Frederico Barbosa de Sousa - Universidade Federal de Pernambuco

Transcription

UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
CONTRIBUIÇÕES AO ESTUDO DE BIOCERÂMICAS DE
FOSFATO DE CÁLCIO FORMADAS EM MODELO IN
SITU DE CÁRIE DENTAL
Frederico Barbosa de Sousa
Recife, novembro de 2005
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UNIVERSIDADE FEDERAL DE PERNAMBUCO
CENTRO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS
CONTRIBUIÇÕES AO ESTUDO DE BIOCERÂMICAS DE
FOSFATO DE CÁLCIO FORMADAS EM MODELO IN
SITU DE CÁRIE DENTAL
Doutorando: Frederico Barbosa de Sousa
Tese apresentada ao Programa de Pósgraduação em Ciências Biológicas como
parte dos requisitos exigidos para obtenção
do grau de Doutor em Ciências Biológicas,
área de concentração em Biotecnologia.
Orientadora: Profa. Dra. Nereide Stela Santos-Magalhães
LIKA, CCB/UFPE
Orientadora externa: Profa. Dra. Sandra Sampaio Vianna
Departamento de Física, CCEN/UFPE
Recife, novembro de 2005
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Sousa, Frederico Barbosa de
Contribuições ao estudo de biocerâmicas de fosfato de cálcio formadas em
modelo in situ de cárie dental / Frederico Barbosa de Sousa. – Recife: O Autor,
2005.
119 folhas : il., fig., tabs.
Tese (doutorado) – Universidade Federal de Pernambuco. CCB.
Ciências Biológicas. Biotecnologia, 2006.
Inclui bibliografia e anexo.
1. Cárie dental 2. Cálculo dental 3. Biocerâmica – Fosfato de
cálcio 4. Microscopial I. Título.
616.314-002
CDU (2.ed.)
UFPE
617.6
CDD (22.ed.)
CCB – 2006–63
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MEMBROS SUPLENTES:
Prof. Dr. Romildo de Albuquerque Nogueira
Depto. de Morfologia e Fisiologia Animal, UFRPE
Profa. Dra. Suely Lins Galdino
Depto. de Bioquímica, CCB/UFPE
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A minha esposa, Leila, ao meu filho,
Henrique, e às minhas mães, Elisa, Amabília
e Maria.
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AGRADECIMENTOS
A Profa. Dra. Nereide Stela Santos-Magalhães, minha orientadora, por ter
assumido e cumprido um compromisso de trabalhar numa área que não era de sua
especialidade, mas que considerou interessante, o que foi fundamental para meu
ingresso no curso e para a conclusão do mesmo.
A Profa. Dra. Sandra Sampaio Vianna, minha orientadora externa, pela
gentileza e atenção com que aceitou trabalhar numa área que também não era de sua
especialidade, colocando seu laboratório à minha disposição e dispensando bastante
tempo para discutir sobre os projetos desenvolvidos, sem cuja ajuda não seria possível
a conclusão do curso.
À Coordenação de Aperfeiçoamento de Pessoal de Nível superior (CAPES)
pelo apoio financeiro e pela concessão de bolsa de estudo para a realização do curso.
A Virgínia, técnica do laboratório de Química do Depto de Física da UFPE,
pela atenção e boa vontade com que me ajudou nos procedimentos laboratoriais.
A Clécio, técnico do laboratório de polímeros não convencionais do Depto. de
Física
da
UFPE,
pela
ajuda
e
pelas
orientações
nos
experimentos
de
espectrofluorescência.
Ao Prof. Jaime Cury, Faculdade de Odontologia de Piracicaba/Unicamp, pelas
críticas feitas ao texto final do artigo sobre modelo in situ de cárie.
Ao Prof. Hélio Magalhães de Oliveira, do Departamento de Eletrônica e
Sistemas da UFPE, pela grande ajuda no processamento dos dados do artigo
matemático.
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Às funcionárias do Programa de Pós-graduação em Ciências Biológicas, em
especial a Adenilda, pelos serviços prestados e pela simpatia.
E a todos que contribuíram, direta ou indiretamente, para a execução deste
trabalho.
SUMÁRIO
Página
AGRADECIMENTOS
LISTA DE FIGURAS
LISTA DE TABELAS
RESUMO
ABSTRACT
1
INTRODUÇÃO
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1.1 Formação e indução de depósitos calcificados
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1.1.1 MODELO IN SITU DE CÁRIE DENTAL
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1.1.2 INDUÇÃO DE DEPÓSITOS CALCIFICADOS EM
MATERIAIS PARA IMPLANTES OSTEOINTEGRADOS
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1.2 Técnicas de análise e de caracterização de cálculo dental e de
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tecido dental duro
1.2.1 MICROSCOPIA ELETRÔNICA DE VARREDURA E
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MICROANÁLISE DE RAIOS X
1.2.2 ESPECTROSCOPIA DE FLUORESCÊNCIA
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1.2.3 O ESMALTE DENTAL CARIADO ANALISADO PELA
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MICROSCOPIA DE LUZ POLARIZADA
2
OBJETIVOS
3
CONCOMITANT
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CARIES
AND
CALCULUS
FORMATION
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2
FROM IN SITU CARIES MODEL IN PERIODS OF 2-14 DAYS
1.2 Abstract
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1.3 Introduction
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1.4 Material and Methods
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1.5 Results
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1.6 Discussion
37
1.7 Acknowledgements
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1.8 References
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COMPARATIVE
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FLUORESCENCE
SPECTROSCOPY
OF
HUMAN DENTAL CALCULUS WITH HEMATOPORPHYRIN
4.1 Abstract
4.2 Introduction
4.3 Material and methods
4.4 Results
4.5 Discussion
4.7 References
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5 A NEW APPROACH FOR IMPROVING THE BIREFRINGENCE
ANALYSIS OF DENTAL ENAMEL MINERAL CONTENT USING
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POLARIZING MICROSCOPY
5.1 Summary
5.2 Introduction
5.3 Methods
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66
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5.4 Results and discussion
5.5 Conclusions
5.7 References
6 CONCLUSÕES
7 REFERÊNCIAS BIBLIOGRÁFICAS
8 ANEXOS
8.1 Normas para publicação na Caries Research
8.2 Normas para publicação no Journal of Periodontal Research
8.3 Normas para publicação no Journal of Microscopy
8.4 Parecer do comitê de ética para artigo n° 1
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102
109
114
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8.6 Dados da literatura usados para aplicação da nova abordagem
matemática para a BRobs teórica do esmalte dental.
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8.7 Comparação entre dados teóricos e experimentais da BRobs do
esmalte dental em diversas condições.
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LISTA DE FIGURAS
CAPÍTULO 3
Figura 1
Fig. 1. Aparelho intra-oral (palatal) removível de resina acrílica com
fragmentos dentais (cabeça de seta), (a). Telas plásticas cobrem os
fragmentos, deixando espaço para o biofilme bacteriano (b).
Pág.
12
4
Figura 2
Fig. 2. Estrutura química da hematoporfirina.
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Figura 3
Fig. 3. Gráfico do comportamento da BRobs teórica dada pelo modelo
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clássico para vários meios de imersão.
CAPÍTULO 3
Figure 1
A, calculus deposits (arrow), of macroscopic size, on a 9-day sample
Pág.
41
with demineralization (Bar = 1 mm). B, detail of the area indicated
by an arrow in “A”, showing opening of dentinal tubules (white
arrow) and dental calculus (black arrow) (Bar = 20 µm). C,
histological aspect of the same sample after hemi-sectioning showing
demineralisation (black arrow) below the experimental surface (Bar
= 1 mm). Opaque outline is demineralization caused by bacterial acid
infiltrated around the sample. D, BSE-SEM image (bar = 300 µm)
showing demineralization (white arrow) in the surface (fractured
apart) indicated by black arrow in “C”.
Figure 2
Fluorescence spectroscopy data of different samples dissolved in
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HCl 27 % and excited with 416 nm: (a), human dentin; (b),
human dental calculus; and (c), sample from the surface of the
sample shown in Fig. 1A, showing fluorescence of dentin
(arrowhead) and dental calculus (arrow).
CAPÍTULO 4
Figure 1
Laser system setup for fluorescence.
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Figure 2
Fluorescence spectra of dental calculus solutions at pHs 2 to 7
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excited with the xenon lamp at 400 nm.
Figure 3
Normalized (at emission maximum) fluorescence spectra of dental
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calculus (a) and hematoporphyrin (b) in 27% HCl solutions (bold
line), at pH 2 (dashed line), and at pH 7 (solid line) excited with
xenon lamp at 400 nm.
Figure 4
Normalized (at 695 nm) fluorescence spectra of four samples of
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solid dental calculus excited with a dye laser at 560 nm. Spectra
are vertically displaced to facilitate comparisons.
Figure 5
Normalized (at emission maximum) fluorescence spectra of
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hematoporphyrin at pHs 7 (line 1) and 2 (line 2), in 27% HCl
solution (line 3), of dental calculus in 27% HCl solution (line 4), and
solid dental calculus (line 5) excited with a dye laser at 560 nm. The
sharp peak in line 5 is the scattered laser light. Spectra are vertically
displaced to facilitate comparisons.
CAPÍTULO 5
Figure 1
Experimental BRobs (scattered data) reported by Angmar et al.(1963),
calculated BRobs with the classical model for n2 and using mineral
density of 3.15 gcm-3 (dashed black line), calculated BRobs with the
classical model for n2 and using mineral density of 2.99 gcm-3
(dashed grey line), and calculated BRobs with our new approach
(continuous line) obtained from the mean values of α1, α2 and
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β (equations 8a-8c). (a), data related to water immersion medium;
(b), data related to air immersion medium. Insert: detail of published
experimental BRobs (scattered data) and calculated BRobs with our
new approach (continuous line).
Figure 2
Plot of the relationship between V1(2.99) and α1, α2 and β within the
analysed range. Linear trends: α1 = 15.3154 – 0.1161V1(2.99), r = 0.7574; α2 = 26.5294 – 0.2740V1(2.99), r = -0.9628; and β = 58.6978
– 0.6157V1(2.99), r = -0.9957.
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LISTA DE TABELAS
CAPÍTULO 3
Table
Occurrence of demineralization and calculus deposits (with mean
Pág.
40
Ca/P ratios).
Tabela 2
Dados experimentais publicados (ANGMAR et al., 1963) de
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volume mineral (V1, %) e BRobs (x 10-4, em água, BRágua, e em ar,
BRar) de esmalte dental maduro obtidos em diferentes distâncias
da superfície (100-1300 µm).
Tabela 3
Comparação entre dados experimentais publicados de BRobs (x
10-4) de esmalte dental (em diversas condições e em diversos
meios de imersão experimental) e os valores correspondentes
teóricos obtidos pelas equações 4, 7 e 8a-8c.
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RESUMO
Biocerâmicas de fostato de cálcio são importantes na Odontologia e na Medicina, no
tocante aos processos de cárie e cálculo dentais, de preenchimento de feridas ósseas e de
recobrimento de implantes metálicos. Este trabalho visa aprimorar o estudo da formação
dessas biocerâmicas em modelos in situ de cárie dental em humanos. Aqui relatamos a
formação concomitante de cárie e cálculo (biocerâmica de fosfato de cálcio) dentais em
modelo in situ de cárie em intervalos de 2-14 dias, mostrando o potencial de se trabalhar
com intervalos relativamente curtos de tempo para formar o cálculo dental. Estudando a
fluorescência do cálculo dental, revelamos que sua fluorescência em soluções aquosas (em
função da concentração de hidrogênio) é similar à da hematoporfirina, e que a fluorescência
do cálculo dental sólido se assemelha mais com a de soluções com alta contração de
hidrogênio, nas quais hematoporfirina bivalente é o principal cromóforo. Para aprimorar a
análise do efeito da formação das biocerâmicas no esmalte dental, um modelo matemático,
que conseguiu, pela primeira vez, apresentar consistência entre dados quantitativos teóricos
e experimentais da birrefringência do esmalte ao microscópio de luz polarizada, foi
proposto. Este modelo tem ampla aplicação na biologia do esmalte por poder analisar os
conteúdos mineral, orgânico e de água.
PALAVRAS-CHAVE: cárie dental, cálculo dental, fosfato de cálcio, microscopia.
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ABSTRACT
Calcium phosphate bioceramics have important applications in Dentistry and
Medicine, considering the processes of caries and calculus formation, the filling of bone
wound defects and the surface coating of metallic bioimplants. The aim of the present work
is to improve the study of calcium phosphate bioceramics formed in in situ caries models.
Here we report the concomitant formation of caries and dental calculus in an in situ caries
model, stressing the importance of short-time intervals to form dental calculus. A study on
the fluorescence of dental calculus showed that, in solutions with different hydrogen
concentrations, it is similar to the fluorescence of hematoporphyrin free base solutions in
the same conditions. In addition, fluorescence of solid dental has been shown to more
closely
resemble the fluorescence of strongly
acid
solutions, indicating
that
hematoporphyrin dication is the main, if not the only, fluorescing chromophore. In order to
improve the analysis of the effect of calcium phosphate bioceramics in dental enamel, we
developed a new mathematical approach for the interpretation of the birefringence of dental
enamel using polarizing microscopy. The new approach enabled, for the first time, a
consistency between quantitative experimental and calculated birefringence of dental
enamel. Such a model has important implications on enamel biology as it enables to derive
quantitative data on the mineral, the organic and the water contents of dental enamel.
KEYWORDS: dental caries, dental calculus, calcium phosphate, microscopy.
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1.
INTRODUÇÃO
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1. INTRODUÇÃO
A formação de depósitos calcificados é um processo que tem sido foco de interesse
na Odontologia e na Medicina. Na primeira, os processo da doença cárie e da formação do
cálculo dental estão diretamente implicados. Na Medicina, depósitos/cerâmicas de fosfato
de cálcio - principalmente hidroxiapatita - sintéticos ou formulados a partir de extratos de
tecidos mineralizados de animais - são usados como materiais de preenchimento em
defeitos ósseos e para recobrimento prévio de implantes de titânio a serem inseridos em
tecido ósseo humano com a finalidade de facilitar a neoformação óssea. Neste contexto, o
controle da formação e inibição da precipitação de depósitos calcificados biológicos tem
sido foco de muitas investigações na área biotecnológica.
Os depósitos calcificados de que tratamos nesta tese são aqueles presentes na
composição do cálculo dental: brushita, fosfato tricálcico, fostato octacálcico e
hidroxiapatita. O cálculo dental constitui o biofilme bacteriano calcificado, formado sobre
os tecidos dentais duros ou qualquer outra superfície dura na cavidade bucal em
decorrência da deposição de sais de fosfato de cálcio (THEILADE, 1992). Sua composição
é de cerca de 70-80% de sais inorgânicos, dos quais dois terços se apresentam na forma
cristalina com uma relação entre o cálcio e o fosfato que varia de 1,6 a 2 (THEILADE,
1992). As principais formas cristalinas encontradas são: hidroxiapatita, fosfato de
octacálcio, whitloquita e brushita (KODAKA & MIAKE, 1991; KAKEI et al., 2000), dos
quais os três primeiros são variantes da matriz de hidroxiapatita e a brushita é uma fase
intermediária produzida durante a formação da hidroxiapatita e/ou whitloquita
(SCHROEDER & BANBAUER, 1966).
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Há indícios de que o cálculo dental poderia se formar a partir de um modelo intraoral, possivelmente em indivíduos que não apresentam tendência natural para formar
depósitos de cálculo a nível clínico (SOUSA, 1996), abrindo espaço para uma eventual
formação controlada de cálculo dental, que teria aplicação em várias áreas da saúde. Nesta
introdução abordaremos a formação de cálculo dental em modelo in situ de cárie e as
técnicas para análise e caracterização do cálculo dental e dos tecidos dentais duros.
1.1 Formação e indução de depósitos calcificados
Dentro da Odontologia, os depósitos calcificados têm repercussão especial na
patologia da cárie dental. As lesões cariosas são desmineralizações dos tecidos dentais
duros expostos na cavidade bucal causadas pela ação de ácidos produzidos por espessos
acúmulos microbianos formados após um certo período de tempo (THYLSTRUP &
BRUUN, 1992). Após a formação inicial do biofilme bacteriano, se não houver forças
mecânicas (contato dental durante mastigação e atrições da mucosa bucal, da escovação e
dos alimentos) que perturbem seu crescimento, sua espessura aumenta e dificulta a
passagem de oxigênio para as bactérias mais próximas da superfície do tecido dental duro;
e essas bactérias, então, passam a apresentar metabolismo anaeróbico com conseqüente
formação de ácido lático e desmineralização mineral (THYLSTRUP et al., 1994).
Há um conjunto de evidências, decorrente de análises em microscopias eletrônica de
varredura e de luz polarizada de estudos in vivo, que descreve a inativação do processo
carioso como o resultado da restauração das forças mecânicas sobre a superfície dental
dura. Estas forças levariam à remoção mecânica do biofilme bacteriano bucal como
também ao polimento da superfície dental, passando esta última a ter uma superfície mais
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lisa e brilhosa, mas não remineralizada (HOLMEN et al., 1987; ÄRTUN & THYLSTRUP,
1989). Apesar da supersaturação da saliva em fosfato de cálcio, algumas proteínas, como as
ricas em prolina e a estaterina, impedem a precipitação espontânea de minerais
(THYLSTRUP et al., 1994). Por outro lado, há um outro conjunto de evidências indicando
que a lesão cariosa se desenvolve como resultado da dinâmica entre fatores que contribuem
para a desmineralização e fatores que contribuem para remineralização, sendo inativação o
resultado da predominância de fatores que contribuem para remineralização (entre eles os
produtos fluoretados) (SILVERSTONE, 1984; FEATHERSTONE, 2004). Com base nos
resultados obtidos com radiomicrografia - a técnica atual mais eficaz para avaliar alterações
minerais nos tecidos dentais duros - há relatos de que a remineralização de lesões cariosas
pode ocorrer (SHEN et al., 2002; LAGERWEIJ & TEN CATE, 2002).
Um viés que deve ser considerado na avaliação da remineralização promovida pelo
flúor na presença de saliva e biofilme bacteriano é a formação de cálculo dental. Num
estudo com modelo in situ de cárie, PEARCE (1982) relatou a formação concomitante de
depósitos calcificados na placa bacteriana e de lesão cariosa no esmalte dental.
Posteriormente, o mesmo autor questionou a possibilidade dessa formação ser cálculo
dental por não haver ligação dos depósitos minerais com células bacterianas (PEARCE et
al., 1991). SOUSA (1996) demonstrou, pela primeira vez, a formação concomitante de
lesão cariosa e de cálculo dental aderido à superfície dental num modelo in situ de cárie
dentinária após um período de trinta dias. A união entre os cristais do cálculo dental e os do
esmalte é considerada muito forte, tanto que para remover todo o cálculo, (HAYASHI,
1993). Além disso, também foi mostrado que o flúor induz a calcificação in vitro de
culturas de Streptoccocus mutans com soluções de fosfato de cálcio (SOUCHAY et al.,
1995).
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No tocante às lesões cariosas inativas, estudos histopatológicos demonstraram que a
camada externa “remineralizada” de lesões cariosas dentinárias inativas representa, na
verdade, bactérias mortas calcificadas - cálculo dental - (SCHÜPBACH et al., 1992).
Assim, há indícios de que o cálculo dental possa estar bastante envolvido na patogênese das
lesões cariosas. Neste contexto, é importante tecer alguns comentários sobre os modelos
experimentais de cárie dental.
1.1.1 MODELO IN SITU DE CÁRIE DENTAL
Uma abordagem sobre modelos de cárie é cabível pelo fato da formação de cálculo
dental ter sido relatada a partir de tais modelos in situ (PEARCE, 1982; SOUSA, 1996). Os
modelos experimentais de cárie dental podem ser divididos em três tipos: in vitro, in vivo e
in situ. Os modelos in vitro são aqueles em que o processo carioso se dá fora da cavidade
bucal em condições bastante controladas. Dos modelos experimentais intra-orais, o modelo
in vivo é aquele em o processo carioso é induzido em dentes naturalmente implantados na
cavidade bucal e que podem oferecer uma resposta do complexo dentina-polpa. Os dentes
testados são normalmente extraídos para análise ou têm suas superfícies moldadas com
materiais plásticos para se obter um molde a ser estudado. Os modelos in situ são aqueles
em que o processo carioso é induzido em fragmentos de dentes extraídos que são inseridos
na cavidade bucal e aí permanecendo através de fixação em aparelhos ou dispositivos
restauradores (ZERO, 1995).
O fragmento de tecido dental duro pode ser fixado em aparelhos removíveis,
adaptados ao maxilar superior ou à mandíbula. Estes aparelhos podem ser projetados
apenas para este fim, ou podem ser próteses funcionais onde o fragmento dental é fixado
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temporariamente. A figura 1 mostra um exemplo de aparelho intra-oral removível, feito
com resina acrílica quimicamente polimerizada, projetado apenas para o protocolo in situ.
Uma tela plástica, fixada à resina, cobre os fragmentos dentais, deixando um espaço de 1-2
mm para a formação de biofilme bacteriano. Alternativamente, o fragmento dental pode ser
fixado num elemento dental normalmente implantado, sobre sua superfície de tecido dental
duro ou sobre uma superfície de uma restauração nele presente.
a
b
Fig. 1. Aparelho intra-oral (palatal) removível de resina acrílica com fragmentos dentais
(cabeça de seta), (a). Telas plásticas cobrem os fragmentos, deixando espaço para o
biofilme bacteriano (b).
Os modelos in situ criam condições intermediárias entre a situação fisiológica
natural e as condições laboratoriais bastante controladas. As vantagens e desvantagens
foram destacadas por ZERO (1995). Como vantagens, podem ser citados: o fato do
processo carioso ser induzido no meio bucal; a possibilidade de controlar as variáveis
experimentais; a flexibilidade no protocolo experimental; a possibilidade de utilizar várias
técnicas científicas analíticas; e a melhor adequação a questões éticas envolvendo humanos
e o menor custo em relação aos estudos clínicos. O modelo apresenta as desvantagens de
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limitar o número de indivíduos participantes (entre cinco e quarenta), é altamente
dependente da colaboração do participante e requer conhecimentos nas áreas clínica e
analítica. Também podem ser citadas como desvantagens o fato de que as condições de
fluxo e composição salivares, composição e espessura do biofilme bacteriano oral e
retenção mecânica são diferentes daquelas que operam naturalmente (FEJERSKOV et al.,
1994). A colaboração do participante é, sem dúvida, o principal problema do modelo in
situ.
Neste contexto, é de fundamental importância delimitar bem o objetivo de um
estudo com modelo in situ para se obter relevância clínica e/ou biológica. É preciso saber se
ambos cárie e cálculo dentais podem se formar concomitantemente em condições
controladas de desafio cariogênico em curtos intervalos de tempo (menores que trinta dias).
Nesta tese, o modelo in situ de cárie com aparelhos removíveis especificamente projetados
para este fim foi escolhido. O foco no modelo in situ de cárie está relacionado à
comprovação de um evento biológico, cujas contribuições dos diversos fatores biológicos
envolvidos ainda serão abordadas. Pelas limitações do modelo in situ, a relevância para a
clínica odontológica vai requerer estudos mais aprofundados par ser bem delimitada.
Uma outra questão que requer investigação é o efeito da formação de cálculo dental
na composição do tecido dental duro. No caso do esmalte dental cariado, esta investigação
pode ser feita empregando a técnica analítica da birrefringência através de microscopia de
luz polarizada, que permite estudar regiões histopatológicas específicas da lesão cariosa
correlacionando-as com a composição bioquímica do tecido.
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1.1.2 INDUÇÃO DE DEPÓSITOS CALCIFICADOS EM MATERIAIS PARA
IMPLANTES OSTEOINTEGRADOS
Depósitos de fosfato de cálcio, principalmente hidroxiapatita (HA), são empregados
na Medicina para cobrir implantes osteointegrados de titânio e servirem de material de
preenchimento em uma gama de procedimentos cirúrgicos envolvendo o tecido ósseo,
ambos com a finalidade de estimular a formação de matriz óssea mineralizada com base na
propriedade da HA de promover adesão biológica com o osso (JARCHO et al., 1977).
Implantes colocados com depósitos de fosfato de cálcio mostraram um melhor reparo ósseo
quando comparado àqueles sem os depósitos (JANSEN et al., 1991). Os depósitos
formados sobre os implantes são sintéticos, enquanto que hidroxiapatitas biológicas são
extraídas de certos animais para preparar materiais com a finalidade de preenchimento.
Dentre as técnicas usadas para formar estes depósitos, podemos destacar as
deposições por laser pulsátil ablasivo (CLERIES et al., 2000; FERNANDEZ-PRADAS et
al., 1999), a técnica eletroforética (DUCHEYNE et al., 1990) e a aplicação de spray de
plasma (DE GROOT et al., 1987). Esta última sendo a mais usada pela vantagem de
promover, em curto espaço de tempo, depósitos espessos em larga escala (SARDIN et al.,
1994). Porém, os depósitos formados pela técnica de spray de plasma apresentam pouca
durabilidade, fraca adesão ao substrato, formação de compostos de fosfato de cálcio não
cristalinos, limitada performance de biocompatibilidade e baixa resistência mecânica
(CLERIES et al., 1999). A técnica do laser pulsátil ablasivo consegue formar camadas de
espessura menor e aumentar a quantidade de cristais de hidroxiapatita, pouco solúveis,
porém estes depósitos apresentam fraca resistência mecânica e são associados com outros
compostos bastante solúveis (CLERIES et al., 1998; CLERIES et al., 2000).
15
O material ideal para substituir osso deveria ser: (1) osteocondutivo, para permitir
uma rápida integração com o tecido ósseo receptor; (2) biodegradável, para ser substituído
por um novo tecido ósseo natural; e (3) suficientemente resistente para suportar as forças
mecânicas sofridas no período inicial do pós-implante, antes de sua substituição por osso
natural (PILLAR et al., 2001).
Recentemente foi demonstrado que os cristais de HA do cálculo dental, assim como
os do osso e outros tecidos mineralizados, também apresentam uma linha escura central
(KAKEI et al., 2000). Esta linha é considerada como o sítio inicial do crescimento de
cristais de tecidos mineralizados (MARSHALL & LAWLESS, 1981; KAKEI et al., 1997) e
não está presente nas HA sintéticas. Esse achado indica que a formação do cálculo pode ser
regida pelos mesmos mecanismos dos tecidos mineralizados (KAKEI et al., 2000).
Também foi demonstrado que o cálculo esterilizado pode ficar encapsulado no tecido
conjuntivo sem causar inflamação acentuada (ALLEN & KERR, 1965) e que, em certas
condições clínicas, pode haver aderência do epitélio gengival mesmo sobre cálculo aderido
à coroa dental (LISTGARTEN & ELLEGAARD, 1973). No tocante à adesão a materiais
não biológicos, o cálculo pode se aderir firmemente à resina acrílica (HAYASHI, 1995), ao
vidro (UYEN et al., 1989), ao amálgama de prata (HOHENWALD et al., 1987) e ao titânio
(SPEELMAN et al., 1992). Mesmo a remoção mecânica ultra-sônica com instrumentos
metálicos pode não conseguir remover todo o cálculo aderido ao titânio (SPEELMAN et
al., 1992).
Assim, o cálculo dental apresenta excelentes características biológicas e físicas para
ser um promissor substituto de osso. Desenvolvimentos nesta área podem ser
impulsionados se for provado que a formação de cálculo dental pode ocorrer (de maneira
controlada) em intervalos de tempo mais curtos que trinta dias e a partir da saliva de
16
indivíduos que não necessariamente têm tendência a formar cálculo. O paciente que teria de
receber um implante metálico, doaria sua saliva para que fosse induzido, em laboratório, o
crescimento de cálculo dental sobre o implante. Este, após ser limpo e esterilizado, seria
implantado no paciente. Não haveria aí o risco de contaminação por príons, por exemplo,
que ocorre no caso dos produtos derivados de matriz óssea bovina.
1.2 Técnicas de análise e de caracterização de cálculo dental e de tecido dental duro
1.2.1 MICROSCOPIA ELETRÔNICA DE VARREDURA E MICROANÁLISE DE
RAIOS X
Na microscopia eletrônica de varredura (MEV), as análises morfológicas podem ser
feitas usando detector de elétrons secundários, que detecta energias de até 50eV e dá
informações de eventos mais superficiais, ou usando detector de elétrons retro-espalhados,
que detecta energias de até 90% da energia incidente e dá informações de eventos em
profundidades com 1-2 ordens de grandeza maiores do que aquelas dos eventos detectados
pelo outro detector (GOLDSTEIN et al., 1999). O primeiro sinal da lesão cariosa visível ao
MEV, com detector de elétrons secundários, é um aumento dos espaços intercristalinos na
superfície do tecido dental duro, que ocorre a partir de dois dias de indução em modelo
experimental de cárie (THYLSTRUP et al., 1994). Usando detector de elétrons retroespalhados, análises em cortes transversais mostram diferença de contraste entre a lesão e a
área normal.
Os tecidos dentais duros requerem uma cobertura de metal ou carbono para serem
observados através dos equipamentos convencionais de alto vácuo. Quanto menor a
17
espessura desta camada, menor a possibilidade de causar artefatos que comprometem a
imagem. Uma espessura de 10 nm é aceitável para esses tecidos. O tipo de secagem
também é um fator que limita a qualidade da imagem. Para se obter boas imagens com
aumentos maiores que 40.000 vezes, é recomendada a secagem ponto crítico e a
metalização em pressão atmosférica em torno de 10-10 Torr, o que é pouco usado na
literatura (HOLMEN et al., 1985).
Os depósitos de cálculo dental apresentam duas morfologias ao MEV: depósitos
associados a corpos bacterianos (tipo A) e depósitos sem associação com corpos
bacterianos (tipos B) - SCHROEDER (1969). Os primeiros são mais facilmente
identificados por apresentarem tabiques de ~ 0,1 µm separando espaços (esféricos,
retangulares, etc) com diâmetro de 1 µm previamente ocupados por corpos bacterianos. Os
segundos são localizados adjacentes aos depósitos tipo A, não apresentam morfologia
compacta irregular e são de difícil diferenciação em relação a impurezas. No primeiro
artigo desta tese, só foram considerados os depósitos tipo A.
As estruturas visualizadas ao MEV podem ser analisadas através de micro-análise
de raios X por dispersão de energia (EDS). Normalmente acoplada ao MEV há uma sonda
de raios X e um detector de radiação secundária. O processo de ionização da camada mais
interna dos elementos químicos pelos raios X primários dá origem à emissão de elétrons
Auger e de raios X característicos. Estes últimos são utilizados pelos detectores de raios X
para se obter informações acerca dos componentes químicos da amostra. As energias
detectadas são comparadas com dados armazenados das energias das camadas de elétrons
dos elementos químicos, sendo o espectro obtido o resultado do ajuste matemático das
energias detectadas com os prováveis elementos (GOLDSTEIN et al., 1999).
18
A micro-análise de raios X pode ser qualitativa ou quantitativa. Normalmente são
consideradas as energias da camada mais interna (camada K) dos elementos. Na análise
quantitativa, devem ser tratados os efeitos de fenômenos de absorção, fluorescência e
número atômico. O efeito de número atômico é a perda de elétrons do feixe incidente por
espalhamento elástico. O efeito da absorção é a diminuição dos raios X que chegam ao
detector. E o efeito da fluorescência é a excitação de elementos leves por raios X derivados
de elementos pesados. Para minimizar os erros decorrentes destes efeitos, um tratamento
matemático adicional é feito nos dados obtidos pela análise qualitativa (GOLDSTEIN et al.,
1999).
As cerâmicas de fosfato de cálcio podem ser inferidas pela razão entre átomos de
cálcio e átomos de fósforo (mol/mol) obtida pela micro-análise de raios X. Durante as
análises, é importante padronizar o tamanho da área analisada, a distância de trabalho, a
energia de irradiação e o tempo em que o detector fica ativo (tempo vivo), a fim de se obter
consistência entre diferentes amostras.
1.2.2 ESPECTROSCOPIA DE FLUORESCÊNCIA
A fluorescência é uma propriedade ótica que alguns materiais possuem de emitir
uma radiação eletromagnética com comprimento de onda maior do que aquele da radiação
que o excitou (LAKOWICZ, 1999). Os tecidos dentais duros e o cálculo dental possuem
auto-fluorescência, que é uma fluorescência decorrente de cromóforos presentes na sua
composição original (ZIJP, 2001). A partir de estudos com luz monocromática não
coerente, foi mostrado que o esmalte dental apresenta fluorescência característica da
ditirosina, com emissão em 410 nm (largura de 60 nm) (BOOIJ & TEN BOSCH, 1982); e
19
que a dentina, que não tem um cromóforo identificado, apresenta fluorescência com
emissão em 440 nm (largura de 100 nm), sendo bem mais intensa que a do esmalte
(MATSUMOTO et al, 1999).
O cálculo dental apresenta fluorescência no vermelho (região entre 580 nm e 700
nm) quando excitado com comprimentos de onda correspondentes às bandas de Soret (390
nm a 440 nm) e Q (500 nm a 600 nm) das porfirinas, tendo sido proposto que essa
fluorescência é devido a algumas espécies de porfirina sem metal (DOLOWY et al., 1995;
REIS et al., 2001; BUCHALLA et al., 2004). A fluorescência do cálculo dental em solução
tem sido estudada com soluções altamente ácidas (27 % v/v de HCl).). Com base em
análise de fatores dos espectros de fluorescência dessas soluções, tem sido proposto que a
fluorescência é decorrente de três cromóforos: um com excitação em 407 nm e picos de
emissão em 604 nm e 656 nm; outro com excitação em 416 nm e picos de emissão em 617
nm e 670 nm; e um terceiro com excitação em 631 nm e picos de emissão em 635 nm e 680
nm, apresentando as seguintes intensidades relativas: 416 nm > 406 nm > 431 nm
(FERREIRA et al., 1999; REIS et al., 2001). Nestes estudos, uma semelhança com a
fluorescência da hematoporfirina (Fig. 2) foi apontada (REIS et al., 2001). Também foi
relatada uma fluorescência com emissão abaixo de 580m nm (DOLOWY et al., 1995), mas
esta ainda não foi relacionada a qualquer cromóforo. Uma vez que a espécie iônica de
porfirina presente nestas soluções é o cátion bivalente (GOUTERMAN, 1973), esses três
cromóforos seriam variantes desta espécie iônica.
A fluorescência do cálculo dental sólido, excitada com luz monocromática não
coerente, também foi relatada como apresentando características similares às da porfirina,
com o máximo de excitação entre 400 nm e 420 nm (BUCHALLA et al., 2004). Mais
recentemente foi mostrado que lesões cariosas de esmalte pigmentadas e lesões cariosas
20
radiculares apresentam fluorescência semelhante à do cálculo dental (BUCHALLA et al.,
2004b; BUCHALLA, 2005), o que indica haver dificuldades no diagnóstico diferencial
entre lesão cariosa e cálculo dental a partir da fluorescência.
OH
CH3CH
CH3
OH
H 3C
N
NH
H3 C
NH
N
CH2
HOOCCH2
CHCH3
CH3
CH2
CH2COOH
Fig. 2. Estrutura química da hematoporfirina.
No tocante à fluorescência de lesões cariosas, duas técnicas têm sido usadas na
literatura odontológica: a da fluorescência quantitativa induzida por luz (FQL) e a do
Diagnodent. A primeira consiste numa fluorescência amarela induzida por lazer azul ou
verde. Um filtro (λ ≥ 540 nm) é usado para eliminar o espalhamento elástico, sendo os
dados coletados por câmera colorida digital e analisados por um software (Inspektor
21
Research Systems BV, Amsterdã, Holanda). As áreas cariadas aparecem como áreas
escuras devido a fluorescência ser menor em relação ao tecido dental duro hígido. Este
sistema tem mostrado bons resultados em estudo in vivo, porém sua sensibilidade parece ser
limitada a uma profundidade de 400 µm (TRAENEUS et al., 2005).
O segundo sistema opera com laser diodo com λ de 650 nm e potência de pico de 1
mW. A fluorescência é coletada por fibra ótica, detectada por um detector foto-diodo e
amplificada para suprimir o sinal da luz ambiente (TRAENEUS et al., 2005). Boas
sensibilidade e reprodutibilidade têm sido relatadas em estudo in vivo (LUSSI et al., 2001).
Neste sistema, a fluorescência das lesões cariosas aumenta em relação aos tecidos hígidos.
Ainda não se sabe o que fluoresce nas lesões cariosas a partir desses dois sistemas.
Espalhamento da luz e supressão e/ou remoção dos cromóforos são uns dos mecanismos
possíveis para explicar a fluorescência detectada pelo sistema FQL. No Diagnodent, tem
sido proposto a ação de porfirinas e de metabólitos de bactérias (TRAENEUS et al., 2005).
O relato da formação de cálculo dental em modelo in situ de cárie dental (SOUSA,
1996) tem levantado uma discussão sobre a necessidade de caracterizar independentemente
da influência de quem analisa. A identificação da morfologia dos depósitos de cálculo
dental através de microscopia depende muito do pesquisador. Uma proposta para resolver
este problema seria incluir a análise de certas características do cálculo dental que não
ocorrem nos tecidos dentais duros. Pelo nosso conhecimento, as técnicas que são voltadas
para analisar a estrutura mineral do cálculo dental não são capazes de diferencia-lo, pelo
menos qualitativamente, dos tecidos dentais duros que possuem as mesmas cerâmicas de
fosfato de cálcio. A alternativa que propomos nesta tese é o uso da fluorescência excitada
com luz não coerente monocromática, focando na ausência de fluorescência no vermelho
22
dos tecidos dentais duros. Conjuntamente com a fluorescência, nossa proposta inclui a
análise prévia das amostras através de microscopia eletrônica de varredura.
Um problema que existe é que não há dados da fluorescência do cálculo dental em
função do pH, dados estes que são importante para a identificação dos cromóforos
presentes. Nesta tese, o nosso terceiro estudo focaliza esta questão. Estes dados podem
aprimorar a caracterização do cálculo dental sólido e em solução através da fluorescência.
1.2.3 O ESMALTE DENTAL CARIADO ANALISADO PELA MICROSCOPIA DE LUZ
POLARIZADA
A birrefringência do esmalte dental vista através das oculares - birrefringência
observada (BRobs) - é o resultado da soma da birrefringência intrínseca (relacionada ao
conteúdo mineral e com sinal negativo) com a birrefringência de forma (relacionada ao
conteúdo não mineral e com sinal positivo). Esta última, que é alterada pelos meios de
imersão, é interpretada através de uma equação clássica formulada para materiais
heterogêneos compostos por diferentes fases, cada uma com seu volume e seu respectivo
índice de refração (IR) - a equação de Wiener (WIENER, 1912) -:
BR form =
V1V2 (n12 − n22 ) 2
,
2(V1 n1 + V2 n2 ). (1 + V1 )n22 + V2 n12
[
]
onde V1 e V2 referem-se aos volumes dos conteúdos mineral e não mineral,
respectivamente; n1 é o IR do conteúdo mineral (1,62) e n2 é o IR do conteúdo não mineral.
A interpretação da BRobs qualitativa do esmalte dental é atualmente feita com base na
aplicação da equação de Wiener de acordo com DARLING (1958), que considerou o
esmalte como sendo composto por duas fases, uma mineral e outra não mineral, esta última
23
apresentando o mesmo IR do meio de imersão (meio em que o esmalte está imerso quando
está sendo analisado ao microscópio de luz polarizada). Todo aumento da birrefringência
positiva era interpretado como perda mineral e vice-versa; e, pela equação de Wiener, a
birrefringência de forma é tão maior quanto maior for a diferença entre os IRs dos volumes
mineral e não mineral. A BRobs teórica poderia, assim como a BRobs experimental, ser
positiva, nula ou negativa. O modelo de DARLING (1958) - deste ponto em diante referido
como modelo clássico - preconiza que a birrefringência de forma é nula quando o esmalte é
imerso num meio aquoso com o mesmo IR da fase mineral, quando a BRobs seria resultado
apenas da birrefringência intrínseca. Foi verificado que o valor máximo da BRobs do esmalte
dental imerso em solução com IR de 1,62 seria de - 0,003. Assim, a BRobs do esmalte
cariado seria dada pela soma deste valor com o da birrefringência de forma calculada pela
equação de Wiener. O comportamento da BRobs em relação ao volume mineral, segundo o
modelo clássico, está ilustrado na figura 3.
O comportamento da BRobs teórica mostra que há amplas faixas de valores
ambíguos para qualquer meio de imersão. A proposta de DARLING (1958) foi a de
correlacionar com a BRobs experimental apenas alguns valores da BRobs teórica. Estes
valores são aqueles próximos de zero e relativos a valores de volume mineral > 50 %. Pela
Fig. 3, BRobs nulas em ar, água e soluções de Thoulet com IRs 1,41 e 1,47 são interpretadas
como indicativas de perda minerais de 1%, 5%, 10% e 25%, respectivamente. Valores
negativos de BRobs neste mesmos meios indicam perdas minerais menores do que aquelas
referentes às BRobs nulas, enquanto que valores positivos de BRobs indicam perdas minerais
maiores.
24
0,08
Ar
0,07
0,06
0,05
BRobs
0,04
0,03
0,02
Água
0,01
Thoulet 1,41
0,00
Thoulet 1,47
-0,01
0,0
0,2
0,4
0,6
0,8
1,0
Volume mineral
Fig. 3. Gráfico do comportamento da BRobs teórica dada pelo modelo clássico para vários
meios de imersão.
Analisando a mesma amostra de esmalte com meios de imersão com diferentes IRs,
DARLING (1958) encontrou uma correlação qualitativa entre a sua interpretação da BRobs e
o conteúdo mineral obtido por radiomicrografia. Assim, foram obtidas as bases para definir
as quatro zonas histopatológicas das lesões cariosas de esmalte com microscopia de luz
polarizada. Duas zonas são visualizadas com imersão em água e as outras duas são
visualizadas com imersão em quinolina. A primeira é uma faixa superficial de BRobs
negativa em água , a camada superficial (perda mineral < 5 %). A segunda é uma área subsuperficial com BRobs positiva em água, o corpo da lesão (perda mineral > 5 %). A terceira
25
é uma faixa de cor escura, observada quando o esmalte é imerso num meio oleoso com IR
de 1,62, a quinolina, a zona escura (perda mineral de 2-4%). Alguns poros não são
penetrados pela quinolina, deixando algumas áreas preenchidas por vapor de água (valor do
IR fica entre o do ar e o da água) com BRobs nula ou levemente positiva. A quarta faixa da
lesão cariosa, conhecida como zona translúcida (perda mineral ~ 1%), também é observada
com imersão em quinolina e é caracterizada por uma BRobs negativa. A zona translúcida
apresenta BRobs levemente positiva com imersão em ar.
Há evidências de que a dinâmica do processo carioso é acompanhada por alterações
nas zonas histopatológicas da lesão cariosa, com o corpo da lesão e a zona translúcida
aumentando com o avanço da perda mineral, e a camada superficial e a zona escura
aumentando com a remineralização (SILVERSTONE, 1984). As alterações nessas zonas
têm sido extensivamente usadas na literatura odontológica para estudar o processo carioso,
e os dados do estudo de DARLING (1958) ainda são citados como os mais completos de
microscopia óptica sobre a patologia da cárie dental de esmalte (DARLING, 1958).
Entretanto, seu modelo apresenta sérias restrições técnicas.
Uma primeira restrição que pode ser mencionada é que o conteúdo não mineral do
esmalte dental apresenta água e matéria orgânica, cada um com seu próprio IR,
diferentemente do conceito de fase única usado pelo modelo clássico. Um aumento na
birrefringência de forma do esmalte dental foi relatado após remoção da matéria orgânica
(HOUWINK, 1971), o que é inconsistente com o modelo clássico. Há, ainda, evidências de
que o conteúdo de matéria orgânica pode aumentar nas lesões cariosas (TERANAKA et al.,
1986; VAN DER LINDEN et al., 1987). Estes dados questionam a maneira de interpretar o
aumento da birrefringência de forma como uma diminuição do conteúdo mineral.
26
Entretanto, o principal problema do modelo clássico é que ele produz uma BRobs
teórica bastante inconsistente com a BRobs experimental do esmalte. O esmalte maduro
imerso em água (87 % de volume mineral, e n1 sendo 1,62, e n2 sendo 1,33), por exemplo,
apresenta uma BRobs teórica de + 0,004, enquanto a BRobs experimental é, em média, 0,002. Por exemplo, uma interpretação de que há 5 % de perda mineral não significa que o
esmalte tenha 82 % de volume mineral.
A formação de cálculo dental em modelo in situ de cárie levanta a questão de quais
seriam as alterações ocorridas na área de esmalte abaixo do cálculo dental em comparação
com as áreas que não são cobertas por cálculo. Haveria uma potencialização da
remineralização? Há uma questão em aberto acerca das razões que levam os minerais do
meio bucal a se depositarem como cálculo dental ao invés de serem totalmente
reincorporados ao interior do esmalte.
A possibilidade de correlacionar a composição bioquímica do esmalte dental
(minerais, matéria orgânica e água) com suas regiões anatômicas microscópicas justifica
um esforço para aprimorar a interpretação da birrefringência através da microscopia de luz
polarizada. A análise do volume de água no esmalte pode ser usada para estudar o
transporte de materiais nas lesões cariosas e para pesquisar como os minerais disponíveis
na placa bacteriana poderiam, ao invés de se precipitar na superfície como cálculo dental,
penetrar no interior do esmalte. O principal problema a ser resolvido é a falta de
consistência entre a birrefringência teórica e a experimental.
Neste trabalho relatamos três estudos, o primeiro sobre a formação concomitante de
cálculo e cárie dentais em modelo in situ de cárie em intervalos de 2-14 dias, o segundo
sobre a avaliação comparativa do cálculo dental à hematoporfirina em diferentes
concentrações de hidrogênio utilizando espectroscopia de fluorescência, e o terceiro sobre
27
uma nova abordagem matemática para aprimorar a interpretação da birrefringência do
esmalte dental através de microscopia de luz polarizada.
28
2. OBJETIVOS
29
2. OBJETIVOS
Os objetivos desta tese consistem em:
1. Investigar se a formação concomitante de cárie e cálculo dentais em modelo
in situ de cárie dental pode ocorrer em intervalos de 2 a 14 dias;
2. Fazer um estudo comparativo da fluorescência do cálculo dental sólido com
a fluorescência de soluções de cálculo dental e de hematoporfirina em
diferentes concentrações de hidrogênio visando obter informações sobre os
cromóforos presentes;
3. Propor um novo modelo matemático que permita obter consistência entre
dados teóricos e experimentais da birrefringência do esmalte dental
utilizando microscopia de luz polarizada.
30
3. CONCOMITANT CARIES AND CALCULUS
FORMATION FROM IN SITU CARIES MODEL IN
PERIODS OF 2-14 DAYS
(SOUSA.
FB,
MANGUEIRA
PJ,
SANTOS-
MAGALHÃES NS, VIANNA SS, TAMES DR)
31
CONCOMITANT CARIES AND CALCULUS FORMATION FROM IN SITU CARIES
MODEL IN PERIODS OF 2-14 DAYS
Artigo submetido à revista Caries Research
SOUSA. FB,1* MANGUEIRA PJ,1 SANTOS-MAGALHÃES NS,2 VIANNA SS,3
TAMES DR.4
1
Departmento de Morfologia, Centro de Ciências da Saúde, Universidade Federal da
Paraíba, Cidade Universitária, 58000-000, João Pessoa, Paraíba; 2Laboratório de
Imunopatologia Keizo-Asami (LIKA), Centro de Ciências Biológicas and 3Departamento
de Física, Centro de Ciências Exatas e da Natureza, Universidade Federal de Pernambuco,
Av. Prof. Moraes Rego, 1235, 50670-901, Cidade Universitária, Recife, Pernambuco; and
4
Faculdade de Odontologia, Universidade do Vale do Itajaí, Itajaí, Santa Catarina, Brazil.
Key words: dental caries, dental calculus, caries model, pathology.
Short title: Caries/calculus formation in situ
*
Corresponding author:
1
Paraíba, Cidade Universitária, 58000-000, João Pessoa, Paraíba, Brazil.
e-mail: [email protected]
Caries/Calculus Formation In Situ
32
Abstract
The aim of this study is to report concomitant formation of calculus deposits and caries on
in situ dentin caries model (with sucrose and fluoride) for short time periods (2-14 days).
Samples were removed after 2, 5, 9 and 14 days in situ and analyzed by SEM, EDS,
stereomicroscopy with image analysis and fluorescence spectroscopy. Each volunteer
presented one or more samples with calculus, on both non-carious and carious surfaces.
Our study shows that more attention must be paid to the formation of calculus from in situ
caries models.
Introduction
In situ caries models are not expected to give rise to calculus formation. However,
the formation of loosely mineralized deposits in dental plaque concomitantly with carious
dissolution of enamel, in an environment with fluoridated mineralizing solution, has been
reported [Pearce, 1982]. In a morphological study with transmission electron microscopy,
those deposits were shown not to be identical to calculus as they were never present within
intact bacterial cells [Pearce et al., 1991]. In addition, it was showed by scanning electron
microscopy that calculus (firmly bound to hard dental tissue) and caries could develop
concomitantly on dentin specimens subjected to an in situ carious attack with a daily
application of a 50% sucrose solution in individuals living in a water-fluoridated area for
periods of four weeks [Sousa, 1996]. In this study, some samples with calculus had the
cariogenic challenge disturbed by weekly mechanical plaque removal, giving an indication
33
that the concomitant development of caries and calculus may occur in shorter periods under
the given conditions.
The aim of this study is to report concomitant formation of calculus deposits and
caries on dentin surfaces submitted to in situ caries model (with sucrose and fluoride) for
short time periods (2-14 days).
Material and Methods
Experimental design
Six volunteers, aged 20-29 yr from non water-fluoridated areas, consuming
fluoridated dentifrices (1000-1500 ppm) daily, with normal salivary function and not taking
any medication, were selected. All volunteers provided signed informed consent. Ethical
approval was obtained from the Universidade do Vale do Itajaí Human Research Ethics
Committee. Human dentin specimens (4 x 4 x 3 mm) from third non-erupted molars had
their surfaces consecutively polished with slurries of decreasing grain size until alumina of
1 µm. After autoclaving, specimens were mounted on acrylic removable palatal appliance
with four specimens. Intra-oral periods lasted fourteen days. Two drops of a 50% sucrose
solution (the same applied by Sousa, 1996) was applied four times a day (only on the
specimens) to mimic meals and a 0.05% NaF solution was also offered for performing one
daily mouthwash lasting about one minute with the appliance in the mouth. For each
volunteer, specimens were removed after 2, 5, 9 and 14 days of intra-oral period. Six other
polished specimens, not exposed to the oral cavity, were also selected for reproducibility
during morphological analysis, in a total of thirty.
34
Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS)
All samples were treated with NaOCl 5% for 15 minutes, to remove organic
coatings, then critical point dried with CO2 - atmosphere of 10-2 Torr - and coated with a 10
nm thick layer of gold. The secondary electron detector of a scanning electron microscope
JEOL 5900HV (voltage of 15 KeV) was used. Each specimen was analyzed under tilting
angles of 0° and 40°. EDS analyses were performed in representative areas by a
VOYAGER system attached to the microscope using the following parameters: spatial
resolution of 1 µm, electron beam area of 10 µm, working distance of 8-9 mm, voltage of
15 KeV and a live time of 60 s. Ca/P (mol/mol) ratios (mean of 5 measurements) were
analyzed using a standardless method - intensity of the standard calculated on the basis of
the spectral properties of the sample [Goldstein et al., 1992] - with corrections for atomic
number, absorption and fluorescence (ZAF correction). SEM analysis of surface changes
(demineralization and dental calculus) was performed twice by blinded examiners with a
time interval of seven days between the two analyses. A set of 3 images from each sample
was chosen. The Kappa test was used to calculate agreement. Only Schroeder’s type A
dental calculus mineralization centers (with mineralized bacterial bodies outlines)
[Schroeder, 1969] were considered.
Stereomicroscopy and backscattered scanning electron microscopy (BSE-SEM)
After SEM, all samples were treated with NaOCl 5% for 15 minutes to remove the
gold coating and then hemi-sectioned centrally on the exposed surface. The surfaces
created after cutting were gently polished with alumina 10 µm and then analyzed under the
stereomicroscope (Leica MZ12, Leica, Switzerland). Image analysis for the automated
35
identification of demineralized areas and lesion depth measurements were performed using
Leica QWIN Plus software (Leica, Switzerland). A standard margin of color discrepancy
detection in the software was used for all samples when the software was asked to
differentiate sound and carious dentin. The only influence of the operator was to choose a 1
mm-wide central area for automated lesion depth measurement.
Fluorescence spectroscopy and backscattered scanning electron microscopy (BSESEM)
In samples that presented thick (> 100µm in height) and extensive calcified
deposits, a scapel was used to remove the surface by scraping after stereomicroscopy. The
scraped material was dissolved in a solution of HCl 27 % (~ 0.2 ml), dispensed in a quartz
cuvette and submitted to fluorescence spectroscopy in a commercial photon-counting
spectrofluorometer (PC1, ISI, USA and Vinci software, ISI, USA) equipped with a xenon
arc lamp operating at 10 mA, using 1 mm slits (bandwidth of 8 nm). Excitation wavelength
(λexcit) was 416 nm and the emission (λemis) was collected from 425 to 800 nm (mean of 15
readings). Fluorescence of other two saturated solutions in HCl 27%, one with human
dental calculus and another with human dentin, were also analyzed.
Next all samples had their cut surface prepared (polishing, drying and metallic
coating as described before) for SEM analysis using backscattered electrons detector
(voltage of 15 KeV) in order to confirm the presence of demineralization.
36
Statistical Analysis
The differences between in situ periods of all volunteers with regard to lesion depth
were evaluated by paired t test (significance limit at 5%).
Results
The occurrence of demineralization and calcified deposits was depicted from SEM
examination (Table). Kappa’s coefficients of intra-examiner agreement were 0.88 and
0.83, and 0.78 for inter-examiner agreement of the diagnosis of demineralization with
SEM. For dental calculus, it was decided to count only cases with full agreement between
examiners. Mean lesion depth values were 0.0, 0.197 (± 0.059), 0.442 (± 0.062) and 1.100
mm (± 0.212) for 2, 5, 9 and 14 in situ periods, respectively (differences were statistically
significant, p < 0.05). BSE-SEM analysis confirmed the presence, not the depth
measurements, of demineralization detected by stereomicroscopy.
Seventeen samples presented calculus deposits (14 with demineralization; Table).
Nine of these presented ~ 50 % or more of the surface area covered by the deposits. In the
other samples, deposits formed isolated or connected islands of 50-500 µm wide. Ca/P
ratios ranged from 1.0 (related to dicalcium phosphate) to 1.7 (related to hydroxyapatite)
(Table). Mean Ca/P ratio of dentin (for all samples) was 1.88 mol/mol (± 0.22), compatible
with hydroxyapatite. Demineralization surrounding calculus deposits occurred in thirteen
samples.
37
As the in situ period proceeded, calculus deposits with increased height,
macroscopic in some cases, were seen on demineralized areas (Fig. 1A-B). The shapes of
intact bacterial bodies (cocci, filaments and rods) remained within extracellular calcified
trabeculae, indicating that the mineralization process was preferably extracellular. Some
deposits were identified only after tilting the sample to 40º. No bacterial cell remnants were
identified within calculus deposits.
Three samples (from two volunteers; in situ times of 9 and 14 days) that presented
“large” deposits of calculus and caries were submitted to fluorescence spectroscopy. All of
them presented fluorescence of dental calculus (Fig. 2: the same sample of Figs. 1A-C) and
histological demineralization (Fig. 1C-D). Preparation for BSE-SEM caused fracture in the
demineralized surfaces of most samples, as can be seen in Fig. 1D.
Discussion
The concomitant development (based on our time intervals) of caries lesions and
calculus deposits using an in situ caries model reported here occurred under conditions
known to cause the formation and growth of dental plaque. Combining SEM, fluorescence
spectroscopy and stereomicroscopy (Figs. 1A-C and 2), caries and calculus developments
in the same dentin sample (sound and not exposed to the oral cavity prior to the in situ
experiment) are shown. Our model was able to promote time-dependent demineralisation
(seen from lesion depth data), which means that it was well accomplished by the
volunteers. To our knowledge, this is the first time that this phenomenon has been reported
on the basis of the combination of such mutually validating techniques.
38
The mean Ca/P ratios resemble the structures of dicalcium phosphate (Ca/P of 1.0),
octacalcium phosphate (Ca/P of 1.3), β-tricalcium phosphate (Ca/P of 1.5) and
hydroxyapatite (Ca/P of 1.7-2.3) [Schroeder, 1969]. The crystalline structure of young
calculus is reported to be mainly of dicalcium phosphate in the early stages, following
which octacalcium phosphate develops and, with further growth, β-tricalcium phosphate
and hydroxyapatite are formed [Schroeder, 1969]. It is known that dicalcium phosphate,
octalcacium phosphate and β-tricalcium phosphate can be precipitated in acidic
environments where hydroxyapatite dissolves [Johnsson & Nancollas, 1992], what may
explain the concomitant formation of dental calculus and caries lesions reported here. The
presence of hydroxyapatite, on the other hand, most probably reflects pH fluctuations (with
neutral and/or basic events) during the in situ period. Crystallite morphology and Ca/P
ratios resembling hydroxyapatite in 10-days old calculus deposits have been reported
previously [Kodaka et al., 1992].
Fluorescence of human dental calculus in acid solution has been shown to present
the highest emission with λexcit in 416 nm and λemis peaks in 620 and 660 nm (originated
from hematoporphyrin) [Reis et al., 2001]. A lower fluorescence emission band below 580
nm has also been reported for human dental calculus [Dolowy et al., 1995]. Human dentin
fluoresces with λemis peak in 440 nm and do not present any emission in the region of 580700 nm [Matsumoto et al., 1999], which is in accordance with our fluorescence data for
λexcit in 416 nm (Fig. 2). The selected samples with “large” calcified deposits showed a
mixture of fluorescence bands of human dental calculus and human dentin (Fig. 2). The
double treatment with NaOCl 5% and the absence of bacterial cell remnants during SEM
39
examination exclude the influence of loosely bound organic material of bacteria on the
observed fluorescence.
Samples with calculus deposits < 100 µm in height were not analysed with
fluorescence spectroscopy because the amount of calculus is too low to be detected by a
spectrofluorometer system with lamp source as excitation light [Lakowicz, 1999].
Calculus formation in active cariogenic dental plaque has important repercussions
on the development and the evaluation of anti-tartar and caries-preventive agents. Our
study shows that more attention must be paid to the identification of calculus on hard dental
tissues exposed to in situ caries models.
Acknowledgements
The first author thanks the Brazilian Ministry of Education (CAPES) for financial
support. The other authors are grateful to CNPq Brazilian Federal Agency of Research.
Authors also wish to thank Prof. Jaime A. Cury for his comments on the manuscript.
40
Table. Occurrence of demineralization and calculus deposits (with mean Ca/P ratios).
Volunteer
Demineralization
Dental calculus (Ca/P, mol/mol)
I
5, 9 and 14 days
5 days (1.04 ± 0.03)
2, 5, 9 and 14
2 (1.12 ± 0.04), 9 (1.40 ± 0.02) and 14*
days
days (1.53 ± 0.04)
II
III
5, 9 and 14 days
IV
5, 9 and 14 days
V
5, 9 and 14 days
VI
2 (1.49 ± 0.07), 5 (1.48 ± 0.05) and 9*
days (1.43 ± 0.06)
2 (1.47 ± 0.02), 5* (1.52 ± 0.04), 9*
(1.66 ± 0.06) and 14 days (1.73 ± 0.05)
2 (1.38 ± 0.02), 5 (1.51 ± 0.05), 9* (1.54
± 0.05) and 14* (1.71 ± 0.06) days
2, 5, 9 and 14
5 (1.56 ± 0.05) and 14* (1.41 ± 0.04)
days
days
* Samples with 50 % or more of their surfaces covered by calculus.
41
A
B
C
D
Fig. 1.
Intensity [arbitrary units]
(a)
(b)
(c)
400 450 500 550 600 650 700 750 800 850
λemiss [nm]
Fig. 2.
42
Fig. 1. A, calculus deposits (arrow), of macroscopic size, on a 9-day sample with
demineralization (Bar = 1 mm). B, detail of the area indicated by an arrow in “A”, showing
opening of dentinal tubules (white arrow) and dental calculus (black arrow) (Bar = 20 µm).
C, histological aspect of the same sample after hemi-sectioning showing demineralisation
(black arrow) below the experimental surface (Bar = 1 mm). Opaque outline is
demineralization caused by bacterial acid infiltrated around the sample. D, BSE-SEM
image (bar = 300 µm) showing demineralization (white arrow) in the surface (fractured
apart) indicated by black arrow in “C”.
Fig. 2. Fluorescence spectroscopy data of different samples dissolved in HCl 27 % and
excited with 416 nm: (a), human dentin; (b), human dental calculus; and (c), sample from
the surface of the sample shown in Fig. 1A, showing fluorescence of dentin (arrowhead)
and dental calculus (arrow).
References
Dolowy WC, Brandes ML, Gouterman M, Parker JD, Lind J: Fluorescence of dental
calculus from cats, dogs, and humans and of bacteria cultured from dental calculus. J
Vet Dent, 1995; 12: 105-109.
Goldstein JI, Newbury DE, Echlin P, Joy DC, Romig Jr AD, Lyman CE, Fiori C, Lifshin E:
Scanning electron microscopy and x-ray microanalysis. 2.ed. New York, Plenum, 1992.
Johnsson, MA, Nancollas, GH. The role of brushite and octacalcium phosphate in apatite
formation. Crit Rev Oral Biol Med, 1992; 3:61-82.
43
Kodaka T, Ohohara Y, Debaru K: Scanning electron microscopy and energy-dispersive xray microanalysis studies of early dental calculus on resin plates exposed to human oral
cavities. Scanning Microsc 1992; 6: 475-486.
Lakowicz JR: Principles of fluorescence spectroscopy, ed 2, revised. New York, Kluwer
Academic/Plenum, 1999.
Matsumoto H, Kitamura S, Araki T: Autofluorescnce in human dentine in relation to age,
tooth type and temperature measured by nanosecond time-resolved fluorescence
microscopy. Arch Oral Biol 1999; 44: 309-318.
Pearce EIF: Effect of plaque mineralization on experimental dental caries. Caries Res 1982;
16: 460-471.
Pearce EIF, Wakefield JStJ, Sissons CH: Therapeutic mineral enrichment of dental plaque
visualized by transmission electron microscopy. J Dent Res 1991; 70: 90-94.
Reis MM, Biloti DN, Ferreira MM, Pessine FBT, Teixeira GM: PARAFAC for spectral
curve resolution: a case study using total luminescence in human dental tartar. Appl
Spectr 2001; 55: 847-851.
Schroeder HE: Formation and inhibition of dental calculus. Berne, Hans Huber, 1969.
Sousa FB: The effect of weekly mechnical plaque removal on the in situ development of
dentin caries (in Portuguese). Master dissertation. 1996. Florianópolis, SC, BR:
Universidade Federal de Santa Catarina.
44
4. COMPARATIVE FLUORESCENCE
SPECTROSCOPY OF HUMAN DENTAL
CALCULUS WITH HEMATOPORPHYRIN
(SOUSA. FB, VIANNA SS, SANTOS-MAGALHÃES
NS)
45
COMPARATIVE FLUORESCENCE SPECTROSCOPY OF HUMAN DENTAL
CALCULUS WITH HEMATOPORPHYRIN
Artigo a ser enviado para o Journal of Periodontal Research
Short title: fluorescence of dental calculus and hematoporhyrin
SOUSA. FB,1* VIANNA SS,2 SANTOS-MAGALHÃES NS.3
1
Paraíba, Cidade Universitária, 58000-000, João Pessoa, Paraíba; 2Departamento de Física,
Centro de Ciências Exatas e da Natureza and 3Laboratório de Imunopatologia Keizo-Asami
(LIKA), Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Av. Prof.
Moraes Rego, 1235, 50670-901, Cidade Universitária, Recife, Pernambuco, Brazil.
1
*
1
46
Abstract
Objectives: The aim of this study was to compare fluorescence of solid dental calculus with
fluorescence of solutions of dental calculus and of hematoporphyrin at different pH values.
Methods and results: Twelve samples of human dental calculus (four in aqueous solutions
with pH from 2 to 7; four in 9 M HCl solutions, and four solid samples) were selected.
Fluorescence of solutions of dental calculus and hematoporphyrin were obtained through
excitation (in the Soret and Q bands) from a xenon lamp and from a coherent light of a
pulsed dye laser (560 nm) using different intensities; solid samples of dental calculus were
investigated only by laser irradiation. Solutions of dental calculus and hematoporphyrin in
the pH range from 2 to 7 presented similar fluorescence spectra for wavelengths > 580 nm
(bands at 595 nm and 655 nm at pH < 4; bands at 615 nm and 675 nm at pH > 4, with the
lowest emission around pH 4). An emission at 460 nm was observed only in dental calculus
and with higher intensity for 9 M HCl solutions. Emission bands in the region of 600 nm,
620 nm and 660 nm were observed in 9 M HCl solutions of dental calculus and
hematoporphyrin, each of which presented specific relative intensities. Under laser
excitation and peak intensity > 300 W/cm2, a new emission band at 700 nm was detected
from all samples. Emission spectra of solid samples more closely resembled those of 9 M
HCl solutions.
Conclusions: Fluorescence of human dental calculus solutions in the pH range 2 to 7 is
similar to the fluorescence of hematoporphyrin at the same pH for emission > 580 nm.
Fluorescence of solid dental calculus more closely resembles the fluorescence of dental
calculus in 9 M HCl solutions.
47
Introduction
The identification of dental calculus through fluorescence has recently received
growing attention in the dental literature. Human dental calculus has been reported to
present fluorescence within the region of 580-720 nm when excited in the Soret band (390440 nm) of porphyrins (1-3). From the analysis of strong acid solutions of dental calculus,
it has been proposed that more than one metal-free porphyrin species is responsible for the
fluorescence of dental calculus (1-2). Fluorescence in the region of 580-700 nm is presented
neither by enamel nor by dentin, (4) and this fact would enable non-invasive spectroscopic
differentiations between dental calculus and hard dental tissues. However, dark-colored
root caries and enamel carious lesions have been reported to present the same fluorescence
emission of dental calculus when their exposed oral surfaces are excited in either the Soret
or the Q bands of porphyrins (5-6). In addition, dental calculus formation has been reported
to occur in the environment created by in situ caries models (7-8). Thus, the reliable
assessment of dental calculus has an important application in periodontal and caries
pathology.
Fluorescence of dental calculus in solutions has been studied only in strongly acid
conditions (1-2). Available data on the influence of pH on dental calculus fluorescence are
lacking. Detailed data on the influence of pH on the fluorescence of human dental calculus
are necessary in order to acquire a deeper understanding of the fluorochromes involved and
as a step towards improving the accurate identification of dental calculus from
fluorescence.
48
The aim of this study was to compare fluorescence spectra of solid human dental
calculus with the spectra obtained from solutions of dental calculus at different pH values.
Hematoporphyrin solutions at the same conditions were used as a reference.
Material and Methods
Specimen selection
Twelve extracted human teeth with dental calculus were immersed in 5% NaOCl for
30 minutes, sonicated for 5 minutes in the same solution, and then washed with distilled
water. Next, they were separated into three groups of four samples each: (1) dental calculus
samples dissolved in 9 M HCl (27% v/v) aqueous solution (9 M); (2) dental calculus
samples in aqueous solution with 10-2M (0.03 %) HCl, which were analyzed at pH ranging
from 2 to 7; and (3) solid dental calculus samples adhered to the teeth. Solutions of dental
calculus had 0.002 g of dental calculus and 3 ml of water, and were gravity filtered before
examination. Solid samples were stored in 0.1% tymol solution at 4°C in the dark until
measurements
were
taken.
Solutions
were
analyzed
soon
after
preparation.
Hematoporphyrin free base [7, 12-Bis (1-hydroxyethyl)-3, 8, - 13, 17– tetramethyl– 21H,
23H–porphine–2, 18–dipropanoic acid; Sigma, USA] was used to prepare the solutions
(concentration of 10-5M) under the same conditions as those of the dental calculus.
Fluorescence
Two systems were used to obtain fluorescence data. The first, just applied for
solutions, was a photon counting spectrofluorometer (PC1, ISS, USA) with a xenon arc
lamp (300 W) as the excitation source, using 1-mm slits (bandwidth of 8 nm) for both
excitation and emission. Solutions were analyzed in a quartz cuvette under excitation in the
49
range of 390 nm to 450 nm and 500 nm to 600 nm (increments of 10 nm). pH
measurements were carried out before and after spectroscopy. Emission was collected from
excitation wavelength + 10 nm to 800 nm (increment of 1 nm). Additionally, samples in 9
M HCl solutions were analyzed under excitation at 416 nm and 431 nm, for comparison
with previous results (2).
In the second system, both solutions and solid samples were irradiated by light at
560 nm from a dye laser pumped by the second harmonic of a Nd:YAG laser (5 Hz
repletion rate and 8 ns time pulse duration). The laser beam was focused on the samples
using a 5-cm focal cilindric lens and the peak laser intensity was 30 kW/cm2. Fluorescence
was analyzed by a monochromator (4 nm resolution) and detected by a photomultiplier tube
and boxcar system (Fig. 1). A 590 nm sharp-cut high-pass filter (2-63 Corning) was used in
front of the monochromator. The intensity of the excitation light was controlled using
neutral density filters of 1% (300 W/cm2), 30% (9 kW/cm2), and 50% (15 kW/cm2).
Fluorescence from solutions was collected at 90° of the excitation light path. Solid samples
had their emission collected at 45° of the excitation light path.
50
Mirror
Nd:YAG
Dye laser
Laser
Lens
Spectrometer
Sample
Lens
Filter
PMT
Fig. 1. Laser system setup for fluorescence analysis.
Results
Fluorescence
Xenon lamp
Emission spectra of dental calculus at pH ranging from 2 to 7, when excited
in the Soret and Q bands, presented two fluorescence maxima. Data obtained with
excitation at 400 nm are shown in Fig. 2. For samples with pH < 4, emission peaks
occurred at 595 nm and 655 nm, with almost the same intensity; and for samples with pH >
4, emission peaks were observed at 615 nm and 675 nm, with the former roughly two times
higher than the latter (Fig. 2). A maximum emission in the region of 600-700 nm was
observed under excitation at 400 nm for all samples. A weak emission band, with no clear
dependence on pH, was also observed at 460 nm, but with a low intensity. For emission in
51
the region of 580 nm to 700 nm, the lowest intensity was reached at pH 4. These features,
except for the 460 nm emission band, were similar to those of hematoporphyrin 10-5M
solutions in the same pH range (Fig 2, insert).
Fluorescence of dental calculus in 9 M HCl solutions presented emission bands with
maxima around 600 nm, 620 nm and 660 nm when excited with 400 nm (Fig. 3a, bold
line), around 620 nm and 660 nm (excitation at 416 nm), and around 635 nm with a
shoulder around 680 nm (excitation at 431 nm). A maximum emission in the region of 600700 nm was observed at 440 nm in two samples (emission peak at 640 nm) and around 416
nm (peaks at 620 nm and 660 nm) in the other two samples. We also observed an emission
band with a maximum at 460 nm, but now with nearly equal intensity of the band at 620
nm (Fig. 3a). Excitation at 560 nm resulted in emission bands with maxima around 620 nm
and 660 nm (the latter being merely a shoulder), but with a lower intensity when compared
to the excitation in the Soret band.
Hematoporphyrin solutions in 9 M HCl, when excited in the Soret and Q bands,
presented an emission with maxima around 600 nm, 620 nm (a shoulder), and 660 nm (the
latter with the highest intensity) (Fig. 3b). No emission was observed at 460 nm.
100
3
Fluorescence intensity (x 10 )
3
Fluorescence intensity (x 10 )
52
90
80
70
60
pH 2
700
600
500
pH 2
400
300
pH 3
200
pH 7
pH 6
pH 5
pH 4
100
0
450
500
550
600
650
700
750
Emission wavelength (nm)
50
40
pH 3
30
20
pH 7
pH 6
10
pH 5
0
450
500
550
600
650
700
750
800
Fig. 2. Fluorescence spectra of dental calculus solutions at pHs 2 to 7 excited with the
xenon lamp at 400 nm. Insert: fluorescence spectra of hematoporphyrin at pHs 2 to 7
excited with xenon lamp at 400 nm.
53
Normalized intensity
1.0
0.8
0.6
0.4
0.2
0.0
1.0
(a)
(b)
0.8
0.6
0.4
0.2
0.0
450
500
550
600
650
700
750
Fig. 3. Normalized (at maximum emission) fluorescence spectra of dental calculus (a) and
hematoporphyrin (b) in 9 M HCl solutions (bold line), at pH 2 (dashed line), and at pH 7
(solid line) excited with xenon lamp at 400 nm.
Laser
54
Fluorescence spectra of solid samples and solutions excited with dye laser are
shown in Fig. 4 and 5, respectively. Fig. 5 also shows the comparative emission spectra of
hematoporphyrin and of dental calculus in 9 M HCl solution with solid dental calculus
excited with dye laser. Solid dental calculus excited with the dye laser showed emission
maxima around 625 nm and 700 nm, under all laser intensity excitations. These samples
presented variations in fluorescence spectra characterized by a more or less sharply defined
emission around 625 nm under high laser intensity excitation (Fig. 4). When excited with a
dye laser at 560 nm, all solutions of hematoporphyrin and dental calculus in the pH range
from 2 to 7 showed a similar behavior to that observed under excitation at 560 nm with the
xenon lamp. The new feature is a band around 700 nm (observed for peak laser intensity >
300 W/cm2). Fluorescence of dental calculus and of hematoporphyrin in 9 M HCl solutions
were rather similar to that excited with the xenon lamp, differing only in the appearance of
a band around 700 nm.
55
1
2
3
4
550
600
650
700
750
Fig. 4. Normalized (at 695 nm) fluorescence spectra of four samples of solid dental calculus
excited with a dye laser at 560 nm. Spectra are vertically displaced to facilitate
comparisons.
56
1
2
3
4
5
550
600
650
700
750
800
Fig. 5. Normalized (at emission maximum) fluorescence spectra of hematoporphyrin at pHs
7 (line 1) and 2 (line 2), in 9 M HCl solution (line 3), of dental calculus in 9 M HCl
solution (line 4), and solid dental calculus (line 5) excited with a dye laser at 560 nm. The
sharp peak in line 5 is the scattered laser light. Spectra are vertically displaced to facilitate
comparisons.
57
Discussion
The assessment of the presence of dental calculus in solutions by means of
fluorescence can be improved in certain circumstances provided that the correlation
between pH and emission spectra is known. Reports on dental calculus formation from in
situ caries models (7-8) have important implications in Periodontology and Cariology,
opening up a novel field of application for fluorescence spectroscopy of dental calculus
dissolved in solutions at different pH values. pH-related effects on dental calculus
fluorescence may thus provide a way to help identifying the fluorescent chromophores in
dental calculus. Here, no differentiation was made between supra- and subgingival calculus
as it has been reported that these two categories present the same fluorescence emission
patterns (3).
Fluorescence of dental calculus at wavelengths < 580 nm, showing an emission
band with a maximum around 460 nm (width of 100 nm), are in agreement with published
data on the fluorescence of dental calculus in solution (1-2) and in solid state (3). This
emission has also been observed from bacterial dental plaque (1) and from
hematoporphyrin in biological tissues (9), and in the latter case it has been proposed to be a
result of a reaction between hematoporphyrin and histidine residues (9). Such an emission
makes a spectrofluoroscopic differentiation between dental calculus and the hard dental
tissues problematic for emission < 580 nm as it partially overlaps enamel fluorescence emission band with maximum at 410 nm; width of 60 nm – (10) and more completely
overlaps dentine fluorescence - emission band with maximum at 440 nm and width of 100
nm – (11).
At wavelengths > 580 nm, fluorescence of dental calculus solutions in the pH range
from 2 to 7 presented emission maxima and pH-related relative intensities similar to those
58
of hematoporphyrin in the same pH range, which, for their part, are in agreement with
published data (12, 13, 14). Solutions with pH > 7 were not studied due to the formation of
precipitates in dental calculus solutions, increasing the light scattering and making emission
intensity comparisons impossible.
Emission spectra from 9 M HCl dental calculus solutions are in agreement with
previous reports (1, 2). Compared with solutions in the pH range of 2 to 7, there are two
new features: (I) the emission bands vary with excitation wavelength; and (II) the excitation
wavelength for maximum emission also varies from sample to sample. These results
corroborate what has already been suggested to the effect that different chromophores (with
relative quantitative variations between samples) are present in human dental calculus (1,
2). The fact that such features are only observed in strongly acid environment might be due
to variations in the side chains of porphyrin molecule, which, in turn, are a result of
variations in the bacterial populations among individuals, as previously suggested (1). The
commercially available hematoporphyrin does not present such variations because it has no
molecules from bacterial populations found in the oral environment.
Fluorescence of hematoporphyrin in water in the pH range from 1 to 13 is known to
be a consequence of different ionic species: dication in two allotropic forms (dication a:
main fluorescence bands at 593 nm and 654 nm; dication b: main fluorescence bands at 596
nm and 658 nm) in the pH range from 1 to 5; monocation (main fluorescence bands at 606
nm and 668 nm) in the pH range from 2 to 7; and the free base (main fluorescence bands at
613 nm and 679 nm) in the pH range from 3.5 to 13, with a lower emission (for all species)
occurring near pH 4 owing to severe molecular aggregation (15). With regard to the 9 M
HCl solutions, fluorescence is known to be a result of hematoporphyrin dication (16).
Fluorescence spectra of dental calculus solutions in the pH range from 2 to 7 are here
59
shown to be similar to those of hematoporphyrin, presenting no variation in the emission
bands with excitation wavelength and no variation in the excitation maximum in the Soret
band, which is in agreement with published data on hematoporphyrin spectra (12, 13, 14).
On this basis of similarity, the possible porphyrin species responsible for the fluorescence
of dental calculus, even in solid state, can be proposed as we do in the following lines.
The choice of 9 M HCl solutions was made because: (I) the most detailed available
data on the fluorescence of dental calculus are based on such solutions (1, 2); and (II), the
chromophore existing under high hydrogen ions concentrations is known to be
hematoporphyrin dication (16). It is interesting to note that, bearing in mind the precision of
our systems, fluorescence emissions around 600nm, 620 nm, and 660 nm of
hematoporphyrin in 9 M HCl solutions are consistent with hematoporphyrin dication
fluorescence bands I, II, and V reported in the literature (15); and the fluorescence
emissions around 620 nm and 660 nm of dental calculus 9 M HCl solutions are consistent
with bands II and V from the same molecule (15). Fluorescence of solid dental calculus
under laser excitation is here reported to more closely resemble the fluorescence of 9 M
HCl solutions, which led us to propose that hematoporphyrin dication (or a similar
porphyrin ionic species) is the main, if not the only, chromophore in solid dental calculus.
Solid samples also presented variations in the fluorescence spectra (Fig. 5), in agreement
with published data (3). It should be emphasized that the different chromophores reported
for dental calculus in strongly acid solutions are related to variations in hematoporphyrin
dication and not to different hematoporphyrin species (1).
We also observed, both in solutions and solid samples, an emission band with
maximum around 700 nm (Fig. 4a-b) that has been reported in porphyrins (17) and in solid
dental calculus (3).
60
In conclusion, for the first time it is reported that: (I) the fluorescence features of
dental calculus solutions in the pH range of 2 to 7 are similar those of hematoporphyrin at
emission wavelengths > 580 nm, presenting maximum emission for excitation at 400 nm;
and (II) fluorescence of solid dental calculus more closely resembles the fluorescence of
dental calculus in 9 M HCl solution.
Acknowledgements
Financial support from CNPq and CAPES, Brazilian Federal Agencies, is largely
appreciated. The authors also wish to thank Dr. Camille Chapados, Département de
Chimie-Biologie, Université du Québec à Trois-Rivières (Canada), for her comments on the
results of this study.
References
1. Dolowy WC, Brandes ML, Gouterman M, Parker JD, Lind J. Fluorescence of dental
calculus from cats, dogs, and humans and of bacteria cultured from dental calculus. J Vet
Dent 1995; 12: 105-109.
2. Reis MM, Biloti DN, Ferreira MMC, Pessine FBT, Teixeira GM. PARAFAC for spectral
curve resolution: a case study using total luminescence in human dental tartar. Appl
Spectrosc 2001; 55: 847-851.
3. Buchalla W, Lennon AM, Attin T. Fluorescence spectroscopy of dental calculus. J
Periodont Res 2004; 39: 327-332.
61
4. Zijp JR. Optical properties of dental hard tissues. Groningen: University of Groningen,
2001. 103pp. Dissertation.
5. Buchalla W, Lennon AM, Attin T. Comparative fluorescence spectroscopy of root caries
lesions. Eur J Oral Sci 2004; 112: 490-496.
6. Buchalla W. Comparative fluorescence spectroscopy shows differences in noncavitated
enamel lesions. Caries Res 2005; 39: 150-156.
7. Pearce EIF. Effect of plaque mineralization on experimental dental caries. Caries Res
1982; 16: 460-471.
8. Sousa FB, Mangueira PJS, Santos-Magalhães NS, Vianna SS, Tames DR. Concomitant
caries and calculus formation from in situ caries models in periods of 2-14 days. Caries Res
(submitted # 27/05).
9. Cao EH, Fan XJ, Chi SJ, Wang YX. Hematoporphyrin derivative photosensitization and
cellular fluorescent photoproduct formation. J Photochem Photobiol B, 1988; 2: 503-513.
10. Booij M, Ten Bosch JJ. A fluorescent compound in bovine dental enamel matrix
compared with synthetic dityrosine. Arch Oral Biol 1982; 27: 417-421.
11. Matsumoto H, Kitamura S, Araki T. Autofluorescence in human dentine in relation to
age, tooth type and temperature measured by nanosecond time-resolved fluorescence
microscopy. Arch Oral Biol 1999; 44: 309-318.
12. Srivastava RC, Anand VD, Carper WR. A fluorescence study of hematoporphyrin. Appl
Spectrosc 1973; 27: 444-449.
13. Pottier R, Laplante JP, Chow Y-FA. Photofrins: a spectral study. Can J Chem 1985; 63:
1463-1467.
62
14. Pottier RH, Kennedy JC, Chow YFA, Cheung F. The pKa values of hematoporphyrin
IX as determined by absorbance and fluorescence spectroscopy. Can J Spectrosc 1988; 33:
57-62.
15. Chapados C, Girard D, Trudel M, Ringuet M. Separation of overlapping spectra from
evolving systems using factor analysis. 4. Fluorescence spectra of hematoporphyrin IX.
Biophys Chem 1995; 55: 289-300.
16. Gouterman M. Optical spectra and electronic structure of porphyrins and related rings.
In: Dolphin J, ed.. The porphyrins. v.III. New York: Academic Press, 1978: 1-161.
17. Seybol PG, Gouterman M. Porphyrins XII: fluorescence spectra and quantum yields. J
Mol Spectrosc 1969; 31: 1-13.
63
5. A NEW APPROACH FOR IMPROVING THE
BIREFRINGENCE ANALYSIS OF DENTAL
ENAMEL MINERAL CONTENT USING
POLARIZING MICROSCOPY
(SOUSA. FB, VIANNA SS, SANTOS-MAGALHÃES
NS)
64
A NEW APPROACH FOR IMPROVING THE BIREFRINGENCE ANALYSIS OF
DENTAL ENAMEL MINERAL CONTENT USING POLARIZING MICROSCOPY
Artigo aceito no Journal of Microscopy
Short title: new approach to enamel birefringence analysis
SOUSA. FB,1* VIANNA SS,2 SANTOS-MAGALHÃES NS.3
1
Paraíba, Cidade Universitária, 58000-000, João Pessoa, Paraíba; 2Departamento de Física,
Centro de Ciências Exatas e da Natureza and 3Laboratório de Imunopatologia Keizo-Asami
(LIKA), Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Av. Prof.
Moraes Rego, 1235, 50670-901, Cidade Universitária, Recife, Pernambuco, Brazil.
2
*
1
65
Summary
The main problem in interpreting birefringence of dental enamel under polarizing
microscopy is the lack of physical constants able to allow the Weiner equation to be applied
directly to the composition of such tissue. The present study introduces a new approach to
circumvent this constraint. Since the non-mineral phase of enamel is heterogeneous, its
refractive index can be computed in terms of its components (namely, water, which is
partially replaced by the immersion medium, and organic matter), thereby providing a more
acceptable refractive index to be used in the Wiener equation. Furthermore, the enamel
mineral volume is ordinarily calculated on the basis of the density 3.15 gcm-3. The density
2.99 gcm-3 has been, however, reported to be more accurate for enamel hydroxyapatite, so
enamel mineral volumes from selected published data are converted using such a density.
The birefringence of mature enamel computed by the Wiener equation taking into account
the above refinements matched, for the first time, published experimental birefringence
values. The theoretical water and organic contents were also consistent with published
experimental data. Thus, a direct application of the Weiner equation to the enamel
composition is now achieved. It is speculated that quantitative data on the mineral, the
water and the organic contents of mature dental enamel can be derived from interpretation
of birefringence in two immersion media (obtained before and after extraction of the
organic matter) with this new approach.
Key words: dental enamel, polarizing microscopy, birefringence, Wiener equation.
66
Introduction
Polarizing microscopy is one of the techniques used to analyse the mineral content
of dental enamel. Enamel birefringence seen under polarizing microscopy, the observed
birefringence (BRobs), is the sum of the intrinsic (related to the mineral phase and with
negative sign) and the form (related to the non-mineral phase and with positive sign)
birefringences. This latter, representing the optical property altered by the immersion
media, is interpreted from a classical equation formulated to heterogeneous materials
composed of different phases, each one with its own refractive index (RI) and volume – the
Wiener equation (Wiener, 1912).
Darling (1958) was the first to apply the Wiener equation to interpret enamel BRobs
assuming the non-mineral phase as a single one presenting the same RI of the immersion
media. On this basis, the higher the form birefringence (due to low RI) the lower the
mineral volume, and vice-versa. This theory allowed obtaining qualitative correlations
between the experimental BRobs of enamel and the mineral volume, and such correlations
were used to define the histopathological zones of carious enamel - surface layer (negative
BRobs under immersion in water), body of the lesion (positive BRobs under immersion in
water), dark zone (isotropic BRobs under immersion in oil medium, quinoline) and
translucent zone (negative BRobs under immersion in oil medium, quinoline) – (Darling,
1958). These zones (and their size modifications) have been extensively used in the dental
literature to analyse the relationship between carious enamel with a number of influencing
factors - microbial species (Marsh et al., 1989), dietary components (Jensen & Wefel,
1990), fluoride-releasing restorative materials (Papagiannoulis et al., 2002), lasers
(Westerman et al., 2002), etc. It is recognized, however, that a huge inconsistency exists
67
between the calculated (yielding too high form birefringence) and the experimental BRobs of
enamel, a result of the lack of physical constants able to allow the application of the Wiener
equation directly to the enamel composition (Carlström et al., 1963). This is still the main
gap in current knowledge.
The aim of this paper is to propose a new approach for improving the interpretation
of enamel BRobs, based on a new model for the calculation of the RI of the non-mineral
phase, in order to achieve a direct application of the Weiner equation to the enamel
composition.
Methods
Wiener Equation for calculation of enamel BRobs
The observed, the intrinsic and the form birefringences are theoretically given by:
obs
BRobs = BRintr
+ BRform,
(1)
obs
corr
= BRintr
V1 A,
BRintr
BR form =
(2)
V1V2 (n12 − n22 ) 2
,
2(V1 n1 + V2 n2 ). (1 + V1 )n22 + V2 n12
[
]
(3)
where A is a factor of crystallite alignment for which the value of 0.85 (± 0.06) is often
used (Carlström et al., 1963). V1 and V2 are volume fractions of the mineral and the nonmineral phases, respectively. n1 (1.62) and n2 are the RIs of the mineral and the non-mineral
obs
phases, respectively. BRintr
is obtained when a medium with the same RI of the mineral
corr
stands for the maximum
phase fills the non-mineral volume, i.e. when BRform is zero. BRintr
68
intrinsic birefringence, for which an experimental value of -0.0065 has been reported for
enamel (Angmar, 1965).
BRform is calculated from the Wiener equation (Wiener, 1912), here presented
according to the modification of Bear et al. (1937) (equation 3). The value adopted for n2 in
the enamel has usually been assumed to be identical to the RI of the immersion medium
(hereafter referred as the classical model for n2) (Darling, 1958). As V2 = 1 – V1 and n1 is
fixed, the only indeterminate is the volume fraction of the mineral content (V1).
The new mathematical approach
Considering that the RI of a heterogeneous material is influenced by its partial
volumes and corresponding RIs (Stokes, 1963), our new model for the calculation of the RI
n2 is:
n2 = 1.33
α1
V2
+ ni
α2
V2
+ 1.56
β
V2
,
(4)
where α1 is the volume fraction of water (excluding structural water) left in enamel after
drying in air at room temperature, and α2 is the volume fraction of water removed by this
latter procedure (Carlström & Glas, 1963). ni is the RI of the immersion medium. β is the
volume of the organic matrix (RI = 1.56; Angmar-Mänsson, 1971). The value of n2 derived
from equation 4 is to be used in equation 3. Clearly,
V2 = α1 + α2 + β
(5)
Since V2 = 1-V1, it is possible to write all the partial volumes of the non-mineral phase (α1,
α2 and β) as a function of V1 if experimental V1, BRobs in water and in air are obtained.
Applying our new model to interpret experimental published data can test this possibility.
69
Experimental BRobs data of a particular mature dental enamel mineralization study
was chosen for interpretation. The study of Angmar et al. (1963) was chosen because it is
still the one with the higher number of measured points in enamel and presents
experimental BRobs data under immersion in water and air combined with experimental
mineral volume derived from microradiography (the 87 % mineral volume for mature
enamel reported in textbooks is based on this study). Besides proposing a new model for
calculating n2, the mineral volume value to be used as V1 in equations 2 and 3 was corrected
to the more precise density of 2.99 gcm-3 for enamel hydroxyapatite, as proposed by Elliott
(1997). The conversion of the published experimental V1 values (denoted by V1(3.15)) to the
new ones derived with a hydroxyapatite density of 2.99 gcm-3 (denoted by V1(2.99)) can be
performed according to parameters related to X-ray linear attenuation coefficients using the
following linear transform (Elliott et al., 1998):
V1(2.99) = 0.954913 V1(3.15)+ 0.1057567.
(6)
Our new mathematical approach includes both the new model for n2 and V1(2.99).
The calculated BRobs with the new approach is written as:
BRobs = − 0.0055V1 +
V1 (1 − V1 )(n12 − n22 ) 2
,
2(V1 n1 + V2 n2 ) (1 + V1 )n22 + V2 n12
[
]
(7)
obs
= – 0.0055V1 (Carlström et al., 1963; a value assumed for all numerical
where BRintr
calculations in this study) and n2 is given by equation 4.
For each of the 106 measuring points (each with a given experimental V1 value)
from 10 teeth of the selected study (Angmar et al., 1963), the new approach was, at first,
applied to fit the experimental BRobs in water (ni = 1.33). In this case, there were two
indeterminates - the total water volume fraction (α; α = α1 + α2) and β - and two conditions
70
– experimental BRobs equals to calculated BRobs and V2 = α + β. The numerical evaluation
of the set of equations 4, 5 and 7 has always provided one only solution (within the limit of
10-5 for BRobs and 10-3 for α1, α2 and β). Afterwards, the same procedure was repeated, now
using the experimental BRobs in air (ni = 1.00). At this step, the only indeterminates were
α1 and α2. Keeping the β value derived in the previous step, values for α1 and α2 were
adjusted in order to match, again, the calculated BRobs with the experimental BRobs. Values
of α1, α2 and β hold for both equation 4 and equation 5 (for ni = 1.33 and for ni = 1.00).
Equations 2 and 3, using both V1(2.99) and V1(3.15), were also applied to interpret the
mean experimental BRobs in water and air from Angmar et al. (1963), in order to test the
restrictions of the classical model for n2.
Results and discussion
Experimental and calculated BRobs were shown to be inconsistent when using the
classical model for n2 with either V1(3.15) or V1(2.99) (Fig. 1a-b). From our new approach, the
numerical calculation was able, for the first time, to fit 97 experimental points via point-topoint fitting of α1, α2, and β, in order to match both BRobs in water and in air at each
individual point. At the remaining 9 points (those for which V1(2.99) > 95.3 %), the model
was not able to achieve positive β values. This means that a direct application of the
Weiner equation to the enamel composition is now achieved. Collected values of V1(3.15)
ranged from 81.25 to 91.25% (step of 0.625%) yielding V1(2.99) values that vary from 88.16
to 97.71% (step of 0.597%).
71
(a)
-4
BRobs (x 10 )
40
20
0
-20
-40
88
90
92
V1
94
(2.99)
(%)
96
98
72
-4
-4
BRobs (x 10 )
(b)
500
BRobs (x 10 )
10
0
-10
-20
400
88
90
92
94
(2.99)
V1
96
98
(%)
300
200
100
0
88
90
92
94
(2.99)
V1
96
98
(%)
Fig. 1. Experimental BRobs (scattered data) reported by Angmar et al.(1963), calculated
BRobs with the classical model for n2 and using mineral density of 3.15 gcm-3 (dashed black
line), calculated BRobs with the classical model for n2 and using mineral density of 2.99
gcm-3 (dashed grey line), and calculated BRobs with our new approach (continuous line)
obtained from the mean values of α1, α2 and β (equations 8a-8c). (a), data related to water
immersion medium; (b), data related to air immersion medium. Insert: detail of published
experimental BRobs (scattered data) and calculated BRobs with our new approach (continuous
line).
73
Mean values of α1, α2 and β as a function of V1(2.99) values were obtained (Fig. 2). A
primary validation can be made assuming that
α1 = 0.15 – 0.11 V1(2.99) ,
(8a)
α2 = 0.26 – 0.27 V1(2.99) ,
(8b)
β = 0.59 – 0.62 V1(2.99),
(8c)
which substituted into equation 5, furnishes V2 = 1- V1(2.99), in spite of the fact that a linear
regression was used to derive the volume distribution in the organic phase as a function of
the mineral volume.
74
5
Volume (%)
4
α1
β
α2
3
2
1
0
88
90
92
V1
(2.99)
94
96
(%)
Fig. 2. Plot of the relationship between V1(2.99) and α1, α2 and β within the analysed range.
Linear trends: α1 = 15.3154 – 0.1161V1(2.99), r = -0.7574; α2 = 26.5294 – 0.2740V1(2.99), r =
-0.9628; and β = 58.6978 – 0.6157V1(2.99), r = -0.9957.
A more precise estimation of the mean refractive index n2 can be derived by inserting
the set of equations 8a-8c into equation 4. This procedure holds when analysing enamel in
air or in aqueous immersion media.
75
For example, for an enamel point with a mineral volume V1(2.99) = 89.35%, the
experimental birefringence BRobs was - 0.002 (in water medium) and 0.00044 (in air
medium). The point-to-point approach closely matched these values with 4.6% for α1, 2.2%
for α2 and 3.8% for β. If we apply the linear fitting from equations 8a-8c, the corresponding
values are 5.15% for α1, 1.88% for α2 and 3.6% for β (V2 = 10.65 %).
Figure 1 also shows BRobs in water (Fig.1a, continuous line) and in air (Fig.1b,
continuous line), calculated with mean values of α1, α2 and β (equations 8a-8c), and the
corresponding published values obtained from Angmar et al. (1963) (scattered data). A
good agreement between the calculated and experimental BRobs is obtained.
The non-mineral phase of dental enamel is composed of the organic matter (RI of
1.56) and water (RI of 1.33), the latter being partially replaced by the immersion medium
(Carlström et al., 1963). Fine-tuned matched points required β values lower than 5% and
the values of β derived from equation 8c ranged from 0.1 to 4.4% in the values of V1(2.99)
fitted by our model. This range of β values is in agreement with the published range of
0.7% to 4.8% v/v for organic content in mature enamel (mineral volume not specified)
obtained from spectrophotometric chemical analysis by Robinson et al. (1971).
Applying our new model for n2 without conversion of V1(3.15) to V1(2.99) allow one to
match all published experimental data, but the resulting values for β are often much higher
than 5% (up to 10%) (data not shown).
Comparing enamel BRobs before and after drying at room temperature for 48 h and
then after heating to 400° C for 2 h, the α2/α1 ratio for a specific point in mature enamel
with 86.4% of V13.15 (94.1% of V1(2.99)) has been reported to be ~ ¼ (Carlström et al., 1963),
which is consistent with the ratio taken from our fine-tuning calculations (Fig. 2). The
76
water content represented here by α2 is the one related to the water content removed by
drying thin enamel sections in air at room temperature (Carlström et al., 1963). Our
theoretical α1 and α2 values can only be compared to experimental ones obtained using the
same procedure. Values obtained using different procedures are prone to be inconsistent
with ours. Drying enamel at, for instance, 200°C results in a value of α1 lower than α2 (the
inverse of what is reported here). On the other hand, data on the total water content of
enamel obtained from different procedures are prone to be similar. In this way, the water
volume of 6% v/v (± 1.2%) for mature enamel reported by Dibdin (1993) (using diffusion
clearance of tritiated water) - without analysing mineral volume - is within the range of
5.5% to 7.5% obtained from the sum of the mean values of α1 and α2 (equations 8a and
8b).
Our approach also explains the results from Houwink (1971), who compared enamel
BRobs before and after extraction of the organic matter and showed more positive (increased
BRform) BRobs in water and more negative (decreased BRform) BRobs in immersion media with
RIs of 1.62-1.74 after extraction of the organic matter. Infiltration of water in the space
previously occupied by organic matter increased the difference between n1 and n2 in
equation 3 (RI of water is lower than the RI of organic matter), leading to increased BRform.
On the other hand, the infiltration of high RI immersion media decreased the difference
between n1 and n2, decreasing BRform. These results are consistent with our new model for
n2 as they confirm the contribution of the organic matter to the RI of the non-mineral phase.
In disagreement with what has been used in the dental literature (based on the study of
Darling, 1958), when the organic matter is preserved, enamel BRobs changes under
immersion in air or in water media cannot be attributed to mineral variations only.
77
The discussion above indicate that applying our new approach to interpret
experimental BRobs of mature enamel in two media (water and air), measured before and
after extraction of the organic matter, V1(2.99), α1, α2 and β can be obtained. V1(2.99)can be
obtained because the correlation between experimental BRobs in water and V1(2.99) is
indicated to be explained by a single phase (n2 = 1.33) after extraction of the organic
matter.
Conclusions
This paper describes a new approach that enables a direct application of the Weiner
equation to the enamel composition and offers the first mathematical model consistent with
experimental data to interpret enamel BRobs. We speculate that, with this new approach,
quantitative data on the mineral, the water and the organic contents of mature dental enamel
can be derived from interpretation of birefringence in two immersion media (obtained
before and after extraction of the organic matter).
Acknowledgements
The first author thanks the Brazilian Ministry of Education (CAPES). The other
authors are grateful to CNPq, Federal Brazilian Agency of Research.
References
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enamel. IV. The mineralization of normal human enamel. J. Ultrastruct. Res. 8, 12-23.
Angmar-Mänsson, B. (1971) A polarization microscopic and micro X-ray diffraction study
on the organic matrix of developing human enamel. Arch. Oral Biol. 16, 147-156.
78
Angmar, B. (1965) Studies on the ultrastructure of dental enamel. VII. A microradiographic
study on developing human enamel. Odontol. Revy. 16, 167-181.
Bear, R.S., Schmitt, F.O. and Young, J.Z. (1937) The ultrastructure of nerve axoplasm.
Proc. Royal Soc. London B. 123, 505-519.
Carlström, D., Glas, J.E. and Angmar, B. (1963) Studies on the ultrastructure of dental
enamel. V. The state of water in human enamel. J. Ultrastruct. Res. 8, 24-29.
Carlström, D. and Glas, J.E. (1963) Studies on the ultrastructure of dental enamel. III.
Birefringence of human enamel. J. Ultrastruct. Res. 8, 1-11.
Darling, A.I. (1958) Studies of the early lesion of enamel caries. Br. Dent. J. 105,119-135.
Dibdin, G.H. (1993) The water in human dental enamel and its diffusional exchange
measured by clearance of tritiated water from enamel slabs of varying thickness. Caries
Res. 27:81-86.
Elliott, J.C. Structure, crystal chemistry and density of enamel apatites. Dental enamel 1997
(ed. by D. Chadwick and G. Cardew), pp.54-72. Ciba foundation Symposium 205.
Wiley, Chichester.
Elliott, J.C., Wong, F.S.L., Anderson, P., Davis, G.R. and Dowker, S.E.P. (1998)
Determination of mineral concentration in dental enamel from X-ray attenuation
measurements. Connec. Tis. Res. 38, 61-72.
Houwink, B. (1971) The effect of organic solvents on the results of imbibition experiments
in sound and carious dental enamel. Caries Res. 5, 279-289.
Jensen, M.E. and Wefel, J.S. (1990) Effects of processed cheese on human plaque pH and
demineralization and remineralization. Am. J. Dent. 5, 217-223.
79
Marsh, P.D., Featherstone, A., McKee, A.S., Hallsworth, A.S., Robinson, C., Weatherell,
J.A., Newman, H.N. and Pitter, A.F. (1989) A microbiological study of early caries of
approximal surfaces in schoolchildren. J. Dent. Res. 68, 1151-1154.
Papagiannoulis, L., Kakaboura, A. and Eliades, G. (2002) In vivo vs in vitro anticariogenic
behavior of glass-ionomer and resin composite restorative materials. Dent. Mat. 18,
561-569.
Robinson, C., Weatherell, J.A. and Hallsworth, A.S. (1971) Variation in composition of
dental enamel within thin ground tooth sections. Caries Res. 5:44-57.
Stokes, A.R. (1963) The theory of the optical properties of inhomogeneous materials. p.35.
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80
6. CONCLUSÕES
81
6. CONCLUSÕES
Considerando os estudos realizados no desenvolvimento desta tese e os resultados
obtidos, podemos destacar as seguintes conclusões:
1. Cárie e cálculo dentais podem ser formados concomitantemente a partir de modelos
in situ de cárie dental em humanos em intervalos de 2-14 dias. Cerâmicas de fosfato
de cálcio com relação Ca/P (mol/mol) variando de 1 a 1,7 foram identificadas sobre
as lesões cariosas induzidas. Em alguns casos, o cálculo dental cobriu quase que
totalmente a extensão da superfície em contato com a placa bacteriana oral. O
ambiente ácido necessário para a formação da desmineralização cariosa não foi tão
intenso a ponto de impedir a formação de depósitos semelhantes a hidroxiapatita
(Ca/P de 1,7 a 2,3). Estes relatos têm importantes repercussões no desenvolvimento
de agentes para ao tratamento da cárie e do cálculo dentais;
2. A fluorescência do cálculo dental na escala de pH de 2 a 7 é muito semelhante à da
hematoporfirina para a mesma região de pH. Numa solução com alta concentração
de hidrogênio, as fluorescências do cálculo dental e da hematoporfirina também
mostram semelhanças, mas com pequenas variações nas intensidades relativas das
bandas de emissão. Dentre as concentrações de hidrogênio estudadas, soluções com
27% de HCl são as que apresentam a maior semelhança com a fluorescência do
cálculo dental sólido, indicando que a hematoporfirina dication é o principal, se não
o único, cromóforo do cálculo dental sólido;
3. O novo modelo matemático proposto para interpretar a birrefringência do esmalte
dental permite, pela primeira vez na literatura, obter consistência entre os dados
teóricos e experimentais da birrefringência do esmalte dental obtidos a partir da
82
microscopia de luz polarizada. Dados sobre os volumes de água, de matéria
orgânica e de mineral podem ser inferidos a partir da aplicação deste novo modelo.
Variações nos volumes de água e/ou de matéria orgânica, assim como as variações
no volume mineral, podem alterar a birrefringência de forma do esmalte dental,
indicando, por exemplo, não ser correto interpretar qualquer aumento da
birrefringência negativa nas lesões cariosas como aumento do volume mineral ou o
contrário.
Dos estudos aqui relatados, alguns avanços foram conseguidos no tocante à
formação de cerâmicas de fosfato de cálcio em modelo de cárie, bem como à caracterização
destas cerâmicas e à avaliação dos seus efeitos no esmalte, sendo possível destacar algumas
perspectivas para estudos futuros. Estes devem investigar se, assim como a
desmineralização cariosa, o cálculo dental também pode ser formado controladamente a
partir da saliva de qualquer indivíduo num modelo in situ de cárie. A formação de cálculo
dental, aqui relatada, em curtos intervalos de tempo, é um ponto positivo para se usar o
cálculo dental como substituo de osso e para recobrimento de implantes.
Dentro da cariologia, é necessário pesquisar como, a partir de microdureza de
superfície e radiomicrografia, a avaliação da condição dos tecidos dentais duros com lesões
cariosas (com vistas a investigar se houve ou não remineralização) pode ser influenciada
pela formação de cálculo dental. Além disso, a contribuição do mineral que forma o cálculo
dental em relação ao montante total de minerais que se incorporam aos tecidos dentais
duros durante o processo de remineralização de lesões cariosas precisa ser investigada.
No tocante à fluorescência do cálculo dental, esta tem a vantagem de ser autovalidada, o que não ocorre com a análise morfológica com microscopia eletrônica. A
indicação de que a hematoporfirina dication pode ser o principal cromóforo do cálculo
83
dental sólido pode aprimorar a diferenciação entre este último e o esmalte e a dentina
desmineralizados, aprimorando o uso da fluorescência na pesquisa odontológica. Um
estudo comparativo entre a fluorescência dos tecidos dentais duros desmineralizados com a
fluorescência de potenciais cromóforos (por exemplo, a ditirosina no esmalte), semelhante
ao desenvolvido nesta tese com cálculo dental, poderá dar uma grande contribuição no
sentido de se descobrir quais componentes apresentam fluorescência nas lesões cariosas.
Enquanto a perda mineral é a característica das lesões cariosas na suas fases de ativa
iniciação e progressão, o aumento do conteúdo orgânico nessas lesões – incluindo aí as
bactérias orais e o cálculo dental – pode acontecer até mesmo quando nenhuma alteração
mineral está ocorrendo. Se a fluorescência de lesões cariosas for mostrada como sendo
devido à hematoporfirina dication, então as alterações dessa fluorescência não podem ser
vinculadas a alterações no conteúdo mineral dos tecidos dentais duros envolvidos.
O modelo matemático proposto oferece parâmetros que podem ser usados para fazer
análises bioquímicas do esmalte dental de uma maneira que nenhuma outra técnica isolada
hoje existente permite. Um exemplo do potencial de aplicação deste modelo é o estudo dos
volumes de água firmemente aderida e fracamente aderida, que são fatores importantes no
transporte de materiais nas lesões cariosas. É interessante ressaltar, também, a possibilidade
de investigar se os volumes de água e de matéria orgânica obedecem a uma certa correlação
entre si e com o volume mineral, quer o esmalte esteja perdendo ou ganhando minerais, e
como a remineralização de lesões cariosas pode se dar em meio às alterações na
composição do esmalte. Essas investigações configuram um bom campo para estudos
futuros. Há agora uma justificativa razoável para que as medidas de birrefringência do
esmalte dental sejam feitas com uma resolução melhor do que aquela comumente usada
(usando luz comum e filtro verde, com um erro de cerca de 20%). O uso de iluminação com
84
laser e a possível utilização de sistemas computadorizados de birrefringência baseados em
cristal líquido são exemplos de como a resolução da quantificação de birrefringência pode
ser melhorada.
O novo modelo matemático tem repercussão não só no estudo da influência da
formação de biocerâmicas de fosfato de cálcio formadas na superfície do esmalte, mas
também na biologia do esmalte dental como um todo (biologia de desenvolvimento,
biologia evolutiva, bioquímica na cavidade bucal, etc), abrindo um leque amplo de
aplicações para o futuro.
85
7. REFERÊNCIAS BIBLIOGRÁFICAS
86
7. REFERÊNCIAS BIBLIOGRÁFICAS
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93
8. ANEXOS
94
8.1 Normas para publicação na Caries Research
Aims
and
Scope
'Caries Research' is an international journal, the aim of which is to promote research
in dental caries and related fields through publication of original research and critical
evaluation of research findings. The journal will publish papers on the aetiology,
pathogenesis, prevention and clinical control or management of dental caries. Papers on
health outcomes related to dental caries are also of interest, as are papers on other disorders
of dental hard tissues, such as dental erosion. Aspects of caries beyond the stage where the
pulp
ceases
to
be
vital
are
outside
the
scope
of
the
journal.
Submission
Manuscripts written in English should be submitted online.
Conditions
All manuscripts are subject to editorial review. Manuscripts are received with the
explicit understanding that the data they contain have not previously been published (in any
language) and that they are not under simultaneous consideration by any other publication.
Submission of an article for publication implies the transfer of the copyright from
the author to the publisher upon acceptance. Accepted papers become the property of
'Caries Research' and may not be reproduced by any means, in whole or in part, without the
written
consent
of
the
publisher.
It is the author's responsibility to obtain permission to reproduce illustrations, tables,
95
etc.,
from
other
Types
publications.
of
Papers
Reports of original work may be Original Papers or Short Communications.
Systematic reviews and meta-analyses are classed as Original Papers. Conventional
Reviews are published either at the invitation of the Editorial Board, or after consultation
with the Editor-in-Chief. Original Papers and Short Communications have the structure
outlined below but for Short Communications the abstract should be less than 100 words
and the manuscript should not exceed 3 printed pages, equivalent to about 9 manuscript
pages
(including
tables,
illustrations
and
references).
Reviews have a freer format but should nevertheless commence with a Title page, an
Abstract and an Introduction defining the scope of the Review. Letters to the Editor,
commenting
on
recent
papers
Preparation
in
the
journal,
are
published
occasionally.
of
Manuscripts
Text should be one-and-a-half-spaced, with wide margins. All pages should be
numbered, starting from the title page. A conventional font, such as Times New Roman or
Arial, should be used, with a font size of 11 or 12. Avoid using italics except for Linnaean
names
of
organisms
and
names
of
genes.
Manuscripts should be prepared as a text file plus separate files for illustrations. The
text file should contain the following sequence of sections: Title page; Declaration of
interests;
Abstract;
Introduction;
Materials
and
Methods;
Results;
Discussion;
Acknowledgements; References; Legends; Tables; Illustrations. Each section should start
96
on a new page, except for the body of the paper (Introduction to Acknowledgements),
which
should
be
continuous.
Title page: The first page of each manuscript should show, in order:
•
the title, which should be informative but concise;
•
the authors' names and initials, without degrees or professional status, followed by
their institutes;
•
a short title, maximum length 60 characters and spaces, for use as a running head;
•
a list of 3-10 key words, for indexing purposes;
•
the name of the corresponding author and full contact details (postal address,
telephone and fax numbers, and e-mail address).
Declaration of Interests: Potential conflicts of interest should be identified for each author
or, if there are no such conflicts, this should be stated explicitly. Conflict of interest exists
where an author has a personal or financial relationship that might introduce bias or affect
their judgement. Examples of situations where conflicts of interest might arise are
restrictive conditions in the funding of the research, or payment to an investigator from
organisations with an interest in the study (including employment, consultancies, honoraria,
ownership of shares). The fact that a study is conducted on behalf of a commercial body
using funds supplied to the investigators' institution by the sponsor does not in itself
involve a conflict of interest. Investigators should disclose potential conflicts to study
participants
and
should
state
whether
they
have
done
so.
The possible existence of a conflict of interest does not preclude consideration of a
97
manuscript for publication, but the Editor might consider it appropriate to publish the
disclosed
information
along
with
the
paper.
Abstract: The abstract should summarise the contents of the paper in a single
paragraph of no more than 250 words (to ensure that the abstract is published in full by online services such as PubMed). No attempt should be made to give numerical results in
detail.
References
are
not
allowed
in
the
abstract.
Introduction: This section should provide a concise summary of the background to
the relevant field of research, introduce the specific problem addressed by the study and
state
the
hypotheses
to
be
tested.
Materials and Methods (or Subjects and Methods): All relevant attributes of the
material (e.g. tissue, patients or population sample) forming the subject of the research
should be provided. Experimental, analytical and statistical methods should be described
concisely but in enough detail that others can repeat the work. The name and brief address
of
the
manufacturer
or
supplier
of
major
equipment
should
be
given.
Statistical methods should be described with enough detail to enable a
knowledgeable reader with access to the original data to verify the reported results. When
possible, findings should be quantified and appropriate measures of error or uncertainty
(such as confidence intervals) given. Sole reliance on statistical hypothesis testing, such as
the use of P values should be avoided. Details about eligibility criteria for subjects,
randomization and the number of observations should be included. The computer software
and the statistical methods used should be specified. See Altman et al.: Statistical
guidelines for contributors to medical journals [Br Med J 1983;286:1489-93] for further
information.
Manuscripts reporting studies on human subjects should include evidence that the
98
research was ethically conducted in accordance with the Declaration of Helsinki (World
Medical Association). In particular, there must be a statement in Materials and Methods
that the consent of an appropriate ethical committee was obtained prior to the start of the
study, and that subjects were volunteers who had given informed, written consent.
Clinical trials should be reported according to the standardised protocol of the
CONSORT
Statement.
In studies on laboratory animals, the experimental procedures should conform with
the principles laid down in the European Convention for the Protection of Vertebrate
Animals used for Experimental and other Scientific Purposes and/or the National Research
Council
Guide
for
the
Care
and
Use
of
Laboratory
Animals.
Unless the purpose of a paper is to compare specific systems or products,
commercial names of clinical and scientific equipment or techniques should only be cited,
as appropriate, in the 'Materials and Methods' or 'Acknowledgements' sections. Elsewhere
in
the
manuscript
generic
terms
should
be
used.
Results: Results should be presented without interpretation. The same data should
not be presented in both tables and figures. The text should not repeat numerical data
provided in tables or figures but should indicate the most important results and describe
relevant
trends
and
patterns.
Discussion: This section has the functions of describing any limitations of material
or methods, of interpreting the data and of drawing inferences about the contribution of the
study to the wider field of research. There should be no repetition of preceding sections,
e.g. reiteration of results. The discussion should end with a few sentences summarising the
conclusions of the study. However, there should not be a separate 'Conclusions' section.
Acknowledgements: Acknowledge the contribution of colleagues (for technical
99
assistance, statistical advice, critical comment etc.) and also acknowledge the source of
funding for the project. The position(s) of author(s) employed by commercial firms should
be
included.
Legends: The table headings should be listed first, followed by the legends for the
illustrations.
Tables: Tables should be numbered in Arabic numerals. Each table should be placed
on a separate page. Tables should not be constructed using tabs but by utilising the table
facilities
of
the
word-processing
software.
Illustrations:
•
Illustrations should be numbered in Arabic numerals in the sequence of citation.
Figure numbers must be clearly indicated on the figures themselves, outside the
image area.
•
Black and white half-tone illustrations must have a final resolution of 300 dpi after
scaling, line drawings one of 800-1200 dpi.
•
Figures with a screen background should not be submitted.
•
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Reference to other publications should give due acknowledgement to previous
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1984]
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101
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Examples
(a) Papers published in periodicals: Lussi A, Longbottom C, Gygax M, Braig F: Influence
of professional cleaning and drying of occlusal surfaces on laser fluorescence in vivo.
Caries
Res
2005;39:284-286.
(b) Papers published only with DOI numbers: Theoharides TC, Boucher W, Spear K:
Serum interleukin-6 reflects disease severity and osteoporosis in mastocytosis patients. Int
Arch
Allergy
Immunol
DOI:
10.1159/000063858.
(c) Monographs: Matthews DE, Farewell VT: Using and Understanding Medical Statistics.
Basel, Karger, 1985
102
(d) Edited books: DuBois RN: Cyclooxygenase-2 and colorectal cancer; in Dannenberg AJ,
DuBois RN (eds): COX-2. Prog Exp Tum Res. Basel, Karger, 2003, vol 37, pp 124-137.
(e) Patents: Diggens AA, Ross JW: Determining ionic species electrochemically. UK
Patent
Application
GB
2
064
131
A,
1980.
(f) World Wide Web: Chaplin M: Water structure and behavior. www.lsbu.ac.uk/water,
2004.
8.2 Normas para publicação no Journal of Periodontal Research
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103
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104
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References must be verified by the author(s) against the original documents.
Examples:
(1)
Standard
journal
article
(List all authors up to 6; for 7 or more list the first 3 and add ''et al.'') Dockrell H,
Greenspan JS. Histochemical identification of T- cells in oral lichen planus. Oral Surg
1979; 48: 42-49. Thomas Y, Sosman J, Yrigoyen O, et al. Functional analysis of human Tcell subsets defined by monoclonal antibodies. I. Collaborative T-T interactions in the
immunoregulation of B-cell differentiation. J Immunol 1980; 125: 2402-2405.
106
(2)
Corporate
author
The Royal Marsden Hospital Bone- Marrow Transplantation Team. Failure of syngeneic
bone- marrow graft without preconditioning in post- hepatitis marrow aplasia. Lancet 1977;
2: 628-630.
(3)
No
author
given
Anonymous. Coffee drinking and cancer of the pancreas [Editorial]. Br Med J 1981; 283:
628-635.
(4)
Journal
supplement
Mastri AR. Neuropathology of diabetic neurogenic bladder. Ann Intern Med 1980; 92 (2 pt
2):
316-
324.
Frumin AM, Nussbaum J, Esposito M. Functional asplenia: demonstration of splenic
activity by bone marrow scan. Blood 1979; 54 (suppl 1): 26- 28.
(5)
Journal
paginated
by
issue
Seaman WB. The case of the pancreatic pseudocyst. Hosp Pract 1981; 16 (Sep): 24-29.
(6)
Personal
author(s)
Eisen HN. Immunology: an introduction to molecular and cellular principles of the immune
response , 5th edn. New York: Harper Row, 1984:406-420.
(7)
Editor,
compiler,
chairman
as
author
Dausset J, Colombani J, eds. Histocompatibility testing 1972. Copenhagen: Munksgaard,
1973: 12-18.
(8)
Chapter
in
a
book
Weinstein L, Swartz MN. Pathogenic properties of invading microorganisms. In: Sodeman
107
WA Jr, Sodeman WA, eds. Pathologic physiology: mechanisms of disease . Philadelphia:
WB Saunders, 1974: 457-480.
(9)
Published
proceedings
paper
DePont B. Bone marrow transplantation in severe combined immunodeficiency with an
unrelated MLC compatible donor. In: White HJ, Smith R, eds. Proceedings of 3rd Annual
Meeting of the International Society for Experimental Hematology. Houston: International
Society for Experimental Hematology, 1974: 44-50.
(10)
Agency
publication
Ranofsky AL. Surgical operations in short-stay hospitals: United States - 1975. Hyattsville,
Maryland: National Center for Health Statistics, 1978; DHEW publication no. (PHS) 781785. (Vital and health statistics; series 13; no. 34.)
(11)
Dissertation
or
thesis
Cairns RB. Infrared spectroscopic studies of solid oxygen. Berkeley, CA: University of
California, 1965. 156pp. Dissertation.
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108
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109
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8.3 Normas para publicação no Journal of Microscopy
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110
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111
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Examples of reference style:
Ashford, A.E. (1998) Dynamic pleiomorphic vacuole systems: are they endosomes and
transport
compartments
in
fungal
hyphae?
Adv.
Bot.
Res.
28,
119-159.
Causton, B. (1984) The choice of resins for electron immunocytochemistry.
Immunolabelling for Electron Microscopy (ed. by J. M. Polak and I. M. Varndell), pp. 2936.
Elsevier,
Amsterdam.
Muller, C. (1966) Spherical Harmonics. Springer-Verlag, Berlin.
Fischer-Parton, S. (1999) Role of pH, calcium and vesicle trafficking in regulating hyphal
tip growth of Neurospora crassa. PhD Thesis, University of Edinburgh, Edinburgh.
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112
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114
115
116
8.6 Dados da literatura usados para aplicação da nova abordagem matemática para a
BRobs teórica do esmalte dental.
Tabela 2. Dados experimentais publicados (ANGMAR et al., 1963) de volume mineral (V1,
%) e BRobs (x 10-4, em água, BRágua, e em ar, BRar) de esmalte dental maduro obtidos em
diferentes distâncias da superfície (100-1300 µm).*
Amostra a
V1
BRágua
BRar
100
200
300
400
85,625 85,625
85
85
-20 -21,25 -23,75 -23,75
-18,75 -17,5
-15
-10
500
85
-22,5
-5
600
83,75
-22,5
-2,5
700
83,75
-22,5
0
800
900
1000 1100
83,75 84,375 84,375 84,375
-21,25 -20 -18,75 -17,5
7,5
2,5
2,5
1,25
1200
85
-17,5
0
1300
85
-18,75
-5
800
900
1000
86,25 86,25 85,625
-26,25 -25 -23,75
-11,25 -11,25 -10
1100
85
-22,5
-7,5
1200
85
-23,75
-5
1300
83,75
-17,5
-5
800
86,725
-18,75
2,5
900
85
-17,5
3,75
1000
84,375
-15
7,5
1100
1200
1300
500
600
700
800
85
84,375 83,75 83,125
-26,25 -22,5 -22,5 -22,5
-5
2,5
5
5
900
82,5
-20
5
1000
82,5
-20
3,75
1100 1200
81,875 81,25
-20 -18,75
3,75
3,75
1300
81,25
-17,5
2,5
1100
83,75
-15
10
1300
Amostra b
200
300
400
500
600
700
100
V1
86,25 86,25 86,875 86,875 86,875 86,875 86,25
BRágua -26,25 -26,25 -27,5 -27,5 -26,25 -26,25 -27,5
-25 -23,75 -20
-17,5
-15
-12,5 -12,5
BRar
Amostra c
200
100
91,25 90,625
-23,75 -23,75
-17,5
-15
300
90
-22,5
-10
400
500
89,375 88,75
-22,5 -22,5
-7,5
-5
Amostra d
100
200
89,375 88,75
V1
-25
-25
BRágua
BRar -21,25 -20
300
87,5
-26,25
-15
400
86,25
-27,5
-10
Amostra e
100
V1
88,175
BRágua -22,5
BRar -21,25
300
400
500
600
700
86,875 86,25 85,625 84,375 83,75
-22,5 -22,5 -23,75 -22,5
-20
-15 -11,12 -7,5
-5
1,25
V1
BRágua
BRar
Amostra f
200
87,5
-22,5
-17,5
600
87,5
-21,25
-2,5
700
87,5
-20
-2,5
800
900
1000
83,75 83,125 83,75
-18,75 -17,5 -17,5
7,5
10
10
1200
117
100
200
300
400
500
600
700
V1
87,5 86,875 86,25 85,625 86,25 85,625 86,25
-25 -26,25 -26,25 -25 -23,75 -23,75 -23,75
BRágua
-5
-6,25 -6,25
BRar -16,25 -15 -11,25 -7,5
800
86,25
-22,5
-6,25
900
85
-20
-5
1000
85
-18,75
-2,5
1100
1200
1300
Amostra g
200
300
400
500
600
100
V1
88,125 87,5 86,875 86,875 86,25
85
-25 -23,75 -22,5 -22,5 -21,25 -21,25
BRágua
-20
-15
-10
-7,5
-5
-1,25
BRar
700
85
-20
-1,25
800
85
-20
0
900
85
,18,75
3,75
1000
1100
1200
1300
Amostra h
200
300
400
500
600
100
88,75 88,125 87,5 86,875 85,625 85,625
V1
BRágua -23,75 -25 -23,75 -22,5
-20 -13,75
-20
-17,5 -11,25 -7,5
-2,5
0
BRar
700
85
-12,5
2,5
800
85
-15
2,5
900
1000
1100
1200
1300
Amostra i
100
91,25
V1
BRágua -27,5
BRar
-25
700
800
900
83,75 81,875 81,25
-21,25 -18,75 -17,5
-5
0
2,5
1000
1100
1200
1300
800
900
1000
83,75 84,375
85
-20
-20 -18,75
5
3,75
3,75
1100
1200
1300
200
90
-25
-22,5
300
88,75
-17,5
400
87,5
-25
-15
Amostra j
100
200
300
400
90,625 89,375 87,5 86,25
V1
BRágua -26,25 -25 -23,75 -23,75
-22,5 -22,5 -17,5
-10
BRar
500
86,25
-23,75
-10
600
85
-22,5
-7,5
500
86,25
-25
-2,5
600
700
85
83,75
-23,75 -21,25
2,5
5
* Estes dados são apresentados em gráficos no artigo de referência.
118
8.7 Comparação entre dados teóricos e experimentais da BRobs do esmalte dental em
diversas condições.
Tabela 3. Comparação entre dados experimentais publicados de BRobs (x 10-4) de esmalte
dental (em diversas condições e em diversos meios de imersão experimental) e os valores
correspondentes teóricos obtidos pelas equações 4, 7 e 8a-8c.
Tipo
Estudo
Carlstrom &
Glas, 1963
Carlstrom &
Glas, 1963
Carlstrom &
Glas, 1963
Angmar,
1965
Angmar,
1965
V1
de
2,99
esmalte
ni
BRobs
experimental
BRobs
teórica
48,8%
developing
1,33
53,0
17,4
48,8%
developing
1,63
-14,1
-14,9
48,8%
developing
1,63
(without
water and
organic
content)
-22,0
-26,7
50,7%
developing
1,64
-18,0
-16,4
developing
1,64
(without
water and
organic
content)
-22,5
-27,3
50,7%
Theuns et al,,
mature
89,8% to 96%
1980
(occlusal)
Theuns et al,,
mature
89,8% to 96%
1980
(occlusal)
Theuns et al,,
87,9% to
mature
1980
95,6%
(mid-coronal)
Theuns et al,,
87,9% to
mature
1980
95,6%
(mid-coronal)
Theuns et al,,
89,8% to
mature
1980
95,2%
(cervical)
Theuns et al,,
89,8% to
mature
1980
95,2%
(cervical)
Theuns et al,,
93,5%
mature
1982
(median)
(occlusal)
Theuns et al,,
93,5%
mature
1982
(median)
(occlusal)
Theuns et al,,
Mature/
86% to 93,7%
1982
artificial
1,33
1,00
1,33
1,00
1,33
1,00
1,33
1,00
1,33
-16,0 to -21,0
-19,2 to –22,3
(± 1,0)
0,0 to –13,5
0,0 to –20,9
(± 2,0)
-16,0 to –23,0
-16,9 to –22,8
(± 1,0)
1,5 to –14,5
7,5 to –19,6
(± 3,0)
-26,0 to –24,0
-19,2 to –23,0
(± 1,0)
-10,0 to –11,0
0,0 to –18,2
(± 3,0)
-22,2
-22,7
(median)
-18,0
-12,2
(median)
-13,0 to –23,0 -14,6 to –22,8
119
carious
(occlusal)
Mature/
Theuns et al,,
artificial
86% to 93,7%
1982
carious
(occlusal)
Theuns et al,,
94,1%
mature
1982
(median)
(mid-coronal)
Theuns et al,,
94,1%
mature
1982
(median)
(mid-coronal)
mature/
Theuns et al,,
artificial
85% to 92,7%
1982
carious
(mid-coronal)
Mature/
Theuns et al,,
artificial
85% to 92,7%
1982
carious
(mid-coronal)
mature
Theuns et al,,
90,8% to
(occlusal and
1983
92,7%
mid-coronal)
Theuns et al,,
maduro/ cárie
20% to 80%
1993
artificial
1,00
1,33
1,00
11,0 to –16,0
-23,0
(median)
-19,0
(median)
13,8 to –12,9
-23,0
-14,4
1,33
-13,0 to –23,0 -13,3 to –22,1
1,00
14,0 to –18,0
1,33
-20,0 to –22,0 -20,3 to –22,1
1,62
-7,3 a –27,8
17,0 to –9,3
-7,0 a -31,8

Frederico Barbosa de Sousa - Universidade Federal de Pernambuco

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