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
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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
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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
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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
63
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.
64
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
65
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).
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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
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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,
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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.
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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
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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-
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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.
131
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
136
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
147
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
149
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
150
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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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
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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).
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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,
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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
179
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).