligatures, which are

different from the expected clinical conditions. However, these variables could be

inserted in the in situ model used in future studies.

Few studies have assessed the mineral loss and caries lesions development

adjacent to orthodontic devices. Gameiro et al.13 evaluated the effect of two different

archwire ligation techniques, elastomeric rings and steel ligature wires, on caries

adjacent to brackets and showed that there was a trend to higher demineralization of

enamel around brackets tied with elastomers than steel ligatures. Our results

corroborate that finds, and also showed that the use of self-ligating brackets reduced

the associated enamel demineralization, probably due to the lower biofilm retention

around this bracket type in the whole experimental set. Also, our results showed that

more complex and retentive brackets and ligatures can promote increased

demineralization, and therefore more attention should be given to the patients using

these devices during orthodontic treatment.

65

CONCLUSION

The design of the bracket and/or type of archwire ligation affects

demineralization adjacent to the devices, as self-ligating brackets and brackets

ligated with stainless steel wires exhibited lower mineral loss adjacent to then.

66

REFERENCES

1. Ousehal L, Lazrak L, Es-Said R, Hamdoune H, Elquars F, Khadija A.

Evaluation of dental plaque control in patients wearing fixed orthodontic appliances: A clinical study. Int Orthod.2011;9:140-155

2. Mattousch TJ, van der Veen MH, Zentner A. Caries lesions after orthodontic treatment followed by quantitative light-induced fluorescence: A 2-year follow-up. Eur J Orthod. 2007;29:294-298

3. van der Veen MH, Mattousch T, Boersma JG. Longitudinal development of caries lesions after orthodontic treatment evaluated by quantitative light-induced fluorescence. Am J Orthod Dentofacial Orthop. 2007;131:223-228

4. Hu W, Featherstone JD. Prevention of enamel demineralization: An in-vitro study using light-cured filled sealant. Am J Orthod Dentofacial Orthop. 2005;128:592-600; quiz 670

5. Behnan SM, Arruda AO, Gonzalez-Cabezas C, Sohn W, Peters MC. In-vitro evaluation of various treatments to prevent demineralization next to orthodontic brackets. Am J Orthod Dentofacial Orthop.2010;138:712 e711-717; discussion 712-713

6. Beerens MW, van der Veen MH, van Beek H, ten Cate JM. Effects of casein phosphopeptide amorphous calcium fluoride phosphate paste on white spot lesions and dental plaque after orthodontic treatment: A 3-month follow-up. Eur J Oral Sci. 2010;118:610-617

7. Suri L, Huang G, English JD, Jr., Owen S, Nah HD, Riolo ML, Shroff B, Southard TE, Turpin DL. Ask us. Topical fluoride treatment. Am J Orthod Dentofacial Orthop. 2009;135:561-563

8. Derks A, Frencken J, Bronkhorst E, Kuijpers-Jagtman AM, Katsaros C. Effect of chlorhexidine varnish application on mutans streptococci counts in orthodontic patients. Am J Orthod Dentofacial Orthop. 2008;133:435-439

9. Doherty UB, Benson PE, Higham SM. Fluoride-releasing elastomeric ligatures assessed with the in situ caries model. Eur J Orthod. 2002;24:371-378

10. Benson PE, Douglas CW, Martin MV. Fluoridated elastomers: Effect on the microbiology of plaque. Am J Orthod Dentofacial Orthop. 2004;126:325-330

11. Mattick CR, Mitchell L, Chadwick SM, Wright J. Fluoride-releasing elastomeric modules reduce decalcification: A randomized controlled trial. J Orthod. 2001;28:217-219

12. Ogaard B, Rolla G, Arends J. Orthodontic appliances and enamel demineralization. Part 1. Lesion development. Am J Orthod Dentofacial Orthop. 1988;94:68-73

13. Turkkahraman H, Sayin MO, Bozkurt FY, Yetkin Z, Kaya S, Onal S. Archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients. Angle Orthod. 2005;75:231-236

15. Gameiro GH, Nouer DF, Cenci MS, Cury JA. Enamel demineralization with two forms of archwire ligation investigated using an in situ caries model--a pilot study. Eur J Orthod. 2009;31:542-546

16. Cury JA, Rebelo MA, Del Bel Cury AA, Derbyshire MT, Tabchoury CP. Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res. 2000;34:491-497

17. Koo H, Hayacibara MF, Schobel BD, Cury JA, Rosalen PL, Park YK, Vacca-Smith AM, Bowen WH. Inhibition of streptococcus mutans biofilm

67

accumulation and polysaccharide production by apigenin and tt-farnesol. J Antimicrob Chemother. 2003;52:782-789

18. Pellegrini P, Sauerwein R, Finlayson T, McLeod J, Covell DA, Jr., Maier T, Machida CA. Plaque retention by self-ligating vs elastomeric orthodontic brackets: Quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence. Am J Orthod Dentofacial Orthop. 2009;135:426 e421-429; discussion 426-427

19. van Gastel J, Quirynen M, Teughels W, Coucke W, Carels C. Influence of bracket design on microbial and periodontal parameters in vivo. J Clin Periodontol. 2007;34:423-431

20. Forsberg CM, Brattstrom V, Malmberg E, Nord CE. Ligature wires and elastomeric rings: Two methods of ligation, and their association with microbial colonization of streptococcus mutans and lactobacilli. Eur J Orthod. 1991;13:416-420

21. Zero DT. In situ caries models. Adv Dent Res. 1995;9:214-230; discussion 231-214

22. Benson PE, Pender N, Higham SM. An in situ caries model to study demineralisation during fixed orthodontics. Clin Orthod Res. 1999;2:143-153

68

Table 1. Percentages of mutans estreptococci and lactobacilli in relation to the total cultivable microorganisms for the biofilms formed adjacent to the brackets.

Type of Bracket / Ligature Method

Mutans estreptococci (%)

Lactobacilli (%)

Self-Ligated 0.08 (0.02 – 0.42) 1.42 (0.32 – 8.33) Steel wire 0.04 (0.005 – 0.07) 2.00 (0.31 – 4.70) Hook 0.02 (0.008 – 0.04) 0.58 (0.01 – 5.61) Elastomeric Rings 0.01 (0.004 – 0.06) 0.41 (0.03 – 2.55)

Values are Median (25% - 75%). There were no statistically significant differences among types of brackets/ligatures for percentages of mutans streptococci (P = 0.754) or percentages of lactobacilli (P = 0.241).

69

Figure 1. Experimental design. Enamel discs obtantion (A); Position of the

brackets and archwires (B); The four experimental conditions randomly assigned to the palatal appliances (C); Sectional view of the appliance and the design for biofilm accumaltion (D); Sucrose exposure regimen during the clinical phase (E); Removal of the samples for biofilm collection (F); Longitudinal section of each enamel disc/bracket for mineral loss assessment (G); Detail of the region evaluated by microhardness (H); Detaiment of the indentations for mineral loss assessment (I).

70

2D Graph 2

Groups

1 2 3 4

Bio

film

Dry

We

igth

(m

g)

0

10

20

30

40

Plot 1

Figure 2. Dry weight (mg) for the biofilms formed adjacent to the brackets / ligature methods.

Self-ligated Stell Wires Hooks Elastomeric

Rings

71

130

150

170

190

210

230

250

270

290

10 20 30 40 50 70 100

Depth (µm)

Mic

roh

ard

ne

ss (

KH

N)

Mineral loss (20 µm from the bracket)

Self-ligated

Elastomeric rings

Hooks

Steel wires

Figure 3. Microhardness values (Average ± standard error) according to type of brackets / method of ligation and depth, at 20 µm from the bracket base.

72

130

150

170

190

210

230

250

270

290

10 20 30 40 50 70 100

Depth (µm)

Mic

roh

ard

ne

ss (

KH

N)

Mineral loss (100 µm from the bracket)

Self-ligated

Elastomeric rings

Hooks

Steel wires

Figure 4. Microhardness values (Average ± standard error) according to type of brackets / method of ligation and depth, at 100 µm from the bracket base.

73

Conclusões

Os estudos de revisão sistemática mostraram dados conflitantes quanto ao

tipo de bráquete e ligadura na prevenção de cárie e acúmulo de biofilme em

pacientes ortodônticos. No entanto, considerando-se os resultados dos estudos in

vitro e in situ conduzidos, o tipo de bráquete e ligadura afeta a perda mineral durante

o tratamento. Ortodontistas devem ficar atentos quando for necessário o uso de

bráquetes com desenho mais complexo para orientar melhor seus pacientes quanto

ao controle do acúmulo de biofilme a fim de previnir o surgimento de lesões de cárie.

74

Referências Complementares

ÅRTUN, J.; BROBAKKEN, B. Prevalence of caries and white spots after orthodontic treatment with multibonded appliances. European Journal of Orthodontics, v.8, n.4, p.229-234, 1986. BISHARA, S.E.; VONWALD, L.; ZAMTUA, J.; DAMON, P. L. Effects of various methods of chlorhexidine application on shear bond strength. American Journal of Orthodontics and Dentofacial Orthopedics, v.114, n.2, p.150-153, 1998. BOERSMA, J. G.; VAN DER VEEN, M. H.; LAGERWEIJ, M. D.; BOKHOUT, B.; PRAHL- ANDERSEN, B. Caries prevalence measured with QLF after treatment with fixed orthodontic appliances: influencing factors . Caries Research, v.39, n.1, p.41 – 47, 2005. BOWEN, M. B.; PRUCHNO, C.; BELLONE, C. J. Characterization of a concanavalin A supernatant-derived idiotype-specific T helper cell factor. Journal of Immunology, v.136, n.4, p.1295-1302, feb.1986. CORBETT, J. A.; BROWN, L. R.; KEENE, H. J.; HORTON, I. M. Comparison of Streptococcus mutans concentrations in non-banded and banded orthodontic patients. Journal of Dental Research, v.60, n.12, p.1936–1942, 1981. FORSBERG, C. M.; BRATTSTRÖM, V.; MALMBERG, E.; NORD, C. E. Ligature wires and elastomeric rings: two methods of ligation, and their association with microbial colonization of Streptococcus mutans and lactobacilli. European Journal of Orthodontics, v.13, n.5, p.416–420, 1991. GAMEIRO, G. H.; NOUER, D. F.; CENCI, M. S.; CURY, J. A. Enamel demineralization with two forms of archwire ligation investigated using an in situ caries model—a pilot study. European Journal of Orthodontics, v.31, n.5, p.542-546, oct. 2009. GOLD, O. G.; JORDAN, H. V.; VAN HOUTE, J. A selective medium for Streptococcus mutans. Archives of Oral Biology, v.18, n.11, p.1357-64, nov.1973. HENAO, S. P.; KUSY, R. P. Frictional evaluations of dental typodont models using four self-ligating designs and a conventional design. The Angle Orthodontist (Appleton), v. 75, n.1, p.75-85, 2004. LEE, S. J.; KHO, H. S.; LEE, S. W.; YANG, W. S. Experimental salivarypellicles on the surface of orthodontic materials. American Journal of Orthodontics and Dentofacial Orthopedics, v.119, n.1, p.59-66, 2001. MIZRAHI, E. Enamel demineralization following orthodontic treatment. American Journal of Orthodontics, v.82, n.1, p.62-67, 1982. PELLEGRINI, P.; SAUERWEIN, R.; FINLAYSON, T.; MCLEOD, J.;COVELL, DA JR.; MAIER, T.; MACHIDA, C. A. Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence. American Journal of Orthodontics and Dentofacial Orthopedics, v.135, n.4, p.426-427, apr. 2009. REDLICH, M.; MAYER, Y.; HARRY, D.; LEWINSTEIN, I. In vitro study of frictional forces during sliding mechanics of reduced friction brackets. American Journal of Orthodontics and Dentofacial Orthopedics (St. Louis), v. 124, n.1, p. 69-73, 2003. ROSENBLOOM, R. G.; TINANOFF, N. Salivary Streptococcus mutans levels in patients before, during, and after orthodontic treatment. American Journal of Orthodontics and Dentofacial Orthopedics, v.100, n.1, p.35–37, 1991. SUKONTAPATIPARK, W.; EL-AGROUDI, M. A.; SELLISETH, N. J.; THUNOLD, K.; SELVIG, K. A. Bacterial colonization associated with fixed orthodontic appliances. A

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scanning electron microscopy study. European Journal of Orthodontics, v.23, n.5, p.475–484, oct. 2001. TABAK, L. A.; BOWEN, W. H. Roles of saliva (pellicle), diet, and nutritionon plaque formation. Journal of Dental Research, v.68, p.1560-1566, 1989. TENUTA, L. M.; RICOMINI FILHO, A. P.; DEL BEL CURY, A. A.; CURY, J. A. Effect of sucrose on the selection of mutans streptococci and lactobacilli in dental biofilm formed in situ. Caries Research, v.40, n.6, p.546-549, 2006. TURKKAHRAMAN, H.; SAYIN, M. O.; BOZKURT, F. Y.; YETKIN, Z.; KAYA, S.; ONAL, S. Archwire ligation techniques, microbial colonization, and periodontal status in orthodontically treated patients. The Angle Orthodontist, v.75, n.2, p.231–236, 2005.

VAN DER VEEN, M. H.; MATTOUSCH, T.; BOERSMA, J. G. Longitudinal development of caries lesions after orthodontic treatment evaluated by quantitative light-induced fluorescence. American Journal of Orthodontics and Dentofacial Orthopedics, v.131, n.2, p.223-228, feb. 2007.

ZACHRISSON, B. U.; ZACHRISSON, S. Caries incidence and oral hygiene during orthodontic treatment. Scandinavian Journal of Dental Research, v.79, n.6, p.394–401, 1971.

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Apêndices

77

APÊNDICE A - Termo de Consentimento Livre e Esclarecido

TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO (TCLE)

Eu __________________________ RG nº ________, concordo em participar da pesquisa

“influencia de bráquetes e tipos de ligadura na aderência de microorganismos e na

desmineralização do esmalte adjacente a dispositivos ortodônticos”

Os materiais que serão utilizados encontram-se disponíveis no mercado e foram previamente

estudados através de testes de biocompatibilidade, não demonstrando nenhum risco à

integridade do ser humano.

Os voluntários utilizarão uma placa palatina contendo discos de esmalte bovino com

dispositivos ortodônticos. Somente sobre esses discos os voluntários gotejarão uma solução

de sacarose a 20% (8 vezes ao dia). Ao final de 14 dias, a placa bacteriana será coletada e os

discos de esmalte serão removidos dos dispositivos para as análises.

A placa palatina pode causar um leve desconforto semelhante a um aparelho ortodôntico

removível. Durante todo o período da pesquisa, acompanhamentos semanais serão realizados,

para verificar a adaptação do dispositivo. Estes serão realizados na clínica da pós-graduação

da Faculdade de odontologia da UFPel, por examinadores, acompanhados por um pesquisador

responsável. Os procedimentos realizados durante a pesquisa não oferecem riscos à saúde.

Qualquer dúvida ou problema entrar em contato com o Marcos Pacce (81240506 – 30254044)

ou com a secretaria da PPGO (32226690).

Assim sendo, dou pleno consentimento à Faculdade de Odontologia de Pelotas para, por

intermédio de seus professores, alunos da pós-graduação e graduação devidamente

autorizados, fazer diagnóstico, planejamento, fotografias e realizar as coletas, visto que não há

danos previsíveis decorrentes desta pesquisa e os pesquisadores asseguram a privacidade

quanto aos dados confidenciais.

Concordo também, que a documentação referente aos exames efetuados e quaisquer outras

informações concernentes ao planejamento de diagnóstico e?ou tratamento, constituem

propriedade exclusiva dessa faculdade, à qual dou plenos direitos de uso para fins de ensino e

divulgação, respeitando os respectivos códigos de ética.

Sei que a minha identidade não será revelada, que as informações sobre os exames somente

serão vistas pelos pesquisadores e que será mantida minha privacidade quando da publicação

dos dados em revistas odontológicas. Os pesquisadores esclareceram todas minhas dúvidas.

Há a possibilidade de retirada do consentimento e desistência da participação em qualquer

momento, mas enquanto estiver participando da pesquisa tenho o compromisso de fornecer

informações atualizadas e o dever de comparecer às consultas. É assegurada a gratuidade de

todas as etapas da presente pesquisa.

Pelotas, 15 de junho de 2010.

Nome e assinatura do participante

Documento nº_________

O presente documento, baseado nos artigos 10 a 16 das Normas de Pesquisa em Saúde, do

Conselho Nacional de Saúde, será assinado em duas vias, de igual teor, ficando uma via em

poder do responsável e outra com o pesquisador (Marcos Pacce).

78

APÊNDICE B – Instruções aos voluntários

AOS VOLUNTÁRIOS

Antes do início de cada fase, todo voluntário receberá um dentifrício fluoretado, uma escova

dental, um frasco conta-gotas contendo a solução de sacarose, um pacote de gaze estéril, um

estojo de dispositivo ortodôntico (acomodação do dispositivo) e um dispositivo intrabucal.

INSTRUÇÕES GERAIS

- utilizar apenas o dentifrício e escova doados pelos pesquisadores.

- não utilizar bochechos, fio dental com flúor, clorexidina.

- não consumir alimentos fontes de flúor. Ex.: chá preto, chá verde

- durante a higiene bucal o dispositivo deverá ser removido, escovado com o dentifrício

fornecido e retomado para a boca imediatamente após a escovação.

- você pode escovar todo INSTRUÇÕES o dispositivo exceto a região onde estão os discos de

esmalte e os dispositivos ortodônticos.

- colocar espuma formada durante a escovação sobre a área dos dispositivos, mas não escovar.

- cuidado ao escovar: não deixe que jatos de água da torneira atinjam diretamente a região

com discos de esmalte e dispositivos ortodônticos. Isso pode causar perda da placa dental ali

acumulada.

- dormir com o dispositivo.

- só remover o dispositivo para fazer as refeições (inclusive mascar chiclete, bala, ou tomar

medicamentos) e higienização.

- quando removê-lo, colocar o mesmo sobre o porta-dispositivo com gaze úmida.

- utilizar uma gota da solução determinada sobre cada bloco de esmalte 08 vezes ao dia nos

seguintes horários: 08:00 – 10:00- 12:00 – 14:00 – 16:00 – 18:00 –20:00 – 22:00.

- antes de gotejar, secar o excesso de saliva com gaze a fornecida.

- após gotejar, aguardar 5 minutos, retirar o excesso de sacarose com gaze, e recolocar o

dispositivo na boca.

INSTRUÇÕES PARA O TÉRMINO DA FASE (14 DIAS)

Ao acordar no dia da entrega do dispositivo:

• não retire o dispositivo da boca,

• não escove os dentes,

•não tome água,

• não se alimente de forma alguma.

Continue usando o dispositivo até o momento de em que chegar ao laboratório e for solicitado

sua remoção.

79

Anexos

80

ANEXO A - Parecer do Comitê de Ética em Pesquisa