Role of Salivary Electrochemistry and Composition in Regulating Physical and Optical ...

151 Pages·2015·4.98 MB·English

by  Yekta Ansari

Role of Salivary Electrochemistry and Composition in Regulating
Physical and Optical Characteristics of Tooth Enamel

YEKTA ANSARI

Faculty of Dentistry

McGill University
Montréal, Québec, Canada
December, 2014

A thesis submitted to McGill University in partial fulfillment of the degree requirements of
Master of Sciences

© Yekta Ansari 2014
Dedication

In memoriam of my dedicated loving father, Bahram Ansari.
He was an inspiration in life and in death.
'To God we belong, and to Him we will return.'(Koran 2:156)

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Table of Contents
List of Figures ............................................................................................................................. viii
List of Tables ................................................................................................................................. x
Abstract ........................................................................................................................................ xii
Résumé ........................................................................................................................................ xiv
Acknowledgments ...................................................................................................................... xvi
Preface: Contribution of Authors........................................................................................... xviii
CHAPTER 1: Introduction and Research Rationale ................................................................ 1
CHAPTER 2: Thesis Hypothesis and Objectives ...................................................................... 3
2.1- Study Hypotheses .................................................................................................................... 3
2.1.1- Implications .......................................................................................................................... 3
2.2- Objectives ................................................................................................................................ 3
CHAPTER 3: Literature Review and Methodology.................................................................. 5
3.1- Literature Review .................................................................................................................... 5
3.1.1- Saliva .................................................................................................................................... 5
3.1.1.1- Salivary Production ........................................................................................................... 5
3.1.1.2- Salivary Composition ........................................................................................................ 6
3.1.1.2.1- Salivary Organic Components ........................................................................................ 7
3.1.1.2.1.1- Salivary Protein Components ...................................................................................... 7
3.1.1.2.2- Saliva Elements ............................................................................................................ 14
3.1.2- Teeth ................................................................................................................................... 23
3.1.2.1- Tooth Anatomy ................................................................................................................ 23
3.1.2.2- Tooth Enamel Structure ................................................................................................... 24
3.1.2.3- Tooth Enamel Composition ............................................................................................. 26
3.1.2.3.1- Elemental and Inorganic Constituents of Tooth Enamel .............................................. 26
3.1.2.3.2- Tooth Enamel Organic Composition ............................................................................ 28
3.1.2.3.3- Tooth Enamel Mineral Composition ............................................................................ 29
3.1.2.4- Tooth Enamel Physical Properties ................................................................................... 29
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3.1.2.4.1- Tooth Enamel Mechanical Properties........................................................................... 29
3.1.2.4.1.1-Tooth Enamel Microhardness ..................................................................................... 30
3.1.2.5- Tooth Enamel Optical Properties .................................................................................... 30
3.1.2.5.1- Color ............................................................................................................................. 30
3.1.2.5.1.1- Color Space................................................................................................................ 31
3.1.2.5.2- Tooth Shade .................................................................................................................. 31
3.1.2.5.2.1- Tooth Shade Distribution........................................................................................... 32
3.2-Methodology ........................................................................................................................... 33
3.2.1- Electrochemical Characterization ....................................................................................... 33
3.2.2- Salivary Protein Quantification Assay ............................................................................... 35
3.2.3- Trace Element Analytical Methods .................................................................................... 35
3.2.4- Tooth Enamel Chemical Composition Analyses ................................................................ 37
3.2.5- Tooth Enamel Hardness Measurement ............................................................................... 39
3.2.6-Contact Angle Measurement ............................................................................................... 39
3.2.7-Tooth Shade Measurement .................................................................................................. 40
CHAPTER 4: Role of Saliva-derived Hydrophobic Organic Layer in protecting Tooth
Surface against Dissolution ........................................................................................................ 42
4.1- Abstract .................................................................................................................................. 42
4.2- Introduction ........................................................................................................................... 43
4.3- Materials and Methods .......................................................................................................... 45
4.3.1- Saliva Collection and Preparation ...................................................................................... 46
4.3.2- Teeth Collection and Preparation ....................................................................................... 47
4.3.2.2- Tooth Sectioning.............................................................................................................. 48
4.3.3- Analytical Techniques ........................................................................................................ 49
4.3.3.1- Wettability of Saliva-treated Tooth Enamel .................................................................... 49
4.3.3.2- Surface Chemical Composition of Tooth Enamel ........................................................... 50
4.3.3.3- Solubility of Saliva-treated Tooth Enamel ...................................................................... 51
4.3.3.4- Tooth Shade Measurement .............................................................................................. 51
4.3.3.5- Tooth Enamel Mechanical Properties.............................................................................. 52
4.3.3.6- Bulk and Surface Enamel Crystallinity Index ................................................................. 52
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4.3.3.7- Qualitative Variations in Elemental Composition within the distance of 30 µm from the
Enamel Surface ............................................................................................................................. 53
4.3.3.8- Quantitative Variations in Elemental Composition across Tooth Enamel Thickness ..... 53
4.3.3.9- Elemental Analysis of Saliva and Tooth Enamel ............................................................ 54
4.3.3.10- Electrochemical Characterization of Saliva .................................................................. 55
4.3.3.11- Salivary Protein Analyses .............................................................................................. 55
4.3.4- Sample Size Calculation and Statistical Analysis .............................................................. 55
4.4- Results ................................................................................................................................... 56
4.4.1- Saliva creates a Hydrophobic Coating on Enamel that prevents Tooth Dissolution .......... 56
4.4.2- Negative Zeta potential of Saliva protects Tooth from Dissolution and saturates Ca and P
in saliva ......................................................................................................................................... 59
4.4.3- Saliva may influence Tooth Shade and Composition ......................................................... 61
Elemental Composition across Tooth Enamel Thickness ............................................................. 62
Surface versus Bulk Tooth Enamel Crystallinity Index ............................................................... 62
4.4.4- Limited Interaction of Saliva with Bulk of Tooth Enamel ................................................. 63
4.4.4.1- Enamel of Erupted and Unerupted Teeth has similar Composition ................................ 63
4.4.4.2-Limited Correlation between the Composition of Bulk Enamel and Saliva Composition64
4.4.4.3- Limited Correlation between Physical Properties of Bulk Enamel and Saliva
Composition .................................................................................................................................. 65
4.5- Discussion .............................................................................................................................. 69
4.5.1-The Organic Layer formed by Saliva on Tooth ................................................................... 70
4.5.2- Saliva Creates a Hydrophobic layer on Tooth Enamel that protects it from Dissolution .. 70
4.5.3.2- Impact of Salivary Zeta potential on Surface Enamel Solubility .................................... 71
4.5.4- Impact of Saliva Composition on Tooth Shade .................................................................. 72
4.5.5- Impact of Saliva Composition on Surface Composition of Tooth Enamel ........................ 73
4.5.6- Impact of Salivary Electrochemical Properties on Tooth Enamel Optical Properties ....... 74
4.5.7- Comparison of Surface and Bulk Enamel Crystallinity Ultrastructure .............................. 74
4.5.8-Clinical Implications and Future Directions ........................................................................ 74
4.6-Conclusion .............................................................................................................................. 75
4.7- Acknowledgements ............................................................................................................... 75
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CHAPTER 5: Age-Related Alterations in Physical and Chemical Characteristics of Saliva
and Tooth Enamel ....................................................................................................................... 76
5.1- Abstract .................................................................................................................................. 76
5.2- Introduction ........................................................................................................................... 77
Oral Health Conditions in Older Adults ....................................................................................... 77
Saliva, Primary Regulator of Oral Health ..................................................................................... 78
Age-related Alterations in Saliva, a Poorly Understood Phenomenon ......................................... 78
5.3- Materials and Methods .......................................................................................................... 80
5.3.1- Saliva Collection and Preparation ...................................................................................... 80
5.3.2- Teeth Collection and Preparation ....................................................................................... 81
5.3.2.2- Tooth Sectioning.............................................................................................................. 81
5.3.3- Analytical techniques ......................................................................................................... 82
5.3.3.1- Tooth Shade Measurements............................................................................................. 82
5.3.3.2- Elemental Analysis .......................................................................................................... 82
5.3.3.3- Electrochemical Characterization of Saliva .................................................................... 83
5.3.4- Sample Size Calculation and Statistical Analysis .............................................................. 83
5.4- Results ................................................................................................................................... 84
5.4.1- Age-related Alterations in Tooth Optical Properties and Composition ............................. 84
5.4.1.1- Aging and Tooth Shade ................................................................................................... 84
5.4.1.2. Aging and Elemental Composition of Tooth Enamel ...................................................... 85
5.4.2- Age-related Alterations in Saliva ....................................................................................... 86
5.4.2.1- Aging and Electrochemical properties of Saliva ............................................................. 86
5.4.2.2-Aging and Elemental Composition of Saliva ................................................................... 87
5.5- Discussion .............................................................................................................................. 88
5.5.1- Saliva and Aging ................................................................................................................ 88
5.5.1.1- Flow Rate of Unstimulated Whole Saliva and Aging ..................................................... 88
5.5.1.2- Salivary Electrochemical Properties and Aging .............................................................. 89
5.5.1.3- Salivary Composition and Aging .................................................................................... 90
5.5.2- Tooth Enamel Elemental Composition and Aging ............................................................. 91
5.5.2.1- Potassium (K) .................................................................................................................. 91
5.5.2.2- Sodium (Na) .................................................................................................................... 91
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5.5.2.3- Magnesium (Mg) ............................................................................................................. 92
5.5.2.4- Lead (Pb) ......................................................................................................................... 93
5.5.2.5- Manganese (Mn) .............................................................................................................. 94
5.5.3- Optical Properties of Enamel and Aging ............................................................................ 94
5.5.3.1- Enamel Shade Lightness .................................................................................................. 94
5.5.4- Strength, Limitation and Future Directions ........................................................................ 95
5.5.5- Clinical Implication ............................................................................................................ 95
5.6- Conclusion ............................................................................................................................. 96
5.7- Acknowledgements ............................................................................................................... 96
CHAPTER 6: Concluding Remarks ......................................................................................... 97
CHAPTER 7: References ........................................................................................................... 98
CHAPTER 8: Appendices ........................................................................................................ 111
i. Abbreviations ........................................................................................................................... 111
ii. Supplementary Data: Chapter 4 .............................................................................................. 114
iii. Supplementary Data: Chapter 5 ............................................................................................. 123

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List of Figures
Figure 3.1 Schematic illustration of the electrical double layer around a charged colloid in solution.
Figure 3.2 Schematic representations of a sessile-drop and three inferential tensions in the contact angle system.
Figure 4.1 Flow chart illustrating the experiments performed on saliva samples.
Figure 4.2 Flow chart illustrating the experiments performed on teeth samples.
Figure 4.3 Photographs demonstrating the change in the contact angle of water on the untreated, saliva-treated and acetone-
treated enamel surfaces (a), XPS spectra showing different peaks of elements on various enamel surfaces (b), bar charts
describing the atomic percentage of the surface tooth enamel chemical composition (c), peak fitting of the XPS high resolution
C1s spectra of different enamel surfaces (d), and box charts describing the [Ca] and [P] released from the untreated, saliva-treated
and acetone-treated tooth enamel surfaces (e). Squared brackets indicate significant differences between groups (P<0.05).
Figure 4.4 Box charts describing the ratio of the [Ca] and [P] released from tooth enamel treated with different zeta potential
salivary secretions. Square brackets indicate significant differences between groups (p<0.01) (a), significant correlations of
electrochemical properties with the elemental composition of saliva (b) and the elemental composition of tooth enamel (c); and
significant correlations of [aPRP] and [statherin] in saliva with salivary elemental composition (d).
Figure 4.5 Significant correlations of tooth shade parameters with trace elements in saliva (a) and the electrochemical properties
of saliva (zeta potential and mobility of salivary ions) (b).
Figure 4.6 Line profile plot depicting the variations in the atomic percentage of 27 trace elements in tooth enamel using SEM-
EDS within 30 µm from the enamel surface.
Figure 4.7 Bar chart describing the crystallinity of surface and bulk tooth enamel. Square bracket indicates a significant
difference between the two groups (p<0.05) (a) and Raman spectra showing carbonate and phosphate peaks of the surface and
bulk tooth enamel (b).
Figure 4.8 Significant correlations of the elemental composition of buccal tooth enamel with saliva composition (a), and
significant correlations of the lingual tooth enamel elemental composition with buccal enamel elemental composition and
carbonate-to-phosphate ratio (b).
Figure 4.9 Significant correlations of the tooth enamel physical properties with saliva elemental composition (a) and tooth
enamel elemental composition (b).
Figure 5.1 Significant correlation of age with tooth shade (lightness).
Figure 5.2 Significant correlations of age with lingual tooth enamel elemental composition (a,b, and c) and buccal tooth enamel
elemental composition (d,e,f, and g).
Figure 5.3 Significant correlations of age with electrochemical properties of saliva: zeta potential (a) and ion mobility of saliva
(b).

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Figure 5.4 Significant correlation of age with the concentration of sodium in saliva.
Figure S4.1 Box plots demonstrating the variations in tooth shade parameters (lightness, chroma and hue) obtained by
calculating the mean and standard deviation of shade parameters. Tooth shade lightness followed a normal distribution; however,
tooth shade chroma and hue were not normally distributed within the analyzed teeth.
Figure S4.2 Histograms describing the variations of enamel physical properties (a-c) among the analyzed teeth; electrochemical
properties of saliva (d-f), protein composition (g and h) and unstimulated whole salivary flow rate (i) among the examined
participants.
Figure S4.3 SEM images showing the selected spots across the enamel thickness in the SEM-EDS analysis (a) and SEM-iXRF
analysis (b) on two different samples.
Figure S4.4 Histograms illustrating the variations in the ratio of calcium (a) and phosphorus (b) released from untreated tooth
enamel to that of saliva-treated enamel. High (HNZ: -31.7 mv), medium (MNZ: -16 mv) and low negative (LNZ: -6.13 mv) zeta
potential saliva. Subscripts B and A denote before and after exposure to saliva.
Figure S4.5 Significant inter-correlations of tooth shade parameters.
Figure S4.6 Significant correlations of buccal tooth enamel elemental composition with tooth shade parameters.
Figure S5.1 Frequency histogram describing the variation in the flow rate of unstimulated whole saliva amongst the examined
volunteers.
Figure S5.2 Frequency histograms illustrating the variations in zeta potential, conductivity and ion mobility of saliva amongst
the examined volunteers.

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List of Tables
Table 4.1 Summary of the correlation analysis between optical and physical properties of tooth enamel and the electrochemical
properties of saliva. For each correlation analysis, the correlation coefficient “r” and the significance of the correlation “P” were
calculated and reported in descending order. The significant correlations were highlighted.
Table 4.2 Summary of the inter-correlation analysis between the concentrations of trace elements in saliva. For each correlation
analysis, the correlation coefficient “r” and the significance of the correlation “P” were calculated and reported in descending
order. The significant correlations were highlighted.
Table 4.3 Summary of the inter-correlation analysis between the average concentrations of trace elements in bucal and lingual
tooth enamel. For each correlation analysis, the correlation coefficient “r” and the significance of the correlation “P” were
calculated and reported in descending order. The significant correlations were highlighted.
Table 5.1 Linear regression analyses between age and optical properties of tooth enamel.
Table 5.2 Linear regression analyses between age and the electrochemical properties of saliva.
Table 5.3 Linear regression analyses between age and trace element's concentrations in saliva, buccal and lingual tooth enamel.
Table S4.1 Number of excluded volunteers and exclusion criteria.
Table S4.2 Frequency of the tooth shade parameters among the examined teeth.
Table S4.3 Linear regression analyses between tooth shade parameters and trace element's concentrations in saliva.
Table S4.4 Frequency of the electrochemical properties of saliva.
Table S4.5 Linear regression analyses between electrochemical properties of saliva and tooth enamel shade lightness.
Table S4.6 Linear regression analyses between electrochemical properties of saliva and trace elements’ concentration in buccal
tooth enamel.
Table S4.7 Frequency of the tooth enamel physical properties amongst the examined teeth.
Table S4.8 Linear regression analyses between enamel physical properties and trace elements’ concentration in saliva.
Table S4.9 Frequencies of the salivary proteins concentration and unstimulated flow rate.
Table S4.10 Linear regression analyses between salivary aPRP and trace elements’ concentration in buccal tooth enamel.
Table S4.11 The frequencies and concentrations of 16 detected trace elements in buccal tooth enamel.
Table S4.12 The frequencies and concentrations of 15 detected trace elements in lingual tooth enamel.
Table S4.13 The frequencies and concentrations of 19 detected trace elements in saliva.
Table S4.14 Linear regression analyses between salivary electrochemical properties and trace elements’ concentration in saliva.
Table S4.15 Linear regression analyses between salivary proteins and trace element’s concentration in saliva.
Table S4.16 Linear regression analyses between [P] in saliva and its corresponding concentrations in buccal enamel.
Table S4.17 Composition and significant correlations and differences between trace elements’ concentration in buccal and
lingual tooth enamel.
Table S4.18 Linear regression analyses between enamel hardness and trace elements’ concentration in tooth enamel.
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