Nanoparticle Guided Dentin Micro-tissue Engineering:

Characterizing Fluid Dynamics for Delivery and

Tissue Mechanical Response

by

Fang-Chi Li

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Faculty of Dentistry

University of Toronto

© Copyright by Fang-Chi Li (2018)

ii

Nanoparticle Guided Dentin Micro-tissue Engineering:

Characterizing Fluid Dynamics for Delivery and

Tissue Mechanical Response

Fang-Chi Li

Doctor of Philosophy

Faculty of Dentistry

University of Toronto

2018

Abstract

Disease process and iatrogenic procedures compromised the mechanical integrity of remaining

dentin, which in turn increased the susceptibility of endodontically treated teeth to vertical root

fracture. Cracks and fracture of endodontically treated teeth diminished the treatment predictability.

Micro-tissue engineering principles are applied to design tissues with improved mechanical and

biological characteristics. Chitosan nanoparticles (CSnp) are bioactive biopolymers, when applied

to dentin displayed antimicrobial and enhanced toughness characteristics. Crosslinking CSnp with

dentin collagen provides additional stabilization towards efficient mechanical load-transfer and

resistance to matrix degradation. However, effective transport of nanoparticles into root canal

remains to be a challenge owing to the complex canal morphology and non-uniform stresses

generated with the syringe-based delivery method. The objectives of this study were: (1) to

characterize the stability of the nanoparticle dispersion and surface ultra-structure of micro-tissue

engineered dentin, (2) to evaluate the fluid-dynamics and delivery efficacy associated with a novel

iii

strategy based on activated microbubbles to deliver nanoparticles into root canal, and (3) to

examine the effect of micro-tissue engineering with crosslinked-CSnp on the mechanical

characteristics of root dentin.

CSnp dispersion of 1mg/ml concentration was selected based on the nanoparticle aggregation

kinetics. This concentration sustained the stability with optimal charge density and dispersity.

Crosslinking CSnp on dentin resulted in a denser/homogeneous coating with altered hardness and

elastic modulus on dentin surfaces. Ultrasonically activated microbubbles induced fluid-dynamics

with high velocity and inertial stress through intensified cavitational bubble dynamics but formed

a coating of CSnp mixed with dentin smear layer on canal wall. Manually agitated microbubbles

generated uniformly high viscous stress with increased particle flux facilitating homogeneous

coating of CSnp on root canal dentin. Micro-tissue engineered root canal dentin with crosslinked-

CSnp and water-soluble chitosan resulted in decreased strain distribution enhancing the stability

of root under physiologically loads. There was an increase in the sustained load at fracture in

specimens treated with photodynamically-crosslinked-CSnp. The findings from this study

emphasized the advantage of manually activated microbubbles to deliver CSnp in root canal and

the impact of micro-tissue engineering with crosslinking CSnp on dentin to enhance the

mechanical characteristics of root. These outcomes have potential application in clinical practice.

iv

But seek first His kingdom and His righteousness, and all these things

will be added to you

Matthew 6:33

v

Acknowledgement

Throughout the process of completing my doctoral thesis, I am full of thanksgiving to everyone

who graciously supports me in all these years. I am with full gratitude towards the persons who

sustain and support me through these years. First and foremost, I am grateful to my God, Who is

the source, Creator, and Savior in my life. He led me to the doctoral program four years ago and

has faithfully supplied me, met my every need, and even granted me His wisdom and inspiration

when I encounter challenging situations. Thank You for Your sovereignty in my every move. May

You preserve me in Your eternal plan.

I am most grateful to my supervisor, Dr. Anil Kishen, who took me on as his student even though

I was a clinician with very little research experience. I thank him for seeing the potential in me and

his tremendous patience as he trained me to be a careful and thoughtful scholar. His enthusiasm

and erudition in both science and education have inspired me to devote myself to learning and

teaching and strive for excellence in research. He is my pattern, mentor, and trusting friend. This

study cannot be completed without his guidance and support. Again, I would like to thank him for

what he has done for me. I would also like to thank my advisory committee, Dr. Arun

Ramchandran, for the valuable feedback, expertise, generous support from his lab and contribution;

as well as Dr. Omar El-Mowafy for his supports and critiques in this project.

Academic and emotional support from members of Dr. Kishen’s lab -- Annie Shrestha, Suja

Shrestha, Arzou Ossareh, Hebatullah Hussein and Anam Hashmi is gratefully acknowledged.

Thanks also to Suraj Borkar, Huai Xi Wang, Ilya Gourevich, Jiang Wang, and Eric Nicholson for

their help in data collections and their professional experiences in experiments.

Finally, I would like to give thanks to my family and friends. My mother Shirley Wu is the most

precious gift that God has given me. Since my childhood, she always encouraged me to read

continuously, study thoroughly, and love the Lord whole-heartedly. I thank her for raising and

shepherding me with her unconditional love, strictness, and unceasing prayers. I will always

remember her, her love, and her words throughout my life. I am also thankful to my father Steve

Lee, who always sustain the whole family and never compromised our educations. His talents,

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perseverance and wisdom in career motivate me to choose and put efforts constantly into the field

which I am passionate about, without hesitation. My dear aunt, Sally Wu, is my foster parent who

supports me to pursue further advancement in my career. Her comprehensive supports sustain me

throughout years. I would not be able to study peacefully without her support behind me. I am also

thankful to my dearest friends Claire Tsai and Grace Liu for their friendship and unceasingly

prayers, in good times and tough times.

Lastly, the generous supports from University of Toronto during my study are greatly appreciated:

Ontario Graduate Scholarship (OGS) Award, Harron Scholarship Award, Dr. Barry Korzen

Scholarship, Student Research Group (SRG) Travel Award, and School of Graduate Studies (SGS)

Conference Grant.

Fang-Chi Li (Alice)

vii

Table of Contents

Acknowledgements ......................................................................................................................... v

Table of Contents .......................................................................................................................... vii

Abbreviations ................................................................................................................................. xii

List of Tables ................................................................................................................................. xiii

List of Figures ................................................................................................................................ xiv

PREFACE .............................................................................................................................................xvii

Dissertation format .......................................................................................................................xvii

Publications ...................................................................................................................................xvii

Scholarship/ Awards .................................................................................................................... xviii

CHAPTER 1 ............................................................................................................................................ 1

INTRODUCTION .................................................................................................................................... 1

1.1 Background .................................................................................................................................. 2

1.2 Hypothesis and Objective ............................................................................................................ 5

1.2.1 Objectives .............................................................................................................................. 5

1.3 Literature Review ........................................................................................................................ 7

1.3.1 Human Teeth: Mechanical considerations and iatrogenic consideration .......................... 7

1.3.1.1 Composition of dentin ....................................................................................................... 7

1.3.1.2 Biomechanical response of dentin .................................................................................. 12

1.3.1.2.1 Biomechanical response in intact teeth .................................................................... 12

1.3.1.2.2 Biomechanical response in root filled teeth ............................................................. 14

1.3.1.3 Mechanism of fracture resistance in dentin ................................................................... 15

1.3.1.3.1 Toughening Mechanisms in dentin ........................................................................... 18

1.3.1.4 Effect of endodontic treatment on dentin ..................................................................... 21

1.3.1.5 Current methods to strengthen dentin in endodontically treated teeth ...................... 23

1.3.1.6 Summary ........................................................................................................................... 24

1.3.2 Fluid Dynamics in Root Canal System ................................................................................ 25

viii

1.3.2.1 Methods to activate irrigants in endodontics ................................................................ 26

1.3.2.2 Experimental methods to study fluid dynamics in root canals ...................................... 30

1.3.2.3 Role of microbubbles to enhance fluid dynamics in root canal .................................... 32

1.3.2.4 Summary ........................................................................................................................... 34

1.3.3 Nanoparticle Guided Micro-Tissue Engineering ................................................................ 35

1.3.3.1 Chitosan nanoparticle ...................................................................................................... 35

1.3.3.2 Crosslinking of dentin collagen ........................................................................................ 38

1.3.3.3 Summary ........................................................................................................................... 40

1.4 References ................................................................................................................................. 41

CHAPTER 2 .......................................................................................................................................... 60

OPTIMIZING THE FORMULATION OF BIOPOLYMERIC NANOPARTICLE VEHICLE ............................ 60

2.1 Abstract ...................................................................................................................................... 61

2.2 Introduction ............................................................................................................................... 62

2.3 Materials and Methods ............................................................................................................. 64

2.3.1 Stabilization of concentration of CSnp solution ................................................................ 64

2.3.2 Characterization of dentin surface conditioned with optimized formulations of

CSnp .............................................................................................................................................. 65

2.3.2.1 FESEM evaluation ............................................................................................................. 67

2.3.2.2 Nanoindentation .............................................................................................................. 67

2.4 Results ........................................................................................................................................ 68

2.4.1 Stabilization of concentration of CSnp solution ................................................................ 68

2.4.2 Characterization of dentin surface conditioned with optimized formulations of

CSnp .............................................................................................................................................. 72

2.4.2.1 FESEM evaluation ............................................................................................................. 72

2.4.2.2 Nanoindentation .............................................................................................................. 72

2.5 Discussion .................................................................................................................................. 73

2.6 Acknowledgement .................................................................................................................... 77

2.7 References ................................................................................................................................. 77

ix

CHAPTER 3 .......................................................................................................................................... 82

CHARACTERIZING FLUID DYNAMIC PARAMETERS WITH ACTIVATED MICROBUBBLES FOR ROOT

CANAL DENTIN COATING WITH NANOPARTICLES ............................................................................ 82

3.1 Abstract ...................................................................................................................................... 83

3.2 Introduction ............................................................................................................................... 84

3.3 Materials and Methods ............................................................................................................. 85

3.3.1 Characterization of fluid dynamics in simulated root canal model .................................. 85

3.3.2 Assessing nanoparticle delivery and nanoparticle-based coating in tooth model .......... 87

3.4 Results ........................................................................................................................................ 88

3.4.1 Characterization of fluid dynamics in simulated root canal model .................................. 88

3.4.2 Assessing nanoparticle delivery and nanoparticle-based coating in tooth model .......... 89

3.5 Discussion .................................................................................................................................. 92

3.6 Acknowledgement .................................................................................................................... 95

3.7 References ................................................................................................................................. 95

CHAPTER 4 .......................................................................................................................................... 99

MICRO-TISSUE ENGINEERING ROOT CANAL DENTIN WITH CHEMICALLY CROSSLINKED-CHITOSAN

NANOPARTICLES FOR MECHANICAL STABILIZATION ....................................................................... 99

4.1 Abstract .................................................................................................................................... 100

4.2 Introduction ............................................................................................................................. 101

4.3 Materials and Methods ........................................................................................................... 102

4.3.1 Sample preparation ........................................................................................................... 102

4.3.2 Digital moiré interferometry (DMI) analysis .................................................................... 103

4.4 Results ...................................................................................................................................... 106

4.5 Discussion ................................................................................................................................ 111

4.6 Conclusion ............................................................................................................................... 114

4.7 Acknowledgement .................................................................................................................. 114

4.8 References ............................................................................................................................... 115

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CHAPTER 5 ........................................................................................................................................ 119

CHARACTERIZING THE MECHANICAL CHARACTERISTICS OF MICRO-TISSUE ENGINEERED ROOT

DENTIN WITH PHOTODYNAMICALLY ACTIVATED CROSSLINKED-CHITOSAN

NANOPARTICLES ............................................................................................................................... 119

5.1 Abstract .................................................................................................................................... 120

5.2 Introduction ............................................................................................................................. 121

5.3 Materials and Methods ........................................................................................................... 123

5.3.1 Part I: Assessment of biomechanical behavior of micro-tissue engineered root

dentin (with DMI) ....................................................................................................................... 123

5.3.2 Part II: Assessment of fatigue resistance of micro-tissue engineered root

dentin (with cyclic fatigue testing) ............................................................................................ 124

5.4 Results ...................................................................................................................................... 126

5.4.1 Part I: Assessment of biomechanical behavior of micro-tissue engineered root

dentin .......................................................................................................................................... 126

5.4.2 Part II: Assessment of fatigue resistance of micro-tissue engineered root

dentin .......................................................................................................................................... 129

5.5 Discussion ................................................................................................................................ 131

5.6 Acknowledgement .................................................................................................................. 138

5.7 References ............................................................................................................................... 138

CHAPTER 6 ........................................................................................................................................ 144

DISCUSSION AND CONCLUSION ...................................................................................................... 144

6.1 General Discussion .................................................................................................................. 145

6.2 Future Studies ......................................................................................................................... 151

6.3 Conclusion ............................................................................................................................... 152

6.4 References ............................................................................................................................... 154

xi

APPENDIX I ........................................................................................................................................ 158

DECIPHERING DENTIN TISSUE BIOMECHANICS USING DIGITAL MOIRÉ INTERFEROMETRY:

A NARRATIVE REVIEW

APPENDIX II ....................................................................................................................................... 178

SUPPLLEMENTARY DATA

xii

Abbreviations

VRF Vertical root fracture

NPs Nanoparticles

CSnp Chitosan nanoparticles

ECM Extracellular matrix

NCP Non-collagenous protein

PGs Proteoglycans

GAG Glycosaminoglycans

MMP Metalloproteinase

NaOCl Sodium hypochlorite

EDTA Ethylenediaminetetraacetic acid

PIV Particle imaging velocimetry

CFD Computational fluid dynamics

GA Glutaraldehyde

EDC 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide

GPn Genipin

RB Rose bengal

PDA Photodynamically activated

CMCS Water soluble carboxy-methyl chitosan/ water soluble chitosan derivatives

CSRBnp Chitosan-conjugated-rose bengal-nanoparticles

DLS Dynamic light scattering

FESEM Field emission scanning electron microscopy

GP Gutta-percha

WM Water-manual

WS Water-sonic

WU Water-ultrasonic

MM MB-manual

MS MB-sonic

MU MB-ultrasonic

DMI Digital moiré interferometry

xiii

List of Tables

Table 2.1 The characteristics of dispersion at each concentration and the peak analysis resulted

from figure 2.3 .......................................................................................................................... 70

Table 2.2 The hardness and elastic modulus resulted before/after treatments of CSnp, EDC-

crosslinked-CSnp and PDA-crosslinked-CSnp .......................................................................... 72

Table 2.3 Approximate values for zeta potential and dispersity parameters ................................. 75

Table 5.1 Experimental design and mean ( SD) of the sustained load (N) and numbers of cycles at

failure ...................................................................................................................................... 129

Table 5.2 Survival rates (probability that the specimens exceeded the respective load or numbers

of cycles without failure (standard deviation)) for the experimental groups (control, EDC-

and PDA-crosslinked-CSnp) ................................................................................................... 131

xiv

List of Figures

Figure 1.1 The role of different constituents on the mechanical integrity of structural dentin..... 10

Figure 1.2 Increasing complexity of the organizational hierarchy of collagen type I ...................... 11

Figure 1.3 Schematic diagrams showing the nature of stress distributions in (A) a column and (B)

an intact tooth .......................................................................................................................... 13

Figure 1.4 Schematic diagram showing a model of natural bio-composites. (A) staggered mineral

crystals embedded in protein matrix and (B) when it is loaded with tension forces ........... 17

Figure 1.5 Schematic diagrams showing the different fracture toughening mechanisms operating

in dentin .................................................................................................................................... 20

Figure 1.6 (A) The molecular structure of chitin and chitosan and (B) the synthesis of chitosan

nanoparticles (CSnp) ................................................................................................................ 37

Figure 2.1 Primary particle and aggregated particles in the dispersion .......................................... 64

Figure 2.2 The measurement of averaged hydrodynamic diameter (Dh) of chitosan nanoparticles

(CSnp) in four concentrations .................................................................................................. 69

Figure 2.3 The hydrodynamic distribution of CSnp dispersion in four different concentrations .. 69

Figure 2.4 The ultrastructure of dentin surfaces, (A) control, (B) treated with CSnp and (C, D)

crosslinked-CSnp ...................................................................................................................... 71

Figure 2.5 The load-displacement curves resulted from each treatment (CSnp, EDC-crosslinked-

CSnp and PDA-crosslinked-CSnp) by nanoindentation .......................................................... 72

Figure 3.1 Schematics of microfluidic experiment set-up (A) and the dimensions of simulated root

canal model (B) ......................................................................................................................... 87

Figure 3.2 The means and standard deviations of (A) velocity, (B) stress and (C) the particle

penetration depth in water/ or microbubbles (MBs) combined manual, sonic and ultrasonic

agitations .................................................................................................................................. 90

Figure 3.3 The SEM images of sectioned root canal dentin model showing the CSnp coating on root

canal dentin in MBs groups: (A) control, (B) manual, (C) sonic and (D) ultrasonic agitation and

the efficacy of the nanoparticle-delivery on dentin (E, F) ...................................................... 91

Figure 4.1 Steps of specimen preparation ....................................................................................... 105

xv

Figure 4.2 The digital moiré interferometry (DMI) experimental setup ........................................ 105

Figure 4.3 Moiré fringe patterns in root dentin. (A) U field at 10N load, (B) V field at 10N load, (C)

U field at 50N load, (D) V field at 50N load ........................................................................... 107

Figure 4.4 Strain values in U and V field generated at the coronal (A) and apical third (B) of root

before (E10) and after (E50) root canal enlargement .......................................................... 108

Figure 4.5 Typical fringe patterns in root dentin model. (A-1, 2, 3) Fringe patterns in U-field at 10,

30 and 50N load in root dentin before micro-tissue engineering with crosslinked-CSnp. (A-4)

Color map of displacement field before micro-tissue engineering with crosslinked-CSnp. (B-

1, 2, 3) Fringe patterns in U field at 10, 30, and 50N load in micro-tissue engineered root

dentin. (B-4) Color map of displacement field after engineering the root dentin with CSnp

.................................................................................................................................................. 109

Figure 4.6 Strain values in U and V field generated from coronal (A) and apical third (B) of root

before and after micro-tissue engineering with crosslinked-CSnp on root dentin surface ......

.................................................................................................................................................. 110

Figure 5.1 Typical fringe patterns in root dentin model. (A-1, 2, 3) Fringe patterns in U-field at 10,

20 and 40N load in root dentin before micro-tissue engineering with PDA-crosslinked-CSnp.

(A-4) Color map of displacement field before micro-tissue engineering. (B-1, 2, 3) Fringe

patterns in U field at 10, 20, and 40N load in PDA-crosslinked-CSnp engineered root dentin.

(B-4) Color map of displacement field after engineering the root dentin with PDA-

crosslinked-CSnp .................................................................................................................... 127

Figure 5.2 Strain values in U and V field generated from coronal (A) and apical third (B) of root

before and after micro-tissue engineering with PDA-crosslinked-CSnp on root dentin surface

.................................................................................................................................................. 128

Figure 5.3 Survival curves according to the steps of loads and numbers of cycles for each failed

tooth ........................................................................................................................................ 130

Figure 5.4 Mean and standard deviation of the sustained load (in N) and numbers of cycles at

failure in each group (control, EDC- and PDA-crosslinked-CSnp) ........................................ 130

Figure 5.5 The mechanisms of EDC- and PDA- crosslinking of collagen molecules....................... 137

xvi

Figure 5.6 The mechanism of collagen crosslinking with the incorporation of water soluble chitosan

derivatives (CMCS) ................................................................................................................. 137

Figure 6.1 (A) The schematics illustrate the push-pull motion of the insert in manual agitation and

the fluid flow. (B) The interaction between CSnp, MBs and root canal dentin during manual

agitation ................................................................................................................................. 147

Figure 6.2 Schematics of micro-tissue engineered dentin collagen demonstrating the crosslinking

within collagen molecules and collagen fibrils, as well as integrating with CSnp and CMCS

................................................................................................................................................. 153

xvii

Preface

Dissertation format

This dissertation is the thesis of a compilation of the research works conducted in University of

Toronto from 2014 to 2018. Some of the chapters have been either published or under reviewed in

peer-reviewed indexed journals. Chapter 1 presents a general introduction including the

background of this research and a detailed literature review of the topics, which related to the

research problem. Chapters 2 to 5 are a compilation of the experimental data including published/

or submitted publications. They are presented in their published form, or with minor changes for

improving readability and including more details. Chapter 6 was prepared as a general discussion

of all the experimental data obtained in the current study. Written permission for reproduction of

all publications has been obtained.

Publications reproduced as dissertation chapters

1. F-C Li, S Borkar, A Ramachandran, A Kishen. Characterizing fluid-dynamic parameters

with activated microbubbles for root canal dentin coating with nanoparticles (Under

review).

2. F-C Li, A Kishen. Micro-tissue engineering root canal dentine with crosslinked

biopolymeric nanoparticles for mechanical stabilization. International Endodontic Journal.

March 2018.

3. F-C Li, A Kishen. Deciphering dentin tissue biomechanics using digital moiré

interferometry: A narrative review. Optics and Lasers in Engineering. 2018;107:273-80.

4. F-C Li, E Nicholson, C Singh, A Kishen. Micro-tissue engineering root canal dentin with

photodynamically crosslinked biopolymeric nanoparticles for enhancing the mechanical

characteristics of root dentin (will be submitted).

Additional publications

1. N Huynh, F-C Li, S Friedman, A Kishen. Biomechanical effects of bonding pericervical

dentin in maxillary premolars. Journal of Endodontics. 2018;44:659-64.

xviii

2. H Lim, F-C Li, S Friedman, A Kishen. Residual microstrain in root dentin after canal

instrumentation measured with digital moire interferometry. Journal of Endodontics.

2016;42:1397-402.

3. F-C Li, A Kishen. Digital moiré interferometric analysis on the effect of nanoparticle

conditioning on the mechanical deformation in dentin. Proc. of SPIE. 2016;969203-1-8.

Scholarship/ Awards

2018 Highest-scoring resident applicant in the Spring 2018 research grant Cycle,

American Association of Endodontics - Foundation for Endodontics

2018 Harron Travel Award, Faculty of Dentistry, University of Toronto

2016, 2018 SGS Conference Grant, University of Toronto

2017-2018 Barry H. Korzen Endodontic Award, Faculty of Dentistry, University of

Toronto

2017-2018 Harron Scholarship Award, Faculty of Dentistry, University of Toronto

2016-2017 Ontario Graduate Scholarship

2016 1st place oral presentation, SRG Travel Awards, Faculty of Dentistry,

University of Toronto

1

Chapter 1

Introduction

2

1.1 Background

Endodontic treatment is the treatment of choice to maintain the long-term functional requirements

of a natural tooth. It is the treatment of choice to conserve an infected tooth, which otherwise

would need an extraction. The goal of endodontic treatment involves disinfecting the infected root

canal system, besides preserving the mechanical integrity of the remaining natural tooth structure

(1-3). It has been reported that even when the highest standards of clinical procedures are followed,

microbial biofilms still persist within the anatomic complexities and uninstrumented portions of

the root canal system (4). Although microbiological factor that lead to the persistent of infection

has been a subject of focus for many years, the loss of mechanical integrity of endodontically

treated teeth has attracted much attention in the recent decades (5-7). Recent studies and clinical

observations have shown that root filled teeth have a higher propensity to vertical root fractures

(VRF) (8, 9). Clinical investigations have highlighted VRF in 6 to 11% of the extracted root filled

teeth (10-12).

Loss of dentin due to disease process or iatrogenic procedures has been suggested to be the primary

cause of diminished fracture resistance in endodontically treated teeth (6). In addition, different

risk factors have also been reported to increase the predisposition of endodontically treated teeth

to VRF. The loss of free water, chemical-based effect of irrigants/medicaments used during root

canal treatment, microdefects induced by instrumentation and root filling procedures,

bacterial/chemical mediated dentin matrix degradation have all been suggested to be risk factors

that increases the propensity of VRF in endodontically treated teeth (6, 7, 10, 13). Thus, developing

a minimally invasive treatment strategy with dual objectives of improving the antibacterial

efficacy within the root canal system, while enhancing the mechanical characteristics of remaining

dentin against VRF would enhance the predictability and improve root canal treatment efficiency.

The principles of micro-tissue engineering aim to design tissues of improved biological and

mechanical characteristics to support tissue function and host integration (14, 15). Nano sized

particles have been introduced in designing a tissue with improved and novel properties, which

revolutionized tissue engineering recently (15-17). In the current study, the above principle was

applied to incorporate bioactive/ antimicrobial nanoparticles to engineer root canal dentin with

3

enhanced mechanical stability for improved tissue function.

Nanoparticles – Biomechanical Consideration

Nanoparticles (NPs) are one of nanomaterials, which are defined as a natural, incidental or

manufactured material containing particles, in an unbound state or as an aggregate or as an

agglomerate and where, for 50 % or more of the particles in the number size distribution, one or

more external dimensions is in the size range 1 nm - 100 nm (18). Nanoparticles display high

surface areas, novel properties and functions. They can be manipulated on the atomic/molecular

scale because of their extremely small size (19, 20). Also, biopolymeric nanoparticles obtained

from natural sources are biocompatible and some of which present to serve as active targets as

nanocarriers (21). Owing to its extreme small size and the very unique properties from their bulk

counterparts, a wide range of applications such as drug/gene delivery, fluorescent labeling for

imaging, tumor destruction, tissue engineering, etc. have been explored with NPs (22). Previous

studies have shown that nanometric particles are capable of being delivered into the anatomic

complexities of the root canal system with the help of high-intensity focused ultrasound (HIFU)

(23).

Chitosan (poly (1, 4), -d glucopyranosamine) is the de-acetylated form of chitin, the second most

abundant natural biopolymer derived from the exoskeleton of crustaceans (24). Chitosan has been

reported to be suitable for the synthesis of nanoparticles and has attracted significant interest in

the field of biomedicine due to its non-toxicity, biocompatibility, antimicrobial properties and

biodegradability (25-28). It is a linear polyamine containing a large number of free amino and

hydroxyl groups that are readily available for crosslinking and has been used for numerous

chemically modified applications (29, 30). Chitosan also possesses structural similarity to the

extracellular matrix components (31), subsequently would mimic the functions of the extracellular

matrix proteoglycans and glycosaminoglycans, by providing mechanical stability and compressive

strength to collagen (32). Recent studies have shown that synthetic and natural chemicals that

increase the number of inter- and intra-molecular collagen crosslinks would enhance the fibrillar

resistance against bacterial enzymatic degradation and provide improved mechanical

characteristics of tissues (33-36).

4

Photodynamic therapy involves the use of photosensitizer and low-level light of appropriate

wavelength to produce singlet oxygen that could eliminate bacteria, viruses and spore (37, 38).

Alternatively, photodynamic therapy has been reported to induce rapid and stable covalent

crosslinking of collagen to stabilize collagen tissue with improved mechanical characteristics (39,

40). Along similar lines, for reinforcing the collagen matrix, crosslinked chitosan nanoparticles

(CSnp) have been used to achieve micro-scale tissue engineering of root dentin. This process of

micro-scale tissue engineering stabilizes the ultrastructure of surface dentin providing the tissue

enhanced mechanical characteristics as well as resistance to host/bacteria mediated enzymatic

degradation (41-44). In addition, CSnp and functionalized-CSnp have been shown to provide a

significant improvement in root canal disinfection by effectively eliminating the residual

adherent/nonadherent biofilms and inactivate bacterial endotoxins (45-47), as well as to enhance

the cell adherence/viability and neo-tissue ingrowth (47, 48), all of which will promote

homeostasis and facilitated post-treatment wound healing.

Delivery of nanoparticles in root canal system

The challenges of bolus delivery of nanoparticles into the root canal system are owing to the

anatomic complexities and the low/non-uniform stresses generated by the fluid flow from the

syringe based delivery. Furthermore, preventing aggregation of nanoparticles and providing

additional forces are required for the application of nanoparticles in root canal dentin (23, 49). The

small dimension of dentinal tubules, accessory canals and complicated root canal morphology

contributed to the challenges associated with the delivery of nanoparticles uniformly within the

root canal systems.

Pressure gradients created by activating the fluid may be used to deliver nanoparticles. However,

optimal stresses at the root canal wall are critical for the homogeneous application of NPs onto the

root canal walls (23). Previous studies have shown that a high-speed jet generated by the collapse

of cavitation bubble helped to deliver the particles toward the channel (23). Also inertial cavitation

produced by ultrasonic agitation during root canal treatment improved the penetration of irrigants

and nanoparticles into anatomic complexities (23). However, inertial cavitation could only be

generated close to the ultrasonically agitating tip when the ultrasonic file oscillated freely in the

canal space, which does not result in optimum forces on the canal wall for NPs delivery (50, 51).

5

Hence increasing the inertial cavitation bubbles may enhance the possibility of NP delivery within

the root canals. Microbubbles, commonly used as carriers for targeted drug delivery, are micro-

sized droplets composed of oxygen carrier and oxidizing agent produced in an emulsion form (37,

52, 53) It generates inertial bubbles when exposed to ultrasonic waves (54). These bubbles

coalesce together decreasing the threshold for bubbles production and increasing bubble dynamics

(55). When the inertial bubbles collapse, they result in high shear stresses from streaming and

shockwave, which facilitate NP delivery into the tissues via micro-jets (56, 57). The intensified

bubble dynamics might be a potential strategy to consider for delivering nanoparticles within root

canal dentin.

1.2 Hypothesis and Objective

It was hypothesized that micro-tissue engineering root dentin with optimized biopolymeric

nanoparticles using activated microbubble based method would enhance the mechanical

characteristics of root dentin.

1.2.1 Objectives

General objectives:

The objective of this study was to assess the ability of novel delivery strategies based on activated

microbubbles to deliver biopolymeric/bioactive nanoparticles within root canal system to enhance

mechanical characteristics of root dentin.

Specific objectives:

(1) Characterize the biopolymeric chitosan nanoparticles formulation for endodontic

application

(2) Study the physical parameters of fluid dynamics associated with manually, sonically,

ultrasonically activated microbubbles in a simulated root canal model

(3) Evaluate the efficacy of a novel microbubble based formulation to deliver chitosan

nanoparticles in root canal dentin using an extracted tooth model

6

(4) Examine the effect of micro-tissue engineering with crosslinked-chitosan nanoparticle on

the biomechanical response of root dentin to physiologically relevant loads

(5) Evaluate the mechanical property of root dentin with / without micro-tissue engineering

with crosslinked-chitosan nanoparticles under fatigue loads

7

1.3 Literature Review

1.3.1 Human Teeth: Mechanical considerations

The primary function of a tooth is chewing and masticating the food. It can be considered as a

mechanical device displaying a distinct manner of distributing the chewing forces. The unique

shape and structure of the root contributes to the load transfer and distribution to the surrounding

alveolar bone (58). Dentin is a natural, hydrated, mineralized hard tissue, which forms the major

bulk of the tooth. The compositions of dentin also provide the essential mechanical properties to

support the structure during functioning. Besides, the structural adaptations of dentin allow

uniform transfer of stress and minimize stress increase or stress concentrations within a tooth (59,

60). A tooth structure generally experiences flexing or bending forces during mastication. Most of

the stress is transferred to the cervical region of the tooth and reduces towards the root apex

resulting in higher compressive stresses in the root structure (60-62). The nature of stress

distribution in tooth is influenced by the coronal tooth structure, the direction of occlusal forces,

the bulk of cervical dentin, shape of the root and the relationship between root and surrounding

alveolar bone (6).

1.3.1.1 Composition of dentin

Dentin forms the bulk of the crown and root of the teeth. It comprises 70% of carbonated apatite

crystals, 20% of protein/collagen with 10% of water content by weight (63). The most prominent

feature of dentin is the dentinal tubules, which resulted from the deposition of dentin around the

odontoblast (dentin), the cells that form dentin matrix. The odontoblasts lie on the inner most

aspect at the dentin-pulp interface, with their long cellular processes termed odontoblastic

processes extending from these cells through the entire thickness of dentin. The lumens of the

dentinal tubules vary in diameter. It ranges from 0.5-0.9 m (dentin-enamel-junction: DEJ) to 2-4

m through the direction toward pulp. The density of the dentinal tubules increases from 20000 to

45000/mm2 from DEJ (outer) to pulp (inner) end (63, 64). In a recent study, it was shown the

diameters of tubules on root canal surface were 4.3-1.7 m from coronal to apical, and the

tubules/dentin surface ranged from 72-13% (65). The portion of the dentin that surrounds the

dentinal tubule/odontoblast process (0.4-0.74m) is termed peritubular dentin, which is 40%

8

higher mineralized dentin than the remaining dentin matrix. The intertubular dentin located

between the dentinal tubules, contains more than 50% organic phase in volume and provides the

elasticity to dentin tissue. Due to the spatial variation of tooth structure and composition, the

mechanical properties, for instance, the elastic modulus varies conspicuously. The elasticmodulus

of enamel is 40-80 GPa whilst it is around 30 GPa in peritubular dentin and 16-21 GPa in

intertubular dentin. It may be even lower (3-19 GPa) at regions in the innermost dentin (500m

from pulp) (66, 67).

The matured dentin constitutes of inorganic, organic and water fraction. Each of these components

supports the critical mechanical characteristics of dentin at the micro-scale (Fig. 1.1) (68). The

crystalline-carbonated hydroxyapatite with a needle- and/or plate-like morphology with size of

approximately 10 x 50 nm is the main constituent of the inorganic phase. These nano-metric scale

minerals are located both within the collagen fibrils (intra-fibrillarly mineralized) and between

collagen fibrils (inter-fibrillarly mineralized). The intra-fibrillar minerals are present inside the

periodically spaced gap zones in the collagen fibrils, whereas the inter-fibrillar minerals occupy

the interstices between the fibrils, which hold 70% of the inorganic fraction (6, 63).

Ninety percentage of the organic fraction in dentin is collagen, which is exclusively Type I

collagen. Minor amount of Type V collagen is also present in the dentin extracellular matrix

(ECM). Type I collagen is a strong, three-dimensional fibrous polymer existing in an aqueous

biological environment. The collagen molecule, also known as tropocollagen, consists of three

polypeptide -chains, and each of them coiled in a left-handed helix. The primary structure of the

polypeptide chain is composed by a regular arrangement of amino acid sequence repeating triplet

(Gly-Pro-X)n or (Gly-X-Hyp)n where X may be one of many amino acid residues. Three

polypeptide chains twist together into a right-handed triple helix which stabilized by hydrogen

bonds (69). Few collagen molecules with gap zones and overlap zones form microfibrils, which

further assemble to collagen fibrils (Fig. 1.2). Collagen fibrils are roughly 50-100nm (0.05-0.1μm)

in diameter and randomly oriented in plane perpendicular to the direction of dentin formation

(inter-tubular dentin). In dentin, the collagen fibers form a scaffold network and are densely filled

with minerals (63, 70). In addition to collagen, the dentin ECM contains multiple non-collagenous

proteins (NCPs), including different proteoglycans (PGs), glycoproteins, enzymes, serum proteins

9

and growth factors. Similar to collagen fibrils, NCPs are synthesized and secreted by odontoblasts.

The roles of NCPs are considered critical to dentinogenesis and responsible of initiating the

mineral deposition/nucleation within ECM, as well as collagen fibril mineralization (70). PGs are

composed of a core protein molecule to which glycosaminoglycans (GAGs) are covalently linked

as side chains, also can interact with various biological active molecules through the GAGs and

the domain of core proteins (70, 71). They play an important role in matrix formation/prevention

of premature mineralization. Enzymes of NCPs in human dentin ECM include metalloproteinases

(MMPs) and cathepsins. Human odontoblasts have been reported to synthesize gelatinases MMP-

2, -9, collagenase MMP-8 and MMP-20 in the tooth with completed formation (72). Degradation

of the dentin collagenous matrix, which is a prerequisite for cavity formation and a finding

associated with root canal infected/and treated dentin, has been attributed to MMP activity (73,

74). MMPs are synthesized as inactive pro-enzymes (or zymogens), and the activation requires the

cysteine-to-zinc switch to be opened by normal proteolytic removal of the pro-peptide domain or

ectopic perturbation of the cysteine-zinc interaction (75).

Water presents 10% of dentin volume. However, water level is varied in locations within dentin.

There are two types of water in dentin: tightly bound and free/ unbound water. Bound water is

associated with the apatite crystals of the inorganic fraction and the collagenous/ non-collagenous

matrix protein of the organic phase. A monolayer of water molecules can be absorbed to the surface

of hydroxyapatite by hydrogen bonds and additional water would be held by weak van der Waals

forces (6, 76). It has been demonstrated that the proteoglycan molecules contain a large amount of

bound water. Besides, two water molecules are incorporated into each tripeptide of the triple helix

of collagen structure. The free water mainly fills the dentinal tubules and other porosities in dentin.

It is related to the transport of inorganic ions such as calcium and phosphate, within the dentin

matrix. Each of these three fractions in the dentin composite supports the essential mechanical

characteristics of dentin. The major roles are listed in Figure 1.1 and the details would be described

in the next sessions.

10

Figure 1.1. The role of different constituents on the mechanical integrity of structural dentin (6). (With

permission from reference)

11

Figure 1.2. Increasing complexity of the organizational hierarchy of collagen type I. (A) Collagen molecules

are composed of three a polypeptide chains; one chain is shown. The repeating (X–Y–Gly)n pattern in

which the X and Y positions are frequently occupied by proline and 4-hydroxyproline residues is

represented by X, Y and G. (B) An illustration of three polypeptide chains twist together forming collagen

triple-helix structure depicting the non-helical N- and C-telopeptides bordering the long, central, helical

domain. (C) Four collagen–ligand binding sites in collagen molecule are indicated. (D) Structure of a

microfibril: each molecule is staggered from its neighbor by a multiple of 67 nm. The gap region is where

there are four collagen molecular segments and the overlap region where there are five. (D-i) The

intermolecular separation is slightly more or slightly less than 1.3 nm inside the hydrated fibrils, yielding

a molecular packing that is quasi-hexagonal. (E) Three interdigitated microfibrils where each red and grey

microfibril bundle represents a single microfibril, as shown in D (ii), forming an intermolecular association

that would resemble thinner microfibrillar bundles. (F) The type I collagen fibril exhibits a characteristic

periodic banded pattern originating from the presence of a gap (black) and an overlap region (white) in

the collagen axial packing (D). (F-i) Atomic force micrograph of a collagen fibril. (F-ii) Lateral view of the

molecular packing within a single fibril, where each circle represents each collagen molecule in cross-

section (77). (With permission from reference: 77, Fig.1 DOI: 10.1016/j.actbio.2012.02.022)

12

1.3.1.2 Biomechanical response of dentin

Dentin from a material science perspective may be suggested as a biological grade composite

material, which facilitates the efficient transfer of mechanical stress and strain to the supporting

bone (59). Thus, in order to achieve a long fatigue life span, the dentin structure should present

minimal stress concentrations and display uniform strain distribution. The crown dentin forms the

interface between enamel and root dentin while the root dentin serves as an interface between the

crown dentin and the supporting alveolar bone (60, 62).

1.3.1.2.1 Biomechanical response in intact teeth

Knowledge of the nature of stress distribution in natural/intact tooth structure would aid in

understanding how natural tooth structure respond to mechanical forces in the mouth (58).

Accordingly, investigation on the nature of stress/strain distribution in endodontically treated teeth

would provide the understanding of the biomechanical behavior of treated teeth, when

experiencing functional forces in the mouth. Such studies would provide the background

knowledge for optimal treatment plans and form the basis for developing new devices / materials,

to maintain the mechanical integrity of the tooth, which is an integral part of the stress-

bearing/generating stomatognathic system (58).

A previous study presented that the intact tooth experiences flexing or bending when subjected to

the chewing forces (62). For instance, if we assume a tooth to be a column structure, when bending

stress distributes in this structure due to an eccentric load shown in Figure 1.3A, the column tends

to bend, resulting in compressive stress on one side and tensile stress on the other side. In a tooth

structure, these stresses are displayed along the facio-lingual plane in dentin, also the highest stress

exhibits at the outer aspect and diminish to zero toward the center of the cross-section (60, 62).

When a tooth structure is subjected to a compressive force, the maximum stress resulting from

bending is observed at the cervical aspect of the root. These stresses are minimal in the inner region

and increase toward the facial and lingual surfaces (Fig. 1.3B). The stress also reduces notably

toward the apical region of the root (60, 61). The decreased stress distribution in the middle/apical

region of the root is attributed to the shape and angulation of the tooth and the interaction with

13

supporting tissues. These findings highlight the importance of cervical root dentin to sustain major

functional stress distribution during chewing/mastication (62).

Figure 1.3. Schematic diagrams showing the nature of stress distributions in a column and an intact tooth.

(A) The applied loads along the axis of symmetry combining the bending forces could result in eccentric

loads on the material (column). This eccentric loads resulted in higher compressive stress when compared

with the tensile stress. The neutral axis shifts from the axis of symmetry. (B) The bending stress distributed

within the tooth showing predominate compressive stress on one side along bucco-lingual plan at cervical

region of the tooth is mainly due to the shape and angulation of the tooth and supporting bone reaction.

The apical region of root showed a notable reduction in bending and manifested particularly compressive

stress (62). (With permission from reference)

14

1.3.1.2.2 Biomechanical response in root filled teeth

The functional stresses distribution in a natural tooth to the supporting bone occurs predominantly

at the cervical region of the root and gradually diminishes toward the apical region (61). However,

the increased loss of dentin and eccentric removal of root dentin resulted from disease and

instrumentation alter the radicular stress distribution patterns resulting in more stress distribution

in the apical direction along bucco-lingual plane of the root dentin. The increased root flexure, or

reduced resistance to root flexure may contribute to vertical root fracture. In addition, the regions

of stress concentration identified in the root filled teeth can be attributed to the location of

endodontic post, stiffness of the post, core restorative materials and directions of the occlusal loads

(78). The stress concentration is often found close to the apex of the endodontic post. This may be

the reason for the presence of numerous microcracks in the inner dentin of clinically fractured

specimens (78, 79).

Studies also demonstrated the stress-strain distribution of inner and outer dentin using finite

element analysis. It displayed high strains and less stresses in the inner core region adjacent to the

root canal, which composed of less mineralized dentin with low elastic modulus. In contrary, it

showed low strains with high stresses at the outer facial/ lingual regions, which consisted of highly

mineralized dentin with high elastic modulus (79). In root filled teeth, the pattern of stress

distribution will be altered. This altered stress distribution is the region of stress concentrations,

attribute to residual stress/strain caused during the treatment procedures or tooth structure

loss/removal and the placed restorations (68). The increased magnitude of tensile stresses and the

stresses concentration in the remaining tooth structure may cause the tooth more susceptible to

fracture (78).

The methodology associated with optic techniques in studying the biomechanical behavior of

biological tissue, such as tooth/ or dentin was reviewed in the published article of “Deciphering

dentin tissue biomechanics using digital moiré interferometry: a narrative review (Appendix I).

15

1.3.1.3 Mechanism of fracture resistance in dentin

Several parameters are used to evaluate the fracture resistance of a material, such as, strength,

elastic modulus, and toughness. Stress is defined as the ratio of applied force to a cross section

area; while strain represents the response of a system to an applied stress. It is defined as the

amount of deformation experienced by a structure in the direction of the applied force divided by

the initial length of the structure. Stress-strain curve is traditionally utilized to understand the load

induced response of structure and to determine mechanical parameters such as elastic limit,

strength and toughness. The elastic modulus (Young’s modulus) of a material is defined as the

ratio of stress to strain within the elastic limit in a stress-strain curve, which is an indicator of

stiffness of a material. The ultimate strength is the maximum stress that a material can withstand

before failure (6, 80). However, toughness, the total energy absorbed by a structure before it fails,

is the true indicator of a material’s ability to resist fracture (6). This section gives an insight of

how the mechanical behavior supports the dentin structure with its distinct compositions and

structure arrangement.

Dental hard tissues have multilevel hierarchical structure with an organic phase and inorganic

phase blended together at the fundamental length scales (81). Dentin is a hydrated bio-composite

composed by hard mineral crystals embedded/ wrapped with soft protein matrix (Fig. 1.4). By

combining soft and rigid building blocks at precisely organized hierarchical level, dentin possesses

strength and toughness with outstanding longevity. This unique arrangement allows the material

to fulfill the need of withstanding life-long cyclic loadings imposed onto the tooth structure (82,

83). Each of the components in dentin works together to provide / support the mechanical

properties of tissue in functions.

Dentin tissue displaces a gradient in mineral deposition/concentration. The mineral concentration

determines the stiffness, static strength (especially compressive strength) and elastic modulus in

dentin (6). These properties would increase with the increase of mineral deposition. Minerals are

released from vesicles in the ECM, accumulating calcium in the amorphous calcium phosphate

form by being stabilized with non-collagenous proteins of the SIBLING family, until fully released

onto the collagenous network (gap zone and inter-molecular spaces of collagen fibrils) (84-86).

16

The intrafibrillar minerals are understood to contribute to the mechanical properties of dentin,

including higher elastic modulus (87, 88). The staggered mineral crystals embedded in protein

matrix also strengthen the organic phase in dentin. The mineral platelets carry the tensile load

when the protein matrix transfers the load between mineral crystals via shear (Fig. 1.4) (89).

Optimum balance between two phases is crucial for the mechanical stability of a biological

structure as dentin. Nevertheless, studies recently suggested that both the interfibrillar

(extrafibrillar) and intrafibrillar minerals should be replenished to achieve affective dentin

remineralization (90).

The toughness or the strain energy absorbed by a tissue during mechanical loading is offered by

the organic fraction, especially the collagen in dentin (88). Collagen is also responsible for

toughening the tissue by rising in crack growth resistance with bridging elements (91). It is

important to know that these structures are tightly bound with water molecules due to the inherent

hydrophilic nature of collagen moieties and the intermolecular spaces separating individual

molecules in a fibril are occupied by water. When the water content exceeds the amount

incorporated within the tripeptides, the water molecules start to swell laterally. At this level of

hydration, water acts as a plasticizer keeping the matrix pliable (6). Proteoglycans (PGs), one of

the non-collagenous proteins (NCPs), plays a fundamental role in structural organization in ECM

of the organic fraction. They constitute a protein core covalently attached to carbohydrate

glycosaminoglycan (GAG) side chains (92) and they are believed to form interfibrillar super-

molecular bridges between collagen in both mineralized and soft tissues (93). GAG component of

PGs is highly negatively charged, allowing it to interact with one another between continuous

fibrils by electrostatic forces and hydrogen bonds. They absorb water and span the spaces between

fibrils effectively interconnecting/ holding together the collagen network contributing to the

stability of ECM (90). Nanoindentation studies demonstrate that the creep strain recovery ability

of dentin decrease conspicuously when either PGs or GAGs are removed (94). PGs and GAGs

provide the strength, ductility, and ability for time-dependent strain recovery in dentin (94).

Water molecules that are tightly associated with minerals and organic phase (including collagen

molecules and NCPs) serve as plasticizers as it is mentioned in previous paragraph. The absorption

of water and fluid flow within the porous collagenous network regulated by PGs and GAGs

17

provide the poroelastic characteristics in dentin, when the tissue is under mechanical stress. The

free water in matrix and dentinal tubules also contribute to the viscoelasticity in dentin (95). The

water in pulp and dentinal tubules in the confined environment, presents a particular hydrostatic

pressure resulting in a stress-strain response characteristic of tough material when the occlusal

loads is applied (96). This facilitates hydraulic transfer and dissipate occlusal forces applied to

teeth. The uniform strain distribution in the inner/ outer dentin is also attributed to the hydration

of tissue (97, 98).

Figure 1.4. Schematic diagram showing a model of natural biocomposites. (A) Shows staggered mineral

crystals embedded in protein matrix. (B) A simplified model showing the load-carrying structure of the

mineral - protein composites when experiencing tension forces. Most of the load is carried by the mineral

platelets whereas the protein transfers load via the high shear zones between mineral platelets (6, 89).

(With permission from references)

18

1.3.1.3.1 Toughening Mechanisms in dentin

Two major toughening mechanisms have been addressed in dentin: (1) the intrinsic and (2)

extrinsic mechanisms (99-102). Basically, toughness is increased by mechanisms that increase the

amount of energy required for failure or methods that prevent strain energy from reaching the

crack tip (6). From a mechanics point of view, dentin contains “defects”, which may act as crack

initiation sites and lead to localized failure (103). The intrinsic toughening mechanism can affect

the inherent resistance to microstructural damage and fracture ahead of the crack tip; while

extrinsic toughening mechanism can promote crack-tip shielding, to reduce the stress intensity

experienced at/ or behind the crack tip. The intrinsic mechanisms, such as crack blunting, tend to

affect the initiation toughness and dominate in ductile materials (101, 102). The extrinsic

mechanisms, such as crack bridging, micro-cracking, and crack deflection, promote crack-growth

toughness (101, 102). The extrinsic mechanisms are the main source of toughening in brittle

materials, which also appear to provide the primary contribution to the toughening of dentin (101)

(Fig. 1.5).

Crack blunting can cause the stresses at the crack tip to be defocused (6). In the region of

intertubular dentin, there are abundant collagens and proteins containing many fine spaces

connected to the main tubules. Fluid is able to migrate into the structure to accommodate both the

dilation ahead of the crack tip and the relaxation of this region behind the crack tip. The collagens

within this region are also able to extend when moist, to accommodate such dilation and shear

strains (102). It is suggested that the viscous effects within the material (dentin), which provided

by water, slow down the rate of delivery of energy to crack tip so that the crack can be propagated

slowly and with difficulty (6, 80). As previous described, the hard mineral plates are embedded in

the soft organic matrix in dentin, in terms of staggered arrangement (81, 89). This unique structure

arrangement facilitates the load transfer and homogenizes the stress distribution within the

composite structure, may also act as an intrinsic toughening mechanism.

The extrinsic toughening mechanisms have been shown to contribute of an average of 26% of the

total energy to fracture compared with 3% of intrinsic mechanism (91). Crack deflection is

promoted by features in the microstructure that deviate the crack path from the plane of maximum

19

driven forces (101). For dentin in the parallel orientation with respect to the dentinal tubules, there

was practically no out-of-plane deflection of the crack. This implies that the contribution to

toughening due to this mechanism is insignificant in this orientation. However, in the orientation

of perpendicular to the direction of dentinal tubules, it is fairly significant (101). In earlier studies,

it was demonstrated that the higher toughness of root dentin is achieved by crack deflection at

incremental lines (the interfaces between mineralized collagen fibrils layer (103, 104). The stiff

peritubular dentin has the ability to induce crack deflection as well. Crack bridging is the most

common form of crack-tip shielding, particularly in fiber composites where intact fibers tend to

bridge the crack and oppose crack opening (101, 105). Because bridges can only form with crack

extension, this mechanism can only affect the crack-growth toughness (105). When crack opens,

fibers extend across the crack dissipating energy by their own deformation or by friction as they

pull out from the bulk of material, so that the energy transferred to crack tip would be less or not

enough for crack propagation. Bridging also occurs where the dominant crack links with smaller

cracks ahead of the crack tip to form uncracked ligaments (Uncracked ligaments bridging). These

bridging mechanisms, however, are only observed in the ‘‘parallel’’ orientation in dentin (101).

Microcracking mechanism is to shield the crack by creating a dilated zone that surrounds the crack

with reduced modulus and is constrained by surrounding tissue/ material, extrinsically toughen the

material. The presence of these microcracks also provides a mechanism of the formation of

uncracked ligament bridges (101, 102, 105).

20

Figure 1.5. Schematic diagrams showing the different fracture toughening mechanisms operating in

dentin.

21

1.3.1.4 Effect of endodontic treatment on dentin

Root canal treatment is for saving/ treating an infected tooth to maintain its function. Generally,

there are 15 million root canal treatments performed in North America every year. In this procedure,

the infected materials would be removed, the root canal space would be enlarged and irrigated with

stiff metal files and liquid chemical irrigants to remove the contaminated dentin and to eliminate

bacteria. The canal space would be sealed with gutta-percha and sealer to prevent reinfection, and

followed by final restoration. However, the endodontically treated teeth hold compromised

mechanical properties caused by both pathologic and iatrogenic processes (6, 7).

Collagenolytic activity can either be the result of specific collagenases activity or non-specific

proteases (106). Specific collagenolytic activity is not expressed by Streptococcus mutans and

Actinomyces species, but Porphyromonas gingivalis strains that are involved in infected root

canals show collagenolytic potential (107). Gelatinolytic activity has not been found in caries-

related bacteria (73), but it has been identified in Enterococcus faecalis in root canals with

persistent infections (108). Those activities can result in the degradation of collagen with lost

helical structure, uncoiling fibrils to microfibrils and being presumed by gelatinases. The acid

produced by microbes or environment conditions (low pH) also activates the host-derived matrix

metalloproteinases (MMPs), which present in latent forms within dentin matrix. Gelatinolytic

(MMP-2 and MMP-9) and collagenolytic (MMP-8 and MMP-20) activities hidden in the dentinal

matrix can be released to participate in sequential degradation processes (73, 74).

The loss of hard dental tissue because of caries/ non-caries lesions and access cavity preparation

(iatrogenic procedures) increases the flexure of possibility of the coronal tooth structure that could

lead to a higher occurrence of coronal fracture in endodontically treated teeth (109). The

subsequent (iatrogenic) procedures including instrumentation and obturation within the root could

diminish the structure integrity (stability) and flexural resistance of the root (110-112). One study

using finite element analysis suggested that the reduction in dentin wall thickness may increase

the susceptibility of VRF (113). Root canal anatomy/morphology also influence the predilection

of VRF in root filled teeth. The canals with oval shape such as premolars, receive greater stresses

at the buccal and lingual extensions, with higher vulnerability to fracture (114, 115). Some studies

22

suggested that the rotary instruments used in canal preparation induced micro-cracks on radicular

root canal dentin. These defects may act as stress concentrators that associate with the crack

initiation and further crack propagation, increasing the risk of root fracture (116, 117).

In root filled teeth, loss of hydrophilic vital pulp tissue and the confined environment of vital pulp

and adjacent dentinal tubules result in loss of free water and the physiologically hydrostatic

pressure. The water-induced effect plus the dentin viscoelasticity, which facilitate energy

absorption and distribution, are lost and therefore the bulk dentin displays increased stiffness and

low plasticity (95, 97, 98, 105). The removal of vital pulp may not result in chemical alteration of

dentin, nevertheless, some of the chemical irrigants/ medicaments used root canal treatment can

interact with the dentin surface and modify its characteristics.

Sodium hypochlorite (NaOCl) is used at the concentration of 0.5-5.25%, to destroy bacteria and

dissolve pulp tissue. Chelators such as 17% of ethylenediaminetetraacetic acid (EDTA), interact

with the mineral content of dentin and are utilized to remove the smear layer formed due to the

canal preparation. Sequential use of 15-17% EDTA for more than 3 minutes and 2.5-6% NaOCl

for more than 3 minutes resulted in decreased microhardness and dentin erosion (118, 119). The

prolonged usage of endodontic irrigants has adverse effect on dentin’s physical properties such as

microhardness, flexural strength, and elastic modulus (120). In contrast, the short exposure to

EDTA as clinically recommended (1 minute), did not affect the mechanical elastic modulus and

flexural strength of dentin (121). NaOCl is a strong base and non-specific oxidizer, which interacts

with the amino acids through neutralization and chloramination reactions (122). The proteolytic

reactions will lead to the degradation of amino acid (collagen). The irrigation of 3% NaOCl for 2

minutes followed by 17% EDTA for 2 minutes caused a slight, but significantly decrease in

calcium and an increase of carbon. However, if NaOCl was followed again after abovementioned

protocol, the calcium and phosphate from dentin would be removed extensively (123). Although

collagen fibrils protected by apatite crystallites in natural mineralized tissue do not degrade over

time, a recent study demonstrated 25 to 35 m dentin collagen degradation zone on mineralized

dentin matrix treated with NaOCl from 30 to 240 minutes (124). It seems that the apatite-

encapsulated mineralized dentin is less vulnerable initially to the destructive effects of NaOCl,

however, displays degradation of its organic fraction, which is deproteinized by NaOCl in a time-

23

dependent manner (124). In water, NaOCl ionizes to produce Na+ and the hypochlorite ion (OCl-),

which establishes an equilibrium with hypochlorous acid (HOCl) (122). The OCl- ion is associated

with higher proteolytic activity, which consequently, increases the degree of destruction of the

collagen component of the mineralized dentin matrix (125).

The effect of various obturation/ root canal filling techniques has been evaluated on the fracture

resistance on tooth/root. The load and deformation of root structure during obturation may generate

wedging effect to decrease the stability of root. Current study also highlights that the higher strain

and the residual strains formed at the apical dentin at the end of obturation are not stored but

diminished gradually (126). In addition, gutta-percha, the root filling material did not adhere to

the root canal walls. Therefore, root canal sealers (cements) are applied to seal the irregularities in

the canal space and improve the interface between the gutta-percha and dentin walls. Recently,

sealers are also developed to strengthen the root to enhance the resistance to root fracture. The

interaction between sealers and root canal dentin matrix will be described in the next session.

1.3.1.5 Current methods to strengthen dentin in endodontically treated

teeth

Compromised mechanical properties of dentin in endodontically treated teeth are caused by

pathological and iatrogenic changes. Several methods are proposed to strengthen the tooth for

improving the resistance to fracture during functions. In addition, several post-endodontic

restorations materials/ devices for post/core and cuspal coverage/crown, several systems of sealer

and core filling materials are developed to strengthen the root dentin in endodontically treated

teeth. Stable adhesion between obturation material and root canal dentin as well as the similarity

in the elastic modulus of the filling material and root dentin are two key factors suggested to

enhance the resistance to fracture in endodontically treated teeth (7, 127). Resin based sealers

including epoxy and methacrylate based resin are proposed to strengthen the dentin by forming a

hybrid layer of exposed collagen fibrils (as a micro-retentive network) interlocking with resin

monomers during polymerization. Epoxy resin sealer is able to react with exposed amino groups

in collagen to form covalent bonds between the resin and collagen when the epoxide ring opens

24

(128). However, their bond strength and elastic modulus are very low and are proven not able to

strengthen the root dentin (127, 129).

Bioceramic sealers are mainly composed of calcium silicates, calcium hydroxide, calcium

phosphate and zirconium oxide. They hold good biocompatibility and the chemical composition/

crystalline structure are similar with bone and dentin. It has been suggested that, the mineral

contents may infiltrate the intertubular dentin resulting a mineral infiltration zone after denaturing

the collagen fibers by this strong alkaline sealer. Also with the dentin moisture, the hydroxyapatite

may form along the mineral infiltration zone (130). Although it is proposed that the bioceramic

sealers may increase the resistance to fracture, current findings did not promise higher fracture

resistance compared with adhesive-based sealers (131, 132). A study also showed that the

bioceramic sealers presented less Ca ion release and did not show Ca and Si incorporation deep in

human root canal dentin (133).

Pericervical dentin is defined as the dentin structure extends 6 mm apical and 4 mm coronal to the

crestal bone (134). Value should be given to the pericervical dentin to reinforce root filled teeth

because intact pericervical dentin allows better transfer of functional forces to radicular portion of

the tooth (135). Since bonded restoration has been suggested to improve the long-term

survivability of root filled teeth (136, 137), the biomechanical effect of bonding pericervical dentin

with composite resin was examined in a recent investigation (138). This study showed that even

though the resin bonded pericervical dentin in endodontically treated teeth impacted in a shift of

strain distribution away from apical region under physiological relevant loads, this effect did not

impact the load at failure when subjected to cyclic followed by continuous mechanical

compressive loading (138).

1.3.1.6 Summary

Tooth serves as a mechanical device to distribute occlusal forces to the stomatognathic system

during mastication. Dentin is a biocomposite, with an organic, inorganic and water fractions. It

forms the major bulk of crown and root, and is critically responsible for the mechanical responses

of tooth to functional forces. Minerals offer the strength and stiffness of the structure while organic

25

phase and water provide the flexibility, toughness, viscoelasticity and the resistance of crack

initiation and propagation to dentin tissue. Nevertheless, compromised mechanical integrity of

dentin structure caused by pathological and iatrogenic factors leads to the higher propensity of root

fracture in root filled teeth. Studying the biomechanical behavior of dentin provides an insight to

its response to functional force and may explain some of the causes for root fracture in root filled

teeth. This information is crucial for designing a treatment strategy for strengthening the root

structure. Photomechanical techniques utilize optical principles to study the biomechanical

response of biological tissues under functional forces in a non-destructive way providing high-

sensitive, and whole-field information of specimens. Currently there is an acute need for treatment

strategy that would reinforce root dentin to functional mechanical forces.

1.3.2 Fluid Dynamics in Root Canal System

Irrigants are liquid chemicals used in root canal treatment. The irrigants used in root canal

treatment are mainly antimicrobials, chelating agents or combination of both. Root canal irrigation

is the process of delivery of irrigant into the root canal. The irrigants are employed in root canal

treatment to lubricate during instrumentation, flush out the debris /organic remnants from the root

canal space and to eliminate microbes and microbial byproducts from the root canal system (139,

140). Chemical irrigation in root canal treatment facilitates the elimination of bacteria and infected

tissue. The anatomical complexities in the root canal system renders thorough cleaning of

biofilm/debris from the root canal system a challenging task. Besides root canal anatomy, the fluid

flow characteristics in a confined geometry such as root canal space makes the replenishment of

irrigant difficult (139). Moreover, the possibility of gas bubble formation at the apical part of the

root canal (apical vapor lock) caused by the gas entrainment during irrigant delivery or the

coalescence of gas bubbles produced from the reaction between NaOCl and organic tissues, could

further block the penetration of irrigants (141, 142). Therefore, effective irrigants delivery and

agitation systems are topics of research in the recent times. Understanding the underlying physical

effects of root canal irrigation would provide a useful insight on the fluid flow characteristics and

the forces generated during irrigation. This information can further bridge the knowledge gap

between clinical outcome and laboratory experiments. Conventionally root canal irrigation

systems are broadly categorized as manual or machine assisted techniques. This section reviews

26

the established agitation methods and fluid dynamics in root canal irrigation with experimental

set-up.

1.3.2.1 Methods to activate irrigants in endodontics

Irrigant is commonly delivered from a syringe through a needle. Different size and the design of

needles ranging from gauge size 23 to 30, open-ended, and side-vent are reported (143). Generally

the rate of irrigant delivery varies tremendously amongst operators from 0.01 to 1.01 mL/s (144).

Different irrigant delivery systems were developed to improve the irrigation efficacy and the fluid

dynamics within the root canals. However, studies did not show convincing evidences (145-147).

The maximum flow velocity using conventional syringe delivery (side-port 30 gauge) determined

using computational fluid dynamics based method, was 2-17 m/s in Boutsioukis’ and 0.12 m/s in

Layton’s investigation when determined using experimental approach. The maximum shear stress

was 0.24 Pa and 2.4 Pa, respectively (148, 149). The stronger flow was only observed near the

needle exit and gradually decreased, within 1 mm away from the tip, showing relative weak fluid

dynamics. It has also been found that syringe delivery system alone did not allow complete irrigant

penetration and exchange for effective root canal cleanliness (150, 151). Agitation during

irrigation promotes the irrigant dispersion and the flushing out of debris from extremities of the

canal system. It aims to “activate” the irrigant in the root canal, which can be achieved by several

methods: (1) manual reciprocation of an instrument; (2) sonic and ultrasonic oscillation of the

instrument placed in canal; (3) mechanically driven rotary instruments inserted in canal; (4) laser

activation (139).

Manual dynamic activation can be performed with hand files, brushes or a well fitted tapered gutta-

percha point (152, 153). The agitation starts with an up and down motion and a 2mm amplitude at

a frequency of 100 strokes during approximately 1 min. It helps the irrigant to interact with the

canal walls, to reach the apical portion of the canal and disrupt the vapor lock effect (a column of

air entrapped in the apical part of canal) providing the effective hydrodynamic effect and the

displacement/ exchange of irrigants (154). The key aspect is that the instrument used must be

tightly fitted with the canal and be pushed close to the end of canal so that it forces the liquid to be

displaced down the tube. When the fluid is not able to be extruded through the canal terminus due

27

to the tissue pressure, it moves sideway and upwards through the gap between the instrument and

the canal wall, promoting better interaction and fluid mixing. Otherwise the liquid would be merely

pulled up and down in a reversible manner when the instrument is not well fitted (139). The master

cone (gutta-percha) is mainly used as the insert during this activation. In brief, the manual dynamic

activation generates higher intracanal pressure changes during the push-pull motion of a well-fitted

gutta-percha, leading to more effective irrigant delivery to the untouched canal surfaces. This

motion of the instrument may promote the fluid mixing by physically displacing, folding and

cutting under the viscosity-dominated flow (153, 155).

Automated systems including sonic and ultrasonic devices are designed for agitation of the

irrigants within the root canal system. Sonic irrigation is different from ultrasonic agitation in that

it operates in lower frequency (1-6 kHz) resulting in less effective microstreaming. The oscillating

pattern of sonically activated insert is also different with ultrasonic agitation. Sonically activated

insert displays only one node (minimum oscillation of amplitude) and antinode (maximum

oscillation of amplitude), while producing an elliptic and lateral movement similar to an

ultrasonically activated insert. However, the oscillation amplitude of the sonically activated tip is

around 1mm while the apical diameter of root canal is smaller than 0.5mm so that sonic agitation

remains in only longitudinal oscillation due to the constrained movement within the micro-space

of root canal (153, 156). This also inhibits free oscillation of the sonic tip reducing the efficient

streaming of the irrigant. The sonic insert can be a stainless-steel file (Sonic Air Endo Hand piece:

1.5-3 kHz) or a polymer tip with various size and tapers (EndoActivator: 160-190 Hz).

The much higher frequencies of ultrasonic oscillation (20-40 kHz) are achieved with either

magnetostrictive or piezoelctric devices. On one hand, magnetostriction is generated by the

deformation of a ferromagnetic material subjected to a magnetic field producing an elliptical

motion at the working tip; on the other hand, piezoelectricity is generated by the stress in dielectric

crystals subjected to an applied voltage producing longitudinal / transverse linear motions (157,

158). The acoustic streaming (or microstreaming) and hydrodynamic cavitation have been claimed

as the working mechanism in ultrasonic oscillation contributing to the effective canal irrigation

(159). Acoustic streaming is a rapid movement of fluid in a circular or vortex-like motion around

a vibrating file, whilst the cavitation is the formation and the implosion of vapor bubbles in a liquid

28

(160). With multiple nodes and antinodes under high frequency of oscillation, ultrasonic activation

induces more intense acoustic microstreamig than sonic activation. It is proposed that acoustic

streaming consists of both a steady and an oscillatory component (161). The presence of an

oscillatory component, dominates near the file with a typical thickness on the order of 1m; and a

steady component is further away from the file with the thickness on the order of 100 m. The

steady component of the flow drives the actual transport and mixing of the irrigant, may also be

able to deliver irrigant into remote locations. The oscillatory component is not directly relevant to

the chemical aspect of cleaning, however, it generates the cavitation which induces sonochemical

effects and plays a role in increasing wall stresses (161).

Cavitation can be described as the impulsive formation of cavities in a liquid through tensile forces

induced by high-speed flows or flow gradients. These bubbles expand and then rapidly collapse

producing a focus of energy that may lead to damage (162). Two types of cavitation could occur

during activation: (1) stable cavitation as linear pulsation of gas-filled bodies in a low amplitude

ultrasonic field; and (2) transient cavitation which occurs when vapor bubbles undergo highly

energetic pulsation (159). The energy generated in transient cavitation leads to the formation of

OH radicals by sonolysis of H2O molecules, which can interact with a chemiluminescent

molecule producing light emission (50). This was used to detect and confirm the transient

cavitation in ultrasonic activation. It is confirmed that the cavitation (transient) can only be shown

in ultrasonic agitation in the root canal system, because the relatively slow movement of the

oscillation in sonic activation is below the threshold needed for cavitation (51, 160). Therefore,

the cavitation bubbles collapse on the file itself and not on the nearby wall in Macedo’ s

investigation may refer to the stable cavitation; and the small individual bubbles observed near the

antinodes which contributes to the sonoluminescence effect may refer to the transient cavitation

(50).

29

Some physical parameters are used to evaluate the fluid dynamics in the root canal irrigation. The

intensity of the acoustic microstreaming is directly related to the streaming velocity. The equation

that describes the liquid streaming velocity (, in m/s) is (158, 161, 163):

𝜈 =2𝜋𝑓𝐴2

𝑅

f is the oscillation frequency (Hz); A is the amplitude of oscillation (meters); R refers to the radius

of the instrument (meters). The steady component of the flow adds a contribution to the shear

stresses along the root canal wall. The shear stress (, in Pa) is expressed in the following equation

(158, 161):

𝜏 = 𝜇𝜈

𝛿=

𝜇 2𝜋𝑓𝐴2

𝛿𝑅

where is the viscosity of fluid (Pa.sec); is the boundary layer thickness (meters).

The cavitation threshold can be determined by estimating the pressures required for the formation,

growth, and collapse of a bubble within a given liquid. Bubbles can grow when the applied pressure

drops from the ambient pressure to below the vapor pressure of the liquid (51). The likelihood of

cavitation occurrence is defined by the cavitation number Ca (51):

𝐶𝑎 =𝑃𝑎𝑡𝑚𝑜𝑠𝑓𝑒𝑟𝑖𝑐 − 𝑃𝑣𝑎𝑝𝑜𝑟

12

𝜌𝑈2

is the density of the liquid (1000 kg/m3 for water, 1100 kg/m3 for NaOCl) and U is the velocity

of the oscillation tip, which can be calculated with the equation U = 2 f A. When the Ca < 1,

cavitation may occur. Usually the ambient pressure is 105 Pa, and the vapor pressure of fluid is

around 2000 Pa. So roughly speaking the 1

2 𝜌𝑈2 should be larger than 105 Pa, results in the

corresponding velocity threshold 15 m/s (160). Therefore, an ultrasonic agitation with a typical 30

kHz frequency and an approximate 100 um oscillation amplitude should be able to generate

cavitation during agitation. On the other hand, a sonic tip oscillating at a frequency of 190 Hz with

30

an amplitude of 1.2mm, only results in the velocity of 1.4 m/s which is not able to generate

cavitation during sonic activation in root canal irrigation (51, 160).

1.3.2.2 Experimental methods to study fluid dynamics in root canals

The most common methods to evaluate the effectiveness of irrigation are scanning electron

microscopy, root canal sectional analysis and microbiological analysis (164-167). These

investigations focus on examining the final static condition rather than the process of fluid

dynamics during the root canal irrigation. However, the irrigation dynamics plays an important

role on the efficacy of irrigation depending on the working mechanism of the fluid flow and the

ability to bring the irrigant in contact with the debris/ microorganisms in the canal system. Studying

the fluid dynamics of irrigation also provides an insight to explain/understand the reason of static

outcomes. Hence, a few studies from 1980s to 2000s have used bead-form gel simulating bacterial

biofilm, red food dye in simulated root canal model (168, 169), or even a thermal image analysis

(170), to evaluate the irrigation with a real-time visual assessment. Methodology has evolved to

high-speed imaging technique with transparent root canal models which provides accurate, real-

time assessment of fluid dynamics at the region of interest during irrigation.

High-speed imaging in flow visualization is primarily aimed at obtaining precise information about

the position and dimensions of the fluid flow at a series of instants in time, i.e. to resolve to the

best possible extent the spatial and temporal scales (171). The most critical component of this

technique is the electro-optical cameral system, so-called image converter camera (high-speed

camera), can capture/ record the images in very short time intervals. The temporal resolution of

the camera depends on the maximum frame rate, while the spatial resolution relates to the pixel

size of the sensor and the minimum length scale that can be imaged in the experiment (171).

Considering the typical flow velocity () inside the root canal is 1 m/s and its typical dimension

() is 100 m, the frame rate required to capture the dynamic flow behavior of needle irrigation

techniques should be higher than 100 k frame-per-second (fps), depending on the applied

magnification (171). Combining with particle imaging velocimetry (PIV), the quantitative analysis

of fluid flow can be achieved efficiently. The tracer particles added into the irrigant enhance the

visualization of flow pattern and enable tracing the flow with accuracy (172, 173). The laser system

31

is usually connected to PIV to illuminate fluorescent tracer particles to visualize and analyze the

flow. A micro PIV system (PIV), with smaller depth of focus and a continuous light source

releases the restriction of recording speed which limited by the amount of light emitted from

fluorescent particles in conventional PIV. Due to the thinner image plane of PIV, the particles

that sufficiently unfocused would not contribute to the velocity field (174). Hence, a higher frame

rate with a prolonged recording time can be accomplished to improve the accuracy of analysis

(173).

Boutsioukis et al. compared the velocity field of a syringe based irrigation in an experimental high-

speed imaging setup to 250 k fps with PIV, to the velocity field generated using computational

fluid dynamics simulation (CFD) as a validation of CFD (148). The velocity close to the outlet of

needle was 0.026-17 m/s with acceptable agreement. Due to the limitation of ensemble averaging

and calculating interrogation area (rather than a single point) in PIV analysis, the velocity field in

CFD is higher. The agreement between CFD and PIV results regarding velocity vectors in the front

view was not exact. This may be attributed to the slight displacements of the needle from the

central portion during irrigation, which are also expected in a clinical procedure (148). Similar

setup used in evaluating the syringe and ultrasonic irrigation demonstrated a maximum velocity of

0.12-0.14 m/s (149). The local strain rates were calculated by PIV software, which were used to

calculate shear stress by classic Newtonian stress strain relationship in the fluid. The maximum

shear stress resulted in this study was 2-2.4 N/m2 (Pa) (149). The agitation of a polymer rotary

finishing file resulted in a maximum velocity of 0.05 m/s, evaluated by a conventional PIV system

(175).

The high-speed imaging setup used to study fluid dynamics, was also utilized in demonstrating the

oscillation flow of sonic, ultrasonic agitation in the simulated transparent root canal model (160,

176, 177), as well as laser-activated irrigation (178-180). These studies focus on demonstrating

the flow patterns and the bubbles formation during activation. Jiang et al. concluded that ultrasonic

activation was significantly more efficient than sonic activation. Based on their study, it was

suggested that sonic oscillation resulted in much wall contact and no cavitation during irrigation

(160). Malki et al. utilized the high-speed imaging technique to evaluate the fluid flow and

penetration length generated in ultrasonic irrigation in both curved and straight canal morphology.

32

It was shown that ultrasonic oscillating could remove dentin debris up to 3 mm in front of the file

tip, coinciding with the extent of the observed flow and the root canal curvature had no influence

on the irrigant flow (176). A hot-film anemometry was applied in addition to high-speed imaging

method to study the oscillation flow and shear stress induced by ultrasonic and polymer rotary

finishing files. The mean/ maximum shear stress exhibited in ultrasonic and polymer rotary files

were 0.86/ 2.2 N/m2 (Pa) and 0.34/ 2.8 N/m2 (Pa) respectively (177).

Laser activated irrigation utilizes the high degree of absorption of Erbium YAG laser by water

molecules. de Groot highlighted improved bubble dynamics and better efficacy of laser-activated

irrigation in removing dentin debris compared to ultrasonic irrigation (178). Matsumoto et al.

clearly demonstrated the expansion/ implosion of laser-induced bubbles and secondary cavitation

bubbles in an artificial root canal model (179). A recent study of Koch et al. evaluated the irrigation

flow activated by photon-induced photoacoustic streaming (PIPS) compared to ultrasonic

activation with a PIV system. PIPS produced higher average fluid speeds (0.3-0.5 m/s) when

compared to ultrasonic irrigation (0.03 m/s), both close and distant from instrument. This may be

relevant to the debriding and disinfecting efficacy during root canal therapy (180). Ultrasonic

systems generate predominately acoustic microstreaming to transport/ mix the irrigant more

efficiently in the root canal system and produce shear stresses along the root canal wall. Even

though transient cavitation is approved to be produced near the antinodes of the ultrasonic agitation

(50, 51), the intensity of the cavitation and bubble dynamics under clinical relevant power setting

are still very low and does not contribute much to the wall stresses.

1.3.2.3 Role of Microbubbles to enhance fluid dynamics in root canal

Microbubble emulsion was first proposed in endodontics as a photosensitizing formulation which

contains a photosensitizer, an oxygen carrier, oxidizer and a surfactant in certain proportions

resulting in significant effects in the disinfection of endodontic biofilm (37). The increased rate of

singlet-oxygen contributes toward the biofilm matrix disruption and bacteria inactivation during

light-activated disinfection. Later the similar formulation of microbubbles was applied to intensify

the cavitation bubble dynamics and the chemical reactivity for enhancing the antibiofilm efficacy

in root canal irrigation (54).

33

Microbubbles (MBs), commonly used as a contrast agent in diagnostic imaging or as a carrier for

targeted drug/ gene delivery, are gas-filled bubbles in micron range produced in an emulsion (55,

181). The diameter of MB is similar as a red blood cell which is lesser than 10 m. The gas core,

which is typically a high molecular weight gas such as a perfluorocarbon or sulfur hexafluoride,

comprises most of the particle volume and provides the mechanism for ultrasound backscatter and

drug delivery (53). Gas bubbles of this size in aqueous media are inherently unstable owing to

surface tension effect, therefore require a stabilizing shell. The shell can be composed of

surfactants, lipids, proteins, polymers or a combination of these materials. In drug/ gene delivery,

nucleic acids and drug molecules are partially or fully incorporated within the shell of MBs (53).

MBs display numerous useful effects when they are insonified by ultrasound because the gas core

expands during the rarefaction phase of the pressure wave and contrasts during the compression

phase (53, 182, 183). At low acoustic pressure, an insonified MB produces a backscattered echo,

which can be used to detect and locate the MB. Hence MB can be utilized as a contrast agent in

ultrasound imaging (181, 182). The steady oscillating MBs generate shear field streamlines of fluid

flow, which may induce shear forces on cellular surfaces that enhance the intercellular and

extravascular transport of nearby macromolecules (184). A more violent activity may occur at

higher acoustic pressure. Inertial cavitation involves the rapid growth and collapse of bubbles

resulting in the shock waves due to the violent implosion. This cavitation induces high fluid

velocities, shear forces, and local temperature increases, thus producing different biological effects

and altered transport kinetics near the site (55, 185). Mainly the cavitation of bubbles increases the

extravascular permeability allowing macromolecules to enter into the targeted tissues from the

blood stream, as sonoporation (186-188).

Ultrasonic activation of MBs decreases the threshold for bubble production and increases bubble

dynamics. This was also confirmed and applied in root canal irrigation (54). Ultrasonic agitation

in combination with MBs generated increased bubble dynamics characterized by larger, strongly

oscillating/ vaporizing bubbles compared with smaller bubbles produced in water. Sonic agitation

in MBs was not able to generate cavitation bubbles. This intensified bubble dynamics enhanced

the apical penetration of MBs and the forming of more turbulent flow along root canal wall.

34

1.3.2.4 Summary

Chemicals (irrigants) are commonly used in root canal treatment to remove debris and for

disinfection. Traditionally syringe-needle based method are used to deliver irrigants (irrigation).

Syringe-needle based method offers different challenges from a fluid dynamics perspective. Thus,

machine assisted and manual methods are employed to activate irrigants within the root canal

space. The activation methods promote (1) fluid interaction with the root canal walls, (2) flow of

irrigant to apical portion of the root canal, and (3) fluid penetration into the irregularities of the

root canal system, to allow better replenishment of the root canal. These benefits would result in

improved disinfection efficacy with cleaner root canal dentin surfaces.

Traditionally static models are used to assess the efficacy of root canal irrigation. These techniques

do not provide dynamic fluid flow information within root canals. Recently, advanced

technologies are used to study fluid flow characteristics using high-speed imaging and

computational modeling in simulated canal geometry/spaces, the physical properties, vectors and

patterns of irrigation flow at the region of interest. These studies were used to understand the fluid

dynamics in root canal irrigation with syringe based method, automated methods and manual

method.

Microbubble composed of micro-size droplet and protein/surfactant shell, is utilized as drug/ gene

carrier and contrast agents in diagnostic imaging. It potentiates the bubble dynamics, decreases the

threshold of cavitation production and facilitates antimicrobial efficacy in the root canal irrigation,

which can be a potential formulation in endodontic application.

35

1.3.3 Nanoparticle Guided Micro-Tissue Engineering

Tissue engineering is an emerging bioscience with the ultimate aim of restoring, maintaining, and

regenerating damaged/ lost tissue with biologically engineered replacements (189). To place a

scaffold as three-dimensional temporary structural framework or to engineer the tissue surface

both aim to design a tissue with optimum biological and mechanical characteristics. This favorable

three-dimensional micro-environment can support cell behavior, tissue function, and host

integration (15, 190). Surface engineering of tissue chemically includes introducing new functional

groups onto the tissue surfaces or coating the surfaces with a thin layer of polymer/ other chemical

species (14). Introduction of nano sized particles has revolutionized the tissue engineering. Owing

to the extremely small size of these particles, the quantum effect and the surface area per unit

volume both greatly increase, which brings in the novel physical/chemical properties to the tissue.

In this section, dentin tissue engineering by strengthening collagen with nanoparticle incorporation

to modify the dentin (tissue) surfaces is introduced.

1.3.3.1 Chitosan nanoparticle

Chitosan is a deacetylated derivative of chitin, the second most abundant natural biopolymer

obtained from the exoskeleton of marine organisms such as crabs, lobsters and shrimps (Fig. 1.6)

(191). The structure of chitin closely resembles that of cellulose, and both act as a structural support

and defense material in living organisms. Structurally chitosan is composed of -(1-4)-linked D-

glucosamine and N-acetyl-D-glucosamine, with a wide range of molecular weight (Mw), degree

and pattern of N-acetylation (191, 192). Depending on the preparation procedure and their sources,

its Mw may vary from 300 to over 1000 kDa with a degree of deacetylation from 30% to 95%

(193). Each repeating glycosidic unit consists of one amino (NH2) group and two hydroxyl (OH)

groups (194). Chitosan is soluble in diluted acidic solutions below pH 6.0 due to the quaternisation

of the amino groups that have a pKa value of 6.3 making it a water-soluble cationic polyelectrolyte

in acidic solution (195). Chitosan with a large number of hydroxyl and free amino groups can be

subjected to numerous chemical modifications and grafting, contributing to its versatility (191,

195). The cationic character, mucoadhesiveness, permeation enhancement, antimicrobial activity,

colon targeting and efflux pump inhibition of chitosan, are also attributed to the primary amine

functional group (193). Due to the favorable properties of chitosan in addition to its non-toxicity,

36

excellent biocompatibility and degradability, it has been extensively utilized in biomedical fields

(193, 196). Nanoparticles of chitosan have been developed mainly for antibacterial and drug/gene

delivery applications.

Chitosan nanoparticles (CSnp) can be synthesized or assembled by different methods, depending

on the end application or physical characteristics required in nanoparticles (197). It is often

prepared using ionotropic gelation method resulting in around 100 nm (Fig. 1.6). The small size

of nanoparticles makes them capable of moving through various biological barrier, bringing drugs

to the target site to enhance its efficacy (198, 199). Besides those abovementioned unique

properties, together with the bioactivity and target specificity triggered by its cationic character,

CSnp has become an excellent drug carrier. It can be functionalized through covalent bonds to

conjugate chemicals/biomolecules for numerous applications (21). CSnp prevents the enzymatic

degradation of encapsulated labile drugs, increases their clearance time and the stability in the

body as well as their release in a controlled manner (193). Recent studies impregnated CSnp as

gene, drug, or growth factors carrier to collagen-based scaffolds for tissue engineering application

(200-202). The controlled release of bioactive molecules of CSnp system also showed an improved

environment for stem cells presenting potential application in dentin pulp regeneration (48, 203,

204).

The antibacterial effect of chitosan is related to the ability of its positively charged amine groups

to bind to the negatively charge surface of bacterial cell membranes. This may lead to the altered

cell wall permeability resulting in the osmotic damage with the efflux of ions and proteins from

the cytoplasm to the extracellular spaces. In fact, the higher the positive charge density of chitosan,

the stronger the electrostatic interactions with the bacterial cell surface. Therefore, the degree of

deacetylation and the degree of substitution on the amino groups play an important role in

antimicrobial property of chitosan. Furthermore, in CSnp, the enhanced antimicrobial effect has

been attributed to the higher density of positively charged amino groups in the nanoparticles, as

well as the increase of surface area to volume ratio and the quantum size effect (196, 205). CSnp

have been shown to provide a significant improvement in root canal disinfection by effectively

eliminating the residual adherent/ nonadherent biofilms (Enterococcus faecalis) and disinfecting/

disrupting Enterococcus faecalis biofilms (27, 45). Studies also demonstrated the photoactivated

37

CSnp presented enhanced ability to eliminate clinically relevant multispecies bacterial biofilm and

were able to inactivate bacterial endotoxins (46, 47, 206).

CSnp also show the ability to enhance the mechanical stability of collagen. The structure of CSnp

is similar as the extracellular matrix glycosaminoglycans. Extracellular matrix proteins such as

proteoglycans and glycosaminoglycans offer the compressive strength and mechanical stability to

the collagen by interwining with the fibrous structure (32). The collagen matrices of bovine

pericardium were reinforced by absorbed chitosan, improving flexibility and stress-strain

properties (207). Chitosan nanomaterials incorporated with collagen scaffolds enhanced the

mechanical properties, structural protection and created a more suitable biomimetic environment

for cells (32, 202, 208). Besides, CSnp and their derivatives interact with and neutralize MMPs or

bacterial collagenase, thereby improving dentinal resistance to collagen degradation (209).

Figure 1.6. The molecular structure of chitin and chitosan (A). (B) Chitosan nanoparticles are synthesized

using ionotropic gelation method and chitosan chains are crosslinked by sodium tripolyphosphate (TPP)

to form chitosan nanoparticles (210, 211).

38

1.3.3.2 Crosslinking of dentin collagen

Tissue stabilization is the process of rendering the ultrastructure of a tissue more stable in order to

provide or enhance its mechanical properties and resistance to chemical-mediated degradation.

Crosslinking is the method/ process for improving physical and mechanical characteristics of

collagenous tissues and scaffolds (212). It is defined as the introduction of chemical or physical

links between polymer (protein) chains, simply to modify mechanical, biological and degradation

properties of tissues (213). Crosslinking the dentin collagens also protects them from host-derived

MMPs and bacterial proteases (44, 214-216).

There are various methods used in biomaterials to crosslink the collagen. Chemical crosslinking

using natural and synthetic reagents, is one of the widely used and effective methods (213).

Glutaraldehyde (GA) reacts with amino or hydroxyl functional groups of protein molecules to

form biopolymeric chains via intra- or inter-molecular reactions. Crosslink of tissue and scaffold

with GA has been the gold standard for many years. It increases the tensile properties and stiffness

of demineralized dentin and increases resistance to enzymatic degradation (206, 216). However,

even though many detoxifying strategies have been proposed to improve the biocompatibility of

GA-crosslinked scaffolds/ tissues, the cytotoxicity and calcification in the host tissue still limit its

application.

1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) is a water soluble carbodiimide that can

react with a variety of chemical groups, such as carboxyl, hydroxyl and sulfhydryl functional

groups. It involves the activation of the carboxyl acid groups of glutamic and aspartic acid residues

in the peptide chains, further links with the amino groups of lysine or hydroxyl lysine residues

forming amide bonds. N-Hydroxylsuccinimide (NHS) can be used in combination with EDC to

activate carboxyl acid groups, which are less susceptible to hydrolysis and can increase the

efficiency of the crosslinking reaction (217). Luo et al. recently showed that collagen nanofibers

crosslinked with EDC/NHS presented optimal mechanical strength, fiber morphology, higher

toughness in wet condition and cell viability, which is a suitable method for tissue restoration (218).

EDC crosslinking showed the ability to prevent host-derived/ and bacterial collagenase induced

collagen degradation in demineralized human dentin (44, 219). Although many studies show

39

increased stiffness, durability, inactivation of dentinal gelatinases in EDC crosslinked dentin-

bonded interfaces (220-222), there are other studies that show inconsistent results (223). In

addition, the adjacent collagen microfibrils are too far apart to be bridged by this zero-length

crosslinker. Oryan et al. showed that EDC-crosslinked scaffolds showed softer surface and more

rapid degradation profiles, compared to GA-crosslinked ones (213).

Natural crosslinking agents, such as genipin, citric acid, tannic acid and proanthocyanidin

(procyanidins) exhibit superiority especially in terms of low cytotoxicity (213). Genipin (GPn) is

a hydrolytic product of geniposide extracted from the fruit of Gardenia jasminoides Ellis. GPn

reacts with free amino groups of lysine, hydroxyl lysine and arginine to form intra- and inter-

molecular crosslinking by polymerization (similar as GA) (224). GP forms annular crosslinking,

which is more stable than the reticular linking formed by GA and the linear crosslinking (225).

Previous study showed that GPn crosslinked demineralized dentin presented increased tensile

strength (35), however, some other studies showed contradictory findings (224, 226). Recently,

Kwon proposed that collagen scaffold crosslinked with GP showed improved compressive strength

and promoted odontogenic differentiation of human dental pulp cells, which may be beneficial to

dentin-pulp regeneration (227). Nevertheless, the blue pigments and complex extraction process

and the high cost limit its application (218).

Photodynamic crosslinking, also called photooxidative crosslinking, is considered a rapid,

efficient method with low cytotoxicity for stabilizing the collagen based biomaterials. It involves

the use of non-toxic dye or photosensitizer, such as riboflavin, rose bengal (RB) and methylene

blue (MB) in combination with ultraviolet (UV) or visible light (43, 228-231). The source of light

depends on the desired penetration of biomaterials and the chromophore of the molecules to be

crosslinked. Photodynamic crosslinking involves two pathways: type I (direct) and type II

(indirect), but the indirect mechanism dominates (232). In this pathway, the excited singlet oxygen

is produced due to the energy transfer from excited-state of photosensitizer to ground-state

photosensitizer. The light-activated photosensitizer transfers its energy to molecular oxygen to

generate singlet oxygen, and returns to its ground state after energy transfer. The highly active

singlet oxygen induces photo-oxidation of photooxidizable amino acid residues such as cysteine,

histidine, tyrosine and tryptophan in one protein molecule resulting in products which, in turn,

40

react with normal/ or photoaltered residues in another protein molecule to produce a crosslink

(233). Photodynamic crosslinking strengthens the mechanical properties of collagen tissues and

artificial scaffold with improved cell viability, as well as water retaining property in tissue

engineering (228, 230, 231).

Photodynamic crosslinking with riboflavin and ultraviolet light on demineralized dentin collagen

inhibited host-derived cysteine cathepsin K and MMP activity significantly (229). This process

also enhanced the flexural strength, elastic modulus of non-demineralized human dentin (234);

improved the stability of dentin-bonded interfaces (235); as well as heightened the resistance to

bacterial collagenase-mediated degradation and mechanical characteristics (apparent-elastic

modulus and tensile strength) of demineralized root dentin (236). Shrestha et al. demonstrated that

photodynamic crosslinking with RB or RB functionalized nanoparticle stabilized root dentin

collagen by increasing tensile strength, toughness and resistance to bacterial collagenase (206).

Though crosslinking may strengthen the collagen based tissues, there are possibilities for the

crosslinked collagen to be stiff or brittle in nature (237). Incorporation of synthetic or natural

polymers could serve as spacers/ fillers in between collagen fibrils preventing the undesired zero-

length crosslinking and improving the stress-strain behavior of tissues (42-44, 216)

1.3.3.3 Summary

Micro-tissue engineering is an interdisciplinary field to design tissues/ scaffolds with ideal

biological and mechanical properties to restore tissue function and host integration. Integrating

CSnp into collagen crosslinking provides not only the enhanced mechanical stability, but also the

attractive advantages of chitosan nanoparticles, which include (1) the structural similarity to the

extracellular matrix glycosaminoglycans, which interwine with the fibrous structure to offer

mechanical stability and compressive strength to collagen. (2) CSnp and their derivatives stabilize

collagen based tissues by neutralizing host-derived / bacterial collagenases. (3) They exhibit

significant antimicrobial ability while producing optimum bioactivity for cell adherence,

proliferation and differentiation. (4) The reactive amino and hydroxyl groups of CSnp can be

utilized for chemical modifications or conjugation bringing multifunctions to the tissues. Micro-

tissue engineering root dentin with optimal collagen crosslinking and simultaneous incorporation

41

of chitosan nanoparticles, would be able to reinforce root dentin. This method will enhance the

mechanical properties of dentin and improve resistances to enzymatic degradation. Addition of

bioactive nanoparticles such as chitosan nanoparticles, also provide the engineered tissue novel

biological properties.

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Chapter 2

Optimizing the Formulation of Biopolymeric Nanoparticle Vehicle

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2.1 Abstract

Aim To characterize and optimize the concentration and formulation of chitosan nanoparticle

(CSnp) for micro-tissue engineering root canal dentin. The stability of the CSnp solution, ultra-

structure and surface mechanical characteristics of the micro-tissue engineered dentin were

determined and characterized.

Methodology CSnp solution in concentration of 0.1 to 2mg/ml was subjected to dynamic light

scattering measurement to evaluate the particle size over an hour, dispersity and zeta potential of

each solution. Sixteen split root canal dentin specimens were finely polished to expose the root

canal surface and subjected to Group-1: control, Group-2: CSnp, Group-3: EDC-crosslinked-CSnp

and Group-4: PDA-crosslinked-CSnp (n=4/ group). The ultrastructure of treated dentin surfaces

was characterized using field emission scanning electron microscopy, while the mechanical

properties of dentin surface such as hardness and elastic modulus were determined using a

nanoindentation method. In the measurement of mechanical properties, two samples from each

group were subjected to nanoindenter before and after treatment. This method allowed each dentin

specimen to serve as its own control. Data of hardness and elastic modulus were subjected to a

statistical analysis with paired sample t-test at 0.05 significance level.

Results The size of nanaoparticles ranged between 360-540 nm. The size from each concentration

remained stable without aggregation overtime. However, the CSnp dispersion showed broad-

polydispersity in concentration above 1 mg/ml and the zeta potential of CSnp decreased drastically

at 2 mg/ml concentration. Root dentin surface coated with CSnp followed by crosslinking

exhibited homogeneous and dense coating of CSnp. The hardness and elastic modulus reduced

28% after treatment with EDC-crosslinked-CSnp (p < 0.01). The elastic modulus increased 17%

in PDA-crosslinked-CSnp group (p < 0.01).

Conclusions This study suggested 1mg/ml chitosan-nanoparticle-formulation followed by

chemical or photodynamic crosslinking was the optimal condition for the micro-tissue engineering

root canal dentin.

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2.2 Introduction

Nanoparticles (NPs) are microscopic particles with one or more dimensions in the range of 1-100

nm, displaying high surface areas, novel properties from their bulk counterparts. They can be

manipulated on the atomic/molecular scale because of their extremely small size (1, 2) and provide

novel and versatile functions in biomedical applications. Moreover, biopolymeric nanoparticles

are biocompatible, stable in biological fluids, and can serve as an active targeting nanocarriers to

improve the therapeutic effects (3, 4).

Chitosan (poly (1, 4), -d glucopyranosamine), a derivate of chitin, is a natural biopolymer derived

from the exoskeleton of crustaceans (5). Owing to its biocompatibility, antimicrobial properties

and biodegradability, significant interest has been given to chitosan in the the field of biomedicine

(6-9). The large number of free amino and hydroxyl groups in the structure of chitosan are

available for crosslinking, being functionalized and has been used for numerous chemically

modified applications (10, 11). In previous studies, chitosan nanoparticle (CSnp) has been

confirmed to provide a significant improvement in root canal disinfection by effectively

eliminating the residual adherent/nonadherent bacteria/biofilms and inactivate bacterial

endotoxins (12, 13), inactivate dentin-bound-LPS (14), stabilize dentin matrix (15), and enhance

stem cell adherence (16) with great potential to be integrated into endodontic procedures.

Crosslinked CSnp also stabilized dentin matrix by resisting host/bacteria mediated enzymatic

degradation and increasing mechanical toughness of dentin (15, 17). In dentin collagen, positively

charged CSnp interacts with the negatively charged dentin collagen, coating the collagen fibrils.

Also, it has been shown that the free reactive groups of chitosan can interact to form chemical

bonds with collagen during crosslinking (10, 18).

Primary nanoparticles tend to aggregate into clusters up to several microns in size (19, 20). Thus,

the usage of stable nanoparticle dispersions is often a prerequisite to maintain their

physicochemical properties during application. This will prevent nanoparticle aggregation in

dispersing media while applied in root canals. Figure 2.1 demonstrates the states and

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configurations of particles when dispersed in liquids. Most of NPs have the tendency to aggregate

once they are hydrated (21). According to the classical DLVO (Derjaguin-Landau-Verwey-

Overbeak) theory of aggregation in colloid science, the sum of attractive and repulsive forces

determines aggregation. This theory presents the only two forces dominating interactions between

particles: van der Waals attractive and electrostatic double layer forces (20). After dispersing

nanoparticles in solution, they would remain as singlets or form agglomerates/ aggregates,

surrounded by an electrical double layer (Fig. 2.1). Nevertheless, if the particle size is lower than

100nm, Brownian motion due to random collisions between the solvent molecules and the particles

may control the long-range forces between individual nanoparticles causing collision between

particles, which makes the interaction more unpredictable (20).

The physical dimension of aggregates formed can affect the reactive surfaces influencing the

reactivity, toxicity, fate, and transport of the nanoparticles (20). The ionic strength, surface charge,

temperature, and pH value of nanoparticle solution would influence the kinetics of NPs in

dispersing media, which affect the stability of the NPs in aqueous solutions (19, 21). The general

criterion to prepare stable dispersion is to increase repulsive forces between particles that the

agglomeration is suppressed or kinetically slow. The zeta potential and the thickness of electrical

double layer are the two important properties of the electrostatic repulsive force between dispersed

particles (19).

Before investigating the delivery of nanoparticle into root canal system, the optimal

concentration/formulation which allows the NPs to remain stable and prevents their aggregation

was determined and evaluated in this section. The particle size and the zeta potential of the CSnp

solution were determined using dynamic light scattering (DLS), and the formulation of the

application was determined according to the surface characterization using field emission scanning

electron microscopy (FESEM) and nanoindentation based method.

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Figure 2.1. Primary particle and aggregated particles in the dispersion (19).

2.3 Materials and Methods

2.3.1 Stabilization of concentration of CSnp solution

Chitosan nanoparticles (CSnp) were synthesized by ionic gelation as described in the literature

(22). In brief, chitosan (low viscosity, Sigma-Aldrich, St Louis, MO) was dissolved in an acetic

acid solution (0.1%) at the concentration of 1.2 mg/ml and the pH was raised to 5-5.2 with 10 N

NaOH. 0.1% sodium triphosphate pentabasic (TPP) solution was prepared in distilled water and

mixed with chitosan solution (1:3 v/v) under magnetic stirring at room temperature. The solution

was centrifuged at 15000 rpm, 20°C for 30 minutes to separate the nanoparticles, then washed

twice with distilled water and freeze dried (-20°C) for 24 hours. The CSnp obtained ranged 80-

120 nm in size as determined using field emission scanning electron microscopy (FESEM) (Quanta

FEG 250, FEI, Hillsboro, OR).

Chitosan nanoparticles conjugated with rose bengal (CSRBnp) were synthesized by conjugating

CSnp with RB. CSnp was synthesized according to the method reported in an earlier work and

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chemically crosslinked to RB using N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (EDC 5

mM) and N-Hydroxysuccinimide (NHS 5 mM) (23). The CSRBnp formed were dialyzed (Sigma,

cellulose tubing, cut off 12000-14000 g/mol) for 1 week, the filtrate was then freeze-dried starting

at −80 °C.

CSnp were dispersed in de-ionized water in four different concentrations, 0.1, 0.5, 1.0 and 2 mg/ml.

The solutions were sonicated before the measurement. The change of CSnp averaged

hydrodynamic diameter (Dh) over time was measured using dynamic light scattering (DLS)

(Zetasizer, Nano ZS, Malvern Instrument, UK) operating with a laser at a wavelength of 633 nm.

The Dh measurement was monitored over a time-period from 1 to 60 minutes with 5-minute-

interval (21, 24). The zeta potential and dispersity of each concentration of CSnp solutions were

determined as well.

2.3.2 Characterization of dentin surface conditioned with optimized

formulations of CSnp

CSnp in unmodified and functionalized forms can be employed to improve antibacterial,

mechanical, and chemical characteristics in the root canal dentin (17). The effect of CSnp to

improve the resistance to enzymatic degradation and increasing the mechanical properties of

dentin collagen by chemical crosslinking and simultaneous incorporation of water-soluble

carboxymethyl chitosan (CMCS) was confirmed previously (17). In this experiment, the

concentration and the formula of CSnp solution were been evaluated to determine the favorable

coating quality on root dentin surface.

Eight upper incisors were decoronated and the root canals of the specimens were enlarged to size

F3 (Tip size 30/variable taper, ProTaper Universal, Dentsply Tulsa Dental Specialties, TN). 6ml

of 2.5% sodium hypochlorite (NaOCl) solution was used to irrigate the canals during cleaning and

shaping. The specimens were sectioned to preserve the coronal third (6mm from orifice) of the

root then split to half along buccal-lingual direction with a slow-speed micromotor. The dentin

sections were polished with SiC abrasive paper from mesh number of #400, 800, 1200, and 2500

followed by final polishing performed with abrasive cloth and diamond particle suspensions of

66

size 6 and 0.05 m (Buehler, Illinois Tool Works Inc., Lake Bluff, IL) to produce a highly polished

surface without imperfections. Sixteen dentin specimens (N=16) were cleaned and sonicated in

17% EDTA for 3 minutes followed with deionized water for 3 minutes at room temperature.

The dentin sections (n=4/each group) were applied with different formula of solutions containing

CSnp and observed under field emission environmental scanning electron microscopy (FESEM,

Quanta FEG250, Oregon, USA.) to examine the surface mechanical characteristics.

1. Group-1: Control, specimens were kept in 100% humidity after the abovementioned

procedures.

2. Group-2: CSnp, each dentin specimen was immersed in CSnp in 1% water soluble

carboxymethyl chitosan (CMCS) solution (1mg/ml) for 30 minutes and kept in 100%

humidity before testing.

3. Group-3: EDC-crosslinked-CSnp, each dentin specimen was immersed in CSnp in 1%

CMCS solution (1mg/ml) for 30 minutes followed with 2 ml 1-Ethyl-3-(3-

dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC/NHS) (4:1, 33mM)

crosslinker for 8 hours. Then washed with deionized water 3 times and stored in 100%

humidity for testing and used in a week.

4. Group-4: PDA-crosslinked-CSnp, CSRBnp were dispersed in water and applied on dentin

surface followed by being activated with a non-coherent light for 10 mins (540nm, 25

J/cm2). After washing, specimen was immersed in CSnp in 1% CMCS solution (1mg/ml)

was applied for 15 mins followed by immersed in RB solution (10 M) for 15 mins. The

specimen was again exposed to the non-coherent light for 10 mins then washed twice.

Samples were kept in 100% before being tested and used in a week.

Two dentin specimens from each group (n=2/each group: one control, three experimental groups)

were subjected to FESEM and another two were sent to nanoindentation. The dentin specimens

were tested under nanoindenter before/ and after treatment in each experimental group, to serve as

its own control for minimizing the variation among individual biological sample.

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2.3.2.1 FESEM evaluation

The dentin specimens were stabilized with a graphite glue on the stand with root canal surface

facing up before being stabilized on the stage in the chamber of microscopy. Images of the root

canal surfaces were taken from low magnification (1000) to high magnification (20 000) under

low vacuum mode with 100 Pa/Voltage 10 kV to evaluate the dentin surface coated with CSnp in

each formulation.

2.3.2.2 Nanoindentation

Nanoindentation testing was performed using a UNHT3 NanoIndenter (Anton Paar, Montreal,

Quebec, CA) equipped with a Berkovich diamond indenter with a 100nm tip radius. The indenter

and probe system were carefully calibrated (the tip area function of the probe tip) by making

repeated indents on a standard fused quartz surface. Specimens were attached to an 18mm sample

mounting metal disc using cyanoacrylate glue (Krazy glue original).

The specimen was subjected to a maximum load of 1 mN in a rate of 3 mN/min with 30s holding

period, which resulted in the penetration of indenter in around 250-350nm. During each test, a 6

7 grid of total forty-two indents, spaced apart at approximately 10 μm were made at root canal

surfaces. The starting points for the indent lines were selected using scanning probe microscopy

to ensure that most of the indents were within the zone being tested. The test area was scanned and

imaged post-indent to confirm that the test was performed on intertubular dentin. The indentation

which was made on dentinal tubules or close to dentinal tubules was excluded from analysis.

Indentation depths varied between 200 to 700 nm of post-treatment. The indentation test data

including hardness and elastic modulus were collected and analyzed. Values of hardness and

elastic modulus from each point were collected and analyzed under paired-sample t test to compare

the difference between treatment (before and after) on dentin at 95% confidence interval (SPSS

Statistics version 20.0, IBM Corp., Armonk, NY).

68

2.4 Results

2.4.1 Stabilization of concentration of CSnp solution

There was a minor decrease in the Dh of CSnp in all concentrations (0.1, 0.5, 1, 2 mg/ml) over

time, especially after 30 minutes. The difference in Dh between 0 minute and 60th minute was

around 50 nm (Fig. 2.2). The CSnp particle size distribution in each concentration was shown in

Figure 2.3. The intensity of the main population of particles decreased with the increase of the

concentration (Fig. 2.3). The average particle size, poplydispersity index (PDI) and zeta-potential

were given by DLS (Zetasizer, Nano ZS, Malvern Instrument, UK). Further analysis was carried

out with OriginLab 8.1 Peak Analyzer (Table 2.1).

2.4.2 Characterization of dentin surface with optimized formulations

2.4.2.1 FESEM evaluation

The root canal dentin surfaces coated with CSnp in each formulation are shown in Figure 2.4.

Crosslinking the dentin surface following the CSnp application resulted in favorable dentin surface

of blocked dentinal tubules and firming intertubular dentin.

2.4.2.2 Nanoindentation

The results of hardness and elastic modulus of 3 experimental groups with its own control are

shown in Table 2.2. The dentin surface treated with only CSnp (Group-2: CSnp) showed increased

hardness and decreased elastic modulus (p < 0.05). However, the hardness and elastic modulus

reduced significantly after EDC crosslinking CSnp on dentin surface (p < 0.01); while the elastic

modulus slightly increased after PDA crosslinking CSnp (p < 0.01). Figure 2.5 shows the

biomechanical response of three treatments (Group-2, 3, 4) compared with its own control.

69

Figure 2.2. The measurement of averaged hydrodynamic diameter (Dh) of CSnp in the concentrations from

0.1 to 2 mg/ml of CSnp dispersion over 60 minutes.

Figure 2.3. The hydrodynamic distribution of CSnp in water with four different concentrations, measured

by Zetasizer.

70

Table 2.1. The characteristics of dispersion at each concentration are listed, as well as the parameters

resulted from peak analysis from Fig. 2.3.

71

Figure 2.4. The dentin surfaces treated with CSnp (B), EDC-crosslinked-CSnp (C) and PDA-crosslinked-CSnp

(D) compared with control (A) in 5k and 20k X magnifications.

72

Table 2.2. The hardness (MPa) and elastic modulus (GPa) resulted before/after each treatment

demonstrated statistically significant (p < 0.01). There was no significant difference of hardness in PDA-

crosslinked-CSnp (blue) (p > 0.05).

Figure 2.5. The load-displacement curves resulted from each group by nanoindentation. The surface

treated with CSnp (A) and PDA-crosslinked-CSnp (C) showed stiffer behavior compared to control. It

resulted in softer behavior in specimens treated with EDC-crosslinked-CSnp (B).

73

2.5 Discussion

The van der Waals attractive forces and repulsive electrostatic double layer forces control the

stability of the particles in a dispersion (20). To produce a stable dispersion, an external force, for

instance, ultrasonication, is commonly used technique to disperse agglomerates pulling the liquid

apart to form evacuated cavities or cavitation bubbles. Bath sonication and probe sonication are

frequently utilized ultrasonication methods and it is suggested to expose the dispersion to bath/ or

probe sonication for 1-2 minutes during preparation (19, 25).

Most nanoparticles show a tendency to aggregate once they are dispersed in a solution (21). This

may be the reasons for the smaller size of nanoparticles when evaluated under SEM or TEM (or

closer to the values reported by vendor) than the hydrodynamic diameter of particles. Dynamic-

light-scattering (DLS) is an established method to access the aggregation kinetics of nanoparticles

in dispersing media over time (21, 24, 26, 27). The dynamic information of particles is derived

from the autocorrelation of the fluctuated scattering intensity caused by small molecules/particles

under Brownian motion in the dispersion. Studies also presented that larger size of particle

distribution resulted from DLS was due to the hydrodynamic shell and even the small amount (1-

2% of volume) of larger particles can significantly influence the DLS derived particle size

distribution (28, 29). This could be the reason of larger size of 350-500 nm of CSnp resulted from

DLS in current study compared to the smaller size derived from TEM (80-120nm) (8, 22). Findings

from the current study showed that the size of CSnp did not increase over time in all four

concentrations. However, it decreased approximately 50nm after 30 minutes of evaluation.

Because the size of CSnp was stable during observation. This result was consistent with the finding

of Rampino et al. that the size of CSnp was increased by 5% “after” the first hour to the next 24

hours and slightly increased by 8% in total in one month (27). Thus, it could be suggested that

there may not be obvious aggregation of CSnp dispersion in the tested formulations.

The value of polydispersity index (PDI) represents the dispersity of the solution (Table 2.3) (30).

It is a measure of the heterogeneity of sizes of particles/molecules in a mixture (30, 31). The value

of the dispersity of CSnp solution can be correlated to the size distribution of particles in each

concentration of CSnp solution (Fig. 2.3, Table 2.1) (32). It was moderate-dispersed (PDI: 0.1-

74

0.4) in the solution of 0.1 and 0.5 mg/ml. The solution of 0.1 mg/ml showed that 100% of the

particles distributed in a close range of size, while there were two peak indexes showing 85% of

particles distributed in around 496nm and 15% of them were approximately 5m. Moreover, in

the concentration above 1mg/ml, the CSnp solution presented broad-polydispersity, showing 3

different ranges of particle size around 100nm, 500nm and 5m.

Zeta potential is the electrokinetic potential in a colloidal dispersion, which is in the interfacial

double layer at the location of slipping plane relative to the point in the bulk fluid away from the

interface (33, 34). Zeta potential is also explained by the potential difference between the

dispersing media and the stationary layer of fluid surrounding the particle (34). This potential value

represents the stability of a dispersion. Also, this electrostatic stabilization is more efficient when

the ionic strength is low, especially when it is lower than 0.1M. The magnitude of the zeta potential

indicates the degree of electrostatic repulsive forces between particles in the solution.

Comparatively, the attractive forces may exceed the repulsive forces in the dispersion of low zeta

potential resulting in the agglomeration/flocculation of particles (Table 2.3) (35, 36). The zeta

potential of CSnp determined in previous studies was 30-35 mV (8, 15, 37). In the current study,

the zeta potential of CSnp solution was around 27mV and it dropped to 17mV when the

concentration reached 2 mg/ml. Charge density is a measure of the amount of electric charge per

unit length/surface area/or volume (38). It associates with the electrostatic interaction between

cationic CSnp and anionic microbial cell membrane, as well as the stability of the CSnp solution

(9, 12, 37). In addition, higher concentration would enhance the efficacy of antibacterial effect and

the delivery in root canal system. Therefore, the optimal concentration of CSnp dispersing media

(in water) was determined as 1 mg/ml for the future applications. For the experimental purpose,

the CSnp dispersion was determined to be used within 30 minutes after the preparation subsequent

to 1-minute sonication.

75

Table 2.3. Approximate values for zeta potential and dispersity parameters (30, 35).

CSnp hold significant potential to improve the root canal disinfection. Dentin treated with

nanoparticles resulted in significantly reduced bacterial adherence as well as ability to disrupt root

canal biofilms. The proposed antibacterial mechanism involves the electrostatic interaction of

positively charged CSnp with negatively charged bacterial cell membranes leading to altered cell

wall permeability, rupture of cells and leakage of proteinaceous and other intracellular components

(9, 12). CSnp eliminates biofilm on a concentration- and time-dependent manner. It also retains

their antibacterial properties after aging for 90 days in saliva (8). It is crucial to note that the tissue

inhibitors such as pulp and serum albumin inhibits the antimicrobial effect of CSnp. While dentin,

dentin matrix and lipopolysaccharides would not affect the efficacy of CSnp. Chitosan has a

structure similar to glycosaminoglycan, the component of extracellular matrix and is therefore

used to reinforce the collagen constructs (39). Studies demonstrated that the oligosaccharides of

chitosan effectively inhibited the metalloproteinases (MMP) -2 due to the chelating property of

binding the Zn2+ (40). These characteristics of CSnp would add in stabilizing dentin matrix.

Dentin crosslinking is another method used to improve mechanical characteristics of hard tissue.

Dentin crosslinking induces the covalent bonds between collagen fibrils in order to improve its

stability including enhanced mechanical properties and the resistance to host-derived MMPs/

bacterial proteases. EDC is one of the chemical crosslinker which links the carboxylic acid groups

with amino groups of protein molecules to form stable amide bonds (41). Photodynamic

crosslinking is a rapid process that occurs via the production of singlet oxygen or radicals by light-

activated photosensitizers. The singlet oxygen interacts with photo-oxidizable amino acid

residuals and the photo-oxidized products react with normal/ or photo-altered residues in another

76

protein molecules to resulting in the crosslink (42). Previous study showed that crosslinking

demineralized dentin beam and mineralized dentin disc with acid-etched surface, with 0.5 M EDC

for 1 min resulted in 1.5-3 times higher stiffness compared with the samples treated with water

(43). Shrestha et al. also demonstrated enhanced tensile strength and toughness resulting from

chemical and photodynamical crosslinking. However, these investigations did not provide

information on the surface properties of such nanoparticle stabilized dentin (17, 23).

The elastic modulus of intertubular dentin is around 16-21 GPa. It may be even lower (3-19 GPa)

at regions as close as 500m from the pulp (44-46). There was a variation in elastic modulus

between cervical, middle and apical dentin of root, which showed 3-10 GPa from the cervical/

middle root including inner and outer dentin (47). The averaged elastic modulus of root canal

dentin from current study before crosslinking was 8-10 GPa, which was comparable with previous

studies. The mean hardness ranged in 300-400 MPa was marginally higher than the data obtained

from Kinney et al. may be due to the difference in the parameters set during measurement (48).

The reduced hardness and elastic modulus of EDC-crosslinked-CSnp dentin surface resulted

mainly from EDC crosslinking. It was not consistent with earlier reports, yet was similar to the

unpublished data of Oryan et al., which demonstrated softer surface in EDC-crosslinked scaffolds

(49). Further investigation is required to explain this mechanism. Conversely, dentin surface

treated with PDA-crosslinked-CSnp resulted in slightly higher mean of elastic modulus (12 GPa),

may due to the induced inter-/intra- molecular covalent bonds between collagen fibrils (50).

Crosslinking CSnp onto dentin matrix (dentin collagen) with water soluble chitosan (CMCS),

CSnp with CMCS will serve as hydrophilic spacer/fillers forming insoluble complex of CSnp-

collagen preventing the brittle behavior of materials that is found subsequent to crosslinking (15,

23, 51). The combination of CSnp incorporated crosslinking facilitates/amplifies molecular bonds

between collagen molecules, enhances the resistance to collagenase degradation, and improves

energy distribution/absorption. Crosslinking applied after CSnp conditioning on dentin surface

may also retain the CSnp coating on the dentin matrix to prolong the efficacy of CSnp. Hence, the

dentin surfaces of coated CSnp with 1mg/ml CSnp solution followed by crosslinking presented

homogeneous and denser appearance of root canal dentin matrix with ideal physical/ chemical

properties of CSnp, which would be the optimal formulation for future applications.

77

2.6 Acknowledgement

Funding from the University of Toronto startup, Natural Sciences and Engineering Research

Council - Discovery grant, Canadian Foundation for Innovation, and Foundation of Endodontics

are gratefully acknowledged. The authors are also thankful to Drs. Suja Shrestha and Annie

Shrestha for their supports and knowledge inputs in the experiments regarding nanoparticles

synthesis and crosslinking procedures.

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Chapter 3

Characterizing Fluid-dynamic Parameters with Activated

Microbubbles for Root Canal Dentin Coating with Nanoparticles

Fang-Chi Li, Suraj Borkar, Arun Ramachandran, Anil Kishen

83

3.1 Abstract

Introduction Activated micro-bubbles (MB) have the potential to deliver nanoparticles in complex

micro-spaces such as root canals. The objective of this study is (1) to determine the fluid-dynamic

parameters associated with ultrasonic, sonic and manual activation of MB in simulated-root canals,

and (2) to assess the effectiveness of surface coating formed by delivering chitosan nanoparticle

(CSnp) using activated MB within root canals in extracted teeth.

Methods Stage-1: Polydimethylsiloxane-models were fabricated to determine the physical effects

of MB agitated manually (MM), sonically (MS), and ultrasonically (MU). Spherical-tracer-

particles were utilized to visualize and record the fluid motion using an inverted-microscope linked

to a high-speed camera. The velocity, wall stress and penetration depth were analyzed at regions

of interest. Stage-2: Thirty-five extracted human incisors were divided into seven groups to

evaluate the effectiveness of CSnp delivery using activated MB (MM, MS, MU groups). Field-

Emission-Scanning-Electron-Microscopy and Energy-Dispersive-X-ray were used to characterize

the nanoparticle-based coating on root canal dentin and the degree of dentinal tubule occlusion.

Results Stage-1: Velocity, wall stress and penetration depth increased significantly in MB groups

compared to the control (p < 0.01). Stage-2: 70% of the dentin surface was coated and 65% of the

dentinal tubule was occluded with nanoparticle-based coating in MM, MU and WU groups.

Element analysis displayed the presence of dentin-smear on the root canal surface for MU and WU

groups.

Conclusion Activated MB enhanced fluid-dynamic parameters when compared to water in

simulated root canal model. Manual activation of MB resulted in uniform and significant

nanoparticle-based surface coating and tubule blockage in root canal dentin without dentin smear

formation.

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3.2 Introduction

The principal objective of root canal treatment is to maintain a healthy, functional and natural tooth

structure in the patient’s mouth (1, 2). In spite of various technological advances, predictable

elimination of root canal biofilms and reestablishment of mechanical integrity of remaining tooth

structure still remains to be major challenge in conventional root canal treatment (2, 3).

Accordingly, developing treatment approaches that combines the advantage of antibiofilm efficacy

in root canal as well as improved mechanical characteristics of remaining root dentin is imperative

to enhance predictability in root canal treatment.

Chitosan is a hydrophilic biopolymer with a molecular structure similar to extracellular matrix

components (4). Previous studies have demonstrated that modified/unmodified chitosan

nanoparticles (CSnp) used as an intra-canal medicament would effectively eliminate/inactivate

residual root canal biofilm/endotoxins (5, 6). Besides, crosslinked CSnp stabilized dentin matrix

by resisting host/bacteria mediated enzymatic degradation and increasing mechanical toughness

of dentin (7-9). Nonetheless, one of the tasks associated with the consistent application of

nanoparticles within root canals is to optimize the mode of delivery. Bolus delivery of

nanoparticles into root canals with syringe-needle may not be a favorable option due to the

complexities of the root canal anatomy and the lack of physical stresses generated by this method

(10). Therefore, optimization of the physical effects that promote nanoparticle-dentin interaction

is a prerequisite for their effective delivery of nanoparticles in root canals. Root canal dentin

surface coated with chitosan nanoparticles will aid in effectively translating their antibacterial and

mechanical benefits in root canal treatment.

Micro-bubbles (MB) are composed of micro-sized droplets with protein/surfactant shell, which

are prepared by an oxygen carrier and oxidizer in an emulsion (11-13). Activated MB have the

ability to potentiate bubble dynamics in micro-spaces, resulting in high particle velocity and

stresses from bubble streaming, cavitation and shockwaves (14, 15). These phenomena generated

by the bubble dynamics provided additional forces toward the wall in constrained micro-spaces,

which could presumably be optimized for the effective application of nanoparticles in root canal

treatment. The objective of this study is twofold: (1) to determine the fluid-dynamic parameters

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associated with ultrasonically, sonically and manually activated MB in simulated-root canals. (2)

To assess the effectiveness of CSnp application within the root canals using the above activation

methods in extracted teeth.

3.3 Material and Methods

3.3.1 Characterization of fluid dynamics in simulated root canal model

Simulated Root Canal Model

Root canal models were prepared simulating a prepared-F2 size-main canal (#25/variable taper,

ProTaper Universal, Dentsply Tulsa Dental Specialties, TN) with a 50um width blocked-end side

channel at the position 4 mm below the orifice of main canal (Fig. 3.1). The design was

reconstructed with AutoCAD (R19.1, Autodesk, San Rafael, CA) followed with photolithography

and soft lithography (16, 17) to fabricate a simulated root canal model with polydimethylsiloxane

(PDMS) (MicroChem®, Westborough, MA). The height of canal space in each section of PDMS

replica was 500μm. Two sectional models were aligned under a stereomicroscope and bonded with

plasma-bonding technique, to obtain a single canal model with a canal space of 1mm height (Fig.

1B). Eighteen models were used to assess the fluid-dynamic parameters in water and microbubble-

based formulation (MB) with ultrasonic, sonic and manual activation methods. The velocity,

inertial/viscous stress and the depth of particle penetrating to the side channel were determined.

MB was prepared using a modified combination of oxygen carrier and non-ionic detergent

surfactant described in previous study (14) and characterized in the present study.

Fluid Dynamic Analysis

In the water-manual (WM), water-sonic (WS) and water-ultrasonic (WU) groups, the canal spaces

in were filled with deionized-water containing hollow glass beads (11μm, Corpuscular Inc., NY)

at 106 particles/ml concentration. In MB-manual (MM), MB-sonic (MS) and MB-ultrasonic (MU)

groups, the canal spaces were filled with MB. In groups WM and MM, a gutta-percha point (GP)

(ProTaper Universal, Dentsply Tulsa Dental Specialties, TN) of size F2 was inserted to 1mm from

the apical terminus, following which push-and-pull strokes were applied (amplitude- 5 mm;

frequency-100 strokes/minute) (18). In groups WS, MS, WU and MU, the tip of the sonic (#25/.04,

86

EndoActivator, Dentsply Tulsa Dental Specialties, OK) and ultrasonic (#20/.02, Endo UltraTM,

VISTA Dental Products, WI) activation device was stabilized to 4mm from the root canal orifice

and was agitated. The real-time fluid flow was viewed on an inverted microscope with 10✕

objective lens (Eclipse Tĭ-S, Nikon Instruments Inc.) and recorded at 22000 frame-per-second (fps)

with a high-speed camera (Phantom V711, Vision Research Inc., NJ) for 826 milliseconds (Fig.

1A). The data obtained from this analysis provided a measure of fluid motion at different locations

in the canal with a resolution of 640 ✕ 480 pixels. The fluid-dynamic parameters were determined

by selecting thirty particles per group and by tracing the displacement of each particle as a function

of time using a MatLab routine (MathWorks, MATLAB8.4, MA). The mean velocity and the wall

stress generated by the fluid movement in the main canal space, as well as the Reynolds number

(Re), which is a characteristic measure of the inertial stresses relative to viscous stress in a velocity

field, were calculated using the following equations (19). Similar to the above protocols, the

penetration depth within side channel were also determined.

𝑉 =∑ 𝑑𝑖𝑛

𝑖=1

𝑛 ∆𝑡

𝑅𝑒 =𝐼𝑛𝑒𝑟𝑡𝑖𝑎𝑙 𝑠𝑡𝑟𝑒𝑠𝑠

𝑉𝑖𝑠𝑐𝑜𝑢𝑠 𝑠𝑡𝑟𝑒𝑠𝑠=

ℓ ⋅ 𝑣 ⋅ 𝜌

𝜇

𝐼𝑛𝑒𝑟𝑡𝑖𝑎𝑙 𝑠𝑡𝑟𝑒𝑠𝑠 = 𝜌 ⋅ 𝑣2

𝑉𝑖𝑠𝑐𝑜𝑢𝑠 𝑠𝑡𝑟𝑒𝑠𝑠 =𝜇 ⋅ 𝑣

12 ℓ

Re < 1: shear stress dominated flow

Re > 1: inertial stress dominated flow

(𝑑 = 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑎 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 𝑜𝑣𝑒𝑟 𝑡𝑖𝑚𝑒 𝑖𝑛𝑡𝑒𝑟𝑣𝑎𝑙 ∆𝑡 (𝑚), ℓ = 𝑤𝑖𝑑𝑡ℎ 𝑜𝑓 𝑐ℎ𝑎𝑛𝑛𝑒𝑙(𝑚),

𝑣 = 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 (𝑚

𝑠) , 𝜌 = 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 (

𝐾𝑔

𝑚3), 𝜇 = 𝑣𝑖𝑠𝑐𝑜𝑠𝑖𝑡𝑦(𝑃𝑎 ∙ 𝑠).)

87

Figure 3.1. Schematics of microfluidic experiment set-up. (A) The fabricated device was mounted on an

inverted microscope attached with a high-speed camera for imaging. (B) The dimensions of the simulated

root canal model. The transparent root canal model was mounted on a glass slide with epoxy adhesive.

3.3.2 Assessing nanoparticle delivery and nanoparticle-based coating in

tooth model

CSnp were synthesized using an ionic gelation method as described in the previous literature (20).

The CSnp obtained were approximately 120 nm, and were dispersed in water/or MB at

concentration of 1.0 mg/ml. The Research Ethics Board of the University approved the use of

extracted teeth for this study. Thirty-five freshly extracted human maxillary incisors were

sectioned at cementoenamel junction to obtain standardized roots for further analysis. The root

canals were enlarged to F3 size (ProTaper Universal, Dentsply®, TN) and irrigated with 6 ml of

2.5% sodium hypochlorite, followed by 17% EDTA (Ethylene-diamine-tetra-acetic acid) for 3

minutes. The specimens were then randomly divided into seven groups (n=5/group) as follows:

Control group: root canal was enlarged without the application of CSnp; the experimental groups:

CSnp in deionized-water delivered into the root canals using manual (WM), sonic (WS) and

ultrasonic agitations (WU) as described in the previous section for a total period of 5 minutes. In

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the remaining three groups, CSnp was dispersed in MB and delivered into the root canals using

manual (MM), sonic (MS) and ultrasonic agitation (MU). The root canal was then rinsed with

water and dried gently with paper points.

The specimens were split mesio-distally into two halves to expose the root canal dentin surface

and subjected to Field-Emission-Scanning-Electron-Microscopy (FESEM) (Quanta FEG250, OR)

to ascertain the coating/penetration of nanoparticles on dentin, whereas an Energy-Dispersive-X-

ray (EDX) (EDAX, AMETEK® Materials Analysis Division) analysis was used to determine the

element characteristics of the chitosan constituents to reconfirm the presence of CSnp and the

quality of the nanoparticle-based coating on the root canal dentin surface. The atomic percentage

of carbon (C), nitrogen (N), oxygen (O), phosphate (P) and calcium (Ca), as well as the ratio of

N/C and O/C atomic mass percent from the dentin surface were recorded. Images were taken from

the region of interest, 4-6mm from the coronal edge of root under 1000✕ magnification in each

sample to analyze the percentage coverage of nanoparticle-based coating on dentin surface. The

percentage of dentinal tubules occlusion was quantified with images under 2000✕ magnification.

ImageJ64 (NIH, Bethesda, MD) was employed for both quantifications.

Means and standard deviations were analyzed with one-way analysis of variance (ANOVA) and

post hoc Dunnett’s T3 test. All tests were carried out with IBM SPSS version 20.0 (SPSS, Chicago,

IL) at significance level of 0.05.

3.4 Results

3.4.1 Characterization of fluid-dynamics in simulated root canal model

The size of MB was ranged from 4 to 20 m; the viscosity and density were 0.59 Pa∙s (water: 10-

3) and 980 Kg/m3 (water: 1000) individually. It was found that the particle velocity and wall stress

due to the fluid/bubble dynamics in proximity to the lateral aspect of the agitating tips were

significantly different for different activation methods (p < 0.01) (Fig. 3.2). In WM, MM and MS

groups, the viscous stress was dominated from the fluid movement (Re < 1); while in WS, WU

and MU groups (Re > 1) the fluid dynamic was governed by inertial stress. MU group consistently

generated the highest velocity (0.74 m/s), wall stresses (658.45 Pa) and deepest penetration of

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particles into side channel (149.14 um) (Fig. 3.2). WM produced lowest velocity (0.025 m/s) and

wall stress (0.51 Pa) as well as particles were not pushed into the side channel during agitation.

3.4.2 Assessing nanoparticle delivery and nanoparticle-based coating in

tooth model

The EDX analysis of the dentin surface coating with CSnp demonstrated lower N/C ratio (0.17-

0.3), O/C ratio (0.55) and percent of Ca and P (0.35-4%), when compared to the control group of

irrigated dentin surface (N/C: 0.24-0.38; O/C: 0.88; Ca, P: 4-8%). FESEM images of the dentinal

tubules from none of the experimental groups showed the presence of CSnp within the dentinal

tubules and CSnp was not observed on the dentin surface in the control group (Fig. 3.3A). The

dentin surfaces from each group under 1000✕ and 10,000✕ magnifications were shown in Figure

3.3 (B-D). Significant difference was found among groups of the percentages of CSnp-covered

dentin and dentinal tubules occlusion (p < 0.01). Specimens from the MM, WU and MU groups

demonstrated nanoparticle-based coating for over 70% of the dentin surface covered. While the

specimens from MM group showed the highest percentage of dentinal tubules occlusion (74.82%).

There was no statistically significant difference between the MM, WU and MU groups (p = 0.96,

0.81) (Fig. 3.3E, F). However, the EDX analysis confirmed a layer of CSnp mixed with dentin

smear layer on root dentin surfaces after agitation in both the ultrasonic groups (WU and MU).

Other groups, which utilized manual and sonic agitations, showed nanoparticle-based coating

without dentin smear layer.

90

Figure 3.2. The means and standard deviations of velocity, stress and the particle penetration depth in

each group. (A) The means of velocity showed significantly different between groups (p < 0.01). (B)

Comparison of inertial/shear wall stress between groups presented significantly different (p < 0.01). (C)

Comparison of penetration depth of particles between groups presented significantly different (p < 0.01).

* same letter: p > 0.05 between groups.

91

Figure 3.3 The images of FESEM from MB groups showing the ultrastructure of CSnp coated root canal

dentin and the efficacy of the nanoparticle-delivery on dentin. (A) The sagittal-section of the dentin

samples with exposed dentinal tubules did not show CSnp within the lumen. In 10 000x magnification,

dentin surface showed no CSnp coating on root canal dentin. (B-D) Images from groups of MM (3B),

MS(3C), and MU(3D) in 1000x and 10 000x magnification. (E) The percentage of dentin surface covered

by CSnp was statistically significant between groups (p < 0.01). MM, WU and WM resulted in over 70%

CSnp-coverage of dentin surface, which was significant compared with other groups. (F) The percentage

of dentinal tubules occlusion was statistically significant between groups (p < 0.01). MM demonstrated

highest dentinal tubules blockage (74.82%) but was not significant with WU (69.4%) and MU (67.22%) (p

= 0.09, 0.81). * same letter: p > 0.05 between groups.

92

3.5 Discussion

The fluid dynamic parameters associated with ultrasonic, sonic and manual agitations in water or

MB, and the final static evaluation of the effectiveness of CSnp delivery with these agitation

techniques/media in root canals were assessed in this study. Soft lithography enabled accurate

replication of instrumented root canals with PDMS. This substrate presented substantial optical

transparency allowing high quality visualization and analysis (14, 21, 22). Characterizing the fluid

dynamics on transparent microfluidic model with a high-speed imaging setup provided real-time

visual assessment of the flow patterns and fluid-dynamic parameters at the region of interest (22,

23). The quantification in current study utilized sequentially acquired digital images with a time

interval between two frames set at a speed up to 45.45 microseconds, which enabled precise

tracking of particles between frames. The application of a transparent root canal model to study

fluid-dynamics and an extracted tooth model to evaluate the effectiveness of nanoparticle-based

coating aided in understanding the impact of physical effects generated by different agitation

methods in water or MB on the delivery of nanoparticles in root canals

The current goal for clinical translation of nanoparticles guided endodontics is to achieve a uniform

coating on the root canal dentin surface. A thin layer of nanoparticle-based coating over both

peritubular and intertubular dentin surfaces with sealed dentinal tubules would be ideal for their

clinical benefits. Previous studies have demonstrated that higher degree of stress/strain distribution

and stress concentrations occurred at the cervical region of the root dentin, particularly in the

bucco-lingual direction (2, 24, 25). In addition, the major changes in the stress/strain distribution

in endodontically treated teeth occurred in the inner dentin at the cervical region mainly due to the

loss of intrapulpal hydrostatic pressure and the free-water from the dentinal tubules. It is also

important to realize the density and diameter of dentinal tubules are higher, while the dentinal

tubules occupies 22% of the total volume at the coronal third of the root (26). Hence, the

examination of physical parameters of fluid and the surface coating of nanoparticles were assessed

at the coronal third of root canal. Accordingly, the tip of the ultrasonic / sonic inserts which created

higher velocity and shear stress was placed at the coronal third for both PDMS and tooth models.

(22, 27).

93

The velocity of activated particles in water was highest in the ultrasonic group followed by sonic

and manual groups in present study. This is because of the higher frequency in this group resulting

in a higher flow velocity (28). On the contrary, fluid-dynamics generated by sonic agitation was

less effective compared to the ultrasonic agitation mainly owing to the lower frequency. In addition,

sonically activated insert displayed only one node and antinode, and the oscillation mostly

remained longitudinal when movement is constrained within the micro-space of root canal (29).

Image analysis of the MS group showed that the particles only moved back and forth following

the movement of insert. It did not show the fluid streaming / circulation in simulated root canal

model. These observations in the sonic group may be attributed to (1) the constrained movement

of the insert, and (2) the higher viscosity in the activated MB. This factors would result in the

inadequate nanoparticle-based coating in MS group (Fig. 3.3E, F).

Ultrasonically activated insert induced intense acoustic microstreaming with multiple nodes and

antinodes (30). The magnitude of velocity observed from WU group in the current study (0.23m/s)

was consistent with Layton et al. (0.04-0.12m/s) at similar experimental setting but with higher

vibration frequency (40k vs. 28k Hz) (22). The velocity in MU was three times higher than in

ultrasonic agitation in WU. This could be due to the intensified cavitational bubble dynamics that

was induced by the concentrated applied energy and reduced threshold for bubble formation in the

MU resulting in the large/strong bubble coalescence (14, 15). The microjets generated from the

collapsing cavitational bubbles in MU may also cause rapid fluid streaming and high stress (15).

Re is an important non-dimensional quantity in fluid mechanics, which is applied to distinguish

the inertial forces dominated flow (Re > 1) from the viscous forces governed fluid dynamics (Re

< 1) (31, 32). Viscous force results from the friction generated between different fluid layers, is

influenced by the viscosity of the fluid whereas the inertial force represents the resistance of a fluid

to any change in momentum which is controlled by density and velocity of fluids (19, 31). In

accordance with Re, the activated fluid-dynamics were governed by inertial forces in WS, WU

and MU. The difference of dominated inertial stresses were mainly contributed by velocity since

the density of MB (980 Kg/m3) was close to water (1000 Kg/m3). Also, the highest Re (Re = 194)

was demonstrated in group MU revealing that the fluid flows generated in all the groups were

laminar flow (33). It is too low to generate turbulent flow. The stresses generated from MM and

94

MS were much higher than in WM, WS and WU group owing to the high viscosity (0.59 Pas) of

MB (water: 10-3 Pas).

Optimization of the pattern of fluid flow and the nature of wall stress may facilitate better

nanoparticle delivery and coating on the root canal dentin. In the current study, the highest wall

stress and the percentage of nanoparticle-based surface coverage were derived from manual

agitation of MB. The push-pull motion of GP in manual agitation resulted in increased fluid efflux

sideways and upwards in spaces between the well-fitted GP and the canal wall under viscously-

dominated flow condition (32). This in turn resulted in uniform and highest viscous stresses along

the canal wall. Also, CSnp dispersed in liquid phase of MB agitated manually with a well-fitted

GP resulted in increased particle flux due to the concentration gradient and the decreased distance

between particles and wall (34). Furthermore, when CSnp is compressed towards the canal wall

during manual agitation, it resulted in stronger electrostatic interaction between cationic CSnp and

anionic dentin promoting uniform coating of CSnp on dentin in MM group (5, 35). In WM, the

fluid-dynamics presented lowest velocity and stresses, however, this group produced fair (50%)

CSnp-coated root canal dentin. This observation probably signifies that the vector of

velocity/stress and the increased particle flux generated in the manual dynamic agitation played a

key role in promoting nanoparticle coating on root canal dentin. In addition, a consistent layer of

coating was also observed on the dentin surfaces with ultrasonic agitation in WU and MU group

(Fig. 3.3D). However, the chemical analysis of the coating formed in both groups, displayed CSnp

mixed with newly formed dentin smear layer. This highlights the unintentional removal of dentin

during ultrasonically oscillation and subsequent packing on canal walls, as shown in previous

studies (36). The CSnp-based coating mixed with smear dentin may also compromise the

antimicrobial efficacy in the root canal system. In summary, the findings from this study

highlighted that activated MB enhanced fluid-dynamics in simulated root canals, while the

application of manually activated MB aided in nanoparticle delivery and subsequent nanoparticle-

based coating of root canal wall.

95

3.6 Acknowledgements

Supported in part by a research grant from the University of Toronto startup (AK). The authors

deny any conflicts of interest related to this study. The authors are also thankful to Dr. Ilya

Gourevich for the great help in instructing the usage of FESEM; and to Dr. Lindsey Fiddes for her

help in fabricating the microfluidic devices.

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Chapter 4

Micro-tissue Engineering Root Canal Dentin with Crosslinked

Biopolymeric Nanoparticles for Mechanical Stabilization

Fang-Chi Li, Anil Kishen. Int Endod J 2018 March

100

4.1 Abstract

Aim To evaluate the functional strain distribution pattern in root dentine following canal

preparation and root canal surface engineering with crosslinked biopolymeric nanoparticles using

digital moiré interferometry (DMI).

Methodology Root dentine specimens were prepared, grating material replicated and tested for

10 N to 50 N, compressive loads in a customized high-resolution, whole-field moiré interferometry

setup. Digital moiré fringes were acquired to determine the strain distribution pattern at specific

regions of interest before and after canal enlargement, and dentine surface engineering with a

chitosan nanoparticle-cross-linker solution. Fringe patterns were acquired and strain distribution

pattern in the direction perpendicular to dentinal tubules (U-field) and parallel to dentinal tubules

(V-field) were analyzed with custom digital-image-processing software. Data were analyzed with

a statistical method on trend analysis at 0.05 significance level.

Results Distinct deformation patterns perpendicular to the dentinal tubules were observed in root

dentine. Root canal dentine removal following instrumentation resulted in an increase in strain

distribution, which increased with an increase in applied loads (p < 0.01). The root canal dentine

engineered with crosslinked-nanoparticles demonstrated a conspicuous decrease in previously

increased strain distribution in both coronal and apical root dentine (p < 0.01). A significant

increase in tensile strain in root dentine was observed subsequent to instrumentation in the

direction parallel to dentinal tubules (p < 0.01). There was a significant reduction in the tensile

strain formed at the apical region of the instrumented root dentine following crosslinked-

nanoparticle treatment (p < 0.05).

Conclusions This study highlighted the potential of root canal dentine micro-tissue engineering

with crosslinked-chitosan-nanoparticle to improve radicular strain distribution patterns in

instrumented canals.

101

4.2 Introduction

Tooth structure plays an important mechanical role during chewing. The biomechanical response

of teeth to forces has been used to understand how they respond to functional forces, and to assess

the mechanical integrity of restored root filled teeth. Previous studies utilized destructive

mechanical testing methods to determine the mechanical characteristics of hard tissues such as

dentine. These experiments were carried under large loads, which drastically exceed the elastic

limit of tooth structures. Additionally, these experiments provided mechanical parameters that

were averaged over the entire specimen. Photomechanical techniques such as digital

photoelasticity, digital moiré interferometry, electronic speckle pattern interferometry and digital

image correlation are applied in dental biomechanics to study stress/strain response in intact (1-3)

and restored root filled teeth (4, 5) in a non-destructive manner under physiologically relevant

loads.

Root canal treatment is the treatment of choice to maintain the long-term functional requirements

of a natural tooth. This treatment aims to disinfect the infected root canal systems, besides

preserving the mechanical integrity of the remaining natural dentine (6-8). In spite of all the

technological advances in the field, recent studies and clinical observations have shown that root

filled teeth have a higher propensity to vertical root fractures (9, 10). Previous studies have

highlighted vertical root fractures in 6 to 11% of the extracted root filled teeth (11-13). Several

risk factors such as disease-mediated changes in dentine and iatrogenic alterations of root canal

surface dentine have been linked with this increased propensity of vertical root fracture in root

filled teeth (14).

Nanoparticles (NP) of bioactive polymers such as chitosan has the ability to enhance the

mechanical properties of the dentine matrix. Chitosan nanoparticles also possess structural

similarity to the extracellular matrix glycosaminoglycans (15), and mimic the functions of the

extracellular matrix proteoglycans and glycosaminoglycans, by providing mechanical stability and

compressive strength to collagen (16). Recent studies have shown that synthetic and natural

chemicals that increase the number of inter- and intra-molecular collagen crosslinks would

enhance the fibrillar resistance against bacterial enzymatic degradation and provide improved

102

mechanical characteristics of tissues (17-20). Along similar lines, crosslinked chitosan

nanoparticles (CSnp) have been used to achieve micro-scale tissue engineering of root dentine.

This process of tissue engineering stabilizes the ultrastructure of surface dentine by providing the

tissue with enhanced mechanical characteristics, bioactivity as well as resistance to host/bacteria

mediated enzymatic degradation (21-24).

Digital moiré interferometry (DMI) utilizes the principles of optical interferometry to measure

micro-level deformations on hard tissue surfaces (25). It is a high-resolution technique, which

allows testing of in situ specimens under physiologically realistic loads. It provides whole-field

strain information of specimens in real-time with high-sensitivity of 0.417 micrometer per fringe

order (26, 27). This method has been applied to study the in-plane mechanical and thermal strain

distribution on dental structures (2, 26, 28-30). The purpose of the current study is to employ DMI

to evaluate the strain distribution pattern in root dentine following canal enlargement with

instruments/chemical irrigation and followed by root canal dentine surface engineered with

crosslinked CSnp. The null hypothesis is that the root canal enlargement and micro-tissue

engineering of root canal dentine would have no effect on the mechanical strain response in the

root.

4.3 Materials and Methods

4.3.1 Sample preparation

The Research Ethics Board of the University approved the collection and use of ten freshly

extracted, non-carious human maxillary incisors for this study. These teeth were transilluminated

before testing to exclude possibilities of cracks or damages during extraction and stored in

deionized water at 4°C. The teeth were decoronated to obtain 12-14 mm length root specimens

(Fig. 4.1). The root canals of the specimens were enlarged with K files (K-Flexofiles, Dentsply

Sirona Endodontics, Tulsa, OK, USA) to size 10 (E10 group) (n=5) and size F3 (ProTaper

Universal F3, Dentsply®) (EF3 group) (n=5). Six mL of 2.5% sodium hypochlorite solution was

used to irrigate the canals during cleaning and shaping, which was followed with 1 mL of 17%

EDTA solution for 3 min as the final irrigant. The tooth specimens were prepared by grinding the

mesial and distal surfaces on wet emery papers of grit size 320, 600, and 800, under constant

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running water to prepare slab shaped, parallel-sided, facio-lingual sections with a uniform

thickness of 2.5 mm for the strain analysis (Fig. 4.1).

4.3.2 DMI analysis

The tooth specimens for DMI analysis were always maintained in 100% humidity environment. A

four-beam DMI setup was used. A high frequency cross-line grating (1200 lines/mm), which is

the deformation sensing element of a DMI was replicated on one sagittal side of the tooth section

using an epoxy adhesive (PC-10, Measurements Group, Raleigh, NC, USA) at room temperature.

During experiments, the specimen grating was interrogated with a reference grating (f = 2400

lines/mm), formed by the interference of two mutually coherent beams incident from a diode laser

(532nm) on the specimen plane at a fixed angle. Moiré fringes resulting from the interference

between the deformed specimen grating and virtual grating were used to determine the in-plane

deformation in dentine (Fig. 4.2). During experiment, compressive loads of 10, 20, 30, 40, and

50N were applied on the coronal edge of the specimen, along the long axis of the tooth. A high-

resolution charge-coupled device (CCD) camera with a spatial resolution of 758(H) ✕ 581(V)

pixels was used to digitize and record the moiré fringe patterns obtained for further analysis. The

normal strains at selected region of interest along different lines (Fig. 4.3) from both axial and

lateral direction were calculated as described below (25, 27):

The displacements in optical metrology is described as:

U (x, y) = g𝑁𝑥 (𝑥, 𝑦) = 1

𝑓𝑁𝑥 (𝑥, 𝑦)

V (x, y) = g𝑁𝑦 (𝑥, 𝑦) = 1

𝑓𝑁𝑦 (𝑥, 𝑦)

where the fringe orders (N) are taken at the corresponding x, y points and where g and f represent

the reference grating. Strain is determined from the displacement fields by using the relationships

for engineering strains:

U-field (axial strain): 휀𝑥= 𝜕𝑈

𝜕𝑥 =

1

𝑓[

𝜕𝑁𝑥

𝜕𝑥] (axial strain: strain perpendicular to dentinal tubules)

V-field (lateral strain): 휀𝑦= 𝜕𝑉

𝜕𝑦 =

1

𝑓[

𝜕𝑁𝑦

𝜕𝑦] (lateral strain: parallel to dentinal tubules)

After the first strain measurements with DMI, root canals of each sample from Group - E10 were

104

enlarged to a size 50 K-file (Group - E50). The root canal space of each sample in Group EF3 were

conditioned with CSnp (1 mg/mL) in water soluble carboxymethyl chitosan (CMCS) solution and

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC/NHS) (4:1)

crosslinker for 8 hrs (named EF3-crosslinked CSnp). The concentration of CSnp was based on the

stability of nanoparticle in solution to maintain appropriate physical properties and prevent

aggregation (31). Size 20 paper points were used to dry the canal after conditioning. All the

specimens were tested again to determine the strain distribution with DMI. In this way, each

sample served as its own control (E10, EF3) before being treated in experimental groups (E50,

EF3-crosslinked CSnp).

The acquired digital moiré fringe patterns were used to determine the in-plane normal strains

(axial/perpendicular to dentinal tubules (U-field) and lateral/parallel to dentinal tubules strains (V-

field)) in the region of interest along the root dentine as shown in Figure 3C. The strain value and

distribution were calculated and analyzed at 10, 20, 30, 40 and 50N loads using image-processing

software (Moiré Analysis V0.949, Beijing, China). Strain values at six relative points of interests

in both coronal 6mm and apical 4mm were recorded from each sample (Fig. 4.3C).

Strain values generated from 0 to 50N in each specimen before and after instrumentation and CSnp

conditioning (E10 and E50; EF3 and EF3-Crosslinked CSnp) were collected and analyzed using a

statistical method on trend analysis. The induced strain values from each point was tested by linear

regression and the slop values from the same region/ field-direction were subjected to subsequent

paired-sample t test to compare the difference between treatment on dentine at 95% confidence

interval (SPSS Statistics version 20.0, IBM Corp., Armonk, NY).

105

Figure 4.1. Steps in specimen preparation. Tooth was first decoronated to obtain 12-14mm length.

Subsequently the mesial and distal surfaces were ground to obtain a 2.5mm thick sagittal slab shaped root

dentine specimen.

Figure 4.2 The digital moiré interferometry experimental setup. It consisted of two mutually coherent

light beams from a green diode laser (λ=532 nm), which were incident on the specimen grating mounted

on a customized loading jig.

106

4.4 Results

The U-field and V-field moiré fringe analysis revealed that the compressive loads resulted in a

distinct distribution of normal strain along the axial and lateral directions in root dentine. There

was a generalized distribution of compressive strain in root dentin, which increased gradually with

increase in applied loads in both axial and lateral directions (Fig. 4.3). The strain distribution in

the direction perpendicular to dentinal tubules (U-field) increased significantly with root canal

enlargement in the coronal region (p < 0.01). This strain difference was not significant in the apical

region of root (p = 0.10) (Fig. 4.4). This alteration in strain distribution was noticeable over coronal

region in the axial direction. The strain in the direction parallel to dentinal tubules (V-field)

increased with root canal enlargement in both coronal and apical regions of the root. A distinct

shifting of strain from a compressive-trend to a tensile-trend was observed with increase in applied

loads after root canal enlargement. The rate of increase in strain in this direction was however

significant at the coronal aspect of the root (p < 0.01). Figure 4.4 shows the strain values for applied

loads from 0N to 50N in both directions.

In the U-field analysis the radicular strain at both the coronal third and the apical third decreased

significantly following CSnp-crosslinking treatment of root canal surface dentine (EF3-

crosslinked CSnp), when compared to the specimens with prepared root canals, without any

nanoparticle-crosslinking treatment (EF3) (p < 0.01) (Fig. 4.5, 4.6). Figure 4.5 shows the U-field

fringe pattern and colour map of the radicular strain distribution patterns before and after root canal

surface micro-tissue engineering with crosslinked CSnp. More bending response was presented at

the cervical area of the root whereas lesser compressive response showed at the middle and apical

regions in micro-tissue engineered dentin compared to its control.

The variations in strain distribution with increase in load at the coronal and apical regions are

shown in the Figure 4.6. The root strain and the rate of strain increase with loads formed after root

canal surface micro-tissue engineered with crosslinked CSnp was significantly less, when

compared with the root strain formed after instrumentation for loads ranging from 10N to 50N in

direction perpendicular to dentinal tubules at both coronal and apical regions of interest (p < 0.01).

107

In the direction parallel to dentinal tubules (V-field), root canal surface engineered with

crosslinked CSnp did not result in significant difference in strain at the coronal region (p = 0.24)

(Fig. 4.6A). Nevertheless, this treatment resulted in significant variation in strain at the apical

region (p < 0.05). A distinct change in the nature of strain distribution from a post-instrumentation

tensile strain to a compressive strain distribution was observed at the apical region following

crosslinked CSnp treatment (Fig. 4.6B).

Figure 4.3. Moiré fringe patterns in root dentine. (A) U field at 10N load, (B) V field at 10N load, (C) U field

at 50N load, (D) V field at 50N load.

108

Figure 4.4. Strain values in U and V field generated at the coronal (4A) and apical third (4B) of root before

(E10) and after (E50) root canal enlargement, when compressive loads ranging from 10N to 50N were

applied. Strain in negative value represented the compressive deformation, while tensional deformation

was shown as positive. The U field analysis showed a significant increase in root dentin strain under the

same loads after root canal enlargement at coronal region (p < 0.01) (Apical: p = 0.10). Distinct tensile

strains were formed in the root dentin in V field at coronal third (p < 0.01) (Apical: p = 0.08).

109

Figure 4.5. Root dentine sample with canal size F3. (A-1, 2, 3) Fringe patterns in U-field at 10, 30 and 50N

load in root dentine before micro-tissue engineering with crosslinked-CSnp. (A-4) Color map obtained the

fringe analysis showing the whole-field displacement of sample before micro-tissue engineering with

crosslinked-CSnp. (B-1, 2, 3) Fringe patterns in U field at 10, 30, and 50N load showed in micro-tissue

engineered root dentine. (B-4) Color map obtained the fringe analysis showing the whole-field

displacement of sample after engineering the root dentine with CSnp.

110

Figure 4.6. Strain values in U and V field generated from coronal (6A) and apical third (6B) of root before

and after micro-tissue engineering with crosslinked-CSnp on root dentine surface. The root strain formed

after root canal surface engineered with crosslinked-CSnp was significantly less compared with root strain

obtained after instrumentation for loads ranging from 10N to 50N, in U field (p < 0.01). Moreover, apical

strain in V field resulted in a shift from tension to compression on micro-tissue engineered root dentin (p

< 0.05).

111

4.5 Discussion

Digital moiré interferometry (DMI) is an established technique that is used to evaluate the

biomechanical response of dentine (2, 32-34). This technique allows the determination of strain

distribution along specific regions of interest in root dentine for loads within physiological limits,

in real-time. The process of application of specimen grating on sample surface was also

standardized by earlier investigations (26, 27). Moiré measurements are performed routinely in the

interferometric domain with fringes representing subwavelength displacements per contour. Since

moiré responds only to geometric changes, it is equally effective for elastic, viscoelastic, and

plastic deformations, for isotropic, orthotropic and anisotropic materials, and for mechanical,

thermal, and dynamic loadings (25). DMI offers a resolution of 0.1μm with accuracy in strain of

approximately 50 strain, which cannot be detected using conventional mechanical testing (25).

DMI permits determination of small-range in-plane deformation in miniature sized biological

samples such as teeth. However, owing to their high sensitivity and the variability in the material

characteristics of dentine that exist among individuals, stress or strain distribution pattern and the

trend of the changes are generally considered more beneficial than the absolute strain value (1,

26).

The functional deformation in dentine hard tissue is predominantly in-plane (x, y) than out-of-

plane (z) (1, 35, 36). The facio-lingual slab shaped specimens utilized in this study allowed to

preserve the major bulk of the tooth (dentine) in the sagittal plane. The validity of this specimen

to evaluate functional in-plane stress/strain distribution in incisors has been previously ascertained

(1, 26). The tooth crown was excluded in the root model so as to avoid any non-axial loads acting

on the root and circumvent the influence of access cavity / access restoration on the radicular strain

distribution (37). Since the primary aim of this study was to examine the biomechanical response

of root canal surface engineering with crosslinked CSnp, only root aspect of the tooth was

considered for the analysis. This model would allow us to examine the biomechanical response of

root dentine without additional variables from the crown such as the access cavity design, type of

coronal restoration or effect of cuspal flexure. Both cervical and apical aspects of the root dentine

were chosen as the regions of interest for strain analysis. This is because the functional stress/strain

from the tooth to the surrounding bone is predominantly distributed at the cervical root dentine (1,

112

35). On the contrary in root filled teeth, higher degree of functional stress/strain is distributed

toward the apical aspect of the root resulting in increased root flexure. This altered biomechanical

response at the apical root dentine with root canal treatment, have been previously implicated as

one of the risk factors for vertical root fracture in root filled teeth (38, 39).

Previous biomechanical experiments demonstrated bending stress distribution in the cervical and

middle third of the root dentine during chewing. The apical aspect of root dentine showed a

noticeable reduction in stress/strain distribution (35, 40). The strain distribution patterns obtained

in the current study were consistent with the previous investigations. The strain displayed in root

dentine along axial direction (U-field) was higher than the strain formed along lateral direction (V-

field). This directional variation in strain distribution was attributed to the orientation of the

dentinal tubules in root dentine (26, 41). The current findings highlighted more than 3 times

increase in the load induced normal strains distribution at the cervical region after canal

enlargement to size 50 when compared to size 10. This indicated that the loss of root canal dentine

resulted in greater deformation of root. On the other hand, surface-engineering the instrumented

root canal dentine with crosslinked CSnp significantly reduced the functional strain distribution in

the cervical and apical root dentine. Compressive loads ranging from 0 to 50N were applied in this

study to simulate a range of physiological level chewing forces (42, 43).

Preserving the underlying biomechanical response of a structural hard tissue is crucial for its

“damage free” longevity upon function (29, 44). Alteration in the functional stress/strain

distribution pattern in a hard tissue will increase the risk of material damage and probability of

structural failure with time. In root canal treatment, dentine loss can result from both iatrogenic

procedures and non-iatrogenic causes. While the purpose of root canal enlargement is to facilitate

disinfection, it is considered as one of the risk factors that decreased the mechanical integrity of

root filled teeth (14). It was also found that teeth were increasingly destabilized by endodontic

procedures, which included access opening, substantial canal enlargement (size 110 on maxillary

central incisors) and post space preparation even under a load of 3.75N (45). Additionally, root

filling procedures are not known to reinforce or enhance the mechanical integrity of remaining

root dentine. Thus it could be suggested that (1) the increased strain distribution (deformation)

associated with canal enlargement, and (2) the tensile strain formed at the coronal/ apical aspect

113

of the root after canal enlargement (EF50) may compromise the mechanical integrity of root

dentine (14, 44). The lateral strain (V-field) prior to canal enlargement (E10) showed a transition

from compressive strain to tensile strain with increase in loads. This may be attributed to the

combination of porous structure of dentine, asymmetrical loading and pre-load applied to secure

the specimen during testing (46).

The principles of micro-tissue engineering aims to design tissues of improved biological and

mechanical characteristics (47). In the current study the above principle was used to engineer root

canal dentine surface to enhance its mechanical stability. 1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC), a biocompatible zero-length cross-linker was used since it is known to induce

collagen crosslinking by forming stable amide-bonds between carboxyl groups and primary

amines (48). Crosslinking of collagen inter- and intra- molecularly with incorporation of chitosan

nanoparticles (CSnp) significantly increases the resistance of dentine collagen to bacterial

mediated enzymatic degradation (24). Chitosan possesses a structural characteristic similar to

glucosaminoglycans, which provides mechanical stability and compressive strength to connective

tissue matrix (16). The incorporation of water-soluble chitosan (CMCS) and CSnp, which served

as hydrophilic spacers providing flexibility as well as preventing zero-length crosslink, may induce

the inter-microfibrillar in addition to inter-fibrillar crosslinks (49, 50). This process further

reinforces the collagen structure by amplifying the number of amino reaction sites, resulting in the

formation of a polyanion-polycation ionic complexes between CSnp and collagen as an insoluble

complex during crosslinking (24). In addition, the introduction of inter-microfibrillar crosslinks is

known to influence the mechanical properties, especially the strain response in collagen tissues

(50). A crosslinking period of 8 hours was chosen in this study based on previous studies (23, 24).

This time frame may not be clinically realistic if it is intended as a chair side procedure. However,

the duration of crosslinking can be reduced by using higher concentration of cross-linker or made

rapid by applying photodynamic crosslinking in lieu of chemical crosslinking method (23).

In a micro-tissue engineered dentine, the dispersed CSnp would act as fillers, which formed

insoluble complex of CSnp-collagen during crosslinking. This ultrastructural arrangement will

facilitate efficient load transfer and energy absorption (24). In addition, the crosslinked CSnp

114

tethered into the collagen structure will act as a plasticizer, absorbing mechanical energy during

deformation (22). Along similar lines, earlier experiments on dentine collagen conditioned with

biopolymeric carboxy-methyl chitosan (CMCS) exhibited improved toughness (23). All the above

factors would contribute to the decreased strain response and distribution in micro-tissue

engineered root dentine, when compared with instrumented root dentine. This reduced rate and

degree of deformation on micro-tissue engineered root dentine may improve the mechanical

integrity of root dentine to chewing forces. The micro-tissue engineering of root canal dentine also

resulted in the reduction of apical tensile strain formed in the root canal prepared teeth, while

uniformly distributing compressive strain in the direction parallel to dentinal tubules. These

changes in the biomechanical response of micro-tissue engineered root dentine may contribute to

enhanced resistance to vertical root fracture (44, 45). Thus, the null hypothesis that the loss of

dentine during canal enlargement and micro-tissue engineering with crosslinked CSnp would have

no effect on the strain distribution in root was rejected.

4.6 Conclusions

Root canal instrumentation resulted in a distinct increase in radicular compressive strain

distribution in the direction perpendicular to dentinal tubules, and produced tensile root strain in

the direction parallel to dentinal tubules. Micro-tissue engineering of the root canal dentine with

crosslinked biopolymeric chitosan nanoparticles permitted homogenous and decreased

compressive strain distribution in the post-instrumented root dentine for the model tested in this

study.

4.7 Acknowledgement

Funding from the University of Toronto startup, Natural Sciences and Engineering Research

Council - Discovery grant and Canadian Foundation for Innovation is gratefully acknowledged.

The authors are also thankful to Dr. Huai-Xi Wang for his inputs during the setting up of digital

moiré interferometry.

115

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46. Lakes R. Deformation mechanisms in negative Poisson's ratio materials: structural aspects.

Journal of Materials Science 1991;26(9):2287-2292.

47. Rashidi H, Yang J, Shakesheff KM. Surface engineering of synthetic polymer materials

for tissue engineering and regenerative medicine applications. Biomaterials Science 2014;2:1318-

1331.

48. Staros JV, Wright RW, Swingle DM. Enhancement by N-hydroxysulfosuccinimide of

water-soluble carbodiimide-mediated coupling reactions. Anal Biochem 1986;156(1):220-222.

49. Everaerts F, Torrianni M, van Luyn M, van Wachem P, Feijen J, Hendriks M. Reduced

calcification of bioprostheses, cross-linked via an improved carbodiimide based method.

Biomaterials 2004;25(24):5523-5530.

50. Sung HW, Chang WH, Ma CY, Lee MH. Crosslinking of biological tissues using genipin

and/or carbodiimide. J Biomed Mater Res A 2003;64(3):427-438.

119

Chapter 5

Characterizing the Mechanical Characteristics of Micro-tissue

Engineered Root Dentin with Photodynamically Activated

Crosslinked-Chitosan Nanoparticles

120

5.1 Abstract

Aim The aim of the study was to evaluate the biomechanical response, fatigue behavior and

resistance to fatigue loadings of micro-tissue engineered root dentin with

chemically/photodynamically crosslinked cchitosan nanoparticles (CSnp) using digital moiré

interferometry (DMI) and cyclic fatigue test.

Methodology Experiments were conducted in two parts. Part-I: Ten root dentine specimens were

prepared, grating material replicated and tested for 10 N to 50 N, compressive loads in a

customized high-resolution, whole-field moiré interferometry setup. Digital moiré fringes were

acquired to determine the strain distribution pattern at specific regions of interest before and after

canal dentine surface engineering with CSnp-crosslinked by chemical or photodynamic method.

Fringe patterns were acquired and strain distribution pattern in axial and lateral directions were

analyzed with custom digital-image-processing software. Data were analyzed with a statistical

method on trend analysis at 0.05 significance level. Part-II: Forty-five root dentin specimens were

prepared and the root canals were instrumented, irrigated and treated with crosslinked chitosan

nanoparticles chemically (EDC-crosslinked-CSnp) and photodynamically (PDA-crosslinked-

CSnp) (n=15/group). Samples were subjected to an accelerated fatigue loadings from 100N to

600N until specimens failed. Load at failure and numbers of sustained-cycles obtained by the

stepwise stress test were recorded and analyzed by Kaplan-Meier and Log Rank (Mantel-Cox)

tests at the significance of 0.05.

Results The root canal dentin engineered with crosslinked-nanoparticles demonstrated a

conspicuous decrease in strain distribution in coronal root dentin from both EDC-crosslinked-

CSnp and PDA-crosslinked-CSnp (p < 0.01). There was a significant reduction in the tensile strain

formed at the apical region of the instrumented root dentin following crosslinked-nanoparticle

treatment (p < 0.05). Survival analysis showed a statistically significant difference (p < 0.05)

among evaluated conditions for load to fracture: PDA-crosslinked-CSnp > EDC-crosslinked-CSnp

> Control.

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Conclusions This study highlighted the potential of root canal dentin micro-tissue engineering

with photodynamically crosslinked chitosan nanoparticles to improve radicular strain distribution

patterns in instrumented canals and resistance to fatigue cycling loadings in root filled teeth.

5.2 Introduction

While tooth serving as mechanical device bearing occlusal loads during mastication, the root

dentin is responsible for transferring function loads from tooth to surrounding alveolar bone (1).

In intact tooth, the cervical dentin experiences conspicuous bending stress along facial-lingual

plane when experiencing chewing forces. This stress reduces notably toward the apical region of

the root (2, 3). This optimized biological structures are the result of a long-term functional

adaptation and evolution (4).

Root canal treatment is the method of treating an infected tooth to maintain its function. Even

though the 10-year survival rate of root canal treated teeth done by endodontists is 80%, tooth

fracture is still a common occurrence which frustrates both clinicians and patients (5). Previous

studies and clinical observations have shown that the prevalence of vertical root fracture in

endodontically extracted teeth were 6 to 11% (6-9). Many physiologic, pathologic and iatrogenic

factors have been attributed to the compromised mechanical integrity of restored root filled teeth

(10). Nevertheless, loss of dentin due to disease process or iatrogenic procedures has been

suggested to be the primary cause of diminished fracture resistance. The increased loss of tooth

structure in endodontically treated teeth altered the radicular stress distribution pattern, resulting

in more stress distribution and root flexure in the apical region particularly the along buccal-lingual

plane of root dentin. This increased root flexure may also contribute to the higher prevalence of

vertical root fracture in non-vital teeth (11). The loss of water-rich pulp tissue and the free water

from the dentinal tubules and dentin matrix diminished the plasticizing and viscoelastic behavior

of fully-hydrated root dentin (12, 13). The intra-pulpal hydrostatic pressure also contributed to the

uniform distribution of stress/strain in intact teeth, which is compromised in root filled teeth (10,

14).

Sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) are used as irrigants

to remove the pulp remnant and smear layer during root canal disinfection. It has been shown that

122

the physical properties of dentin such as flexural strength, elastic modulus and micro-hardness

were significantly reduced in a prolonged usage of these chemicals, which might increase the risk

of root fracture (15, 16). When EDTA is used on the root canal dentin, a layer of collagen and

other extracellular matrix proteins will be exposed on the surface of root canal, which may

facilitate the binding of bacteria on dentin collagen. The collagenolytic activity of bacteria

resulting in the degradation/ modification of collagen can cause the deterioration of the mechanical

property of root dentin (10). Moreover, the microdefects may also be formed because of the

instrumentation and the wedging effect generated during canal obturation (10, 17). These

microcracks tend to grow with time eventually resulting in fracture as fatigue failures (11).

Several procedures are used after root canal treatment to strengthen the coronal and root structure

of endodontically treated teeth (18). These involve prosthetic consideration such as using carbon/

glass fiber posts to retain the coronal restoration (19, 20); utilizing the adhesive core materials,

luting cements and dentin bonding agents (21); as well as temporary or final cuspal coverage with

crowns (22-24). Additional steps to conserve tooth structure, which includes contracted access

design (25, 26), preserving pericervical dentin (25, 27) and avoiding potential iatrogenic risk

factors for fracture in root filled teeth have been considered (10, 17). However, to date there is still

no treatment in current methods which neutralizes the adverse effects resulted from disease/

treatment procedures while strengthening the remaining dentin structure in endodontically treated

roots.

The principles of micro-tissue engineering aim to design tissues of improved biological and

mechanical characteristics to support tissue function, cell behavior and host integration (28, 29).

Nanoparticles have been introduced into tissue engineering bringing novel properties to the

scaffold/designed tissues, which engineered the tissue on a micro-scale and have tremendously

revolutionized tissue engineering. Recent studies have shown that synthetic and natural chemicals

that increase the number of inter- and intra-molecular collagen crosslinks would enhance the

fibrillar resistance against bacterial enzymatic degradation and provide improved mechanical

characteristics of tissues (30-32). Alternatively, photodynamic activated (PDA) crosslinking has

been reported to induce rapid and stable covalent crosslinking of collagen by exposing

photosensitizer to an appropriate wavelength of light (33, 34). In addition, previous studies also

123

confirmed that developing collagen composites with chitosan or other glycosaminoglycan-like

components to create more suitable biomimetic microenvironments providing biological and

mechanical benefits in tissue engineering (35). Along similar lines, crosslinked chitosan

nanoparticles (CSnp) have been used to achieve micro-scale tissue engineering of root dentin. This

process of micro-scale tissue engineering stabilizes the ultrastructure of surface dentine providing

the tissue enhanced mechanical characteristics, bioactivity as well as resistance to host/bacteria

mediated enzymatic degradation (36-39).

However, the mechanical integrities have not been characterized on a root canal model. The aim

of the study was to evaluate the biomechanical response, fatigue behavior and resistance to fatigue

loadings of micro-tissue engineered root dentin.

5.3 Material and Methods

5.3.1 Part I: Assessment of biomechanical behavior of micro-tissue

engineered root dentin (with Digital moiré interferometry (DMI))

Five of maxillary incisors were decoronated to obtain 12-15mm length root specimens. The root

canals of the specimens were enlarged with ISO K files (K-Flexofiles, Dentsply Maillerfer, Tulsa,

OK, USA) to size F3 (ProTaper Universal F3, Dentsply®) (EF3). Six ml of 2.5% sodium

hypochlorite solution was used to irrigate the canals during cleaning and shaping, which was

followed with 1ml of 17% EDTA solution as the final irrigant. The tooth specimens were prepared

to slab shaped, parallel-sided, facio-lingual sections with a uniform thickness of 2.5mm for the

strain analysis. Samples were subjected to a compressive load from 0 to 50 N with 10N increment

under digital moiré interferometry (DMI) to study the strain distribution of canal preparation under

physiological relevant loads. After first strain investigation conducted with DMI, root canals of

the samples in EF3 were conditioned with photo-dynamically activated crosslinked-CSnp (PDA-

crosslinked-CSnp). All the specimens were again subsequently tested with the DMI.

In Group: EDC-crosslinked-CSnp, the canal space of each dentin specimen was conditioned with

CSnp in water soluble carboxymethyl chitosan (CMCS) solution (1mg/ml) using manual agitation

for 5 minutes followed with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-

124

hydroxysuccinimide (EDC/NHS) (4:1, 33mM) crosslinker for 8 hours. Then washed with

deionized water 3 times and stored in 100% humidity for testing and used in a week.

In Group: PDA-crosslinked-CSnp, (CSRBnp) were dispersed in water and applied on root canal

hedentin surface using manual agitation for 5 minutes followed by being activated with a non-

coherent light for 10 mins (540nm, 25 J/cm2). After washing, the canal was conditioned with CSnp

in 1% CMCS solution (1mg/ml) by manual agitation for 15 mins followed by filled with RB

solution (10 M) for 15 mins. The specimen was again exposed to the non-coherent light for 10

mins then washed twice. Samples were kept in 100% before being tested and used in a week.

Strain values generated from 0 to 50N in each specimen after instrumentation (EF3) and

crosslinking CSnp (EDC (Ch4)/ or PDA-crosslinked-CSnp (Ch5)) were collected and analyzed

using a statistical method on trend analysis. The induced strain values from each point was tested

by linear regression and the slop values from the same region/ field-direction were subjected to

subsequent paired-sample t test to compare the difference between treatment on dentine at 95%

confidence interval (IBM SPSS Statistics version 20.0).

The details of the principle and experimental procedures of DMI has been demonstrated in the

published paper (Chapter 4 and Appendix-additional paper).

5.3.2 Part II: Assessment of fatigue resistance of micro-tissue engineered

root dentin (with Cyclic Fatigue Testing)

Sample Preparation

Forty-five lower premolar teeth were collected, transilluminated before testing to exclude

possibilities of cracks or damages during extraction as well as subjected to x-ray to confirm the

geometry of canal system, and stored in deionized water at 4°C for this study. The teeth were

decoronated to obtain 12-14mm length root specimens. The root canals of the specimens were

enlarged with to size F3 (ProTaper Universal F3, Dentsply®). 6-10 ml of 2.5% sodium

hypochlorite solution was used to irrigate the canals during cleaning and shaping, which was

125

followed with 2ml of 17% EDTA solution as the final irrigant accompanied with ultrasonic

agitation to remove smear layer. The tooth specimens were prepared by grinding the mesial and

distal surfaces on wet emery papers of grit size 320, 600, and 800, under constant running water

to prepare slab shaped, parallel-sided, facio-lingual sections with a uniform thickness of 3.0mm

for the experiment. Specimens were subjected to three groups: Control, EDC-crosslinked-CSnp,

and PDA-crosslinked-CSnp. The root canal of specimens in two experimental groups were treated

following the protocol mentioned as below.

In Group: EDC-crosslinked-CSnp, the canal space of each dentin specimen was conditioned with

CSnp in water soluble carboxymethyl chitosan (CMCS) solution (1mg/ml) using manual agitation

for 5 minutes followed with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-

hydroxysuccinimide (EDC/NHS) (4:1, 33mM) crosslinker for 8 hours. Then washed with

deionized water 3 times and stored in 100% humidity for testing and used in a week.

In Group: PDA-crosslinked-CSnp, (CSRBnp) were dispersed in water and applied on root canal

hedentin surface using manual agitation for 5 minutes followed by being activated with a non-

coherent light for 10 mins (540nm, 25 J/cm2). After washing, the canal was conditioned with CSnp

in 1% CMCS solution (1mg/ml) by manual agitation for 15 mins followed by filled with RB

solution (10 M) for 15 mins. The specimen was again exposed to the non-coherent light for 10

mins then washed twice. Samples were kept in 100% before being tested and used in a week.

Fatigue Testing

Root specimens were mounted on brass rings with the roots embedded in self-curing resin (SR-

Ivolen Standard Kit, Ivoclar Vivadent, Schaan, Lichtenstein) up to 6 mm apical to the CEJ with a

0.2 mm-thick silicone rubber barrier (Aquasil LV, Dentsply Detrey GmbH, Konstanz, Germany)

surrounding the root surfaces to mimic the periodontal ligament. Root specimens were mounted

in the Instron Universal Testing Machine (Instron, Canton, MA) maintained in 100% humidity.

Samples were submitted to stepwise mechanical cycling in a frequency of 15 Hz and the stress

ratio of 0.1 with a ball indenter. The stepwise procedure began with a load of 100N, followed by

200, 300, 350, 400, 450, 500, 550, 600, and 650N at a maximum 27000 load cycles each (30 mins)

till failure. Load at failure (N) and numbers of sustained-cycles obtained by the stepwise stress test

126

were recorded and analyzed by Kaplan-Meier and Log Rank (Mantel-Cox) tests at the significance

of 0.05 (IBM SPSS Statistics version 20.0).

5.4 Results

5.4.1 Part I: Assessment of biomechanical behavior of micro-tissue

engineered root dentin

The U-field and V-field moiré fringe analysis revealed that the compressive loads resulted in a

distinct distribution of normal strain along the axial and lateral directions in root dentin. There was

a generalized distribution of compressive strain in root dentin, which increased gradually with

increase in applied loads in both axial and lateral directions.

In samples of PDA-crosslinked-CSnp, the radicular strain at the coronal third decreased

significantly in U-field, following CSnp-crosslinking treatment of root canal surface dentin (PDA-

crosslinked-CSnp) (p < 0.01), as well as in the apical region of specimens (p < 0.01). Figure 5.1

shows the U-field fringe pattern and color map of the radicular strain distribution patterns before

and after root canal surface micro-tissue engineering with PDA-crosslinked-CSnp.

In V-filed, root canal surface engineered with PDA-crosslinked-CSnp did not result in significant

difference in strain at the coronal region (p = 0.24) (Fig. 5.2A). However, in the apical third of

specimen, it also resulted in significant variation of a distinct change in the nature of strain

distribution from a post-instrumentation tensile strain to a compressive strain distribution

following crosslinked CSnp treatment with PDA (p < 0.01) (Fig. 5.2B).

127

Figure 5.1. Root dentin sample with canal size F3. (A-1, 2, 3) Fringe patterns in U-field at 10, 20 and 40N

load in root dentin before micro-tissue engineering with PDA-crosslinked-CSnp. (A-4) Color map obtained

the fringe analysis showing the whole-field displacement of sample before micro-tissue engineering with

PDA-crosslinked-CSnp. (B-1, 2, 3) Fringe patterns in U field at 10, 20, and 40N load showed in micro-tissue

engineered root dentin. (B-4) Color map obtained the fringe analysis showing the whole-field

displacement of sample after engineering the root dentin with CSnp.

y

x

A-1 A-2 A-3

B-1 B-2 B-3

- 2.23e-04

3.92e-04

1.01e-03

1.62e-03

2.24e-03

2.85e-03

3.47e-03

4.08e-03

4.70e-03

5.31e-03

5.92e-03

-3.74e-04

1.04e-04

5.81e-04

1.06e-03

1.54e-03

2.01e-03

2.49e-03

2.97e-03

3.45e-03

3.92e-03

4.40e-03

A-4

B-4

128

Figure 5.2. Strain values in U and V field generated from coronal (A) and apical third (B) of root before and

after micro-tissue engineering with PDA-crosslinked-CSnp on root dentin surface. The root strain formed

after root canal surface engineered with PDA-crosslinked-CSnp was significantly less compared with root

strain obtained after instrumentation for loads ranging from 10N to 50N, in U field (p < 0.01). Moreover,

apical strain in V field resulted in a shift from tension to compression on micro-tissue engineered root

dentin (p < 0.01).

-0.001

-0.0008

-0.0006

-0.0004

-0.0002

0

0.0002

0 10 20 30 40 50

Str

ain

Loads

Strain - Coronal

EF3 (U field) PDA-Crosslinked-CSnp (U field)

EF3 (V field) PDA-Crosslinked-CSnp (V field)

(A)

-0.001

-0.0008

-0.0006

-0.0004

-0.0002

0

0.0002

0 10 20 30 40 50

Str

ain

Loads

Strain - Apical

EF3 (U field) PDA-Crosslinked-CSnp (U field)

EF3 (V field) PDA-Crosslinked-CSnp (V field)

(B)

129

5.4.2 Part II: Assessment of fatigue resistance of micro-tissue engineered

root dentin

Survival analysis (Kaplan-Meier and Log Rank (Mantel-Cox tests) showed a statistically

significant difference among evaluated conditions for load to fracture (Log Rank (Mantel-Cox)

test, X 2 = 6.02, df = 2, p < 0.05), as well as for numbers of sustained cycles until fracture (Log

Rank (Mantel-Cox) test, X 2 = 4.3, df = 2, p = 0.12) (Table 5.1, Fig. 5.3). The PDA-crosslinked-

CSnp had the highest fatigue failure loads and numbers of sustained cycles to failure, whereas the

control group showed the lowest values (Table 5.1, Fig. 5.4). From the pairwise comparisons in

comparing the load at failure, there was statistically significant between control and PDA-

crosslinked-CSnp groups (p < 0.05), while no significant difference between control and EDC-

crosslinked-CSnp (p = 0.066).

Table 5.2 summarizes the probability that the root dentin specimens exceeded the respective load

and number of cycles without failure for group control, EDC-crosslinked-CSnp and PDA-

crosslinked-Csnp.

Table 5.1. Experimental design and mean ( SD) of the sustained load (N) and numbers of cycles at failure.

130

Figure 5.3. Survival curves according to the steps of loads and numbers of cycles for each failed tooth.

Figure 5.4. Mean and standard deviation of the sustained load (in N) and numbers of cycles at failure in

each group.

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Table 5.2. Survival rates (probability that the specimens exceeded the respective load or numbers of

cycles without failure (standard deviation)) for the experimental groups.

5.5 Discussion

Biomechanical behavior of dentin (tooth) provides an understanding on the response of dentin

(tooth) to functional/ parafunctional forces, especially after therapeutic intervention. Digital moiré

interferometry (DMI) is an established technique that is used to evaluate the biomechanical

response of dentin (40-43). This technique allows the determination of strain distribution along

specific regions of interest in root dentin for loads within physiological limits, in real-time. The

deformation patterns of specimen provide not only the quantitative information of the

biomechanical response of materials, but also the qualitative (nature) of the deformation. It has

been demonstrated that the functional stress/strain from the tooth to the surrounding bone is

predominantly distributed at the cervical root dentin (1, 2). This maximum stress / bending stress

reduces notably toward the apical region of root (1). On the contrary in root filled teeth, higher

degree of functional stress/strain is distributed toward the apical aspect of the root resulting in

increased root flexure (11, 44). This altered biomechanical response at the apical root dentin with

root canal treatment, have been previously implicated as one of the risk factors for vertical root

fracture in root filled teeth (11, 45). With the application of high sensitive DMI, the deformation

of both cervical and apical dentin can be studied under physiological level chewing forces. (More

details of the strength, methodology design of DMI and the biomechanical behavior of

instrumented root canal dentin are discussed in Chapter 4)

Previous investigations showed that instrumented root canal dentin exhibited reduced stability with

increased deformation and tensile strain formed along the specimen (Ch4). This alteration in the

functional stress/strain distribution pattern in a hard tissue will increase the risk of material damage

Loads (N) Number of Cycles *

Groups 300 350 400 450 500 550 600 80,000 120,000 160,000 200,000

Control 0.82 (0.12) 0.55 (0.15) 0.55 (0.15) 0.18 (0.12) 0.09 (0.09) 0.00 (0.00) 0.00 (0.00) 0.82 (0.12) 0.64 (0.15) 0.46 (0.15) 0.09 (0.09)

EDC crosslinked-CSnp 1 1 0.55 (0.15) 0.55 (0.15) 0.27 (0.13) 0.18 (0.12) 0.00 (0.00) 1 0.64 (0.15) 0.55 (0.15) 0.18 (0.12)

PDA crosslinked-CSnp 1 1 0.78 (0.14) 0.56 (0.17) 0.44 (0.17) 0.22 (0.14) 0.00 (0.00) 1 0.89 (0.11) 0.67 (0.16) 0.33 (0.16)

132

and probability of structural failure with time. The micro-tissue engineered dentin showed the

decreased strain response and distribution, when compared with instrumented root dentin. This

reduced rate and degree of deformation on micro-tissue engineered root dentin were consistent in

the axial direction (U-field) at the coronal region in both chemical (EDC; Ch4 Fig. 4.6) and PDA

crosslinked-CSnp root dentin, which may improve the mechanical integrity of root dentin to

chewing forces. The micro-tissue engineering of root canal dentin also resulted in the reduction of

apical tensile strain formed in the root canal prepared teeth, while uniformly distributing

compressive strain in the direction parallel to dentinal tubules (V-field). This may lessen the

increased root flexure in root filled teeth, contributing to the enhanced resistance to vertical root

fracture (12, 46). The lesser degree of decreased deformation and the absence of strain reduction

in the axial direction of apical third in the group of PDA crosslinked-CSnp can be due to the

difference between EDC/NHS-mediated crosslinking and rose-bengal-mediated photodynamic

crosslinking demonstrated in Chapter 2. The elastic modulus and hardness are reduced (28%) on

the dentin surface after EDC-crosslinked-CSnp treatment; while they are slightly increased after

treated with PDA-crosslinked-CSnp. The root specimen with softer (reduced elastic modulus) root

canal dentin surface may cause more bending fringes at the cervical root, which restricted the

compressive fringes generated from apical end of root further resulting in lesser deformation at

both cervical and apical region of tested specimen (Ch4 Fig. 4.5).

Failure in root filled teeth is considered as a fatigue process, mainly by cyclic fatigue-induced

subcritical crack growth (47). Fatigue refers to the response of a material to repeated application

of stress/ or strain. In a fatigue failure, microscopic cracks which may be produced during disease/

treatment procedures tend to grow with time, eventually result in fracture under functioning. These

cracks behaving as stress concentrators, are the initiation point of crack propagation when the

stress magnitude is high enough to induce microscopic plastic deformation at the crack tip (11).

Fatigue strength is an important parameter for assessing the ability of a root to resist crack

propagation/ fracture. And mechanical cycling, which simulates mastication cycles observed in

the mouth, is an intermittent loading set-up with controlled parameters such as load, numbers of

cycles, and frequency utilized in the laboratory (48). From previous studies, one million-cycles of

chewing was proposed of simulating 1-5 years of chewing function (49, 50). According to the

preliminary test, subjecting the specimen to a clinical level of loads (50-150N) for one million of

133

cycles was not able to induce the crack propagation leading to catastrophic failure of teeth.

Therefore, the step-wise loading methodology starts from 100N with 50/ 100N increment every

27,000-cycles was more applicable to the investigation (51-53). Evaluation of the resistance to

root fracture using DMI and fatigue cyclic loading provides a better clinical insight in comparison

with mechanical static loading, which represents an overestimation of the strength value under

very large testing loads (54). Tooth selection was based on the reported higher incidence of root

fractures in root filled lower premolars (55). The application of X-ray images allowed

nondestructive standardization of root canal geometry of specimens used in this study.

Current study also showed that the load at failure was significantly higher in PDA-crosslinked-

CSnp compared to control group (p < 0.05). The load at failure was higher in EDC-crosslinked-

CSnp compared to control group, however, the difference was not statistically significant. The

sustained cycles at failure from high to low was also presented as PDA-crosslinked-CSnp > EDC-

crosslinked-CSnp > control, though without significant difference. The findings showed that root

specimen with prepared root canal treated with PDA-crosslinked-CSnp resulted in enhanced

resistance to the accelerated fatigue cyclic loadings. This suggests the ability of crosslinking the

root canal dentin collagen with CSnp photodynamically in improving the resistance to crack

propagation/ fracture. Root canal dentin treated with EDC-crosslinked-CSnp resulted in improved

biomechanical response under physiological level loads (lower loads), however, did not engender

the increased resistance to fatigue loading significantly.

EDC (1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide) crosslinking exhibits low cytotoxicity

and is washable with water, therefore it is a popular chemical crosslinker utilized to increase the

mechanical and structural stability of collagen scaffold and tissue (38, 39, 56-58). It is also used

often in restorative dentistry to create a durable/ stable hybrid layer (59, 60). This zero-length

crosslinker contains a functional group with the formula RN=C=NR. It activates the carboxyl

groups of glutamic and aspartic acid to form an O-acylisourea intermediate that reacts with a non-

proteinated amino groups in protein molecules to create a stable covalent amide bond between two

proteins with the only product, urea (Fig. 5.5) (61, 62). Dentin structure can be reinforced,

strengthened and stabilized with increased resistance to enzymatic/ hydrolytic degradation over

time by the formation of intra- and inter molecular bonds (39, 57). The drawbacks of EDC

134

crosslinking are: (1) Time required to form stable crosslinking by chemical methods is much

longer than photodynamic crosslinking which may not be applicable in clinical usage. (2) A zero-

length crosslinking agent like EDC can only crosslink adjacent molecules in around 1nm, which

is not able to bridge microfibrils. However, the introduction of inter-microfibrillar crosslinks is

known to influence the mechanical properties, especially the strain response in collagen tissues

(63).

Photodynamic crosslinking is considered a rapid, efficient method with low cytotoxicity to induce

covalent bonds for stabilizing the collagen based biomaterials. Rose Bengal is one of the

photosensitizers that can be activated with green light ( = 520-560 nm) to form photodynamic

crosslinking. During this process, the singlet oxygen or radical is produced by light-activated

photosensitizer. The highly active singlet oxygen induces photo-oxidation of photooxidizable

amino acid residues such as cysteine, histidine, tyrosine and tryptophan in one protein molecule

resulting in products, which further react with normal/ or photo-altered resides in another protein

molecule to induce a crosslink (Fig. 5.5) (64). Shrestha et al. demonstrated that photodynamic

crosslinking with RB or RB functionalized nanoparticles stabilized root dentin collagen by

increasing tensile strength, toughness and resistance to bacterial collagenase (65).

In addition, collagen crosslinking can also result in crosslinking of other classes of

macromolecules within the collagen structure, such as proteoglycans, either to one another or to

collagen molecules (66). In the current study, the dispersed CSnp would act as fillers, which

formed insoluble complex of CSnp-collagen during crosslinking. The hydrophilic CSnp,

functionalized CSnp and water soluble carboxy-methyl chitosan (CMCS) infiltrated/ tethered into

the collagen structure before crosslinking collagen matrix (Fig. 5.5, 5.6). These hydrophilic

components, which hold structural similarity of proteoglycans and glycosaminoglycans, will act

as plasticizers absorbing mechanical energy during deformation to provide mechanical

compressive strength and tissue flexibility (35, 37, 67). This ultrastructural arrangement will

facilitate efficient load transfer and energy absorption (39). Along similar lines, earlier

experiments on crosslinking dentine collagen with chitosan derivatives exhibited improved

toughness (38, 65).

135

It has been proved that crosslinking collagen based tissue improved the mechanical properties such

as tensile/ compressive strength, toughness or elastic modulus. In dental research, crosslinking

demineralized dentin beam with EDC resulted in 3 times higher stiffness compared with the

samples treated with water (68); Shrestha et al. also presented enhanced tensile strength and

toughness resulted from chemical and photodynamical crosslinking (38, 65). A study also showed

the crosslinked lumbar intervertebral discs exhibiting improved stiffness modulus, ultimate tensile

strength and toughness (69). However, these studies used dentin beams and crosslinked the entire

specimen for the evaluation. Further research of using a clinically relevant model is needed to

evaluate the effect on improving the mechanical properties of tissue. Therefore, the present study

examined the ability of micro-tissue engineering using crosslinked biopolymeric nanoparticles to

restore the compromised mechanical characteristics in root canal prepared tooth. Only the root

canal dentin surface was engineered with crosslinked-CSnp while the whole root structure was

subjected to the testing. Hence the effect may not be as significant as previous in vitro studies.

Current study did not show significantly improved resistance to fatigue mechanical loading of the

root dentin with EDC-crosslinked CSnp. This may be caused by several reasons. One limitation

could be the small sample size. Since tooth is a biological tissue, the variation among individuals

can be considerable. In the strain evaluation, the specimen served as its own control, however, this

was not possible in accelerated fatigue testing. Secondly, as aforementioned disadvantages of EDC

crosslinking, this zero-length may not be able to induce bonds between protein molecules further

away than 1nm (63). Even when the incorporation of CSnp and CMCS may facilitate the

crosslinking, the effect may not manifest under large loadings as in mechanical testing.

The current study indicates that micro-tissue engineered root canal dentin enhanced the mechanical

characteristics of the root structure. Crosslinking the instrumented root canal with both chitosan

nanoparticles chemically and photodynamically improved the biomechanical response of root

dentin with significantly decreased compressive strain in the direction perpendicular to dentinal

tubules at the cervical region of post-instrumented root. It also resulted in the reduction of apical

tensile strain formed in the root canal prepared teeth, in the direction parallel to dentinal tubules.

Moreover, root canal dentin photodynamically crosslinked with the incorporation of chitosan

nanoparticles resulted in enhanced load at failure during fatigue cyclic loading. These changes in

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the biomechanical response of micro-tissue engineered root dentin may contribute to enhanced

resistance to crack propagation and vertical root fracture in endodontically treated teeth.

137

Figure 5.5. Mechanism of EDC- and PDA- crosslinking of collagen molecules.

Figure 5.6. Mechanism of collagen crosslinking with the incorporation of water soluble chitosan

derivatives (CMCS).

138

5.6 Acknowledgement

Funding from the University of Toronto startup, Natural Sciences and Engineering Research

Council - Discovery grant, Canadian Foundation for Innovation, and Foundation of Endodontics

are gratefully acknowledged. The authors are also thankful to Dr. Huai-Xi Wang for his inputs

during the setting up of digital moiré interferometry; and to Jiang Wang for his support on

conducting the mechanical testing.

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Chapter 6

Discussion and Conclusion

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6.1 General Discussion

Different aspects of nanoparticles delivery strategy which includes the characterization of the fluid

dynamic parameters associated with root canal irrigation with MB and determination of the

mechanical effect of micro-tissue engineered root canal dentin with crosslinked CSnp have been

addressed in the current project. The experiments were conducted in three phases: In phase-1,

experiments were conducted to characterize the kinetics and formulation of CSnp dispersion. In

phase-2, experiments were conducted to evaluate the effectiveness of using activated MBs to

deliver CSnp into root canal dentin. In phase-3, experiments were conducted on micro-tissue

engineered root dentin to assess the strain distribution pattern when subjected to continuous loads

within physiological limits and mechanical characteristics under cycles of fatigue loads. The MB

effectively improved fluid dynamic parameters and facilitated improved coating of CSnp on root

canal dentin. Micro-tissue engineering of root canal dentin with crosslinked CSnp improved

biomechanical and fatigue behavior of root dentin. The proposed nanoparticle based treatment

strategy have the potential to offer twofold advantages in root canal treatment: antibacterial

efficacy and root strengthening.

Application of CSnp in this study is based on some of its unique advantages when applied to

infected dental hard tissue. CSnp on infected root dentin provides (a) effective elimination of

residual surface adherent/nonadherent biofilms; (b) inactivation of bacterial endotoxins (1, 2) and

dentin-bound-LPS (3); (c) stabilization effect on the root dentin matrix (4), and (d) enhanced the

ability of stem cells to adhere, proliferate and promote wound healing (3, 5, 6). Its nano-ranged

size possesses high positive charge density and surface area, while its distinct difference in the

uptake mechanisms between prokaryotic and eukaryotic cells promotes targeted antimicrobial

effect (7). These advantages make these nanoparticles an ideal material for application in the

treatment of infected tissue, which is in close proximity to host tissue. Although the benefits of

nanoparticles in the treatment of infected dentin have been highlighted in previous in vitro and

animal models (1-3), optimization of its delivery methods into the root canal dentin was not

investigated in detail in the past. A stable nanoparticle dispersion is a crucial prerequisite to

preserve the physicochemical properties of nanoparticles in the formulation. Since two major

forces, van der Waals attractive and electrostatic double layer repulsive forces determine the

146

interaction between particles collision, to increase the repulsive forces between dispersed particles

will increase the stability of the dispersion (8). Consequently, the chosen concentration of CSnp

dispersion should be able to sustain the charge density of nanoparticles. The experiments

conducted in the chapter 2 enabled us to determine the maximum concentration of CSnp, which

sustains the charge density as well as stability of the nanoparticles dispersion.

Ultrasonic or sonic activation of MBs was proposed in this study to generate uniform wall stress

distribution along the canal system by potentiating cavitational bubbles, which were not adequately

produced with water based formulations (9-11). The findings from the current study showed that

intensified cavitational bubbles resulted from ultrasonically activated MBs, which in turn induced

high fluid velocity and inertial stresses towards the canal wall. This high fluid velocity and inertial

stresses may facilitate CSnp delivery in root canals. However, in the current study ultrasonic

activation of MB resulted in the formation of dentin smear layer mixed with CSnp. The formation

of dentin smear layer during the ultrasonic activation was attributed to the inadvertent touching of

root canal wall by the ultrasonically agitating tip. Similar observation of dentin smear layer on root

canal walls with ultrasonic activation was reported in the literature (12). It was interesting to note

that manual agitation of MBs with gutta-percha points did not produce intensified flow with only

0.1 m/s of velocity in the simulated root canal model. The dominant high viscous forces produced

by the manual agitation of MBs was attributed to (a) the high-viscosity of MBs and (b) frictional

forces generated between the MBs and canal wall. The fluid vector was parallel along the root

canal wall, yet, the narrow space between the well-fitted gutta-percha point and the lateral root

canal wall increased the fluid efflux forcing the particles sideways and upwards along the canal

wall (13) (Fig. 6.1A). It was noted that CSnp were dispersed in the water-phase of MBs in between

micro-droplets (data not shown). During the manual agitation, the particle flux density was

increased due to the increased concentration gradient, and the decreased distance between particles

and canal wall (14). In addition, when CSnp is manually pushed/compressed in between MBs and

canal wall, the electrostatic attraction between cationic CSnp and anionic dentin surface may

further facilitate CSnp coating (1) (Fig. 6.1B).

The fluid dynamics associated with the manual agitation of gutta-percha point in water presented

lowest velocity and stresses, however, this group produced fair (50%) CSnp-coated root canal

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dentin wall. This observation probably signifies that the vector of velocity/stress generated in the

manually activated water played a key role in promoting nanoparticle coating on root canal dentin.

The sonic activation groups did not exhibit effective coating of CSnp on root canal dentin. The

sonic agitation of MBs showed acceptable fluid dynamics parameters with an averaged shear stress

of 316 Pa, however, the fluid circulation in this group was limited to the back and forth movement

near the tip of the sonically agitating insert. This phenomenon may be because of (a) the

constrained longitudinal oscillation of the tip within the root canal lumen and (b) combined with

the high viscosity of MBs, resulting in the restricted flow and inadequate CSnp coating (15, 16).

Figure 6.1. (A) Push-pull motion of GP of manual agitation resulted in fluid efflux sideways and upwards

in spaces between the insert (gutta-percha) and wall. (B) The interaction between anionic dentin surface

and cationic CSnp. CSnp are dispersed in the water-phase of MBs formulation. The particle flux density

was increased with the presence of microbubbles, and when CSnp being pushed/compressed in between

microbubbles during manual agitation. The dominated viscous forces along the dentin wall enhanced the

electrostatic attraction between CSnp and dentin resulting in uniform coating of CSnp. (PFC:

perfluorocarbon)

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CSnp can interact well with dentin and form a conditioning layer on root canal dentin (1, 4).

Negatively charged dentin collagen and positive charged CSnp form a polyanion-polycation ionic

complexes (4, 17). The coated CSnp would impart the afore-mentioned advantages such as

antibacterial properties, and improved biological characteristics on root canal dentin. In addition

to coating, crosslinking the CSnp to dentin tissue may further amplify the effect brought about by

CSnp to strengthen dentin matrix. The process of crosslinking introduces covalent bonds between

CSnp (or CMCS)-collagen and collagen-collagen. Crosslinking of CSnp to dentin collagen will

retain the antimicrobial properties and resistance to bacterial mediated enzymatic degradation even

for longer duration in root canal dentin (4, 7, 18, 19). The integration of CSnp and water soluble

chitosan derivatives (CMCS) in between collagen molecules and the collagen fibrils will serve as

hydrophilic spacers to facilitate water holding characteristics, flexibility and load transfer/ energy

absorption characteristics during mechanical functions (4, 20-22) (Fig. 6.2).

CSnp have been shown to neutralize host-derived proteases (MMPs) and bacterial collagenases (4,

23). These characteristics will increase the resistance to host derived enzymatic degradation of

CSnp conditioned dentin. CSnp, due to their structural similarity with extracellular matrix

glycosaminoglycans, which intertwines the fibrous collagen network will support the mechanical

stability of collagen (24). These findings are in agreement with earlier studies that demonstrated

improved flexibility and stress-strain properties in collagen matrices with absorbed chitosan/ or

chitosan nanoparticles (20-22, 24-26). Moreover, integrating CSnp into the crosslinked collagen

would amplify the numbers of amine reaction sites resulting in the formation of ionic complexes

between CSnp/ chitosan derivatives during crosslinking (22, 27). CSnp may also bridge the

microfibrils in addition to intra- and inter-fibrillar crosslinks to influence the mechanical response

(28). Thus, delivering and coating CSnp or functionalized CSnp onto the root canal dentin would

bring these attributes to support tissue functioning and host integration.

The findings from our study showed that there was alteration of load-displacement behavior and

surface mechanical properties such as hardness, elastic modulus on CSnp coated root canal dentin

surface (without crosslinking). 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), when

used for crosslinking, activates the carboxyl acid groups of glutamic and aspartic acid residues in

the peptide chains, further links with the amino groups of lysine or hydroxyl lysine residues

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forming amide bonds (29, 30). In PDA-crosslinking, the highly active singlet oxygen resulted from

the light-activated photosensitizer (rose bengal), induces photo-oxidation of photooxidizable

amino acid residues such as cysteine, histidine, tyrosine and tryptophan in one protein molecule

resulting in products which, in turn, react with normal or photoaltered residues in another protein

molecule to produce crosslinks (31). Collagen tissue often becomes stiff after crosslinking owing

to the amplified covalent bonds between intra-molecules, inter-molecules or even inter-

microfibrils (28). Unexpectedly, the dentin block treated with 33mM EDC for 8 hours showed 18-

45% reduction of hardness and elastic modulus. The reduction was lesser with the incorporation

with CSnp. In contrast, photodynamically crosslinking with chitosan-conjugated-rose bengal-

nanoparticles (CSRBnp)/CSnp resulted in 16% higher elastic modulus in dentin (Appendix II, Fig.

a). The softer dentin surface (“more elastic”) resulted from EDC crosslinking was not consistent

with most of the previous studies (28, 32, 33). The reason of this unexpected effect needs more

investigations, however, was consistent with a currently unpublished data described in Oryan’s

group (30). This effect may be related to the swelling effect on collagen during the crosslinking

(34), as well as only the surface of treated dentin (< 700 nm) was evaluated by indenter. It may

also be one of the reasons for the improved tensile strength and toughness in EDC-treated dentin

than PDA-treated dentin beams in the earlier study from our group (22).

Biomechanical response of endodontically treated tooth is altered from an intact tooth with vital

pulp (35). In an intact natural tooth, the stress distribution is predominant in the cervical region of

root and it gradually diminishes toward the apical region of root (36). However, the increased loss

of dentin and eccentricity in the removal of root dentin during instrumentation will alter the

radicular stress distribution patterns resulting in more stress distribution towards the apical

direction and in the bucco-lingual plane of root dentin (35, 37). From the investigations in chapter

4, it was observed that canal enlargement decreased the stability of cervical root dentin and

generated distinct tensile strain in the apical root dentin. In addition, the strain distribution in the

middle third of root was increased showing more deformations at this region following root canal

instrumentation (Appendix II, Fig. b). Both EDC- and PDA-crosslinked-CSnp resulted in

decreased deformation in the axial direction at cervical area of the root, and distributed relatively

low levels of tensile strain in the lateral direction at the apical region of root. Similar findings of

decreased stability and increased tensile strain in endodontically treated/restored teeth were

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demonstrated in earlier studies (38, 39). In the CSnp crosslinked specimens, moiré fringe patterns

showed distinct bending response at the cervical region along the bucco-lingual direction with

lesser compressive deformation at cervical/ and middle regions. This bending effect may be due

to the incorporation of CSnp and CMCS, since this effect was not observed in the specimens

treated with only EDC crosslinking (Appendix II, Fig c, d). The reduction in strain distribution at

the cervical region was not also observed in root treated with only EDC crosslinking.

Typically, in vitro fatigue loading of specimens is used to simulate chewing forces in vivo (40,

41). In the current study, under accelerated fatigue cycling, the micro-tissue engineered dentin

using both PDT and EDC crosslinked CSnp, sustained higher loads before catastrophic failure

when compared to the instrumented root canal specimens. Although the survival analysis showed

an increased load at failure in the EDC-crosslinked-CSnp group, it was not statistically significant.

This finding may correspond with the softening effect observed in the EDC-crosslinked dentin

collagen. However, both methods of crosslinking CSnp resulted in improved strain distribution

over root specimens when static loads at the physiological levels were applied in digital moiré

experiments. In this case, the difference in the surface mechanical properties in EDC-/ PDA-

crosslinked-CSnp did not influence the strain distribution pattern. This result may be because of

(1) the incorporated CSnp induced bending response of the cervical root dentin in both crosslinking

methods resulting in similar strain distribution patterns, and (2) the relative low level of loads (0-

50N) may have resulted in an early root dentin response to applied force (elastic deformation). It

is also demonstrated that hardness represents the resistance to plastic deformation on the surface

of a specimen, which does not influence the strain distribution in bulk tooth (42). Nevertheless,

the effect of improved resistance to the fatigue cycling was significant in group of PDA-

crosslinked-CSnp. The fatigue analysis also simulates the mastication and represents the resistance

to crack propagation during functioning (41, 43). Considering the efficiency (time-consuming) and

the findings resulted from present study, photodynamic crosslinking CSnp was preferable than

EDC crosslinking CSnp. Photodynamic crosslinking strengthens the dentin collagen with stable

crosslinks between collagen molecules providing enhanced stiffness (and elastic modulus) and the

resistance to plastic deformation (hardness). In addition, with the incorporation of CSnp and

CMCS, the flexibility and the ability to absorb energy can be enhanced. This further improved the

resistance to fracture of tissue. The proposed micro-tissue engineering strategy using biopolymeric

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CSnp followed by crosslinking procedures, will have potential in clinical application to enhance

the mechanical integrity of root filled teeth.

6.2 Future Studies

1. The future studies based is proposed to assess the feasibility of applying engineered CSnp with

additional benefits for micro-tissue engineering. In this line, CSnp functionalized with

hydroxyapatite precursor (CS-HAnp) in lieu unmodified CSnp. The application of CS-HAnp

would promote remineralization of demineralized dentin matrix and subsequently aid in

integrating dentin with root canal sealers and filling materials used to fill the root canals. This

strategy would further enhance the mechanical integrity of root filled teeth.

2. Preexisting cracks in teeth in non-endodontically treated teeth has been an important concern.

These cracks were attributed to history of trauma, orthodontic treatment, excursive occlusion and

restorative procedures. Application of CS-HAnp on dentin matrix can provide the additional

benefit repairing microcracks using the principles of biomineralization. This would prevent crack

propagation in root dentin and further improve the mechanical properties in root filled teeth.

3. Free water loss in endodontically treated teeth compromises the plasticizing and toughening

effect in dentin, further causes the decrease of resistance to fracture. The structure of hydrophilic

CSnp is similar to the component of ECM, which supports the water holding property and

mechanical strength of collagen. Micro-tissue engineering with crosslinked CSnp may enhance

the resistance to free water loss further assisting the mechanical characteristics of root filled teeth.

4. In future, experiments may also be conducted in clinically relevant animal model with simulated

infection. The effect of micro-tissue engineering with CSnp based application and delivery on

targeted antimicrobial, neo-tissue formation, cell proliferation/adherence, and wound healing

would be evaluated in vivo. This investigation would confirm the improved biological

characteristics of micro-tissue engineered dentin in a clinically relevant model, which can be

further applied in endodontics for enhancing treatment efficacy and predictability.

152

6.3 Conclusion

Present study aims to develop an effective strategy for NP delivery in root canal dentin, and to

micro-tissue engineer the dentin with crosslinked CSnp for enhancing the mechanical

characteristics of endodontically treated teeth. Within the evaluation of current findings, the

optimum CSnp dispersion was determined at the concentration of 1mg/ml, which sustained the

stability of solution. Crosslinking CSnp on dentin resulted in a denser/homogeneous coating with

increased hardness and elastic modulus on dentin surfaces treated with PDA-crosslinked-CSnp.

MBs are introduced to facilitate NP delivery in combination with manual, sonic and ultrasonic

agitations. Ultrasonically activated MBs induced fluid dynamics with high velocity and inertial

stress through intensified cavitational bubble dynamics, however formed a coating of CSnp mixed

with dentin smear layer on canal wall. Manually agitated MBs generated uniform high viscous

stress with increased particle flux and electrostatic attraction between agitation-insert and canal

wall, facilitating homogeneous coating of CSnp on root canal dentin. Micro-tissue engineered root

canal dentin with crosslinked CSnp and water-soluble chitosan resulted in decreased strain

distribution and a decrease of tensile strain improving the stability of root under static loads in

physiological level. Also, root dentin model treated with PDA-crosslinked-CSnp showed an

increase in the sustained load at fracture when experienced fatigue cycling. Current findings

underlined the advantage of manually activated MBs to deliver CSnp in root canal and the

significant impact of micro-tissue engineering with crosslinking CSnp on dentin to enhance the

mechanical characteristics of root. These outcomes will have potential application in clinical

practice.

153

Figure 6.2. (A) Schematics of micro-tissue engineered dentin collagen demonstrating intra-, inter-

molecular crosslinking within the collagen molecules and collagen fibrils. The integration of CMCS and

CSnp amplifies the sites of crosslinking between molecules and proteoglycans. It may also facilitate the

inter-microfibrillar crosslinking to support the collagen tissue. (B) The mechanism of collagen crosslinking

in molecular level. In EDC-crosslinking, the activation of the carboxyl acid groups of glutamic and aspartic

acid residues would link with the amino groups of lysine or hydroxyl lysine residues forming amide bonds.

In PDA-crosslinking, the highly active singlet oxygen induces photo-oxidation of photooxidizable amino

acid residues such as cysteine, histidine, tyrosine and tryptophan in one protein molecule forming the

products, which would react with normal/ or photoaltered resides in another protein molecule to produce

a crosslink. The presence of CMCS may integrate into this process to amplify the site of crosslinking and

to prevent the zero-length crosslinking. (C) Schematics of intra-, inter- molecular and inter-microfibrillar

crosslinking and the incorporation with CMCS during crosslinking.

(A)

(B) (C)

154

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158

Appendix I

Deciphering dentin tissue biomechanics using digital moiré

interferometry: a narrative review

Fang-Chi Li, Anil Kishen. Opt Lasers Eng 2018;107:273-80

159

Abstract

The functional integrity of human teeth to different thermo-mechanical stresses relies on the

characteristics of dentin. Dentin is a biocomposite and biologically adapted material that is

composed of mineral, organic and water phases. It displays conspicuous gradients in its

ultrastructural, physical and mechanical characteristics, which experiences further variations with

age and disease. Thus the biomechanical behavior of tooth / dental hard tissues under different

functions is considered extremely complex. Photomechanical techniques utilize optical principles

to study the biomechanical response of biological tissues under functional forces. They are largely

non-destructive techniques, which provide high-sensitive, and whole-field information of

specimens in situ. They have been applied extensively in dentistry to understand the biomechanical

principles underlying the responses of dental hard tissue, tooth-bone relationship and restorative

appliances to different forces generated within the mouth. In this line, moiré interferometry is an

established method to study the deformation patterns in tooth and dental hard tissues under water-

loss / thermo-mechanical loads. This article aims to provide a comprehensive review on the

application of moiré interferometry to understand the biomechanical response of teeth/dentin hard

tissue.

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1. Introduction

Photo-biomechanics is a discipline of science that utilizes optical techniques to study the behavior

of biological structures under function or external forces (biomechanics). It includes the

investigations of biological structure/ function at any level, ranging from organisms to organs,

cells and organelles using the knowledge and methods of mechanics and photonics (1). In dentistry,

these investigations are crucial to comprehend the biomechanical principles of dental tissues and

to approximate the properties of artificial materials with natural tooth structure. Such studies would

provide the knowledge for optimal treatment plans and basis for developing new devices /

materials, to maintain the integrity of the tooth and stomatognathic system as a stress-

bearing/generating system (2).

An ideal biomechanical technique should be able to test complex human specimens under

physiologically realistic loads/conditions (2). The sophisticated anisotropy exhibited by biological

structures such as enamel, dentin and periodontium is one of the major challenges in these

experiments. This varied material characteristics in the biological structures, along with the

complexity in geometry, as well as the nature of external forces, all can contribute to the challenges

of characterizing biomechanical response of dento-osseous structures (2). Thus, optimal

experimental designs are important prerequisites in biomechanical studies, mainly to simulate a

physiologically/clinically realistic condition. Conventional mechanical property measurements

tend to represent properties that are averaged over a large volume of the regional tissue. Typically,

it is challenging to test biological specimens under physiologically realistic loads. A miniature-

sized strain gauge may be able to offer the strain information at one point of interest, mostly in one

direction. However, they cannot demonstrate the strain information of the whole specimen (3). An

ideal biomechanical technique should be able to test complex biological specimens under

physiologically realistic loads and conditions (2).

Photonics is the technology of generating and sensing radiant energy over the whole

electromagnetic spectrum mostly in the range of visible and near-infrared light. While, photo-

biomechanics applies optic-based experimental techniques for measuring certain physical

quantities such as displacement or strain in biological system (4). Photo-biomechanics builds a

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synergy between the fields of photonics and mechanics (4). These are non-destructive techniques

that provide complete-field stress and strain information even in a micro-range. They permit

determination of specific deformation such as in-plane or out-of-plane with the aid of a particular

experimental configuration, which is sensitive to specific displacement component. They allow

testing and visualization of strain gradients in biological structures. With recent advancements in

digital image processing techniques and fiber optics, optical fringe analysis has become robust and

less time consuming, while optical systems have become more flexible and compact (5). Common

photomechanical experiments that are applied to dentistry are: photoelasticity, moiré

interferometry, digital image correlation and holographic interferometry.

Photoelasticity utilizes the principle of load-induced birefringence property exhibited by the

transparent models to study the stress distribution in structure. It was applied to study the

orthodontic movements (6), tooth-bone supporting structure (7, 8) and restorative methods (9-11).

To apply this method, the shape of the model and the manner in which the model is supported

(boundary conditions) must be similar to the in vivo situation. The photoelastic models simulating

dental structures are fabricated using photoelastic resins that possess the birefringence property

(12). However, the variation of the structure components and anisotropic in material properties of

the biological structure are difficult to simulate in a transparent photoelastic model.

Digital image correlation (DIC) uses a series of images of the specimen acquired with charged-

couple device (CCD) cameras during experimentation. It tracks and analyses individual spots

(speckle) on the specimen surface with customized software to determine the displacement fields

(13). Typically this technique compares the speckle patterns at two different states of the object

(deformed and un-deformed) to obtain the desired displacement information. Since the sensitivity

of the displacement measurements correlated with the size of surface speckles, formed by the spray

paint. DIC is the least sensitive among different techniques discussed, while it is easy to use when

compared to other optical methods (14).

In holographic interferometry, during testing, the holograms of the two states of an object are

acquired. The reconstructed image results from the interference of two waves of holographic

recording (before and after loadings), which offers the displacement field of the object (15). To

162

avoid limitations associated with chemical development and precisely repositioning of the

holographic plates, holographic applications employs a video camera for image acquisition

coupled to a computer image processing system. This setup is technically called electronic speckle

pattern interferometry (ESPI). ESPI provides less clear images and the fringe patterns are

intrinsically noisy, but it is very easy to obtain quantitative measurements of deformations than

holography (15, 16). These techniques are able to provide measurements of surface deformation

of structures in a non-destructive manner with different advantages (4, 12).

Moiré interferometry is a non-destructive method, offering the information of whole-field, real-

time displacement field of tested objectives. It is capable of measuring in-plane displacements in

micron range and distinct from the contour maps produced by classical interferometry and

holographic interferometry which are most effective for determining the out-of-plane

displacements (17). The distinction is important for strain-stress analysis, since the functional

deformation in hard tissue of tooth is predominantly in-plane than out-of-plane (18). Owing to its

high strain sensitivity and spatial resolution, it is considered an effective method for studying

deformation of miniature, complex-shaped biological structures, such as human teeth. The

application of moiré interferometry to determine the deformation pattern in dental hard tissues

during water-loss and thermo-mechanical loading has been reviewed in this article.

2. Bio-composite Nature of Dental Hard Tissues

A schematic diagram of a human tooth is shown in Figure 1. Enamel covers the anatomical crown

of a tooth and mature enamel has a mineral content of around 96% hydroxyapatite by weight; 1-

2% organic substrates particularly enamelins; and water makes up the remained composition of

enamel. Mineral crystals are arranged in long, thin structures called enamel rods ranged from 4-8

m. There is rod sheath surrounding each rod composed by a protein matrix, enamelin. The space

between rods was inter-rod enamel and pores exist where crystals do not form between rods which

allow fluid movement or diffusion to occur in the structure (19, 20).

Dentin forms the bulk of the crown and root of the teeth and comprises 70% of carbonated apatite

crystals, 20% of collagen and proteins with 10% of water content by weight (21). The most

prominent feature of dentin is the dentinal tubules which resulted from the deposition of dentin

163

around the odontoblast (dentin) cell, the cells that form dentin. The cells lie on the inner most

aspect at the dentin-pulp interface, with their long cellular processes called odontoblastic processes

extend from these cells through the entire thickness of dentin. The lumens of the tubules vary in

diameter: from 0.5-0.9 m (dentin-enamel-junction: DEJ) to 2-4 m through the direction toward

pulp. The densities of the tubules are 20000-45000/mm2 from DEJ to pulp end (Fig. 2) (21, 22).

In a recent study, it was shown the diameters of tubules on root canal surface were 4.3-1.7 m

from coronal to apical, and the tubules/dentin surface ranged from 72-13% (23). According to the

structure of the dentinal tubules, dentin surrounds the tubules (0.4-0.74m) termed peritubular

dentin, which is 40% higher mineralized dentin in proximity to the odontoblastic process. The

intertubular dentin located between the tubules, contains more than 50% organic phase in volume

and proves the elasticity of dentin. Due to the spatial variation of tooth structure and composition,

the mechanical properties, for instance, the elastic modulus varies conspicuously. The elastic

modulus of enamel is 40-80 GPa whilst it is around 30 GPa in peritubular dentin and 16-21 GPa

in intertubular dentin. It may be even lower (3-19 GPa) at regions as close as 500m from the pulp

(24, 25) .

Figure 1. The schematic of tooth structure with surrounding bone (Credit image @ OpenStax college,

Wikimedia commons).

164

Figure 2. The distribution of dentinal tubules in tooth structure. (a, b) The density and diameter of tubules

vary in crown portion of tooth structure: (a) close to dentin-enamel junction; (b) close to pulp end. (c-f)

The density and diameter of tubules vary in root portion of tooth structure: (c, d) a scanning-electron-

microscopy image (5000x) showing the structure of dentinal tubules from root canal wall at coronal region;

(d) a cross section of dentinal tubules of the coronal region of root (5000x); (e, f) the root canal dentin

surfaces of middle (e) and apical (f) region of root (5000x).

3. Moiré Interferometry

3.1. General concept

The moiré method is based upon the optical phenomenon of the superposition of two gratings, for

example, two arrays of uniformly spaced lines. Moiré interferometry and shadow moiré are two

versatile methods for determining the in-plane (x and y axis) and out-of-plane (z axis) displacement

fields, respectively. High sensitive moiré interferometry was evolved from the low-sensitivity

geometric moiré, used on solid mechanics of measuring the in-plane surface deformations. Since

moiré responds only to geometric changes, it is equally effective for elastic, viscoelastic, and

plastic deformations, for isotropic, orthotropic and anisotropic materials, and for mechanical,

thermal, and dynamic loadings. Shadow and projection moiré methods are two techniques which

determine the out-of-plane displacement, the topography of the specimen surface. Although U and

V may also be present, the methods sense and measure only W (z axis) (17]. The sensitivity of

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shadow and projection moiré is lower, but fortunately, satisfies the engineering practical

applications (4). However, it has been greatly enhanced by shifting / moving the reference grating

(z) relative to the specimen grating (w) (fringe shifting) and the optical/digital fringe multiplication

method to increase the resolution with optimal accuracy (26, 27).

Moiré interferometry involves the principle of optical diffraction and interference. It provides the

whole-field patterns with high spatial resolution, sensitivity and excellent clarity. The general

scheme of 4 beams moiré interferometry is illustrated in Figure 3. A high-frequency (fs = usually

1200 lines/mm) cross-line grating is firmly adhered to the specimen by a thin layer of adhesive

and deforms together with the specimen. Two beams (B1, B2) of coherent laser light illuminate

the specimen grating obliquely at angle . Two beams of B1 and B2 intersect at a region to form

a wall of constructive and destructive interference, as a reference/ virtual grating (f = 2400

lines/mm), without a physical presence (Fig. 3a). At the beginning of the experiment, the specimen

grating and reference grating should be adjusted and matched to form a null field, which

demonstrates neither interference nor fringe pattern generated. When the specimen grating is

deformed as a result of the applied loads, the interaction of two gratings produces interference

patterns of dark and light bands, so called moiré patterns. The fringe pattern is viewed and recorded

by a charge-coupled device (CCD) camera (Fig. 3a, b).

The optical systems for moiré interferometry usually consist of three parts (Fig. 3c). (1) the

illumination system, consisting of a coherent light source, beam expander and collimator (2) the

moiré interferometer, which divides the input beam into two or four separate beams and directs

them onto the specimen grating, and (3) the camera system. Low power lasers such as He-Ne, Ar+,

diode, dye, CO2 or Nd-YAG laser types can be used as the beam sources and a 5 to 50 mW laser

has generally been adequate (17).

The sensitivity of the displacement measurement is determined by the number of fringes generated

per unit displacement. Therefore, the displacement sensitivity is equal to the frequency f of the

reference grating. When f = 2400 lines/mm, the displacement per fringe order is 0.417 m. The

reliability of displacement measurement depends on the displacement resolution, which is 1/5f or

1/10f. Then the displacement at any point can be resolved to at least 0.1 m (17). Consequently,

166

moiré interferometry method provides whole-field displacement patterns over the tested specimen.

It allows the determination of strain distribution along specific regions of interest in the specimens

for loads within physiological limits. It is a real-time technique as the displacement field can be

viewed as loads are applied. This system offers a resolution of 0.1μm with accuracy in in-plane

deformation of approximately 50 strain along x (U field) and y (V field) axis. These outstanding

points of moiré interferometry permits the determination of small-range in-plane deformation in

miniature biological samples such as teeth.

Figure 3. Schematic illustration of moiré interferometry (Post et al. 1994). (a) The interaction of a virtual

reference grating formed by 2 beams of B1/B2 and a specimen grating to create moiré fringe pattern. (b)

A four-beam moiré interferometry to demonstrate the Nx and Ny fringe patterns which represent the U

and V displacement fields. (c) The apparatus of four-beam moiré interferometry comprises three

subsystems. (Beams corresponding to B3 and B4 of figure(b) are not shown here) D: spatial filter; C: plane

mirror; M: moiré interferometer (reprinted/adapted by permission from Sringer Nature, High Sensitivity

Moiré, 1994 [17]).

3.2. The analysis of moiré fringes

The normal strain () and shear strain () at selected region of interest along different lines from

both axial (x-axis) and lateral direction (y-axis) were calculated as described below (17, 28):

167

The displacements in optical metrology at each point (x, y) is described as:

U (x, y) = g𝑁𝑥 (𝑥, 𝑦) = 1

𝑓𝑁𝑥 (𝑥, 𝑦)

V (x, y) = g𝑁𝑦 (𝑥, 𝑦) = 1

𝑓𝑁𝑦 (𝑥, 𝑦)

where the fringe orders (N) are taken at the corresponding x, y points and where g and f represent

the reference grating. Strain is determined from the displacement fields by using the relationships

for engineering strain:

U-field (axial normal strain): 휀𝑥= 𝜕𝑈

𝜕𝑥 =

1

𝑓[

𝜕𝑁𝑥

𝜕𝑥]

V-field (lateral normal strain): 휀𝑦= 𝜕𝑉

𝜕𝑦 =

1

𝑓[

𝜕𝑁𝑦

𝜕𝑦]

Shear strain: 𝛾𝑥𝑦 = 𝜕𝑈

𝜕𝑦 +

𝜕𝑉

𝜕𝑥 =

1

𝑓 (

𝜕𝑁𝑥

𝜕𝑦+

𝜕𝑁𝑦

𝜕𝑥)

Fringe order Nx determines the in-plane displacements U at each point in the field. Nx pattern is a

simple function of U while Ny pattern is a function of V.

Thus, the strain is determined by the rate of change of fringe orders in the patterns or the fringe

gradient surrounding each point.

For fringe gradients: 𝜕𝑁𝑥

𝜕𝑥≈

Δ𝑁𝑥

Δ𝑥 , the change of the fringe order that occurs in a finite distance x.

Currently, with the application of CCD camera and advanced image processing software, the fringe

acquisition and analysis become extremely robust. By defining the fringe patterns and fringe orders

with parameters input, the displacement field and strain can be calculated and demonstrated by the

software rapidly (Fig. 4) (29-31).

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Figure 4. Fringe patterns of the root specimen in axial (x) (a-1) and lateral direction (y) (b-1). Color map

obtained in the fringe analysis shows the whole-field displacement of sample in axial (a-2) and lateral

direction (b-2). Color map of strain distribution of sample in axial (a-3) and lateral direction (b-3) (data

were from our un-published studies, Li F-C & Kishen A).

4. Applications of moiré interferometry in dental biomechanics

Moiré interferometry was used to study the strain distribution in tooth structure since 1990s. In

this section, ten principle articles of applying moiré method on studying the biomechanics of tooth

structure were discussed (Table 1). The thin layer of specimen grating was usually bonded to one

of the flat surfaces of prepared tooth specimen, which were 2-3 mm thickness, with epoxy adhesive.

Reference grating was formed by source of diode laser ( = 532, 670nm) or argon laser ( = 514nm)

in a frequency double that of the specimen grating.

y

x

a-1

b-1

a-2 a-3

b-2 b-3

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Table 1. Research articles on studying tooth structure with moiré interferometry, in chronological order.

4.1. Mechanical strain gradients in tooth structure

Wang and Weiner (32) studied the strain-structure relationship in crown of human canines and

premolars under a constant load to maximum 500N. The frequency of the specimen grating of 200

lines/mm used in this study was lower than regular investigations (1200 lines/mm). They

concluded that the strain exhibited in the enamel is significantly lower than that in the dentin. A

200µm-thick zone in the dentin beneath the dentino-enamel junction (DEJ), which experienced

larger strain than the remaining bulk of the coronal dentin, was identified in this study. The moiré

maps in this investigation distinctly demonstrated a whole-field displacement field over the entire

specimen, which can be well-related to the spatial variation of the biological structure.

Moiré interferometry was applied to understand the strain gradient on dentin structure under

mechanical function in the study of Kishen in 2005 (8). The lower incisors were studied under

physiologically realistic loads of 10-30N to evaluate the strain distribution in dentin tissue and

correlate to the stress distribution of structure examined with photoelasticity. There was a

conspicuous reduction in strain from the cervical to the apical third of the root dentin as well as no

shear strain formation in root dentin. At the same time, the distinct bending stress along facio-

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lingual plane in the coronal and cervical region of tooth reduced toward the apical third of the root.

The research established the application of moiré method on biological tissue under physiologic

level of loads and able to measure micro-range deformation of structure.

In 2006, Kishen et al. studied the biomechanical principles on tooth structure to understand the

biomechanics of the cause of non-caries cervical lesions (NCCL) in teeth (33). Figure 5 shows the

U field and V field moiré fringe patterns formed on the tooth sections loaded at 30N. This

experiment showed that the enamel and dentin displayed a unique in-plane deformation in the

direction perpendicular (U- axial direction) and parallel to the long axis (V- lateral direction) of

the teeth. The strain in the lateral direction (V) within the enamel and the strain in the axial

direction (U) within the dentin concentrated with higher loads towards the cervical region adjacent

to the cemento-enamel junction (CEJ) on the facial side. This study supported the hypothesis that

biting forces (10-30N) will contribute to the loss of dental hard tissue in the cervical region.

Figure 5. It is shown the V-field (left) and U-field (right) moiré fringe patterns conducted to study the cause

of biting forces on non-carious cervical lesions (with permission from reference [33]).

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4.2. Thermal property gradients in dentin

Deformation over tooth structure or dentin caused by thermal changes were also investigated. The

experiments were conducted to observe the displacement of specimen subjected to thermal

variation without other external applied loads. Kishen and Asundi (34) confirmed the close

agreement between the response of dentin to thermal changes observed by moiré interferometry

and that detected by thermomechanical analysis. Hence, by obtaining the thermal strain from moiré

analysis, the linear coefficient of thermal expansion (LCTE) at every point in the dentin could be

determined as well. Besides, the moiré interferometric patterns showed an initial phase of

expansion over dentin specimen was followed by contraction at higher temperatures (34). Shrestha

et al. (35) found that the application of both heat and cold stimulus of tooth surface resulted in

significantly higher strain in partially dehydrated bovine teeth than in the fully hydrated ones. The

trend of strain produced from enamel to dentin in fully hydrated samples was not much different

after thermal changes [35, Fig. 3] emphasizing the important role of free water in biomechanical

behavior of tooth. The strain close to DEJ in both fully hydrated and partial hydrated teeth

presented highest strain after thermal stimulations, this finding was similar to the study of Wang

and Weiner (32) that the DEJ underwent larger strain than the central dentin during mechanical

loads.

4.3. Hydromechanics in dentin

Moiré interferometry was utilized to study the effect of moisture change / water-loss induced

deformation of dentin. Specimens were observed under dehydration and rehydration process

without subjected to external loads in two of the studies (36, 37). It was concluded the dehydration

process (directly and indirectly) induced strain formation in dentin and it was reversed during

rehydration process. Kishen and Rafique emphasized that even the 80% of water loss occurred in

the first 2 hours of dehydration, the dehydration-produced strain showed after an initial latent phase

(37). Wood et al. focused on the different level of strain induced and distributed over enamel-

constrained dentin and unconstrained dentin during moisture changes (36). The water content also

influenced the stress-strain response of structural dentin especially in the direction of the axis of

tooth (38). The increased deformation in hydrated dentin under 5-60N were more than dehydrated

dentin showing more characteristic of a tough material. Loss of water of hydration resulted in

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brittle response and strain hardening in dentin. These investigations demonstrated that free-water

and surface adsorbed water in dentin was significant for the toughness and the uniform distribution

of strain within dentin structure.

4.4. Other applications

Recent studies from our group have applied moiré method on other clinical-relevant investigations

to obtain an insight of the biomechanical behavior of tooth influenced by dental treatment

procedures. Root canal treatment is basically to remove/disinfect the infected pulp tissue and

bacteria-contaminated dentin from the canal space, then seal the space with a biocompatible

material. The disinfection process during treatment involves mechanical instrumentation and

chemical irrigations. Various metallic instruments were designed to provide efficient root canal

cleaning and shaping without disturbing the morphology of it. The residual strain formed around

the canal wall during the root canal treatment by three different systems of instruments was

examined by Lim et al. (39). The dentin instrumented with Wave One performed highest residual

strain compared to those was shaped with ProTaper Universal and hand files. In addition, the

localized concentration of post-instrumentation microstrain was mainly induced in non-hydrated

dentin, whereas it was not observed on fully hydrated root dentin specimens (Fig. 6).

Instrumentation of non-hydrated roots caused localized microstrain concentration and diminished

stress relaxation due to the reduced viscoelasticity resulting from the loss of free-water from dentin

matrix (40).

Moiré interferometry was applied to study the effect of using bonded resin composite to restore

the pericervical dentin (PCD: 6mm below/ 4mm above crestal bone) after root canal treatment

compared to regular filled roots (41). In addition, it was also employed to examine the mechanical

characteristics after microtissue-engineering the dentin tissue with crosslinked-biopolymeric

nanoparticles. This method allowed each specimen to be served as its own control and analyzed

before/after treatment as well as to extract information from regions of interest. These experiments

highlighted the potential advantages of dentin microtissue-engineering and the challenges

associated with bonding composite resin to strengthen tooth root. Moiré method was likewise

advanced to study the objects in a microscopic scale. A high sensitivity microscopic moiré

interferometry system has been carried out to measure the microstrain of a range of 1-10 ()

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across loaded resin-dentin interfaces (42). Other studies also presented the strength of utilizing

moiré technique to detect the deformation on micro/nano structures (43, 44).

Figure 6. The typical fringe pattern in root dentin of a specimens maintained in non-hydrated environment.

(a) U-field moiré fringe patterns before instrumentation and (b) after instrumentation (with permission

from reference [39]).

5. Conclusion

Moiré interferometry has been used to examine the biomechanical behavior of human teeth under

mechanical, thermal and hydro stimulations. This technique allows to detect the deformation of

0.1-1 micrometer displacement on the specimen, which also provides the reliability of studies the

strain distributions in tooth structure ranged from 10-1000 microstrain under physiologically

realistic conditions. In conclusion, moiré interferometry with the improvements in optics and

digital image processing is a useful method to study deformation characteristics in anisotropic and

complex dental structures with sensitivity and accuracy.

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Appendix II

Supplementary Data

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Figure a. The hardness (MPa) and elastic modulus (GPa) resulted before/after each treatment

demonstrated statistically significant (p < 0.01). There was no significant difference of hardness in PDA-

crosslinked-CSnp (p > 0.05).

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Figure b. Root dentin sample with canal size #10. (A-1, 2, 3) Fringe patterns in U-field at 10, 30 and 50N

load in root dentin before canal enlargement (dentin loss). (B-1, 2, 3) Fringe patterns in U field at 10, 30,

and 50N load showed in root canal enlarged till #50.

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Figure c. Root dentin sample with canal size F3. (A-1, 2, 3) Fringe patterns in U-field at 10, 30 and 40N load

in root dentin before EDC crosslinking on root canal dentin. (B-1, 2, 3) Fringe patterns in U field at 10, 30,

and 40N load showed in EDC crosslinking root dentin (without CSnp incorporation).

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Figure d. Strain values in U field generated from cervical (A) and apical third (B) of root before and after

EDC crosslinking on root dentine surface. The apical strain formed after root canal surface crosslinked

with EDC was significantly less compared with root strain obtained after instrumentation for loads ranging

from 10N to 50N, in U field (p < 0.05). However, there was no difference of strain distribution at the

cervical region of root (p > 0.05).