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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