role of dentin compositional changes and structural loss on … · 2015-08-27 · and to prevent...

Role of Dentin Compositional Changes and Structural

Loss on Fracture Predilection in Endodontically Treated

Teeth

by

Arezou Ossareh

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Dentistry

University of Toronto

© Copyright by Arezou Ossareh (2015)

ii

Role of Dentin Compositional Changes and Structural Loss on Fracture Predilection in

Endodontically Treated Teeth

Arezou Ossareh

Master of Science

Faculty of Dentistry, University of Toronto

2015

Abstract

The aim of this study was to examine the role of chemical compositional changes and iatrogenic

dentin structural loss on the mechanical response of teeth to force and resistance to fracture. The

experiments were divided into three phases. In phase 1, experimental studies were performed to

evaluate the effect of chemicals used during treatment on ultrastructure, composition and

resistance to fracture of dentin. In phase 2, experimental studies were used to evaluate the

influence of dentin removal and remaining dentin volume on the resistance to fracture and

microcrack formation in root dentin. In phase 3 finite element analysis was carried out to

examine the influence of dentin loss on the stress distribution in root dentin. The combination of

experimental and numerical analysis highlighted the role of remaining dentin volume and

moment of inertia on root dentin biomechanics.

iii

Acknowledgments

First and foremost, I would like to express my deepest gratitude to my supervisor Dr. Anil

Kishen for his invaluable guidance, support and dedication throughout this research project. His

direct involvement and excellent command of the research area helped me at every stage. His

constructive feedbacks, motivation and enthusiasm have been a driving force in my graduate

career at the University of Toronto and helped me complete this project. His exceptional support,

encouragement and guidance not only helped me academically, but also made a better and

stronger individual and gave me great insight into life. I am also grateful to my thesis committee

members, Dr. Cari Whyne and Dr. Grace De Souza for their constant support and encouragement

throughout my research endeavors.

I would like to thank Dr. Whyne’s Biomechanics Lab in Sunnybrook Research Institute and Dr.

Rosentritt at the University Hospital Regensburg UKR, for their invaluable help and support for

using of their laboratory and facilities. I would also like to thank Drs. Badle and Bellamy for

their valuable insights and assistance with the root canal preparations. I am also thankful for the

technical support provided by Jian Wang and Audrey Darabie

A number of individuals in Dr. Kishen’s lab were extremely helpful in this learning process and

supported me in executing laboratory procedures and data collection. These include, but are not

limited to Drs. Annie Shrestha, Suja Shrestha and Alice Li. Their contribution was crucial in the

completion of this Master’s thesis research.

Lastly, and most importantly I express my forever gratitude to my parents and my sister. I thank

them for their love, emotional support and for all they have done for me.

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Table of Contents

Acknowledgments ........................................................................................................................... ii

List of Tables ................................................................................................................................ vii

List of Figures .............................................................................................................................. viii

List of symbols ................................................................................................................................ x

Chapter 1 ......................................................................................................................................... 1

1 Introduction ................................................................................................................................ 1

1.1 Background ......................................................................................................................... 1

1.2 Objective and Hypothesis ................................................................................................... 3

1.2.1 Hypothesize ............................................................................................................. 3

1.2.2 Objective ................................................................................................................. 3

1.2.3 Specific Objectives ................................................................................................. 3

1.3 Literature Review ................................................................................................................ 4

1.3.1 Endodontic Treatment ............................................................................................. 4

1.3.2 Dentin as a Biomaterial ........................................................................................... 4

1.3.3 Fracture in Endodontically Treated Teeth ............................................................ 10

1.3.4 Effect of Endodontic Chemicals on Dentin Structure and Composition .............. 12

1.3.5 Effect of Iatrogenic Dentin Structural Loss on Mechanical Integrity of Dentin .. 15

1.3.6 Critique of Literature ............................................................................................ 16

Chapter 2 ....................................................................................................................................... 18

2 Effect of Chemicals on ultrastructure, composition and mechanical properties of dentin ...... 18

2.1 Introduction ....................................................................................................................... 18

2.2 Materials and Methods ...................................................................................................... 19

2.2.1 Sample Selection ................................................................................................... 19

2.2.2 Sample Preparation ............................................................................................... 19

v

2.2.3 Ultrastructure Analysis ......................................................................................... 20

2.2.4 Compositional Analysis ........................................................................................ 20

2.2.5 Mechanical Analysis ............................................................................................. 21

2.2.6 Statistical Analysis ................................................................................................ 21

2.3 Results ............................................................................................................................... 22

2.3.1 Ultrastructural Analysis ........................................................................................ 22

2.3.2 Compositional Analysis ........................................................................................ 23

2.3.3 Mechanical Analysis ............................................................................................. 24

2.4 Summary ........................................................................................................................... 26

Chapter 3 ....................................................................................................................................... 27

3 Effect of Dentin Loss on Fracture Resistance of Root Dentin ................................................. 27

3.1 Introduction ....................................................................................................................... 27


3.2.1 Sample Selection ................................................................................................... 28

3.2.2 Sample Preparation and Groups ............................................................................ 28

3.2.3 Determination of Dentin Volume and Moment of Inertia .................................... 29

3.2.4 Thermal and Mechanical Cyclic Testing .............................................................. 29

3.2.5 Determination of the Load to Fracture .................................................................. 30

3.2.6 Micro-Crack Analysis ........................................................................................... 31

3.2.7 Statistical Analysis ................................................................................................ 31

3.3 Results ............................................................................................................................... 32

3.3.1 Determination of Dentin Volume Removed ......................................................... 32

3.3.2 Determination of Load to Fracture ....................................................................... 33

3.3.3 Load to Fracture and Remaining Dentin Volume Analysis .................................. 33

3.3.4 Load to Fracture and Moment of Inertia Analysis ................................................ 34

3.3.5 Micro-crack Analysis ............................................................................................ 35

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3.4 Summary ........................................................................................................................... 37

Chapter 4 ....................................................................................................................................... 38

4 Effect of Structural Loss on Stress Distribution Pattern in Dentin .......................................... 38

4.1 Introduction ....................................................................................................................... 38


4.2.1 Sample Preparation and Micro-CT Imaging ......................................................... 39

4.2.2 Segmentation of Tooth and Generation of FEA models ....................................... 39

4.2.3 Finite Element Analysis ........................................................................................ 39

4.3 Results ............................................................................................................................... 41

4.3.1 Von Mises Stress Distribution .............................................................................. 41

4.4 Summary ........................................................................................................................... 42

Chapter 5 ....................................................................................................................................... 43

5 Discussion ................................................................................................................................ 43

Chapter 6 ....................................................................................................................................... 49

6 Conclusion................................................................................................................................ 49

6.1 Conclusion ........................................................................................................................ 49

6.2 Future studies .................................................................................................................... 50

Chapter 7 ....................................................................................................................................... 51

7 References ................................................................................................................................ 51

vii

List of Tables

Table 1. Material Properties .......................................................................................................... 40

viii

List of Figures

Figure 1. Tooth Structure [11] ........................................................................................................ 5

Figure 2. Intertubular and Peritubular structure in dentin ............................................................... 7

Figure 3. SEM images of root canal surface after irrigation with (a) 5.25% NaOCl for 30 min, (b)

17% EDTA pH 7.0 for 15min, (c) 5.25% NaOCl for 10min followed by 17% EDTA pH 7.0 for 2

min as a final rinse, (d) 5.25% NaOCl for 10min followed by 17% EDTA pH 7.0 for 1 min

followed by 5.25% NaOCl for 1 min as a final rinse and (e) water for 30 min ............................ 22

Figure 4. Amide/phosphate ratio on dentin surface treated with (1) water for 30min, (2) 5.25%

NaOCl for 30min, (3) 17%EDTA pH 7.0 for 15min, (4) 5.25% NaOCl for 10min followed by

17% EDTA pH 7.0 for 2 min as a final rinse, and (5) 5.25% NaOCl for 10min followed by 17%

EDTA pH 7.0 for 1 min followed by 5.25% NaOCl for 1 min as a final rinse ............................ 24

Figure 5. The compressive strength and b) toughness of dentin samples treated with (1) water for

10min, (2) 5.25% NaOCl for 10min, (3) 17%EDTA pH 7.0 for 2min, (4) 5.25% NaOCl for

10min followed by 7% EDTA pH 7.0 for 2 min as a final rinse, and (5) 5.25% NaOCl for 10 min

followed by 7% EDTA pH 7.0 for 1 min followed by 5.25% NaOCl for 1 min as final rinse .... 25

Figure 6. The percentage reduction in the dentin volume simulated with different level of dentin

removal mean±SE (n=10) ............................................................................................................. 32

Figure 7. Load to fracture, values are reported as mean± SE (n=7, * statistically significant with

control, ** statistically significant with Low) .............................................................................. 33

Figure 8. Correlation between remaining dentin volume and load to fracture .................... 34

Figure 9 Correlation between load to fracture and moment of inertia .......................................... 35

Figure 10. Microcrack analysis of samples after mechanical/thermal cycling load based on

different degree of dentin removal ................................................................................................ 36

ix

Figure 11. Von Mises Stress Distribution on root dentin (B: Buccal, L: Lingual, M: Mesial and

D: Distal) ....................................................................................................................................... 41

x

List of symbols

VRF

Vertical Root Fracture

NaOCl

Sodium Hypochlorite

EDTA

Ethelenediaminetetraaccetic acid

CHX

Chlorohexidine

FTIR

Fourier Transform Infrared Spectrocopy

FEA

Finite Element Analysis

ATR- FTIR

Attunated Total Reflectance- Fourier Transform Infrared Spectroscopy

SEM

Scaning Electron Microscopy

HMDS

Hexamethyl disilizane

CEJ

Cemento-enamel junction

PDL

Periodontal Ligament

1

Chapter 1

1 Introduction

1.1 Background

Tooth fracture is considered to be the third most common cause of tooth loss after dental caries

and periodontal disease [1, 2]. The diagnosis and management of fractures in teeth are difficult

and tooth extraction is often the only solution. Root canal treatment is indicated to treat the

infected teeth and maintain them to be healthy and functional. Currently, more than 20 million

root canal treatments are carried out in the United States alone yearly [3]. However, 10% of the

root canal treated teeth or root-filled teeth that were referred for extraction showed root fracture

[4, 5]. This percentage of root fracture is very significant considering the prevalence of root

canal treatment world-wide. The occurrence of root fracture may be reduced by analyzing the

structural and compositional changes that occur in the mechanical integrity of dentin during

treatment procedure. This knowledge may be used to develop treatment strategies and treatment

planning that reduces fracture predilection with increasing the functional longevity of root canal

treated teeth.

Mechanical failures of endodontically treated teeth are likely to result from excessive stresses

and/or fatigue, which is a cumulative process of crack initiation and its propagation [6]. In this

study, in order to understand the mechanical response of dentin to forces and resistance to

fracture, the experiments were divided into three phases. In phase-1 experimental studies were

performed to evaluate the effect of compositional changes (due to chemicals used during

treatment) on ultrastructure, composition and resistance to fracture of dentin. In phase-2

2

experimental studies were used to evaluate the influence of three different simulated dentin

structural losses on resistance to fracture of root dentin. In phase-3 of the study finite element

analysis were carried out to evaluate the influence of simulated dentin loss on stress distribution

in root dentin.

There are still lack of information on the effect of both dentin loss and compositional changes on

the mechanical integrity of endodontically treated teeth. This information is important to

understand the mechanism of fracture predilection in endodontically treated teeth.

3

1.2 Objective and Hypothesis

1.2.1 Hypothesis

Compositional changes and iatrogenic dentin structural loss would influence the biomechanical

response of teeth, which may subsequently predispose teeth to fracture

1.2.2 Objective

To examine the role of chemical compositional changes and iatrogenic dentin structural loss on

the mechanical response of teeth to force and resistance to fracture

1.2.3 Specific Objectives

The proposed study has 3 specific objectives:

1. Analyze the effect of endodontic chemicals on ultrastructure, compositional changes and

fracture resistance of root dentin

2. Calculate the amount of dentin loss, remaining dentin volume and moment of inertia after

instrumentation to simulate three degrees of dentin removal and analyze their effect on

resistance to fracture of root dentin

3. Analyze the effect of simulated dentin loss on stress distribution patterns in root dentin

4

1.3 Literature Review

1.3.1 Endodontic Treatment

Apical periodontitis or endodontic infection is the host defense response to a microbial challenge

from the root canal system [7]. It is a destructive inflammatory process that can cause caries,

periodontal disease or trauma [7]. The goal of endodontics treatment is to prevent or eliminate

apical periodontitis by disinfecting the canal system, using various chemo-mechanical methods

and to prevent reinfection using a root canal filling [8-10]. After accessing the root canal,

mechanical instruments and chemical solutions clean and shape the root canal [8-10].

Subsequently, the canal space is filled and sealed with a core filling materials and a cement

sealer [8-10]. The prognosis of root canal treated teeth not only depends on the success of the

endodontic treatment but also on the amount of remaining dentin tissue, disinfection of the canal

with endodontic chemicals and restoration of the mechanical integrity of dentin [3-6]. Therefore,

early identification of iatrogenic risk factors will aid in treatment planning, diagnosis and

estimating treatment prognosis of root canal treated teeth. Understanding the effect of iatrogenic

dentin structural loss and compositional changes on tooth response to forces may provide better

insight into iatrogenic risk factors that predispose endodontically treated teeth to fracture.

1.3.2 Dentin as a Biomaterial

Dentin is a mineralized and hydrated hard tissue that forms the major bulk of the human tooth. It

is surrounded by enamel in the crown and by cementum tissue in the root (Figure.1). It is a

composite material that consists of oriented tubules. The tubules are surrounded by a highly

mineralized peritubular dentin and are contained in an intertubular matrix that primarily consists

of Type I collagen, which has embedded in apatite crystals [6]. The tubules contain dentinal fluid

5

and to a varying extent, an odontoblastic process [12]. Dentin is formed by odontoblastic cells

that secrete an organic matrix which becomes mineralized to form dentin [13].

Figure 1. Tooth Structure, http://anatafizziology.files.wordpress.com/2012/03/tooth.jpg [11]

1.3.2.1 Dentin Composition

By weight, dentin is composed of approximately 70% inorganic materials, 18% organic materials

and 12 % water [14-17]. By volume, the inorganic constituent makes up 50%, while the organic

matrix constitutes 30% and the water makes up 20% of the dentin [18]. Other organic

components are present in small amounts and consist of proteoglycan, non-collagenous protein,

citrate, lactate and lipid which account for approximately 2% by weight [14-17]. It has been

determined that in dentin 75.2% of the water is in the tubules and 24.8% is in the mineralized

matrix [18].

http://anatafizziology.files.wordpress.com/2012/03/tooth.jpg

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1.3.2.2 Dentin Structure

The structural integrity of dentin is provided by the inorganic and organic fraction of its

constituents which is crucial to retain the function of restored teeth. The inorganic component of

dentin is mainly carbonated nanocrystalline apatite minerals [19]. These minerals are closely

associated with the collagen scaffold either intrafibrillar or extrafibrillar [19]. The apatite crystals

are 5 nm in thickness. They have a needle like morphology near the pulp and a plate like

morphology towards the dentin-enamel junction [19]. The organic component is mainly type I

collagen which composes up to 90% of the organic fraction [20]. Type I collagen is 100nm in

diameter and exists as fibrils in dentin [21]. The collagen fibrils are oriented in a plane

perpendicular to the plane of dentin formation or dentinal tubules [22].

The water content of dentin exists as free or bound water. The free water is found in dentinal

tubules and other porosities in dentin [22]. This water can be lost by heating at 1000C and 85%

of water is lost in the first 30 min of dehydration [17, 23]. Bound water is associated with

inorganic apatite crystals and the organic phase and requires a much higher temperature to be

removed (6000C) [17].

The water forms a mono-layer of its molecules on the surface of hydroxyapatite via hydrogen

bonds, as well as weak van der Waals forces [25]. Bound water also forms an integral part of the

matrix and stabilizes the triple helix architecture of collagen molecules. Each tripeptide is known

to contain two water molecules [26]. As the number of water molecules increases per collagen

molecule, it swells laterally. In addition, water also acts as a plasticizer [26]. Dehydration of

collagen leads to increased stiffness mainly due to the formation of additional interpeptide

hydrogen bonds that were previously inhibited by the hydrogen bonding with water [25]. The

7

overall diameter of the collagen fibrils reduces as the interfibrillar spaces shrink and additional

bonds are formed [25].

The presence of dentinal tubules is the most characteristic structural feature of dentin (Figure.2)

[27]. Dentin is transversed by dentinal tubules that run continuously from the dentino enamel

junction towards the pulp in the root. The tubules are surrounded by highly mineralized

peritubular dentin and embedded in an intratubular matrix [12]. Dentin, as a result of its tubular

structure, is very porous [28]. The tubular framework of dentin may contribute to the physical

properties of dentin, as the fluid filled dentinal tubules may function to hydraulically transfer and

dissipate the occlusal forces that are applied to the tooth [6]. The alignment of the dentinal

tubules is also known to govern the mechanical properties of dentin depending on the direction

of the force applied [22].

Figure 2. Intertubular and Peritubular structure in dentin

Peritubular dentin is a highly mineralized aspect of dentin, consisting mainly of hydroxyapatite

and less organic matrix when compared to intertubular dentin [17, 23]. It is harder than

intertubular dentin and may provide structural support for the intertubular dentin [17].

Peritubular

Intertubular

Scott taylor DDS

8

1.3.2.3 Mechanical Properties of Dentin

The mechanical properties of dentin depend on the optimum balance between toughness and

stiffness [29]. The mechanical properties of dentin such as young’s modulus, tensile and

compressive strength and fracture toughness are the result of the complex interactions of its

constituents as well as the microstructural arrangement [29]. The collagen fraction of dentin

contributes to its toughness and ultimate tensile strength, while the mineral fraction contributes

to its elastic modulus and compressive strength [30, 31].

Dentin elasticity varies between peritubular and intertubular dentin and is also affected by

location within the tooth [32-37]. Studies have shown that peritubular dentin has a modulus of

elasticity of 29.8 GPa, whereas intertubular dentin have a modulus of elasticity of 17.7 GPa close

to the pulp and 21.1 GPa close to root surface [32,33]. The average modulus elasticity of bulk

dentin is considered to be in the range of 16.5 to 18.5 GPa [34, 35]. Palamara et al found the

lowest value for elastic modulus of around 10.4 GPa by using optical imaging [36].

Microindentation measurements have shown that the modulus of elasticity is significantly larger

in dentin with the orientation of the dentinal tubules running parallel to the direction of force

than in those running perpendicular to the direction of force [37].

Craig and Peyton reported a proportional limit and ultimate compressive strength of 167MPa and

297MPa respectively in dentin [34]. Stanford et al showed no significant differences in

compressive strength between the coronal dentin of different teeth. The orientation of the coronal

dentin was also found to be not affecting the compressive properties [38]. However, they

reported a lower value for compressive strength of root dentin [38]. There were no significant

differences in the compressive properties of the root dentin from vital and pulpless teeth [38].

9

The tensile strength of dentin was found to be 41.4MPa, which is lower than its compressive

strength [39]. Sano, Ciucchi Mathews and Pashley found that the ultimate tensile strength of

dentin was 104MPa, which is much larger than previous reports [40]. Lertchirakarn et al reported

that ultimate tensile strength was lowest (36.7 MPa) when the tensile force was parallel to the

tubule orientation and greatest at 90 degree to tubule orientation (60.3 MPa) [41]. Huang et al.

reported that the ultimate tensile strength of wet root dentin of both vital and root canal treated

teeth was not significantly different [42].

Dentin fracture, as a result of occlusal load is a result of microcrack initiation and propagation

with subsequent macrocrack growth [6]. Mastication and parafunctional activity produce cyclic

stresses that promote fatigue crack propagation. Fracture initiates from a defect and is due to a

localization of high stress concentration [31, 43]. Dentin possesses inherent toughness which aids

in resisting fracture [31]. This is due to the orientation of the collagen fibrils to the

hydroxyapatite that counter the directional effect of the dentinal tubules [31]. The dentinal

tubules may act as a weak interface, thereby exhibiting a crack stopping behavior [31].

Propagation of the fracture would require sufficient energy to re-initiate the fracture process [44].

Toughness, being the energy required to induce fracture, has been shown to be significantly

reduced by dehydration [6]. Dehydrated dentine demonstrates a lack of plastic flow [6]. This

suggests that the presence of fluid in dentin increases the energy that is required to induce

fracture [6, 45]. Therefore, dentinal fluid provides biomechanical integrity to the tooth and the

fluid-filled tubules that may function to hydraulically transfer and relieve the stresses that are

applied to the tooth [27, 45].

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1.3.3 Fracture in Endodontically Treated Teeth

Endodontically treated teeth are weakened due to decreased or altered tooth structure [31]. These

changes are attributed to caries, endodontic access and instrumentation, placement of previous

restorations, decreased moisture and fracture or trauma [31]. A typical fracture in endodontically

treated teeth is vertical root fracture (VRF). VRF is defined as a complete or incomplete fracture

initiated in the root at any level, which is usually directed buccolingually. [4, 20, 26, 46-55]. The

prevalence of VRF in root-filled teeth has been reported over a wide range depending on the

method of evaluation (2-20%). The studies that identified VRF using radiographs reported a

lower percentage of prevalence (2-5%) [56, 57] in contrast to the studies that used extracted root

filled teeth to determine VRF (11-20%) [58, 59]. Studies have shown that VRF most commonly

occurred within the maxillary second premolar (27%) and the mesial roots of the mandibular

molar (24%) [4]. Vertical root fracture occurred more often in women (52%) than in men (47%)

and were more common in individuals between the ages of 41 and 50 [60].

G.V Black (1895) was the first to propose the hypothesis that increased fracture predilection of

root-filled teeth is due to brittleness resulting from the changes in biomechanical properties due

to loss of moisture [61]. This was later confirmed by Helfer et al., who reported that the moisture

content of dentin from root-filled teeth was about 9% less than their vital counterpart [62].

However, there are other studies that contradict this view [63]. Papa and Messer reported an

insignificant difference in the moisture content between root-filled teeth and vital teeth, and

emphasized the importance of conserving the bulk of dentin in maintaining the structural

integrity of root-filled teeth [63]. In another study, Jameson et al. reported that dehydration of

dentin at 20ºC (50% relative humidity) brought about a 30% loss of moisture, and this moisture

loss resulted in a significant decrease in the toughness and an increase in the stiffness of dentine

11

bars [23]. Kruzic et al. conducted a simulation study and found that dehydrated specimens

showed significantly lower crack-initiation toughness compared to the hydrated specimens [64].

Khaler et al. found that the work of fracture of hydrated specimens was significantly higher than

dehydrated specimens [6]. Kishen and Asundi used digital moiré interferometry to study the role

of free water on the mechanical deformation of structural dentin [65]. They tested fully hydrated

and dehydrated specimens, dehydrated at 20ºC for 72 hours [65]. They found a strain response

characteristic of a tough material in fully hydrated dentin, while dehydration resulted in a

response characteristic of a brittle material [65]. In a study by Kinney et al., resonant ultrasound

spectroscopy was used to calculate the isotropic elastic modulus of dry dentin as 28.1 GPa and

wet dentin as 25.1 GPa [66]. Kinney et al. conducted nanoindentation based experiments as well

and suggested that dry dentin exhibited an elastic modulus of 23.9 GPa, while wet dentin

exhibited an elastic modulus of 20 GPa [67]. Several studies have also highlighted time-

dependent properties or viscoelastic behaviour in dentin [36].

During Root Canal treatment, the infected pulp tissue is removed, and the root canal is

disinfected and dehydrated before obturation [8-10]. Various factors have been found as

causative in the development of a VRF [31, 68]. These factors can be broadly classified into

iatrogenic and non-iatrogenic factors [31, 68]. Iatrogenic factors are the factors which are within

the control of clinicians during the treatment procedure [31, 68]. The two most common

iatrogenic risk factors are dentin structural loss and changes in the constituent of dentin [31].

Dentin structural loss can occur during access cavity preparation and instrumentation [31]. The

composition of dentin can be altered due to the usage of endodontic chemicals and medicaments

on dentin during the treatment procedure [31]. In this project, the effect of iatrogenic dentin loss

(structural changes) and endodontic chemicals (compositional changes) on the mechanical

integrity of dentin will be analyzed.

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1.3.4 Effect of Endodontic Chemicals on Dentin Structure and

Composition

Endodontic irrigation flushes dentinal debris and reduces the number of canal bacteria [69-71]. It

also disinfects and penetrates into dentin and its tubules [69-71]. These solutions should offer a

long term antibacterial effect and be nonantigenic, nontoxic and noncarcinogenic [69-71]. None

of the irrigants available in the market has all of these properties. Therefore, at least two irrigants

are usually used to reduce organic and inorganic matter and debris from the dentin [69]. The two

most widely used irrigants are sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid

(EDTA). There are other irrigants on the market, including chlorohexidine, MTAD and QMix.

Chlorohexidine (CHX) is used in endodontics as both irrigant and intracanal medicament [72]. A

few studies have evaluated its properties (antimicrobial activity and biocompatibility) with the

purpose of it being an alternative to sodium hypochlorite [72, 73]. However it has little to no

tissue dissolving capacity, it is unable to kill all bacteria and cannot remove the smear layer. It

should therefore only be used after irrigation with NaOCl [73-75].

MTAD irrigation solution contains, Tetracycline, acid and detergent [76]. The citric acid and

tetracycline remove the smear layer and the detergent helps in penetrating the irrigation solution

into the tubules [76]. In addition it has sustained antibacterial activity, is biocompatible and

enhances bond strength [76]. Its effectiveness to completely remove the smear layer is enhanced

when a low concentration of NaOCl is used as an intracanal irrigant [76].

QMix contains EDTA, CHX and detergent and it is recommended to be used at the end of

instrumentation after NaOCl irrigation [77, 78]. According to Grossman et al, low surface

13

tension is one of the ideal characteristics of this irrigant [79]. This is because it may help in

better penetration of the irrigating solution into the dentinal tubules [80].

Further details about NaOCl and EDTA will be discussed below.

1.3.4.1 Sodium Hypochlorite (NaOCl)

The use of NaOCl solution in endodontics as the main irrigant was recommended by Coolidge in

1919 [81]. Walker (1936) later on introduced the use of 5% NaOCl solution as a root canal

irrigant [82]. NaOCl is an efficient organic solvent that causes dentin degeneration because of

the dissolution of collagen by the breakdown of the bonds between carbon atoms and

disorganization of the protein’s primary structure [72, 83, 84]. Free chlorine in NaOCl dissolves

vital and necrotic tissue by breaking down proteins into amino acids [72, 83, 84]. It has been

used in dilutions ranging from 0.5% to 5.25% [72]. Decreasing the concentration of the solution

reduces its toxicity, antibacterial effect and ability to dissolve tissues [72]. Increasing its volume

or warming it increases its effectiveness as a root canal irrigant [72].

NaOCl solutions may affect mechanical dentin properties via the degradation of organic dentin

components [85]. Marending et al., evaluated the effects of NaOCl on the structural, chemical

and mechanical properties of human root dentin [85]. They found that NaOCl caused a

concentration-dependent reduction of elastic modulus and flexural strength in human root dentin

[85]. Mountouris et al., evaluated the deproteination of 5% NaOCl solution on the composition

and morphology of the smear layer covered and acid-etched human dentin surfaces [86]. They

found that NaOCl treatment reduced the organic matrix (amide I, II, III peaks) but did not affect

carbonates and phosphate [86]. Di Renzo et al. evaluated chemical alterations on the dentin

surface after treatment with NaOCl using Fourier transform infrared spectroscopy (FTIR) [87].

14

They found that NaOCl treated dentin samples demonstrated a slow and heterogeneous removal

of their organic phase, leaving calcium hydroxyapatite and carbonate apatite unchanged [87].

1.3.4.2 Ethylenediaminetetraacetic acid (EDTA)

EDTA was first introduced to endodontics by Nygaard-Østby (1957), who recommended the use

of a 15% EDTA solution (pH 7.3) with the following composition: disodium salt of EDTA (17

g); distilled water (100mL) and 5M sodium hydroxide (9.25 mL) [88]. EDTA is often suggested

as an irrigation solution because it can chelate and remove the mineralized portion of smear

layers. According to Nygaard-Østby, the main mineral components of dentin, phosphate and

calcium, are soluble in water [88]. When the disodium salt of EDTA is added, calcium ions are

removed from the solution [88]. This leads to the dissolution of further ions from dentin so that

the solubility product remains constant [88]. Thus, chelators cause decalcification of dentin.

EDTA alone cannot completely remove the smear layer. This is because smear layer contains

organic matter as well, and EDTA only affects the inorganic part of the layer [80]. For complete

removal of the smear layer, use of hypochlorite is necessary [89].

EDTA diffuses through the dentinal tubules and leads to a primary decalcification of the

peritubular and then intertubular dentin [90, 91]. After 2 hours, 150µm of the exposed dentin

surface will be completely decalcified and softened [90, 91]. Eri et al have shown that when root

dentin is exposed to 17% EDTA solution for 15 minutes, its microhardness significantly

decreases [92]. Dentin rods immersed in 17% EDTA for 2 hours lose approximately 30% of their

flexural strength and 50% of their modulus of elasticity [93]. However, in practice, a long term

infiltration of the pulp cavity with EDTA is not indicated. In a study by Marending et al. , it was

shown that there was no significant effect of a 3 minute application of 17% EDTA on the

modulus of elasticity or the flexural strength of dentin rods [94].

15

1.3.4.3 Combination

The combinations of EDTA and NaOCl have been shown to be an effective irrigation regime to

remove the organic and inorganic matter [89, 95]. But there is no clear consensus regarding the

ideal irrigation sequence, volume or application time in the literature. While NaOCl is used

during instrumentation, EDTA is preferably used at the end of instrumentation to complete the

removal of the smear layer. Grawehr et al. studied the interactions of EDTA with NaOCl [96].

They concluded that EDTA retains its calcium- complexing ability when mixed with NaOCl

[96]. However, EDTA causes NaOCl to lose its tissue dissolving capacity, and virtually no free

chlorine was detected in the combinations [96]. Clinically, this suggests that EDTA and NaOCl

should be used separately. In an alternating irrigating regimen, copious amounts of NaOCl

should be administered to wash out remnants of EDTA.

1.3.5 Effect of Iatrogenic Dentin Structural Loss on Mechanical Integrity

of Dentin

Changes in mechanical properties due to loss of tooth structure as a result of endodontic

procedures have been reported to influence the resistance of endodontically treated teeth to

fracture [1-4]. The main cause is considered to be changes in gross canal morphology, including

loss of dentin thickness, altered canal curvature, and altered canal cross-sectioned shape [97-

105]. It is likely that these factors interact with one another to influence the distribution of

stresses and tooth vulnerability to loading. This ultimately increases the possibility of a

catastrophic failure. In a study by Bender et al., it was shown that excessive enlargement of the

root canal may weaken the tooth and increase its susceptibility to vertical root fracture [97].

16

Using finite element analysis (FEA), it was found that the magnitude of generated radicular

stress was directly correlated with simulated canal diameters [98]. Wilcox et al. found that root

surface craze lines formed on roots that have a greater percentage of the canal wall removed

[99]. Hong et al., constructed FE models of the mandibular first molar with a diameter of root

canal modified to 1/4, 1/3 and 1/2 of that of the root [100]. They revealed that enlargement of the

root canal results in an increased concentration of stress on the root canal surface at the orifice

and coronal 1/3 [100]. In the lower part of the root canal, the stress distribution disparity

smoothened out with the decrease of stress on the root canal surface [100]. Chen et al., studied

the stress distribution with FEA models of normal wall thickness (12 mm) and roots with 75% ,

50% and 25%, respectively, of normal wall thickness [101]. They found that the enlargement of

root canal diameter increased the stress of the root canal wall up to 37% under lateral loading

[101]. Sathorn et al., built eight FE models indicating that the more dentin removed, the greater

the fracture susceptibility [102, 103]. These results concur with those reported by Wilcox et al.

[99]. However, reduced dentin thickness does not necessarily result in an increased fracture

susceptibility. Lertchirakarn et al, logically speculated that the reduction of the degree of the

curvature inside the root canal could reduce fracture susceptibility and that changing the canal

shape from oval to round actually relieves internal stresses despite the substantial thinning of

proximal dentin [104, 105].

1.3.6 Critique of Literature

Experiments should be designed to simulate clinical conditions to evaluate the impact of changes

in mechanical/ chemical properties [68]. Conventional mechanical tests tend to represent

properties that are averaged over a large volume of regional tissue [68]. Static compressive

loading is often used as it is simply applied through universal testing machines [68]. It allows

17

evaluation of the mechanical properties of a material including stiffness and strength [98-101]. In

comparison fatigue or cyclic mechanical test require much more time and effort, but are able to

characterize the mechanical performance of materials over time [98-101]

Modeling efforts, aimed at understanding stress distribution patterns in the tooth, have utilized

many simplifying assumptions including: 1) not considering the effect of periodontal ligament

and bone, 2) constant elastic moduli, and 3) simplified geometry [68]. The effect of storage

media during the mechanical test have also not been considered in previous studies, where

samples were only hydrated by irrigating with water during testing and not fully hydrated.

Finally, no studies have done both numerical and experimental analyses to look at the role of

iatrogenic dentin structural loss on the fracture of endodontically treated teeth. The goal of this

study is to examine the effect of compositional changes on fracture resistance of dentin and

combine the experimental and numerical models to understand the effect of iatrogenic dentin

structural loss on the mechanical integrity of dentin by providing more sensitive and accurate

information using both numerical and experimental models.

18

Chapter 2

2 Effect of Chemicals on ultrastructure, composition and

mechanical properties of dentin

2.1 Introduction

Irrigation has a major role in endodontic treatment. The primary role of irrigation solutions is to

facilitate the removal of microorganisms, tissue remnants and the smear layer from the root canal

space during and after instrumentation. However, none of the current irrigation solutions alone

can be used to achieve all the requirements of irrigation. Therefore, irrigants are currently used in

combination to achieve the desired outcome. Hence, the precise mechanism of interaction of

irrigants (individually/ in combination) with root dentin and their effect on ultrastructure and

mechanical properties needs to be further investigated. The objective of this study is to examine

the effect of different irrigation protocols on the ultrastructure, composition and mechanical

properties of dentin.

19

2.2 Materials and Methods

2.2.1 Sample Selection

Ethics approval was attained from University of Toronto and seventy extracted mandibular

premolar teeth with one root canal were selected for this study. Debris and soft tissue remnants

on the roots were removed with a sharp scalpel. The tooth specimens were transilluminated and

examined under a stereomicroscope to exclude those specimens with cracks or craze line. The

teeth were stored in deionized water at 40C after the root surface was cleaned until use.

2.2.2 Sample Preparation

The crowns were removed at the cement-enamel junction using a low speed diamond disk under

water-cooling. For ultrastructure and chemical composition analysis, each root was sectioned

longitudinally in the buccol- lingual plane and separated into two halves (seventy sections). Root

sections were prepared by grinding through a series of SiO2 papers (320-, 800-, and 1400- grit

emery papers) under running water. Subsequently the specimens were polished with water-based

diamond paste to 0.25µm under distilled water to remove any surface irregularities. All the

sections were ultrasonicated in water for 30 minutes to clean the surface. Sections were divided

into 5 groups (n= 14) and treated with (1) water for 30min, (2) 5.25% NaOCl for 30min, (3) 17%

EDTA pH 7.0 for 15min, (4) 5.25% NaOCl for 10min followed by 17% EDTA pH 7.0 for 2 min

as a final rinse, and (5) 5.25% NaOCl for 10min followed by 17% EDTA pH 7.0 for 1 min and

5.25% NaOCl for 1min as a final rinse. The chemical composition of ten specimens in each

group was determined by attentuated total reflectance fourier transform infrared spectroscopy

(ATR-FTIR) analysis. The ultrastructure analysis was conducted for the remaining four

specimens using scanning electron microscopy (SEM). For mechanical testing premolar teeth

20

were cut into cubic shape blocks with a low speed diamond disk under water-cooling. The

average size of the blocks was 2×2×2 mm3. All the samples were ultrasonicated in water for 30

minutes to clean the surface. Fifty dentin blocks were then divided into 5 groups (n=10) and

treated with (1) water for 10 min, (2) 5.25% NaOCl for 10 min, (3) 17%EDTA pH 7.0 for 2 min,

(4) 5.25% NaOCl for 10 min followed by 17% EDTA pH 7.0 for 2 min as a final rinse, and (5)

5.25% NaOCl for 10 min followed by 17% EDTA pH 7.0 for 1 min and 5.25% NaOCl for 1 min.

Each tooth block was placed in a 24 well plate containing 2 mL of the above solutions on shaker.

The sections were rinsed with distilled water between and after treatments.

2.2.3 Ultrastructure Analysis

The ultrastructure changes on the root dentin subsequent to treatment with different irrigation

protocols were examined with SEM. The samples were dehydrated through a graded series of

ethanol to remove water from the samples. To remove ethanol from the sample, the samples

were dried chemically with hexamethyldisilizane (HMDS) and air dried in room temperature

under fume hood. The samples were then mounted in the SEM sample holding stub and sputter

coated with a layer of gold-palladium. Each sample was observed with a scanning microscope

operating at 15kV at a magnification of 1K and 5K (Hitachi S-2500, Sapporo, Japan). The area

to be analyzed was selected at low magnification at which dentinal tubule openings could not be

seen. The magnification was then increased to 5K.

2.2.4 Compositional Analysis

For compositional analysis, the samples were dehydrated within a vacuum desiccator for 24

hours. In the ATR-FTIR spectra (Shimadzu, Kyoto, Japan), the specimens were placed on the

diamond crystal of the ATR chamber. They were adjusted so that the pointed tip would be just

21

pressed onto the root canal wall. Three different spots on the surface of each specimen were

randomly chosen on the canal wall for this analysis. The spectra were collected in the range from

600 to 4,000/cm-1

at 4/cm-1

resolution using 28 scans. The ATR-FTIR system was calibrated

before conducting the analysis. The effect of NaOCl and EDTA on collagen depletion and

apatite depletion were evaluated using the collagen and apatite ratio (the ratio of absorbance of

amide I peak to phosphate v3 peak) [86, 87].

2.2.5 Mechanical Analysis

The samples were compressively loaded in a universal testing machine at a rate of 0.1 mm/min

under water (Instron, Canton, MA). Test was stopped as soon as a drop of load appeared on the

force –displacement curve. The force-displacement curve was used to generate a stress-strain

curve. The normal stress on the plane perpendicular to the longitudinal axis of the samples was

obtained by dividing the force over the original cross section area. The normal strains in the

longitudinal direction were obtained by dividing the displacement values over original height.

From the stress-strain curve, the compressive strength and toughness of dentin samples were

calculated. Compressive strength is the maximum stress the material can withstand under

compressive loading. Toughness is the total energy absorbed by a structure before it fractures,

and is determined by calculating the area under the stress-strain curve.

2.2.6 Statistical Analysis

The compressive strength and toughness between different groups were analyzed by One-way ANOVA

and Tukey post-hoc test (p<0.05). All statistical analysis was performed to a 95% level of

confidence (α= 0.05).

22

2.3 Results

2.3.1 Ultrastructural Analysis

Examination of the surface of root canal walls exposed to different irrigation protocols showed

differences in dentin surface structure. The use of both irrigation sequences (EDTA as a final

irrigation or NaOCl as a final irrigation) resulted in complete removal of the smear layer (Figure.

3 c, d) respectively. Root sections irrigated first with NaOCl followed by a final rinse with

EDTA had a smooth intertubular dentin surface ( Figure.3c). The dentinal tubule orifices were

regular and open in this case. However, erosion of peritubular and intertubular dentin was

detected when EDTA was used as an initial rinse followed by NaOCl as a final rinse (Figure.3

d). This erosion was extensive in some regions with merging of adjacent tubules.

Figure 3. SEM images of root canal surface after irrigation with (a) 5.25% NaOCl for 30

min, (b) 17% EDTA pH 7.0 for 15min, (c) 5.25% NaOCl for 10min followed by 17% EDTA

pH 7.0 for 2 min as a final rinse, (d) 5.25% NaOCl for 10min followed by 17% EDTA pH

7.0 for 1 min followed by 5.25% NaOCl for 1 min as a final rinse and (e) water for 30 min

a) b) c) d) e)

23

2.3.2 Compositional Analysis

In group 2 (NaOCl 30min), the amide/phosphate ratio was compared with the untreated dentin

(water 30min). For groups 4 and 5, the amide/phosphate ratios were compared with both

untreated dentin and 17% EDTA. It can be observed from Figure.4 that the amide I/phosphate

ratio of the EDTA treated dentin (control for NaOCl/EDTA treated dentin of group 4 and 5) was

higher than the amide I/ phosphate ratio of untreated dentin (control for NaOCl treated dentin for

group 2).

The amide I /phosphate ratio of 5.25% NaOCl treated for 30min was lower than that of untreated

dentin. This observation suggests that most of the organic fractions are depleted. The amide

I/phosphate ratios of both groups 4 and 5 were lower than the corresponding value of the 17%

EDTA control. However when compared with untreated dentin, it can be observed that the amide

I /phosphate ratio for both groups 4 and 5 was higher. When groups 4 and 5 were compared, it

was observed that the amide/phosphate ratio of group 5 was lower than that of group 4. This

observation indicated that most of the organic fraction is depleted after the final rinse with

NaOCl.

24

Figure 4. Amide/phosphate ratio on dentin surface treated with (1) water for 30min, (2)

5.25% NaOCl for 30min, (3) 17%EDTA pH 7.0 for 15min, (4) 5.25% NaOCl for 10min

followed by 17% EDTA pH 7.0 for 2 min as a final rinse, and (5) 5.25% NaOCl for 10min

followed by 17% EDTA pH 7.0 for 1 min followed by 5.25% NaOCl for 1 min as a final

rinse

2.3.3 Mechanical Analysis

In this study the effect of different irrigation solutions on the compressive strength and toughness

of dentin was evaluated. It was observed that the compressive strength of the NaOCl treated

group was 15.47% lower than the untreated group, Figure.5a. EDTA treatment produced a

decrease of 55.77% in compressive strength compared to the untreated group, Figure.5a. This

reduction in compressive strength can also be seen between groups 4 and 5. Figure.5b, showed

that toughness of NaOCl treated dentin was 74% lower than controls. The toughness of group 5

was 31.95% lower than that of group 4.

25

Figure 5. The compressive strength and b) toughness of dentin samples treated with (1)

water for 10min, (2) 5.25% NaOCl for 10min, (3) 17%EDTA pH 7.0 for 2min, (4) 5.25%

NaOCl for 10min followed by 7% EDTA pH 7.0 for 2 min as a final rinse, and (5) 5.25%

NaOCl for 10 min followed by 7% EDTA pH 7.0 for 1 min followed by 5.25% NaOCl for 1

min as final rinse

a)

b)

*

**

*

26

2.4 Summary

The purpose of this study was to examine the effect of chemicals used in root canal treatment on

the ultrastructure, chemical composition and mechanical properties of root dentin. Dentin

sections were divided into five groups and were treated with (1) 5.25%NaOCl, (2) 17%EDTA,

(3) 5.25%NaOCl followed by 17%EDTA, (4) 5.25%NaOCl followed by 17%EDTA and a final

irrigation with 5.25%NaOCl, and (5) water. The ultrastructure of dentin was analyzed by

Scanning Electron Microscopy, the chemical composition (amide/phosphate ratio) was

determined qualitatively using ATR-FTIR and the mechanical properties (toughness and

compressive strength) were measured using compressive testing. The SEM analysis showed that

the irrigation sequences in groups 3 and 4 resulted in complete removal of smear layer, whereas

in group 4, erosion of peritubular and intertubular dentin was observed. The amide/phosphate

ratio determined by the ATR-FTIR analysis for group 4 was lower than that of group 3.The

group treated with EDTA alone showed a reduction in the compressive strength. The group

treated with NaOCl as a final irrigation showed reduction in the toughness properties when

compared to the untreated control. The findings of this study highlight that the application of

different chemical protocols lead to distinct changes in the dentin ultrastructure, amide/phosphate

ratio and mechanical properties of dentin.

27

Chapter 3

3 Effect of Dentin Loss on Fracture Resistance of Root

Dentin

3.1 Introduction

Endodontically treated teeth show more susceptibility to vertical root fractures [4, 111-113].

Different iatrogenic and non-iatrogenic risk factors that remove substantial amounts of root

dentin or produce micro-defects in root dentin have been suggested to be risk factors that

increase susceptibility of vertical root fractures in root-filled teeth [30]. Previous static and cyclic

mechanical testing has emphasized the significance of preserving root dentin to retain the

mechanical integrity of root-filled teeth. However, the role of iatrogenic dentin removal on the

respone of dentin to mechanical loading is not well understood [114-116]. The goal of this study

was to understand the effect of three different degrees of root dentin removal on the resistance to

fracture of endodontically treated teeth.

28


3.2.1 Sample Selection

Ethics approval was attained from University of Toronto and forty non-carious, extracted

(orthodontic reason) human premolar of adult patient (20-40 years of age) with mature root,

single canals and straight roots were selected for this study. The tooth specimens were

transilluminated and examined under a stereomicroscope to exclude any specimens with cracks

or craze lines. The presence of single root canals and angle measurements were verified

radiographically [117]. These specimens were stored in deionized water at 40

C until use.

3.2.2 Sample Preparation and Groups

All the teeth were mounted in a custom made device and µCT imaged (pre-treatment scan)

(18µm voxel size, 100kV, 100uA; 1172 High resolution µCT, SkyScan, Belgium). The

specimens were divided randomly into 4 groups based on the degree of dentin removed using

three instrumentation protocols. Following was performed for all the groups except for Control

group. Access cavities were prepared using diamond burs under water-cooling as per

conventional guideline [118]. All canals were negotiated with size 10 K-type files (Lexicon:

Dentsply Tulsa Dental Specialist, Germany). The working length was measured from the pre-

treatment scan at a reference point 0.5mm short of the portal of exit, and confirmed

radiographically. A glide path was established with a size 15 K-type file and each canal

instrumented to the working length with instruments as indicated below.

In Group-1 (Low), the canals were enlarged up to ISO K- type file # 20 to simulate a low amount

of dentin removed. In Group-2 (Medium), the canals were enlarged up to ISO size 20 and Gates

Glidden drills # 1 to 2 (Lexicon: Dentsply Tulsa Dental Specialist, Switzerland) were then used

29

at 600 rpm to enlarge the coronal third of the root canal. This step was followed by apical

enlargement up to K-type file # 35 to simulate a medium amount of dentin removal. In Group-3

(Extreme), the coronal third of the canals were further enlarged up to Gates Glidden drill # 4 and

apical enlargement up to K-type file # 50 to simulate an extreme amount of dentin removal. In

Group-4 (Control) the root canals were uninstrumented. During the instrumentation procedures,

the root canals were irrigated with distilled water using a ProRinse side-vented30 G needle

(Dentsply Tulsa Dental Specialties, Tulsa, OK) at standardized intervals and the canals were

dried with paper points.

3.2.3 Determination of Dentin Volume and Moment of Inertia

Micro-CT scanning was repeated (as above) on the root canal instrumented teeth (post-treatment

scan). Manual volumetric segmentation of the pre and post treatment images were used to

determine the amount of dentin removed (Amira 5.2.2, Visage Imaging, San Diego, CA, USA).

Amira software was used to calculate the cross-sectional area and radius of gyration of 100 slices

in the middle region of the post instrumentation root dentin in order to determine the moment of

inertia using the following equation.

k = √I/A I= k2A

k= radius of gyration I= moment of Inertia A= Cross-sectional area

Moment of inertia is a geometric property of a structure that measures the distribution of material

about a given axis, representing the ability to resist bending or torsion.

3.2.4 Thermal and Mechanical Cyclic Testing

Thermal and mechanical cycling was performed to simulate aging and mastication of root dentin

during function. Prior to the thermal and mechanical cycling, the crowns of the teeth were

30

sectioned off under water-cooling with a diamond disk at the cemento-enamal junction (CEJ).

Teeth were embedded in cylindrical mold of polymethylmethacrylate (Palapress Vario, Heraeus-

Kulzer, Germany) with a 200 μm thick layer of polyether material (Impregum, 3M Espe,

Seefeld, Germany) surrounding the root surfaces to mimic the periodontal ligament. The CEJ

was positioned approximately 1.5mm above the level of mold to simulate the bone crest [119].

All the teeth were then loaded/aged under thermal and mechanical cycling. Thermal cycling

consisted of 6000 cycles x 5°/55°; each cycle was 2 minutes (TCML, Chewing Simulator, EGO,

Regensburg, Germany) [125-128]. The specimens were also subjected to simultaneous

mechanical cycling of 1.2x106

cycles of 50N at frequency of 1.6Hz to simulate 5 years of clinical

function ( TCML, Chewing Simulator, EGO, Regensburg, Germany) [125-128]. These values

are based on masticatory loads, speed of mandibular movements and rate of chewing as reported

in the literature [125, 126]. All specimens were kept hydrated in deionized water throughout the

experiments.

3.2.5 Determination of the Load to Fracture

The load to fracture was determined in samples subjected to thermal and mechanical cycling.

Twenty eight samples (n=7 from each group 1-4), were subjected to compressive loading to

failure (Zwick 1446, Ulm, Germany) using a cross head speed of 1 mm/min, within a custom

stainless steel loading fixture. Vertical load was applied via a cylindrical tip (radius=4mm)

centered over the occlusal aspect of the tooth. The specimens were loaded till the load dropped

suddenly observed in load-displacement curve.

31

3.2.6 Micro-Crack Analysis

Scanning electron microscopy (Quanta FEG 400, FEI Company, Oregon, USA) was used to

determine the presence of micro-cracks in root dentin for the remaining 3 specimens per group,

previously subjected to mechanical/thermal cycling. Cross-sectional specimens (3 mm thick)

were prepared under water-cooling with a diamond disk from the root specimens at apical,

middle and coronal levels. Only microcracks that initiated from the root canal wall were

analyzed as representative of vertical root fractures.

3.2.7 Statistical Analysis

The mean dentin volume removed and load to fracture was compared between the Low,

Medium, and Extreme groups using a one way ANOVA and post-hoc Tukey testing. All

statistical analyses was performed to a 95% level of confidence (α= 0.05).

32

3.3 Results

3.3.1 Determination of Dentin Volume Removed

Based on the µCT segmentations, the volume of dentin removed was lowest in the Low group

and highest in the Extreme group (2.63 ± 0.24% to 7.34 ± 0.69%, p<0.05, Figure.6). The dentin

volume removed in the Medium group showed a wide variation ranging from 1.05% to 12.36 %.

This dentin volume removed in the medium group did not exhibit a statistically significant

difference when compared to the Extreme or Low groups.

Figure 6. The percentage reduction in the dentin volume simulated with different level of

dentin removal mean±SE (n=10)

33

3.3.2 Determination of Load to Fracture

The load to fracture was significantly lower in the Extreme group as compared to the Low and

the Control groups (p<0.05). There were no statistically significant differences in the load to

fracture for the Medium group as compared to the other groups (p>0.05).

Figure 7. Load to fracture, values are reported as mean± SE (n=7, * statistically significant

with control, ** statistically significant with Low)

3.3.3 Load to Fracture and Remaining Dentin Volume Analysis

The load to fracture and the remaining dentin volume after instrumentation were compared

between different groups, Figure.8. A linear relationship was seen between the load to fracture

and remaining dentin volume in the Low group in which increase in remaining dentin volume led

to an increase in load to fracture. For the Medium and Extreme groups there was no correlation

between the remaining dentin volume and load to fracture.

**

*

34

Figure 8. Correlation between remaining dentin volume and load to fracture

3.3.4 Load to Fracture and Moment of Inertia Analysis

The correlation of the moment of inertia with load to fracture was calculated for all the samples.

It was observed that there was a linear relationship (R2= 0.52) between the load to fracture and

moment of inertia. This indicated that by decreasing the load to fracture, the moment of inertia of

root dentin decreased, leading to increase flexure of root dentin during loading.

35

Figure 9 Correlation between load to fracture and moment of inertia

3.3.5 Micro-crack Analysis

The fractographic analysis showed that many cracks were initiated from the root canal wall in

the Extreme group. These microcracks in the root dentin of the Extreme groups were mostly

observed in the apical and cervical aspects of the root dentin (Figure.10). However, no cracks

were observed in other groups which initiated from the root canal surface.

36

Figure 10. Microcrack analysis of samples after mechanical/thermal cycling load based on

different degree of dentin removal

Apical Middle Coronal

Co

ntro

l Low

Med

ium

Ex

treme

37

3.4 Summary

This study aimed to understand the mechanism by which iatrogenic root dentin structural loss

would influence resistance to vertical root fracture in teeth. Extracted premolar teeth were

instrumented to simulate three degrees of dentin removal (Low, Medium, and Extreme). Micro-

CT analysis was performed to quantitatively determine the amount of dentin removed and

moment of inertia of apical root dentin in order to correlate it with the remaining dentin volume.

Experimental studies were also conducted to evaluate the influence of dentin removal and

remaining dentin volume on the resistance to fracture and microcrack formation of root dentin.

This study highlighted that the changes in dentin volume and fracture resistance after root canal

preparation were dependent on the remaining dentin volume rather than the amount of dentin

removed or instrumentation technique used. Also higher dentin removal of the root canal resulted

in greater number of microcracks and root fractures

38

Chapter 4

4 Effect of Structural Loss on Stress Distribution Pattern

in Dentin

4.1 Introduction

Some of the key factors that predispose root-filled teeth to fracture are iatrogenic or induced by

clinical procedures [31]. Although clinicians have realized this issue by empirical knowledge, the

exact mechanism by which these risk factors influence the biomechanical response of teeth is

still not well understood. Previous studies have indicated that tooth structural loss would alter the

nature of the functional stress distribution within the root dentin, which would eventually

increase the propensity of vertical root fractures in endodontically treated teeth [97- 105].

Finite Element (FE) Analysis has been used widely to study the stress/strain responses of the

dental structures to various functional loads. These investigations have demonstrated that dentin

loss will increase fracture susceptibility in endodontically treated teeth [102, 103]. However,

previous numerical modeling studies examining the stress-strain distribution patterns in teeth

used many simplifying assumptions including: no consideration of the effect of the periodontal

ligaments and bones, or the complex geometry of teeth. The objective of this study was to

analyze the effect of dentin structural loss on the stress distribution pattern on root dentin using a

Micro Computed Tomography (µ-CT) based specimen specific FE analysis.

39


4.2.1 Sample Preparation and Micro-CT Imaging

Ethics approval was attained from University of Toronto and an extracted human premolar tooth

with a single and straight root canal was used. The tooth was positioned in a custom-made mount

and µCT imaged intact. Root canal instrumentation was performed sequentially and µCT images

were acquired at each stage to simulate three different degrees of dentin removal on a single

tooth (Control, Low, Medium and Extreme group, as described in chapter 3).

4.2.2 Segmentation of Tooth and Generation of FEA models

For each µ-CT scan, intensity based manual image segmentation of the tooth and supporting

structures (the periodontal ligament and bone) was performed on a slice by slice basis (Amira

5.2.2). The surfaces were saved as a STL file and then were imported into ICEM CFD 14.5 and

4-noded tetrahedral meshes generated (Ansys Inc. Southpointe, Canonsburg, PA, USA). Four

root meshes were generated representing different levels of dentin removal, Control, Low,

Medium and Extreme.

4.2.3 Finite Element Analysis

The meshes were imported into ABAQUS 6.12 (Providence, RI, USA) where material properties

and boundary condition were applied and the FE analysis run. All materials were considered to

be homogenous, isotropic and linearly elastic [129-132]. The mechanical properties of the

dentin, periodontal ligament (PDL) and bone used in the model are given in Table.1. The

interfaces between the components were treated as perfectly bonded interfaces [129-136]. The

base of the model was constrained to a zero displacement boundary (restraining all forms of

40

translational movements). A load of 100 N was applied on the coronal aspect of the root as

described in earlier studies [136, 138, 139]. The resulting Von Mises stresses were determined at

the cervical, middle and apical portions of the root.

Table 1. Material Properties

Materials Elastic Modulus Poisson’s ratio

Dentin [133] 18.6 GPa 0.31

Bone [132, 137] 14.5 GPa 0.323

PDL [130] 0.0689 GPa 0.45

41

4.3 Results

4.3.1 Stress Distribution

With low removal of dentin, the highest stress was located at the cervical region (Figure 11 – top

row). The stress distribution was transferred into middle region of the root as more dentin was

removed (Figure 11 – middle and bottom rows). In all of the groups, stress was distributed

predominantly around the canal wall and circumferentially in the mesio-distal direction.

However, with increase in loss of dentin volume from the root canal, the stress distribution

pattern became more conspicuously in the bucco-lingual direction.

Figure 11. Von Mises Stress Distribution on root dentin (B: Buccal, L: Lingual, M:

Mesial and D: Distal)

M B

L D

42

4.4 Summary

This study aimed to understand the mechanism by which iatrogenic root dentin structural loss

influences the stress distribution in teeth. Finite element analysis was carried out with simulation

of three degrees of dentin removal to examine the influence of dentin removal on stress

distribution of root dentin. This study showed that the Von Mises stress was predominantly

distributed circumferentially around the coronal region. However with increasing dentin

removal, there were more noticeable signs of root flexure, which resulted in the stress

distribution along the buccal-lingual plane.

43

Chapter 5

5 Discussion

Understanding the interaction of endodontic irrigant with dentin would aid in maintaining a

stable hard tissue environment in endodontically treated teeth for long term function. Previous

studies have highlighted deleterious effect caused by different irrigants on root dentin [85, 140-

146]. NaOCl is the most commonly used endodontic antimicrobial irrgiant which has the ability

to dissolve organic materials [72, 83, 84]. EDTA is a chelating agent that is used to remove

inorganic fractions of smear layer from the dentin [89].

Baumgartner & Mader reported that when EDTA and NaOCl solutions were alternatively

applied to uninstrumented root canal wall, dentin showed an eroded appearance, and tubular

orifice diameters were enlarged [89]. In our study, from the structural analysis with SEM, it was

observed that when NaOCl was used before EDTA, the hydroxyapatite seemed to have protected

the collagen fibers from the dissolving action of NaOCl. However, when NaOCl followed

EDTA, NaOCl directly attached to the collagen which had already been exposed by

demineralization. A previous study monitored dentin mineralization dynamically with

synchrotron radiation computed tomography. It was suggested that dentin demineralization

occurred in two stages. Each stage was governed by a unique rate [147]. About 70- 75% of the

mineral was removed rapidly, while the remaining mineral etched at a significantly slower rate.

Hence, dentin erosion occurred markedly when NaOCl followed EDTA [147].

Previous ATR-FTIR analysis showed that in intact human dentin the amide I, II and III bands of

the spectrum are directly related to the molecular conformation of the polypeptide chains of

44

intact type I collagen [78, 148]. The amide I (C=O stretching vibration at 1600-1700 cm -1

) in

particular, is a sensitive marker for the collagen component of dentin [78, 148]. The phosphate

vibration between 900 and 1200 cm -1

is typically assigned to the apatite-related band [78, 148].

Mountouris et al. evaluated the deproteination potential of 5% aqueous NaOCl solution applied

with a rubbing action on the molecular composition and morphology of smear-layer covered and

acid-etched human coronal dentin surfaces [86]. They found that in both groups, NaOCl

treatment reduced the organic matrix (amide I, II, III peaks), but did not affect carbonates and

phosphates [86]. Di Renzo et al. evaluated chemical alterations on the dentin surface after the

treatment with NaOCl using a photoacoustic FTIRS (PA-FTIRS) technique [87]. Results showed

that NaOCl-treated dentin samples demonstrated a slow and heterogeneous removal of their

organic phase, leaving calcium hydroxyapatite and carbonate apatite unchanged [87]. In our

study it was observed that the organic fraction is mostly depleted after the final rinse with

NaOCl. When EDTA alone is used for irrigation, the organic matrix of dentin is the limiting

factor on the dissolution of dentin, because it accumulates on the canal surface, preventing

further dissolution [84]. Therefore, additional irrigation with NaOCl may facilitate further

exposure of the inorganic material through removal of the organic matrix [149-151] and hence

increase the demineralizing effect.

Changes in the organic and inorganic components of dentin will also affect the mechanical

properties of dentin. It is shown that the mineral component in hard connective tissues

contributes to strength and elastic modulus, whereas the collagen component is responsible for

toughness of the tissues [30, 31].

In this study, the main cause of the decrease in compressive strength of the samples treated only

with NaOCl might be due to the deproteination of dentin which involves the dissolution of

45

collagen, leaving behind the hydroxyapatite crystals which are very brittle [145]. However, this

was not statistically significant. In a study by Sim et al, a 2 hour exposure of dentin to NaOCl

solutions significantly decreased the elastic modulus and flexural strength of human dentin

compared to a control which was physiological saline [145]. Marending et al. evaluated the

effects of NaOCl on the structural, chemical and mechanical properties of human root dentin

[85]. They found that NaOCl caused a concentration-dependent reduction of elastic modulus and

flexural strength in human root dentin [85].

The reduction in compressive strength of dentin treated with EDTA, is due to the fact that EDTA

demineralizes the inorganic components of dentin by chelating calcium ions, which reduces the

compressive strength of dentin [88, 90, 91]. This can be due to the irrigation with 2 minute

EDTA as a final irrigation. EDTA reacted with calcium ions in the hydroxyapatite crystals and

resulted in changes in the microstructure of dentin and changes in the calcium/phosphorus ratio

[88]. Calcium and phosphorous present in the hydroxyapatite crystals are the main inorganic

elements of dentin [88]. Therefore, due to mineral loss, the compressive strength may decrease.

The reduction in toughness is likely due to generation of a brittle layer of apatite crystallites that

are not supported by the collagen matrix. Destruction of the collagen matrix in mineralized

tissues results in a less tough, more brittle substrate that might precipitate fatigue crack

propagation during cyclic loading [64].

Micro-CT is a non-destructive technique that allowed evaluation of the tooth in series of cross-

sectional slices, which are later reconstructed to determine various parameters such as root canal

morphology, volume of dentin removed and remaining dentin thickness [118-121, 152-154].

Ikram et al, showed that the largest loss of dentin hard tissue was caused by caries removal

(~8%), while the root canal preparation did not result in a significant dentin hard tissue loss (1%)

46

[152]. Elnaghy et al, also showed that there is no significant difference among the tested groups

instrumented with ProTaper Next instruments with and without Glide Path regarding the volume

of removed dentin and centering ratio [153]. In the current study, the micro-CT based analysis

showed that the amount of root dentin removed during instrumentation differed only between the

Low and Extreme instrumentation groups. There was no significant difference between the

amounts of dentin removed between the Medium and the Low/Extreme groups. This finding

suggested that the amount of dentin removed during root canal instrumentation was not only

influenced by the instrumentation protocol, but also depended on the initial root canal geometry

and remaining dentin volume [118, 120, 121, 154, 155].

The resistance to fracture of root-filled teeth is a particular concern, because the mechanical

integrity of the remaining tooth structure may be compromised by different pathological and

iatrogenic reasons [31]. In this study, the teeth were loaded without filling the endodontic access

cavities to avoid the confounding effects of different bonded filling materials. In this manner, the

effect of dentin structural loss on the mechanical integrity of remaining root dentin could be

directly assessed. In the current study, the teeth were subjected to cyclic loading under fully

hydrated conditions to simulate functional chewing forces in an oral environment. The cyclic

loading of 1,200,000 cycles utilized in this study simulated about 5 years of clinical functioning

[125-128, 156]. The findings from the cyclic loading experiments followed by static loading

demonstrated that there is a significant difference in the loads to fracture of Extreme group as

compared to Low and Control groups. However, there is no significant difference between the

load to fracture for Medium group compared to other groups.

Correlation between the remaining dentin volume and load to fracture showed that even though

remaining dentin volume was associated with fracture resistance for low group, the lack of

47

correlation in the Medium and Extreme case, in spite of a significant difference in dentin

removal in Extreme group, suggested that measurement of dentin removal should not be

considered the only alternate outcome for the fracture resistance of teeth. Hence, other factors

such canal geometry, canal volume and the remaining dentin volume can affect the fracture

resistance of tooth to loading. Correlating the moment of inertia with load to fracture shows that

the role of dentin removal on fracture resistance, not only depends on the remaining dentin

volume but also on the distribution of materials around the canal wall.

Vertical root fracture is defined as longitudinally oriented fracture of the tooth that originates

from the apical region of the root and propagates towards the coronal aspect of the root [46, 47,

157]. Though they originated in proximity to the root canal lumen, they may be complete or

incomplete in nature. They are generally found in the bucco-lingual direction of the root [46, 47,

157]. Therefore only the cracks that initiated from the root canal were recorded using SEM. The

micro-crack analyses of root dentin samples with different degrees of dentin removal highlighted

that higher dentin removal from the apical and cervical region of the root canal resulted in

greater number of microcracks and root fractures [48, 158].

Teeth in the oral cavity serve as a mechanical device for mastication of food [159]. The intact

natural teeth are understood to experience flexing or bending stress when biting forces act on

them [31, 159]. Since dentin forms the major bulk of the tooth structure, examining the nature of

stress distribution within an intact tooth structure will aid in understanding how natural tooth

structure resists mechanical forces during function. The pattern of stress distribution on the root

canal dentin is critical in initiating and propagating cracks, which would lead to vertical root

fractures [68]. The current numerical analysis and micro-crack experiments demonstrated that

functional stresses were predominantly distributed circumferentially at the cervical dentin.

48

However with increasing root dentin removal, the functional stresses were distributed more

apically and along the bucco-lingual plane. The increased stress distribution in the apical

direction and in the bucco-lingual direction can be attributed to the increasing root flexure with

cervical dentin removal. Previous clinical studies on vertical root fractures have suggested these

types of fractures to propagate from the apical portions of the tooth root to coronal portions in

the bucco-lingual direction [160-163].

In summary, the susceptibility of teeth to fracture needs to be taken into consideration when

endodontic treatment protocols are established. Iatrogenic risk factors that increase the

susceptibility of root-filled teeth to fracture are important to consider since it is under the control

of the clinicians to identify those factors. Two of the risk factors examined in this study, (1)

effect of irrigation solution on structure/composition of dentin and (2) effect of iatrogenic dentin

structural loss on mechanical integrity of dentin, are very relevant factors in endodontics.

Findings from this study highlight the effect of each of these factors individually on the

mechanical integrity of dentin.

49

Chapter 6

6 Conclusion

6.1 Conclusion

Changes in constituent of dentin produced ultra-structural features characteristic of dentin

surface erosion, reduction in amide/phosphate ratio and reduction in toughness of dentin

Changes in the dentin volume and fracture resistance after root canal preparation were

dependent on the remaining dentin volume and the distribution of material

Increasing dentin removal resulted in stress distribution more apically and in the bucco-

lingual plane of root dentin

50

6.2 Future studies

The influence of interaction of two iatrogenic risk factors on fracture resistance of root dentin

need to be analyzed experimentally

A non-invasive techniques need to be performed in order to analyze the cracks formation

after the mechanical/thermal cycling test

Numerical analysis can be improved by taking into consideration the elastic modulus

gradient, the multi rooted tooth, the whole tooth including crown, and also the presence of

dentinal tubules and hydrostatic pressure within them

51

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