dentin substrate modification with chitosan-hydroxyapatite ...€¦ · anam hashmi master of...
TRANSCRIPT
Dentin Substrate Modification with Chitosan-Hydroxyapatite
Precursor Nanocomplexes
by
Anam Hashmi
A thesis submitted in conformity with the partial fulfillment of the
requirements for the degree of Master of Science
Graduate Department of Dentistry
University of Toronto
© Copyright by Anam Hashmi (2019)
ii
Dentin Substrate Modification with Chitosan-Hydroxyapatite
Precursor Nanocomplexes
Anam Hashmi
Master of Science 2019
Faculty of Dentistry, University of Toronto
Abstract
The purpose of the study was to develop and characterize Chitosan-Hydroxyapatite precursor (C-
HA) nanocomplexes and to evaluate the effect of dentin conditioning with C-HA nanocomplexes
on (1) tricalcium silicate sealer (TCS) penetration, (2) interfacial remineralization and (3) dentin
mechanical property. In Phase-I, physico-chemical/bioactivity characterization of C-HA
nanocomplexes was done along with assessment of ultimate tensile strength (UTS) of dentin. In
Phase-II and III, tubular penetration of TCS and chemical characterization after conditioning of
dentin was evaluated using fluorescent imaging and time-of-flight secondary ion mass
spectrometry respectively. The results showed polyanionic, hydrophilic and bioactive nature of
C-HA nanocomplexes along with increased TCS penetration and UTS of conditioned dentin.
Conditioned dentin was modified with formation of ion-rich layer constituting of abundant
phosphates, calcium, calcium-phosphates and chitosan fragments at the TCS-dentin interface and
subsurface. The study highlighted the potential role of C-HA nanocomplexes in enhancing TCS
penetration, interfacial remineralization and UTS of dentin.
iii
Acknowledgements
I am grateful to all of those with whom I have had the pleasure to work during this project.
I would like to extend my greatest and foremost gratitude to my supervisor, Dr. Anil Kishen, who
provided me the opportunity to join the Master’s program at UofT in 2017. Since then, he has
been instrumental in transforming my aptitude, both towards research and life at large.
The extent of support and supervision he has extended in completing my program is
unparalleled. Dr. Kishen’s dedication and passion towards research, intellect and eloquence,
continue to inspire me to be perseverant in my efforts and achieve higher scholarship standards.
To say the least, I could not have imagined having a better counsellor and mentor for my
research degree who during the process brought out the best of character and professional
attributes that shall stay with me for a lifetime. It is not an understatement to say, the “Kishen
lab” was an institution within itself.
I would also like to thank other members of my advisory committee, Dr. Bernhard Ganss and Dr.
Anuradha Prakki for their expert insights and encouragement during this project. I appreciate
the feedback offered by Dr. Rana N.S Sodhi during the countless hours spent on TOF-SIMS data
analysis.
I would like to thank Dr. Suja Shrestha, who I still remember as my first acquaintance in the
Kishen lab and Dr. Alice Li for her help during research work. A special mention of Dr.
Hebatullah Hussein, for her constant support and friendship. Amongst many others, I would like
to mention Nancy Valiquette, Jian Wang, Ilya Gourevich, Jared Mudrik, Audrey Darabie and
George Kretschmann for their much-valued technical expertise.
I am grateful to God Almighty, for having bestowed me with a strong support system of my
family. This journey would not have been possible without the countless prayers of my parents
and the drive they have instilled in me to pursue academic excellence. I am at a loss of words to
express the unconditional support, motivation, optimism and love of my husband (Najeeb), who
has been my greatest comfort zone. Lastly, the untiring patience of my daughters (Aisha and
Maria), their brimming smiles and door-step warm hugs were enough to get me going.
I shall forever cherish this BOUNDLESS intellectual experience at University of Toronto.
Anam Hashmi
iv
Table of Contents
Chapter 1 ....................................................................................................................................... 1
Introduction ................................................................................................................................... 1
Background ................................................................................................................................ 1
Hypothesis .................................................................................................................................. 4
Objective ..................................................................................................................................... 4
Specific Aims .............................................................................................................................. 4
Chapter 2 ....................................................................................................................................... 5
Literature Review ......................................................................................................................... 5
Dentin.......................................................................................................................................... 5
Dentin Alteration ..................................................................................................................... 11
Dentin remineralization .......................................................................................................... 16
Tricalcium Silicate based materials ....................................................................................... 24
Chitosan .................................................................................................................................... 26
Characterization of bioactivity ............................................................................................... 34
Critique of Literature .............................................................................................................. 40
Chapter 3 ..................................................................................................................................... 42
Manuscript (I) ............................................................................................................................. 42
Abstract .................................................................................................................................... 42
Introduction ............................................................................................................................. 45
Materials and Methods ........................................................................................................... 46
Results ....................................................................................................................................... 50
Discussion ................................................................................................................................. 56
References ................................................................................................................................ 60
Chapter 4 ..................................................................................................................................... 64
Manuscript (II) ..................................................................................................................................... 64
4.1 Abstract ........................................................................................................................................... 64
4.2 Introduction .................................................................................................................................... 67
4.3 Materials and Methods .................................................................................................................. 69
4.4 Results .............................................................................................................................................. 71
4.5 Discussion ........................................................................................................................................ 78
4.6 References........................................................................................................................................ 83
Chapter 5 ..................................................................................................................................... 86
Discussion..................................................................................................................................... 86
Chapter 6 ..................................................................................................................................... 92
Conclusion ................................................................................................................................... 92
Chapter 7 ..................................................................................................................................... 93
v
Future Studies ............................................................................................................................. 93
Chapter 8 ..................................................................................................................................... 94
References .................................................................................................................................... 94
Supplementary Data ................................................................................................................. 109
..................................................................................................................................................... 110
vi
List of Tables
Table 2.1. List of NCP analogs [adapted and modified (80)]. ..................................................... 23
Table 2.2. Comparison of characterization techniques ................................................................ 38
Table 3.1. Contact angle measurements for C-HA nanocomplexes with significant reduction in
C-HA nanocomplexes conditioned group…………………………………… ………………….62
vii
List of Figures
Figure 2.1. Hierarchical structure of dentin. Dentinal tubules with mineralized peritubular dentin
(PTD) act as fiber reinforcements within the less mineralized intertubular dentin (ITD)
matrix. Mineralized collagen fibrils intersect perpendicularly with the dentinal tubules.
Nanometric structure of collagen fiber with staggered collagen molecules and intrafibrillarly
deposited hydroxyapatite (HAP)............................................................................................. 6
Figure 2.2. Iatrogenic alteration in dentin. A) Schematic showing role of dentin components
towards mechanical integrity (adapted & modified from (1) and B) shows how individual
components become altered with use of chemical irrigants affecting dentin interaction with
materials. ............................................................................................................................... 15
Figure 2.3. Proposed nucleation theories for collagen mineralization. Extrafibrillar
mineralization is an ion-based pathway explained by classical nucleation theory whereas
amorphous precursor-based pathway has been proposed for intrafibrillar mineralization. .. 17
Figure 2.4. A) Molecular structure of N-acetyl chitin. B) Side view of a chitin fibril with Acetyl
groups outlined in rectangles showing inter-fibril associations (114). ................................. 29
Figure 2.5. Deacetylation of chitin and modified chitosan derivatives. Adapted and modified
(122). ..................................................................................................................................... 29
Figure 2.6. Proposed role of Chitosan-Hydroxyapatite precursor (C-HA) nanocomplexes in
dentin biomineralization. ...................................................................................................... 33
Figure 2.7. Principle of TOF-SIMS operation. Adapted and modified (129). Incident primary ion
beam on sample surface causes surface ionization. The secondary ions are extracted into a
flight tube and identified by the mass detector based on the time of flight and mass to
charge ratio............................................................................................................................ 36
Figure 3.1. Optimal C-HA nanocomplexes formulation determination. (A) Scatter plot showing
mean zeta potential values for surface charge measurements (B) Bar chart with pH values
for serial C-A nanocomplexes concentrations and (C) Bar chart showing polydispersity
index for assessing aggregation. (D) Table 3.1. Water contact angle measurements for
dentin with/without C-HA nanocomplexes conditioning showing significant reduction in C-
HA nanocomplexes conditioned group. (E) Bar graph showing increased UTS in
nanocomplexes conditioned…………………………………………………………….......62
Figure 3.2. Bioactivity characterization of C-HA nanocomplexes. (A) SEM image showing
mineral islands integrated with organic chitosan matrix, with EDX (inset) confirming rich
presence of C, O, Ca and P. (B) FTIR spectrum showing presence of bifid phosphate peaks
(red) and carbonate bonds in C-HA nanocomplexes films after SBF incubation suggestive of
mineral formation. (C) Sharp peaks on XRD spectrum suggest crystalline nature of formed
mineral (red) after bioactivity assessment in SBF compared to
complex…………………………………………………………………………………...…63
Figure 3.3. (A) Calculation of outcome measures of sealer penetration depth into dentinal
tubules………………………………………………………………………………………64
viii
Figure 3.3. (B). Bar charts (b-g) showing Mean, Maximum and % Area Penetration at 4 and
6mm levels from root apex in C-HA nanocomplexes conditioned group and control groups.
The % Area penetration at 6mm level and all outcome parameters at 4mm are significantly
higher than the control……………………………………………………………………...65
Figure 4.1. (A and B) Total ion maps for control and C-HA nanocomplexes conditioned dentin.
Interfacial layer with differences in pixel intensity can be seen (broken line marks the
interfacial boundary). (C and D) CN- chemical ion maps showing intense CN- signals of
integrated C-HA nanocomplexes at interface/subsurface in conditioned dentin. No signal
zone suggestive of degraded collagen is seen in control (yellow arrows). (E and F)
Showing Red-Green overlays of organic fragment (C2H4NO---red) with phosphates (PO2
-,
PO3- and PO4
- --green). (E) Minimal presence of phosphate ions at the sealer-dentin interface
(broken line) in control dentin. (F) Well-defined interfacial band of phosphates (PO2-, PO3
-) in
C-HA nanocomplexes conditioned dentin. (G) Red-Green overlay of chitosan fragment
(C5H6NO+ --red) with calcium (Ca+ --green) showing presence of calcium islands in
conditioned dentin in areas lacking chitosan matrix (yellow arrows)………………….…..83
Figure 4.2. Normalized mass spectral intensities for negative and positive polarity at interfacial
and total dentin region of interests. (A) Negative polarity non-specific protein fragments at
interface and (B) total dentin. (C) Negative polarity characteristic chitosan fragments at
interface and (D) total dentin. (E) Positive polarity characteristic chitosan fragments and (F)
non-specfic organic fragments at interface. (G) Phosphate fragments at interface and (H)
total dentin. (I) Presence of tricalcium silicate hydration products (Ca+, CaO+ and CaOH+)
at interface. (J) Higher molecular mass fragments (CaPO2+, CaPO3
+, CaPO4+, Ca2PO3
+ ) of
calcium phosphates at interfacial dentin…………………………………………………....85
Figure 4.3. TEM micrographs showing sealer-dentin (D) interface (IF) with presence of
interfacial layer (IL). (A) Control dentin shows a thin, discontinuous interfacial layer with
collagen fraying. (B) Conditioned dentin shows a closely adapted interfacial layer with
markedly increased thickness……………………………………………………………....87
ix
Preface
Dissertation format
This dissertation is submitted in form of a manuscript-based thesis. The research work done over
the course of Master’s program has been divided in two manuscripts, which are in the process of
being submitted for review in peer-reviewed indexed journal (Journal of Endodontics).
Chapter 1: provides a general introduction to the subject followed by hypothesis and objectives.
Chapter 2: presents a literature review of the topics related to the research problem.
Chapters 3 and 4: comprise of the experimental data in the form of manuscripts.
Chapter 5: provides a general discussion of the study rationale and combined experimental data
presented in the study.
Chapter 6: concludes the findings of the study.
Chapter 7: refers to potential future studies.
Scholarships/Awards
2017 Dr. Jaro Sodek Merit Award, Faculty of Dentistry, University of Toronto
2018 Harron Scholarship Award, Faculty of Dentistry, University of Toronto
2019 CADR-NCOHR Student Research Award (Travel Award )
x
Abbreviations
ACP Amorphous Calcium Phosphate
BSP Bone Sialoprotein
CEJ Cemento-Enamel Junction
Ca(OH)2 Calcium hydroxide
CMCS Carboxymethyl chitosan
C-HA Chitosan-Hydroxyapatite precursor nanocomplexes
DA Degree of acetylation
DD Degree of deacetylation
DEJ Dentino-Enamel Junction
DMP-1 Dentin Matrix Protein
DSP Dentin Sialoprotein
EDTA Ethylenediamine tetraacetic acid
GAGs Glycosaminoglycans
GTR Guided Tissue Remineralization
HA Hydroxyapatite
MEPE Matrix Extracellular Phosphoglycoprotein
NaOCl Sodium hypochlorite
NCP Non-Collagenous Proteins
OPN Osteopontin
PAA Polyacrylic acid
PAMAM Polyamidoamine dendrimer
P-chi Phosphorylated chitosan
PILP Polymer Induced Liquid Precursors
PVPA Polyvinylphosphonic acid
STMP Sodium trimetaphosphate
TCS Tricalcium Silicate Sealer
1
Chapter 1
Introduction
Background
Dentin forms the major proportion of tooth structure. It is a hydrated composite consisting of an
organic and inorganic fraction (1). The organic component of dentin contributes chiefly towards
toughness and tensile strength whereas the inorganic fraction adds to stiffness (1). The structural
and chemical characteristics of dentin can be compromised due to iatrogenic (treatment -
mediated) and non-iatrogenic (disease-mediated) causes (1) .
Non-surgical root canal treatment (NSRCT) is an established procedure for treatment of pulpal
infections causing the inflammation of peri-radicular tissues (2). The primary objectives for a
successful root canal treatment include complete bacterial elimination, prevention of reinfection
and preservation of structural integrity (3). Mechanical instrumentation and chemical irrigants
are routinely employed to achieve root canal debridement. However, with their use subsequent
alteration of dentin substrate is a common occurrence (4). Currently, there is extensive
documented evidence that links the use of root canal irrigants with compromised compositional,
mechanical and physical characteristics of dentin (5, 6). Reduced dentin hardness, flexural and
tensile strength have all been reported (1, 7). The use of caustic chemicals, such as sodium
hypochlorite and ethylenediaminetetraacetic acid, as root canal irrigants results in the loss of
mineral ions and denudation of collagen matrix (5). Since, the collagen has a weakly polar
character the surface properties of demineralized dentin become unfavorable for crystal
nucleation (8). Such ultrastructural changes also result in compromised interaction with root-
filling materials affecting interfacial integrity and associated predilection to fracture (9).
2
Currently, reconstitution of dentin matrix has been focusing towards mineral incorporation with
solutions of calcium and phosphate ions, such conventional approaches are limited to crystal
growth from residual seeding crystals that grow uninterruptedly as extrafibrillar precipitates (10).
A deeper understanding of structural hierarchy and mechanistic exploration of dentin
biomineralization have inspired tissue engineering of materials for hard tissue repair (11). This
has shifted the interest towards strategies capable of achieving functional remineralization which
encompasses extrafibrillar and intrafibrillar mineral deposition integrated with organic matrix
resulting in biomechanical behavior similar to dentin (12). These strategies work as bottom-up
schemes and are therefore independent of seeding crystals (13). Guided tissue remineralization
has surfaced as a novel biomimetic strategy that involves polyelectrolytic polymers that function
to stabilize amorphous calcium phosphate precursor phases and provide a template to achieve
intrafibrillar mineralization (14). This has been attempted with the use of polyacrylic acid and
polyvinylphosphonic acid polymers for dentin remineralization (15).
Chitosan is an attractive biopolymer owing to its biocompatibility, biodegradability and anti-
bacterial characteristics (16) to achieve guided mineralization of dentin. Its popularity in
biomedical field is largely due to the availability of free pendant groups on its backbone, such as
OH and NH2, that can be modified to conduct specific functions with optimized physical,
chemical and biological characteristics (17, 18). It provides an added advantage of mechanical
stability due to presence of parallel H-bonded chains compared to other polymers (11).
Phosphorylated chitosan has been used to achieve surface modification of demineralized dentin
and facilitate mineral deposition (8). Chitosan-hydroxyapatite blends as cements and pastes have
also been employed in context of temporary scaffolding in bone substitutes that ultimately
become resorbed and replaced with mature bone (18). Chitosan-Hydroxyapatite precursor (C-
3
HA) nanocomplexes, is one such development for guided remineralization of dentin. Its role as a
non-collagenous protein (NCP) analog is channeled through presence of a dense surface charge
allowing calcium sequestration and subsequent stabilization of precursor amorphous calcium
phosphate phase. Based on a bottom-up mineralization strategy, it is able to achieve synergistic
intrafibrillar and extrafibrillar mineralization of demineralized dentin. Combining such a
biopolymer with hydroxyapatite precursors would allow a multi-functional modification of
dentin substrate with respect to interaction with materials as well as provide structural support to
the HA precursors as they crystallize both intrafibrillarly and extrafibrillarly to strengthen
radicular dentin.
4
Hypothesis
Chitosan-Hydroxyapatite (C-HA) nanocomplexes conditioning of altered dentin would improve
chemical/mechanical characteristics of dentin.
Objective
The purpose of the study is: To develop and characterize a Chitosan-HA precursor (C-HA)
nanocomplex and to assess the ability of C-HA nanocomplexes conditioning on (1) sealer
penetration, (2) interfacial remineralization and (3) dentin mechanical property.
Specific Aims
Specifically, the study aims to:
1) Characterize physico-chemical and bioactivity potential of C-HA nanocomplexes and
assess dentin mechanical property.
2) Evaluate the effect of C-HA nanocomplexes conditioning of root canal dentin on depth of
tricalcium silicate sealer penetration.
3) Conduct chemical characterization of tricalcium silicate sealer-dentin interface.
5
Chapter 2
2 Literature Review
Dentin
Composition, structure and mechanical properties
Dentin is a mineralized tissue that has the greatest proportion amongst other tissues in a tooth
(12). It is a composite structure that consists of an 60% inorganic fraction, 30% organic matrix
and 10% water by weight (19, 20). The mineral is present in the form of calcium deficient
carbonated hydroxyapatite crystals (50 x 10nm), which are arranged intrafibrillarly and
extrafibrillarly with respect to collagen fibrils (Fig. 2.1) (11). The apatite crystals are rod/needle
like near the pulp and become more plate like towards the dentin-enamel junction with an
approximate thickness of 5nm (1).
The organic matrix is largely composed of Type I collagen (90%), which is also the most
abundant protein. Type I collagen exists as a fibrillar structure with the fibrils oriented in a plane
perpendicular to that of dentinal tubules (21). The non-collagenous proteins (NCPs) which are a
heterogeneous group of proteoglycans, phosphoproteins, glycoproteins, serum proteins, enzymes
and growth factors make up the remaining 10% of the organic matrix (22-24) and serve
important roles in biomineralization of dentin (25).
The water in dentin is present in bound and free states. The bound water is also known as
structural water, is associated with the inorganic apatite crystals, collagen and non-collagenous
proteins such as glycosaminoglycan’s (1). On the other hand, the unbound water is freely found
6
in dentinal tubules and other porosities in the dentin structure. The unbound water serves as a
source to keep dentin bathed in mineral constituents (26).
Figure 2.1. Hierarchical structure of dentin. Dentinal tubules with mineralized peritubular dentin (PTD)
act as fiber reinforcements within the less mineralized intertubular dentin (ITD) matrix. Mineralized
collagen fibrils intersect perpendicularly with the dentinal tubules. Nanometric structure of collagen fiber
with staggered collagen molecules and intrafibrillarly deposited hydroxyapatite (HAP).
http://www.dunand.northwestern.edu/research/images/bio/dentin1.png (retrieved on 18-11-2018)
As a distinct micro-structural feature, dentin consists of a network of tubules containing dentinal
fluid, that extend outward from the pulp towards the dentin-enamel junction (DEJ) with a typical
density 10,000 to 96,000 tubules per mm2 (1). Dentin shows anisotropic behavior as reflected by
regional differences in tubular density and alignment (27). At a structural level, dentin is
described as a “fiber-reinforced composite”, wherein a less mineralized intertubular dentin forms
the matrix, and the highly mineralized peritubular dentin forms the fiber reinforcements. The
matrix is traversed by dentinal tubules that run through bulk of the dentin (21, 28).
The mechanical properties of dentin are governed by the alignment of the dentinal tubules with
respect to direction of force applied (27). Mechanical integrity of dentin is maintained by an
7
interplay of organic and inorganic components. The mineral fraction mainly contributes towards
stiffness and compressive strength while the organic component is responsible for ultimate
tensile strength and toughness which is linked to the extent of tensile strain it can absorb before
failure (29). Dentin is therefore regarded as a tough material owing to the soft-wrapping like
effect of organic matrix around mineral platelets that dissipates stress concentrations within the
structure to inhibit fracture initiation and propagation (1).
Bound water plays a crucial role in stabilizing triple-helix of collagen structure. Each tripeptide
consists of two water molecules, and as this number increases the collagen swells laterally and
acts as a plasticizer. Proteoglycans are non-collagenous proteins that are constituted of a protein
core covalently attached to carbohydrate side chains of unbranched repeating units of
disaccharides called as Glycosaminoglycans (GAGs) (30) . GAGs are known to form
intrafibrillar supramolecular bridges with mineralized collagen fibrils (11). GAGs are highly
negatively charged and are surrounded by a shell of water molecules that allows them to occupy
a large hydrodynamic volume in solution, filling the inter fibrillar spaces within the collagen
matrix (31). GAGs interact with one another by forming tape-worm like aggregates due to
repulsions arising from the negative charge, their associations are further stabilized by
electrostatic and hydrophobic interactions and H-bonds. Therefore, they maintain a hydrostatic
pressure allowing them to stretch and unfold to help regulate mechanical stress (12, 31).
Dentin Biomineralization
Biomineralization is the formation or accumulation of minerals by organisms especially into
biological tissues or structures (such as bones and teeth) as defined by Merriam-Webster
dictionary (32, 33). This process is based on a highly dynamic environment orchestrated by
8
organic matrix components, importantly non-collagenous proteins (NCPs) (25). Dentin
formation is a tissue-specific process that occurs at spatially independent sites within the organic
matrix (34). Odontoblasts extend their processes through non-mineralized zone of pre-dentin
into the mineralization front. In the pre-dentin layer, odontoblastic cell bodies actively secrete
collagen and proteoglycans. A second pool of non-collagenous proteins is secreted close to the
mineralization front whereby they interact with collagen fibrils and trap calcium ions to initiate
nucleation (34).
2.1.2.1 Hierarchical Structure of Mineralized Collagen
Tropocollagen molecules form the building blocks of mineralized collagen. Each tropocollagen
molecule consists of three helical polypeptide chains with a length of ≈300 nm and a diameter of
about 1.5 nm, arranged in a staggered configuration. The most recent model describing the
molecular organization of collagen type I shows that the space between triple helical molecules
averages at about 1.3 nm (35). According to the quarter-staggered model, each collagen molecule
is off-set by 67 nm from the neighboring molecule creating a periodicity called the D-period.
There is a 40 nm gap zone, between the ends of each of these units owing to greatest
intermolecular and hydrophobic interactions of the charged side chains. Also, there is a 27 nm of
overlap zone between adjacent units. This spacing gives rise to the basic 67 nm repeat unit and
banding observed by electron microscopy that corresponds to approximately 234 amino acid
residues (36, 37). Five tropocollagen molecules bundle to form a microfibril (5-6 nm in
diameter) with respect to hydroxyapatite crystals, that grow on their surface with their c-axes
oriented along the longitudinal axes of the fibrils, as the next hierarchical level. Numerous
9
mineralized microfibrils organize into a collagen fibril. At the highest level, multiple collagen
fibrils combine to form the mineralized collagen fiber with a diameter of 80-100 nm (38).
2.1.2.2 Role of Collagen
The active role of collagen in biomineralization is generally well accepted (39, 40). Specific
sites in the type I collagen gap zones were reported by Silver and Landis, which have the
potential to sequester and bind calcium ions (41). It is also conceived that the three-dimensional
arrangement of charged groups on collagen may present a template for epitaxial nucleation as
well as contribute towards controlling the crystallographic orientation of minerals (39).
Subsequently, Nudelman et al. demonstrated charged interactions between the C-terminus end
region (+ve) of collagen gap zones and the negatively charged pAsp-ACP complex (40). These
findings emphasize the role of collagen in mineral growth.
2.1.2.3 Role of Non-Collagenous Proteins
In dentin, biomineralization is chiefly regulated by non-collagenous proteins. The NCPs are first
localized by its interaction with the type I collagen fibril, which then directs crystal nucleation
and templates the apatite crystals to grow (40). They act as promoters and inhibitors of
nucleation as well as have control over mineral orientation (25). They are classified as either
phosphorylated or non-phosphorylated proteins. Amongst them, SIBLING (Small Integrin-
Binding Ligand N-linked Glycoprotein) family of proteins consisting of bone sialoprotein (BSP),
osteopontin (OPN), matrix extracellular phosphoglyco protein (MEPE), dentin matrix protein
(DMP-1) and dentin sialophosphprotein (DSPP) is of significance. These proteins are highly
phosphorylated, acidic and rich in serine residues. Dentin sialophosphoprotein is the major NCP
10
of dentin which is secreted only at the mineralizing front and is not present in predentin. This
highlights its specific role in crystal growth behind mineralization front (34). It is rapidly cleaved
into its sub-domains, DSP (dentin sialoprotein) and DGP (dentin glycoprotein) and is very rich in
serine residues that develop a strong negative surface charge on phosphorylation and therefore
acts like a virtual sink for calcium ions (25).
DMP-1 has been shown to play a dual role in biomineralization such that it acts as mineral
nucleation site at specific collagen binding domains and on the other hand inhibits uninterrupted
crystal growth to occur (42, 43). The binding between collagen and DMP-1 is electrostatically
driven based on the presence of acidic residues in its peptide domains. The high negative charge
density created by presence of aspartic acid residues facilitates stabilization of amorphous nuclei
(42). Further, in-vivo and in-vitro studies have demonstrated the role of DMP-1 in both
promotion and regulation on mineralization with their lacking presence associated with
osteomalacia and rickets development (43, 44). Another important aspect of the process is
transport and availability of ion sources of calcium and other minerals at the mineralization front.
Calcium is transported within the odontoblastic process to the active mineralization front by
means of numerous intracellular transport mechanisms such as Ca+2-activated ATPase and
Na+/Ca+2 exchangers. While, phosphate is made available by non-specific cleavage of different
compounds by alkaline phosphatase to form inorganic phosphate (34). Biomineralization of
dentin is a complex process that involves an interplay between organic-inorganic matrix
interaction and availability of mineral ions.
11
Dentin Alteration
Iatrogenic effects during root canal treatment supplement the previously incurred disease
mediated changes in dentin causing further alterations in dentin ultrastructure (1).
Complementary use of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid
(EDTA) for root canal debridement is a standard practice in root canal treatment. Dentin
alteration (Fig. 2.2) attributed to the use of root canal irrigants have been extensively reported in
literature (9). Their individual effects on dentin composition, mechanical and physico-chemical
characteristics are reviewed in the following paragraphs.
Compositional alteration
Sodium hypochlorite (NaOCl), is a commonly used root canal irrigants because of its anti-
microbial and organic tissue dissolution properties (7). It is a strong oxidizing agent that exerts
an irreversible, nonspecific, digestive effect on vital and necrotic organic tissues as a result of
direct contact between free available chlorine and organic matter (45). The damaging effects of
NaOCl on dentin increase with time and concentration (9, 46) ranging from 0.5% to full strength
of 6%. (9). Penetration up to 77 μm was measured after incubation with 1% NaOCl for 2 minutes
at room temperature whereas a 300 μm depth of penetration was obtained with 6% NaOCl for 20
minutes at 45°C, evaluated with bleaching of crystal violet dye (47).
Sodium hypochlorite causes protein denaturation by breakage of peptide links and salt formation
through neutralization reactions (48). It may also cause collagen dissolution by oxidation of
cysteine amino acid residues present in dentin matrix to form cysteic acid (CysO3) (48). These
alterations result in disturbed chemical profile of dentin, with reported significant reduction in
carbon and nitrogen contents (49). Deproteination effect of 5% NaOCl on acid-etched dentin
12
resulted in reduced organic matrix with no differences in carbonate and phosphate using infrared
spectroscopy (50). Although, NaOCl is not a chelating agent, experimental evidence suggests
that it has a possible role in removal of inorganic ions such as calcium, magnesium and
carbonate (Ca+2, Mg+2 and CO3-2) from root dentin (46). The small size of hypochlorite anion
allows it to penetrate the water compartments of apatite-encapsulated collagen fibrils and thereby
continue its degradative action on the collagen molecules (4).
Ethylenediaminetetraacetic acid (EDTA), is a hexadentate ligand that is routinely employed for
chelation of calcium ions during smear removal from instrumented root canals. It is commonly
used in concentration of 5- 17% as disodium salt of EDTA. A 5-min. exposure of the root canal
to EDTA would remove the smear layer and open the dentinal tubules to a depth of 20-30 um
(51). It is capable of removing not only the calcium ions from HA structure of dentin but also the
ones that are bonded to NCPs (51). EDTA can also denature proteins that require Zn binding
such as type I collagen of dentin. EDTA is a strong Zn chelator and may cast irreversible
denaturation and aggregation of particular Zn binding domains (52).
The action of EDTA is described as self-limiting (51), based on studies that did not show an
increase in the demineralization depth beyond 50um even after a prolonged duration (53).
Combined use of EDTA followed by NaOCl has been shown to result in significant reduction in
calcium and phosphorous contents (6). Removal of magnesium and calcium from dentin after
EDTA irrigation was also reported in a recent time-of-flight secondary ion mass spectroscopy
study (54). It has been suggested that EDTA induced demineralization and collagen exposure
enhances bacterial adherence to dentin which may incur further toxin mediated damage (55).
Differences in irrigation methods, sequence and chemicals have shown different levels of
13
erosion. Dentin surface erosion on root canal dentin has been suggested to be an indicator of loss
of calcium and phosphorous from dentin matrix. Use of EDTA followed by NaOCl, has shown
conspicuous erosion of peritubular/intertubular dentin, increased surface roughness and widened
tubular orifices (56).
Mechanical alteration
The use of endodontic irrigants also alters mechanical characteristics of dentin. Exposure to
5.25% NaOCl for 2 hours was shown to significantly reduce the modulus of elasticity and the
flexural strength of standardized bars of dentin when compared to exposure to saline or 0.5%
sodium hypochlorite (49). A 59% decrease in strength of dentin cylinders has also been reported
with its use after 5 weeks of exposure with 5.25% solution (57). This is chiefly explained by the
damage incurred to the organic matrix of dentin that is responsible for contributing towards
dentinal strength. Significant reduction in microhardness of root dentin exposed to 17% EDTA
for 15 minutes has been reported (68). A 30% reduction in flexural strength and a 50% loss of
elastic modulus was shown for dentin beams exposed to EDTA over a 2 hours period
(58). Several in vitro studies have shown that combined use of NaOCl and EDTA can remove
the inorganic phase as well as the organic phase of dentin, giving rise to detrimental effects and
most pronounced reduction in microhardness of dentin. The chemically altered radicular dentin
can also form potential sites for crack initiation and subsequent fatigue failures (1).
14
Physico-chemical alteration
Use of chemical disinfectants alters the physico-chemical properties of root dentin which is
associated with disturbances in organic and inorganic proportions of dentin (59). Significant
reduction in dentin wettability assessed by static water contact angle measurements was reported
for dentin treated with combined use of NaOCl and EDTA compared to untreated dentin. EDX
analysis confirmed the presence of increased inorganic phase (calcium and phosphorus) in
groups with lower contact angle while increasing proportion of organic content (nitrogen and
carbon) was recorded for higher contact angle groups (60). Earlier findings have also reported
decreased dentin wettability after use of EDTA alone (61, 62). Induced surface roughness and
widening of dentinal tubules that allow air pockets to form within the tubules have been
proposed as causes of decreased dentin wettability (63). In another study, association between
increase calcium concentration with enhanced surface wettability was also established (64). Use
of NaOCl as a final flush following chelating agents also resulted in compromised wetting of the
root canal dentin (65). As discussed above, the use of irrigants alter the chemical and mechanical
properties of root dentin. One sequelae of these alterations is reduced bond strength between root
dentin and root filling materials that endangers interfacial integrity (9).
15
Figure 2.2. Iatrogenic alteration in dentin. A) Schematic showing role of dentin components towards
mechanical integrity (adapted & modified from (1) and B) shows how individual components become
altered with use of chemical irrigants affecting dentin interaction with materials.
A
B
16
Dentin remineralization
Conventional dentin remineralization strategies
Conventionally, dentin remineralization has been attempted using solutions with calcium and
phosphate ions in various fluoride concentrations (10). It is well established that these
remineralization strategies are based on the concept of classical nucleation kinetics through ion-
mediated pathway (66), whereby precritical nucleus crystallizes to a primary crystal without any
amorphous precursors (67). In context of dentin remineralization, it means epitaxial growth over
existing seed crystallites and a passive role of collagen fibrils in inducing mineral nucleation (23,
68). Therefore, in the absence or sub-critical presence of seeding crystallites, remineralization of
dentin surface would be halted (69, 70).
In conventional remineralization strategies, the mineral nucleation is dependent on local
supersaturation governed by pH, amount of seeding crystals and surface area (34). Although,
these strategies are successful in surface deposition of calcium phosphate crystals, the size of
these crystals grows uninterruptedly such that they can only be deposited extrafibrillarly. The
rapid accumulation of ions results in surface hyper-mineralization and presence of a sustained
mineral source becomes a limiting factor for remineralization at greater depths (34).
For every two fluoride ions, six phosphate ions and ten calcium ions are needed to form one unit
cell of fluorapatite (Ca10(PO4)6F2) (71). Recovery of dentin tissue entails a complex process as it
involves a bi-phased reconstitution of type-I collagen and apatite which have a specific spatial
connection that has invoked interest towards newer strategies (72).
17
Paradigm shift in dentin remineralization
Growth of apatite crystals from a super-saturated solution was initially viewed as a classical
precipitation reaction from studies (Fig. 2.3). However, studies by Eanes et al. (1965)
demonstrated that early precipitates are amorphous in nature which later transform into
octacalcium phosphate (OCP) and then Hydroxyapatite (HA) (73). This was followed by a model
for phase transformation that stressed that the governing factor for these transformations is
kinetically driven i.e. the most energetically feasible phase, amorphous calcium phosphate (ACP)
forms first followed by the most stable forms (HA) later. With lack of in-vivo evidence, it
became a matter of general acceptance that although ACP and other HA precursors form in in-
vitro models but are not likely to form in in-vivo mineralization (74). However, recent in-vivo
studies have reported formation of transient ACP phase in growing zebrafish bones (75), mouse
enamel as well as rat calvaria (76). This rekindled the interest towards mechanistic exploration of
biomineralization with non-classical pathways.
Figure 2.3. Proposed nucleation theories for collagen mineralization. Extrafibrillar mineralization is an
ion-based pathway explained by classical nucleation theory whereas amorphous precursor-based pathway
has been proposed for intrafibrillar mineralization.
Classical Pathway
Extrafibrillar
Mineralization
Ion-based
Non-Classical
Pathway
IntrafibrillarMineralization
Precursor- based
18
2.3.2.1 Proposed Models for Intra-fibrillar Mineralization
For the intrafibrillar mineralization two main models have been proposed to investigate the
possible routes of infiltration in the collagen fibrils for the ACP precursors.
The first in-vitro study to successfully demonstrate intrafibrillar mineralization of collagen was
reported by Olszta et al (2007). They proposed a “polymer-induced liquid precursor” (PILP)
hypothesis, that suggested that polyaspartic acid (pAsp) inhibits nucleation of apatite in solution
and stabilizes the formation of a liquid-like, highly hydrated ACP phase. The polymer-stabilized
ACP then infiltrates into the collagen through capillary action, and undergoes transformation into
oriented apatite crystals overtime (77). In contrast to previous, Deshpande et al. (2008) proposed
that binding of pAsp to collagen occurs, creating a local supersaturation of calcium ions,
allowing mineralization to occur directly in the fibril as calcium phosphate aggregates (76).
Later studies supplemented these models and showed that besides forming complexes with ACP,
the pAsp could also interact with collagen. This was attributed to the presence of rich carboxyl
groups of the polymer that are able to chelate calcium ions, creating a charge to sequester
phosphate ions forming a negatively charged complex that can interact with positive C-terminal
domains of collagen gap zone (78). However, this has been challenged by a recent study,
suggesting other long range forces may also be involved since electropositivity or
electroneutrality of polymer employed does not change the dynamics of ACP stabilization and
infiltration into collagen (79).
Regardless of the mechanism for ACP infiltration, these results were significant in introducing a
new concept and providing some mechanistic details regarding the potential of intrafibrillar
collagen mineralization through biomimetic models. The most significant deduction being the
19
role of polyelectrolytic nature of NCPs that plays a critical role in ACP stabilization and
subsequent intrafibrillar mineralization (80).
Biomimetic dentin remineralization approaches
Merriam-Webster dictionary defined Biomimetic as, “the study of the formation, structure, or
function of biologically produced substances and materials and biological mechanisms
especially for the purpose of synthesizing similar products by artificial mechanisms which
mimic natural ones”.
Guided tissue remineralization (GTR) strategy as a bottom up scheme, makes use of the
amorphous nanoprecursors as the critical nuclei of crystallization mimicking the process of
nature (14, 40, 80). Guided Tissue remineralization is based on the electrostatic interactions of
macromolecules (NCP analogs) that function as sequesters, stabilizers and templates for initially
formed ACP precursors to guide towards the gap zone of collagen matrix achieving ordered
intrafibrillar mineralization. For biomimetic mineralization, the in-vitro models employ use of
bioactive molecules, synthetic polymers and recombinant proteins as NCP analogs. Biomimetic
studies are carried out under ambient conditions without the use of special equipment making
their use an economically viable option (34).
A noteworthy aspect of adopting guided tissue remineralization strategy is the impact on
mechanical properties. Kinney et al. found the hardness and elastic modulus of dentin lacking
intrafibrillar mineral was significantly reduced owing to only a minor difference in mineral
quantity from normal dentin (81). This was a significant finding to highlight the importance of
quality of remineralized dentin in terms of microstructure, mineral density and most importantly
location of mineral formed with respect to the organic matrix (11). Ideally, mineral incorporation
20
in intrafibrillar and extrafibrillar locations would lead to a full mechanical recovery of
demineralized dentin as it allows collagen matrix reinforcement and efficient stress transfer (12).
Intrafibrillar mineral also provides added resistance towards collagenolytic enzymes and
demineralization as the loss of mineral from intrafibrillar location is slower, (82, 83) as well as
the partial demineralization of dentin may contain residual minerals associated with NCPs, that
would subsequently act as nucleation sites for mineral growth (12).
2.3.3.1 Non-Collagenous Protein analogs
Non-collagenous protein analogs (NCPs) play an important role in biomineralization of
dentin, therefore it becomes a natural strategy to employ their use in biomimetic schemes.
Extraction of intact NCPs from animal sources is difficult and has not yet become economically
viable. Moreover, concerns for allied immunogenicity and allergens have also restricted their use
(84).
Polyvinylphosphonic acid (PVPA) and Polyacrylic acid (PAA): Amongst pioneering analogs
were Polyvinylphosphonic acid (PVPA) and Polyacrylic acid (PAA) (10, 14, 85, 86). Poly
aspartic acid (PAA) has been used as the sequestering NCP analog found in bone alongside
Polyvinylphosphonic acid (PVPA) as the templating analog to stabilize ACP nanoprecursors
with evidence of both extrafibrillar and intrafibrillar remineralization of acid etched dentin.
These studies evidenced similar formation and crystallographic alignment of apatite crystal
inside collagen. Remineralization was shown to occur as the result of slow release of calcium
ions from the hydration of Portland cement and the interaction with phosphate containing
fluid. At 2 weeks, partial zones of remineralized dentin were observed starting from the base of
the mineralized dentin outward toward the dentin surface. At 4 weeks, more continuous zones of
21
intra and extrafibrillar mineralization were observed. Only by the end of 8 weeks, complete
remineralization of 5um demineralized region was achieved. When the templating analogue was
not incorporated, a very limited interaction with collagen matrix was seen (14).
Sodium trimetaphosphate (STMP): Is an industrial phosphorylating agent. The phosphate groups
of STMP can be transferred onto collagen by reacting with hydroxyl groups of proteins,
providing a strong negative surface charge which allows calcium nucleation (68). Sodium
trimetaphosphate (STMP) along with PVPA was also employed as a templating molecule to
attract ACP-nanoprecursors to nucleate in the collagen fibrils (8).
Chitosan: Phosphorylated chitosan (P-chi) has a strong affinity to bind to calcium ions and hence
induces calcium phosphate formation. (P-chi) was used as a NCP analog and demonstrated
calcium phosphate deposits on demineralized dentin (8).
Polyamidoamine dendrimer (PAMAM): In artificial saliva promoted formation of PAMAM-
collagen matrix complexes and resulted in intrafibrillar mineral deposition which was shown
with apparent collagen periodicity (72, 87).
Casein phosphopeptide-Amorphous calcium phosphate (CPP-ACP): It contains the cluster
sequence of -Ser (P)-Ser (P)-Ser (P)-Glu-Glu from casein which gives the ability to stabilize
ACP and thereby maintains high concentration gradients on tooth surface (13). Compared to its
role in enamel remineralization which is extensively documented and is also available as
commercial products, its effect on dentin intrafibrillar remineralization is less documented. It has
been employed in one study as a root canal irrigant to improve micro-hardness of dentin (88).
Modifications to functional groups on polymers have also been used to enhance their
phosphorylation potential. The order of binding affinity of some functional groups to calcium ion
is highest for phosphates followed by carboxyl, amido and hydroxyl groups (8, 89).
22
2.3.3.2 Remineralizing Medium
Biomimetic studies employ use of multiple supersaturated solutions that are metastable with
respect to HA are reported in literature. They are prepared to match the ionic concentrations of
body fluid in an attempt to provide a facilitative environment for mineral formation and growth
in bioactivity studies by providing calcium and phosphate.
The most commonly used calcium-containing remineralizing medium was Portland cement,
which acted as the dual source for calcium and hydroxyl ions (14, 85, 90, 91). In addition,
calcium phosphate remineralizing solution, casein phosphopeptide-amorphous calcium
phosphate or CPP-ACP paste (92), artificial saliva (93), bioactive glass (91), calcium chloride
solution (94), and metastable calcium phosphate solution (95) were also used. As phosphate
source, remineralizing mediums such as simulated body fluid (SBF) (90, 96, 97), phosphate-
containing solution/gel, calcium phosphate remineralizing solution, artificial saliva and
phosphate-buffered saline (PBS) were used (13).
23
Table 2.1. List of NCP analogs adapted and modified (80).
NCP Analogues Function of NCP Analogues Approach
Polyacrylic acid (PAA) Simulating CaPO4 binding sites
of DMP1
Stabilizing ACP
Inhibiting nucleation for ACP
stabilization
Prolonging the lifetime of ACP
PAA-containing SBF
PAA-STMP-MTA
PAA/PVPA & PO4 solution
Sodium trimetaphosphate
(STMP)
Phosphorylating of type I
collagen
Binding to demineralized
collagen matrix
Forming covalent bonds
Attracting ACP-nanoprecursors
STMP-collagen matrix
PAA-STMP-MTA
Phosphorylated chitosan
(P-chi)
Binding to collagen
Introducing functional groups
onto the collagen
P-chi collagen matrix
Peptide Binding calcium ions
Initiating mineral deposition
Binding collagen by electrostatic
interactions
Peptide-collagen matrix
Agarose gel Binding to collagen molecules Agarose gel-PO4-collagen
matrix
Polyamidoamine dendrimer
(PAMAM)
Binding to collagen fibrils
Recruiting ACP nanoprecursors
into collagen matrix
Guiding meso-crystals to
assemble into large ones
Inducing the periodicity of the
mineralized fibrils
PAMAM-collagen matrix
L-glutamic acid (Glu) Triggering crystallization
Promoting calcium phosphate
crystallization
Substituting Glu-rich domain of
DMP1
PAA/L Glu-CaPO4 solution
24
Tricalcium Silicate based materials
In context of mineralization, bioactivity is described as the ability of materials to form
carbonated apatite on their surface when exposed to simulated body fluids in vitro (98). Inspired
from the use of bioactive materials in restorative dentistry, (such as incorporation of silica or of
calcium phosphate materials in filling materials) mineral trioxide aggregate (MTA) was
introduced as a root-end filling material. Owing to its biocompatibility and osteogenic abilities, it
gained popularity for use in extensive applications such as vital pulp therapy, apexogenesis,
perforation repairs etc. (99).
MTA as a Portland cement, mainly consists of a mixture of tricalcium silicate, tricalcium
aluminate, silicon oxide and bismuth oxide as radio-opacifier (100). The 1st generation MTA
based materials were alumina based with drawbacks of difficulty in handling, discoloration on
contact with NaOCl and extended setting time. To overcome some of these challenges, a 2nd
generation of MTA based materials was introduced with modifications of cement formulation
from Portland to calcium silicate, alternative radiopacifiers and the introduction of additives.
Another change to the material, which improved its characteristics was reduction in the particle
size of the powder (101). Use of tricalcium silicate based cements as sealers was proposed by
Gandolfi et al. (102). Sealer penetration into dentinal tubules is considered to be a clinically
positive outcome as it may facilitate deeper anti-bacterial action inside dentinal tubules for
sealers having anti-bacterial potential as well as improve sealer retention due to an extended area
of interface (103).
25
Hydration Reaction of Tricalcium Silicate based Sealers
In comparison to conventional base/catalyst based sealers, tricalicum silicate sealers (TCS) are
hydraulic in nature. This means moisture present in environment (dentinal tubules) is essential to
activate their bioactivity resulting in apatite formation while the sealer continues to set. Studies
conducted to assess sealer setting in different conditions of humidity showed a lack of TCS
setting in dry environments (104). Bioactivity of TCS is attributed to the formation of calcium
hydroxide in presence of phosphate containing fluids through the active components of tri and
di-calcium silicates.
The setting reaction has been described as follows:
(A) 2 (3CaO . SiO2) + 6H2O --->- 3CaO . 2SiO2 . 3H2O + 3Ca (OH )2 Hydration
(B) 2 (2CaO . SiO2) + 4H2O --->- 3CaO . 2SiO2 . 3H2O + Ca (OH )2
(C) 7Ca(OH )2 + 3Ca(H2PO4)2 --->- Ca10(PO4)6(OH )2 + 12H2O Precipitation
The setting reaction has been described as a “dissolution-precipitation” reaction whereby a
gradual dissolution of the unhydrated calcium-silicate particles occurs to result in formation of
calcium hydroxide and calcium-silicate crystals (elongated, needle-like) dispersed in an
amorphous, hydrated calcium-silicate gel (CSH). The second reaction occurs in contact with
phosphate containing medium or if a phosphate source is present among the constituents of TCS
sealer (such as in Endosequence BC Sealer, Brasseler, Savannah, GA or iRoot SP, Innovative
BioCreamix Inc., Vancouver, Canada) (15, 99, 104).
Such an environment allows for a rapid ion exchange with a prolonged calcium and hydroxyl ion
leaching into the environment. Since most of the TCS formulations available lack a phosphate
phase among their constituents, need for a phosphate source exists to allow formation of calcium
phosphate phases (HA). This forms the basis for most of the in-vitro characterization studies to
26
incorporate a mineralizing medium (as discussed above). Lack of formation of phosphate phases
has been reported when TCS was allowed to set in contact with root canal dentin (105). This
highlights the importance of phosphate presence in the local environment to allow optimal
tapping of bioactivity potential of tri-calcium silicate sealers in approximation to dentin surface.
Chitosan
Chitin was isolated for the first time by H. Brancota, in 1811. Chitin is a polyaminosaccharide
that is primarily derived from the shells of crustaceans, such as crabs, shrimps, and lobsters
(106). The deacetylated form of chitin, called chitosan, was described by Rouget in 1859.
Chitosan is the second most abundant biopolymer, next in line to cellulose.
Structure, chemistry and properties
Conversion of chitin to chitosan involves a multi-step process involving demineralization,
deproteination and deacetylation with strong alkalis. The deacetylation of chitin is difficult to
achieve in totality mainly because of unreachable amide groups, therefore it consists of varying
amounts of linked residues of N-acetyl-2 amino-2-deoxy-D-glucose (N-acetyl-glucosamine) and
2-amino-2-deoxy-D-glucose (glucosamine) residues (Fig. 2.4) (107). The ratio of glucosamine
to N-acetyl glucosamine is referred to as the degree of deacetylation (DDA). The DDA is a
factor of both the source of the chitosan as well as the preparation methods, and may range from
as low as 30% to almost 90% (108). DDA is reliably calculated using infrared spectroscopy with
most commercially available forms in range of 75-85%. Chitin chains can assemble into larger
bundles in at least three different conformations that have been termed α, β and γ chitin. α-Chitin
is composed of antiparallel chains of N-acetyl-glucosamine, allowing for strong intermolecular
27
bonding. It is commonly found in the shells of crustaceans like shrimp and crabs, and also in
fungi (109). β-Chitin, which is commonly found in squid pens, contains chains that are aligned in
a parallel fashion, resulting in weaker intermolecular interactions (109) . Finally, γ-chitin has two
chains going in the same direction while a third chain goes antiparallel to them (109). In these
crystalline structures of chitin, acetyl groups serve as essential interactive sites that connect to
adjacent chains via hydrogen bonds as well as other non-bonded interactions. These interactions
between micro-fibrils provide tensile strength and toughness which makes chitosan a superior
biopolymer providing means of structural support in organisms (110). The physical and chemical
properties of chitosan such as crystallinity, charge density, surface energy, degradation are
highly dependent on its molecular weights and degree of deacetylation (111). Like most of the
biopolymers, chitosan is well-known for its tendency to self-aggregate in aqueous solution. The
higher molecular weight and hydrophobic moieties in chitosan, i.e., acetyl groups and glucosidic
rings, play a significant role towards this. However, it can dissolve in acidic medium due to the
protonation of –NH2 groups at the C2 position. It has a pKa of 6.3. Due to the strong electrostatic
repulsion between protonated amino groups, chitosan adopts an extended conformation in acidic
solution (112). The solution conformation of chitosan has been described as “flexible worm-like
chains” (113). The strong functionality of chitosan due to presence of (two hydroxyl groups (C3,
C6) and one primary amine group (C2) per-repeat unit) gives it a considerable opportunity for
chemical modification (107) compared to chitin that lacks the amino groups.
Carboxymethylation is one of the many modifications for chitosan, which results in introduction
of carboxymethyl groups (–CH2-COOH) onto chitosan. Since there is more than one reactive site
in chitosan’s molecular structure, three carboxymethyl derivatives (Fig. 2.5) with different
substitution sites, i.e., O-CMC (with –OH being substituted), N-CMC (with –NH2 being
28
substituted), N-O-CMC (with both –OH and –NH2 being substituted), can be prepared through
various synthesis routes (17, 114). Important common properties shared by these derivatives
include their water solubility over a large pH range with minimal degree of substitution,
amphoteric behavior and metal chelation abilities. Chitosan is polycationic owing to presence of
-NH2 groups while addition of carboxyl groups renders it anionic, therefore depending on pH of
the medium, it can react with both acids and bases conferring amphoteric properties.
Carboxymethyl chitosan is water soluble due to the substitution of carboxymethyl group in place
of acetyl group which limits the extent of inter-chain H-bonding, dissociation of carboxyl groups
(COO-) and H-bonding between COO- groups and water (115).
Chitosan is a non-toxic, anti-microbial, bioadhesive, metal chelating, biodegradable and a
biocompatible polymer, therefore it presents as an excellent material for biological applications
(116). It is FDA approved for wound dressings. The biodegradation in vivo takes place by
lysozyme (117). Presence of higher concentration of nitrogen and the ability to synthesize
chitosan in various forms such as paste, membranes, sponges, fibers and spatial porous structures
like scaffolds further adds to its widespread utilization. It is currently being explored for a
multitude of applications like drug carriers, bone substitutes as well as wound dressings amongst
others in several fields of pharmacy, biomedicine, agriculture, food industry and biotechnology
(17, 18).
29
Figure 2.4. a) Molecular structure of N-acetyl chitin. b) Side view of a chitin fibril with Acetyl groups
outlined in rectangles showing inter-fibril associations (114).
(O-Carboxymethyl Chitosan) ( (N,O-Carboxymethyl Chitosan)
(N-Carboxymethyl Chitosan)
Figure 2.5. Deacetylation of chitin and modified chitosan derivatives. Adapted and modified (122).
30
Amorphous Calcium Phosphate (ACP)
Amorphous calcium phosphate (ACP) is a unique form of calcium phosphate minerals in
organisms that lacks the long-range periodic array found in crystalline calcium phosphates. It has
a basic structural unit - 9.5 Å dia, spherical Posner’s clusters Ca9 (PO4 )6 which contains 10–20%
by weight of water within the interstices. These clusters pack randomly to form ACP particles. It
has a molar Ca/P ratio of 1.18–2.50 and forms in neutral to basic pH with sizes of about 20–
300 nm (73, 118, 119).
ACP is a metastable phase, i.e. it is thermodynamically unstable and is formed as the initial solid
phase that precipitates from a highly-supersaturated calcium phosphate solution. It then
undergoes transition pathways to form the more stable crystalline hydroxyapatite (HA) phase
(118). ACP has gained tremendous significance since its role in biomineralization has been
established. It fulfills three roles during this process. Foremost, is its role as a mandatory
precursor towards HA formation. Secondly, it is employed as a temporary storage for apatite as
Ca and P sources. Thirdly, it has an isotropic nature giving it flexibility to conform to available
spatial geometry. This allows overcoming of directional restrictions of crystals and facilitates the
transport of large amount of the mineral to the growth front that are necessary to build
mineralized structures (40). This forms the basis for intrafibrillar mineralization to take place.
Moreover, it has better osteoconductivity and biodegradability compared to apatite, owing to
its disordered structure that gives ACP high reactivity with body fluid, causing substantial
dissolubility and fast apatite precipitation (120).
ACP has been used to prepare the crystalline phases such as hydroxyapatites, tricalcium
phosphates and calcium pyrophosphates in the form of powders, coatings, ceramics and fillers.
In combination with dopants and resins, ACP has been employed as a sustained phosphate
31
release source in composite restorations (121). Due to inherent high solubility of ACP, its use as
a dental material becomes restricted. As opposed to the use of inorganic additives, which
potentially change the chemical composition and characteristics of ACP, use of organic
stabilizers can endow ACP with versatile bioactivities. This is where the stabilizing role of
polymers such as chitosan and polyaspartic acid as NCP analogs has been explored (80).
Chitosan-Hydroxyapatite Precursor Nanocomplexes
Chitosan-Hydroxyapatite precursor nanocomplexes have carboxymethyl chitosan as the organic
fraction and the amorphous calcium phosphate as the precursor to hydroxyapatite. Due to its
outstanding biocompatibility and biodegradability, challenges associated with potential risk of
lactose intolerance with traditional casein phosphopeptide-amorphous calcium phosphate
complex are not a concern (122). Moreover, unlike hydrophobic stabilizers such as dextrins, the
hydrophilicity of water soluble chitosan may attract more water to facilitate HA formation
through dissolution-precipitation process (123). Chitosan-HA composites have been extensively
explored as bone substitutes and role of phosphorylated chitosan in remineralization of dentin
has also been documented (8).
Inspired from natural composites (bones, teeth), chitosan-hydroxyapatite precursor
nanocomplexes (C-HA) consists of a polymer with mineral-precursor filler to provide the
necessary strength and flexibility of polymer and the hardness and stiffness from the mineral
formed over time (18, 124). It may serve as an initial matrix to hold the ACP during early phase
of mineralization and then eventually degrade to allow seamless integration of newly formed
mineral with the underlying dentin, similar to the concept of bone scaffolds.
32
The main driving force behind the use of chitosan and amorphous calcium phosphate as tooth
substitute material is their chemical similarity to glycosaminoglycan in dentin structure. In
addition to being non-toxic, they are not recognized as foreign materials in the body and, most
importantly, both exhibit bioactive behavior and can integrate into living tissue by the same
processes as those active in biomineralization (18, 125). C-HA nanocomplexes are functionally
inspired by the in-vivo role of DMP-1 that inhibits spontaneous precipitation and aggregation of
calcium phosphate from a supersaturated solution and owing to its polyelectrolytic nature of
carboxyl groups stabilizes them as pre-nucleation clusters (126). In line with proposed models of
intrafibrillar mineralization they can infiltrate within the collagen gap zones to allow intrafibrillar
mineralization (Fig. 2.6) followed by extrafibrillar mineralization to take place (127-129). The
potential for sustained release of calcium phosphate would allow sufficient time for newly
formed minerals to integrate with the collagen matrix, a major aspect lacking in conventional
mineralization (10). The nanometric length scale has important bearing on the functional role of
this complex as they are specifically engineered to be able to penetrate the collagen gap zones.
33
Figure 2.6. Proposed role of Chitosan-Hydroxyapatite precursor (C-HA) nanocomplexes in dentin
biomineralization.
34
Characterization of bioactivity
Characterization involves the study of material’s features such as its composition, structure,
physico-chemical, mechanical and biological properties that shall impact its physiological
behavior and therapeutic efficacy. Therefore, a combination of analytical tools is routinely
employed for an in-depth characterization.
In electron microscopy, when a collimated beam of primary electrons comes in contact with
matter, multiple interactions occur generating secondary electrons (SEM) and transmitted
electrons (TEM) which are detected by their respective detectors to produce images. SEM is
commonly used for morphological assessment of surface topography and couple energy
dispersive spectroscopy detector (EDS) allows elemental identification and calcium phosphate
ratios for compositional profiling.
Use of transmission electron microscopy (TEM), is employed when very high resolution is
required such as for structure and location of mineral formation or their growth directions.
However, it involves the use of very small tissue volumes that may not represent the bulk
material (12). Further, TEM imaging does not enable the distinction of mineral that is closely
positioned to the organic matrix from the one that is chemically bound to it (8, 12, 14).
Fourier transformation infrared spectroscopy (FTIR) is an optical instrument useful for
identifying organic and inorganic compounds through detection of chemical bonds. It allows
assessment of nature of mineral formed by identification of phosphate and carbonate as well as
provides quantitative information on changes in mineral to matrix ratio (130).
X-ray diffraction analysis (XRD) is a non-invasive, crystallographic technique that provides
information about degree of mineralization. Since, atoms in a crystal are arranged in a long-
periodic array they allow constructive interference of incident x-rays to occur at specific angles
35
(2 theta) which appear as sharp peaks on the diffractogram as opposed to an amorphous solid. It
is a bulk characterization technique that allows identification of crystals through matching of
crystal planes against a standard database. It has been frequently used for identification of apatite
precursor phases (8, 131). Transverse microradiography, polarized light microscopy and
thermogravimetric analysis have also been used to provide a net assessment of mineral quantity
(12). A comparative analysis of characterization techniques is presented in Table 2.2.
Time of Flight-Secondary Ion Mass Spectrometry
Time-of-flight Secondary Ion Mass Spectrometry (TOF-SIMS) is a chemical characterization
tool. In Secondary Ion Mass Spectroscopy (SIMS), the sample surface is bombarded with high
energy ions (1keV to 25keV) using a primary ion source. The particle energy is transferred to
the atoms of the solid by collision cascade providing sufficient energy to ionize many elements.
The secondary ions released are accelerated across a voltage gradient and extracted into a time of
flight mass detector, which works on the principle highlighted in the below equation. The arrival
times of ions is dependent on their mass to charge ratio i.e. the lightest ones will arrive the
earliest and vice versa (132).
The primary ion dose is a significant factor in determining if static or dynamic SIMS analysis
would be conducted. Static SIMS uses a primary ion beam dose which minimizes the interaction
of the primary ion beam with the top layer of atoms or molecules (Fig. 2.7), such that less than
1% of the surface is removed (133). Thus, provides monolayer sensitivity with high spatial
m/z = mass to charge ratio
V= velocity of ion
t = time of flight
L= length of vaccuum
36
resolution. Whereas, in dynamic SIMS the static ion limit is bypassed, this analytical mode is
more suited for bulk analysis and trace elements. Data output is in form of high-resolution mass
spectra, chemical ion maps and possibility of depth profiling with 3-D reconstruction with
dynamic SIMS. Supremacy of TOF-SIMS over other chemical characterization techniques
comes from a combination of extreme surface sensitivity along with the ability to detect trace
amounts of materials (133).
Figure 2.7. Principle of TOF-SIMS operation. Adapted and modified (129). Incident primary ion beam
on sample surface causes surface ionization. The secondary ions are extracted into a flight tube and
identified by the mass detector based on the time of flight and mass to charge ratio.
https://www.ifwdresden.de/fileadmin/_processed_/9/a/csm_31_me_sims2_skizze_7a8632709a.jpg ( retrieved on 17-11-2018)
37
Moreover, it gives complete elemental, isotopic and molecular information for all elements of
the periodic table unlike limitations of energy dispersive spectroscopy. Although, TOF-SIMS
was originally developed as a technique for inorganic characterization with advancements in
secondary cluster sources it has been used to characterize phospholipids, peptides and other
biomolecules on the cellular level (134).
Frequently cited limitations of TOF-SIMS include the excessive amount of data generated such
that a typical 256 x 256 pixel image contains 65,536 spectra. Also, differences in fragmentation
patterns reported with use of different instruments. Another, challenge with data analysis of
organic substrates occurs that show overlapping signals (133). Use of TOF-SIMS in dental
studies is still in infancy with only a few (approximately 9) studies reported in literature. These
studies mostly concentrated on characterization of dentin composition (135, 136), analysis of
hypomineralized and normal enamel (137), detecting presence of esters in carious dentin (138),
assessment of fluoride uptake after remineralization treatment (139), fluoride incorporation depth
at resin-dentin interface (grey literature) and qualitative analysis of precipitate formation on
dentin post irrigation protocols (54, 140).
38
Table 2.2. Comparison of characterization techniques.
Characteristics TOF-SIMS XRD EDX FTIR
Principle Mass
spectrometry
Diffraction by
atomic planes
Characteristic
x-rays
Molecular
vibrational
frequency
matching the IR
Assessment Chemical (ion)
mapping
Crystallographic Elemental Chemical bonds
(composition)
Sample diversity Powder, Films,
Fixed/frozen
hydrated
Powder
Films
Powder
Films
Powder
Liquid
Primary excitation Ions X-Rays Electron Mid-IR
Identify Organics Yes Some crystalline No Yes
39
Characteristics TOF-SIMS XRD EDX FTIR
Detection limit ppm 1% 0.1% wt ppm
Resolution Lateral R -100nm
Depth profiling -1nm
Mass R 10,000 m/z
Lateral -30um Lateral R- 1um
Depth Profiling-
1-2um
10-30um
Elemental range All Crystalline
materials
6-92 N/A
Technique Time Min
(hours for analysis)
Hours Quick Sample prep
takes time
Quantitative Semi Semi Yes Yes
40
Critique of Literature
The currently accepted notion in dentin remineralization is that mineral nucleation occurs neither
by spontaneous precipitation nor by nucleation on the organic matrix instead by the remnant
crystals in the demineralized dentinal matrix (34, 69). Conventional dentin remineralization
strategies using solutions with calcium and phosphate ions in various fluoride concentrations are
unable to achieve spontaneous precipitation and intrafibrillar mineralization. However, these
strategies result in a rapid buildup of extrafibrillar mineral that results in surface hyper
mineralization with further supply of ions becoming scarce (34) (71). Greater in depth
understanding on the role of organic matrix in the dentin biomineralization has highlighted that
collagen could play a key role in the remineralization of dentin (39, 40). Thus, remineralization
could be attempted by modifying collagen, while foregoing the conventional concept of sole
dependence on the inorganic matrix to induce remineralization of dentin (34). Inspired by the
stabilization effect of DMP-1 on ACP, materials that can simulate this role have been designed
such as polyacrylic acid and polyaspartic acid (14). It is highlighted here that numerous NCP
analogs are capable of mineral extrafibrillar deposition on collagen surface by mimicking surface
phosphorylation role of NCPs for calcium sequestration but their ability to infiltrate collagen gap
zones for intrafibrillar mineralization remains largely unexplored (83, 92).
The mechanical properties should be assessed as an end-point of successful remineralization as
opposed to qualitative characterization of mineral formation alone (12). Existing studies mostly
evaluate surface hardness, which serves as a surrogate marker of increased mineral quantity.
However, it may not ascertain coupling of mineral with collagen matrix (21, 88). In an earlier
study, it was demonstrated that use of CPP-ACP as final irrigant after EDTA and NaOCl did not
have a difference in bond strength values compared to EDTA and NaOCl treated samples but
41
showed increased microhardness of root dentin. This may highlight the lack of organic
reinforcement by the CPP-ACP amongst other reasons (88). Therefore, other techniques such as
tensile, flexural, compressive strength measurements should also be considered (12, 141).
The focus of previous studies has largely been restricted towards modifying properties of
materials coming in contact with dentin instead of modifying the dentin substrate characteristics
such as use of surfactants to improve dentin wetting by irrigants (59, 142, 143). In general,
dentin surface modifications must aim at improving/stabilizing its physico-chemical
characteristics. One earlier study attempted to modify dentin surface using silanization to
provide better interaction between a hydrophobic sealer and dentin surface with higher
hydrophobic characteristics (142). However, the hydrated nature of dentin and the potential
damage due to toxic solvents used and products derived from the chemical reaction of
silanization was not taken into consideration. Thus, experiments should be designed to keep the
end point as clinical application in mind. Moreover, extrapolation of wettability of dentin slabs
as enhanced sealer penetration may seem speculative as factors such as thickness of dentinal
slabs and presence of smear layer can play a major role in confounding the results (142, 144).
Therefore, a root canal model should be incorporated to test this association.
Surface wettability is a physical phenomenon, dependent on surface chemical composition,
therefore chemical characterization must also be considered with surface sensitive techniques.
Moreover, no study has evaluated chemical characterization of dentin substrate to explain the
physico-chemical changes at TCS-dentin interface. Finally, TCS-dentin interfacial
characterization has not been attempted with a highly surface sensitive technique such as Time-
of-Flight Secondary Ion Mass Spectrometry, that can provide simultaneous information on
chemical profile and spatial distribution of chemical constituents.
42
Chapter 3
3 Manuscript (I)
Impact of Dentin Substrate Modification with Chitosan-Hydroxyapatite Precursor
Nanocomplexes on Sealer Penetration and Dentin Mechanical Property
Anam Hashmi1, Xu Zhang2, Anil Kishen 1
1Kishen Lab, Faculty of Dentistry, University of Toronto, Toronto, ON 2School of Stomatology, Hospital of Stomatology, Tianjin Medical University, China
Abstract
Objective: The purpose of the study was to evaluate the effect of dentin conditioning with
Chitosan-Hydroxyapatite precursor (C-HA) nanocomplexes on depth of tricalcium silicate sealer
(TCS) penetration into tubules and dentin mechanical property.
Methods: Phase-I Surface charge and size distribution for C-HA nanocomplexes formulation
was evaluated followed by bioactivity assessment of films (6.5x2.5cm2) of C-HA
nanocomplexes (n=15) incubated in simulated body fluid. Mineralization potential was assessed
with x-ray Diffraction and Fourier Transformation Infrared Spectroscopy while Scanning
Electron Microscopy was used for ultrastructural evaluation. Static water contact angles and
Ultimate tensile strength (UTS) was measured on dentin discs (n=2/group) and dentin beams
(n=10/group) respectively treated with/without NaOCl/EDTA and C-HA nanocomplex
conditioning. Phase-II: Depth of sealer penetration after C-HA nanocomplexes conditioning was
evaluated using fluorescent imaging (n=12). Percent penetration area, mean/maximum
penetration depth was calculated at 4 and 6mm levels from root apex.
43
Data from contact angle measurements, mechanical testing and penetration assessment
parameters were subjected to independent samples T-test with significance level set at p<0.05.
Results: 2mg/ml of C-HA nanocomplexes was chosen as polyanionic, hydrophilic, non-
aggregating formulation having bioactivity potential established through formation of
phosphate/carbonate bonds and crystalline nature of the formed minerals. Significantly lower
contact angle and higher UTS was registered for C-HA nanocomplexes conditioned group
(p<0.05). Statistically significant (p<0.05) greater sealer penetration was recorded at 4mm level
for all assessment parameters and percent area penetration at 6mm for C-HA nanocomplexes
group.
Conclusion: C-HA nanocomplexes conditioning enhances dentin surface wettability to facilitate
greater TCS sealer penetration and UTS of dentin.
Keywords: Chitosan, Nanocomplexes, Dentin, Ultimate tensile strength, Sealer penetration
depth
44
Highlights
Dentin conditioning with C-HA nanocomplexes increases surface wettability for greater
sealer penetration into the dentinal tubules.
C-HA nanocomplexes enhance ultimate tensile strength of dentin with simultaneous
remineralization and organic matrix reinforcement.
Significance
C-HA nanocomplexes conditioning of dentin may facilitate sealer-dentin interaction along with
strengthening of dentinal matrix for reinforced interfacial integrity.
45
Introduction
Dentin plays a crucial role in maintaining the bulk structural integrity of teeth. As a
biocomposite, it strikes the balance between stiffness and toughness through an interplay
between inorganic and organic fractions (1). Iatrogenic application of chemicals that
demineralizes or deproteinates dentin matrix are the most common factors that alters the
ultrastructural and mechanical characteristics of dentin (1). Denudation of collagen through the
loss of mineral protection due to the use of chelating agents such as ethylenediaminetetraacetic
acid (EDTA), ensues a time dependent degradation, that affects the integrity of dentin-root filling
interface and the mechanical properties of dentin (2). Sodium hypochlorite (NaOCl) application
reduces mechanical strength, increases dentin surface roughness, while eroding the surface
dentin to create a ghost layer (3). The above features may contribute to the risk of vertical root
fractures in root-filled teeth (4).
Exposed collagen surface is a challenging substrate for mineral nucleation owing to its weak
polarity and surface charge (5). Altered dentin ultrastructure not only affects the physiochemical
properties with resultant compromised wettability of the dentin surface (3) but also the extent of
interaction with root-filling materials, particularly the root canal sealers (6). From a clinical
stand-point, this would dictate the degree of penetration and sealer-root dentin integrity (7).
Enhanced sealer penetration into tubules is considered desirable as it forms a physical barrier,
allows residual bacterial entombment and provides improved interfacial integrity between the
sealer and dentin, all the above would enhance the mechanical integrity of the root dentin (8).
46
Biomineralization is an effective strategy for restoring the physico-chemical characteristics of
dentin (5, 9, 10). Guided tissue mineralization is based on polyelectrolytic nature of polymers
that can achieve intrafibrillar mineralization through amorphous precursor mediated pathway
(11), as reported previously using Chitosan-Hydroxyapatite precursor (C-HA) nanocomplexes
(13). Chitosan is a linear biopolymer of glucosamine with a 1-4 linkage that has established
biocompatibility, biodegradability, anti-bacterial and metal chelation attributes (4). Incorporation
of chitosan for improved mechanical properties of collagen has also been used (12). Carboxyl
groups on carboxymethyl chitosan sequesters, stabilizes and act as a template for grafted
amorphous calcium phosphate (ACP) precursors to achieve intrafibrillar mineralization (13).
Chitosan shares structural approximations with glycosaminoglycan’s and in this case, is also
functionally inspired by the in vivo role of non-collagenous proteins (13).
The purpose of the study is to evaluate the effect of dentin conditioning with Chitosan-
Hydroxyapatite precursor (C-HA) nanocomplexes on depth of tri-calcium silicate sealer
penetration and dentin mechanical property.
Materials and Methods
All the chemicals used in this study were of analytical grade and were purchased from Sigma
Aldrich (St Louis, MO). Chitosan-hydroxyapatite precursor nanocomplexes were synthesized as
per previous protocol (13). Extracted human teeth used in the study were collected abiding
ethical approval guidelines of the University (Protocol ID- 35073).
47
Physico-chemical and bioactivity characterization
Aggregation Kinetics
Zeta potential as a measure of surface charge and polydispersity index to establish non-
aggregating concentration was measured for a series of C-HA nanocomplexes formulations in
deionized water using a zetasizer (Malvern Instruments Ltd, Malvern, UK) at 25C. pH values
were also measured for the same with a pH probe (ORION-8157BNUMD, Thermoscientific,
USA).
Contact Angle Measurements
Static water contact angle measurements were carried out on dentin discs (8x5x0.5 mm) using a
goniometer (Rame-Hart instrument Co, USA) connected to a DROPimage software at room
temperature. Discs (n=2/group) were treated with NaOCl (6% for 20min) and EDTA (17% for 5
min) in control group and treatment followed by C-HA nanocomplexes conditioning with
2mg/ml of C-HA nanocomplexes for 30 min was done for the experimental group. Ten readings
were averaged and subjected to independent samples T-test at p<0.05 significance level.
Mechanical Property Assessment
Dentin beams (8x1.5x0.5mm) were prepared from the coronal aspects of extracted human molar
teeth and treated with NaOCl (6% for 20min) and EDTA (17% for 5min). The beams were
randomly distributed (n=10/group) between control and C-HA nanocomplexes conditioned
group followed by incubation in SBF at 37C with 5% CO2 for a 7 days’ period. For
conditioning, beams were immersed in 300ul of 2mg/ml of C-HA nanocomplexes for 30min.
48
During testing the dentin beams were rinsed with deionized water, attached to the jig of a micro-
tensile tester (Bisco Inc., Schaumburg, IL, USA) with cyanoacrylate glue and loaded until
failure. Tensile forces were applied parallel to long axis of each specimen at a crosshead speed of
0.5 mm/min (15). The specimens were kept well-hydrated during the test. Statistical analysis was
done using Independent Samples T-test with p < 0.05 significance.
In-vitro Bioactivity Evaluation
Films (n=15) of 2mg/ml C-HA nanocomplexes (6.5 x 2.5cm2) were drop-casted on glass slides
and incubated in simulated body fluid (SBF) for 3 days at 37C and 5% CO2. SBF was prepared
according to earlier protocol (14). The films were washed with deionized water (5ml), dried
under laminar flow, ground to powder and stored in a desiccator until used.
Morphological changes were characterized using scanning electron microscopy (SEM)
(QUANTA FEG 250, USA) under low vacuum coupled with an energy dispersive X-ray
spectrometer (EDS) for chemical analysis of the film surfaces. For Fourier transformation
infrared spectroscopy (FTIR), samples were mixed with KBr powder in 1:4 ratio by volume.
Data were recorded in transmittance mode over 4000 to 500 cm-1 range at 16 cm-1 resolution
and 32 times scan using an infrared spectrophotometer (Perkin Elmer One Spectrometer, IL,
USA). The x-ray diffraction (XRD) analysis was done to determine crystal phase of the
minerals using a Philips x-Ray diffractometer operated with a Cu tube (wavelength Kα1
=1.540562), at at 40 kV and 30 mA with a step size of 0.02° from 20-50 2θ range.
Depth of Sealer Penetration
Sample preparation
49
Mesio-distal and bucco-lingual x-rays were taken for human extracted premolars for orthodontic
reasons, to select teeth with single, straight canals, mature apices, lacking resorptive defects,
caries or endodontic treatment. Teeth (n=24) having similar length (191 mm) were pre-screened
with transillumination for presence of cracks and maintained in 0.9% saline at 4C until use. A
double varnish layer was applied externally avoiding the root apex to simulate impervious
cementum. Access into the root canal was gained using a No.2 round bur (Dentsply Sirona,
Tulsa, OK, USA). Canal patency was established using a size #10 K-file (K-Flexofiles,
Dentsply). Canals were instrumented with full sequence of S1, S2, F1, F2 and F3 rotary
Ni-Ti files (ProTaper Universal, Dentsply). 2ml of 6 % NaOCl was used per instrument
change. 5 ml of 17% EDTA with sonic activation was used as last irrigant (16). After final
rinse with deionized water (5ml), teeth were randomly allocated (n=12/group) to control and
C-HA nanocomplexes group. 2mg/ml solution of C-HA nanocomplexes was introduced into
the canal using a 3cc syringe (1.5, 27G) needle up to 2-3mm of the working length. Manual
dynamics agitation (3 strokes/sec) was employed with a matching F3 cone (ProTaper Universal,
Dentsply) for 3min. C-HA nanocomplexes slurry was allowed to stand in the canal for 30min
with the apex temporarily secured with wax.
Root filling of specimens
Tricalcium silicate based sealer, (iRoot SP, Innovative BioCreamix Inc., Vancouver, Canada)
was mixed with trace amount of 0.25% of rhodamine B solution and placed in the canal using a
lentulo spiral. Single cone obturation with F3 cones (ProTaper Universal, Dentsply) was done
and the samples were temporarily restored and incubated in 100% humidity at 37C with 5%
CO2 for 10 days to allow complete sealer setting (16).
50
Evaluation of depth of sealer penetration
The samples were resin-embedded and sectioned at 4 and 6mm levels from root apex using a
water-cooled, diamond wafering blade (4x0.12x1/2) mounted on a slow-speed saw (Isomet
Low Speed Saw, Buehler, IL, USA). The specimens were polished with abrasive discs,
ultrasonicated (deionized water for 5mins), placed on a glass slide and imaged using a
fluorescent microscope (5x) (Leica DM 2500, Leica Microsystems GmbH, Mannheim,
Germany) to match the excitation/emission (540nm/590nm) wavelengths of rhodamine dye.
Each section was imaged using 9-12 frames to reconstruct the whole image. For analysis Image J
software (National Institutes of Health, Bethesda, MD, USA) was used. Reconstructed image
was calibrated and total area perimeter of the section and canal circumference was measured
followed by image thresholding to calculate percentage area penetration (17). To measure mean
penetration depth, four pre-designated points (mesial, distal, buccal and lingual) at canal center
were (Fig. 3.3A) used. For maximum penetration, any point with farthest penetration depth was
selected.
For statistical analysis, Shapiro Wilk test for Normality was conducted. Since, data were
normally distributed, independent samples T-test was conducted for all assessment parameters at
4 and 6mm levels with p < 0.05 significance.
Results
Physico-Chemical and Bioactivity Characterization
Fig. 3.1A. Shows high negative surface charge and alkaline pH (Fig. 3.1B) for all formulations
except 0.5mg/ml. Formulations above 2mg/ml and below 1.0mg/ml showed (Fig. 3.1C)
51
polydispersity (>0.3). Optimal formulation of 2mg/ml for the C-HA nanocomplexes was chosen
as having a stable surface charge (-27mV), least potential for aggregation (PDI:0.25) and high
pH stability (pH:8.7). Contact angle measurements (D) Table. 3.1 on dentin showed a
significant decrease following C-HA nanocomplexes conditioning (5.030.1), compared to
treated dentin (46.22.0) alone.
Highly significant difference in UTS was noted (Fig. 3.1E) between conditioned dentin (359)
MPa and the control group (229) MPa.
SEM (Fig. 3.2A) showed mineral formation in islands on chitosan matrix with EDX confirming
their presence with evident C, O, Ca and P peaks. In-vitro bioactivity potential was established
with respect to detection of phosphates and carbonates. Fig. 3.2B showed splitting of the 577 cm-
1 phosphate bond to form a bifid peak at 600 and 560 cm-1 (bending mode) (18). Vibration band
at 885cm-1 was derived from HPO4-2 group (P-OH stretching). Presence of 1467 cm-1 band
(stretching mode) confirmed the presence of CO3-2
(19). Other major phosphate bonds were
recognized at 1070 and 577 cm-1 associated with ACP component of the synthesized
nanocomplexes. XRD spectrum in Fig. 3.2C confirmed the presence of crystalline phase of
minerals through presence of 2 peaks at 26, 32 and 45 angles.
Depth of Sealer Penetration
Significantly increased sealer penetration for all measurement parameters at 4mm in C-HA
nanocomplexes conditioned group was recorded. Increased penetration was seen for conditioned
group for all measurements at 6mm level with percentge area penetration having a significant
increase in comparison to the control group (Fig. 3.3B).
52
Figure 3.1. Optimal C-HA nanocomplexes formulation determination. (A) Scatter plot showing mean
zeta potential values for surface charge measurements (B) Bar chart with pH values for serial C-HA
nanocomplexes concentrations and (C) Bar chart showing polydispersity index for assessing aggregation.
(D) Table 3.1. Water contact angle measurements for dentin with/without C-HA nanocomplexes
conditioning showing significant reduction in C-HA nanocomplexes conditioned group. (E) Bar graph
-40
-35
-30
-25
-20
-15
-10
-5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
Zet
a P
ote
nti
al (
mV
)
Serial Formulations (mg/ml)
Zeta Potential
Group Mean Contact
Angle
Treated
46.212.0
Treated and
conditioned
5.03 0.1*
0
10
20
30
40
50
u-T
ensi
le S
tren
gth
/Mp
a
Mechanical Testing
Control Conditioned
6.5
7
7.5
8
8.5
9
9.5
pH
pH
0.5mg/ml 1.0mg/ml 1.5mg/ml
2.0mg/ml 3.0mg/ml 5.0mg/ml
Table 3.1 Contact angle measurements.
0
0.1
0.2
0.3
0.4
0.5
0.6
Po
lyd
isp
ersi
ty i
nd
ex (
PD
I)
Polydispersity Index
0.5mg/ml 1.0mg/ml 1.5mg/ml
2.0mg/ml 3.0mg/ml 5.0mg/ml
A B
C
D
E
*
53
showing increased UTS in nanocomplexes conditioned group. (*) Statistically Significant with p< 0.05.
(Error bars represent 1S. E).
Figure 3.2. Bioactivity characterization of C-HA nanocomplexes. (A) SEM image showing mineral
islands integrated with organic chitosan matrix, with EDX (inset) confirming rich presence of C, O, Ca
and P. (B) FTIR spectrum showing presence of bifid phosphate peaks (red) and carbonate bonds in C-HA
nanocomplexes films after SBF incubation suggestive of mineral formation. (C) Sharp peaks on XRD
spectrum suggest crystalline nature of formed mineral (red) after bioactivity assessment in SBF compared
to complex (black) not immersed in SBF.
A
B C
*
FTIR Spectrum XRD Spectrum
Post incubation Pre-incubation
Post incubation
Pre-incubation
(211) (112) and (300)
(002)
54
Figure 3.3 (A) Calculation of outcome measures of sealer penetration depth into dentinal tubules.
A
55
Figure 3.3 (B). Bar charts (b-g) showing Mean, Maximum and % Area Penetration at 4 and 6mm levels
from root apex in C-HA nanocomplexes conditioned group and control groups. The % Area penetration at
6mm level and all outcome parameters at 4mm are significantly higher than the control. (*) Statistically
Significant with p< 0.05. (Error bars represent 1S. E)
0
100
200
300
400
500
Mea
n P
enet
rati
on
/u
mMean Penetration at 4mm
Control Conditioned
p<0.05
*
0
100
200
300
400
500
Mea
n P
enet
rati
on
/ u
m
Mean penetration at 6mm
Control Conditioned
0
200
400
600
800
Max P
enet
rati
on
/ u
m
Max Penetration at 4mm
Control Conditioned
p<0.01
0%
10%
20%
30%
40%
50%
60%
% A
rea P
enet
rati
on
/ u
m2
% Area penetration at 4mm
Control Conditioned
p<0.05
f
0
200
400
600
800M
ax P
enet
rati
on
/ u
m
Max penetration at 6mm
Control Conditioned
0%
10%
20%
30%
40%
50%
60%
% A
rea P
enet
rati
on
/ u
m2
% Area penetration at 6mm
Control Conditioned
*
b c
d e
g
B
56
Discussion
Dentin substrate modification to enhance interaction with sealers and simultaneous
reinforcement of dentin mechanical property may serve to improve interfacial integrity.
The characterization experiments showed that synthesized C-HA nanocomplexes possessed a
highly stable, polyanionic surface charge in an alkaline medium. These characteristics are
imperative to establish higher ratio of ionized carboxyl groups (COO-) in solution to prevent
aggregation of dispersed nanocomplexes that forms the basis of their role in guided dentin
mineralization (20). The bioactivity potential was established using a standard protocol (14).
This formation of bi-fid phosphate peaks as observed in the FTIR analysis has been associated
with the gradual transition from a poorly crystalline into well crystallized hydroxyapatite (21).
The XRD reflections were similar to those reported earlier (22) with the most prominent peak at
32 2. This may be regarded as a composite peak ascribed to collective contributions from HA
diffraction planes, (211), (112), (300) that would become resolved as the crystallinity matures
over time (19). This observation can be explained in the way carboxymethyl chitosan stabilizes
the precursor phase i.e. through adsorption on to the nascent crystal nuclei and binding of
calcium ions by ion-pair formation causing a prolonged ACP stabilization phase (23).
Micro-tensile testing facilitates standardization of testing sample and limits dentin anisotropy
induced confounding (24). In the current study, values of treated dentin were in range of those
previously noted using similar methodology (25). Exposure to NaOCl/EDTA may have resulted
in collagen demineralization/degradation owing to the small size of its oxidizing species (3, 26).
In control group, subsequent immersion in SBF would have resulted in dentin surface
57
precipitation that was not supported by structurally intact collagen, resulting in lower UTS values
(27). Whereas in conditioned group, a simultaneous organic-inorganic reinforcement of dentin
matrix resulted in higher UTS. C-HA nanocomplexes may have facilitated both extra and intra-
fibrillar collagen mineralization as established in earlier study through formation of collagen
cross-banding with transmission electron microscopy (13). Previous studies have shown that
tensile forces applied perpendicular to the tubular direction required disruption of collagen fibers
(24), this may explain the requirement of higher loads to fracture in C-HA nanocomplexes
conditioned beams that were reinforced with chitosan. The improved tensile strength was
attributed to the inherent toughness of chitosan due to inter-fibrillar hydrogen bonding, its
hydrophilic nature facilitating collagen adsorption and dentin remineralization (12, 20, 28).
Depth of sealer penetration was evaluated as an assessment measure of physico-chemical dentin
surface modification. Experimental design was adapted from earlier established protocols (16,
29). Choice of a fluorescent imaging technique compared to SEM is regarded superior owing to
difficulties in obtaining an over-all low-resolution image and subsequent analysis along with
concern for artefacts during sample processing (8). Possible factors affecting penetration were
considered, therefore sonic activation was employed to ensure adequate smear removal. To avoid
presence of sclerotic dentin/anatomical complexities in apical third, first root section was chosen
at 4mm level from apex (30). Percentage of area penetration along with other parameters was
measured to account for the heterogeneity in dimensions of samples. The results are in
accordance with earlier studies that show increasing sealer penetration towards coronal sections
(29).
58
An earlier study concluded poor TCS penetration depth in chitosan nanoparticles irrigated group
without smear layer removal (31). However, in current study all penetration parameters assessed
were noted to be higher in C-HA nanocomplexes conditioned root canals. Sealer penetration at
6mm level was not statistically significant in conditioned group unlike at 4mm. This might be
because of the closer adaptation of GP cone at 4mm level that resulted in a generation of higher
shear stress on canal walls allowing a denser and deeper nanocomplex coating with use of
manual dynamics. In contrast to another study, near zero contact angle measurements were
recorded for C-HA nanocomplexes conditioned dentin (32). Both the earlier studies used a non-
functionalized chitosan, which has a hydrophobic character due to the high molecular weight and
presence of hydrophobic acetyl groups that tend to aggregate in solution (28). However, in this
study, due to presence of rich carboxyl groups in water soluble chitosan and ACP as a hydrated
molecule provided overall hydrophilicity. This was established by the wettability experiments.
Irrigant-induced mineral loss exposed hydrophobic collagen matrix (33), which did not
complement the hydrophilicity of the sealer used and may explain the lower degree of sealer
penetration observed in the control group.
A hydrophilic layer of chitosan-hydroxyapatite precursor nanocomplexes formed along the root
canal walls presented carboxyl groups from chitosan and interstitial water from ACP which
contributed to the increased dentin wettability by the tricalcium silicate sealer (20). Whereas the
formation of intrafibrillar minerals and their simultaneous encapsulation by chitosan allowed the
integration of organic-inorganic dentin matrix components for enhanced mechanical strength.
In brief, the findings from this study highlighted the dentin substrate modification potential of C-
HA nanocomplexes conditioning of previously irrigated root canal dentin by restoring the
59
surface wettability with synchronous collagen reinforcement. These findings may find potential
application in establishing improved TCS sealer-dentin interaction and enhanced interfacial
integrity in root-filled teeth.
Acknowledgements The authors deny any conflict of interest.
60
References
1. Kishen A. Mechanisms and risk factors for fracture predilection in endodontically treated
teeth. Endodontic Topics. 2006;13:57-83.
2. Ferrari M, Mason PN, Goracci C, Pashley DH, Tay FR. Collagen degradation in
endodontically treated teeth after clinical function. J Dent Res. 2004;83:414-419.
3. Gu LS, Huang XQ, Griffin B, Bergeron BR, Pashley DH, Niu LN, et al. Primum non
nocere - The effects of sodium hypochlorite on dentin as used in endodontics. Acta Biomater.
2017;61:144-156.
4. Kishen A, Shrestha S, Shrestha A, Cheng C, Goh C. Characterizing the collagen
stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation. Dent
Mater. 2016;32:968-977.
5. Xu Z, Neoh KG, Lin CC, Kishen A. Biomimetic deposition of calcium phosphate
minerals on the surface of partially demineralized dentine modified with phosphorylated
chitosan. J Biomed Mater Res B Appl Biomater. 2011;98:150-159.
6. Dogan Buzoglu H, Calt S, Gümüsderelioglu M. Evaluation of the surface free energy on
root canal dentine walls treated with chelating agents and NaOCl. Int Endod J. 2007;40:18-
24.
7. Topçuoğlu HS, Tuncay Ö, Demirbuga S, Dinçer AN, Arslan H. The effect of different
final irrigant activation techniques on the bond strength of an epoxy resin-based endodontic
sealer: a preliminary study. J Endod. 2014;40:862-866.
8. Mamootil K, Messer HH. Penetration of dentinal tubules by endodontic sealer cements in
extracted teeth and in vivo. Int Endod J. 2007;40:873-881.
9. Shao C, Zhao R, Jiang S, Yao S, Wu Z, Jin B, et al. Citrate Improves Collagen
Mineralization via Interface Wetting: A Physicochemical Understanding of
Biomineralization Control. Adv Mater. 2018;30:1-7.
10. Gandhi B, Bollineni S, Janga RK, Saraswati D, Babu MR. Evaluating the Effect of CPP-
ACP as a Final Irrigant in Improving the Micro-Hardness of Erosive Root Dentin and its
Influence on the Bond Strength of Self Etch Resin Sealer - An In-vitro Study. J Clin Diagn
Res. 2016;10:ZC53-56.
61
11. Tay FR, Pashley DH. Guided tissue remineralisation of partially demineralised human
dentine. Biomaterials. 2008;29:1127-1137.
12. Shrestha A, Friedman S, Kishen A. Photodynamically crosslinked and chitosan-
incorporated dentin collagen. J Dent Res. 2011;90:1346-1351.
13. Chen Z, Cao S, Wang H, Li Y, Kishen A, Deng X, et al. Biomimetic remineralization of
demineralized dentine using scaffold of CMC/ACP nanocomplexes in an in vitro tooth model
of deep caries. PLoS One. 2015;10:e0116553.
14. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials. 2006;27:2907-2915.
15. Park JH, Choi YS. Microtensile bond strength and micromorphologic analysis of surface-
treated resin nanoceramics. J Adv Prosthodont. 2016;8:275-284.
16. Al-Haddad A, Abu Kasim NH, Che Ab Aziz ZA. Interfacial adaptation and thickness of
bioceramic-based root canal sealers. Dent Mater J. 2015;34:516-521.
17. Menezes M, Prado M, Gomes B, Gusman H, Simão R. Effect of photodynamic therapy
and non-thermal plasma on root canal filling: analysis of adhesion and sealer penetration. J
Appl Oral Sci. 2017;25:396-403.
18. Drouet C. Apatite Formation: Why It May Not Work as Planned, and How to
Conclusively Identify Apatite Compounds. BioMed Research International 2013;2013:12.
19. Li X, Lan J, Ai M, Guo Y, Cai Q, Yang X. Biomineralization on polymer-coated multi-
walled carbon nanotubes with different surface functional groups. Colloids Surf B
Biointerfaces. 2014;123:753-761.
20. Kalliola S, Repo E, Srivastava V, Heiskanen JP, Sirviö JA, Liimatainen H, et al. The pH
sensitive properties of carboxymethyl chitosan nanoparticles cross-linked with calcium ions.
Colloids Surf B Biointerfaces. 2017;153:229-236.
21. Sun J, Chen C, Pan H, Chen Y, Mao C, Wang W, et al. Biomimetic promotion of dentin
remineralization using L-glutamic acid: inspiration from biomineralization proteins. Journal
of Materials Chemistry B. 2014;2:4544-4553.
22. Wang Y, Van Manh N, Wang H, Zhong X, Zhang X, Li C. Synergistic
intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic
62
strategy to promote the regeneration of bone defects. Int J Nanomedicine. 2016;11:2053-
2067.
23. Andrew CA, Khor E, Hastings GW. The influence of anionic chitin derivatives on
calcium phosphate crystallization. Biomaterials. 1998;19:1309-1316.
24. Inoue S, Pereira PN, Kawamoto C, Nakajima M, Koshiro K, Tagami J, et al. Effect of
depth and tubule direction on ultimate tensile strength of human coronal dentin. Dent Mater
J. 2003;22:39-47.
25. Cecchin D, Soares Giaretta V, Granella Cadorin B, Albino Souza M, Vidal CMP, Paula
Farina A. Effect of synthetic and natural-derived novel endodontic irrigant solutions on
mechanical properties of human dentin. J Mater Sci Mater Med. 2017;28:141.
26. Fuentes V, Ceballos L, Osorio R, Toledano M, Carvalho RM, Pashley DH. Tensile
strength and microhardness of treated human dentin. Dent Mater. 2004;20:522-529.
27. Smith LJ, Deymier AC, Boyle JJ, Li Z, Linderman SW, Pasteris JD, et al. Tunability of
collagen matrix mechanical properties via multiple modes of mineralization. Interface Focus.
2016;6:20150070.
28. Yu Z, Lau D. Flexibility of backbone fibrils in α-chitin crystals with different degree of
acetylation. Carbohydr Polym. 2017;174:941-947.
29. Wang Y, Liu S, Dong Y. In vitro study of dentinal tubule penetration and filling quality
of bioceramic sealer. PLoS One. 2018;13:e0192248.
30. Kinney JH, Nalla RK, Pople JA, Breunig TM, Ritchie RO. Age-related transparent root
dentin: mineral concentration, crystallite size, and mechanical properties. Biomaterials.
2005;26:3363-3376.
31. Aydın ZU, Özyürek T, Keskin B, Baran T. Effect of chitosan nanoparticle, QMix, and
EDTA on TotalFill BC sealers' dentinal tubule penetration: a confocal laser scanning
microscopy study. Odontology. 2018.
32. Ururahy MS, Curylofo-Zotti FA, Galo R, Nogueira LF, Ramos AP, Corona SA.
Wettability and surface morphology of eroded dentin treated with chitosan. Arch Oral Biol.
2017;75:68-73.
63
33. Ballal NV, Tweeny A, Khechen K, Prabhu KN, Satyanarayan, Tay FR. Wettability of
root canal sealers on intraradicular dentine treated with different irrigating solutions. J Dent.
2013;41:556-560.
64
Chapter 4
Manuscript (II)
Interfacial Characterization of Dentin Conditioned with Chitosan Hydroxyapatite
precursor Nanocomplexes using Time-Of-Flight Secondary Ion Mass Spectrometry
Anam Hashmi1, Rana N.S. Sodhi2, Anil Kishen 1
1Kishen Lab, Faculty of Dentistry, University of Toronto, Toronto, ON 2Ontario Centre for the Characterization of Advanced Materials (OCCAM),
Department of Chemical Engineering and Applied Chemistry, University of Toronto, ON
4.1 Abstract
Introduction: The purpose of the study was to evaluate the effect of chitosan-hydroxyapatite
precursor (C-HA) nanocomplexes conditioning on chemical modifications at the tricalcium
silicate sealer-dentin interface using time-of-flight secondary ion mass spectrometry (TOF-
SIMS).
Methods: Dentin slabs from human premolar root dentin were prepared, demineralized and
randomly distributed between control and C-HA nanocomplexes conditioned groups. Tricalcium
silicate sealer was applied and slabs were allowed to set in 100% humidity for 10 days. Cross-
sectional area was exposed, and the sealer-dentin interface was characterized for
chemical/ultrastructural evaluation with TOF-SIMS and transmission electron microscopy
respectively.
Results: Chemical analysis revealed presence of an ion-rich layer constituting of abundant
phosphates (PO2-. PO3
-, PO4-), hydroxide (OH-) and chitosan fragments (C2H4NO-, C3H4NO2
- ,
65
C2H5O2+, C2H6NO+, C4H6NO2
+, C5H6NO+, C5H5O2+) on the dentin surface at sealer-dentin
interface and subsurface dentin following conditioning with C-HA nanocomplexes. In contrast,
decreased interfacial presence of calcium (Ca+), calcium-phosphates (CaPO2+
, CaPO3+
, CaPO4+
,
Ca2PO3+) and absence of phosphate fragments in the control was noted. Ultrastructural
evaluation showed an interfacial layer (<1um) with interrupted mineral aggregates in control as
opposed to a continuous (5um) mineral layer formation on conditioned dentin.
Conclusions: C-HA nanocomplexes conditioning of dentin formed a chemically modified dentin
substrate with presence of an ion-rich layer that chemically modified the dentin
surface/subsurface with the presence of phosphate, calcium, calcium phosphates and chitosan.
Keywords: Chitosan, Amorphous calcium phosphate, Nanocomplexes, Dentin, Time-Of-Flight
Secondary Ion Mass Spectrometry
66
Highlights:
Interfacial chemical characterization of tricalcium silicate sealer-dentin interface was
evaluated with monolayer sensitivity of TOF-SIMS.
Dentin surface conditioning with C-HA nanocomplexes resulted in chemical
modification of dentin with formation of modified ion-rich layer at interface.
Interfacial remineralization in C-HA nanocomplexes conditioned dentin was enhanced
with presence of well-defined phosphate band.
Significance:
Dentin substrate conditioning with C-HA nanocomplexes may find potential application in
enhancing chemical interaction of sealers with dentin for reinforcement of interfacial integrity.
67
4.2 Introduction
Dentin is a biocomposite composed of carbonated hydroxyapatite mineral crystallites, collagen
fibrils (mostly type I) and non-collagenous macromolecules over several length scales (1).
However, iatrogenic application of chemicals and or medications for chemo-mechanical
debridement during root canal therapy induces structural and compositional damages to dentin
altering its physico-chemical and mechanical characteristics (2, 3). Use of
ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite (NaOCl), commonly used root
canal irrigants, results in loss of mineral and irreversible, non-specific digestive action on the
organic contents of dentin (4). Such ultrastructural/chemically altered dentin with denuded
collagen shows poor surface polarity and becomes a challenging substrate for remineralization
(5). Root canal dentin surface with collapsed collagen network can increase the risk for
interfacial failure. Moreover, dentin possesses an inherent tubular structure that encourages fluid
movement and contaminant ingress even when frank interfacial gaps are lacking, through
anastomosing network of its secondary dentinal tubules (6). Therefore, materials that set in the
presence of moisture have been introduced.
Tricalcium silicate based materials (TCS), are hydraulic in nature and are known for their
bioactivity through carbonated apatite formation on contact with physiological fluids (7, 8), that
forms the basis of their interaction with dentin. Therefore, physico-chemical profile of dentin
plays a crucial role in directing the mechanics of interaction and subsequent mineral formation
(5, 9). Differences in nature of precipitate formation in the bulk of TCS and that formed in
contact with dentin have been reported, chiefly due to the inadequacy and accessibility to
phosphorous ions (10). Availability of a sustained mineral source and subsequent nucleation on
68
hydrophobic, denuded collagen surface are current challenges in dentin remineralization (5).
Chitosan-Hydroxyapatite precursor (C-HA) nanocomplexes, may serve as an effective strategy.
Chitosan is an abundant biopolymer consisting of ,1-4 glucosamine units (11). Chitosan and its
derivatives have been found to offer different advantages due to their biocompatible, bioactive
and antibacterial nature (11). C-HA precursor nanocomplex is an organic-inorganic composite
that is functionally inspired by the role of non-collagenous proteins. Their bioactivity is
facilitated through polyanionic surface charge on water soluble chitosan backbone that allows
sequestration and stabilization of hydroxyapatite precursor phases (amorphous calcium
phosphate) with its carboxyl groups. This facilitates synergistic intra- and extra-fibrillar collagen
mineralization to take place (12, 13), similar to how it occurs in biomineralization.
Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) is a well-established technique
in material sciences that allows analysis and identification of both organic proteins and inorganic
fractions such as hydroxyapatite with milimass precision and monolayer sensitivity (14). It
provides high mass resolution spectra along with their spatial ion maps (15-17). Recently, TOF-
SIMS analysis have been utilized to study precipitate formation on dentin surface and in dentinal
tubules subsequent to the application of different irrigants (18). However, no study has
characterized the sealer-dentin interface using TOF-SIMS and there is a lack of knowledge on
how dentin surface chemistry affects the interfacial characteristics of tricalcium silicate sealers.
The aim of the study was to chemically characterize the tricalcium silicate sealer-dentin
interface, after dentin conditioning with C-HA nanocomplexes.
69
4.3 Materials and Methods
Five extracted human premolar teeth were collected under the University Ethical guidelines and
stored in 0.9% saline until use. Slabs of root dentin were prepared by sectioning the root along
the long axis of the tooth using a diamond wafering blade (4x 0.12 x 1/2) (Presicion Smart
Cut, UKAM Industrial Superhard Tools, Valencia, CA) mounted on a slow-speed saw (Isomet
Low Speed Saw, Buehler, IL, USA) under running water. Each slab (n=6) was then shaped and
grinded with carbide paper discs to final dimensions of 6 x 4 x 0.2 mm. Slabs were
demineralized, (17% EDTA for 7 days), ultrasonicated (10 min with deionized water) and
randomly distributed between control and C-HA nanocomplexes conditioned group.
Carboxymethyl chitosan was synthesized according to earlier protocol (19). Amorphous calcium
phosphate was then grafted by addition of K2HPO4 and CaCl2 (12). Dentin slabs were
conditioned in 1ml of 2mg/ml solution of C-HA nanocomplexes for 30min. Sealer (iRoot SP,
Innovative BioCreamix Inc., Vancouver, Canada) was allowed to spread into a uniform thickness
layer in between dentin slabs and incubated in 100% humidity at 37C with 5% CO2 for a 10
days’ period. The samples were sectioned to expose the sealer-dentin interface using a
microtome (Leica EM UC6/FC6 Ultra-cryomicrotome, Leica Microsystems GmbH, Germany).
One sample from each group was processed for ultrastructural analysis with transmission
electron microscopy (TEM).
70
TOF-SIMS Analysis
Dentin surface analysis was performed by TOF-SIMS (TOF-SIMS V; ION-TOF GmbH,
Münster, Germany). Bi3++ cluster primary ion source was used with a bismuth (Bi) liquid metal
ion gun operated in a high mass resolution bunched mode over an area of 500μm × 500μm for
100 seconds. Additionally, a high spatial resolution imaging mode (“burst alignment”) was used
to obtain spectral images (256 pixels × 256 pixels) from 20 scans over an area of 150μm ×
150μm. A pulsed electron flood gun was used for charge neutralization. The calibration of the
mass scale was performed using standard, identifiable, and well-spaced peaks found in all the
spectra. Both positive and negative polarity mass spectra and spatial chemical maps for
molecular fragments of interest were generated through regions of interest (ROI) at interface (5 x
25um) and total dentin (150 x 70um). To allow a semi-quantitative analysis, spectral
comparisons were carried out after normalization of the intensity, proportionally to the total
intensity of each spectrum (20). Thus, data from both normalized spectral intensities and spectral
chemical maps are presented below.
TEM Evaluation
Specimens were fixed with Karnovsky’s fixative (2.5 wt.% glutaraldehyde buffered to pH 7.3)
for 3 days at 4C, and post-fixed in 1% osmium tetroxide for 1 h. The specimens were
dehydrated in an ascending ethanol series (30–100%), immersed in propylene oxide as a
transition medium and ultimately embedded in pure epoxy resin. Ninety-nanometer thick
sections were prepared. Sections containing the material-dentin interface were stained with 2%
aqueous uranyl acetate and Reynold’s lead citrate. The sections were examined along the cross-
section using a JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV.
71
4.4 Results
TOF-SIMS Analysis
Total ion and CN- maps are shown in Figure. 4.1A-D. Conditioned dentin showed a
distinguishable interfacial zone (5um), owing to differences in pixel intensity as opposed to the
control (Fig. 4.1A and B). Ion maps for CN- on (Fig. 4.1C and D) show low intensity with no
signal areas in control whereas increased signals are seen in conditioned dentin. Red-Green
overlays of chemical ion maps for organic and inorganic fragments are shown in Figure 4.1E-G.
Presence of PO3- and PO4
- fragments was non-existent at the interfacial zone in control sample
while greater intensity of PO2- was found (Fig. 4.1E). In contrast, presence of phosphate was
seen in well-defined interfacial bands of PO2- and PO3
- in conditioned dentin (Fig. 4.1F). The
PO4- map although less defined depicted dense phosphate presence. Distribution of Ca+ in islands
can be noticed from Fig. 4.1G in conditioned dentin.
The results from normalized spectral intensities are shown in Figure 4.2. Higher intensities for
non-specific (negative polarity) organic fragments of O-, CN-, CNO- at both ROIs (Fig. 4.2A and
B) were seen for conditioned dentin with the exception of cysteine residue (SH-) in comparison
to the control. OH- intensity was higher in total dentin of conditioned group only. Conditioned
dentin (Fig. 4.2C-E), showed increased intensity for specific organic fragments of chitosan with
negative polarity (C2H4NO- and C3H4NO2- at both ROIs) and positive polarity (C2H5O2
+,
C2H6NO+, C4H6NO2+, C5H6NO+, C5H5O2
+ and C5H6NO2
+ fragments at interface), compared to
control. C2H4NO- was used as a characteristic marker fragment of carboxymethyl group to tag
chitosan presence. CHO2- was seen to be higher at interface of conditioned dentin. Other non-
72
specific organic fragments (NH4+, CH4N
+ and C4H8N+) were noted to be higher (Fig. 4.2F) at
interface in conditioned dentin.
Inorganic fragments of phosphates as PO2-, PO3
- and PO4- (Fig. 2G & H), registered higher
intensity at interface and total dentin ROIs in conditioned group whereas PO3- and PO4
-
fragments were absent in the control. Increased intensity for CaO+ and CaOH+ (Fig. 4.2I), was
noted in control sample with Ca+ and all other higher molecular mass fragments (CaPO2+,
CaPO3+, CaPO4
+ and Ca2PO3
+), to be greater in intensity in the conditioned dentin (Fig. 4.2J).
TEM Evaluation
TEM micrographs are shown in Figure. 4.3A and B. Isolated mineral aggregates as thin (1um)
discontinuous layer, with denuded collagen matrix is noted in control (Fig. 4.3A). Fraying of
collagen fibril ends as a sign of degradation can also be noted on the same. Whereas, a consistent
interfacial layer with appreciably increased thickness (5um) and (Fig. 4.3B) abundant in coarse
mineral aggregates encapsulated in a gel like matrix was seen in conditioned dentin. The
nanometric sized aggregates were seen deposited in close adaptation to the underlying collagen
matrix.
73
2 um
500 nm
Sealer
Sealer-Dentin Interface
Dentin
B
C D
A
C2H4NO- PO2- Overlay
C2H4NO- PO3- Overlay
C2H4NO- PO4- Overlay
E Sealer
Sealer-Dentin Interface
Dentin
74
Figure 4.1 (A and B) Total ion maps for control and C-HA nanocomplexes conditioned dentin.
Interfacial layer with differences in pixel intensity can be seen (broken line marks the interfacial
boundary). (C and D) CN- chemical ion maps showing intense CN- signals of integrated C-HA
nanocomplexes at interface/subsurface in conditioned dentin. No signal zone suggestive of
degraded collagen is seen in control (yellow arrows). (E and F) Showing Red-Green overlays of
organic fragment (C2H4NO---red) with phosphates (PO2
-, PO3- and PO4
- --green). (E) Minimal
presence of phosphate ions at the sealer-dentin interface (broken line) in control dentin. (F)
Well-defined interfacial band of phosphates (PO2-, PO3
-) in C-HA nanocomplexes conditioned
dentin. (G) Red-Green overlay of chitosan fragment (C5H6NO+ --red) with calcium (Ca+ --green)
showing presence of calcium islands in conditioned dentin in areas lacking chitosan matrix
(yellow arrows).
C2H4NO- PO4- Overlay
C5H6NO+ Ca+ Overlay
C2H4NO- PO2- Overlay
C2H4NO- PO3- Overlay
Sealer
Sealer-Dentin Interface
Dentin
F
G
75
0
0.2
0.4
0.6
0.8
1
1.2
No
rmal
ized
inte
nsi
ty (
a.u
)
Control Conditioned
OH- O- CN- CNO- SH-
0
0.2
0.4
0.6
0.8
1
1.2
No
rmal
ized
inte
nsi
ty (
a.u
)
Control Conditioned
0
0.05
0.1
0.15
0.2
No
rmal
ized
Inte
nsi
ty (
a.u
)
Control Conditioned
C
0
0.05
0.1
0.15
0.2
No
rmal
ized
Inte
nsi
ty (
a.u
)
Control Conditioned
D
0
0.1
0.2
0.3
0.4
0.5
No
rmal
ized
Inte
nsi
ty (
a.u
)
Control Conditioned
E
0
0.2
0.4
0.6
0.8
1
No
rmal
ized
Inte
nsi
ty (
a.u
)
Control Conditioned
NH4+ CH4N+ C4H8N+
F
A B
Interface Dentin
CO2H- C2H4NO- C3H4NO2-
CO2H- C2H4NO- C3H4NO2-
Interface Dentin
C2H5O2+
C2H6NO+ C4H6NO2+ C5H6NO+ C5H5O2
+ C5H6NO2
+
Interface
OH- O- CN- CNO- SH-
Interface
76
Figure 4.2. Normalized mass spectral intensities for negative and positive polarity at interfacial
and total dentin region of interests. (A) Negative polarity non-specific protein fragments at
interface and (B) total dentin. (C) Negative polarity characteristic chitosan fragments at
interface and (D) total dentin. (E) Positive polarity characteristic chitosan fragments and (F) non-
specfic organic fragments at interface. (G) Phosphate fragments at interface and (H) total dentin.
(I) Presence of tricalcium silicate hydration products (Ca+, CaO+ and CaOH+) at interface. (J)
Higher molecular mass fragments (CaPO2+, CaPO3
+, CaPO4+, Ca2PO3
+ ) of calcium phosphates
at interfacial dentin.
0
0.01
0.02
0.03
0.04
No
rmal
ized
Inte
nsi
ty (
a.u
)
Control Conditioned
G
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
No
rmal
ize
d In
ten
sity
(a.
u)
Control Conditioned
H
0
0.05
0.1
0.15
0.2
0.25
0.3
No
rmal
ized
Inte
nsi
ty (
a.u
)
Control Conditioned
I
0
0.05
0.1
0.15
0.2N
orm
aliz
ed In
ten
sity
(a.
u)
Control Conditioned
CaPO2+ CaPO3
+ CaPO4+ Ca2PO3
+
J
PO2- PO3
- PO4
-
Dentin
Ca+ CaO+ CaOH+
Interface
Interface Interface
PO2- PO3
- PO4
-
77
Figure 4.3. TEM micrographs showing sealer-dentin (D) interface (IF) with presence of
interfacial layer (IL). (A) Control dentin shows a thin, discontinuous interfacial layer with
collagen fraying. (B) Conditioned dentin shows a closely adapted interfacial layer with markedly
increased thickness.
500 nm
2 um
500 nm
2 um
D
D
IF
IF IL IL
78
4.5 Discussion
In TOF-SIMS, a primary ion beam directed onto the sample causes surface ionization (1-2
monolayers) (16). The time-of-flight based on molecular mass is then used for fingerprint
identification of these secondary ions (21). Previously, interfacial characterization for mineral
trioxide (MTA) based cements has been attempted using scanning electron microscopy (SEM)
coupled energy-dispersive spectroscopy (EDS) and electron probe micro analysis (EPMA),
which have limited resolution requiring peak deconvolution such as reported for phosphorus
masking by zirconium peaks (11). Further sample preparation steps, the inability to detect trace
elements and organic materials are other reported disadvantages. Techniques like confocal laser
scanning microscopy (CLSM) are unable to provide chemical information that warrants the use
of additional analysis (22). Use of fluorescent dyes has also been reported problematic, since
rhodamine is preferentially imbibed by MTA based materials therefore its movement across
sealer-dentin interface is difficult to locate while fluorescein having small sized particles is easily
taken up by dentin, again making the interfacial gradient difficult to follow (23). Hence, use of
TOF-SIMS as a label free technique was employed in this study.
Specific advantages for employing TOF-SIMS in the current study included its sensitivity to
identify, spatially resolved chemical characteristics of elemental and molecular fragments of the
thin modified layer on conditioned dentin. This is significant as chemical associations provide a
far greater understanding of nature of mineralization (24). Retrospective analysis granted
complete freedom to choose specific areas with precision for analysis. Operating parameters are
crucial as they dictate yield of secondary ions, therefore control and conditioned dentin samples
were run under same experimental parameters (25).
79
Presence of Ca, P and Si ion rich deposits along the calcium silicate cement-dentin interface and
dentin subsurface to varying depths were reported by previous studies (26, 27). Variation in
interfacial layer thickness (4.8 um to 14.5 um) from SEM measurements has been attributed to
the decreased length of interaction between the phosphate from the supersaturated solution and
the calcium leached from the calcium silicate cements (10). Along similar lines, a 5-um thick
layer was identified on conditioned dentin from the total ion chemical maps and TEM
micrographs. However, it was difficult to measure a consistent layer in control.
Presence of phosphate as a well-delineated band in conditioned dentin but lacking in the control
group was an important finding in this study. TCS, which is a hydraulic cement was tested as a
calcium source based on the earlier biomineralization studies (8, 28). Presence of a calcium
phosphate monobasic phase in the sealer could have been a potential source for phosphate
fragments. However, this contribution was very nominal and virtually non-existent at the
interface for PO3- and PO4
- fragments in control sample and was also reflected in lower calcium
phosphate intensities registered. As the TCS hydration continued, more Ca(OH)2 was formed but
due to low phosphate availability remained unutilized in control and recorded a higher intensity
(7, 8). This is in contrast to earlier studies wherein large quantity of phosphate-based fluid is
provided (27, 29). Such an approach clearly established the source of phosphate to be the C-HA
nanocomplexes without any confounding factors.
Demineralized dentin allowed mapping of calcium gradients from the interface into the dentin.
Registration of Ca+ in conditioned dentin as dense islands maybe explained by the lack of
80
chitosan fragment in those areas as is evident on the RG overlay, whereas in the rest of the image
the Ca+ signals were masked by the near homogenous presence of chitosan of ~5 µm, which is
way larger than the 1 – 2 monolayer sampling depth of TOF-SIMS. Amongst others, high O- and
OH- signals was noted in the conditioned sample, which represented substrate signals as well as
suggestive of calcium phosphate precipitation (25).
An overlap of signals occurred from the dentin matrix and the chitosan layer. Therefore,
chemical nature of the conditioned layer was established through presence of characteristic
positive and negative chitosan fragments along with non-specific fragments (20, 25). Presence of
SH- as a cysteine residue was identified as an intense signal in control in comparison to the
conditioned group, whereby its presence was almost negligible. Since, cysteine residue is not
present in chitosan structure (25), this further supported the adsorption/integration of chitosan as
an additional, modified layer at the sealer-dentin interface. Also, the minor SH- signals in
nanocomplex group may also point at the close adaptation of the modified layer to underlying
dentin.
As reported in an earlier study, formation of Ca(OH)2 by calcium silicate cement results in
caustic etching and degradation of collagen matrix (22). Such a finding may also be supported
from the lack of CN- signals seen (black zone) in the few microns nearest to the interface in
control sample while strong CN- signals can be clearly seen emanating from integration of C-HA
nanocomplexes in conditioned dentin. This is supplemented by TEM images showing collagen
fraying while a much homogenous surface is seen in conditioned dentin, highlighting a potential
protective effect on dentin collagen.
81
Tricalcium silicate-based cements have been reported to interact with dentin through formation
of ion-rich layer and sealer tags (26). Use of C-HA nanocomplexes in this study, provided a basis
for restoring the chemical characteristics of demineralized dentin. This maybe explained such
that as the TCS continued to set, Ca(OH)2 was produced and underwent hydrolysis to provide a
continued supply of calcium and the OH- ions (30). Hydrophilic nature of carboxymethyl
chitosan would have attracted more water molecules close to the C-HA nanocomplexes at the
sealer-dentin interface forming a micro-environment allowing ACP dissolution and precipitation
(31), that aided in providing a phosphate source. The accumulation of calcium, hydroxyl and
phosphate ions inside the chitosan microenvironment formed the ion-rich layer, which would
facilitate subsequent phase transformation towards thermodynamically stable phase assisted by
medium alkalinity (8). Presence of C-HA nanocomplexes could have also provided added
heterogeneous nucleation sites to sequester Ca+ ions with its carboxyl groups as well and
hastened the reaction kinetics.
All biomimetic mineralization schemes are composed of fundamental components requiring
organic-inorganic interaction to initiate nucleation and an uninterrupted mineral replenishment
source, which was provided by C-HA nanocomplexes conditioning of dentin. By mimicking the
role of NCPs, adsorption of C-HA nanocomplexes on dentin collagen could produce a negatively
charged surface and therefore reduced interfacial energy between the aqueous microenvironment
and dentin, allowing mineral deposition (5). Since, nucleation and growth of minerals is
proportional to the concentration of the available ions (32), enhanced bioactivity of TCS was
seen with C-HA nanocomplexes conditioning, which resulted in a modified interaction between
the TCS and dentin as established in the current study.
82
The findings from this study highlighted the possible contributions of C-HA nanocomplexes
conditioning in chemically modifying dentin surface and subsurface by forming an ion rich layer.
This may facilitate enhanced interfacial integrity of sealer-dentin interfaces and improved dentin
mechanical integrity in root-filled teeth.
Acknowledgements
OCCAM gratefully acknowledges the support of the Canadian Foundation for Innovation, the
Ontario Research Fund and the University of Toronto. The authors deny any conflict of interest.
83
4.6 References
1. Bertassoni L. Dentin on the nanoscale: Hierarchical organization, mechanical behavior
and bioinspired engineering. Dental Materials. 2017;33:637-49.
2. Gu LS, Huang XQ, Griffin B, Bergeron BR, Pashley DH, Niu LN, et al. Primum non
nocere - The effects of sodium hypochlorite on dentin as used in endodontics. Acta Biomater.
2017;61:144-56.
3. Dogan Buzoglu H, Calt S, Gümüsderelioglu M. Evaluation of the surface free energy on
root canal dentine walls treated with chelating agents and NaOCl. Int Endod J. 2007;40:18-24.
4. Tartari T, Bachmann L, Maliza AG, Andrade FB, Duarte MA, Bramante CM. Tissue
dissolution and modifications in dentin composition by different sodium hypochlorite
concentrations. J Appl Oral Sci. 2016;24:291-8.
5. Xu Z, Neoh KG, Lin CC, Kishen A. Biomimetic deposition of calcium phosphate
minerals on the surface of partially demineralized dentine modified with phosphorylated
chitosan. J Biomed Mater Res B Appl Biomater. 2011;98:150-9.
6. Rechenberg DK, Thurnheer T, Zehnder M. Potential systematic error in laboratory
experiments on microbial leakage through filled root canals: an experimental study. Int Endod J.
2011;44:827-35.
7. Xuereb M, Vella P, Damidot D, Sammut CV, Camilleri J. In situ assessment of the
setting of tricalcium silicate-based sealers using a dentin pressure model. J Endod. 2015;41:111-
24.
8. Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, Weller RN. Calcium phosphate
phase transformation produced by the interaction of the portland cement component of white
mineral trioxide aggregate with a phosphate-containing fluid. J Endod. 2007;33:1347-51.
9. Shao C, Zhao R, Jiang S, Yao S, Wu Z, Jin B, et al. Citrate Improves Collagen
Mineralization via Interface Wetting: A Physicochemical Understanding of Biomineralization
Control. Adv Mater. 2018;30(8).
10. Kim JR, Nosrat A, Fouad AF. Interfacial characteristics of Biodentine and MTA with
dentine in simulated body fluid. J Dent. 2015;43:241-7.
84
11. Kishen A, Shrestha S, Shrestha A, Cheng C, Goh C. Characterizing the collagen
stabilizing effect of crosslinked chitosan nanoparticles against collagenase degradation. Dent
Mater. 2016;32:968-77.
12. Chen Z, Cao S, Wang H, Li Y, Kishen A, Deng X, et al. Biomimetic remineralization of
demineralized dentine using scaffold of CMC/ACP nanocomplexes in an in vitro tooth model of
deep caries. PLoS One. 2015;10:e0116553.
13. Wang Y, Van Manh N, Wang H, Zhong X, Zhang X, Li C. Synergistic
intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to
promote the regeneration of bone defects. Int J Nanomedicine. 2016;11:2053-67.
14. Malmberg P, Nygren H. Methods for the analysis of the composition of bone tissue, with
a focus on imaging mass spectrometry (TOF-SIMS). Proteomics. 2008;8:3755-62.
15. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without
collagen: role of a phospholipid-proteolipid complex. Calcif Tissue Int. 2007;81:191-205.
16. Eriksson C, Malmberg P, Nygren H. Time-of-flight secondary ion mass spectrometric
analysis of the interface between bone and titanium implants. Rapid Commun Mass Spectrom.
2008;22:943-9.
17. Gotliv BA, Veis A. The composition of bovine peritubular dentin: matching TOF-SIMS,
scanning electron microscopy and biochemical component distributions. New light on
peritubular dentin function. Cells Tissues Organs. 2009;189:12-9.
18. Kolosowski KP, Sodhi RN, Kishen A, Basrani BR. Qualitative analysis of precipitate
formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a
final rinse of chlorhexidine or QMiX. J Endod. 2014;40:2036-40.
19. Chen X, Park H. Chemical characteristics of O-carboxymethyl chitosans related to the
preparation conditions. Carbohydrate Polymers. 2003;53:355-9.
20. D'Almeida M, Attik N, Amalric J, Brunon C, Renaud F, Abouelleil H, et al. Chitosan
coating as an antibacterial surface for biomedical applications. PLoS One. 2017;12:e0189537.
21. Sodhi RN. Time-of-flight secondary ion mass spectrometry (TOF-SIMS):–versatility
in chemical and imaging surface analysis. Analyst. 2004;129:483-7.
22. Atmeh AR, Chong EZ, Richard G, Festy F, Watson TF. Dentin-cement interfacial
interaction: calcium silicates and polyalkenoates. J Dent Res. 2012;91:454-9.
85
23. Kebudi Benezra M, Schembri Wismayer P, Camilleri J. Interfacial Characteristics and
Cytocompatibility of Hydraulic Sealer Cements. J Endod. 2018;44:1007-17.
24. Fearn S. An Introduction to Time-of-Flight Secondary Ion Mass Spectrometry (ToF-
SIMS) and its Application to Materials Science. Morgan & Claypool Publishers; 2015.
25. Wagener V, Boccaccini AR, Virtanen S. Protein-adsorption and Ca-phosphate formation
on chitosan-bioactive glass composite coatings. Applied Surface Science. 2017;416:454-60.
26. Han L, Okiji T. Uptake of calcium and silicon released from calcium silicate-based
endodontic materials into root canal dentine. Int Endod J. 2011;44:1081-7.
27. Han L, Okiji T. Bioactivity evaluation of three calcium silicate-based endodontic
materials. Int Endod J. 2013;46:808-14.
28. Gandolfi MG, Taddei P, Siboni F, Modena E, De Stefano ED, Prati C. Biomimetic
remineralization of human dentin using promising innovative calcium-silicate hybrid "smart"
materials. Dent Mater. 2011;27:1055-69.
29. Loushine BA, Bryan TE, Looney SW, Gillen BM, Loushine RJ, Weller RN, et al. Setting
properties and cytotoxicity evaluation of a premixed bioceramic root canal sealer. J Endod.
2011;37:673-7.
30. Prati C, Gandolfi MG. Calcium silicate bioactive cements: Biological perspectives and
clinical applications. Dental Materials. 2015;31:351-70.
31. Yang T, Xiao W, Chen W, Sui L. Effect of Carboxymethyl Chitosan and Aging Time
on Synthesis and Storage of Amorphous Calcium Phosphate. Journal of Nanoscience and
Nanotechnology. 2016;16:12582-9.
32. Weng J, Liu Q, Wolke JG, Zhang X, de Groot K. Formation and characteristics of the
apatite layer on plasma-sprayed hydroxyapatite coatings in simulated body fluid. Biomaterials.
1997;18:1027-35.
86
Chapter 5
Discussion
Dentin substrate modification may serve to enhance interfacial integrity with simultaneous
reinforcement of dentin mechanical property in root-filled teeth. This study evaluated the
potential of effect of dentin surface modification using chitosan-hydroxyapatite precursor
nanocomplexes on physical and chemical interaction with a tricalcium silicate sealer and
mechanical property of dentin. The experiments were conducted in three phases. Phase I and II
are covered by manuscript I and manuscript II is based on the third experimental phase. Chitosan
is a polyelectrolytic biopolymer that has extensive applications due to its biocompatibility,
biodegradability and anti-bacterial characteristics (16). Its popularity in biomedical fields is
largely due to the availability of free pendant groups on its backbone, such as OH and NH2, that
can be engineered to conduct specific functions with optimized physical, chemical and biological
characteristics (17, 18). Chitosan-Hydroxyapatite (C-HA) precursor nanocomplexes, is one such
development for guided tissue remineralization of dentin that comprises of a carboxymethyl
derivate of chitosan. The closely regulated synthesis parameters allow the carboxymethylation to
take place on the pendant hydroxyl group allowing formation of O-Carboxymethyl chitosan
(128). Such functionalization also allows the amino group mediated non-specific anionic-
cationic anti-bacterial action of chitosan to remain viable. This confers added versatility to C-HA
nanocomplexes compared to polymers being currently employed for guided mineralization. The
role of C-HA nanocomplexes as a NCP analog is channeled through presence of a dense surface
charge and amphoteric nature of chitosan backbone allowing calcium sequestration and
subsequent stabilization of precursors of amorphous calcium phosphate via potential self-
87
assembly mechanisms. Based on a bottom-up mineralization strategy, it is able to achieve
synergistic intrafibrillar and extrafibrillar mineralization of demineralized dentin/collagen
through dissolution of ACP precursors in remineralizing medium which are then able to infiltrate
collagen gap zones as established in earlier studies by our group using a collagen model and
subsequent ultrastructural evaluation with transmission electron microscopy (128).
The first phase of the experiments involved physico-chemical and bioactivity evaluation to
optimize the formulation of C-HA nanocomplexes employing routine assessment methods.
Surface charge measurements were conducted with zeta potential, which is a measurement of net
surface charge of nanoparticles dispersed in solution. The higher value of -27mV noted in the
current study, gives a measure of stability of the nanoparticles in a suspension (145). Self-
assembly potential of these nanocomplexes was also observed that can be explored fiuther in
future studies. Qualitative assessment methods of FTIR and XRD were used to establish the
nature of formed minerals. Bioactivity potential was established with formation of phosphate
bonds and carbonate incorporation as demonstrated by FTIR spectrum (131). Whereas transition
towards crystallinity of the formed minerals was confirmed both by the formation of bi-fid
phosphate bands (FTIR) and peaks on XRD (146). The assessment of mechanical recovery as an
indicator of successful dentin remineralization was also evaluated (12, 81). Conventional
mechanical tests tend to represent properties that are averaged over a large volume of regional
tissue. However, considering the effect of tubular direction and regional differences in
mechanical properties of dentin, a standardized sample preparation approach was chosen and
micro-tensile testing was done (27). The significant increase in UTS of conditioned dentin was
an important finding as it highlights the close nature of association and interaction between the
collagen and newly formed mineral. As the dentin beams were pre-incubated in NaOCl and
88
EDTA, the dissolution and damage to collagen fibrils could not be restored by mere precipitation
on the collagen surface from super saturated calcium phosphate solution in control group. In
contrast, in C-HA nanocomplexes conditioned dentin mineral formation occurred. The
nanocomplexes may have infiltrated into the collagen matrix and within collagen gap zones for
intrafibrillar mineralization to occur, regaining better coupling with the collagen matrix (18,
128). Moreover, role of chitosan alone in reinforcement of collagen with increased UTS and
toughness is supported by earlier studies (2). Hence, the increased UTS noted for conditioned
dentin beams may be explained interms of a combined organic-inorganic reinforcement of
dentinal matrix. Such a strengthening may play a significant role by providing resistance to
micro-cracking and increased toughening mechanisms of dentin towards fracture (1).
In the second phase of experiments, assessment of depth of TCS penetration was conducted with
a fluorescent labelling technique in order to demonstrate the effect of physico-chemical
modification of the dentin surface post conditioning with C-HA nanocomplexes. Established
earlier protocols were employed to measure the penetration depth parameters (147). Fluorescent
imaging technique enabled easy distinction of sealer penetration and did not require sample
processing in comparison to SEM used in previous studies (148). Results from a recent study by
Aydin et al. showed least penetration of a TCS in chitosan irrigated group (149). In contrast, to
our results that showed increased penetration depths for the conditioned group for all penetration
parameters measured at both 4 and 6mm levels from the root apex. The difference in results may
be explained in terms of physico-chemical character of the chitosan nanoparticles used in the
earlier study. Chitosan is insoluble in water owing to presence of hydrophobic acetyl groups
(150), this necessitates the functionalization and characterization with respect to surface charge
and pH in the dispersing medium, as conducted in the first phase of the experiments. A
89
carboxymethyl chitosan (water-soluble) derivate of chitosan was employed in this study, that
showed a stable negative charge in alkaline pH, suggesting maximum ionization of carboxyl
groups (123) giving intense hydrophilicity. This was also demonstrated with contact angle
measurements with water that showed formation of a super-hydrophilic dentin surface after C-
HA nanocomplexes conditioning. Dentin topography and chemical structure as surface
properties of dentin play an important role in dictating surface wettability characteristics (60).
These results are clinically relevant, as for materials employed in root canal treatment such as
sealers, it is important that they come in close contact with dentin to facilitate chemical adhesion
or penetration for micromechanical interlocking (65).
In the third phase of experiments, chemical characterization of the TCS-dentin interface was
conducted using TOF-SIMS. Use of SEM-EDX in earlier characterization studies offers a poor
comparison with TOF-SIMS that provides spatial mapping of the organic and inorganic
constituents with monolayer surface sensitivity (151). Additionally, precipitated, adsorbed and
contaminant species on samples require sufficient sensitivity and selectivity to detect and
discriminate between the very similar chemistry and closely related mineral phases (152) of
dentin, TCS and layer formed by C-HA nanocomplexes conditioning which was achievable with
TOF-SIMS. Moreover, other reported techniques such as confocal laser scanning microscopy
lack chemical identification (153), warranting the use of allied techniques. TOF-SIMS has been
used to characterize mineralized tissues like bone and dentin, and implant-bone interfaces (135,
136, 151). An in-depth analysis of all the material components involved was run individually
before analyzing the sealer-dentin interface (such as demineralized dentin, set sealer, C-HA
nanocomplexes amongst others).
90
A demineralized dentin model was used to facilitate identification of calcium formed and TCS
setting on dentin was allowed to occur only in 100% humidity. This was based on earlier studies
that showed hydration of tricalcium silicate based materials can occur in contact with distilled
water and resulted in formation of Ca(OH)2 and calcium carbonates (154). However, this is in
contrast to most of the studies that have employed large volumes of calcium-phosphate
supersaturated solution as source of calcium and phosphate (104, 155, 156). A TCS was used to
allow a better comparison of dentin surface modification as it is hydrophilic and has established
bioactivity unlike resin based sealers (99). The ultrastructural evaluation with TEM
complemented the findings from TOF-SIMS analysis for thickness of interfacial layer (5um) and
presence of coarse aggregates encapsulated in a matrix. The chemical identity of which was
resolved with TOF-SIMS analysis as an organic-inorganic ion rich layer.
A well-delineated interfacial phosphate band was seen in the conditioned sample in comparison
to the control. The TCS sealer as per manufacturer’s description, contains a calcium phosphate
monobasic phase as one of the constituents (157), however its contribution towards formation of
Ca-PO4 was minimal as no phosphate fragments (PO3, PO4) were found at interface. This
established the role of C-HA nanocomplexes as a phosphate source. This was also supported by
the high interfacial presence of CaO and Ca(OH)2 in control sample due to lacking phosphate
presence. Hence, the calcium ions being leached by the hydrating sealer remained unutilized as
they were not consumed to form calcium phosphate phases (104) (157). Studies on calcium and
silica incorporation into dentin have been done but similar data for phosphate is not gathered.
This can be implied to limitations of analytical tools employed as well as difficulty in delineating
it from the dentin matrix. Moreover, studies have shown lack of formation of calcium phosphate
phases by these materials even when physiological simulation of phosphate source was
91
employed (105). This only reinforces the importance of phosphate reserve needed in close
association with dentin.
The demineralized dentin surface becomes a challenging substrate due to increased surface
hydrophobicity and low polarity. From the results of this study, it can be deduced that surface
conditioning of dentin with C-HA nanocomplexes modified the surface physico-chemical
characteristics with its hydrophilic and polyelectrolytic nature. This may have complemented the
hydrophilic nature of TCS and facilitated its deeper penetration as well as lowering of energy
requirements that would have facilitated mineral nucleation to occur on C-HA nanocomplexes
conditioned dentin (8). ACP dissolution served to provide release of phosphate into the medium
that combined with calcium ions leached from setting TCS to form calcium phosphates.
The findings from this study can be combined to provide an overall scheme. Tricalcium silicate
sealers have been reported to interact with dentin both physically and chemically. We
demonstrated enhanced TCS interaction with dentin post C-HA nanocomplexes conditioning
with respect to both the physical and chemical aspects. Enhanced sealer penetration was seen as
a physical evidence of dentin surface modification that can potentially add to increased
mechanical interlocking. On the other hand, TOF-SIMS analysis highlighted increased interfacial
mineralization, which is the basis of chemical interaction of TCS with dentin. Additionally, the
role of C-HA nanocomplexes conditioning in reinforcement of collagen with increased UTS was
also shown.
92
Chapter 6
Conclusion
Formulation of Chitosan-Hydroxyapatite precursor nanocomplexes was developed
and characterized to have a polyanionic surface charge, hydrophilic nature, non-
aggregating characteristics and bioactivity potential.
Dentin conditioning with C-HA nanocomplexes resulted in reinforced mechanical
property (UTS) of dentin.
Improved dentin surface wettability characteristics modified the interaction of
tricalcium silicate sealer with C-HA nanocomplexes conditioned dentin resulting in
increased TCS penetration depth into the dentinal tubules.
C-HA nanocomplexes conditioned dentin surface was chemically modified with
formation of an organic-inorganic layer (5um) at the TCS-dentin interface consisting
of abundant calcium, phosphate, calcium phosphate and chitosan fragments.
93
Chapter 7
Future Studies
Studies to gain more temporal and spatial control of dentin remineralization using C-HA
nanocomplexes should be conducted. Approaches to potentiate amorphous to crystalline
apatite transformation may be considered with assessment over longer duration and/or
use of modifiers such as amino acid residues and crosslinking with dentin collagen. Self-
assembly potential of C-HA nanocomplexes may be investigated further.
Effect of C-HA nanocomplexes conditioning on TCS sealer-dentin interfacial integrity
may be evaluated with bond-strength tests and expanded to other sealer classes.
Future studies may consider assessment of sealing ability of C-HA nanocomplexes
conditioned interface with dye-penetration studies.
94
Chapter 8
References
1. Kishen A. Mechanisms and risk factors for fracture predilection in endodontically treated
teeth. Endodontic Topics 2006;13(1):57-83.
2. Shrestha A, Friedman S, Kishen A. Photodynamically crosslinked and chitosan-
incorporated dentin collagen. J Dent Res 2011;90(11):1346-1351.
3. Siqueira JF. Aetiology of root canal treatment failure: why well-treated teeth can fail. Int
Endod J 2001;34(1):1-10.
4. Gu LS, Huang XQ, Griffin B, Bergeron BR, Pashley DH, Niu LN, et al. Primum non
nocere - The effects of sodium hypochlorite on dentin as used in endodontics. Acta Biomater
2017;61:144-156.
5. Hulsman M. Effects of mechanical instrumentation and chemical irrigation on the root
canal dentin and surrounding tissues. Endodontic Topics 2013(29):55-86.
6. Doğan H, Qalt S. Effects of chelating agents and sodium hypochlorite on mineral content
of root dentin. J Endod 2001;27(9):578-580.
7. Hulsman M. Effects of mechanical instrumentation and chemical irrigation on the root
canal dentin and surrounding tissues. Endodontic Topics 2013(29):55-86.
8. Xu Z, Neoh KG, Lin CC, Kishen A. Biomimetic deposition of calcium phosphate
minerals on the surface of partially demineralized dentine modified with phosphorylated
chitosan. J Biomed Mater Res B Appl Biomater 2011;98(1):150-159.
9. Hulsman M. Effects of mechanical instrumentation and chemical irrigation on the root
canal dentin and surrounding tissues. Endodontic Topics 2013(29):55-86.
10. Hernández M, Cobb D, Swift EJ. Current strategies in dentin remineralization. J Esthet
Restor Dent 2014;26(2):139-145.
11. Bertassoni L. Dentin on the nanoscale: Hierarchical organization, mechanical behavior
and bioinspired engineering. Dental Materials 2017;33(6):637-649.
95
12. Bertassoni LE, Habelitz S, Kinney JH, Marshall SJ, Marshall GW. Biomechanical
perspective on the remineralization of dentin. Caries Res 2009;43(1):70-77.
13. Cao CY, Mei ML, Li QL, Lo EC, Chu CH. Methods for biomimetic remineralization of
human dentine: a systematic review. Int J Mol Sci 2015;16(3):4615-4627.
14. Tay FR, Pashley DH. Guided tissue remineralisation of partially demineralised human
dentine. Biomaterials 2008;29(8):1127-1137.
15. Tay FR, Pashley DH, Rueggeberg FA, Loushine RJ, Weller RN. Calcium phosphate
phase transformation produced by the interaction of the portland cement component of white
mineral trioxide aggregate with a phosphate-containing fluid. J Endod 2007;33(11):1347-1351.
16. Kishen A, Shi Z, Shrestha A, Neoh K. An Investigation on the Antibacterial and
Antibiofilm Efficacy of Cationic Nanoparticulates for Root Canal Disinfection. Journal of
Endodontics 2008;34(12):1515-1520.
17. Muzzarelli RA. Carboxymethylated chitins and chitosans Carbohydrate Polymers
1998;1(8):1-21.
18. Muzzarelli C, Muzzarelli RA. Natural and artificial chitosan-inorganic composites. J
Inorg Biochem 2002;92(2):89-94.
19. Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall GW. In situ atomic force
microscopy of partially demineralized human dentin collagen fibrils. J Struct Biol
2002;138(3):227-236.
20. Kishen A, Ramamurty U, Asundi A. Experimental studies on the nature of property
gradients in the human dentine. J Biomed Mater Res 2000;51(4):650-659.
21. Kinney JH, Marshall SJ, Marshall GW. The mechanical properties of human dentin: a
critical review and re-evaluation of the dental literature. Crit Rev Oral Biol Med 2003;14(1):13-
29.
22. Ito S, Saito T, Amano K. In vitro apatite induction by osteopontin: interfacial energy for
hydroxyapatite nucleation on osteopontin. J Biomed Mater Res A 2004;69(1):11-16.
23. Saito T, Yamauchi M, Crenshaw MA. Apatite induction by insoluble dentin collagen. J
Bone Miner Res 1998;13(2):265-270.
96
24. Tjäderhane L, Buzalaf MA, Carrilho M, Chaussain C. Matrix metalloproteinases and
other matrix proteinases in relation to cariology: the era of 'dentin degradomics'. Caries Res
2015;49(3):193-208.
25. George A, Veis A. Phosphorylated proteins and control over apatite nucleation, crystal
growth, and inhibition. Chem Rev 2008;108(11):4670-4693.
26. Agee KA, Prakki A, Abu-Haimed T, Naguib GH, Nawareg MA, Tezvergil-Mutluay A, et
al. Water distribution in dentin matrices: bound vs. unbound water. Dent Mater 2015;31(3):205-
216.
27. Inoue S, Pereira PN, Kawamoto C, Nakajima M, Koshiro K, Tagami J, et al. Effect of
depth and tubule direction on ultimate tensile strength of human coronal dentin. Dent Mater J
2003;22(1):39-47.
28. Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: structure, composition and
mineralization. Front Biosci (Elite Ed) 2011;3:711-735.
29. Budiraharjo R, Neoh KG, Kang ET, Kishen A. Bioactivity of novel carboxymethyl
chitosan scaffold incorporating MTA in a tooth model. Int Endod J 2010;43(10):930-939.
30. Prydz K. Determinants of Glycosaminoglycan (GAG) Structure. Biomolecules
2015;5(3):2003-2022.
31. Gandhi NS, Mancera RL. The structure of glycosaminoglycans and their interactions
with proteins. Chem Biol Drug Des 2008;72(6):455-482.
32. Elsharkawy S, Al-Jawad M, Pantano MF, Tejeda-Montes E, Mehta K, Jamal H, et al.
Protein disorder-order interplay to guide the growth of hierarchical mineralized structures. Nat
Commun 2018;9(1):2145.
33. Liu Y, Kim Y, Dai L, Li N, Khan S, Pashley D, et al. Hierarchical and non-hierarchical
mineralisation of collagen. Biomaterials 2011;32(5):1291-1300.
34. Xu Z, Xiao Z, Wang H, Kitten A. Biomineralization and Biomaterial Considerations in
Dentin Remineralization. Journal of Operative Dentistry and Endodontics 2016;1:7-12.
97
35. Okuyama K, Bächinger HP, Mizuno K, Boudko S, Engel J, Berisio R, et al. Re:
Microfibrillar structure of type I collagen in situ. Acta Crystallogr D Biol Crystallogr 2009;65(Pt
9):1007-1008; author reply 1009-1010.
36. Orgel JP, Irving TC, Miller A, Wess TJ. Microfibrillar structure of type I collagen in situ.
Proc Natl Acad Sci U S A 2006;103(24):9001-9005.
37. Chapman JA, Tzaphlidou M, Meek KM, Kadler KE. The collagen fibril--a model system
for studying the staining and fixation of a protein. Electron Microsc Rev 1990;3(1):143-182.
38. Gautieri A, Vesentini S, Redaelli A, Buehler MJ. Hierarchical structure and
nanomechanics of collagen microfibrils from the atomistic scale up. Nano Lett 2011;11(2):757-
766.
39. Silver FH, Landis WJ. Deposition of apatite in mineralizing vertebrate extracellular
matrices: A model of possible nucleation sites on type I collagen. Connect Tissue Res
2011;52(3):242-254.
40. Nudelman F, Bomans PH, George A, de With G, Sommerdijk NA. The role of the
amorphous phase on the biomimetic mineralization of collagen. Faraday Discuss 2012;159:357-
370.
41. Landis WJ, Hodgens KJ, Arena J, Song MJ, McEwen BF. Structural relations between
collagen and mineral in bone as determined by high voltage electron microscopic tomography.
Microsc Res Tech 1996;33(2):192-202.
42. He G, Gajjeraman S, Schultz D, Cookson D, Qin C, Butler WT, et al. Spatially and
temporally controlled biomineralization is facilitated by interaction between self-assembled
dentin matrix protein 1 and calcium phosphate nuclei in solution. Biochemistry
2005;44(49):16140-16148.
43. He G, Dahl T, Veis A, George A. Dentin matrix protein 1 initiates hydroxyapatite
formation in vitro. Connect Tissue Res 2003;44 Suppl 1:240-245.
44. Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, et al. Loss of DMP1 causes rickets and
osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet
2006;38(11):1310-1315.
98
45. Abuhaimed TS, Abou Neel EA. Sodium Hypochlorite Irrigation and Its Effect on Bond
Strength to Dentin. Biomed Res Int 2017;2017:1930360.
46. Sayin TC, Cehreli ZC, Deniz D, Akcay A, Tuncel B, Dagli F, et al. Time-dependent
decalcifying effects of endodontic irrigants with antibacterial properties. J Endod
2009;35(2):280-283.
47. Zou L, Shen Y, Li W, Haapasalo M. Penetration of sodium hypochlorite into dentin. J
Endod 2010;36(5):793-796.
48. Mohammadi Z. Sodium hypochlorite in endodontics: an update review. Int Dent J
2008;58(6):329-341.
49. Sim TP, Knowles JC, Ng YL, Shelton J, Gulabivala K. Effect of sodium hypochlorite on
mechanical properties of dentine and tooth surface strain. Int Endod J 2001;34(2):120-132.
50. Mountouris G, Silikas N, Eliades G. Effect of sodium hypochlorite treatment on the
molecular composition and morphology of human coronal dentin. J Adhes Dent 2004;6(3):175-
182.
51. Hülsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode
of action and indications for their use. Int Endod J 2003;36(12):810-830.
52. Nyborg JK, Peersen OB. That zincing feeling: the effects of EDTA on the behaviour of
zinc-binding transcriptional regulators. Biochem J 2004;381(Pt 3):e3-4.
53. Ostby N. Chelation in root canal therapy: ethylenediaminetetraacetic acid for cleansing
and widening of root canals. OdontologyiskTidkrift 1957;65:3-11.
54. Kolosowski KP, Sodhi RN, Kishen A, Basrani BR. Qualitative Time-of-Flight Secondary
Ion Mass Spectrometry Analysis of Root Dentin Irrigated with Sodium Hypochlorite, EDTA, or
Chlorhexidine. J Endod 2015;41(10):1672-1677.
55. Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on the
adherence of Enterococcus faecalis to root canal dentin. J Endod 2008;34(7):850-854.
56. Niu W, Yoshioka T, Kobayashi C, Suda H. A scanning electron microscopic study of
dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J
2002;35(11):934-939.
99
57. White JD, Lacefield WR, Chavers LS, Eleazer PD. The effect of three commonly used
endodontic materials on the strength and hardness of root dentin. J Endod 2002;28(12):828-830.
58. De-Deus G, Reis CM, Fidel RA, Fidel SR, Paciornik S. Co-site digital optical
microscopy and image analysis: an approach to evaluate the process of dentine demineralization.
Int Endod J 2007;40(6):441-452.
59. Ballal NV, Ferrer-Luque CM, Sona M, Prabhu KN, Arias-Moliz T, Baca P. Evaluation of
final irrigation regimens with maleic acid for smear layer removal and wettability of root canal
sealer. Acta Odontol Scand 2018;76(3):199-203.
60. Yassen GH, Sabrah AH, Eckert GJ, Platt JA. Effect of different endodontic regeneration
protocols on wettability, roughness, and chemical composition of surface dentin. J Endod
2015;41(6):956-960.
61. Attal J-PA, Erik.Degrange, Michel. Effects of surface treatment on the free surface
energy of dentin. Dent Materials 1994;10:259-264.
62. Tani C, Manabe A, Itoh K, Hisamitsu H, Wakumoto S. Contact angle of dentin bonding
agents on the dentin surface. Dent Mater J 1996;15(1):39-44.
63. Ramos SM, Alderete L, Farge P. Dentinal tubules driven wetting of dentin: Cassie-Baxter
modelling. Eur Phys J E Soft Matter 2009;30(2):187-195.
64. Panighi M, G'Sell C. Influence of calcium concentration on the dentin wettability by an
adhesive. J Biomed Mater Res 1992;26(8):1081-1089.
65. Dogan Buzoglu H, Calt S, Gümüsderelioglu M. Evaluation of the surface free energy on
root canal dentine walls treated with chelating agents and NaOCl. Int Endod J 2007;40(1):18-24.
66. Cölfen H, Mann S. Higher-order organization by mesoscale self-assembly and
transformation of hybrid nanostructures. Angew Chem Int Ed Engl 2003;42(21):2350-2365.
67. Zahn D. Thermodynamics and Kinetics of Prenucleation Clusters, Classical and Non-
Classical Nucleation. Chemphyschem 2015;16(10):2069-2075.
68. Zhong B, Peng C, Wang G, Tian L, Cai Q, Cui F. Contemporary research findings on
dentine remineralization. J Tissue Eng Regen Med 2015;9(9):1004-1016.
100
69. Kawasaki K, Ruben J, Stokroos I, Takagi O, Arends J. The remineralization of EDTA-
treated human dentine. Caries Res 1999;33(4):275-280.
70. Koutsoukos PG, Nancollas GH. Crystal growth of calcium phosphates - epitaxial
considerations. Journal of Crystal Growth 1981;53(1):10-19.
71. Reynolds EC. Calcium phosphate-based remineralization systems: scientific evidence?
Aust Dent J 2008;53(3):268-273.
72. Li J, Yang J, Chen L, Liang K, Wu W, Chen X. Bioinspired intrafibrillar mineralization
of human dentine by PAMAM dendrimer. Biomaterials 2013;34(28):6738-6747.
73. Termine JD, Posner AS. Infra-Red Determination of the Percentage of Crystallinity in
Apatitic Calcium Phosphates. Nature 1966;211:268-270.
74. Johnsson MS, Nancollas GH. The role of brushite and octacalcium phosphate in apatite
formation. Crit Rev Oral Biol Med 1992;3(1-2):61-82.
75. Mahamid J, Sharir A, Addadi L, Weiner S. Amorphous calcium phosphate is a major
component of the forming fin bones of zebrafish: Indications for an amorphous precursor phase.
Proc Natl Acad Sci U S A 2008;105(35):12748-12753.
76. Deshpande AS, Beniash E. Bio-inspired Synthesis of Mineralized Collagen Fibrils. Cryst
Growth Des 2008;8(8):3084-3090.
77. Olszta MJ, Cheng XG, Jee SS, Kumar R. Bone structure and formation: a new
perspective. Material Science and Engineering:R 2007(58):77-116.
78. Zeiger DN, Miles WC, Eidelman N, Lin-Gibson S. Cooperative calcium phosphate
nucleation within collagen fibrils. Langmuir 2011;27(13):8263-8268.
79. Niu L-nSJ, Sang. Ashley , David H. Tay, Franklin R. Collagen intrafibrillar
mineralisation as a result of the balance between osmotic equilibrium and
electroneutrality Nature Materials 2017;16(3):370-378.
80. Nudelman F, Lausch AJ, Sommerdijk NA, Sone ED. In vitro models of collagen
biomineralization. J Struct Biol 2013;183(2):258-269.
101
81. Kinney JH, Habelitz S, Marshall SJ, Marshall GW. The importance of intrafibrillar
mineralization of collagen on the mechanical properties of dentin. J Dent Res 2003;82(12):957-
961.
82. Balooch M, Habelitz S, Kinney JH, Marshall SJ, Marshall GW. Mechanical properties of
mineralized collagen fibrils as influenced by demineralization. J Struct Biol 2008;162(3):404-
410.
83. Niu LN, Zhang W, Pashley DH, Breschi L, Mao J, Chen JH, et al. Biomimetic
remineralization of dentin. Dent Mater 2014;30(1):77-96.
84. Yang Y, Lv XP, Shi W, Li JY, Li DX, Zhou XD, et al. 8DSS-promoted remineralization
of initial enamel caries in vitro. J Dent Res 2014;93(5):520-524.
85. Gu LS, Kim YK, Liu Y, Takahashi K, Arun S, Wimmer CE, et al. Immobilization of a
phosphonated analog of matrix phosphoproteins within cross-linked collagen as a templating
mechanism for biomimetic mineralization. Acta Biomater 2011;7(1):268-277.
86. Dai L, Liu Y, Salameh Z, Khan S, Mao J, Pashley DH, et al. Can Caries-Affected Dentin
be Completely Remineralized by Guided Tissue Remineralization? Dent Hypotheses
2011;2(2):74-82.
87. Liang K, Zhou H, Weir M, Bao C, Reynolds M, Zhou X, et al. Poly( amido amine) and
calcium phosphate nanocomposite remineralization of dentin in acidic solution without calcium
phosphate ions. Dental Materials 2017;33(7):818-829.
88. Gandhi B, Bollineni S, Janga RK, Saraswati D, Babu MR. Evaluating the Effect of CPP-
ACP as a Final Irrigant in Improving the Micro-Hardness of Erosive Root Dentin and its
Influence on the Bond Strength of Self Etch Resin Sealer - An In-vitro Study. J Clin Diagn Res
2016;10(8):ZC53-56.
89. Li X, Lan J, Ai M, Guo Y, Cai Q, Yang X. Biomineralization on polymer-coated multi-
walled carbon nanotubes with different surface functional groups. Colloids Surf B Biointerfaces
2014;123:753-761.
90. Gu L, Kim YK, Liu Y, Ryou H, Wimmer CE, Dai L, et al. Biomimetic analogs for
collagen biomineralization. J Dent Res 2011;90(1):82-87.
102
91. Reyes-Carmona JF, Felippe MS, Felippe WT. Biomineralization ability and interaction of
mineral trioxide aggregate and white portland cement with dentin in a phosphate-containing
fluid. J Endod 2009;35(5):731-736.
92. Cao Y, Mei ML, Xu J, Lo EC, Li Q, Chu CH. Biomimetic mineralisation of
phosphorylated dentine by CPP-ACP. J Dent 2013;41(9):818-825.
93. Zhou Y, Yang J, Lin Z, Li J, Liang K, Yuan H, et al. Triclosan-loaded poly(amido amine)
dendrimer for simultaneous treatment and remineralization of human dentine. Colloids Surf B
Biointerfaces 2014;115:237-243.
94. Ning TY, Xu XH, Zhu LF, Zhu XP, Chu CH, Liu LK, et al. Biomimetic mineralization of
dentin induced by agarose gel loaded with calcium phosphate. J Biomed Mater Res B Appl
Biomater 2012;100(1):138-144.
95. Cao Y, Liu W, Ning T, Mei ML, Li QL, Lo EC, et al. A novel oligopeptide simulating
dentine matrix protein 1 for biomimetic mineralization of dentine. Clin Oral Investig
2014;18(3):873-881.
96. Liu Y, Li N, Qi Y, Niu LN, Elshafiy S, Mao J, et al. The use of sodium trimetaphosphate
as a biomimetic analog of matrix phosphoproteins for remineralization of artificial caries-like
dentin. Dent Mater 2011;27(5):465-477.
97. Qi YP, Li N, Niu LN, Primus CM, Ling JQ, Pashley DH, et al. Remineralization of
artificial dentinal caries lesions by biomimetically modified mineral trioxide aggregate. Acta
Biomater 2012;8(2):836-842.
98. Kim JR, Nosrat A, Fouad AF. Interfacial characteristics of Biodentine and MTA with
dentine in simulated body fluid. J Dent 2015;43(2):241-247.
99. Prati C, Gandolfi MG. Calcium silicate bioactive cements: Biological perspectives and
clinical applications. Dental Materials 2015;31:351-370.
100. Torabinejad M, Pitt Ford TR, McKendry DJ, Abedi HR, Miller DA, Kariyawasam SP.
Histologic assessment of mineral trioxide aggregate as a root-end filling in monkeys. J Endod
1997;23(4):225-228.
103
101. Camilleri J. Is Mineral Trioxide Aggregate a Bioceramic? Int. J. Dental Sc., 2015;18-1:
13-17.(1659-1046).
102. Gandolfi MG, Taddei P, Siboni F, Modena E, Ginebra MP, Prati C. Fluoride-containing
nanoporous calcium-silicate MTA cements for endodontics and oral surgery: early fluorapatite
formation in a phosphate-containing solution. Int Endod J 2011;44(10):938-949.
103. Kokkas AB, Boutsioukis ACh, Vassiliadis LP, Stavrianos CK. The influence of the smear
layer on dentinal tubule penetration depth by three different root canal sealers: an in vitro study.
J Endod 2004;30(2):100-102.
104. Loushine BA, Bryan TE, Looney SW, Gillen BM, Loushine RJ, Weller RN, et al. Setting
properties and cytotoxicity evaluation of a premixed bioceramic root canal sealer. J Endod
2011;37(5):673-677.
105. Xuereb M, Vella P, Damidot D, Sammut CV, Camilleri J. In situ assessment of the
setting of tricalcium silicate-based sealers using a dentin pressure model. J Endod
2015;41(1):111-124.
106. Kishen A, Shrestha A. Nanoparticles for Dentin Tissue Stabilization. In: Kishen A,
editor. Nanotechnology in Endodontics. Springer; 2015. p. 121-138.
107. Aranaz I, Harris R, Heras A. Chitosan Amphiphilic Derivatives. Chemistry and
Applications. t Organic Chemistry, 2010;14:308-330.
108. Chesnutt BM, Yuan Y, Brahmandam N, Yang Y, Ong JL, Haggard WO, et al.
Characterization of biomimetic calcium phosphate on phosphorylated chitosan films. J Biomed
Mater Res A 2007;82(2):343-353.
109. Alvarez FJ. The effect of chitin size, shape, source and purification method on immune
recognition. Molecules 2014;19(4):4433-4451.
110. Yu Z, Lau D. Flexibility of backbone fibrils in α-chitin crystals with different degree of
acetylation. Carbohydr Polym 2017;174:941-947.
111. Kaş HS. Chitosan: properties, preparations and application to microparticulate systems. J
Microencapsul 1997;14(6):689-711.
104
112. Wang X, Du Y, Ding S, Wang Q, Xiong G, Xie M, et al. Preparation and third-order
optical nonlinearity of self-assembled chitosan/CdSe-ZnS core-shell quantum dots multilayer
films. J Phys Chem B 2006;110(4):1566-1570.
113. Schatz C, Viton C, Delair T, Pichot C, Domard A. Typical physicochemical behaviors of
chitosan in aqueous solution. Biomacromolecules 2003;4(3):641-648.
114. Kong X. Simultaneous determination of degree of deacetylation, degree of substitution
and distribution fraction of –COONa in carboxymethyl chitosan by potentiometric titration. .
Carbohydrate Polymers 2012;1(88):336-341.
115. Zhu A, Chan-Park MB, Dai S, Li L. The aggregation behavior of O-
carboxymethylchitosan in dilute aqueous solution. Colloids Surf B Biointerfaces 2005;43(3-
4):143-149.
116. Hamman JH. Chitosan based polyelectrolyte complexes as potential carrier materials in
drug delivery systems. Mar Drugs 2010;8(4):1305-1322.
117. Kean T, Thanou M. Biodegradation, biodistribution and toxicity of chitosan. Adv Drug
Deliv Rev 2010;62(1):3-11.
118. Dorozhkin SV. Calcium orthophosphates: occurrence, properties, biomineralization,
pathological calcification and biomimetic applications. Biomatter 2011;1(2):121-164.
119. Zhao J, Liu Y, Sun W, Yang X. First detection, characterization, and application of
amorphous calcium phosphate in dentistry. Journal of Dental Sciences 2012;7(4):316-323.
120. Li Y, Weng W. In vitro synthesis and characterization of amorphous calcium phosphates
with various Ca/P atomic ratios. J Mater Sci Mater Med 2007;18(12):2303-2308.
121. Zhang F, Allen AJ, Levine LE, Vaudin MD, Skrtic D, Antonucci JM, et al. Structural and
dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental
composites. Dent Mater 2014;30(10):1113-1125.
122. Yang T, Xiao W, Chen W, Sui L. Effect of Carboxymethyl Chitosan and Aging Time
on Synthesis and Storage of Amorphous Calcium Phosphate. Journal of Nanoscience and
Nanotechnology 2016;16:12582-12589.
105
123. Kalliola S, Repo E, Srivastava V, Heiskanen J, Sirvio J, Liimatainen H, et al. The pH
sensitive properties of carboxymethyl chitosan nanoparticles cross-linked with calcium ions.
Colloids and Surfaces B-Biointerfaces 2017;153:229-236.
124. Venkatesan J, Kim SK. Chitosan composites for bone tissue engineering--an overview.
Mar Drugs 2010;8(8):2252-2266.
125. Kumar R, Prakash KH, Cheang P, Gower L, Khor KA. Chitosan-mediated crystallization
and assembly of hydroxyapatite nanoparticles into hybrid nanostructured films. J R Soc Interface
2008;5(21):427-439.
126. Andrew CA, Khor E, Hastings GW. The influence of anionic chitin derivatives on
calcium phosphate crystallization. Biomaterials 1998;19(14):1309-1316.
127. Wang H, Xiao Z, Yang J, Lu D, Kishen A, Li Y, et al. Oriented and Ordered Biomimetic
Remineralization of the Surface of Demineralized Dental Enamel Using HAP@ACP
Nanoparticles Guided by Glycine. Sci Rep 2017;7:40701.
128. Chen Z, Cao S, Wang H, Li Y, Kishen A, Deng X, et al. Biomimetic remineralization of
demineralized dentine using scaffold of CMC/ACP nanocomplexes in an in vitro tooth model of
deep caries. PLoS One 2015;10(1):e0116553.
129. Wang Y, Van Manh N, Wang H, Zhong X, Zhang X, Li C. Synergistic
intrafibrillar/extrafibrillar mineralization of collagen scaffolds based on a biomimetic strategy to
promote the regeneration of bone defects. Int J Nanomedicine 2016;11:2053-2067.
130. Boskey AL, Mendelsohn R. Infrared spectroscopic characterization of mineralized
tissues. Vib Spectrosc 2005;38(1-2):107-114.
131. Dorset C. Apatite Formation: Why It May Not Work as Planned, and How to
Conclusively Identify Apatite Compounds BioMed Research International 2013;2013:12.
132. Sodhi RN. Time-of-flight secondary ion mass spectrometry (TOF-SIMS):–versatility
in chemical and imaging surface analysis. Analyst 2004;129(4):483-487.
133. Fearn S. An Introduction to Time-of-Flight Secondary Ion Mass Spectrometry (ToF-
SIMS) and its Application to Materials Science Morgan & Claypool Publishers; 2015.
106
134. Henss A, Hild A, Rohnke M, Wenisch S, Janek J. Time of flight secondary ion mass
spectrometry of bone-Impact of sample preparation and measurement conditions. Biointerphases
2015;11(2):02A302.
135. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without
collagen: role of a phospholipid-proteolipid complex. Calcif Tissue Int 2007;81(3):191-205.
136. Gotliv BA, Veis A. The composition of bovine peritubular dentin: matching TOF-SIMS,
scanning electron microscopy and biochemical component distributions. New light on
peritubular dentin function. Cells Tissues Organs 2009;189(1-4):12-19.
137. L M, J L, P M, JG T, DH C. XRMA and ToF-SIMS Analysis of Normal and
Hypomineralized Enamel. Microsc Microanal 2015;2:407-421.
138. Almhöjd US, Norén JG, Arvidsson A, Nilsson Å, Lingström P. Analysis of carious
dentine using FTIR and ToF-SIMS. Oral Health Dent Manag 2014;13(3):735-744.
139. Daskalova A, Kaspersky G, Rousseau R, Lawicki A. Interaction of slow highly charged
ions with hard dental tissue: studies of fluoride uptake and remineralization efficacy. Journal of
Physics: Conference Series 2014(514).
140. Kolosowski KP, Sodhi RN, Kishen A, Basrani BR. Qualitative analysis of precipitate
formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a
final rinse of chlorhexidine or QMiX. J Endod 2014;40(12):2036-2040.
141. Anil K. Mechanisms and risk factors for fracture predilection in endodontically treated
teeth. Endodontic Topics 2006;13(1):57-83.
142. Gaitan-Fonseca C, Collart-Dutilleul PY, Semetey V, Romieu O, Cruz R, Flores H, et al.
Chemical treatment of the intra-canal dentin surface: a new approach to modify dentin
hydrophobicity. J Appl Oral Sci 2013;21(1):63-67.
143. Iwanami M, Yoshioka T, Sunakawa M, Kobayashi C, Suda H. Spreading of root canal
irrigants on root dentine. Aust Endod J 2007;33(2):66-72.
144. Zhang H, Shen Y, Ruse ND, Haapasalo M. Antibacterial activity of endodontic sealers by
modified direct contact test against Enterococcus faecalis. J Endod 2009;35(7):1051-1055.
145. Clogston JD, Patri AK. Zeta potential measurement. Methods Mol Biol 2011;697:63-70.
107
146. Sun J, Chen C, Pan H, Chen Y, Mao C, Wang W, et al. Biomimetic promotion of dentin
remineralization using L-glutamic acid: inspiration from biomineralization proteins. Journal of
Materials Chemistry B 2014;2(28):4544-4553.
147. Al-Haddad A, Abu Kasim NH, Che Ab Aziz ZA. Interfacial adaptation and thickness of
bioceramic-based root canal sealers. Dent Mater J 2015;34(4):516-521.
148. Van Meerbeek B, Vargas M, Inoue S, Yoshida Y, Perdigão J, Lambrechts P, et al.
Microscopy investigations. Techniques, results, limitations. Am J Dent 2000;13(Spec No):3D-
18D.
149. Aydın ZU, Özyürek T, Keskin B, Baran T. Effect of chitosan nanoparticle, QMix, and
EDTA on TotalFill BC sealers' dentinal tubule penetration: a confocal laser scanning microscopy
study. Odontology 2018.
150. Kalliola S, Repo E, Srivastava V, Heiskanen JP, Sirviö JA, Liimatainen H, et al. The pH
sensitive properties of carboxymethyl chitosan nanoparticles cross-linked with calcium ions.
Colloids Surf B Biointerfaces 2017;153:229-236.
151. Eriksson C, Malmberg P, Nygren H. Time-of-flight secondary ion mass spectrometric
analysis of the interface between bone and titanium implants. Rapid Commun Mass Spectrom
2008;22(7):943-949.
152. Hart B, Biesinger M, Smart RSC. Improved statistical methods applied to surface
chemistry in minerals flotation. Minerals Engineering 2006;19:790-798.
153. Kebudi Benezra M, Schembri Wismayer P, Camilleri J. Interfacial Characteristics and
Cytocompatibility of Hydraulic Sealer Cements. J Endod 2018;44(6):1007-1017.
154. Han L, Okiji T, Okawa S. Morphological and chemical analysis of different precipitates
on mineral trioxide aggregate immersed in different fluids. Dent Mater J 2010;29(5):512-517.
155. Camilleri J, Formosa L, Damidot D. The setting characteristics of MTA Plus in different
environmental conditions. Int Endod J 2013;46(9):831-840.
156. Han L, Okiji T. Bioactivity evaluation of three calcium silicate-based endodontic
materials. Int Endod J 2013;46(9):808-814.
108
157. Koch K, Brave D, Nasser AA. A review of bioceramic technology in endodontics. In.;
2013.
109
Supplementary Data
Figure 1. Real time self-assembly of C-HA nanocomplexes captured using optical microscope at
63x. Dewetting followed by interfacial self-assembly, the formed “fractal trees’’ are separated
from each other having distinct boundaries.
t=0 sec t=1 sec
t=2 sec t=3 sec
110
Conditioned COntrol
Figure 2. Positive polarity chemical ion maps for C-HA nanocomplexes conditioned dentin and
control dentin from TOF-SIMS analysis.
125
100
75
50
25
0
12080400μm
totalMC: 372; TC: 7.238e+006
210
110
010
125
100
75
50
25
0
12080400μm
sum of restMC: 284; TC: 5.204e+006
210
110
010
125
100
75
50
25
0
12080400μm
Na+MC: 45; TC: 2.468e+005
110
010
125
100
75
50
25
0
12080400μm
K+MC: 58; TC: 3.109e+005
110
010
125
100
75
50
25
0
12080400μm
Ca+MC: 56; TC: 1.642e+005
110
010
125
100
75
50
25
0
12080400μm
P+MC: 4; TC: 1.159e+004
010
125
100
75
50
25
0
12080400μm
SiMC: 12; TC: 1.167e+005
110
010
125
100
75
50
25
0
12080400μm
SiOMC: 19; TC: 1.667e+005
110
010
125
100
75
50
25
0
12080400μm
CaOHMC: 13; TC: 1.239e+005
110
010
125
100
75
50
25
0
12080400μm
CaOMC: 22; TC: 1.457e+005
110
010
125
100
75
50
25
0
12080400μm
NH4
MC: 9; TC: 3.447e+004
010
Sample: DM-CON-XS ̶ Field of View: 150 x 150 µm² ̶ Polarity: Positive ̶ File: DM-XS-CON_p1_peaks.ita150
125
100
75
50
25
0
12080400μm
totalMC: 241; TC: 4.590e+006
210
110
010
150
125
100
75
50
25
0
12080400μm
sum of restMC: 167; TC: 3.001e+006
210
110
010
150
125
100
75
50
25
0
12080400μm
Na+MC: 8; TC: 3.604e+004
010
150
125
100
75
50
25
0
12080400μm
K+MC: 27; TC: 8.676e+004
110
010
150
125
100
75
50
25
0
12080400μm
Ca+MC: 26; TC: 5.079e+004
110
010
150
125
100
75
50
25
0
12080400μm
PMC: 4; TC: 1.235e+004
010
150
125
100
75
50
25
0
12080400μm
CaOHMC: 14; TC: 8.210e+004
110
010
150
125
100
75
50
25
0
12080400μm
SiMC: 11; TC: 8.777e+004
110
010
150
125
100
75
50
25
0
12080400μm
CaOMC: 13; TC: 1.143e+005
110
010
150
125
100
75
50
25
0
12080400μm
SiOMC: 19; TC: 1.844e+005
110
010
150
125
100
75
50
25
0
12080400μm
NH4+MC: 9; TC: 3.589e+004
010
Sample: DM-NC-XS ̶ Field of View: 151 x 151 µm² ̶ Polarity: Positive ̶ File: DM-XS-NC_p2_peaks.ita
150
125
100
75
50
25
0
12080400μm
totalMC: 241; TC: 4.590e+006
210
110
010
150
125
100
75
50
25
0
12080400μm
sum of restMC: 167; TC: 3.001e+006
210
110
010
150
125
100
75
50
25
0
12080400μm
Na+MC: 8; TC: 3.604e+004
010
150
125
100
75
50
25
0
12080400μm
K+MC: 27; TC: 8.676e+004
110
010
150
125
100
75
50
25
0
12080400μm
Ca+MC: 26; TC: 5.079e+004
110
010
150
125
100
75
50
25
0
12080400μm
PMC: 4; TC: 1.235e+004
010
150
125
100
75
50
25
0
12080400μm
CaOHMC: 14; TC: 8.210e+004
110
010
150
125
100
75
50
25
0
12080400μm
SiMC: 11; TC: 8.777e+004
110
010
150
125
100
75
50
25
0
12080400μm
CaOMC: 13; TC: 1.143e+005
110
010
150
125
100
75
50
25
0
12080400μm
SiOMC: 19; TC: 1.844e+005
110
010
150
125
100
75
50
25
0
12080400μm
NH4+MC: 9; TC: 3.589e+004
010
Sample: DM-NC-XS ̶ Field of View: 151 x 151 µm² ̶ Polarity: Positive ̶ File: DM-XS-NC_p2_peaks.ita
125
100
75
50
25
0
12080400μm
totalMC: 372; TC: 7.238e+006
210
110
010
125
100
75
50
25
0
12080400μm
sum of restMC: 284; TC: 5.204e+006
210
110
010
125
100
75
50
25
0
12080400μm
Na+MC: 45; TC: 2.468e+005
110
010
125
100
75
50
25
0
12080400μm
K+MC: 58; TC: 3.109e+005
110
010
125
100
75
50
25
0
12080400μm
Ca+MC: 56; TC: 1.642e+005
110
010
125
100
75
50
25
0
12080400μm
P+MC: 4; TC: 1.159e+004
010
125
100
75
50
25
0
12080400μm
SiMC: 12; TC: 1.167e+005
110
010
125
100
75
50
25
0
12080400μm
SiOMC: 19; TC: 1.667e+005
110
010
125
100
75
50
25
0
12080400μm
CaOHMC: 13; TC: 1.239e+005
110
010
125
100
75
50
25
0
12080400μm
CaOMC: 22; TC: 1.457e+005
110
010
125
100
75
50
25
0
12080400μm
NH4
MC: 9; TC: 3.447e+004
010
Sample: DM-CON-XS ̶ Field of View: 150 x 150 µm² ̶ Polarity: Positive ̶ File: DM-XS-CON_p1_peaks.ita
Conditioned Control
Interface Sealer
Dentin Dentin
Interface Sealer
Sealer
111
Figure 3. Negative polarity chemical ion maps for C-HA nanocomplexes conditioned dentin.
High signal intensity of O-, OH-, CN- can be noted. Defined bands of phosphate fragments at
interfacial boundary can be seen.
150
125
100
75
50
25
0
12080400μm
totalMC: 199; TC: 1.649e+006
210
110
010
150
125
100
75
50
25
0
12080400μm
sum of restMC: 138; TC: 1.109e+006
210
110
010
150
125
100
75
50
25
0
12080400μm
O-MC: 24; TC: 1.369e+005
110
010
150
125
100
75
50
25
0
12080400μm
CN-MC: 29; TC: 1.502e+005
110
010
150
125
100
75
50
25
0
12080400μm
Cl-MC: 35; TC: 6.968e+004
110
010
150
125
100
75
50
25
0
12080400μm
OH-MC: 25; TC: 6.400e+004
110
010
150
125
100
75
50
25
0
12080400μm
PO2
MC: 7; TC: 3.314e+003
010
150
125
100
75
50
25
0
12080400μm
PO4
MC: 3; TC: 2.368e+003
010
150
125
100
75
50
25
0
12080400μm
PO3
MC: 5; TC: 5.260e+003
010
Sample: DM-NC-XS ̶ Field of View: 151 x 151 µm² ̶ Polarity: Negative ̶ File: DM-XS-NC_n2_peaks.ita
Interface
Dentin
Interface
112
Figure 4. Negative polarity chemical ion maps for control dentin. Absence of phosphate
fragments can be evidently seen.