CEP SENIOR PROJECTS
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CEP SENIOR PROJECTS

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CEP Senior Projects, first draft 2/97, -Updated January 3, 2012 CEP SENIOR PROJECTS What is a Senior Project? The Senior Project is the capstone project in the major and the culmination of your time at the UW. As such, it is an opportunity to show people (including yourself) what you have learned and what you are capable of doing, as well as a way to assess your own abilities at the end of your time in CEP and the UW.
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Nombre de lectures 48
Langue Português
Poids de l'ouvrage 13 Mo

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Structural and mechanical characterization of sialoliths



Pedro Almeida Nolasco



Dissertação para obtenção do Grau de Mestre em

Mestrado de Bolonha em Bioengenharia e Nanossistemas







Júri:
Presidente: Doutor João Pedro Estrela Rodrigues Conde
Orientador: Doutora Patrícia Maria Cristovam Cipriano Almeida de Carvalho
Co-orientador: Doutora Maria Margarida Fonseca Rodrigues Diogo
Vogais: Doutor António Pedro Alves de Matos


Outubro 2011 Acknowledgements


It would not have been possible to produce this dissertation without the help of several people
who I would like to thank.

I would like to give my thanks to my supervisor Doutora. Patrícia Carvalho for all her support,
endless patience, dedication and availability. Also, I would like to thank to Doutor Antonio Alves
de Matos, for his help and suggestions throughout this work.

I also must thank to Daniela Nunes and Enginheira Isabel Nogueira for their availability and help
on operating the scanning electron microscope and the transmission electron microscope.

For their help in samples preparation to use on the Transmission Electron Microscope, I would
like to thank to the Anatomic Pathology Department, Curry Cabral Hospital.

ii
Agradecimentos


Não seria possível a realização desta dissertação sem a ajuda de várias pessoas a quem gostaria
de agradecer.

Gostaria de agradecer à minha orientadora. Doutoura Patrícia Carvalho por todo o apoio,
dedicação e disponibilidade. Gostaria também de agradecer ao Doutor António Alves de Matos
pela sua ajuda e sugestões ao longo de todo o trabalho.

Não podia deixar de agradecer à Daniela Nunes e à Enginheira Isabel Nogueira pela
disponibilidade e ajuda na operação do microscópio electrónico de varrimento e do microscópio
electrónico de transmissão.

Pela ajuda na preparação das amostras para o Microscópio Electrónico de Transmissão gostaria
de agradecer ao Departamento de Anatomia Patológica do Hospital Curry Cabral.







iii
Abstract


Sialolithiasis, the formation of calculi in salivary glands, affects 1.2% of the general
population, and non-invasive management of this condition shows a growing interest.
Extracorporeal shock waves can be used to shatter sialoliths however, the method has achieved
moderate success in sialolith elimination and the subject demands therefore further investigation.
In the present work, the microstructure, local chemistry, crystallography and mechanical
properties of submandibular sialoliths have been characterized using powder X-ray diffraction,
electron microscopy combined with X-ray spectroscopy and ultramicro-indentation assays.
The results have shown that the sialoliths are composed of highly-mineralized, lamellar
and globular regions. The mineralized regions are fairly homogeneous and constituted by
hydroxyapatite, whitlockite and brushite. Lamellar regions consisted of alternating layers of
mineralized material and organic matter, presenting a concentric morphology that points to a
cyclic chronologic formation. Globular regions are composed of organic globules with high-
sulphur content. Some of the larger globules exhibited internal mineralized granules and
teardrop-like morphologies surrounding their boundaries which indicate an extrusion process,.
Bacteria in distinct degrees of mineralization were observed in the sialoliths microstructure
demonstrating that infection occurs at early and late stages of the sialolith formation process,
possibly recurrently.
The Young modulus and hardness increased with the mineralization degree of the
sialoliths. The relatively high amount of compliant and soft organic matter in these calculi seems
to play a major role in the modest success of the shock wave therapies for sialolith fragmentation.
Identification of the sulphur origin, characterization of the mechanical properties by
indentation at the nanoscale and optimization of shock wave parameters for sialolith
fragmentation is planned for future work.


Key words: Sialolith, X-ray powder diffraction, Scanning electron microscopy, Transmission
electron microscopy, Ultramicro-indentation, Extracorporeal shock wave lithotripsy.
iv

Resumo


A sialolitíase, ou formação de cálculos em glândulas salivares, tem uma incidência de
1,2% na população mundial e o interesse em abordagens não-invasivas para o tratamento desta
patologia tem vindo a aumentar. Ondas de choque extracorpóreas podem ser usadas para eliminar
sialolitos, no entanto, o método atingiu apenas um sucesso moderado e requer investigação
adicional.
Neste trabalho efectuou-se a caracterização microestrutural, química, cristalográfica e
mecânica de sialolitos submandibulares através de microscopia electrónica combinada com
espectroscopia de raios-X e medidas de ultramicro-indentatação. Mostrou-se que os sialolitos são
constituídos por regiões mineralizadas, globulares e lamelares. As regiões mineralizadas, mais
homogéneas, são compostas por hidroxiapatite, bruchite e witloquite. As lamelares consistem em
lamelas alternadas de matéria mineralizada e orgânica, e exibem uma arquitectura concêntrica
que aponta para um processo de formação cíclico. As regiões globulares apresentam glóbulos de
material orgânico com grande quantidade de enxofre. Alguns glóbulos de maiores dimensões
possuem grânulos mineralizados no seu interior e nas suas fronteiras apresentam estruturas em
forma de lágrima resultantes de um processo de extorsão. Foram ainda encontradas bactérias em
vários níveis de calcificação, reflectindo a ocorrência de infecção em estádios iniciais e finais do
processo de formação dos sialolitos, possivelmente de forma recorrente. O estudo das
propriedades mecânicas demonstrou que o modulo de Young e a dureza aumentam com o nível
de mineralização dos sialolitos. No entanto, a relativamente elevada quantidade de matéria
orgânica macia parece ter um papel importante na baixa eficácia da terapia por ondas de choque
aplicada à fragmentação de sialolitos.
De futuro pretende-se identificar a origem do enxofre presente na matéria orgânica,
caracterizar as propriedades mecânicas à escala nanométrica e optimizar os parâmetros das ondas
de choque para fragmentação de sialolitos.


Palavras-chave: Sialolito, Difracção de raios-X de pós, Microscopia electrónica de varrimento,
Microscopia electrónica de transmissão, Ultramicro-indentação, Litotripsia extracorporal por
ondas de choque.










v


Table of Contents

ABSTRACT IV
1. INTRODUCTION 1
2. MATERIALS AND METHODS 4
2.1. X-RAY POWDER DIFFRACTION 5
2.2. SCANNING ELECTRON MICROSCOPY 9
2.3. TRANSMISSION ELECTRON MICROSCOPY 12
2.4. ULTRAMICRO-INDENTATION ASSAYS 16
3 RESULTS AND DISCUSSION 19
3.1 X-RAY DIFFRACTION 19
3.2 ELECTRON MICROSCOPY 21
3.3 ULTRAMICRO-INDENTATION 34
4 CONCLUSIONS 39
5. ON GOING AND FUTURE WORK 40
6 REFERENCES 40
vi


List of Figures

Figure 1.1 - Salivary gland system.----------------------------------------------------------------------------2
10
Figure 1.2 - Mechanism proposed for sialolith formation .--------------3
Figure 2.1 - Braag law schematic: diffraction will only occur if the path length difference
23
between the two waves is a multiple of λ .-------------------------------------------------------------------6
Figure 2.2 - Scheme of a diffractometer. S, T and C are, respectively, the sample stage, the X-ray
source and the detector. The movement of the detector and the sample stage is coordinated to
23
scan all the possible orientations of the atomic planes .--------------------------------------------------6
Figure 2.3 - Scanning electron microscope.---------------------------------9
Figure 2.4 - Interaction volume of a beam of electrons with a surface. ------------------------------ 10
Figure 2.5 - Relation between the intensity of SE signal and the topography of the specimen.-- 11
Figure 2.6 - Transmission electron microscope.---------------------------------------------------------- 13
Figure 2.7 - Bright- and a dark-field TEM imaging modes.------------ 14
32
Figure 2.8 - The rays path in TEM imaging and diffraction modes . -------------------------------- 15
Figure 2.9 - (a) Variation of the applied force during an indentation. (b) Methods for measuring
the projected indented area with triangular and pyramidal indenters. For both indenters the
35
diagonals length is measured (d) .-------------------------------------------------------------------------- 18
35Figure 2.10 - Force vs displacement plot registered during an indentation .---------------------- 18
26Figure 3.1 Experimental X-ray diffractograms together with simulations for hydroxyapatite ,
27 28whitlockite and brushite . The core and the periphery of sample I have been analyzed
separately. The stars indicate non-identified peaks.----------------------------------------------------- 20
Figure 3.2 - Microstructure of polished median cross-sections.------- 23
Figure 3.3 - Cross-sectional morphology of sample H. Generally the sialoliths are composed of
mineralized (yellow), lamellar (green) and globular (red) regions. In this case the core is absent
due to a preparation hazard.---------------------------------------------------------------------------------- 24
Figure 3.4 – Highly mineralized regions.------------------------------------------------------------------- 25
Figure 3.5 - Typical globular regions.------ 25
Figure 3.6 - Lamellar regions.-------------------------------------------------------------------------------- 26
Figure 3.7 - SEM image (BSE signal) and EDS spectra obtained in a globular region.---------- 26
Figure 3.8 - SEM image (BSE signal) and corresponding X-ray maps of globular (a) and
lamellar (b) regions, showing a negative distribution of S when compared with Ca and P
distributions. ----------------------------------------------------------------------------------------------------- 27
Figure 3.9 - TEM bright-field image of a globular region and point analysis EDS spectra.----- 28
viiFigure 3.10 - TEM bright-field and SEM images of globular regions. (a) and (b) Mineralized
material scattered inside globules. (c) Teardrop-like structures observed by TEM. (d) and (e)
40
Teardrop-like structures observed by SEM .-------------------------------------------------------------- 29
Figure 3.11 - TEM bright-field image of mineralized structures: A - hydroxyapatite fibers and B -
crystals with other shapes.----------------------------------------------------- 30
Figure 3.12 - (a) Fiber-like crystals. (b) Ring diffraction pattern of fiber-like crystals with
superimposition of the theoretical pattern of hydroxyapatite------------------------------------------- 31
Figure 3.13 - (a) and (b) Single crystals in mineralized regions, (c) and (d) experimental
microdiffraction patterns, (e) and (f) corresponding simulated diffraction patterns--------------- 32
Figure 3.14 - (a) Non-mineralized and (b) mineralized bacteria in sialoliths observed by TEM.
(c) Bacteria observed by SEM in an indented region. The arrows point to cell division
occurrences.------------------------------------------------------------------------------------------------------ 33
Figure 3.15 - Typical indentations performed in different regions with the corresponding force vs
displacement curves (underneath). (a) Mineralized, (b) lamellar and (c) globular regions.----- 35
Figure 3.16 - (a) Young modulus and (b) hardness as a function of the indented region.-------- 37
Figure 3.17 - Box plots of (a) Young modulus and (b) hardness measured on each sample
(mineralized regions).------------------------------------------------------------------------------------------ 38
Figure 3.18 - SEM images of crack profiles at indentations performed in globular regions. (a)
and (b) SE images. (c) and (d) BSE images of the same regions. The cracks show preference for
globule/mineralized interfaces (see (a) and (c)) and were deflected or ceased progression at the
organic globules (see (b) and (d)).--------------------------------------------------------------------------- 38

viii


List of Tables


Table 2.1 - Sample set analyzed. -------------------------------------------------------------------------------4
Table 2.2 - General overview of the techniques employed.----------------5
Table 2.3 - Structural parameters of hydroxyapatite (Ca (PO ) (OH) ): Space group P6 /m, Z=2, 10 4 6 2 3
a=9.4218 Å and c= 6.8813 Å and atomic positions listed below.----------------------------------------7
Table 2.4 - Structural parameters of whitlockite (Ca (PO ) ): Space group R3c, Z=3, 3 4 2
a=10.350(5) Å and c= 37.085(11) Å and atomic positions listed below.-------------------------------8
Table 2.5 - Structural parameters of brushite (Ca (PO ) ): Space group C2/c, Z=4, a=5.812 ± 3 4 2
0.002 Å, b= 15.180 ± 0.003 Å, c= 6.239 ± 0.002 Å and atomic positions listed below.-------------8
Table 3.1 - Mineral phase proportion and crystallographic data determined from the
experimental XRPD data.-------------------------------------------------------------------------------------- 21
Table 3.2 – Average hardness and Young modulus. 35
Table 3.3 - Comparision of sialolith experimental Young modulus and hardness with literature
values.------------------------------------------------------------------------------------------------------------- 39



ix


List of Abbreviations



XRPD X-ray powder diffraction
SEM Scanning electron microscopy
TEM Transmission electron microscopy
SE Secondary electrons
BSE Back scattered electrons
EDS Energy dispersive X-ray spectroscopy
x