Effects of heat organic matrix on enamel demineralization diffusion

EFFECTS OF HEAT AND ORGANIC MATRIX ON ENAMEL DEMINERALIZATION AND DIFFUSION HUANG LI B.D.S A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PREVENTIVE DENTISTRY N

EFFECTS OF HEAT AND ORGANIC MATRIX ON

ENAMEL DEMINERALIZATION AND DIFFUSION

HUANG LI B.D.S

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF PREVENTIVE DENTISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2006

ACKNOWLEDGEMENTS

I wish to express my deepest gratitude to my supervisor Prof Hsu Chin-Ying Stephen,

whose academic and professional motivation stimulated my continued pursuing of

knowledge on this journey

I am greatly indebted to my family for their understanding, spirit and financial support,

especially my mother, for her attention devoted to my son during my long and frequent

absences from home

I would like to thank my colleagues in the cariology lab, Ms Liu Yuanyuan, Ms Deng

Ying, Dr Gao Xiaoli, for their invaluable help during this working, and lab technician, Mr

Chan, for his patient technical support in the use of equipments

Special thanks go to Dr Nyi Lay Maung, who cheerfully endured my numerous lengthy

discussions and conversations with patient reply

I would also like to thank the following individuals for their help and assistance with

professional information: Dr Deng Xudong, Department of English, has not only helped

me revise this thesis, but also teach me writing skills; Prof Thorsten Wohland,

Department of Chemistry, has kindly guided me to acquire new knowledge and handle

equipments; Dr Chan Yiong Huak, Biostatistics Unit, has dedicated to instruct me to do

the statistical analysis

Finally, financial assistance provided by the Faculty of Dentistry, National University of

Singapore in the form of research scholarship toward the completion of the research is

thankfully acknowledged

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS iii

SUMMARY viii

LIST OF FIGURES x

LIST OF TABLES xii

LIST OF ABBREVIATION xiii

CHAPTER I INTRODUCTION 1

CHAPTER II LITERATURE REVIEW 5

2.1 Human Dental Enamel 6

2.1.1 Structure of Enamel 6

2.1.1.1 Enamel Rod 6

2.1.1.2 Enamel Crystals 7

2.1.1.3 Enamel Tufts, Lamellae, Spindles and Cracks 9

2.1.1.4 Striae of Retzius 10

2.1.1.5 Hunter-Schreger Bands 10

2.1.1.6 Dentino-Enamel Junction (DEJ) 10

2.1.1.7 Water Structure of Enamel 11

2.1.1.8 Porosity 11

2.1.2 Chemical Properties 13

2.1.2.1 Inorganic components 13

2.1.2.2 Organic components 14

2.2 Heat-Induced Effect on Human Enamel 15

2.2.1Optical, Morphological, and Crystallographic Changes 16

2.2.2 Chemical Reaction 18

2.2.3 Heat-Induced Reduction of Enamel Demineralization 19

2.3 Caries Formation and Progression 22

2.3.1 Introduction 22

2.3.2 Protective Role of Sound Enamel Surface Chemistry 22

2.3.2.1 Fluoride, Carbonate and Magnesium 23

2.3.2.2 Organic Matrix 23

2.3.2.3 The pH value 24

2.3.3 In vitro Demineralization 25

2.3.4 Polarized Light Microscopy (PLM) and structure of carious enamel 27

2.3.4.1 The Translucent zone 29

2.3.4.2 The Dark zone 30

2.3.4.3 The Body of lesion 30

2.3.4.4 The Surface zone 30

2.4 Diffusion Phenomena of Enamel 31

2.4.1 Diffusion Phenomena and Diffusion Pathways 31

2.4.2 The Published DC of Enamel 32

2.4.3 Factors Affecting Enamel Diffusion 33

2.4.3.1 Pore Size 33

2.4.3.2 Charge of Surface Enamel 33

2.4.3.3 Organic Matrix in Enamel 33

2.4.3.4 Chemical Reaction 34

2.5 Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Application on Dental Enamel 34

2.5.1 Introduction 34

2.5.2 Confocal Laser Scanning Microscopy 35

2.5.2.1 Principles and Theoretical Background 35

2.5.2.2 Basic Structure 39

2.5.2.3 Application of CLSM in Dentistry 41

2.6 Fluorescence Recovery After Photobleaching (FRAP) 42

2.6.1 Introduction 42

2.6.2 Basic Principles of Fluorescence 42

2.6.3 Autofluorescence and Photobleaching in Dental Enamel 45

2.6.4 Fluorescent Dyes Commonly Used for Dental Enamel 45

2.6.5 FRAP Principles and Applications 48

2.6.5.1 Two-Dimensional Model 50

2.6.5.2 Three-Dimensional Model 51

CHAPTER III MATERIALS & METHODS 53

3.1 Overview 54

3.2 Sample Preparation 54

3.2.1 Tooth Selection and Preparation 54

3.2.2 OM Extraction 54

3.2.3 Enamel Sections Preparation 58

3.2.4 Heat Treatment 58

3.2.5 Demineralization Process 58

3.3 Measurement of Lesion Depth (LD) 59

3.4 Measurement of DC 60

3.4.1 Labeling Samples 60

3.4.2 Theoretical Model For FRAP 61

3.4.3 FRAP Procedures 61

3.4.4 Data Collection and Analysis 64

3.5 Measurement of Birefringence 65

3.6 Statistical Analysis 65

3.6.1 Evaluation of OM Extraction and Heat Treatment Effects on LD 65

3.6.2 Evaluation of OM Extraction and Heat Treatment Effect on DC 65

3.6.3 Evaluation of OM Extraction and Heat Treatment Effect on Birefringence 66

CHAPTER IV RESULTS 67

4.1 Characterization of Lesion Depth (LD) using PLM 68

4.1.1 Qualitative Results 68

4.1.2 Quantitative Results 68

4.2 Characterization of DC by FRAP/CLSM 72

4.2.1 Qualitative Characterization of Enamel 72

4.2.2 Quantitative Characterization of Enamel 75

4.3 Characterization of Birefringence under PLM 80

4.3.1 Qualitative Characterization of Enamel 80

4.3.1.1 Stereomicroscopy (SM) 80

4.3.1.2 Polarized Light Microscopy (PLM) 80

4.3.2 Quantification of Birefringence of Enamel 84

CHAPTER V DISCUSSION 87

5.1 Effect of Heat and OM on Enamel Demineralization 88

5.2 Effect of Heat and OM on Enamel DC 90

5.2.1 Heat Effect 91

5.2.2 OM Effect 92

5.3 Effect of Heat and OM on Enamel Birefringence 93

5.4 Summary 95

5.5 Confounding factors and Future Studies 98

CHAPTER VI CONCLUSSION 100

6.1 Conclusion 101

REFERENCES 102

APPENDIX A 114

APPENDIX B1 116

APPENDIX B2 117

APPENDIX B3 118

SUMMARY

The reduced subsurface enamel demineralization caused by heat and the laser treatments

have been studied in the past few decades But the mechanism was not completely clear

The purpose of this experiment was to investigate the changes of the birefringence, ion

diffusion, and lesion formation in enamel with & without organic matrix following the

temperature increment during heating

Seven sound teeth were cut into halves and prepared into two groups: the normal and

Organic Matrix (OM)-extracted groups Six sound sections were chosen from each tooth

half and heated in the temperature range of 100℃ to 500℃, with one section kept at

room temperature as the control group Their birefringences before and after heating were

measured using Polarized Light Microscope (PLM) Thereafter, these sections were cut

into two segments — the coronal and the cervical The coronal segments were subjected

to the measurement of Diffusion Coefficient (DC) quantitatively using Flurescence

Recovery After Photobleaching (FRAP) coupled with Confocal Laser Scanning

Microscpe (CLSM) The cervical segments were subjected to demineralization treatment

and the Lesion Depth (LD) was evaluated by Polarized Light Microscope (PLM)

The OM-extracted groups showed an increased LD, DC, and birefringence compared

with those of the normal group These results confirmed the previous assumption that the

organic matrix has an inhibitory effect on the ion diffusion and demineralization in the

enamel

LD reached its minimum at 300℃ in both normal and OM-extracted groups Cracks

appeared in surface enamel when temperature was above 300 ℃ Therefore this

temperature may be the limit of heating There was an abrupt decrease of DC in the

normal group in the temperature range 300℃ to 400℃ But not for the OM-extracted

group The DC continued to decrease in the temperature range 400℃ to 500℃ in both

groups This finding suggested that the DC decrease was not just caused by the decrease

of permeability in the enamel after heating, especially above 300℃ It could also have

been affected by the inorganic changes in the enamel

The increased birefringence in both groups indicated the increased space in both heated

and OM-extracted enamel The birefringence increase in the OM-extracted group at 200

℃ was more than that of the normal group The results suggested that the micropores in

the enamel were related to the organic matrix

In conclusion, the reduced demineralization induced by heat was attributed to the mixed

effect of both permeability reduction and compositional changes in enamel The organic

matrix has the retardation effect on the diffusion of ions in enamel The heating in the

temperature range 200 ℃ to 300 ℃ was considered beneficial to the prevention of

incipient caries The FRAP technique provided a novel approach to study ion diffusion in

hard tissue

LIST OF FIGURES

Figure 2-1 Crystal structure of hydroxyapatite 8

Figure 2-2 Schematic model of subsurface demineralization 27

Figure 2-3 Diagram of the retardation formation 28

Figure 2-4 Schematic structure of a CLSM 35

Figure 2-5 Diagram of the 3D-PSF 37

Figure 2-6 Section through the 3D-PSF 38

Figure 2-7 The convolution and deconvolution used in the theoretical 3D-PSF 38

Figure 2-8 Diagram of the optical slicing in a specimen (www.zeiss.com) 39

Figure 2-9 Basic structure of CLSM 39

Figure 2-10.Wavelength of laser spectrum 40

Figure 2-11 Jablonski diagram illustrating fluorescence 43

Figure 2-12 Stokes shift 44

Figure 2-13 Optical spectra of fluorescein 47

Figure 2-14 Ionization equilibria of fluorescein 48

Figure 3-1 Flowchart of experimental procedures 55

Figure 3- 2 OM extraction procedures 57

Figure 3-3 Coronal and cervical segments of one tooth section 59

Figure 3-4 Lesion Measurement 60

Figure 3-5 Picture of ROI under CLSM 62

Figure 3-6 Diagram of FRAP setup in tooth section 63

Figure 3-7 The recovery curve from the image sequence 64

Figure 4-1 Representative PLM photograph of lesions after demineralization 68

Figure 4-2 LD of two groups with heat treatment at different temperature 69

Figure 4-3 Representative CLSM image of ROI in enamel 73

Figure 4-4 Reprehensive T-series images of FRAP under CLSM 74

Figure 4-5 Fluorescent intensity curve 75

Figure 4-6 DC changes of normal and OM-extracted groups after heat treatment 77

Figure 4-7 Enamel sections after heat treatment under SM 81

Figure 4-8 Enamel sections after heat treatment under PLM (lower magnification 2X) 82 Figure 4-9 Respective pictures under PLM of enamel after heating (highrt magnification 10X) 83

Figure 4-10 Birefringence changes after OM-extracted and heat treatment 85

LIST OF TABLES

Table 2-1: Summary of the heating effect 21

Table 4-1 Comparison between normal and OM-extracted groups 69

Table 4-2 Percentages of LD reduction 70

Table 4-3 Statistical significance of Tests Between-Subject Effects of LD 71

Table 4-4 Pairwise comparisons in Post-hoc Tests of LD 71

Table 4-5 Pairwise comparisons of Post-hoc Test in subgroups of LD 72

Table 4-6 Means and standard deviations of K, τ and f in FRAP model 76

Table 4-7 DC with heat treatment in normal and OM-extracted groups 76

Table 4-8 Percentages of DC reduction 78

Table 4-9 Statistical results of Tests of Between-Subject Effects of DC 78

Table 4-10 Multiple Comparisons of Post hoc Test of DC 79

Table 4-11 Pairwise Comparisons of Post-hoc Test in subgroups of DC 79

Table 4-12 Raw Birefringence data in normal and OM-extracted groups 84

Table 4-13 Statistical results of Tests Between-Subject Effects of birefringence 84

Table 4-14 Heating effect on birefringence 85

Table 4-15 Statistical results of heat-induced birefringence change 86

LIST OF ABBREVIATION

CLSM Confocal Laser Scanning Microscopy

DCPD Dicalcium phosphate dihydrate

FRAP Flurescence Recovery After Photobleaching

PLM Polarized Light Microscope

CHAPTER I INTRODUCTION

The caries prevalence and caries experience have been improved significantly in the past

century This is attributed to the development of oral health and the use of fluoride in

water and oral health products In recent years, early dental caries has attracted increased

attention in the dental community Diagnosis and treatment of the early caries would

prevent the development of caries, and greatly improve people’s living quality

The primary caries preventive agents have been fluoride and fissure sealants Recently,

with the development of the laser technique, laser has been used in the research and

treatment of caries The first laser application in dentistry was reported by Stern and

Sognnae (1964) and Goldman et al (1964) Several early researchers (Stern, 1969; Stern

and Sognnaes, 1972) reported that laser irradiation might increase caries resistance of

enamel and has the potential capability in cares prevention Several types of lasers

including CO2 laser (Stern et al., 1972; Featherstone, et al., 1998), Nd-YAG laser

(Yamamoto and Ooya, 1974), Argon laser (Oho and Morioka, 1990), and Ho:YLF laser

(Bachmann et al., 2004) have been studied and were reported to have the effect of

increased acid resistance and reduced subsurface demineralization in the enamel

However, the mechanism of laser-induced physical and /or chemical changes that cause

the reduced demineralization is not clear Stern et al (1966) and Yamamoto and Sato

(1980) attributed this reduction to the reduced permeability of enamel But Borggreven et

al (1980) suggested that the reduced rate was caused by the chemical modifications

Fowler and Kuroda (1986) suggested that the formation of pyrophosphate might reduce

the enamel solubility Nelson et al (1986) suggested that the inhibitory effect of laser was

probably due to a combination of surface sealing, compositional changes, and effect on

organic matrix As the laser-irradiated enamel would normally have a temperature

gradient that decreases towards the DEJ, identification of the changes along this

temperature gradient in tooth enamel will help us to understand the inhibitory mechanism

on subsurface demineralization

As for the development of a subsurface cariogenic lesion, there exist two processes First,

enamel apatite crystals are dissolved by the acid ions Second, the dissolved ions such as

calcium, phosphate ions and hydrogen ions would diffuse out and into enamel,

respectively, through an apparently intact surface (Moreno and Zahradnik, 1974) Several

studies have revealed the compositional, structural, and phase changes of heated enamel

(Holcomb and Young, 1980; Kuroda and Fowler, 1984; Palamara et al., 1987), and the

changes of lesion depth (LD) (Sato, 1983) The minimum LD was found to occur at 300

℃

There are relatively few studies to identify the diffusion phenomenon in enamel

(Featherstone and Rosenberg, 1984), since it is a complicated process Several methods

have been used to study this process including diaphragm cell method (Moreno and

Burke, 1974), penetration profile study (de Rooij et al., 1980), and conductometry

measurement (Scholberg et al., 1984) These methods are time-consuming and difficult to

reproduce

With the development of Confocal Laser Scannng Microscopy (CLSM) coupled with

Fluorescence Recovery After Photobleaching (FRAP), it becomes straightforward to

measure intracellular ions diffusion in the biomedical field But there has been no

application in the dental hard tissue until recently

In this experiment, the quantitative measurement of diffusion coefficient (DC) and

birefringence in enamel would be carried out Simultaneously, the Organic Matrix (OM)

and diffusion changes after heating and their roles in reducing the subsurface

demineralization would be explored The main objective of this study is to quantitatively

evaluate the effect of temperature and /or organic matrix on the carious-like lesion

formation, diffusion coefficient (DC) and birefringence of tooth enamel

CHAPTER II LITERATURE REVIEW

2.1 Human Dental Enamel

Enamel is the most highly mineralized tissue in human body By weight, it consists of

96% of mineral, 4% of organic material and water (ten Cate, 1985) Embryologically,

enamel is derived from cells of the oral epithelium This cell, called ameloblast, is quite

different from the internal dental epithelium Anatomically, enamel is the outer layer of

tooth structure, coving the anatomic crown of the tooth Functionally and physiologically,

enamel and its anatomical configuration provide a durable surface for tearing and

chewing of food They also help protect the underlying tissues, i.e dentin and pulp

2.1.1 Structure of Enamel

2.1.1.1 Enamel Rod

The enamel prism or rod is the basic structural unit of enamel In permanent teeth, its size

ranges from 4 to 7 micrometer in diameter (Gwinnett, 1992) Initially enamel rods

originate from the region that is quite close to the dentino-enamel junction (DEJ) Then

they decussate into the two-thirds of the enamel and finally arrive at their parallel

alignment in the outer third of the enamel Osborn (1965) stated that this decussation

produces an optical artifact that is known as Hunter-Shreger bands The parallel rods are

slightly oblique to a tangent of the natural surface of enamel In the outmost side of

permanent teeth, there are rodless enamels, usually in the pit, fissure and cervical regions

(Gwinnett, 1967) In the cross sections, the prisms appear somewhat like “keyholes”

Usually the head of prism is oriented to the occlusal surface of the tooth and its tail

toward the cervical region of the cross section (Meckel et al., 1965)

Within a prism, crystallites of hydroxyapatite are preferentially arranged (Schmidt and

Keil, 1958) In the center of the prism, the crystals are tightly packed compared to those

in the periphery and interprism enamel Around the periphery of each prism there is a

zone with relatively high organic contents where crystals are oriented in a different

direction from the central axis of rod The zone is known as rod sheath or interprismatic

substance, and is believed to have the function of holding the rods together

2.1.1.2 Enamel Crystals

Crystallites of hydroxyapatite occupy 80-90% of the overall volume of the enamel The

remaining 10-20% consists of fluids and organic, usually proteinaceous materials

(Robinson et al., 1971) Within a crystal, by weight, 37% is calcium, 52% is phosphate,

and 3% is hydroxyl The size of a crystal is approximately 0.03 by 0.04 by 0.2µm (Larsen

and Bruun, 1986) They are the main compositions of apatite and are called major

elements Their relative amounts are quite stable in the enamel In contrast to these major

components, the minor components are scarcely distributed evenly through the enamel

Some of the components, e.g fluoride, zinc, and lead, show a high concentration in the

surface layers below which the concentration drops dramatically Some components, e.g

sodium, carbonate, and magnesium, exhibit a reverse gradient Still there are components,

e.g strontium and copper, are not affected by the depth (Larsen and Bruun, 1986)

Each crystal has a long axis, called crystal c-axis or fiber axis in shape (Thewlis, 1940) It

usually sits in parallel with the direction of the prisms, but has a tendency to deviate from

the prism axes from the cusp to the cervical margin (Poole and Brooks, 1961) The

carbonated-apatite crystals have a trend to extend from the dentine toward the enamel

The stoichiomatric formula for hydroxyapatite is Ca10 (PO4)6(OH) 2 (Kay et al., 1964)

This is the unit cell that repeats in all directions to form the single enamel crystal that is

approximately 50 nm wide by 25 nm thick, extending from the dentin toward the enamel

surface (possibly up to 1mm) (Johansen, 1965)

Robinson et al., (2000) described this stoichiomatric structure in arrangement of main

ions of the crystals (Figure 2-1) The following statements are excerpted from his

descriptions:

The stoichiometric structure is most easily appreciated by a consideration of the

arrangement of ions around the central hydroxyl column, which extends in the

c-axis direction through the long axes of the crystals In the plane of the diagram,

the hydroxyl ion is enclosed by a triangle of calcium ions (calcium II) This is in

turn surrounded by a triangle of phosphate ions rotated out of phase by 60° These

triangles are in turn surrounded by a hexagon of calcium ions (calcium I) The

entire crystal structure can be envisaged as a series of such hexagonal plates

stacked one on top of another, each rotated 60° in relation to its immediate

neighbors

P

P P

central c-axis hydroxyl column can be seen (Robinson et al., 2000)

However, there are a number of variations on this theme in enamel crystal structure Such

variations include missing ions, particularly calcium and hydroxyl, which dissolve from

the crystals and diffuse into surrounding fluid At the same time, extraneous ions such as

carbonate, fluoride, sodium, and magnesium are frequently found to precipitate within the

crystal structure of surface enamel (Robinson et al., 2000) Such defects and substitutions

do have a profound impact on the behavior of apatite, especially with regard to its

solubility at low pH In other words, it has a very close relationship with the enamel

dissolution and initiation of the incipient caries

2.1.1.3 Enamel Tufts, Lamellae, Spindles and Cracks

In ground sections, both enamel tufts and lamellae are best demonstrated Enamel tufts

extend from the DEJ to the enamel in a short distance These lower-mineralized tufts

appear to be branched and contain greater concentrations of enamel protein than the rest

of enamel Lamellae project from the surface of enamel to the deeper enamel, consisting

of linear, longitudinally oriented defects, which are filled with enamel protein or organic

debris from the oral cavity

Origination of enamel tufts is the result of abrupt changes in the direction of packed rods

during development But the reason for the development of lamellae could be related to

blocking of the relief of enamel internal strains produced by dimensional changes during

enamel maturation Another reason for the high concentration of organic contents in tufts

and lamella is likely due to faulting of blocking of the exit for enamel protein after

maturation

Enamel spindles are formed by some newly formed odontoblast processes that are

trapped between adjoining ameloblast when enamel formation begins (ten Cate, 1985)

2.1.1.4 Striae of Retzius

As the human enamel precipitates at a rate of approximately 4 µm per day, there appear

periodic bands or cross striations, occurring 4 µm intervals across rods, on the ground

enamel sections Therefore, the striae of Retzius are actually incremental lines of this

daily growth in enamel They appear as concentric growth rings running from DEJ

toward the occlusal surface and appear brownish under illuminating light in longitudinal

sections Accentuated incremental lines are produced by systemic disturbances, such as

fever and nutritional changes The lines’ surface manifestation is called Perikymata

(Boyde, 1997)

2.1.1.5 Hunter-Schreger Bands

The Hunter-Schreger bands are an optical phenomenon, not a real structure of the enamel

Under incident of polarized light, there appear dark and light alternating band in the inner

four fifths of the enamel in the longitudinal ground sections It was assumed that

Hunter-Schreger bands are formed due to the changes of prism orientation When groups of

prism are cut transversely, they are known as diazones; those, cut more longitudinally,

are known as parazones They can also be reversed by an alteration of the direction of the

incident illumination

2.1.1.6 Dentino-Enamel Junction (DEJ)

The DEJ is a junction area between enamel and dentine It can be easily seen as a series

of depressions or concaves towards surface of enamel In scanning electron microscopy

(SEM), the DEJ shows as a series of ridges that increase the surface area and probably

enhance adhesion between enamel and dentine

2.1.1.7 Water Structure of Enamel

The water content in enamel has been reported around 12% by volume (Carlstrom et al.,

1963) and 1 ~ 6% by weight (Brudevold et al., 1960) The water content appears to vary

with the relative location of the enamel in the tooth and may also vary with age and tooth

type The amount of water in enamel could be reflected by the magnitude of the form

birefringence (Carlstrom and Glas, 1963) Normally enamel has a negative birefringence

under polarized light investigation Angmar et al., (1963) found that after heating or

drying, enamel shows a positive birefringence It was believed that water was lost and air

was substituted into the submicroscopic spaces It increased the form birefringence and

compromised the intrinsic birefringence and eventually changed the observed

birefringence from negative to positive

Little et al., (1962) found that water was bound to enamel in two different ways - loosely

and firmly He also demonstrated that the firmly bound water occupied a greater part,

associated with minerals of enamel Carlstrom et al (1963) stated that the ratio of

loosely and firmly bound water should be around 1:4 The loosely bound water, at least

part of it, was related to the organic matrix whereas the firmly bound water was related to

the mineral phase The evidence for the relationship of the water and organic matrix is

that the enamel completely loses the ability of reimbibition in water at 200℃, while the

organic matrix begins to carbonize in 200℃, and both water and organic matrix increase

from the surface to DEJ (Carlstrom and Glas, 1963)

2.1.1.8 Porosity

The crystal arrangement of enamel gives rise to two main categories of porosity,

prisms Moreno and Zahradnik (1973), using the isothermal water vapour sorption

technique, have demonstrated that the intact enamel has a bimodal pore volume

distribution They also stated that the larger pores are related to the interprismatic regions

and the smaller ones are probably associated with the intraprismatic spacing of dental

enamel in this geometrical model The bimodal distribution in the core body has peaks at

9 to 25 Å A few other researchers have stated that the enamel possess a range of pores

diameters from 30 to 140 Å (Ying et al., 2004) or 10 to 250 Å (Medema and Houtman,

1969) Orams et al (1974) showed that the two dominant pores have sizes in a range

from 1 to 10nm, observed by SEM All these range coincided with each other

The pore size of mature enamel is very small The function of the pores was described in

different ways such as “a molecular sieve” (Darling et al., 1961 and Poole et al., 1961), a

semipermeable and osmotic membrane (Atkinson, 1947) So only air and water (Angmar

et al., 1963), methanol (Darling et al., 1961), and molecules and ions of the same order of

magnitude (Fosdick and Hutchinson, 1965) could be diffused into these microscopic

spaces of mature enamel

However, Moreno and Zahradnik (1973), with the help of vapour uptake technique,

suggested that dental enamel did not contain micropores and the sieve behavior was

probably due to the presence of pore constrictions that in turn are related with the organic

matter Brudevold et al (1960) found that the enamel sorption capacity increased from

the enamel surface to near the DEJ, which probably reflected the fact that the organic

matrix increased from surface to DEJ as well So Brudevold used a simple experiment to

prove this assumption: the low-temperature (<60℃) ashed enamel samples differ from

the intact enamel samples only in the integrity of organic matter The ashed samples did

not exhibit the phenomenon of activated diffusion So it can be concluded that the pore

constriction has a close relationship to organic matter of the enamel

2.1.2 Chemical Properties

2.1.2.1 Inorganic components

The main mineral component of enamel is hydroxapatite, nearly 80-90% by weight

Since the surface of tooth is immersed in an environment of saliva with supersaturated

calcium and phosphate relative to hydroxapatite, analysis of successive layers of enamel

has demonstrated that the chemistry of the surface enamel differs from that of the interior

enamel in several respects: Fluorine, zinc, lead and iron accumulate in the surface enamel

and the concentrations of these elements virtually depend on those of the external

environment of the tooth Conversely, some constituents, including carbonate, sodium

and magnesium increase in concentration from the surface inward and are found in

greatest amounts at DEJ At the same time, some other elements (strontium and copper)

are evenly spreading in the enamel (Thylstrup and Fejerskov, 1994) The apatites also

exhibit a number of variations, which include the missing of ions, such as calcium

(Winand et al., 1961) and hydroxyl (Young and Spooner, 1969), and substitutions with

other ions (Young, 1974)

The incorporation of the non-apatitic and apatite minerals of surface enamel has different

impact on the stability and solubility of the enamel to the acid attack Robinson et al

(2000) and Simmer and Fincham (1995) have stated their main effects on crystal

structure as following:

1) Fluoride incorporation is classically thought to occur by fluoride ions filling

hydroxyl vacancies in the c-axis columns or displacing hydroxyl ions (Kay et

to a much closer fit for fluoride within the Ca II triangles This has the effect

of lowering lattice energy and effectively stabilizing the crystal structure It is

observed significantly higher in the surface enamel compared with the

subsurface enamel

2) Carbonate can replace hydroxyl or phosphate/acid phosphate These

substitutions of a poorer fit of carbonate in the lattice generate a less stable

and more acid-soluble apatite phase Carbonate substitution for hydroxyl

occurs almost exclusively near DEJ and cannot be detected near the surface of

the tooth

3) Magnesium can replace calcium to some extent, to about 0.3% (Fertherstone

et al., 1983) Magnesium is thought to be located on crystal surface or in

separate, more acid-soluble Magnesium has large density, would have a

destabilizing effect on the apatite lattice It diminishes from 0.4% at the DEJ

to 0.1% at the surface of the tooth

4) Carbonate and magnesium compete for the same adsorption sites, and also

have a positive synergistic effect, both on their incorporation by the

hydroxyapatite lattice and in their ability to increase the acid solubility of

apatite mineral (LeGeros, 1984)

In addition, Bachra et al (1963) has stated that the dental enamel is a quite stable crystal

as it contains less amount of impurities, e.g Mg, Na, CO3, HPO4, citrate, etc

2.1.2.2 Organic components

The organic contents of mature dental enamel are at very low concentrations,

approximately 1.2% by weight or 2% by volume, consisting of 58% of protein, 40% of

lipids, and other trace elements (Odutuga and Prout, 1974) It usually exists in high

concentration in the enamel tufts, near the dentine and in areas where crystal packing is

less compact i.e the cusps, fissure regions Melfi and Alley (2000) has showed the

organic substance to be a fine fibrillar latticework of rods, rod sheaths, and inter-rod

substance by Scanning Electron Microscopy (SEM) All these organic constituents play

important roles during development and calcification of enamel, as well as in the

formation of caries

The protein of enamel is classified as the amelogenins and the enamelins (Simmer and

Fincham, 1995; Melfi and Alley, 2000) During the formation of the crystallite, both

amelogenins and enamelins are believed to regulate the shape of the crystals (Doi et al.,

1984) and presumablely bind to crystals through multiple electrostatic interactions

(Simmer and Fincham, 1995) Nylen et al (1963) delineated the protein as gel-like

materials, randomly arranged molecules in enamel

The lipid has been recognized as an important constituent during calcification of enamel,

especially the phospholipids that can bind calcium and stabilize amorphous calcium

phosphate (Dirksen and Vogel, 1976) The lipid is also thought as an important

component in inhibiting the progress of demineralization in enamel Featherstone and

Rosenberg (1984) found that the lesion progression was more than doubled in

lipid-extracted enamel compared with normal enamel

In the intact enamel, up to 4% by weight and 11% by volume is water (Larsen and Bruun,

1986), with a ratio of 1:4 between loosely and firmly bound water (Carlstrom et al., 1963)

Featherstone et al (1979) has assumed that both components could diffuse the ions into

and out of enamel and play an important role in the dissolution and remineralization of

crystals of enamel Deakins (1942) suggested that the process of mineralization of enamel

involves the displacement of water by minerals Therefore, the water concentration and

the degree of mineralization have an inverse relationship

2.2 Heat-Induced Effect on Human Enamel

Previous studies have shown that enamel after heat treatment and enamel with laser

irradiation had reduced subsurface demineralization when enamel was exposed to acid

dissolution reduction mechanism for this phenomenon is not clear at the moment

Nevertheless, three explanations have been suggested:

1 The sealing effect on enamel pores and irregularities to reduce permeability (Sato,

1983);

2 The phase changes in the inorganic materials and chemical changes (Flowler and

Kuroda, 1986);

3 The change of crystallite size (Sato, 1983)

The identification of the temperature-induced changes in enamel would help us better

understand the mechanism of the reduced demineralization in heated subsurface enamel

2.2.1Optical, Morphological, and Crystallographic Changes

Heat-induced tissue changes in enamel depend on the heating temperature and duration of

heating Markolf (2002) stated step-by-step the heat effect in different temperatures

From 42℃ ~ 50℃, the thermal effect on hard tissue contributed to the conformational

changes of molecules, accompanied by bond destruction and membrane alterations If

heating lasted for a few minutes, necrosis and reduction in enzyme activity were observed

At 60℃, protein and collagen were denaturized, thus making the tissue to be coagulation

and look like whitish in visualization At 100℃, water inside tissue started to vaporize

and the volume of tissue increased dramatically This increasing gas volume would in

turn decompose and collapse the hard tissue At >100℃, carbonization was observed as

the blackening of tissue and smoke escape from the tissue And finally, at >300℃,

melting occurred in the hard tissue

These observed changes were related to the interaction between laser-induced heating

effect and hard tissue In addition, there are some techniques used to observed different

enamel changes with different temperatures

Firstly, Sato (1983) and Palamara et al (1987) used polarized light microscopy (PLM) in

their studies They observed the heat-induced birefringent changes with different

temperatures In their findings, between the temperature ranges of 100℃ to 300℃, the

first thermal change was seen in the inner region of the enamel At 300℃, the positively

birefringent region extends to the middle enamel and outer enamel shows a bluish color

At 400℃, the entire enamel showed positive birefringence with slight opacity At 600℃,

all enamel became completely opaque

Secondly, Sato (1983) used scanning electron microscope (SEM) to observe the changes

of heated enamel Below 300℃, it showed there were no noticeable changes in the

samples At 400℃, enamel crystals are sharply outlined and there were dotty microspores

At 500 ℃ , a great number of pores appeared and some of them were enlarged

Furthermore, fusion between neighboring enamel crystals was clearly observed

Thirdly, Sakae (1988) used x-ray diffraction to study the changes of crystallites of enamel

From 200℃ to 400℃, crystallites in enamel initially became smaller along the hexagonal

a-axis direction Beyond 400℃, these crystallites grew bigger That was quite consistent

with the observations made by of LeGeros et al (1978) and Young and Holcomb (1984)

In these latter studies, they also showed crystallites gradually became bigger along the

c-axis (beyond 240℃)

Lastly, transmission electron microscope (TEM) was used by Palamara et al (1987) His

study showed inter- and intra-crystalline voids formation in heated enamel at

temperatures as low as 200℃ Between 200℃ and 350℃, the negative birefringent

regions of the near surface enamel showed minimal or nearly no changes in void volume

However at 350℃, intra-crystalline voids appeared mainly From 350℃ to 600℃, the

increase in the number and size of voids was more significant in the positive birefringent

area

In summary, all these techniques have contributed to the research of the heating effect on

enamel and the results are summarized in Table 2-1

2.2.2 Chemical Reaction

The chemical changes of heated enamel have been studied by several researchers (Corcia

and Moody, 1974; Sakae, 1988; Hsu et al, 1994; Fowler and Kuroda, 1986) Their

findings can be summarized as follows:

1 The water content decreases with increasing temperature and an abrupt decrease

happens at 250 ℃ to 300 ℃ losing about one-third of the amount initially

incorporated water in enamel The decrease of water content coincides with a

sharp contraction of lattice parameter (a-axis) at the same temperature range

Therefore, Holcomb and Young (1980) stated that the enlargement of the a-axis

length is related to the structurally incorporated water

2 With the increasing temperature, there is a consistent loss and rearrangement of

CO32- ions: the substitution of CO32- for PO43- decreases and the substitution of

CO32- for OH-increases (Holcomb and young, 1980)

3 The content of OH- increases progressively to a maximum in the range 300℃ to

500℃(Fowler and Kuroda, 1986)

4 The endothermic water maximum is in a range from 100℃ to 140℃, up to a peak

at 140℃ The pyrolysis and volatilization of organic constituents change from

250℃ to 400℃, up to a peak at 350℃ (protein decompose) The oxidation of

carbon content attends the peak at 500℃ A glass phase is formed between 400℃

and 460℃ (Corcia and Moody, 1974)

5 The acid phosphate ions condense to form pyrophosphate (P2O74-) ions and its

content progressively increases in the temperature range 200℃ to 400℃

6 The formation of more soluble Beta-TriCalcium Phosphate (β-TCP) in the enamel

after 400℃ (Palamara et al., 1987)

7 The decrease of solubility from 150℃ to 400℃ is the main reason for the

increased acid resistance (Hsu et al, 1994)

From above chemical changes, enamel gradually lost its translation materials (water and

protein) and becomes more stable and more resistant to the acid solution

2.2.3 Heat-Induced Reduction of Enamel Demineralization

After heat treatment, surface enamel showed an increased birefringence with increasing

temperatures Oho and Morioka (1990) attributed it to the loss of the protein and the

formation of microspaces in enamel acting as a trap for the deposition of ions released by

acid attack This trap could compromise acid effect and reduce the demineralization rate

Sakae (1988) used X-ray diffraction to study the size change of crystallite after heat

treatment of the enamel samples Contraction of the a-axis length was observed and

attributed to the loss of structurally incorporated water from the apatite lattice Holcomb

and Young (1980) found that in the range 300℃ to 500℃ OH- content increases and at

500℃ pyrophosphate (P2O74-) appears in the hydroxyapatite Sato (1983) found the

minimal calcium dissolution at 350℃ and attributed to the products of pyrolysis of

organic matrix, which closed up the porosity newly formed in heated enamel All these

changes could affect the ions diffusion and dissolution of enamel during acid attack

The heating effects on the constituents of the tooth enamel, birefringence, and artificial

lesion depth are summarized in Table 2-1 The arrows indicate the trend of the changes,

whereas the numbers specify the articles in which the specific effect was studied

Table 2-1: Summary of the heating effect

Soluble Carbonate Factors

Pyro-e

Enamel melting / fusing

TCP BF LD

Note 1: “+” indicates increment with temperature Note 2: “-” indicates decrement with temperature Note 3: “x” indicate no change

Note 4: “x” indicates no change Note 5: “*” indicates no data Note 6: Number of relevant articles is as follows:

1 ─ Fowler and Kuroda, 1986 2 ─ Sato, 1983 3 ─ Herman H, Dallemagne MJ, 1961 “

4 ─ Hsu et al., 1994 5 ─ lin et al., 2000 6 ─ Arends j, Davidson CL ,1975

7 ─ Sakae, 1988 8 ─ Palamara et al., 1987 9 ─ Holcomb and Young, 1980

10 ─ Corcia and Moody, 1974 11 ─ Oho and Morioka, 1990 12 ─ Ying et al, 2004

2.3 Caries Formation and Progression

2.3.1 Introduction

In dental caries, the disease occurs within the tooth hard tissues where the mineral

substance of the tooth is dissolved by acid, and subsequently the organic substance is

destroyed by proteolysis The acid is created by oral bacteria that metabolize and convert

carbohydrates, especially sugars, into acid Caries-susceptible individuals have many

such kind of acidogenic (acid-producing) bacteria in their saliva and dental plaques

Because the acids in the plaque are in contact with the tooth surface, enamel beneath the

plaque is slightly dissolved This process is the beginning of a caries lesion The presence

of an apparently intact surface layer overlying a subsurface demineralization is the

feature of the early lesion A slight increase in enamel porosity changes the optical

properties of the enamel in such a way that light is scattered Because of this, enamel

gradually becomes less and less translucent with increasing tissue porosity in the early

demineralization Clinically, this can be observed as the appearance of whitish (opaque)

changes of the enamel, or “white spot” Because carious dissolution follows the direction

of the rods, the lesion appears triangular in sections cut through the central lesion part As

the mineral loss increases, the surface enamel loses the support of the beneath structure

and finally collapses The cavities consequently appear on the enamel surface (Fejerskov

O and Kidd EAM, 2003)

2.3.2 Protective Role of Sound Enamel Surface Chemistry

The physico-chemical integrity of dental enamel in the oral environment is heavily

hinged upon the composition and chemical behavior of the surrounding fluids

Normally, saliva and plaque fluid are supersaturated with respect to enamel apatite

Under this chemical equilibrium, it not only prevents enamel from dissolving, but also

tends to precipitate apatite, partly as calculus in the form of crystal growth on surface

enamel But this equilibrium could be broken if the exterior ions added or the

concentration of ions changed (Moreno and Zahradnik, 1974) Factors affected the

stability on enamel apatite are the free active concentrations of calcium, phosphate,

fluoride, organic matrix and pH in solution

2.3.2.1 Fluoride, Carbonate and Magnesium

The surface enamel contains, for example, high concentrations of fluoride Fluoride is of

particular importance in stabilizing the surface enamel by the reaction of fluoride with the

dental apatite The major product is called calcium fluoride (CaF2) (Moreno and

Zahradnik, 1974):

2

10( 4 6) ( )2 20 11 10 2 3 2 4 3 4 2 2

Ca PO OH + F−+ H+→ CaF + H PO −+ HPO −+ H O

The surface enamel also contains low concentrations of carbonate and magnesium, which

have a destabilizing effect When moving inward away from the surface, gradients of

fluoride decrease, while gradients of both carbonate and magnesium increase together

with increasing porosity (Hallsworth et al., 1972) Therefore, as caries process

progresses inward toward the deeper enamel, the chemistry of dissolution will change,

and the solubility will increase

2.3.2.2 Organic Matrix

The presence of organic matrix on or in the enamel surface is a contributor to surface

zone formation by reducing mineral loss or acting as a barrier When protein material has

been removed, the natural lesion is able to take up more calcium from the external

environment (Robinson et al., 1990) This supports the view that the protein layer can

slow the transmission of mineral ions through the enamel surface

2.3.2.3 The pH value

When pH falls, the solubility of the enamel apatite will increase dramatically The

rationale is that: the hydroxyl concentration is inversely proportional to the hydrogen

concentration and the concentration of the phosphate ionic species depends on the pH of

the solution When the pH decreases, more PO43- ions are transformed to HPO42- that in

turn reacts to H2PO4- The conversion of phosphate and the effect on the solubility can be

illustrated by the reaction (Fejerskov O and Kidd EAM, 2003):

The pH at which the fluids are exactly saturated with respect to enamel apatite is defined

as the “critical pH” It is usually around 5.5 However it is not a fixed number It is

determined by the concentration of calcium and phosphate presented in the oral fluids

(Dawes, 2003)

When the pH goes below the critical level the aqueous phase is unsaturated with respect

to hydroxyapatite due to the decreased activity of PO43- and of hydroxyl

If the pH increases again, the aqueous environment of the enamel surface will gradually

return to a state of supersaturation with respect to hydroxyapatite and/or fluorapatite

Caries progression from ultrastructural changes to visible decay should therefore be

regarded as the cumulating effect of a long series of alternating dissolution at a low pH

and a partial reprecipitation when pH rises

2.3.3 In vitro Demineralization

To study the formation of lesion, many laboratory models (Darling,1956; Gray and

Francis, 1963; Holly and Gray, 1968) have been built, trying to understand the

mechanism behind this phenomenon It has been related with the driving forces and the

kinetics in the carious process and the pertinent equations based on diffusion theory have

been developed The initial enamel dissolution rate is largely a function of the total buffer

concentration, buffer acid strength, and pH (Gray, 1962) Several theories have been

proposed to explain the phenomenon of preferential subsurface dissolution occurred

when the dental enamel is subjected to acid attack These theories are usually focused on

discussion of a specific factor However it is increasingly clear that many factors are

simultaneously contributing to the process of demineralization

Firstly, the anatomical variations in the structure and composition of enamel are the cause

of the subsurface demineralization The outer enamel surface, which is intact in early

caries, is protected by inhibitors, for example, F+, derived from the oral environment and

adsorbed on the outer surface It makes the surface less soluble (Gray, 1965) Conversely

fluoride ions are less in subsurface enamel But the concentration of carbonate and

magnesium, which are related to the solubility of enamel, increases from the surface of

enamel to DEJ The gradients favoring dissolution of mineral content stimulated the

subsurface demineralization (Theuns et al., 1986a)

Secondly, the initial stage of lesion formation is described as the dissolution of mineral

with outward diffusion of calcium and phosphate ions As this diffusion ions accumulated

at the surface, the surface enamel was in a dynamic process of

demineralization/remineralization Margolis and Moreno (1985) suggested that the

precipitation of CaHPO4·2H2O (DCPD) from dissolved enamel in the subsurface region

was the cause of intact surface layer Moreno and Zahradnik (1974) stated that the

quasi-equilibrium was maintained by a kinetic balance between the rate of transfer of dissolved

ions across the enamel-solution interface and the rate of precipitation of DCPD, HA and

FA Theuns et al (1986a) attributed the formation of surface layer to the transformation

of the apatite to a more stable calcium phosphate in the presence of acid, as more F+ or

less carbonate contained in an apatite

Thirdly, following the formation of the incipient caries, a model acting as a pumping

mechanism can be formulated as schematically illustrated in Figure 2-2 (Moreno and

Zahradnik, 1974) The model works as follows: Firstly, the plaque bacteria, particularly

Streptococcus mutans and lactobacilli, produced organic acids (HB) Second, the acids

diffuse through the pellicle into the surface enamel Third, the plaque pH drops to maybe

5.5, the critical pH Some phase transformation occurs in the surface enamel, including

dissolution of the enamel surface, followed by precipitation of the solid phases

CaHPO4·2H2O (DCPD) and Ca5F(PO4)3 (FA) Therefore there are three solid phases,

DCPD, FA and the bulk of the tooth enamel, in the surface enamel Last, basic

constituents will diffuse from the inner region into the surface zone, and from the surface