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UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS DA SAÚDE
PÓS-GRADUAÇÃO EM ODONTOLOGIA
DOUTORADO EM ODONTOLOGIA
ÁREA DE CONCENTRAÇÃO EM CLÍNICA INTEGRADA
ANA MARLY ARAÚJO MAIA
APLICAÇÃO DE TÉCNICAS ÓPTICAS PARA ANÁLISE
QUALITATIVA E QUANTITATIVA DE PERDAS
MINERAIS DO TECIDO DENTÁRIO
RECIFE - PE
2013
ANA MARLY ARAÚJO MAIA
APLICAÇÃO DE TÉCNICAS ÓPTICAS PARA ANÁLISE
QUALITATIVA E QUANTITATIVA DE PERDAS
MINERAIS DO TECIDO DENTÁRIO
Tese apresentada ao Colegiado da Pós Graduação em
Clínica Integrada do Centro de Ciências da Saúde da
Universidade Federal de Pernambuco, como requisito
para obtenção do grau de doutora em Odontologia.
Orientador: Prof. Dr. Anderson S. L. Gomes
Co-orientador: Prof. Dr. Cláudio Heliomar Vicente
RECIFE - PE
2013
UNIVERSIDADE FEDERAL DE PERNAMBUCO
REITOR
Prof. Dr. Anísio Brasileiro de Freitas Dourado
VICE-REITOR
Prof. Dr. Sílvio Romero de Barros Marques
PRÓ-REITOR DE PESQUISA E PÓS-GRADUAÇÃO
Prof. Dr. Francisco de Sousa Ramos
CENTRO DE CIÊNCIAS DA SAÚDE
DIRETOR
Prof. Dr. Nicodemos Teles de Pontes Filho
COORDENADOR DA PÓS-GRADUAÇÃO EM ODONTOLOGIA
Profa. Dra. Jurema Freire Lisboa de Castro
PROGRAMA DE PÓS-GRADUAÇÃO EM ODONTOLOGIA
MESTRADO EM CLÍNICA INTEGRADA
COLEGIADO
MEMBROS PERMANENTES
Profa. Dra. Alessandra Albuquerque Tavares Carvalho
Prof. Dr. Anderson Stevens Leônidas Gomes
Prof. Dr. Arnaldo de França Caldas Júnior
Prof. Dr. Carlos Menezes Aguiar
Prof. Dr. Danyel Elias da Cruz Perez
Prof. Dr. Edvaldo Rodrigues de Almeida
Profa. Dra. Flávia Maria de Moraes Ramos Perez
Prof. Dr. Jair Carneiro Leão
Profa. Dra. Jurema Freire Lisboa de Castro
Profa. Dra. Liriane Baratela Evêncio
Prof. Dr.Luiz Alcino Monteiro Gueiros
Prof.Dra. Maria Luiza dos Anjos Pontual
Prof.Dr. Paulo Sávio Angeiras Goes
Profa. Dra. Renata Cimões Jovino Silveira
Dra. Simone Guimaraes Farias Gomes
Dr. Tibério César Uchoa Matheus
MEMBRO COLABORADOR
Profa. Dra. Lúcia Carneiro de Souza Beatrice Prof. Dr. Cláudio Heliomar Vicente da Silva
SECRETARIA
Oziclere Sena de Araújo
APLICAÇÃO DE TÉCNICAS ÓPTICAS PARA ANÁLISE
QUALITATIVA E QUANTITATIVA DE PERDAS
MINERAIS DO TECIDO DENTÁRIO
ANA MARLY ARAÚJO MAIA
Tese defendida e aprovada em: _17_/_01_/_2013__
MEMBROS DA BANCA EXAMINADORA:
Prof. Dr. John Michael Girkin __________________________________
Professor Titular Biophysics Durham University – UK
Prof. Dra. Denise Maria Zezell __________________________________
Pesquisadora e professora do Instituto de Energia Nuclear IPEN – CLA/ USP
Prof. Dr. Gustavo Pina Godoy __________________________________
Professor Adjunto Odontologia da Universidade Estadual da Paraíba - UEPB
Profª. Dra. Maria Luiza dos A. Pontual ____________________________________
Professora Adjunto Odontologia da Universidade Federal de Pernambuco- UFPE
Prof. Dr. Danyel E. da C. Perez ____________________________________
Professor Adjunto Odontologia da Universidade Federal de Pernambuco- UFPE
RECIFE - PE
2013
DEDICATÓRIA
... a Deus pelo dom da vida, por ter me proporcionado nascer em uma família abençoada
por Seus ensinamentos.
... aos meus pais José Maia e Maria Zilda pela dedicação e doação aos filhos, no sentido
mais puro e verdadeiro existe. É uma honra ter pai e mãe cheios de amor, saúde, familiares
incríveis, com princípios éticos e exemplares em vários aspectos. Nesse contexto de família,
estendo minha gratidão a minha irmã Maria Rosa e meu irmão José Neto, pela cumplicidade
eterna, depois de casados e morando distante.
AGRADECIMENTOS
A defesa de uma tese é similar a uma despedida. Para mim uma despedida saudosa de
uma companhia e motivação diária de quase quatro anos. A transformação do gerúndio
(doutoranda), em infinitivo (doutora), foi moldada ao longo dos anos, através da mais íntima
busca pelo conhecimento e questionamento contínuo.
Durante os anos de formação, a compreensão de como o conhecimento continua sendo
descoberto, questionado, provado e relatado, faz o doutorando sentir-se numa fronteira
buscando pontos ou vírgulas ausentes em bilhões de palavras. Uma tese escrita é como
uma peneira, que filtra alguns % dos testes e aprendizados para serem organizadamente
relatados com inicio e fim, infelizmente excluindo um meio que incluiria folhas e folhas de
cadernos de laboratório. Um meio cheio de personagens, de tentativas, de insucessos, de
trabalhos incompletos, mas de extrema aprendizagem. Um meio sem qualis, sem
pontuação, sem direito a ser relatado, mas de grande riqueza para o que se forma doutor.
Nesse meio tempo vivido, inúmero personagens se fizeram presentes e importantes nessa
caminhada, alguns participantes efetivos na construção dos artigos que a compõem, outros
colaboradores de experimentos laboratoriais, que foram também fundamentais.
Relembrando o primeiro ano de doutorado, agradeço a heterogênea primeira turma de
doutorado do Programa de Odontologia UFPE, da qual tive a honra de fazer parte, com
anseios e experiências diferentes, que tornaram nossas aulas mais interessantes;
Aos professores do colegiado de Odontologia, que tiveram cada um sua contribuição
através de disciplinas ou através de exemplos fora de sala de aula. Ressaltando os
coordenadores do Programa, Professor Jair Leão e Professora Jurema Lisboa, pelo
empenho em aperfeiçoar a Pós Graduação, e ao Professor Cláudio Heliomar, pelos
momentos de escuta enquanto coorientador; Aos funcionários da pós-graduação de
Odontologia da UFPE, especialmente Oziclere e Tânia, sempre disponíveis para ajudar; A
secretaria Ieda pela disponibilidade e dedicação de representar o Programa de Pós no dia
da minha defesa de tese;
Aos companheiros de formação do CEPLO, partilhando informações e atividades práticas
para aplicações do laser, em especial ao Prof. Jair Leão, Prof. Luiz Alcino, Luiz Mário,
Cláudia e Igor; A funcionária Rita pelos cuidados com o CEPLO, além da disponibilidade
para aguardar a utilização do laser em horários extras;
Apesar de aluna de um programa de odontologia, passei 85% do tempo em Departamentos
de Física, vivendo uma realidade divergente, que fez crescer e visualizar a construção do
conhecimento de uma forma ampla. Sou grata:
Ao meu orientador Professor Anderson, por mesmo acreditando e confiando em minhas
atividades, estava sempre motivando um desafio a ser vencido. Grata pelas inúmeras
oportunidades oferecidas, pelo incentivo e recursos sempre disponíveis;
Aos professores Cid Araújo e Renato Araújo, pelo exemplo de busca do conhecimento em
família, fontes de inspiração. Além das colaborações com equipamentos e instrumentais
óticos entre Laboratórios;
Aos funcionários da Secretaria e Setor Financeiro do Departamento de Física pelo exemplo
de dedicação ao bem público, em especial a Claudésio pelo esforço e disponibilidade; Aos
técnicos dos laboratórios de Física, Marcos, Sérgio, João, Virgínia e Tarsila pela
disponibilidade e confiança; Além dos funcionários terceirizados da limpeza e segurança,
pela disponibilidade em ajudar;
Ao meu amor, Bebeto Amorim, que disfarçado de melhor amigo conquistou meu coração no
Laboratório de Física. Acreditando mais em meu potencial do que eu mesma, uma
motivação intensa e verdadeira, cheia de amor e companheirismo. A sua paixão e
curiosidade pela eletrônica foi também fonte de inspiração; Grata a sua família pelo carinho.
Aos companheiros do Laboratório de Optoeletrônica e Fotônica, importantes em diferentes
fases dos últimos 4 anos;
A Patrícia Cassimiro, que numa relação doutoranda-mestranda, tornou-se uma grande
amiga pra vida toda, com quem eu espero dar continuidade na nossa “eficiente” parceria;
A Sérgio Campello pelos debates de conceitos físicos, parceria em trabalhos, além da
amizade construída;
A Cláudia Brainer, que mesmo da Odontologia, foi sempre uma companhia de laboratório,
com quem desenvolvi estudos também importantes;
A Gabriela Monteiro, pela colaboração nos meses que esteve presente nessa caminhada;
A Monica Schaffer pela agradável companhia, com quem vivi a primeira experiência informal
de co-orientação enquanto doutoranda;
A Marco Sacilotti e Kátia Calligaris pela atenção e disponibilidade em ajudar quando
solicitados;
A Eusébio, do DPQ, pela paciência em ceder espaço para os primeiros testes de diluições
de dentifrícios;
A Daene Tenório e Angelinne Ângelo pela amizade, e colaboração em diferentes pesquisas;
A Jamil Saade pelas inúmeras tentativas de praticar os conceitos da espectroscopia Raman;
Aos fomentos de bolsista de doutorado e Processo AMD cedidos pela Fundação de Amparo
à Ciência e Tecnologia do Estado de Pernambuco que me permitiram dedicação exclusiva e
a experiência de estágio na Inglaterra por seis meses, em 2010; I would like to especially
thank:
To Professor John Girkin for the lovely example of how to be a master. You are worthy of all
respect, with unique simplicity. A nature researcher, who makes optical physics something
magical;
To Dr. Chris Longbottom for all attention and discussions, for supplying me with articles that
are true relics and always have a sincere sympathy;
To Lena Karlsson for the friendship and all contributions and discussions about paper I.
To my “hinny” friend Laura Fleming, who welcomed me as a sister, and lent me his mother
and grandparents as my family in England. Six months of a friendship that will last a lifetime;
To Brazilian friends, Tarsila Burity and Rafael Vilar, for all tips to uncover the best way to
solve the silliest things;
To my roommates (125b) Jonny Taylor, James Osborn, Luke Tyas who respected my
shyness and have always been available to help me;
To the other housemates that made my experience more fun and enjoyable, with different
cultures, and methodological discussions of the simplest household chores, Rachel Sedman,
Nuria Polo and Federico Casari;
Ao voltar ao Brasil, em 2011, uma nova experiência me aguardava para ser vivida, a
experiência docente na Faculdade Mauricio de Nassau, sou grata: A coordenadora Regina
que me acolheu com carinho e serenidade da experiência; E a coordenadora Patrícia
Leiming pela motivação em continuar, e compreensão quando chegou a necessidade de
deixar a faculdade;
Aos professores companheiros de trabalhos, como Igor, Amanda, Paulinho, Daiane,
Eduardo, Gabriela, Conceição; Em especial ao amigo Pierre Andrade, companheiro de
graduação e de trabalho como professor na Nassau, e hoje na UEPB – Campus IV;
Aos meus alunos pela motivação constante de descobrir a melhor forma de guiar a busca
pelo conhecimento; Aos funcionários técnicos de laboratório, por toda a disponibilidade e
auxílio nas aulas práticas;
Além do Departamento de Física, outros centros como o CETENE e o CLA/IPEN, e as
pessoas que o compõem, foram fundamentais para o desenvolvimento dessa tese;
Ao professor Anderson Zanardi CLA/IPEN, pela atenção nas discussões de processamentos
de imagens via Skype;
A professora Denise Zezell CLA/IPEN, pelas diversas colaborações e pelo acolhimento em
seu laboratório; e a Patrícia da Ana, por desmistificar e concretizar metodologias descritas
em artigos publicados;
Aos funcionários e pesquisadores do CETENE, pela disponibilidade e prontidão em ajudar a
pesquisa ser realizada da melhor forma, especialmente para Edwin, Francisco, Maurício,
Josie, Gabriela, Hans, Conceição e Juliet;
Dentre os agradecimentos indiretos:
Aos meus avós paternos e maternos, tios, tias, primos, primas, com quem partilho as intimas
e divertidas reuniões familiares aos finais de semana, e que direta ou indiretamente
ajudaram a finalização desse sonho;
As moradoras do apartamento Andrezza, Ana Cristina, Thayze e Marcela, que em diferentes
fases partilharam momentos inesquecíveis;
As minhas cadelinhas (Sammy 15 anos, Blubby 10 anos, e Nina 1 ano) que me recebem
com tanta festividade;
Aos estudantes companheiros de viagem Campina Grande <> Recife <> Maceió, pela
partilha de experiências de pesquisas com as mais diversas áreas do conhecimento.
A TODOS que contribuíram para meu crescimento pessoal e profissional durante esses
anos de doutoranda.
RESUMO
A detectação precoce e monitoramento de perdas minerais da estrutura do tecido dentário
requerem técnicas conservativas, não invasiva ou minimamente invasivas, como os
métodos ópticos, baseados nos propriedades físicas da luz. Esta tese demonstra a
aplicabilidade de três técnicas ópticas qualitativas e quantivativas para perda mineral por
cárie ou erosão dentária. Uma das técnicas é comercialmente estabelecida, a Quantificação
de fluorescência por luz-laser induzida (QLF), e outros dois métodos vem sendo aplicados
para estudos experimentais, a Tomografia por coerência óptica (OCT) e a Microscopia
confocal por escaneamento a laser (CLSM). Objetivo geral : Avaliar e caracterizar a
aplicabilidade de técnicas ópticas para qualificar e quantificar a perda mineral por cárie ou
erosão dentária, elucidando a importância do conhecimento integrado das propriedades
ópticas da interação laser-tecido (in vitro). Materiais e Métodos : No artigo I , um modelo de
cárie artificial desenvolvido em 6 amostras de esmalte dentário humano, foi quantificado
quanto a perda de fluorescência quantificada pelo QLF e correlacionado com as alterações
no coeficiente de atenuação (A-Scan) detectada pelo OCT. Cerca de 200 cortes
tomográficos (B-Scan) por amostra foram processados para formação de um novo mapa da
lesão (C-Scan) com informações da curva de decaimento da luz em tecido sadio e cariado.
No artigo II , a microscopia confocal com escaneamento a laser (CLSM), analisou e
comparou amostras de esmalte dentário humano submetidos a ciclagem erosiva e tratados
com dentifrícios com diferentes mecanismos de flúor, o fluoreto de estanho e a caseína
fosfopeptídea com cálcio amorfo. O artigo III através de quatro protocolos de ciclagens
erosivas múltiplas em esmalte bovino demonstrou a aplicação da Tomografia por Coerência
Óptica, similar a análise de perfilometria, como um meio de quantificação de perda mineral,
comparando as interfaces entre a região sadia de referência e a região erodida.
Resultados : O processamento do valor de coeficiente de decaimento exponencial da luz em
cada A-Scan (artigo I ), resultou em um mapa C-Scan com eficiência para quantificação da
lesão de atenuação gerado através da técnica de OCT. No artigo II , a análise das imagens
3D e secções transversais da microscopia confocal permitiu comparar áreas sadias e
erodidas, visualizar detalhes morfológicos como a irregularidade do esmalte com exposição
dos cristais de esmalte, além de mensurar o desnível entre as áreas de interface, e a
deposição da camada protetora de fluoreto de estanho. O artigo III demonstrou a viabilidade
de utilizar a OCT para comparar as médias dos batentes de perda minerais decorrente de
múltiplas imersões em solução de ácido cítrico ou refrigerante coca-cola sob protocolos
erosivos variados. Tanto o OCT como a perfilometria mostraram maior perda mineral
promovida pela solução de ácido cítrico, e diretamente proporcional ao maior tempo de
exposição. Conclusões : Em geral, concluiu-se que as técnicas ópticas tem alto potencial
para quantificação da perda mineral, seja por cárie ou erosão dentária, além da
possibilidade de monitorar a evolução da lesão dentária visto que são não destrutivas.
Descritores: Diagnóstico precoce, Cárie Dentária, Erosão Dentária.
ABSTRACT
The early detection and monitoration of mineral loss of dental tissue structure needs
conservative techniques, non-invasive or minimally invasive, as optical methods, based on
physics properties of the light. This Thesis describes application of three Optical Techniques
qualitative and quantitative of mineral loss from dental caries and erosion. One of the
techniques is commercial established, Quantitative Light Induced Fluorescence (QLF), and
the other two techniques has been applied on experimental studies, the Optical Coherence
Tomography (OCT) and the Confocal Scanning Laser Microscope (CLSM). General Aim :
Analyze and characterize the application of optical techniques to qualify and quantify mineral
loss for dental caries and erosion, highlighting the importance of integrated knowledge of
optical techniques of laser material interacts (in vitro). Materials and Methods : On paper I ,
an arthificial carie model was developed in 6 samples of human enamel dental, and loss of
fluorescence was quantified through QLF and correlated with the attenuation coefficient
alterations (A-Scan) detected by OCT. Almost 200 tomographic sections (B-Scan) from each
sample were processed to plot a new map of caries lesion (C-Scan) with information of
decay curve of light interacts with health and carious tissue. On paper II , the Confocal Laser
Scanning Microscope, was used to analyze and compare samples of human enamel
structure submitted to erosive cycle and treated with dentifrices with different fluoride
mechanism, stannous fluoride and phosphor-peptide casein with amorphous calcium
phosphate. Using four protocols of multiple exposure erosive cycles and bovine enamel
samples, the paper III demonstrated an application of OCT as a quantitative method, similar
to profilometry, comparing interface between sound reference and eroded regions. Results :
The value of exponential decay coefficient of each A-Scan (paper I ) resulted in one C-Scan
map quantifying caries lesion with better efficiency. On paper II , images 3D and transversal
sections obtained by Confocal Microscopy allowed: comparison between sound and eroded
areas with interface levels measurements, visualization of morphological details as the
irregularity of enamel rods exposition, and deposition of the stannous fluoride protective
layer. The paper III demonstrated the feasibility of using OCT to compare the means of
mineral loss steps caused by multiple immersions in erosive solution of citric acid or coca-
cola, under various protocols. Both techniques, OCT and profilometry, showed greater
mineral loss promoted by citric acid solution, and also proportional to the exposure time.
Conclusions : In general, it was found that optical techniques have high potential for
quantification of mineral loss, either by caries or dental erosion, besides the possibility to
monitor the progress of dental lesions as they are not destructive.
Descriptors: Early diagnosis, Dental Caries, Tooth Erosion.
LISTA DE FIGURAS REVISÃO DA LITERATURA
Figura 1: Micrografia por microscopia eletrônica de varredura evidenciando os danos
causados pela ponta de diamante da perfilometria de contato em região de esmalte dentário
previamente erodido. Micrografia MEV CETENE. 34
Figura 2: Espectro eletromagnético, com ênfase para os comprimentos de onda da física
óptica. Incluindo o ultravioleta e infravermelho. Ilustração adaptada por Ana Marly A. Maia.
35
Figura 3: Esquema de propriedades ópticas na interação laser-tecido. Ilustracão adaptada
por Ana Marly A. Maia. 36
Figura 4: (a) Imagem da superfície lingual dos dentes superiores refletida no espelho bucal;
(b) Imagem da superfície lingual dos dentes infeirores após luz transmitida, observar
translucidez do esmalte. Fotografia arquivo pessoal. 37
Figura 5: (a) Aparência do contraste da superfície sadia e desmineralizada (MB) com luz
branca refletida, com reflexos de saturação (R) e baixo contraste; (b) Aparência do contraste
na mesma região, através da luz transmitida de dentro da amostra para o detector óptico,
logo sem reflexos (R). Fotografia arquivo pessoal. 38
Figura 6: Secção dentária transiluminada. (a) Laser infravermelho 1300nm; (b) Luz branca.
Fotografia arquivo pessoal. 39
Artigo I Figure 1: The commercial SR-OCT, OCP930SR, schematic diagram (adapted from Thorlabs
New Jersey, USA). 50
Figure 2: a) The handheld scanning probe from the OCT system (Thorlabs) and the tooth in
a micrometer translation stage controlled by a Motor Move system, about 0.5mm/s; b) off-
axis images, A-Scan, B-Scan and C-Scan images. 51
Figure 3: Diagram of the whole sequence processing. 52
Figure 4: a) Image obtained by reflection visible light; b) Image obtained by transilluminated
visible light; c) Image from QLF software; d)The C S-can image processed. 54
Figure 5: Image reference and A-scan showing the decrease of light in evaluated by
exponential decay, illustrating the alterations of attenuation coefficient on carious signal.
Fitting in decay signal curve of sound tooth (green line) and carious region (blue line). 55
Figure 6: (a) Enamel surface and dentine enamel junction (DEJ); (b) same region after
arthificial demineralization, not possible to see DEJ below carious surface. 56
Figure 7: (a) Polarized optical microscope image of 200um section; (b) Tomographic B-Scan
image of the caries lesion. 56
Artigo II Figure 1: CLSM typical images of sound enamel surface (a); soft eroded surface (b); and
areas of aggressive eroded surface (c). Figures (d), (e) and (f) show XZ sections taken from
the reconstructed images from samples a, b and c, respectively. 71
Figure 2: CLSM of typical enamel surfaces treated with solutions; a) G2: CPP-ACP NaF
Solution; b) G4: Oral B SnF2 Solution; c) G3: CPP-ACP NaF Tooth-brushed; and d) G5: Oral
B SnF2 tooth-brushed effects. Below each image XZ sections taken from the reconstructed
images. 72
Figure 3: CLSM image on XZ section representative interface of each tested group at the
end of cycle regime. 73
Artigo III Figure 1: Example of an OCT image profile analyzed. The total distance used as reference to
get value of step was 3mm, meaning 1,5 mm of each surface. For measurement of tissue
loss, the box tool helps to identify the distance between both superficial face. 89
Figure 2: Comparison of OCT images of the enamel surface reference and eroded are for
different solution and time periods. Images identified with lowercase represent 3D OCT; and
images by uppercase cross-sectional. (A/a) Citric Acid, 10d:6x5min; (B/b) Citric Acid,
7d:5x3min; (C/c) Coca-Cola®, 7d: 5x3min; and (D/d) Coca-Cola®, 5d:4x90sec. 90
Figure 3: Scanning electron micrographs (all in the same magnification level of 10.000X) of
the surface of groups (A) sound enamel; (B) Citric Acid, 10d:6 x 5min; (C) Citric Acid,
7d:5x3min; (D) Coca-Cola® 7d:5x 3min and (E) Coca-Cola® 5d:4x90sec. 92
LISTA DE TABELAS Revisão da Literatura
Tabela 1: Parâmetros de pesquisa utilizados em ensaios in vitro de erosão em esmalte
(adaptado de WIEGAND, ATTIN, 2011). 31
Artigo I
Table 1: Data of fluorescence Intensity reduction (%) and attenuation coefficient increase (%)
of each sample. 55
Artigo II Table 1: Tissue loss (µm) in all groups (mean + SD) after three days of in vitro
demineralization and relative mineral loss (percentage of control group). 74
Artigo III
Table 1: Mean of tissue loss (µm) in all groups and by Profilometry and OCT techniques.
Mean roughness values and standard deviations of reference and eroded surfaces. 91
LISTA DE ABREVIATURAS E SIGLAS
ACP - amorphous calcium phosphate/ fosfato de cálcio amorfo
AFM - atomic force microscopy/ microscopia de força atômica
APF - acidulated phosphate fluoride/ flúor fosfato acidulado
CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CCD - Charge-Coupled device/ Dispositivo de câmera acoplada
CETENE - Centro de Tecnologias Estratégicas do Nordeste
CLSM - confocal laser scanning microscopy/ microscopia confocal por
escaneamento a laser
CPP - casein phosphor-peptide/ caseína fosfopeptídea
DEJ - Dentine enamel junction/Junção esmalte dentina
DES-RE - Demineralization and Remineralization/ Desmineralização e
Remineralização
DIFOTI - Digital Imaging fibre-optic trans-illumination
EDS - Energy-dispersive X-ray spectroscopy/ Espectroscopia de energia
dispersiva
FACEPE - Fundação de Amparo à Ciência e Tecnologia do Estado de
Pernambuco
IR - Infrared/ infravermelho
Laser - Light Amplification by Stimulated Emission of Radiation
LUT - Lookup tables
MB - Mancha branca/ White spot
MET - Microscopia eletrônica de transmissão
MEV - Microscopia eletrônica de varredura
MHDP - methanehydroxydiphosphonate
MO - Microscópio Optico
NA - numeric aperture/abertura numérica
NIR - Near Infra-Red/ Infravermelho próximo
OCT - Optical Coherence Tomography
OM - Optical Microscope
PC - Personal computer
PS OCT - Polarization Sensitive Optical Coherence Tomography
QLF - Quantitative Light-induced fluorescence
Ra - Roughness average/ Rugosidade média
SA - Saliva artificial
SD-OCT - Spectral Domain Optical Coherence Tomography/ Tomografia
por coerência óptica no domínio espectral
SLD - superluminescent diode/Diodo superluminescente
SPSS - Statistical Package for the Social Sciences
SEM - Scanning Electron Microscope
SE - Elétron Secundário/Secondary electron
STM - Scanning Tunneling Microscope/microscopia de tunelamento com
varredura
TCO - Tomografia por coerência óptica
UV - Ultraviolet / Ultra-violeta
UFPE - Universidade Federal de Pernambuco
∆F - average change in fluorescence
∆Q - área x ∆F
LISTA DE SÍMBOLOS
ºC - graus Celcius
~ - Aproximadamente
% - Porcentagem
g - Gramas
ml - Mililitros
mm - Milímetros
nm - Nanômetro
µm - Micrômetro
pH - Potencial de hidrogênio
ppm - Partes por milhão
qsp - Quantidade suficiente para
mW - Miliwatts
kV - Quilovolts
N - Newton
CaF2 - Fluoreto de Cálcio
NaF - Fluoreto de Sódio
F - Flúor
AmF - Amino fluoreto
SnF2 - Fluoreto de estanho
SUMÁRIO
LISTA DE ILUSTRAÇÕES
LISTA DE QUADROS E TABELAS
LISTA DE SIGLAS E ABREVIATURAS
LISTA DE SÍMBOLOS
1.0 INTRODUÇÃO 23
2.0 REVISÃO DA LITERATURA 25
2.1 Perda Mineral por Cárie e Erosão Dentária 25
2.2 Mecanismos de prevenção para erosão dentária 27
2.3 Metodologia de Ensaios Erosivos 29
2.4 Métodos Ópticos para detecção de perda mineral 32 2.4.1 Princípios Físicos dos Métodos Ópticos para detecção de perda mineral 34
2.4.2 Quantificação da Fluorescência induzida por Luz (QLF) 40
2.4.3 Tomografia por Coerência Óptica (OCT) 40
2.4.4 Microscopia por Confocal de escaneamento a laser (CLSM) 42 3.0 OBJETIVOS ARTIGO I
43
Abstract 45 Introduction 46 Materials and Methods 48 Results 54 Discussion 57 Conclusions 59 References 60
ARTIGO II
Abstract 64 Introduction 65 Materials and Methods 68 Results 71 Discussion 74 Conclusions 77 References 77
ARTIGO III
Abstract 83 Introduction 84 Materials and Methods 86 Results 90 Discussion 93 Conclusions 96 References
96
CONSIDERACOES FINAIS 100 REFERENCIAS BIBLIOGRÁFICAS 101
23
1 INTRODUÇÃO
O panorama atual da saúde bucal no Brasil apresenta um cenário de declínio
de doenças orais de maior prevalência, como a cárie dental. No entanto, a
prevalência da cárie ainda é alta, em torno de 57%, entre crianças aos 12 anos
(SBBrasil, 2010). Paralelamente a abordagem de diagnóstico precoce da lesão de
cárie, há uma maior preocupação com a perda de tecido dental não decorrente da
doença cárie, isto é, sem a presença de bactérias cariogênicas. A perda mineral por
erosão química, podendo estar associada à abrasão, tem se tornado um fator de
risco para danos a estrutura dentária (JAEGGI, LUSSI, 2006), com registros de
prevalência de 64% em jovens adultos (MULIC et al., 2012). O alto consumo de
bebidas ácidas, como refrigerantes e sucos de frutas cítricas, torna o esmalte
poroso, dissolvendo cristais de esmalte camada por camada, até uma perda
volumétrica do esmalte e possível exposição de dentina. Este processo tem o
potencial de tornar o esmalte ainda mais vulnerável à abrasão durante a escovação
dentária.
Dessa forma, mesmo diante de uma população com altos índices de perda
dentária, pesquisas vêm ganhado espaço em buscas da prevenção para as mínimas
perdas dentárias por cárie ou erosão, visto que a ultima é decorrente de hábitos
comuns difíceis de serem excluídos da rotina diária. Nesse contexto, inúmeras
pesquisas buscam elucidar métodos de diagnóstico para detectar a perda mineral
mínima, interceptar o processo com meios reparadores, e conscientizar o paciente
de sua responsabilidade. O diagnóstico precoce é unânime em vantagens para o
tratamento de doenças em todas as áreas da saúde, e os métodos diagnósticos
buscam cada vez mais, melhor qualidade e objetividade, sem promover danos ao
paciente.
Dentre os meios diagnósticos, além dos recursos imaginológicos por meio dos
raios X, pesquisas nas áreas de interface multidisciplinar buscam utilizar outras
faixas do espectro eletromagnético, como o infravermelho e ultravioleta, para
aplicações em diagnóstico. A utilização dos raios infravermelho e ultravioleta foi
expandida com a emissão em forma de laser, que significa uma amplificação do
sinal por emissão estimulada da radiação eletromagnética, e detém características
24
peculiares que permitem diferentes aplicações na área biológica. Para aplicações
em diagnóstico, a tecnologia óptica lança mão de lasers com baixa potência, o que
permite explorar tecidos vivos de forma não destrutiva, além de exigir mínimo
preparo da amostra a ser analisada. Através da quantificação das variadas
interações da luz com o tecido, como a reflexão, absorção, fluorescência e
espalhamento, o laser tem sido utilizado principalmente como uma ferramenta de
diagnóstico precoce associados a diferentes detectores ópticos.
Dentre as técnicas ópticas, algumas são fundamentadas em diferentes
propriedades da interação luz - tecido biológico, como o QLF (Quantitative Light
Induced Fluorescence) que quantifica alterações da fluorescência dentária
(JOSSELIN de JONG et al., 1995), o CLSM (Confocal Laser Scanning Microscope)
que também quantifica a fluorescência, permitindo analisar em melhor resolução
através de cortes transversais em profundidade, e o OCT (Optical Coherence
Tomography) que analisa alterações ópticas a partir da reflexão e retroespalhamento
do tecido biológico (OTIS et al., 2000). As referidas técnicas foram avaliadas em
diferentes experimentos de simulação de cárie e erosão dentária visando
estabelecer novos parâmetros para caracterização qualitativa e quantitativa da perda
mineral mínima da estrutura dentária.
A inovação de técnicas biomédicas ressalta a importância de interação entre
as mais diversas linhas de pesquisa, tornando-se multiplicador de opções e
soluções. Resgatando a evolução das técnicas, encontra-se razão para acreditar na
importância da busca pelo diagnóstico precoce e prevenção, visando evitar o
surgimento de novos doentes. O desenvolvimento de pesquisas experimentais,
quanto às mínimas perdas minerais do tecido dentário, visa migrar além das lentes
de microscópios e bancadas de laboratório para as páginas das revistas científicas,
tornando-se realidade social aplicável clinicamente, modificando o alto índice de
perdas dentárias na sociedade, particularmente das camadas sociais menos
favorecidas.
25
2 REVISÃO DA LITERATURA
2.1 Perda Mineral por Cárie e Erosão Dentária
O processo carioso é contínuo sempre em busca de um equilíbrio iônico que
se desloca de acordo com a variação de reagentes e produtos presentes na
cavidade oral. É passível de regressão quando detectado precocemente, com
intervenção apenas de agentes preventivos, como o fluoreto, que pode interferir na
superfície dente-biofilme-cálculo, remineralizando a integridade do esmalte, como
mostrado em diversos trabalhos (TENUTA et al., 2009; CURY, TENUTA, 2008). A
doença cárie em seu estágio inicial manifesta-se como mancha branca discreta no
esmalte, resultante da desmineralização parcial do tecido mineralizado (BESIC,
WIEMANN, 1972).
O processo de diagnóstico da lesão de cárie iniciou com importantes técnicas
como a visual e tátil, com espelho e sonda, e vem sendo usada ao longo dos anos
até hoje, após limpeza e secagem da superfície como preconizado por Black,
(1908). A radiografia intrabucal interproximal foi um marco no diagnóstico
complementar desde 1925, preconizada por Raper. Entretanto, essas técnicas
atualmente consideradas convencionais, detectam principalmente lesões cavitadas,
demonstrando baixa sensibilidade e especificidade para detectar precocemente
lesões cariosas (ISMAIL, 2004).
Como exemplo, radiografias interproximais são excelentes para diagnosticar
lesões extensas, avançadas e possivelmente cavitadas, mas tem resolução limitada
e baixo contraste radiográfico para lesões de mancha branca. Além dos fatores
limitantes descritos, é importante ressaltar que a técnica de raios X interproximal,
mesmo com as doses mínimas necessárias, trata-se de uma radiação
eletromagnética ionizante, portanto formadora de radicais livres, e
consequentemente não indicada para análises de monitoramento para proservação
de uma lesão de cárie (KO et al., 2008).
26
Nos dias atuais, principalmente em centros urbanos desenvolvidos, a
incidência da cárie dental tem declinado (LUSSI et al., 2011). Entretanto, a
manutenção dos dentes, os predispõe a outras lesões dentárias, tal como a erosão.
A erosão dental é definida como a perda progressiva de tecido duro dental por um
processo químico que não envolve bactérias (AMAECHI; HIGHAM, 2005;
DUGMORE; ROCK, 2004; LEVITCH et al., 1994; LUSSI; JAEGGI; SCHARER, 1993)
podendo ocorrer em qualquer superfície do dente. A erosão se diferencia de outras
lesões tais como abrasão, atrição e abfração (IMFELD, 1996; TEN CATE, IMFELD,
1996), bem como da cárie dentária, por não haver envolvimento bacteriano na perda
do tecido dentário, no entanto dificilmente estes fenômenos ocorrem,
simultaneamente, no mesmo sítio (IMFELD, 1996; TEN CATE, IMFELD, 1996).
Há vários fatores comportamentais que influenciam no processo de erosão,
como o consumo frequente e excessivo de bebidas dietéticas específicas, bem
como alcoólicas. Hábitos não usuais de beber e deglutir – por exemplo, reter um
refresco ácido na boca antes de deglutir ou fazer um bochecho - podem também
aumentar o tempo de contato de uma substancia ácida com o dente,
consequentemente aumentando o risco de erosão (MILLWARD et al., 1997;
EDWARDS et al., 1998; JOHANSSON et al., 2004). Sucos de frutas, refrigerantes,
vinagre e chá gelado são bebidas conhecidas como altamente erosivas, pois são
compostas por ácidos, como o cítrico, fosfórico, acético, e por apresentar pH abaixo
de 4.5 (LUSSI, JAEGGI, ZERO, 2004).
A desmineralização por erosão depende do tipo de ácido e do tempo que o
esmalte fica exposto, o que pode deixá-lo poroso, dissolvendo cristais de
hidroxiapatita camada por camada, até uma perda volumétrica do esmalte e possível
exposição de dentina (LUSSI et al., 2011). O ácido pode ser de fontes intrínsecas
como refluxo gástrico, ou extrínsecas, a depender da frequência de consumo de
bebidas ácidas, o que pode ser um hábito comum, difícil de ser excluído, o que leva
a ciência a buscar estratégias preventivas e visar o diagnóstico precoce (PARKER,
2009). Vários estudos mostram que o esmalte amolecido é muito susceptível a
riscos e danos (EISENBURGER et al., 2003; JAEGGI, LUSSI, 1999; LIPPERT et al.,
2004), altamente instável e facilmente removido mesmo com delicado ato físico
(EISENBURGER et al., 2003). Portanto, a escovação dentária do esmalte erodido
lidera as menores alterações na morfologia de superfície e propriedades mecânicas
27
(LIPPERT et al., 2004), e foi ainda comprovado que a perda de esmalte causada
pela escovação depende do tempo decorrente entre o desafio erosivo e a escovação
dentária (JAEGGI, LUSSI, 1999).
Com o aumento da prevalência de erosão dental (JAEGGI, LUSSI, 2006), o
manejo clínico deste quadro vem se tornando um importante aspecto de saúde
dentária, junto aos cuidados da cárie e da doença periodontal. Neste contexto,
acredita-se que além do atendimento clínico, o contato com o profissional de saúde
bucal, deve ser um momento de motivação e conscientização para que o paciente
compreenda que é o principal agente responsável por fornecer flúor a cavidade oral,
através de 90% dos dentifrícios. É extremamente válido que o paciente seja
conscientizado do estágio inicial de sua condição oral, para avaliar possíveis danos
provocados ao esmalte por hábitos prévios.
2.2 Mecanismos de prevenção para erosão dentária
O impacto do tratamento com fluoretos na progressão da erosão do esmalte e
dentina vem sendo analisado em vários estudos. Tem sido demostrado in vitro que
tratamentos com fluoretos, como fluoreto de sódio, amino fluoreto ou flúor fosfato
acidulado, forma a precipitação do CaF2 na superfície erodida (GANSS, et al., 2004;
GANSS, KLIMEK, STARCK, 2004). A formação da camada de CaF2 e seu efeito
protetor na desmineralização depende do pH, da concentração de F, e do tipo de sal
do agente fluoreto (SAXEGAARD, ROLLA, 1988). Entretanto, todo o processo de
aplicação do fluoreto na prevenção da erosão continua em discussão repleta de
controvérsias (WIEGAND, ATTIN, 2003), desde que o depósito de CaF2 a partir de
aplicações de fluoretos tópicos parece ser dissolvido pela maioria das bebidas
ácidas (GANSS, et al. 2007), removendo traços de um prévio tratamento de flúor
tópico (LARSEN, RICHARDS, 2002).
A eficácia dos fluoretos no processo de desmineralização e remineralização é
relacionada com a concentração e o pH do agente fluoreto. Além de que, a alta
concentração de agentes fluoretantes como enxaguantes orais, géis e vernizes, tem
demonstrado aumento na resistência à abrasão e diminuição do desenvolvimento
28
dos processos erosivos no esmalte e dentina em ensaios in vitro e in situ (GANSS,
et al. 2004). Dependendo do desenho do estudo, a aplicação de altas concentrações
do agente flúor pode resultar em redução completa do processo de erosão dentária.
A maioria dos estudos focando o efeito preventivo do fluoreto na erosão,
avalia compostos de flúor indicados na prevenção da cárie, como o NaF, AmF, SnF2
e o flúor fosfato acidulado (APF) (de 12,300 a 22,600 ppmF, pH 1.0 para 7.0). Várias
estratégias têm sido testadas para controlar a erosão no esmalte, como a aplicação
tópica de formulações de fluoreto ou de fosfato de cálcio. Outras opções consistem
na adição de cálcio, fosfato, ferro, sulfato ferroso, ions de estanho e/ou
hexametafosfato de sódio a enxaguantes orais ou dentifrícios (LUSSI, 2009).
Dentre esses, um agente que tem se mostrado também promissor sob
condições médias e severas de erosão é o íon estanho (TINANOFF, 1995, GANSS
et al, 2004). Pouco se sabe sobre a interação entre o íon estanho e a dureza do
tecido dentário; razão pela qual seu potencial anti-erosivo continua não descrito. A
aplicação de soluções contendo estanho é direcionada a depositar-se na superfície
do dente (WILLUMSEN, 2004; HOVE, 2008) e há indicações que esta deposição é
relativamente resistente a dissolução ácida (MOAZZEZ, 2004; HJORTSJO, 2008). É
sabido que o íon de estanho reage com a hidroxiapatita pura (YOUNG, 2006;
SCHLUETER, 2007) na superfície do tecido dentário, (WILLUMSEN, 2004;
BIRKHED, HEINTZE, 1989) resultando na redução da solubilidade da hidroxiapatita
ou do esmalte (MOAZZEZ, 2004; SREEBNY, 1996; GANSS, 2004).
Em um novo conceito de remineralização dentária, baseado na molécula da
fluorpatita Ca10(PO4)6F2, foi questionado se a disponibilidade de cálcio e fosfato pode
ser um fator limitante, visto que a molécula da hidroxiapatita é formada por dez
elementos de Ca, e seis de fosfato (REYNOLDS, 2008). No entanto, a maioria dos
tratamentos visa à aplicação tópica apenas de flúor, o qual representa o mínimo de
elementos químicos na hidroxiapatita.
Alguns testes com aplicações clínicas de cálcio e fosfato mostraram
insucesso devido à baixa solubilidade do fosfato de cálcio, principalmente na
presença do flúor. Dessa forma, foi necessário associar leve acidez para aumentar a
solubilidade e difusão desses íons na lesão de subsuperfície do esmalte. Por outro
lado, visto que os íons de cálcio e fosfato tem alta tendência de precipitar e não se
29
manter disponível na cavidade oral, a aplicação torna-se limitada a baixas
concentrações (REYNOLDS, 2008). Diante dessas limitações, sistemas
remineralizadores baseados em fosfato de cálcio vêm sendo desenvolvidos e
comercializados, com o principal intuito de aumentar a biodisponibilidade dos íons
cálcio e fosfato para o processo de remineralização dentária. A aplicação dessas
técnicas vem ganhando espaço em diferentes produtos de higiene oral, com a
prosposta de ampliar o uso para chicletes e outros alimentos.
O sistema conhecido como CPP-ACP (Recaldent™) foi desenvolvido por E.C.
Reynolds, professor da Universidade de Melbourne, Austrália, com estudos desde
1997. Consiste em nanocomplexos de fosfato de cálcio amorfo (ACP) estabilizada
pela caseína fosfopeptídea (CPP), impedindo que nano aglomerados de ACP
cresçam para tamanhos críticos e se transformem. Dessa forma, consegue manter
altas concentrações de íons cálcio e fosfato, junto com íons flúor, na superfície do
dente seja através da película ou do biofilme dental em estado biodisponível. Em
estudo clínico com enxaguante oral, a tecnologia CPP-ACP apresentou-se superior
a não estabilizada ACP (REYNOLDS, 1999), mostrando a importância da CPP para
estabilizar altos níveis, estado supersaturado, de ions cálcio e fosfato, e
disponibilizá-los à superfície dental, fazendo possível a remineralização (RAHIOTIS,
VOUGIOUKLAKIS, 2007). Fundamentalmente a tecnologia CPP-ACP promete
remineralizacao sem adição de flúor, no entanto foi demonstrado em estudos que o
fosfato de cálcio amorfo pode interagir com o flúor, formando a fase ACFP
(REYNOLDS, 2008).
2.3 Metodologia de Ensaios Erosivos
Com o aumento da prevalência da erosão, muitas pesquisas passaram a ser
desenvolvidas para avaliar o potencial erosivo de algumas bebidas ou produtos, ou
mesmo para testar meios de prevenção. Essas pesquisas deveriam ser idealmente
conduzidas clinicamente in vivo, utilizando técnicas não destrutivas e de alta
resolução para quantificação intra-oral. Entretanto muitas das técnicas disponíveis
para clínica não dispõem de resolução e acurácia suficientes para mensurar a perda
30
mineral (HUYSMANS et al., 2011). Portanto, os modelos in situ e in vitro ainda são
predominantes, permitindo controles estritos de testes de variáveis específicas e
tempo de exposição, além de permitir o uso de tecnologias padrões para mensurar o
desgaste do tecido dentário (WEST, DAVIES, AMAECHI, 2011).
A diferença quantitativa de perda mineral entre estudos in situ e in vitro,
ocorre provavelmente devido aos efeitos naturais de proteção como a presença de
proteínas na saliva e a película adquirida nos estudos in situ (WEST et al., 1999;
YOUNG et al., 2006). Ressalta-se ainda que a simplicidade das pesquisas in vitro é
fundamental para se definir a trajetória inicial, e as tendências de novos estudos,
visto o controle e o maior número de variáveis que pode ser testado (WEST,
DAVIES, AMAECHI, 2011). No entanto, os estudos in vitro devem buscar mimetizar
os reais desafios erosivos observados no dia-a-dia, através de modelos
representativos como a ciclagem erosiva com múltiplas exposições ao ácido. Essa
metodologia de ciclagem por utilizar múltiplas imersões durante minutos é também
viável para reavaliações em estudos in situ, os quais simulam condição oral mais
efetiva.
No entanto, mesmo considerando apenas pesquisas que utilizaram ciclagem
erosiva com múltiplas exposições, observa-se ainda grande variedade quanto ao
número de dias, número de ciclagens por dia, duração de cada ciclo, e tipo de
solução erosiva e amarzenamento intermediário, como pode ser constatado na
tabela 1 abaixo.
31
Tabela 1: Parâmetros de pesquisa utilizados em ensaios in vitro de erosão em
esmalte (adaptado de WIEGAND, ATTIN, 2011).
Estudo
Esmalte Ciclagem Erosão
Meio
intermediário
armazenamento
Origem Dias, ciclos/dia Duração/
ciclo Meio Duração Meio
HARA et al. [2009] Humano 3 d, 3x/dia 2 min Ácido cítrico,
pH 3.75 60 min SA
LAGERWEIJ et al.
[2006] Bovino 14 d, 6x/dia 30 s
Ácido cítrico,
pH 2.3
Alguns
minutos SA
MORETTO et al.
[2010] Bovino 7 d, 4x/dia 5 min
Sprite, pH
2.8 Nenhum
YU et al. [2009] Humano 10 d, 6x/dia 1 min Ácido cítrico 30 min SA
ROCHEL et al.
(2011) Bovino 7d, 4x/dia 2 min Coca-Cola 2 hs SA
WEGEHAUPT,
ATTIN, (2010) Bovino 20d, 6x/dia 20 s
Ácido
clorídrico 1h SA
GANSS et al., 2008 Humano 10d, 6x/dia 2 min Ácido Cítrico 1,5hs SA
SCHLUETER., 2009 Humano 10d, 6x/dia 2 e 5 min Ácido Cítrico 1h SA
MAGALHAES et al.,
2012 Bovino 5d, 4x/dia 90s Coca-Cola 1h SA
LEVY et al., 2011 Bovino 5d, 4x/dia 90s Coca-Cola 1h SA
SA: Saliva artificial.
Como observado, modelos de estudos erosivos in vitro têm sido
extremamente reproduzidos, pois podem ser realizados em curto período de tempo,
requerem menor número de participantes, além de menor custo. No entanto, os
resultados não podem representar a realidade da condição oral, visto que outros
fatores estariam envolvidos. Considerando que os estudos in vitro podem ter
variáveis da ciclagem erosiva controladas, como a temperatura, o pH e a
concentração da solução ácida, o uso e tipo de saliva artificial, bem como a agitação
e o tempo de exposição da amostra na solução, torna-se indispensável que esses
fatores sejam descritos na publicação dos resultados, visto que são cruciais para a
comparação dos métodos erosivos e preventivos (WEST, DAVIES, AMAECHI,
2011).
32
A diversidade de soluções e modelos erosivos dificulta a comparação dentre
as pesquisas desenvolvidas, logo se busca padronizar ciclagens erosivas para
estabelecer um desafio erosivo suficiente para visualizar os possíveis danos da
solução ácida, bem como quantificar o efeito protetor dos fluoretos. Como
demonstrado posteriormente por Ganss et al. (2012), o protocolo erosivo pode não
ser suficiente para quantificar o efeito preventivo do produto testado. Além disso, é
indispensável que a técnica de caracterização a ser utilizada para comparação dos
métodos disponha de resolução suficiente para visualizar as diferenças entre os
efeitos erosivos e preventivos.
2.4 Métodos Ópticos para detecção de perda mineral
Visando possibilitar uma melhor comunicação no diálogo de motivação entre
profissionais e pacientes, métodos diagnósticos vêm sendo desenvolvidos para a
detecção de lesões precocemente, e monitoramento da lesão pelo profissional e
pelo paciente. Acredita-se que ao visualizar a lesão através de imagens
diagnósticas, o paciente terá maior compromisso em proporcionar meios de higiene,
como escovação e fio dental, para regressão da lesão, além de retornar para a
próxima consulta para verificar se os procedimentos estão sendo efetivos.
Na última década, novas técnicas ópticas para detecção precoce de cárie
foram desenvolvidas, dentre as tecnologias, algumas se encontram comercializadas
em âmbito clínico, e outras estão em fase experimental em laboratório de pesquisas.
Dentre as mais populares e comercializadas clinicamente em alguns países,
enumeramos: a DIFOTI (Digital Imaging fibre-optic trans-illumination)
(SCHNEIDERMAN et al., 1997), a qual se baseia em transiluminação com luz visível
comercializada pela Electro-Optical Sciences, Inc. (Irvington, NY, EUA); o QLF
(Quantitative Light-induced fluorescence) (JOSSELIN de JONG et al., 1995) baseia-
se na quantificação da fluorescência excitada com luz azul em torno de 380nm,
desenvolvido e comercializado por grupo holandês, Inspecktor Research System
(Amsterdam, Holanda); e o DIAGNOdent baseado no sinal fluorescência apos
33
excitação com vermelho, em torno de 680nm, sendo o mais popular no Brasil,
comercializado pela KaVo (Charlotte, Carolina do Norte, EUA).
Outras técnicas de maior complexidade tecnológica mostram bons resultados
laboratoriais in vitro, e estão em fase de desenvolvimento para maior aproximação
entre as quantificações matemáticas e o diagnóstico clínico. Dentre as diversas
técnicas, destaca-se: a NIR Transillumination imaging baseada em transiluminação
com laser no infravermelho (JONES et al., 2003; BUHLER et al., 2005; KARLSSON
et al., 2010); a OCT - Tomografia por coerência óptica (OTIS et al., 2000) baseada
em secções internas da amostra através da analise do sinal de retroespalhamento
da luz, usando laser no infravermelho, em torno de 830nm a 1300nm; e a
espectroscopia Raman associada a OCT (KO et al., 2005), que analisa a amostra
bioquimicamente através da vibração molecular, e detecta perda mineral na
estrutura.
Para estudos in vitro, e desenvolvimento metodológico para quantificação de
perda mineral decorrente também de lesões erosivas, outros métodos qualitativos
laboratoriais, maioria de formas de microscopia, tem sido aplicados de forma
independente ou combinados a resultados quantitativos. Dentre estes métodos, cita-
se: a microscopia de luz transmitida, a microscopia confocal de varredura a laser
(CLSM – confocal laser scanning microscopy), microscopia eletrônica de
transmissão (MET), microscopia eletrônica de varredura (MEV/SEM – scanning
eletronic microscope), espectroscopia de energia dispersiva (EDS - energy-
dispersive X-ray spectroscopy), microscopia de força atômica (AFM - atomic force
microscopy) e microscopia de tunelamento com varredura (STM - scanning tunneling
microscope), e a espectrometria de massa de íon secundário (SCHLUETER, HARA,
SHELLIS, GANNS, 2011).
Cada um dos métodos existentes apresentam vantagens, desvantagens e
limitações. Como exemplo a perfilometria de contato, que apesar da ampla utilização
por pesquisadores e boa reprodutibilidade, apresenta a desvantagem de ser
destrutiva, como observado na figura 1. Entretanto, a utilização conjunta dos
resultados de diversos métodos pode preencher adequadamente a maior parte das
necessidades das pesquisas envolvendo perda mineral dentária. Não obstante,
ainda há grande necessidade para o desenvolvimento, avaliação e, principalmente,
34
validação de novos métodos que podem vir a melhorar sobremaneira o estudo da
perda mineral em todas as fases e nos diversos modelos de estudo, in vitro, in situ,
bem como em ensaios clínicos (SCHLUETER, et al., 2011).
Figura 1: Micrografia por microscopia eletrônica de varredura evidenciando os danos
causados pela ponta de diamante da perfilometria de contato em região de esmalte
dentário previamente erodido. Micrografia MEV CETENE.
2.4.1 Princípios Físicos dos Métodos Ópticos para detecção de perda mineral
Em geral, as técnicas ópticas por apresentarem a vantagem de serem não
destrutivas, tem ganhado espaço dentre as técnicas desenvolvidas para detectar
perda mineral dentária. Baseado nos conceitos de interação da energia aplicada ao
dente, ou na observação da energia que é emitida a partir do dente (HALL, GIRKIN,
2004), os métodos ópticos usam a energia da luz visível, incluindo ainda o
ultravioleta e infra-vermenlho (figura 2).
35
Figura 2: Espectro eletromagnético, com ênfase para os comprimentos de onda da
física óptica. Incluindo o ultravioleta e infravermelho. Ilustração adaptada por Ana
Marly A. Maia.
Ao interagir com a estrutura dentária, essa energia pode interagir enquanto
onda eletromagnética, de acordo com os conceitos de reflexão, absorção que pode
ser seguida de fluorescência, transiluminação, espalhamento e retroespalhamento
(HALL, GIRKIN, 2004). Por ser de fundamental importância conhecer as
propriedades da onda eletromagnética e da estrutura observada, buscou-se
relembrar os conceitos de interação luz com a matéria. Em geral, a área
desmineralizada torna-se porosa, o que aumenta o espalhamento da luz incidente,
levando essa região a parecer mais esbranquiçada, portanto nomeada de mancha
branca. Na figura 3, por meio de um desenho esquemático, são demonstradas as
interações ópticas entre o elemento dentário e a onda eletromagnética. Estas
diferentes propriedades ópticas podem ser quantificadas de diferentes formas, a
depender do comprimento de onda e dos detectores ópticos.
36
Figura 3: Esquema de propriedades ópticas na interação laser-tecido. Ilustracão
adaptada por Ana Marly A. Maia.
A propriedade da reflexão é a mais popularmente conhecida no meio clínico
odontológico, obtida macroscopicamente através do uso do espelho oral plano ou
convexo. É um fenômeno de superfície que resulta na mudança de direção da onda,
um exemplo clássico dessa propriedade na clínica odontológica é quando o espelho
bucal proporciona a iluminação e visualização das superfícies linguais ou palatinas
dos dentes, como mostrado na figura 4 (a), sendo nossos olhos os detectores óticos
naturais. A estrutura cristalina do esmalte permite a passagem da luz, propriedade
chamada de transmissão, neste caso difusa. A figura 4 (b) demontra a observação
da luz transmitida da face vestibular para a lingual dos incisivos inferiores.
37
Figura 4: (a) Imagem da superfície lingual dos dentes superiores refletida no espelho
bucal; (b) Imagem da superfície lingual dos dentes infeirores após luz transmitida,
observar translucidez do esmalte. Fotografia arquivo pessoal.
O espalhamento consiste na mudança de direção do feixe de luz sem perda
de energia, visto que a luz é forçada a desviar quando interage com partículas
menores ou objetos que provoquem essa propriedade. A intensidade do
espalhamento da luz depende também do comprimento de onda e do material,
podendo levar a dissipação de energia. A cárie dentária inicial, devido a maior
porosidade estrutural e grande número de obstáculos difusores, é um meio
espalhador, e por provocar maior espalhamento da luz apresenta-se como uma
mancha branca (ANGMAR-MANSSON, TEN BOSCH, 1993). O espalhamento é
dependente do comprimento de onda, de forma que ondas menores apresentam
maior espalhamento (HALL, GIRKIN, 2004). E vários métodos ópticos quantificam a
quantidade de luz espalhada por transiluminação ou por retroespalhamento.
Os efeitos do espalhamento podem ser ressaltados ao comparar a luz
refletida e transmitida do dente cariado, como demonstrado nas figuras 5 a e b. A luz
branca refeletida sofre maior espalhamento no tecido demineralizado, processo
chamado também de retroespalhamento, apresentando-se como mancha branca,
figura 5 (a). No entanto, observando a luz transmitida através do dente, observa-se
que o tecido sadio que apresenta cristais translúcidos e organizados, permite a
passagem efetiva da luz, no entanto, na região do tecido cariado, ou mancha branca
38
(MB) que sofreu modificações estruturais com presença de poros, bactérias, e outros
constituintes, a luz é pouco transmitida, como pode ser observada na figura 5 (b).
Observar como o contraste entre a mancha branca e o esmalte sadio é maior na
amostra com luz branca transiluminada 5 (b).
Figura 5: (a) Aparência do contraste da superfície sadia e desmineralizada (MB) com
luz branca refletida, com reflexos de saturação (R) e baixo contraste; (b) Aparência
do contraste na mesma região, através da luz transmitida de dentro da amostra para
o detector óptico, logo sem reflexos (R). Fotografia arquivo pessoal.
Devido à diminuição do espalhamento com o aumento do comprimento de
onda, o infravermelho próximo mostra-se como eficiente fonte de luz para iluminar a
estrutura dentária como meio de diagnóstico. Visto que o esmalte sadio promove
baixa atenuação na faixa do infravermelho, e a cárie é um meio espalhador,
observa-se um aumento do contraste óptico entre o sadio e o cariado. Métodos de
detecção de cárie são baseados nas diferenças de espalhamento entre o sadio e
cariado, através da quantificação da onda eletromagnética no infravermelho
transmitida, como publicado em Maia et al., 2011, figura 6 (a) e (b).
39
Figura 6: Secção dentária transiluminada. (a) Laser infravermelho 1300nm; (b) Luz
branca. Fotografia arquivo pessoal.
A absorção é o processo de interação da luz em que o fóton é interrompido
por um objeto e sua energia dissipada para a estrutura. A energia perdida pode ser
convertida e posteriormente transformada em calor, ou em outros comprimentos de
onda de menor energia e maior comprimento de onda. Dentre exemplos na
Odontologia observamos a propriedade de absorção da luz durante a
fotopolimerização da resina composta contendo canforquinona, no clareamento com
géis fotoabsorvedores e na terapia fotodinâmica.
Após a absorção, a energia pode ser emitida em maior comprimento de onda,
através do processo de fluorescência, decorrente da interação da luz com os
fluoróforos da estrutura dentária. A energia é convertida para um nível mais alto de
energia, no qual o elétron permanece por pequeno intervalo de tempo. Em seguida,
o elétron pode decair para um estágio energético menor e liberar a energia
acumulada em um comprimento de onda maior, resultando na emissão da
fluorescência. A estrutura dentária dispõe de autofluorescência, sem necessitar da
adição de substância luminescente (BENEDICT, 1928), e a desmineralização resulta
em perda da referida autofluorescência (BORISOVA et al., 2006), a qual pode ser
quantificada por métodos ópticos.
40
2.4.2 Quantificação da Fluorescência induzida por Luz (QLF)
A aplicação clínica do QLF, traduzido como quantificação da fluorescência
induzida por luz, promove imagens por fluorescência da lesão de cárie, podendo
esta ser quantificada em extensão e perda mineral. Um dos meios de se obter
fluorescência do elemento dentário, é usar uma fonte de luz fluorescente, que para o
QLF foi escolhido azul visível e ultra-violeta, com comprimento de onda em torno de
380nm, visto que o dente quando excitado com ultra violeta, emite fluorescência no
azul, e quando excitado com verde e azul, emite fluorescência no amarelo e laranja
em diferentes intensidades (ANGMAR-MANSSON, TEN BOSCH, 2001).
Como meio de bloquear a reflexão da luz azul, utiliza-se um filtro amarelo,
que proporciona apenas a observação do comprimento de onda emitido como
fluorescência. No entanto, nas regiões cariadas há diminuição da emissão da
fluorescência, ressaltando o contraste entre a região sadia e cariada. A diminuição
da fluorescência da região de cárie ocorre devido a alguns mecanismos: como o alto
espalhamento da luz que impede a penetração da luz para que seja absorvida e
convertida, emitindo consequentemente pouca fluorescência; além do espalhamento
atuar como uma barreira impedindo que a luz penetre até a dentina, responsável por
grande parte da fluorescência; e ainda devido a possíveis alterações moleculares
nas proteínas cromóforas responsáveis pela fluorescência natural do elemento
dentário (ANGMAR MASSON, TEN BOSCH, 2001).
Dessa forma a imagem captada por uma câmera CCD, apresenta-se com um
maior contraste entre o tecido sadio (fluorescente), e o meio cariado espalhador e
com menor fluorescência. Imagem esta que evidencia a lesão cariosa podendo ser
observada facilmente pelo paciente durante o exame clínico, e assim motivá-lo para
melhorar a higiene oral. O sistema permite repetidas análises, sem danos ao
paciente, o que proporciona um monitoramento da lesão ao longo do tempo.
2.4.3 Tomografia por Coerência Òptica (TCO)
A Tomografia por Coerência Óptica (TCO), no entanto mais conhecida pela
sigla em inglês OCT, é também considerada não invasiva e não destrutiva. Baseada
na propriedade do retroespalhamento da luz laser após interação com o material
41
analisado, a TCO utiliza laser no infravermelho 850 nm a 1300nm, sendo o maior
comprimento de onda aplicado para obter maior penetração nas amostras dentárias,
como demonstrado anteriormente (FONSECA et al, 2008). As imagens da TCO são
obtidas através de uma série de processamentos decorridos da interação óptica
básica de interferência entre um feixe retroespalhado com um feixe de referencia.
Por se tratar de um tomógrafo fornece imagens seccionadas de estruturas internas,
em tempo real, e com alto poder de resolução espacial (FUJIMOTO, 2003).
Enquanto imagem tomográfica, a TCO tem sido utilizada para diversos fins, como
avaliação da interface de restaurações (MELO et al., 2005; MONTEIRO et al., 2011),
para o diagnóstico de cárie (FREITAS et al., 2006; MAIA et al., 2010), para análise
de materiais dentários (KYOTOKU et al., 2007; BRAZ et al., 2008), detecção
precoce de câncer oral (JUNG et al., 2005), detecção de cáries recorrentes e
adaptação marginal de restaurações (OTIS et al., 2000) e caracterização de
estruturas periodontais (COLSTON et al., 1998; OTIS et al. 2000).
A configuração óptica da TCO consiste em um interferômetro de Michelson, o
qual requer uma fonte de luz de banda larga de baixa coerência, podendo ser laser
ou diodo superluminescente. Os fundamentos da interferometria avaliam a
sobreposição de ondas eletromagnética em um sistema, com um divisor de feixes de
radiação que pode ser um prisma ou através de fibras ópticas, que divide a radiação
da fonte em duas direções com 50% cada. Metade da intensidade da luz interage
com a amostra estudada, e a outra metade é totalmente refletida por um espelho
móvel, tendo suas características preservadas. A radiação retroespalhada da
amostra estudada carrega os sinais de alterações, e se recombina com a radiação
refletida 100% pelo espelho de referência, sendo então comparadas quanto aos
padrões de interferência quando analisadas pelo espectrômetro. Para melhor
resolução da imagem a ser formada, a radiação passa por uma grade de difração
que divide o feixe de luz para que o sinal seja analisado pontualmente. Deste modo,
diferentes graus de interferência da radiação advindos da estrutura estudada podem
ser observados, dando origem a uma imagem de corte tomográfico, conhecida como
B Scan, a qual é formada por sinais de interferência pontual, conhecida como A
Scan (FUJIMOTO, 2003).
A análise do sinal óptico pode ser quantificada através de processamentos da
intensidade ou do decaimento da luz no tecido analisado. A análise matemática do
42
comportamento da luz observado deve permitir mínima influência das habilidades do
operador, e compensar qualquer deficiência do mesmo (ANGMAR MASSON, TEN
BOSCH, 2001). Considerando a necessidade de proporcionar sistemas cada vez
mais independentes do subjetivismo clínico, buscou-se processar imagens para
calcular alterações do tecido dentário com maior precisão. No caso do esmalte
dentário, observa-se que regiões cariadas que são meio espalhadores, apresentam
um coeficiente de atenuação maior, não permitindo a passagem da luz através da
estrutura.
2.4.4 Microscopia por Confocal de escaneamento a laser (CLSM)
A técnica baseia-se na redução da detecção da quantidade de luz espalhada
através do isolamento do sinal de resposta do foco do scanner. Um microscópio
convencional de fluorescência de amplo espectro coleta luz de todo o volume do
espécime, resultando na aquisição de fluorescência por partes do espécime que não
se encontram exatamente no plano focal que está sendo examinado, o que leva a
uma perda severa dos detalhes da imagem. Um microscópio confocal tem a
habilidade de remover a fluorescência de quase toda a extensão do espécime que
não se encontra no plano focal, resultando em uma imagem mais clara e definida
(SHEPPARD, SHOTTON, 1997).
Como o ponto de foco encontra-se escaneando em torno do tecido, o sinal
detectado deste é usado para construir uma alta resolução (<1micron) de imagem do
tecido (BOYDE, 1985). O foco de luz visível no tecido não pode ser mantido em
profundidades maiores de 300 micrometros, visto que o desafio do espalhamento
torna-se bem maior. Portanto, imagem 3D de alta resolução pode ser obtida por
escaneamento de posições profundas com pontos focais sequenciados, até uma
profundidade limítrofe em torno de 300µm (HILLMAN, BURGESS, 2009).
43
OBJETIVO GERAL
Avaliar e caracterizar a aplicabilidade de técnicas ópticas para qualificar e quantificar
a perda mineral por cárie ou erosão dentária, elucidando a importância do
conhecimento integrado das propriedades ópticas da interação laser-tecido (in vitro).
Objetivos Específicos
� Estabelecer comparações quanto aos métodos de QLF, com a diminuição de
fluorescência, e da OCT, com o aumento do coeficiente de atenuação, na
análise de detecção de cárie precoce no esmalte dentário (artigo I );
� Analisar o sinal de decaimento da luz (A-Scan) segundo o coeficiente de
decaimento da luz para diferenciar o comportamento da luz em áreas sadias
e cariadas quando analisadas pela Tomografia por coerência optica (artigo I );
� Caracterizar a superfície erodida de esmalte através da microscopia confocal
com escaneamento a laser (artigo II );
� Quantificar o efeito protetor do fluoreto de estanho e o fluoreto de sódio, e da
caseína fosfopeptidea associada ao fluoreto de sódio, contra a erosão
dentária através de mensurações de batentes entre a área sadia e erodida,
utilizando as imagens seccionais da microscopia confocal com escaneamento
a laser (artigo II );
� Aplicar a tomografia por coerência optica como método para análise de perfil
ótico, a ser comparado com a perfilometria de contato, na mensuração de
perda mineral entre região sadia e erodida (artigo III );
� Comparar protocolos de múltipla exposição previamente realizados em
ensaios erosivos para estabelecer parâmetros de julgamento quanto ao
protocolo adequado para visualizar o dano real e o efeito protetor do fluoreto
testado (artigo III );
ARTIGO IARTIGO IARTIGO IARTIGO I
45
Dental caries assessment by two optical techniques: Quantitative Light Induced
Fluorescence (QLF) and Optical Coherence Tomography (OCT)
Ana Marly Araújo Maia 1, Anderson Zanardi de Freitas2, Sergio de L. Campello3,
Lena Karlsson4, Anderson Steven Leônidas Gomes5
1 Post-graduate Program in Dentistry, Universidade Federal de Pernambuco – UFPE,
Recife, PE, Brazil.
2 Nuclear Energy Research Institute, IPEN-CNEN/SP, São Paulo, SP, Brazil.
3Post-graduate Program in Material Science, Universidade Federal de Pernambuco –
UFPE, Recife, PE, Brazil.
4Departament of Dental Medicine, Karolinska Institutet, Sweden.
5 Physics Department, Universidade Federal de Pernambuco – UFPE, Recife, PE,
Brazil.
Short title: Dental Caries detection: QLF and OCT
Key words: Dental Caries, Quantitative Light Fluorescence, Optical Coherence
Tomography.
Corresponding Author: Msc. Ana Marly Araujo Maia, Universidade Federal de
Pernambuco, Physics Departament, Av. Prof Luis Moraes Rego, S/N, Cidade Universitária,
Recife, Pernambuco, Brazil. CEP 50670-901; e-mail: anamarlyamaia@gmail.com; Fax: +55-
81-32710359
Abstract
A conservative, non-invasive, or minimally invasive approach to management of
caries lesions requires diagnostic methods which can quantify very small changes in
enamel structure. In this context, optical techniques seem to promote advantages to
minimize subjective diagnostic by the clinical dentist and promoting early interceptive
methods to control caries progression. The aim of this work was to exploit two
important photonics based techniques to characterize alterations between sound
46
dental structure and artificially induced caries lesions in human teeth, through the
loss of fluorescence by Quantitative Light-Induced Fluorescence (QLF) and the
alterations of attenuation coefficient of light signal by Optical Coherence Tomography
(OCT). Six vestibular surfaces of premolar teeth were submitted to demineralization
cycle to develop artificial caries lesions. Then, each tooth was analyzed by QLF and
OCT techniques, detecting average changes and lesion area. Lesion severity in
terms of fluorescence loss and backscattered increase were calculated by
commercial and home-made software, respectively. Samples were then sectioned in
slices (~200µm), and analyzed transversally by optical microscope. Carious damage
showed correlation between images of each system, although comparing the
percentage of alteration, the attenuation of light calculated by OCT image processing
showed difference with high intensity. QLF images are easily obtained, however OCT
images processed by tomographic sections showed higher differences of optical
characteristics between sound and caries regions.
Keywords: Optical Coherence Tomography, Fluorescence, Dental Caries
Introduction
Caries-related clinical decision-making remains a centrepiece of clinical
dentistry. To arrest or reverse the disease process and to intervene before operative
restorative dentistry is needed requires most often an early detection of the carious
lesion. Clinically applicable methods for detection of a very early phase of mineral
loss and quantification of caries lesions have therefore emerged [Featherstone,
2008].
The evolution of biomedical research and industry has developed several
optical techniques that exploit different interactions between light and dental hard
tissue. This development has been possible due to the evolution in the knowledge
about optical properties and interactions inherent to the complex inhomogeneous
dental structures [Darling, 2006]. Enamel consists of approximately 96% inorganic
material, constituting biological hydroxyapatite crystals. Remnants of organic matter
(proteins 0.6%) from the period of development and water (3,5%) are also found in
the enamel [Ten Cate 1994; Ehrlich et al. 2009]. The crystals are clustered together
and roughly perpendicularly to the tooth surface, due to the scattering distributions
47
are generally anisotropic and depend on tissue orientation relative to the irradiating
light source in addition to the polarization of the incident light [Fried, 1995; Zijp, Ten
Bosch, 1993; Zijp, Ten Bosch, 1998].
One optical method for an objective assessment of early incipient changes in
the enamel mineral content is the quantitative light-induced fluorescence (QLF). The
method is based on a visible blue-green light system with an excitation wavelength of
370 nm that is applied to the enamel. The resultant auto-fluorescence is detected by
filtering out the excitation light using bandpass filter at >540nm by a small intra-oral
camera [Pretty, 2006]. The high sensitivity of QLF has been confirmed in several
studies [Tranæus et al., 2002; Gmur et al., 2006] and the method has been rapidly
adopted as a standard reference measure in clinical tests of the efficacy of
preventive measures [Pitts and Stamm, 2004], with established correlation between
the mineral loss and the fluorescence loss in enamel demineralized.
The other system evaluated, Optical Coherence Tomography (OCT) consists
of an emerging diagnostic method for creating nondestructive cross-sectional
imaging of internal biological structures due to the scattering and absorption of laser
light [Huang et al., 1991; Fujimoto, 2008]. The light source used are near-IR lasers or
broadband incoherent radiation sources from 780 to 1550nm that offer a great
potential for optical imaging modalities also in dentistry due to the weak scattering
and absorption in dental hard tissue [Hall and Girkin, 2004].
Optical coherence Tomography allows several comparisons between sound
and caries lesions enamel. For instance, caries lesions can be detected by the
reduction in enamel reflectivity [Amaechi et al., 2003]; by the increase of scattering in
the analyzed image [Maia et al., 2010]; by the mineral loss correlation with the
increase in reflectivity [Douglas, Fried, Darling, 2010]; or by the refractive index
alterations [Hariri et al, 2013]. It also has been demonstrated that PS OCT, which
stands for polarization sensitive OCT, can be more efficient than conventional OCT,
even though the last one has also potential to detect early demineralization [Douglas,
Fried, Darling, 2010]. Furthermore, other researchers have compared carious
surfaces by changes of attenuation coefficient of signal light exponentially decay
[Popescu et al., 2008; Cara et al., 2012].
48
The aim of the present study was to analyze the correlation between the loss
of fluorescence by QLF and the alterations of attenuation coefficient of light signal by
OCT, in artificially caries lesions. OCT cross sectional images were analyzed by
attenuation coefficient and a new map transverse to caries lesions were generated
promoting comparison between QLF and OCT images.
Materials and Methods
Ethics
As the biological material comprising the study sample could not be traced to
an individual donor, the regional Ethics Committee in Stockholm, Sweden determined
that the study was not subject to the law of ethical approval (2006/3:4). Eight intact
premolar teeth, extracted for orthodontic reasons, were collected and stored in
saturated thymol saline under refrigeration before the experiment. Extrinsic deposits
were gently removed with a soft toothbrush and water, and the teeth were thereafter
photographed with a digital camera (COOLPIX 4500, Japan), to detect cracks or
other inhomogeneity on the buccal surface. Two of them were excluded from the
study because of crack findings.
Demineralisation procedure
Each of the 06 samples were embedded in wax leaving a 2x3 mm open
window on the buccal surface, and artificial caries lesions were created on all
samples using a demineralising solution (pH=5,0) described by Buskes et al. [1985].
The solution also contained protective agents as 2–50 µM MHDP
(methanehydroxydiphosphonate), which leads to the formation of subsurface lesions
and inhibits demineralization in vitro. The waxed teeth were placed in separate small
tins filled with a demineralising solution and placed in an incubator (Electrolux,
Sweden) in 37°C. The solution was replaced every th ird day for 9 days, but due to
natural anatomical structure, different stages of non-cavitated artificial incipient
carious lesion on smooth surfaces was produced. The teeth were rinsed with ionized
water at the occasion of replacing the solution.
49
After the artificial caries induction procedure, the wax was removed from the
teeth and cleaned with deionized water. The artificial white-spot lesions were
investigated by optical techniques quantifying: visible light reflected and trans-
illuminated by an stereomicroscope, levels of fluorescence by Quantitative Light-
induced Fluorescence (QLF™) and intensity of backscattering by the Optical
Coherence Tomography (OCT). In the final step each sample was transversally
sectioned and evaluated by polarized optical Microscopy to confirm lesion depth.
Experimental set up
Quantitative Light Induced Fluorescence (QLF)
The artificial caries lesions were examined automatically by the QLF
commercial software. The sample was illuminated by violet-blue light (wavelengths
290-450 nm, average pick 380 nm) from a handpiece, and image was obtained using
a camera fitted with a yellow 520-nm high-pass filter. The filter is necessary to
capture only the wavelengths emitted by the fluorescence, and blocked all violet-blue
light reflections of surface (QLF; Inspektor™ Research Systems, Amsterdam, the
Netherlands). The image was captured, saved and processed.
The image was digitally stored on a computer for analyses. The difference
between fluorescence intensity values gives three quantities; ∆F (average change in
fluorescence, %), lesion area (mm2), and in later versions of the QLF software, ∆Q
(area x ∆F), which gives a measure of the extent and severity of the lesion, but is not
extensively used. The average loss of fluorescence, highlighted through color’s
degree of yellow (high fluorescence loss), red, pink and purple (low fluorescence
loss), was observed and dimensions calculated. The parameters ∆F and lesion size
were obtained, first to objectively support/confirm the presence and the extent of the
white-spot lesion. The image was stored, and the spot within the lesion with highest
loss of fluorescence was used as a reference for the subsequent analyze.
50
Optical Coherence Tomography (OCT)
A commercially available OCT system was used (Spectral Radar SR-OCT: SR
930/Thorlabs, New Jersey, USA), operating in the spectral domain using a
superluminescent diode (SLD) light source with central wavelength of 930 nm. This
system consists of three main parts: a handheld scanning probe, a base unit that
contains the SLD light source and a personal computer (PC) (Figure 1).
Figure 1: The commercial SR-OCT, OCP930SR, schematic diagram (adapted from
Thorlabs New Jersey, USA).
The whole system is based on fiber optics couplers to direct the light from a
broadband SLD source to the Michelson interferometer, which is located inside the
handheld probe. After that, the light that travels back from sample and from the
reference mirror, goes through the same fiber to the spectrometer and the image
sensor located in the base unit. The base unit was connected to the PC, which was
equipped with two high-performance data acquisition PC-cards.
51
OCT image acquisition
The system was configured to save images in automatic model, as stream
mode, making possible to capture about 2.3 frames per second. Some other
parameters were set under the following conditions: files saved as numeric array
matrix; images composed by 2000 columns and 512 lines, providing a pixel
resolution of 3µm x 2.88µm. Each cross-sectional image is a tomogram, known as
the “B-scan”, with 6µm of transversal resolution and 4µm of axial resolution
composed of several “A-scans”, along line produces information from a 'slice' of tooth
tissue.
The handheld scanning probe from the OCT system (SR-OCT
930nm,Thorlabs) was firmly fixed into a stand perpendicular to the floor, and each
sample was positioned in a micrometer translation stage controlled by a Motor Move
system, about 0.5mm/s (Figure 2.a). Each sample had the surface scanned, through
4mm from mesial to distal on tooth surface, counting about 200 B-scans of 6mm
cervical-incisal, totalizing an scanned area around 24mm2 captured in 8 seconds. All
these B-scans were processed making possible to project data into a 2D map,
named as C-scan, as schematically shown in Figure 2.b.
Figure 2: (a) The handheld scanning probe from the OCT system (Thorlabs)
and the tooth in a micrometer translation stage controlled by a Motor Move system,
about 0.5mm/s; (b) off-axis images, A-Scan, B-Scan and C-Scan images.
52
OCT Image Processing
A software was developed in Labview specifically to analyze/calculate the
attenuation coefficient of all A-scan of each image. This procedure develops an
attenuation coefficient bi-dimensional map of analyzed area, named C-Scan. Each
new processed map gives information of particularly optical characteristic of the
internal tissue structures evaluated. The whole sequence described can be explained
by the block diagram below:
Figure 3: Diagram of the whole sequence processing.
Several B-Scan images were analyzed through each A-Scan, by the
calculation of the attenuation signal that verify the distribution of the scattering of light
that penetrated into each analyzed sample. The inherent curvature of the tooth
surface was corrected graphically for each image, by aligning each A-Scan using the
peak of the reflection of enamel/air interface as reference. The peak was excluded
from attenuation coefficient calculation, as it represents only the abrupt change of
refraction index between air and enamel. For the new C-Scan image, each B-scan
represents one line and each A-Scan only one point that shows the value of
attenuation coefficient, as an artbitrary number. The data which compose the C-Scan
image was obtained by fitting its curve with a Beer-Lambert type function:
�(�) = ��� + ��
where Y(x) is the OCT signal intensity, µ the attenuation coefficient, and x is the
depth of light penetration (Cara et al., 2012;). The “x” number of points included on
the exponential curvature line was 40 µm below reflect peak, and also the last 500µm
was excluded as is a noise region. The points used to fit the exponential curvature
line should be described as the value of coefficient can be modifiable. The C-Scan
image was composed of the value of attenuation signal coefficient, of a total of 2000
53
A-scans (columns), from each B-scan. Through 6mm to be scanned, approximately
200 B-Scans were necessary, generating images that after process represented the
new lines of the C-Scan image.
To better understand optical principles by OCT signal and images, it is
important to consider that the system detector cannot identify huge difference
between in homogenous material that has high absorption or high transmission. So a
high attenuation coefficient signal can also represents loss of signal light by
transmission. This fact enforces how important is to previously than OCT analyze,
have samples optically characterized.
Stereomicroscope
As a complementary analysis simple images were captured by a
stereomicroscope (magnification X10, Olympus), using the reflection light of the
system, and also the transmitted extra light perpendicular to surface captured. These
images added information about macroscopic effects of light properties of reflection
and trans-illumination, between soud and caries regions.
Optical Polarized Microscope
After all techniques evaluation described, sections of each tooth were
prepared by the Low Speed diamond Whellsaw, model 650, SBT inc., with water
irrigation. The sections were obtained by cutting sample perpendicular to the buccal
face. Selected sections were then ground using grinding stones until the required
thickness of 200µm. The depth of caries lesions was observed under 50X of
magnification using a transmitted Polarized Light Microscope (Olympus, USA) and
through the measurements software it was possible to measure the real value of
lesions depth, used as gold standard technique.
54
Results
The map reconstructed by the value of attenuation coefficient of OCT images
permitted both quantitative and qualitative comparison with the QLF technique. As a
representative guide, images by visible light were captured for better samples
evaluation, as in figure 4. Comparing the lesion extension by both techniques, it was
observed that OCT as a tomographic mode of capture showed better resolution and
delimited contours.
Figure 4: a) Image obtained by reflection visible light; b) Image obtained by
transilluminated visible light; c) Image from QLF software; d) The C- Scan image
processed.
OCT processing model presented in figure 5 represents the calculation of light
behaviour in each A-Scan. The value of attenuation coefficient excluded data of the
peak first interference and it value consist an arbitrary number, although it is possible
to compare the light behavior of the light on structure and shows difference between
values for sound and carious points of the same sample.
(a)
(b) (c) (d)
55
Figure 5: Image reference and A-scan showing the decrease of light in
evaluated by exponential decay, illustrating the alterations of attenuation coefficient
on carious signal. Fitting in decay signal curve of sound tooth (green line) and
carious region (blue line).
Similar to QLF software, for each teeth structure analyzed, it was created a
particular scale of colors through coefficient value with different intensity of colors
between orange, yellow and blue. Considering the loss of fluorescence and the
variation of the attenuation coefficient, it was possible to compare intensity of
damages. Results from QLF were obtained by commercial software, and new
parameters were established for OCT software, as presented in table 1.
Table 1: Data of fluorescence Intensity reduction (%) and attenuation
coefficient increase (%) of each sample.
Sample Lesion Area (mm2) Fluorescence Intensity
Reduction (%)
Attenuation coeficiente
increase (%)
A 16.1 -28.3% 116%
B 20.7 -34.2% 187%
C 18.5 -27.2% 261%
D 7.79 -19.7% 239%
E 10.2 -13.3% 161%
F 4.25 -11.9% 109%
56
QLF images identified caries lesions by the contrast of these areas that showed
loss of fluorescence of 21.75% due to the increase of scattering and decrease of
absorption. Although OCT images after data processing identified caries lesions by
an increase of 178% on the value of attenuation coefficient µ. Cross sectional (B-
scan) OCT images also shows that the higher attenuation of caries also represents
an increase of reflectivity due to the increase of scattering in the first points of A-Scan
signal, although the last points of the A-Scan signal shows that high attenuation of
caries lesions, as observed when the enamel-dentine junction is near surface caries
lesions doesn’t allow capture of this structure, as observed in figure 6.
Figure 6: (a) Enamel surface and dentine enamel junction (DEJ); (b) same region
after artificial demineralization, not possible to see DEJ below carious surface.
Highlighting the optical principles, the tomographic technique based on
backscattering, analyzed by B-Scan images (figure 7), allows measurements of how
deep the light is still scattered by caries alterations on surface and subsurface,
comparing sound and carious enamel. Measurements of caries lesion depths
showed value about ~130micrometers.
Figure 7: (a) Polarized optical microscope image of 200um section; (b) Tomographic
B-Scan image of the caries lesion.
57
Discussion
In this paper it was shown how optical methods offer advantages to observe
and characterize the dental structure, mainly because of translucence, crystalline and
regular structure of enamel, which has specifically interactions with radiation from UV
to IR. Consequently, early alterations, as carious lesions, can be detected by
alterations of reflected, back-scattered and absorbed light.
QLF and OCT optical fundamentals are interrelated because both are based
at first on the increase of light scattering by caries lesion, which is much stronger
than in sound enamel [Pine, ten Bosch, 1996]. In general, scattering causes the light
path in the lesion to be much shorter than in sound enamel [Angmar Manson, 2001],
although physics fundamentals of interactions between light and material depends on
wavelength [Darling et al., 2006]. Used as a map guide of lesion, macroscopic
images captured by visible light were just observed by contrast and brightness. And
as expected, reflected visible light image shows the worst contrast and highest
brightness, what made difficult detection of details. Instead of transmitted visible light
that showed better contrast, without brightness, and also definition of caries lesions
contour extensions. However, it is important to consider that as the tooth has been
already sectioned, light transmission was more effective, as was demonstrated by
Karlsson and co-authors using IR laser [2010].
The commercial QLF system analyzes samples by blue and UV light to excite
yellow or orange fluorescence, and its physical principles requires absorption to
consequently emit fluorescence. As commented before, regions of demineralization
present higher porosity that increases scattering and decrease absorption, showing
the first sign of visual appearance as small white spot [Arends, Christoffersen, 1986].
So demineralized areas analyzed by QLF technique, observe the same increase of
scattering previously described, that decrease absorption resulting in a consequently
loss of fluorescence, that is quantified proportionally.
OCT system has advantages to be applied on enamel structure, due to the
source that in general is a laser near infrared (830nm ~ 1300nm). This wavelength is
a great option because of the low absorption of light by enamel structure close to
58
1300nm [Maia et al., 2010]. Images of sound enamel surfaces generally shows low
scattering and high transmission of laser, although as commented before, the A-Scan
signal of high transmission or high absorption is similar when analyzed by OCT
system.
In this paper, our images were analyzed by optical attenuation coefficient of
light signal received by OCT system detector. Excluding the interface peak of
reflections it was possible to better understand what exactly happen when light
penetrates the sample. It was observed by the exponential decay of signal that on
carious surface the light decreases faster due to increase of scattering that limits the
observation of deeper structure below carious. The effects of attenuation difference
between carious and sound teeth promoted an interesting contrast image. However
this difference of light attenuation can be also observed in a depth observation of B-
Scan with the presence of another interface below enamel, as the dentine-enamel
junction, that it is easily observed through sound regions and not observed below
caries lesion, due to the increase of scattering.
One critical point on detection of caries disease by computer techniques
based on images is the high definition and resolution of alterations. QLF and OCT
were applied to detect artificial caries lesions, the first technique captured and
compared alterations of fluorescence induced by blue light coupled to an CCD
camera, but OCT, as a tomographic technique used property of laser coherent to
detect and quantify increase of light backscattered. OCT is a punctual capture
technique, what explain better resolution, although more complexity of image analyse
and high cost.
Nowadays, OCT system also generates 3D images that can be also named C-
Scan and en face images, what allows a direct visualization of the caries lesion. In
this research, en face images obtained through OCT systems were based on the
attenuation coefficient of the A-Scan signal information. Caries surface has been
analyzed and interpreted by different physicals and mathematics concepts, as
changes of reflectivity intensity [Amaechi et al., 2003; Le, Darling, Fried et al., 2010],
increases of scattering [Maia et al., 2010], through optical attenuation [Popescu et al.,
2008], but the best evaluation is still not established. All these calculations analyze
59
has the same aim to facilitate detection of caries of mathematical parameters what
also avoid the subjective factor from professional evaluation.
Another point that should be discussed is the ability to determine the depth of
lesion, as a tomographic technique, OCT B-Scan section, allows measurements of
lesion depth, as demonstrated in figure 6 a and b. Comparing a B-Scan image with a
histologic section of teeth by polarized optical microscope, it was observed that
superficial lesions of 150µm can be delimited by OCT. Even for OCT technique, the
high increase of scattering prejudice image details, as low light come back to CCD
detector. In the other hand, QLF based on parameter Delta Q, analyze depth by
volume, but is not very useful, as it estimate the severity of the caries lesion,
comparing caries lesion as a cylinder (geometry), which is not likely the way a caries
lesion develop in shape.
Most of the techniques developed provide static information of the caries
lesion, but the evolution of techniques to detect enamel caries as a dynamic
phenomenon hasn’t stopped, as lesion progress and repair are strongly time-
dependent [Arends, Christoffersen, 1986]. So the decision to remineralize or restore
a caries lesion depends also on the depth of lesion, and OCT system, with non-
destructive morphologic studies, using contour lines, longitudinal sections, or depth
scattering seems to be an alternative to monitor caries lesion configuration.
Conclusion
The more comprehension of a technique better results can be extracted, data
calculation of light behavior avoid subjective judgment about the presence of early
caries. Both techniques even divergent are connected by optical scattering
alterations of caries lesions, and OCT showed better accuracy when analyzed by
attenuation coefficient to differentiate sound and carious surfaces.
Acknowledgement
The authors acknowledge the support of PRONEX/FACEPE/CNPq CNPq grants.
60
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ARTIGO IARTIGO IARTIGO IARTIGO IIIII
64
Enamel erosion and prevention effects characterized by
Confocal Laser Scanning Microscope
Maia, Ana Marly Araújo1; Longbottom, Christopher2; Gomes, Anderson Stevens
Leonidas3; Girkin, John Michael4
1PhD student, Dentistry Department, Federal University of Pernambuco, Recife,
Brazil
2PhD, Centre for Clinical Innovations, Dundee University, Dundee, UK
3PhD Professor, Department of Physics, Federal University of Pernambuco, Recife,
Brazil
4PhD Professor, Department of Physics, Durham University, County Durham, UK
Short title: Eroded enamel characterized by CLSM
Key words: Tooth Erosion, Confocal Laser Scanning Microscopy.
Corresponding Author: Msc. Ana Marly Araujo Maia, Universidade Federal de
Pernambuco, Physics Departament, Av. Prof Luis Moraes Rego, S/N, Cidade
Universitária, Recife, Pernambuco, Brazil. CEP 50670-901; e-mail:
anamarlyamaia@gmail.com; Fax: +55-81-32710359
Abstract
Background: Fluoride toothpastes have shown caries protective properties, but their
preventive effect against erosive/abrasive enamel wear is unclear. Aim: To evaluate
the erosion-inhibiting effect of two fluoride toothpastes on the development of
erosion-like lesions, measured using a Confocal Laser Scanning Microscope
65
(CLSM). Materials and Methods: Forty human enamel molar blocks were mounted in
acrylic, and divided into five groups (n=8), in accordance with following specifications:
G1: Reference control; G2: CPP-ACP NaF (S); G3: CPP-ACP NaF (TB); G4: SnF2
(S) and G5: SnF2 (TB). Before starting the erosion protocol, specimens were
immersed in artificial saliva (Orthana S.A.) for 24 hours, then submitted to an erosion
challenge from citric acid (0,5%, pH=2,8), for 5 minutes, 6 times a day. The
specimens were exposed to slurries of the toothpaste under test (3ml of a 1:3
water/paste mixture) twice daily for two minutes, after the first and last erosive
challenges, in an attempt to mimic human daily dentition exposure. The samples
from the groups treated with tooth brush (TB), were abraded, for 30 seconds during
the two minutes of immersion, using an electrical toothbrush (Phillips Essence) with a
force of approximately 2 Newtons. The enamel surfaces were characterized for their
morphology and fluorescence emission using CLSM images, with mineral loss being
measured using the resulting 3D images referenced to an un-challenged portion of
the sample. Step values were compared using the One Way ANOVA test. Results:
CLSM was shown to be a viable, non-contact and simple technique to characterize
eroded surfaces as well as show changes in fluorescence due to differences in
scattering in the enamel, and the protective effects of treatments. The statistical
difference in the step size was significant between the groups (p=0.001) and using
multiple comparisons a statistically significant protective effect of SnF2 (p=0.001) and
CPP-ACP NaF (p=0.041) was shown when these were applied as toothpaste
slurries.The SnF2 and CPP-ACP NaF solutions treatments showed similar efficiency
(p>0.05), although groups submitted to tooth brush showed mineral loss similar to
reference control group, due to the damages of abrasion associated.
Introduction
There is evidence that the prevalence of dental erosion is steadily increasing
[Jaeggi and Lussi, 2006] and its management has becoming an important aspect of
the long-term health of dentition around the world. The management of dental
erosion consists of prevention, avoiding risk factors when possible, and therapy to
prevent irreversible damage from occurring. In the light of the difficulties involved in
66
clinically detecting, monitoring and managing dental erosion, researchers are actively
searching for new agents for the prevention, or repair, of dental erosion lesions and
recently several strategies have been tested aiming to limit enamel erosion
[Huysmans, et al 2011; Moretto et al, 2010; Ranjitkar et al., 2009, Rios et al., 2006].
It has been shown in vitro that fluoride treatments, such as sodium fluoride,
amine fluoride or acidulated phosphate fluoride, form CaF2-like layers on the tooth
surface, which is unlikely to provide a preventive effect against erosion, as an acidic
drink will rapidly dissolve the accessible CaF2 and remove traces of any previous
topical fluoride treatment [Larsen and Richards, 2002]. In recent years several
research groups [Ganss et al., 2011; Wiegand et al., 2010; Schuelter et al, 2009;
Rees, Loyn, Chadwick, 2007; Magalhaes et al., 2007] have investigated the
preventive effects of different fluoride formulations on dental erosion in order to
identify preparations or compounds that form precipitates other than CaF2-like layers.
Agents based on milk products have been investigated for many years and
currently, several different paste formulations are available as variations of Tooth
Mousse (GC Tokyo, Japan), which is based on a nano-complex of the milk protein
casein phosphor-peptide (CPP) with amorphous calcium phosphate (ACP). CPP
binds to form nano-clusters of ACP preventing their growth to the critical size
required for nucleation and phase transformation [Reynolds et al., 1998]. The
complex compound thus formed has demonstrated preventive and re-mineralization
properties in the caries process [Reynolds et al., 1999]. It has been claimed that
CPP-ACP promotes a supersaturated state close to dental hard tissues, making
remineralisation of surface enamel possible [Rahiotis and Vougiouklakis, 2007].
One other agent that has shown promise under both mild and severe erosive
conditions is the stannous ion [Tinanoff N, 1995, Ganss et al, 2004]. The application
of tin-containing solutions leads to deposits on the tooth surface [Willumsen et al,
2004; Hove et al, 2008] and there are indications that these deposits are relatively
resistant to acid dissolution [Hjortsjö et al, 2008]. It is known that the stannous ion
reacts with pure hydroxyapatite [Young et al., 2006; Schlueter et al., 2007] on the
surface of the dental hard tissue [Willumsen et al, 2004], resulting in reduced
solubility of hydroxyapatite or enamel [Tinanoff N, 1995].
67
While good oral hygiene is of proven value in the prevention of periodontal
disease and dental caries, frequent tooth brushing with abrasive oral hygiene
products may enhance tooth damage [Lussi et al, 2011]. Several studies have shown
that softened enamel (such as that caused by acidic drinks) is very susceptible to
scratching [Eisenburger et al., 2003; Jaeggi, Lussi, 1999; Lippert et al., 2004], is
highly unstable and can be easily removed by short and relatively gentle physical
action [Eisenburger et al., 2003]. Tooth brushing of eroded enamel thus leads to
minor changes in its surface morphology and mechanical properties [Lippert et al.,
2004].
The structural changes resulting from different challenges and anti-erosive
treatments can be studied by qualitative methods, such as optical or electron
microscopy, which can be used either alone or combined with quantitative
measurements [Schlueter et al., 2011]. Confocal laser scanning microscopy (CLSM)
is a non-destructive 3D technique commonly used in biological imaging, capable of
producing high-resolution images, by scanning the surface with a highly focused
laser beam and using the principle of confocal imaging to reject light returned from
out of focus layers, thus effectively optically sectioning the sample [Sheppard,
Shotton, 1997]. This has been recently applied to the analysis of eroded enamel
surfaces to assess quantitative of tissue loss [Heurich et al, 2010]. The advantages
of CLSM are the high resolution (sub micron) images which are similar to low
magnification Scanning Electron Microscopy (SEM) but without any of the problems
of specimen preparation [Field, Waterhouse and German, 2010]. Systems are
routinely capable of imaging at in excess of 10 frames per second and thus rapidly
record the surface topography allowing quantification of the interface step between
an eroded area and a sound enamel reference.
Therefore, the purpose of the present study was to perform an in vitro
evaluation of enamel surfaces subjected to citric acid attack and to quantify the
erosion-inhibiting and/or re-mineralising potential of specific anti-erosive agents
applied with and without toothbrush abrasion. We sought (a) to visualize the enamel
structure using confocal laser scanning microscopy and then (b) to evaluate the
extent of any material deposition and incorporation to the anti-erosive effect of the
test regime/paste.Materials and methods
68
Sample preparation
Permission was granted by the Ethical Committee of the Federal University of
Pernambuco - Recife PE, according to approval form (038/2010), and 20 third molar
teeth were acquired from the tooth bank from the same Institution. All teeth were kept
in 0.05% Chloramine T for 1 week for disinfection and stored in a humid environment
during the experimental stages. The selection criteria of included the absence of
caries, cracks, fractures, grooves or surface decalcification under visual observation
in natural light. Forty transverse-sectioned enamel specimens were prepared from
the facial and/or lingual surface of the freshly extracted molars.
Each sample was embedded in acrylic resin and the natural surfaces were
ground flat in a water-cooled mechanical grinder and carefully polished with
sandpaper of decreasing grit (600 and 1200) until the preparation resulted in an
experimental surface area of at least 4 x 4mm2. Final polishing was performed with a
felt disk and diamond suspension in water (5 µm) to produce a completely smooth
test surface.
Specimens were randomly divided into five groups initially [n=8]. Each sample
was attached to a single holder and around one third of the experimental area of
each specimen was covered by waterproof transparent adhesive tape (3M) to protect
the reference area of un-etched enamel from the test regimens. To permit
simultaneous immersion of all samples in the solution the teeth were attached by
wire to the caps of the falcon tubes into which the test solutions were placed. The
caps were then attached to a rod so that the samples could be inserted and removed
from the solutions simultaneously.
Erosion Cycle
Before the erosion cycle, samples were soaked in commercially available
artificial saliva (A.S Orthana Saliva) for 24 hours. All specimens (Groups 1-5) were
subjected to a cyclic demineralization and remineralisation procedure, with six
demineralisation periods per day (5-min each; 0,5% citric acid, pH 2.8, anhydrous
citric acid; Merck), as an adaptation of the methods of Schlueter et al, [2009]. There
was a gap of around one and a half hours between each immersion and this cycle
was repeated over three days.
69
The control reference samples were submitted only to cyclic demineralization
and reinsertion in artificial saliva pH=7,0, for one and a half hours between acid
attacks to simulate the oral environment. After each immersion, the specimens were
taken out from the solution, and carefully washed using deionised water for 30
seconds to remove any residual acid or saliva.
Two preventive products were used: Casein Phosphor Peptide Amorphous
Calcium Phosphate (CPP-ACP) plus Sodium Fluoride 900ppm available in GC MI
Tooth Mousse Plus (Mint flavour, Recaldent, UK); Stannous Fluoride 1100ppm and
Sodium Fluoride 350ppm available in Oral B Pro Health (Proctor & Gamble,
Weybridge, UK).
To compare the best treatment for the control or arrest of erosion based
lesions, samples were subjected to the preventive solution cycling treatment. The
toothpastes under test were combined into slurries in 1:3 ratio of deionized water and
the slurries placed on the samples after the first and last erosion period each day, for
2 min on each occasion. Specimens of two groups were also submitted to abrasion
by tooth brush. For the abrasion test, groups were also brushed for 30 seconds
within the slurry during the two minute immersion time, using an electrical toothbrush
fixed in a mechanical set-up to control the brushing force to 2N.
Procedures were started in the morning, with the erosive solution renewed at
the beginning of each day. The pH of all solutions was measured and controlled on
each experimental day. All procedures were performed, avoiding agitation, at room
temperature (20°C).
CLSM Measurements
Each sample was analysed by CLSM after the three days of erosive and
preventive regime. Moving the microscope objective through the optical axis, it was
possible to produce successive focal optical section at 1 micron step intervals and
thus reconstruct a 3D image of the tooth. From the image stack it was then possible
to quantify the height differences between the eroded and reference area.
70
The images were taken using a Nikon D-Eclipse C1 confocal microscope, with
a 405 nm, 25 mW laser used to illuminate and excite the tooth samples with the
power on the sample limited to around 100 µW. Fluorescence from the sample was
detected in two different channels: blue (515-530 nm) and green (590-650 nm). An
apochromatic 60x water dipping objective lens with an NA of 1.0 was used unless
otherwise stated and optical sections were recorded at 1 micron depth intervals
(accuracy of depth sections being +/- 50 nm). From the resulting image stack
measurements could then be made on the height differences between the eroded
and un-damaged areas along with a qualitative assessment of the surface finish of
the samples.
Images analysis
Images were plotted using Image J public domain software
[http://rsbweb.nih.gov/ij]. The individual image slices were initially combined into an
image stack for each series of confocal sections and the resulting stacks then used
for the subsequent image description. Using the 3D reconstructed images the XZ (Z
being defined as into the tooth) profile was examined and the average height
difference between the eroded and un-eroded sections measured. No other image
processing was undertaken on the images and the standard “autumn” look-up table
is used in all images presented.
Statistical Analysis
Data were organized into an Excel spreadsheet (Microsoft Office 2007) and
analyzed using SPSS 13.0 (Statistical Package for the Social Sciences, Chicago,
USA) for Windows. Statistical tests were guided after a Komogorov-Smirnov test was
used to evaluate the normality of the data. The One Way ANOVA test was performed
for comparison among groups. All tests were applied with 95% confidence.
71
Results
Around 350 images were analyzed and typical images are shown below in
figure 1. Figure 1a, shows a representative sample of a polished area of sound
enamel (areas under the protective tape) with the surface appearing quite smooth,
the prisms and organic matrix not well defined. Both the returned fluorescence and
reflected light shows little scattering even to a depth of around 25 microns below the
surface. The absence of clear enamel structure seems to correspond to the
aprismatic layer of enamel produced during the polishing procedure.
Figure 1: CLSM typical images of sound enamel surface (a); soft eroded surface (b);
and areas of aggressive eroded surface (c). Figures (d), (e) and (f) show XZ sections
taken from the reconstructed images from samples a, b and c, respectively.
The typical appearance of eroded enamel submitted only to citric acid attack is
shown in figure 1b and 1c. Samples subjected to the preventive treatments of
toothpaste slurries showed differences in the resulting enamel morphology. As
shown in figure 2a, the sample treated with CPP-ACP NaF demonstrated a similar
appearance to the control-eroded group (Figure 1c), with areas of mineral loss,
though the XZ section is perhaps not as rough. Samples subjected to toothpaste
slurries containing SnF2 present a lower level of fluorescence (compared to the
control eroded sample) and it appears as though a thin layer of stannous fluoride is
covering the enamel surface. The layer is not uniform and appears as a series of
swirls, as observed on figure 2b.
72
Figure 2: CLSM of typical enamel surfaces treated with solutions; a) G2: CPP-ACP
NaF Solution; b) G4: Oral B SnF2 Solution; c) G3: CPP-ACP NaF Tooth-brushed; and
d) G5: Oral B SnF2 tooth-brushed effects. Below each image XZ sections taken from
the reconstructed images.
In the groups where samples were abraded for 30 seconds during the
toothpaste slurry treatment, it was possible to distinguish toothbrush effects on the
eroded enamel. In the CPP-ACP NaF group (G3) lines of brushing in specific
directions can be observed, with greater mineral loss at the top of enamel rods,
leaving the rod boundary well defined (figure 2c). Samples submitted to tooth
brushing during the Stannous Fluoride treatment showed brushing effects, as a
mixed appearance with areas of etched prisms combined with areas where a surface
layer appears to cover enamel (Figure 2d).
73
As can be seen in figure 3 at the interface between the exposed and protected
enamel during the acid attack a significant step develops. Visually it is clear that the
stannous containing compound alone (with no tooth brushing) shows less damage
than the samples protected by tooth mouse and no protection.
Figure 3: CLSM image on XZ section representative interface of each tested group at
the end of cycle regime.
In order to quantify these visual differences the average height change was
measured using the XZ projection. Table 1 shows the average height change for
each group, together with the standard deviation and percentage mineral loss. The
statistical difference in the step size was significant between the groups, as
demonstrated by One Way ANOVA test (p=0.001). The Mann-Whitney test analyzing
by multiple comparisons showed statistically significant protective effect of SnF2
(p=0.001) and CPP-ACP NaF (p=0.041) when applied as toothpaste slurries.
74
Table 1: Tissue loss (µm) in all groups (mean + SD) after three days of in vitro
demineralization and relative mineral loss (percentage of control group).
One Way ANOVA test p=0.001.Data sharing the same superscript letter are not
significantly different.
Discussion
This study aimed to elucidate the effects of two different fluoride toothpastes
on dental hard tissue using confocal laser scanning microscope and quantifiable
differences were recorded in line with the expected findings. At present there is no
generally accepted standard protocol used in erosion studies in vitro, nor a previously
reported reliable method of quantifying mineral loss non-destructively. A
representative acidic challenge was necessary to promote alterations and facilitate
demonstration of the effects of preventive agents. Therefore, an immersion time of 5
min cycles was selected to simulate clinical conditions, though the precise timings
may require further optimisation. The erosive cycling model can be considered to be
of medium severity, with for a daily exposure of 30 minutes - this was repeated for
three days.
The confocal images provide some evidence of the processes taking place
during the etching cycles. The initial polishing of the samples, to produce a uniform
starting point left, as anticipated, an aprismatic area, as observed on sound reference
region. However, the results of eroded surfaces show variations within the group, in
which, even allowing for the same etch time, the emerging enamel rods have
different shapes. This variation can be partly explained by the nature of the enamel
rod following an S-shape course on the horizontal plane from DEJ to the surface.
Control
Group
CPP-ACP NaF
(S)
CPP-ACP NaF
(TB)
SnF2 + NaF
(S)
SnF2 + NaF
(TB)
Average Height
Loss
15.3 +4.8A 9.3 +4.9
B 14.2 +6.8
A 4.6 +1.3
B 10.3 +3.1
A
% Reduction of
enamel Loss
- 39.2% 7.2% 70% 32.7%
75
When the enamel specimens are prepared, the grinding should ideally occur at 90° to
the enamel rods in order to achieve an evenly etchable surface [Hjortsjo et al., 2010].
Due to the nature curvature of the teeth this may not always occur when an area of
around 3 x 3 mm is required, hence the alignment of the exposed enamel rods in the
various samples may be different, thus affecting their etch susceptibility and their
response to fluoride treatment.
The eroded surface showed a clearly visible increase in fluorescence, with
areas of mild alteration of the enamel organic matrix, evidenced by the more
apparent ‘honeycomb’ morphology. In areas where the eroded enamel prisms were
clearly exposed, the ‘honeycomb’ morphology was better defined, with an apparently
greater loss of organic matrix. Erosion of the rod boundary appears as a lower level
of detected fluorescence from around each prism. The interaction of the light with
such microscopic surfaces with large changes in the refractive indices of materials is
complex and the exact reasons for the appearance of features is open to debate but
it is clear that the method of fluorescence confocal microscopy clearly shows the
changes in surface morphology. Surface roughness can also be confirmed through
the transverse section image and this can again be quantified using more advanced
image processing methods than used in these preliminary measurements.
Previously, studies have assumed that tissue loss values of the order of 10-15
µm compared to the negative control group are sufficient for demonstration of
differentiation of agent effects [Ganss et al., 2012]. In this study, the erosive
procedure was more intense, due to an acid etch time of five rather than two minutes,
and the cycles completed in 3 days rather than 10. However, this resulted in a step
height of 15.3µm (+/-4.8) of similar magnitude to the previous slower etch and
perhaps more suited to a high throughput initial screen.
In this study, casein phospho-peptide as a component of a tooth cream in
combination with ACP, although not indicated for daily use, was investigated for anti-
erosive effects and abrasion prevention. It is known that CPP-ACP limits the free
calcium and phosphate ion activities, thus helping to maintain a state of super-
saturation, which decreases demineralization [Reynolds et al, 2008], although there
are conflicting results about its effectiveness [Wang et al., 2011; Wegehaupt, Attin,
2010; Rees, Loyn, Chadwick, 2007]. We observed a weaker protective effect, of 39%
76
mineral loss reduction, against acid challenge, although still statistically significant
(p=0.041).
There is preliminary evidence that toothpastes containing the Sn2+ ion could
be promising agents in the prevention of acid based erosion [Ganss et al., 2008;
Huysmans et al., 2011]. In this study a toothpaste containing stannous fluoride was
tested and a continuous surface coating appeared on the treated samples. One of
the suggested mechanisms of erosion prevention of SnF2 is the promotion of a
protective layer on the tooth surface [Huysmans et al., 2011] and stannous fluoride
has been demonstrated to be capable of depositing appreciable levels of tin on
enamel. Using CLSM for surface analysis, we observed an area of minimum
fluorescence, similar to a dehydrated surface, and as previously described, even
when analysed using the water dipping objective lens through distilled water the tin
protective layer was stable.
As noted, surfaces treated additionally with tooth-brushing showed decreased
protection, probably due to the abrasion effects of physical forces. The stannous
layer was abraded and totally removed in some areas. CLSM X-Z sections supported
the evidence that the SnF2 does form a thin, but not strong, protective layer on the
tooth. Comparing interface values of the step no statistical difference was found
between the negative control and the tooth-brushed groups.
CLSM has high resolution that was sufficient to evaluate erosion effects on
samples with minimum sample preparation. CLSM was shown to be an alternative to
scanning electron microscope (in environmental mode) that facilitated evaluation
without damage. The non-contact method is a significant advance as there is no risk
of damaging the delicate protein matrix left exposed after erosive attack, which may
well play a role in supporting the re-mineralisation process. In this limited study
toothpaste containing SnF2 showed a significant (p=0.001) protective ability of 70%
against acid based erosion, though the use of a toothbrush reduced its effectiveness.
Abrasion promoted by toothbrush procedures also reduced the protective effects of
CPP-ACP NaF.
77
Conclusions
Eroded samples showed loss of the organic matrix and exposure of enamel
rods, increasing the irregularity of the enamel surface. The stannous fluoride within
the Oral B toothpaste and the CPP-ACP NaF in the Tooth Mousse Plus demonstrate
mineral loss reduction of 70%, and 39%, respectively. However, abrasion damage
decreased those protective effects. Confocal microscopy is an excellent non-contact
method for the monitoring of acid erosion and re-mineralisation on enamel. This
study also shows the potential of this method to quantify the effect of acid based
erosion in vitro, which may be suitable for high throughput screening of new
toothpaste formulations in relation to protection against acid attack.
Acknowledgements
Financial support to this work from AMD PROJECT FACEPE/CNPq and PRONEX-
FACEPE/CNPq, Brazilian Agencies, are gratefully acknowledged.
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ARTIGO IARTIGO IARTIGO IARTIGO IIIIIIIII
83
Investigation of the effect of various erosive prot ocols by
profilometry and optical coherence tomography
A. M. A. Maia1; P. Cassimiro-Silva1; G. Q. M. Monteiro1; A.S.L.Gomes3
1PhD student, Dentistry Department, Federal University of Pernambuco, Recife,
Brazil
3PhD Professor, Department of Physics, Federal University of Pernambuco, Recife,
Brazil
Short title: Erosive protocols by optical coherence tomography
Key words: Enamel erosion, profilometry, Optical Coherence Tomography.
Corresponding Author: Msc. Ana Marly Araujo Maia, Universidade Federal de
Pernambuco, Physics Departament, Av. Prof Luis Moraes Rego, S/N, Cidade
Universitária, Recife, Pernambuco, Brazil. CEP 50670-901; e-mail:
anamarlyamaia@gmail.com; Fax: +55-81-32710359
Abstract
Variations in erosive solutions and cycling protocols make comparison of fluoride
efficiency difficult and the aim of this study was to evaluate erosive potential of
common acidic solutions tested previously, using multiple exposure times,
additionally validating optical coherence tomography (OCT) as a quantitative
measurement technique of mineral loss. A total of 28 enamel bovine specimens were
flattened and polished to a uniform mineral structure before erosive protocols.
Samples then were divided in four groups (n=7), two of them submitted Citric Acid
84
(CA), and the other Coca-Cola® (CC). And the erosive cycles consisted on 3
protocols: Group 1 (CA): 10days with 6 exposures per day of 5 minutes; Group 2
(CA): 7d: 5 x 3min; Group 3 (CC®): 7d: 5 x 3min; and Group 4 (CC®): 5d: 4 x 90sec.
After erosions procedures, specimens were analyzed by OCT and profilometry. Data
were statistical analyzed. Values average obtained by both methods for the tissue
loss on the enamel interface between the reference and eroded surfaces just not
showed statistical agreement (p=0.018) on measurements of G4 protocol, with the
lowest material loss. Furthermore, the G1 erosion cycle was significantly greater than
all other groups and more erosive than Coca Cola when normalized for exposure
time. Statistical data also showed difference between erosive solutions, based on
comparison of mineral loss of G3 (17.2µm +3.4) and G4 (6.9 µm +1.1) for the same
cyclic design (p=0.002) which was also followed for when etch rates were normalized
for time. Roughness increased significantly after the erosion cycles in all groups and
morphological analyzes by scanning electron microscopy (SEM) micrographs
confirmed the results of mineral loss. OCT also showed potential to quantify mineral
loss as non-destructive technique with high resolution.
Introduction
Dental erosion, an irreversible loss of dental hard tissue [Imfeld, 1996], has
been associated with the excessive consumption of acidic beverages [Dugmore,
Rock, 2004], being one of the major extrinsic causes. Researchers have evaluated
commercial drinks to test erosive potential and the influence of time on alterations
[Jager et al., 2012], however the principle aim of most of studies was to elucidate
which preventive or interceptive method is more effective against erosion [Nogueira
et al, 2000; Lussi et al. 2008; Lippert, Parker, Jandt, 2004; Wongkhantee et al, 2006],
as erosion processes requires preventive and therapeutic strategies that are different
from the established approaches in cariology [Ganss et al., 2012].
Even considering a simple in vitro model, variables such as erosive agent, pH,
agitation, buffering, and constancy of composition, temperature and duration of
erosive challenge have been discussed by Shellis et al., [2011]. The most commonly
85
tested extrinsic erosive agents are soft drinks and fruit juice, or a simple acid
solution. The commercial available products offer the advantage of being more
realistic, but also may vary between batches, and contain other substances as sugar,
gums and polyphenols. However, acid solutions are reproducible, inexpensive and
can be formulated to produce a consistent response [Shellis et al., 2011].
Some of the factors listed above should be considered in order to make in vitro
studies as close as possible to clinical conditions. In this context, multiple-exposure
of low pH cycles, of short time duration with the acid immersion alternating with saliva
immersion and has been considered the best model to reflect erosive challenges
faced by the dentition [West, Davies, Amaechi, 2011]. Even only examining multiple
exposure erosive cycling models, it is also possible to find a huge variation of
protocols, covering time of exposition to acid, saliva or remineralizing solution,
temperature etc. Furthermore, the lack of an adequate control group is a major
problem of numerous studies on new compounds as demonstrated by Schlueter et
al., [2009a].
Despite such disadvantages, in vitro models should be a primary method to
define the trajectories of new products or new techniques for reducing the erosive
effects of acid exposure. One of the main advantages of in vitro studies is the
opportunity to analyze erosive effects by a range of different techniques each
providing new information on the erosive effect such as surface hardness, surface
profilometry, longitudinal or transversal microradiography and scanning electron
microscopy associated with energy-dispersive X-ray. In this context, the Optical
Coherence Tomography (OCT), is a non-invasive technique gaining significant
interest in the dental community that uses near-infrared electromagnetic radiation
(light), instead of the ionizing radiation used in X-rays. It has been shown to have
potential for clinical erosion studies in terms of detection and lesion preservation
[Huysmans et al., 2011]. Although, the technique based on low coherence
interference has shown the potential to determine the locations and depths of lesions
alterations, it has yet to be used in vitro studies to validate the optical tomogram as
an accurate technique to measure mineral loss due to erosive challenges.
The aim of this study was validate the use of Optical Coherence Tomography
as a technique to quantify mineral loss, comparing measurements of the step profile
86
between a reference and eroded area. The study design also used three different
intensities of the demineralization protocol, testing time of exposure and erosive
potential of each solution.
Materials and Methods
Enamel Block Preparation
Permission to use bovine tooth samples was granted by the Ethical Committee
of the Federal University of Pernambuco - Recife PE, according to approval form
(00874/2012). The experimental samples were prepared from 28 freshly bovine
incisors previously inspected for physical damage, specifically cracks. Further
cleaning was conducted with distilled water and a dental brush, and then submitted
two weeks in cloramina 0,5%. The teeth were sectioned with a water-cooled
diamonded disc and enamel blocks were obtained.
Samples had the outer enamel surface flattened and polished with water-
cooled sandpaper of decreasing grit (600 and 1200 ground discs Buhler, Illinois,
EUA) until a flat area of approximately 4x4mm was produced. This area was then
polished with a metallographic polishing cloth (SUPRA – Arotec, Sao Paulo-SP,
Brazil) moistened with 1µm diamond polishing oil suspension (Buehler, Illinois, EUA).
Subsequently each polished section was ultra-sonication in distilled water twice for
10 minutes on each ocassion. For each sample, half the enamel surface was coated
with cosmetic nail varnish (non-pigmented color) and a sticky wax layer, leaving an
area about 8.0mm2 exposed during erosive challenge. This procedure ensured the
establishment of a reference surface for further evaluation of enamel loss.
A total of 28 enamel blocks were mounted on sample holders attached to
falcon tubes suitable for individual solutions (25ml), and also transposition between
solutions to ensure consistent immersion times. The storage solution was an artificial
saliva containing CaCl2 0,2205g, KCl 3,735g, NaH2PO4, HPMC 0,4%, sorbitol 6%,
nipagin 0,2 and had a pH of 7.4. Temperature was controlled by using an incubator
at 37°C.
87
Test Products and Erosive Cycle Protocols
The acidic solutions were two erosive demineralization solutions, previously
tested: citric acid monohydrate diluted in distilled water to a concentration of 0,5%
and pH=2,6 (Schlueter et al., 2009); and the commercial beverage broadly
consumed, Coca-Cola®, also previously used as a drink erosive option in research
protocols, known pH=2,47 (Magalhaes et al., 2011; Levy et al., 2012).
Over the experimental period, the enamel samples were subjected to a cyclic
demineralization and saliva procedure including daily multiple erosive attacks,
varying numbers of exposures, duration of exposure and number of cycle days.
When solutions were changed all samples were rinsed in distilled water for 30 sec,
gently dried with hanky paper and stored in 25 ml artificial saliva for 1h.
In experimental Group 1, the erosive solution was citric acid, samples were
challenged for a total of 30 minutes a day through 6 repetitions of 5 minutes each, for
10 days. Group 2, also submitted to citric acid, was immersed 5 times for 3 minutes
for 7 days. Samples from groups 3 and 4 were immersed in Coca-cola. Group 3
exposed with exactly the same protocol as group 2, and group 4 immersed 4 times
for 90 seconds for 5 days.
Tissue loss measurement
The nail varnish and stick wax were removed from the reference areas with
the aid of alcohol 99% and soft paper tissues. During removal, each specimen was
inspected under a stereomicroscope (magnification x25) for checking the complete
removal of this layer and protect the interface. Two systems were used to quantify
mineral loss scanning surface to generate a two-dimensional profile.
Profilometry
This analysis was performed using profilometer (SJ-400, Mitutoyo, Japan)
connected to a PC with the software SURFPACK – SJ Version 1.300. The contact
profilometer scanned the surface with a diamond tip, analyzing data of five readings
88
on each fragment from the reference to eroded surface of the specimen. Each
surface steps at intervals of 0,6mm between each profile captured, covering a
scanned line about 1,7mm, and vertical range of 800µm.
Each baseline profile of interface were processed ad interpreted with the
system software (Origin 8.5), and the average depth of wear was obtained by the
distance in micrometers between the linear regression achieved from 500 points of
the reference surface and a midpoint of the linear regression achieved from 500
points of the treated-eroded surface. It is important to elucidate that a total of 250
points near interface of each line were excluded, as mineral loss near interface
seems to be not representative.
To measure the surface roughness, two traits were conducted in a
representative line of 1,7 mm of each specimen . The parameter used was the Ra
(roughness average).
Optical Coherence Tomography
The same procedure of scanning surface was done using the Optical
Coherence Tomography (OCT), from a commercially available system (Spectral
Radar SR-OCT:OCP930SR / Thorlabs, New Jersey, USA), a non-destructive and
non-contact technique with high resolution of 6µm that is based on interference of
coherence laser light. As distances can be effectively measure by OCT, cross
sections images of interface area showed to be able to measure depth promoted by
mineral loss. Specimens were positioned on a manual stage that allows XYZ
translation. An scanning line of 5mm was obtained including interface of the
reference and eroded surfaces of each specimen, resulting in five tomogram images,
at intervals of 0,6mm.
These cross sections images (fig. 1a) were processed scaled and a LUT tool, called
edges, was applied to image to allow better definition of surface reflections (Fig 1b).
To better analyze of the interface area, a zoom was added on image for
measurements by public JAVA domain Image J (fig 1c). The discrepancy between
the height of the reference surface and the treated-eroded surface was measured. A
89
bar tool promote the measurement of length that represents the step of mineral loss
between referred surface, and the average depth of wear was calculated for each
specimen (µm).
OCT three-dimensional images were also captured to allow better description
by this technique, and the signal decay of light of one point from each are was also
evaluate to compare optical changes on eroded surfaces.
Figure 1. Example of an OCT image profile analyzed. The total distance used as
reference to get value of step was 3mm, meaning 1,5 mm of each surface. For
measurement of tissue loss, the box tool helps to identify the distance between both
superficial face.
SEM
Scanning electron microscope images has proven to be fundamental for
characterization of structural changes after erosion challenges of enamel. So after all
profiles analysis, two randomly selected samples from each group were again
submitted to citric acid for 2min, rinsed with distilled water (Ganss et al., 2008), than
immersed three minutes in ultra-sonication. Samples were dried at 37°C for 5
minutes, and sputter coated with lightly gold-sputtered. Representative eroded areas
of the surface were analyzed in a Scanning Electron Microscope (SEM, FEI
Company, Quanta 200 MK2 FEG, Oregon, USA). The acceleration voltage was set
to 20kv. Images were obtained using a secondary electron detector with an increase
of 10.000X.
b
c
a
90
Statistics Analysis
The statistical procedures were realized with SPSS 13.0 (Statistical Package
for the Social Sciences, Chicago, USA) for Windows. Statistical measures were
obtained and Komogorov Smirnov test was used to assess the normality of the data.
The Kruskal Wallis test for compare groups in general, and the Mann-Whitney test to
compare group vs. group. Wilcoxon test was performed to compare different times.
All tests were applied with 95% confidence.
Results
Cross sectional OCT images representative of the four groups observed on
enamel surface step after acid challenge in figure 2. The results for the tissue loss on
the enamel interface between reference and eroded surfaces following the several
cycling design and measured techniques are presented in Table 1.
Figure 2: Comparison of OCT images of the enamel surface reference and eroded
are for different solution and time periods. Images identified with lowercase represent
91
3D OCT; and images by uppercase cross-sectional. (A/a) Citric Acid, 10d:6x5min;
(B/b) Citric Acid, 7d:5x3min; (C/c) Coca-Cola®, 7d: 5x3min; and (D/d) Coca-Cola®,
5d:4x90sec.
Values obtained by OCT images haven’t showed differences between surface
profilometer values of interface. Regarding the measured techniques, significant
difference was only found in the G4 (p=0.018) that evaluate acid potential of Coca-
Cola® in less exposure time. Furthermore, comparing cyclic erosion groups,
significant differences were observed between the group exposed to citric acid for 10
days (G1) and all other protocols groups, G2 (p=0.002), and Coca-Cola® groups
(G3; p=0.002) and (G4; p=0.002). Statistical data also showed difference between
erosive solutions, based on comparison of mineral loss of samples submitted to citric
acid (G3: 17.2µm +3.4) and Coca-Cola® (G4: 6.9 µm +1.1) for the same cyclic
design (p=0.002).
Table 1: Mean of tissue loss (µm) in all groups and by Profilometry and OCT
techniques. Mean roughness values and standard deviations of reference and
eroded surfaces.
Mineral Loss, µm Roughness
Groups Profilometry OCT P value *¥ Sound Ra Eroded Ra p value
G1 47.6 +5.2a 52.5 +6.2a 0.176 0.13 +0.05 0.57 +0.16 0.018
G2 17.2 +3.4b 14.1 +6.0b 0.176 0.11 +0.02 0.43 +0.12 0.018
G3 6.9 +1.1c 7.7 +3.2c 0.735 0.12 +0.04 0.43 +0.24 0.018
G4 3.8 +0.9c 2.8 +0.4c 0.018 0.11 +0.05 0.28 +0.17 0.018
p-
value** €
<0.001** <0.001** 0.956** 0.056**
(*) Wilcoxon Signed Rank Test
(¥) Comparison between means of the types of techniques
(**) Kruskal Wallis Test
(€) Comparison between groups, and letters represent statistical difference.
92
All data about roughness of eroded enamel following the different protocols
are also presented in Table 1. Roughness increase significantly after eroded cycles,
although comparing eroded roughness it was not observed significantly differences
between groups. However, it was confirmed statistical differences when comparing
G1 (Citric Acid) and G4 (Coca-Cola®), designs with high and low exposure time.
SEM images (figure 3) revealed different phases of typical sound (figure 3A)
and eroded enamel surfaces. Samples exposed to citric acid (figure 3B) for longer
time showed an aggressive eroded surface, with exposed honey comb structure, as
surface were not dissolved uniformly, as prisms, inter-prisms areas and organic
sheats seems to present different resistance to acid. An opposite aspects, samples
exposed to shorter time of Coca-Cola® (figure 3C) showed areas of initial erosion.
Comparing effects of Citric Acid (figure 3D) and Coca-Cola® (figure 3E) by the same
protocol time, it was observed more exposition of enamel nano rods and mineral loss
on samples submitted to Citric Acid.
Figure 3: Scanning electron micrographs (all in the same magnification level of
10.000X) of the surface of groups (A) sound enamel; (B) Citric Acid, 10d:6 x 5min;
(C) Citric Acid, 7d:5x3min; (D) Coca-Cola® 7d:5x 3min and (E) Coca-Cola®
5d:4x90sec.
93
Discussion
In the present study, bovine enamel was used as substitutes for human teeth.
Even human tissue been the substrate of choice, bovine tissue are suitable for most
purposes [Shellis et al., 2011], as previous studies have found no significant
difference in either mineral content, distribuction or mechanical properties between
both enamel structures [Davidson et al., 1973]. Several studies have used bovine
enamel for erosion tests [Rios et al., 2009; Wiegand et al., 2009; Kato et al., 2010],
providing valid relative data, even not directly extrapolated to the human situation.
It is also clear that tooth surface change is a complex process that can be
measured in a variety of ways [Field, Waterhouse, German, 2010]. In this paper,
erosive effects resulted from multiple-exposure models were analyzed by roughness
and the measure of steps detected by the contact profilometry and non-contact
Optical Coherence Tomography (OCT). These techniques have different levels of
vertical resolution, and the last one hasn’t been applied to this aim before.
Considering the advantages of OCT to measure distances of high coherence
(resolution of 6um), non-contact and non-preparation of sample and also its potential
to be applied in vivo, the hypothesis to be tested was that there are no differences
between OCT and Profilometry to measure steps of mineral loss. Comparing average
value of steps of each group by both techniques, statistical differences between
erosion protocols effects was detected. Although it was observed statistical
difference in Coca-Cola Group low exposures time between steps values found by
Profilometry and OCT. It is our suggestion that this difference can be explained
because of the minimum mineral loss observed in this group, what made OCT
images analyze imprecise.
Surfaces aspects visualized by OCT 3D showed an idea of irregularity of
enamel erosion, due to variation on optical superficial scattering characterization o
eroded surface. SEM micrographs from each group corroborator with OCT images
but also showed surface details as the typical honeycomb structure of etched prisms
been exposed depends on the exposure time of each protocol group. Group 1
showed higher mineral loss by step measurements and it was confirmed by more
94
intense contrast between nanorods and interprismatic region around. Although
groups submitted to Coca-Cola drink showed a mixed appearance, with areas where
a surface layer seems to not have been eroded.
In the way of new developments to evaluate minimal mineral loss, the OCT
was compared with other two techniques mostly used surface profilometry and SEM,
presenting an optical non-destructive technique to improve the study of erosion at all
stages, not only in vitro or in situ, but also in clinical settings, as has been suggested
by Schlueter et al., [2011]. The optical coherence tomography seems to be an
interesting method to be applied, but it is also important to consider that the amount
of tissue loss might be below the detection limit of the technique [Attin, 2006].
These research was also motivated to authors also consider that a higher level
of standardization among experiments would allow better comparison of study as
discussed by Wiegand and Attin, [2011]. There are several options of erosive
solutions to test, although no standard protocol for experimental studies has been
established. This absence of standard protocols prejudices the comparison between
anti-erosive agents, been the lack of adequate control group a problem to compare
fluoride compound.
Although, it was previously discussed by Ganss et al., [2008], that the
experimental set-up should provide erosive conditions that are mild enough to
identify differences between various fluoride formulations. However, these mineral
loss and preventive effects would also depend on which technique will identify and
quantify changes, as best resolution and magnification techniques can register
minimal alterations.
Most of experiments that submitted samples to multiple exposure time, as
cycle protocols, use artificial saliva and fluoride application between acidic exposure,
although as our aim was to evaluate just the effects of erosive solutions, any
treatment was applied. Our results of tissue loss can be compared to negative control
group of other experiments, also realized in vitro and with the use of a saliva
substitute. Being important to highlight that artificial saliva was used to better
simulate oral conditions, although it is known that results must be analyzed with
95
precaution, because of the absence of acquired pellicle that is present only under in
situ and in vivo conditions.
Comparing our results of tissue loss with negative control group of other
experiments also measured by contact profilometric, it was observed similar average
of mineral loss when citric acid was tested 5x3min, during 7 days in Ganss`s
experiments [2012], although huge variations of mineral loss was observed
comparing our higher exposure time group with the 7 days, 6x5min protocol tested by
Schlueter et al., [2009] with an average of mineral loss probably twice greater than
our which was developed during 10days, what could be explained by a low pH
solutions. Experiments that tested Coca-Cola® showed mineral loss similar [Rochel
et al., 2011].
The time to sample be exposed to erosive agent depends on the type of acidic
challenge and its pH and buffer capacity. As observed by Jager and collaborators
[2012], comparing several commercially beverages, the short and long exposure
times showed different erosive effect on surface. In our research, time exposure was
compared between groups 1 and 2, testing citric acid and also between groups 3 and
4, using Coca-Cola. And significantly differences of step measurements were
observed. These effects can result in divergent conclusions about erosion and also
preventive potential, as observed by Schuelter et al., 2009 when compared the same
preventive agent by 3 experiments, one acidic exposure intensive enough to be
measured by profilometric, but mild enough to observe differences in the
effectiveness of the test compounds. Based on those and our results, we suggested
that more researches need to be develop to evaluate if a preventive fluorides effects
could be observed in different exposure time experiments. Evaluating if a fluoride
with known protective effect can demonstrate its properties exposed as samples of
group 1 with the high exposure total of 30 min/day during ten days, and also if
exposed to group 4 with low exposition total of 6min/day during five days.
Surface roughness of eroded surface seems to be useful only for early stages
of erosion [Schlueter et al., 2011]. However it was done roughness before erosion
cycle to check the flatness of polished specimens, an average of specimens with
0.11um was included. After eroded cycle roughness showed statistical difference
96
only between citric acid and Coca-Cola®, high and low exposure, respectively.
Results confirmed by SEM micrograph.
Erosive potential of both solutions tested were compared between effects of
groups 2 and 3, which samples were multiple exposed by the same time, during 7
days. Both groups compared by Mann Whitney test showed statistical differences, as
citric acid (pH=2.5) (P: 17.2µm; OCT 14.1µm) promoted a mineral loss at least twice
severity than Coca-Cola (pH=2.4) (P: 6.9µm; OCT 7.1µm). This difference can be
partially correlated with difference of viscosity, that as discussed by Jager et al.,
[2012], the viscosity of a drink, together with contact angle and surface tension,
determines its penetration coefficient into a capillary space such as pores [Perdok,
Van der Mei, Busscher, 1990], even not tested in this research the solution of citric
acid seems to be less viscosity than Coca-Cola. Our results seems to highlighted the
necessity of standard an erosive protocol reproducible for researchers around the
world, as it influences directly on prevention conclusion of fluorides.
Conclusions
Based on comparison with Contact Profilometry, the Optical Coherence Tomography
even with different levels of vertical resolution showed potential to be applied as a
non-contact, with less preparation of sample, to measure steps of mineral loss. Our
results also enforce the necessity previously discussed about the development of
similar erosive cycles protocols to establish better comparisons between fluoride
efficacy.
Acknowledgements
The authors acknowledge the support of PRONEX/FACEPE and CNPq grants.
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100
CONSIDERAÇÕES FINAIS
A aplicabilidade das técnicas ópticas foi demonstrada frente a quantificação
de perda mineral decorrente de cárie ou erosão dentária, elucidando a importância
do conhecimento integrado das propriedades ópticas da interação laser-tecido (in
vitro).
A Tomografia por Coerência Óptica mostrou efetividade quando analisada
segundo a comparação do valor do coeficiente de atenuação entre a região sadia e
cariada, permitindo ainda uma visualização da lesão em extensão.
A Microscopia Confocal com escaneamento a laser demonstrou resolução
suficiente para a caracterização morfológica da superfície erodida de esmalte, além
de ter proporcionado a quantificação do efeito protetor do fluoreto de estanho e o
fluoreto de sódio, e da caseína fosfopeptidea associada ao fluoreto de sódio.
Podendo ser utilizado em uma quantidade de energia que não promova danos a
superfície analisada.
A aplicação da Tomografia por Coerência Óptica como meio de análise do
perfil ótico, mostrou-se efetivo para perdas maiores que 6 micrometros, quando
comparado a perfilometria de contato, na mensuração de perda mineral entre região
sadia e erodida. O estudo ressaltou as diferenças existentes entre os protocolos de
múltipla exposição previamente realizados em ensaios erosivos, estimulando a
busca por parâmetros para maior controle quanto ao dano real e ao efeito protetor
do fluoreto testado;
101
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