BASIC RESEARCH – TECHNOLOGY

Anam Hashmi, BDS, MSc,*

Impact of Dentin Substrate Xu Zhang, PhD,† and
Anil Kishen, BDS, MDS, PhD*z
Modification with Chitosan-
Hydroxyapatite Precursor
Nanocomplexes on Sealer
Penetration and Tensile
Strength

ABSTRACT
SIGNIFICANCE
Introduction: The purpose of this study was to evaluate the effect of dentin conditioning with
chitosan-hydroxyapatite precursor (C-HA) nanocomplexes on the depth of tricalcium silicate Dentin conditioning with C-HA
sealer penetration into dentinal tubules and ultimate tensile strength (UTS). Methods: surface nanocomplexes promoted
charge and size distribution for C-HA nanocomplex formulation was evaluated followed by TCS sealer-dentin interaction
bioactivity assessment of standardized films of C-HA nanocomplexes (n 5 15) incubated in through increased surface
simulated body fluid. Mineralization potential was assessed with X-ray diffraction and Fourier- wettability, greater sealer
transform infrared spectroscopy, whereas scanning electron microscopy was used for penetration, and enhanced
ultrastructural evaluation. Static water contact angles and UTS were measured on dentin UTS of endodontic irrigant-
discs (n 5 2/group) and dentin beams (n 5 10/group) treated with/without sodium hypo- treated dentin.
chlorite/EDTA and C-HA nanocomplex conditioning. In phase 2, the depth of sealer pene-
tration after C-HA nanocomplex conditioning was evaluated using fluorescent imaging
(n 5 12/group). The percent area penetration and mean/maximum penetration depth were
calculated at 4- and 6-mm levels from the root apex. Data from contact angle measurements,
mechanical testing, and penetration assessment parameters were subjected to the inde-
pendent samples t test with a significance level set at P , .05. Results: A formulation of C-HA
nanocomplexes (2 mg/mL) was chosen as a polyanionic, hydrophilic, nonaggregating
concentration having bioactivity potential established through the formation of phosphate/
carbonate bonds and the crystalline nature of the formed minerals. A significantly lower
contact angle and higher UTS were registered for the C-HA nanocomplex–conditioned group
(P , .05). Statistically significant (P , .05) greater sealer penetration was recorded at the
4-mm level for all assessment parameters and percent area penetration at 6 mm for the C-HA
nanocomplex group. Conclusions: C-HA nanocomplex conditioning enhances dentin
surface wettability to facilitate greater tricalcium silicate sealer penetration and UTS of
From the *Kishen Lab, Faculty of
dentin. (J Endod 2019;-:1–8.)
Dentistry, University of Toronto, Toronto,
Ontario, Canada; †School and Hospital of
KEY WORDS: Stomatology, Tianjin Medical University,
Tianjin, Shi, China; and zDepartment of
Chitosan; dentin; nanocomplexes; Sealer penetration depth; ultimate tensile strength Dentistry, Mount Sinai Health System,
Mount Sinai Hospital, Toronto, Ontario,
Canada
Dentin plays a crucial role in maintaining the bulk structural integrity of teeth. As a biocomposite, it strikes Address requests for reprints to Dr Anil
the balance between stiffness and toughness through an interplay between inorganic and organic Kishen, Faculty of Dentistry, University of
fractions1. Iatrogenic application of chemicals that demineralize or deproteinate the dentin matrix is the Toronto, Toronto M5G 1G6, Ontario,
most common factor that alters the ultrastructural and mechanical characteristics of dentin1. Denudation Canada.
E-mail address: anil.kishen@utoronto.ca
of collagen through the loss of mineral protection with the use of chelating agents such as EDTA ensues a 0099-2399/$ - see front matter
time-dependent degradation that affects the integrity of the dentin–root filling interface and the
Copyright © 2019 American Association
mechanical properties of dentin2. Sodium hypochlorite (NaOCl) application reduces mechanical strength, of Endodontists.
increases dentin surface roughness, and creates a ghost layer by depletion of collagen3. These features https://doi.org/10.1016/
may contribute to the risk of vertical root fractures in root-filled teeth1,2. j.joen.2019.03.021

JOE  Volume -, Number -, - 2019 Dentin Substrate Modification 1

 An exposed collagen surface is a
challenging substrate for mineral nucleation
because of its weak polarity and surface
charge4. An altered dentin ultrastructure not
only affects the physiochemical properties with
resultant compromised wettability of the dentin
surface3 but also the extent of interaction with
root filling materials, particularly the root canal
sealers5. From a clinical standpoint, this would
dictate the degree of penetration and sealer–
root dentin integrity6. Enhanced sealer
penetration into tubules is considered
desirable because it forms a physical barrier,
allows residual bacterial entombment, and
provides improved interfacial integrity between
the sealer and dentin. The aforementioned
characteristics would enhance the mechanical
integrity of the root dentin7.
Biomineralization is an effective strategy
for restoring the physicochemical
characteristics of dentin4,8,9. Guided tissue
mineralization is based on the polyelectrolytic
nature of polymers that can achieve intrafibrillar
mineralization through an amorphous
precursor mediated pathway10, as reported
previously using chitosan-hydroxyapatite (C-
HA) precursor nanocomplexes11. Chitosan is a
linear biopolymer of glucosamine with a b-(1-4)
linkage that has established biocompatibility,
biodegradability, antibacterial, and metal
chelation attributes12. Chitosan shares
structural approximations with
glycosaminoglycan. In case of C-HA, the role
of chitosan is functionally inspired by the in vivo
role of noncollagenous proteins in dentin
biomineralization11. The incorporation of
chitosan for improved mechanical properties
of collagen has also been used12. The
presence of carboxyl and hydroxyl groups on
chitosan backbone confers a surface
hydrophilicity, polarity, and polyanionic nature,
which is prime to establish calcium
sequestering sites and favorable nucleation
parameters11. Thus, conditioning of root canal
dentin with C-HA nanocomplexes before
obturation could modify dentin characteristics,
resulting in improved sealer-dentin interaction
and mechanical integrity. The purpose of the
study was to evaluate the effect of dentin
conditioning with C-HA nanocomplexes on the
depth of tricalcium silicate sealer (TCS)
penetration and ultimate tensile strength (UTS)
of dentin.
MATERIALS AND METHODS
All the chemicals used in this study were of
analytical grade and were obtained from
Sigma-Aldrich (St Louis, MO). C-HA precursor
nanocomplexes were synthesized according
to a previous protocol11. Simulated body fluid
(SBF) was prepared according to an earlier
2 Hashmi et al.
protocol13. The extracted human teeth used in
the study were collected abiding ethical
approval guidelines of the university (protocol
ID- 35073).
Physicochemical and Bioactivity
Characterization
Aggregation Kinetics
Zeta potential as a measure of surface charge
and the polydispersity index to establish a
nonaggregating concentration was measured
for a series of C-HA nanocomplex formulations
in deionized water using a Zetasizer (Malvern
Instruments Ltd, Malvern, UK) at 25 C. pH
values were also measured for the same with a
pH probe (Orion 8157BNUMD; Thermo
Scientific, Waltham, MA).
Contact Angle Measurements
Longitudinal dentin sections (8 ! 5 ! 0.5 mm)
from premolar roots avoiding the root canal
were prepared using a slow-speed saw
(Isomet Low Speed Saw, Buehler, IL) under
water. Static contact angle measurements
were performed with a water droplet using a
goniometer (Rame-Hart Instrument Co,
Succasunna, NJ) connected to DROPimage
software (Rame-Hart, Succasunna, NJ) at
room temperature. Discs (n 5 2/group) were
treated with NaOCl (6% for 20 minutes) and
EDTA (17% for 5 minutes) in the control group,
and treatment followed by C-HA nanocomplex
conditioning with 2 mg/mL C-HA
nanocomplexes for 30 minutes was performed
for the experimental group. Ten readings were
averaged and subjected to the independent
samples t test at a significance level of P , .05.
UTS Assessment
Dentin beams (8 ! 1.5 ! 0.5 mm) were
prepared from the coronal aspects of
extracted human molar teeth and treated with
NaOCl (6% for 20 minutes) and EDTA (17% for
5 minutes). The beams were randomly
distributed (n 5 10/group) between the control
and C-HA nanocomplex–conditioned group
followed by incubation in SBF at 37 C with 5%
CO2 for a 7-day period. For conditioning,
beams were immersed in 300 mL 2 mg/mL
C-HA nanocomplexes for 30 minutes. During
testing, the dentin beams were rinsed with
deionized water, attached to the jig of a
microtensile tester (Bisco Inc, Schaumburg, IL)
with cyanoacrylate glue, and loaded until
failure. Tensile forces were applied parallel to
the long axis of each specimen at a crosshead
speed of 0.5 mm/min14. The specimens were
kept well hydrated during the test. Statistical
analysis was performed using the
independent samples t test with a significance
level of P , .05.
In Vitro Bioactivity Evaluation
Films (n 5 15) of 2 mg/mL C-HA
nanocomplexes (6.5 ! 2.5cm2) were drop
casted on glass slides and incubated in SBF
for 3 days at 37 C and 5% CO2. The films were
washed with deionized water (5 mL), dried
under laminar flow, ground to powder, and
stored in a desiccator until used.
Morphologic changes were
characterized using scanning electron
microscopy (SEM) (Quanta FEG 250, Thermo
Scientific) under the low vacuum mode
coupled with an energy-dispersive X-ray
spectrometer for chemical analysis of the film
surfaces. For Fourier-transform infrared
spectroscopy, samples were mixed with KBr
powder in 1:4 ratio by volume. Data were
recorded in the transmittance mode over a
4000 to 500 cm21 range at 16 cm21 resolution
and 32 times scan using an infrared
spectrophotometer (One Spectrometer;
PerkinElmer, Waltham, MA). X-ray diffraction
(XRD) analysis was performed to determine the
crystal phase of the minerals using an X-ray
diffractometer (Philips, Amsterdam,
Netherlands) operated with a Cu tube
(wavelength Ka1 51.540562) at 40 kV and 30
mA with a step size of 0.02 from the 20–50 2q
range.
Depth of Sealer Penetration
Sample Preparation
Mesiodistal and buccolingual radiographs
were taken for human extracted premolars for
orthodontic reasons to select teeth with single
straight canals, mature apices, and lacking
resorptive defects, caries, or endodontic
treatment. Teeth (N 5 24) of a similar length
(19 6 1 mm) were prescreened using
transillumination for the presence of cracks
and maintained in 0.9% saline at 4 C until use.
A double varnish layer was applied externally
avoiding the root apex to simulate cementum.
Access into the root canal was gained
using a no. 2 round bur (Dentsply Sirona,
Tulsa, OK). A size #10 K-file (K-Flexofiles,
Dentsply Sirona) was introduced into the root
canal until it was visible at the apical foramen,
and the working length of each root canal was
determined to be 1 mm short. Canals were
instrumented with a full sequence of S1, S2,
F1, F2, and F3 rotary nickel-titanium files
(ProTaper Universal; Dentsply Maillefer,
Ballaigues, Switzerland). A 6% NaOCl solution
was used (2 mL per instrument change), and
17% EDTA (5 mL) with sonic activation was
used as the last irrigant15. After a final rinse with
deionized water (5 mL), teeth were randomly
allocated (n 5 12/group) to the control and C-
HA nanocomplex groups. A 2-mg/mL solution
of C-HA nanocomplexes was introduced into
JOE  Volume -, Number -, - 2019
 A Zeta Potential B pH
Serial Formulations (mg/ml) 9.5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 9
-5
8.5

Zeta Potential (mV)

-10

pH
8
-15
7.5
-20
7
-25
6.5
-30
-35 0.5mg/ml 1.0mg/ml 1.5mg/ml
2.0mg/ml 3.0mg/ml 5.0mg/ml
-40

C Polydispersity Index D Ultimate Tensile Strength

0.6 45
Polydispersity index (PDI)

40 *
0.5

u-Tensile Strength/Mpa
35
0.4
30
0.3 25
0.2 20
15
0.1
10
0 5
0.5mg/ml 1.0mg/ml 1.5mg/ml 0

2.0mg/ml 3.0mg/ml 5.0mg/ml Control Conditioned

FIGURE 1 – Physicochemical characterization of C-HA nanocomplexes. (A) A scatterplot showing the mean zeta potential values. (B) A bar chart with pH values and (C) the poly-
dispersity index for C-HA nanocomplex concentrations. (D) A bar graph showing increased UTS in the nanocomplex–conditioned group. *Statistically significant with P , .05. (Error
bars represent 6 1 standard error.)

the canal using a 3-mL syringe (1.5-inch, 27-G)
needle up to 2–3 mm from the working length.
Manual dynamics agitation (3 strokes/s) was
used with a matching F3 cone (ProTaper
Universal) for 3 minutes. The C-HA
nanocomplexes slurry was allowed to stand in
the canal for 30 minutes with the apex
temporarily secured with wax. Each canal was
dried using a single F1 paper point without
intimate contact with the canal walls.
Root Filling of Specimens
Tricalcium silicate–based sealer (iRoot SP;
Innovative BioCreamix Inc, Vancouver,
Canada) was mixed with a trace amount of
0.25% rhodamine B solution and placed in the
canal using a Lentulo spiral. Single-cone
obturation with F3 cones (ProTaper Universal)
was performed, and the samples were
temporarily restored (Coltosol F; Coltene/
Whaledent AG, Altsta €tten, Switzerland) and
incubated in 100% humidity at 37 C with 5%
CO2 for 10 days to allow complete sealer
setting15.
Evaluation of Depth of Sealer
Penetration
The samples were resin embedded and
sectioned at 4- and 6-mm levels from the root
apex using a water-cooled, diamond wafering
blade (4 ! 0.12 ! 1/2 inches) mounted on a RESULTS
slow-speed saw (IsoMet Low Speed Saw;
Physicochemical and Bioactivity
Isomet, Buehler, IL). The specimens were
Characterization
polished with abrasive discs, ultrasonicated
A high negative surface charge (Fig. 1A) and
(deionized water for 5 minutes), placed on a
alkaline pH (Fig. 1B) for all formulations except
glass slide, and imaged using a fluorescent
0.5 mg/mL were noted. Formulations above 2
microscope (5!) (Leica DM 2500; Leica
mg/mL and below 1.0 mg/mL showed
Microsystems GmbH, Mannheim, Germany) to
(Fig. 1C) polydispersity (.0.3). Optimal
match the excitation/emission (540 nm/590
formulation of 2 mg/mL for the C-HA
nm) wavelengths of rhodamine dye. Each
nanocomplexes was chosen as having a
section was imaged using 9 to 12 frames to
stable surface charge (227 mV), least potential
reconstruct the whole image. For analysis,
for aggregation (polydispersity index 5 0.25),
Image J software (National Institutes of Health,
and high pH stability (pH 5 8.7). Contact angle
Bethesda, MD) was used. The reconstructed
measurements on dentin showed a significant
image was calibrated, and the total area
decrease after C-HA nanocomplex
perimeter of the section and canal
conditioning (5.03 6 0.1) compared with
circumference were measured followed by
treated dentin (46.2 6 2.0) alone. The highly
image thresholding to calculate the percentage
significant difference in UTS was noted
area penetration16. To measure the mean
(Fig. 1D) between conditioned dentin (35 6 9
penetration depth, 4 predesignated points
MPa) and the control group (22 6 9 MPa).
(mesial, distal, buccal, and lingual) at the canal
A scanning electron microscopic image
center were used. For maximum penetration,
showed (Fig. 2A) mineral formation in islands
any point with the farthest penetration depth
on the chitosan matrix along with an energy-
was selected.
dispersive X-ray confirming their presence with
For statistical analysis, the Shapiro-Wilk
evident C, O, Ca, and P peaks. In vitro
test for normality was conducted. Because
bioactivity potential was established with
data were normally distributed, the
respect to the detection of phosphates and
independent samples t test was conducted for
carbonates. Splitting of the 577 cm21
all assessment parameters at 4- and 6-mm
phosphate bond to form a bifid peak at 600
levels with a significance level of P , .05.

JOE  Volume -, Number -, - 2019 Dentin Substrate Modification 3

FIGURE 2 – Bioactivity characterization of C-HA nanocomplexes. (A) SEM–energy-dispersive X-ray image showing mineral formation with organic chitosan matrix and EDX confirming
a rich presence of C, O, Ca, and P. (B) The presence of bifid phosphate peaks and carbonate bonds on the Fourier-transform infrared spectroscopic spectrum. (C) Sharp peaks on the
XRD spectrum suggest a crystalline nature of the formed mineral.

and 560 cm21 (bending mode) is noted in
Figure 2B. A vibration band at 885 cm21 was
derived from the HPO422 group (P-OH
stretching). The presence of a 1467 cm21
band (stretching mode) confirmed the
presence of CO322. Other major phosphate
bonds were recognized at 1070 and 577 cm21
associated with the amorphous calcium
phosphate (ACP) component of the
synthesized nanocomplexes. The XRD
spectrum in Figure 2C confirmed the presence
of crystalline phase of minerals through the
presence of 2q peaks at 26 , 32 and 45
angles.
Depth of Sealer Penetration
Significantly increased sealer penetration for all
measurement parameters at 4 mm in the C-HA
nanocomplex–conditioned group was
recorded by image analysis as shown in
(Fig. 3A). An increased penetration trend was
observed for the conditioned group for all

FIGURE 3 – (A) Calculation of outcome measures of sealer penetration depth into dentinal tubules. (B) Bar charts (b–g) showing the mean, maximum, and % area penetration at 4- and
6-mm levels from the root apex in the C-HA nanocomplex–conditioned group and the control group. *Statistically significant with P , .05. (Error bars represent 6 1 standard error.)

4 Hashmi et al. JOE  Volume -, Number -, - 2019

 B
b Mean Penetration at 4mm c Mean penetration at 6mm
500 500

Mean Penetration /um

Mean Penetration/ um
*
400 p<0.05 400
300 300
200 200
100 100
0 0
Control Conditioned Control Conditioned

d Max Penetration at 4mm e Max penetration at 6mm

800 800
Max Penetration/ um

Max Penetration/ um
p<0.05
600 600

400 400

200 200

0 0
Control Conditioned Control Conditioned

f % Area penetration at 4mm g % Area penetration at 6mm

60% 60%
% Area Penetration/ um2

% Area Penetration/ um2

*
50% p<0.05 50% p<0.05

40% 40%
30% 30%
20% 20%
10% 10%
0% 0%
Control Conditioned Control Conditioned

FIGURE 3 – Continued

measurements at the 6-mm level as well.
However, a statistically significant difference
was noted for the percentage area penetration
in comparison with the control group (Fig. 3B).
DISCUSSION
The characterization experiments conducted
in this study showed that the synthesized C-
HA nanocomplexes possessed a highly stable,
polyanionic surface charge in an alkaline
medium. These characteristics are imperative
to establish a higher ratio of ionized carboxyl
groups (COO2) in solution to prevent
aggregation of dispersed nanocomplexes that
forms the basis of their role in guided dentin
mineralization17. The bioactivity potential was
established using a standard protocol13. This
formation of a bifid phosphate peak18 as
observed in the Fourier-transform infrared
spectroscopic analysis has been associated
with the gradual transition from a poorly
crystallized hydroxyapatite into a well
crystallized hydroxyapatite19. The XRD
reflections were similar to those reported
earlier20 with the most prominent peak at
32.2q. This may be regarded as a composite
peak ascribed to collective contributions from
HA diffraction planes (211, 112, and 300) that
would become resolved as the crystallinity

JOE  Volume -, Number -, - 2019 Dentin Substrate Modification 5

matures over time21. This observation can be
explained in the way carboxymethyl chitosan
stabilizes the precursor phase (ie, through
adsorption onto the nascent crystal nuclei and
binding of calcium ions by ion-pair formation
causing a prolonged ACP stabilization
phase)22.
Natural biocomposite such as dentin at
an elementary structure level exhibits a generic
microstructure, which consists of staggered
mineral plates in an organic matrix. It is
postulated that under tensile loads, the mineral
platelets will carry the tensile load, whereas the
organic matrix will transfer the load between
mineral crystals. Thus, the tensile strength of
the protein phase in a natural biocomposite
material is amplified by the large aspect ratio of
mineral plates. Therefore, UTS was used to
evaluate the effect of C-HA nanocomplexes on
dentin23. In addition, microtensile testing
facilitates standardization of the testing sample
and limits dentin anisotropy–induced
confounding24. In the current study, values of
treated dentin were in the range of those
previously noted using similar methodology25.
Exposure to NaOCl/EDTA may have resulted in
collagen demineralization/degradation
because of the small size of its oxidizing
species3,26. In the control group, subsequent
immersion in SBF would have resulted in
dentin surface precipitation that was not
supported by structurally intact collagen,
resulting in lower UTS values27; in the
conditioned group, a simultaneous organic-
inorganic reinforcement of dentin matrix
resulted in higher UTS. The C-HA
nanocomplexes may have facilitated both
extra- and intrafibrillar collagen mineralization
as established in an earlier study through the
formation of collagen crossbanding with
transmission electron microscopy11. Previous
studies have shown that tensile forces applied
perpendicularly to the tubular direction
required disruption of collagen fibers24. This
may explain the requirement of higher loads to
fracture in C-HA nanocomplex–conditioned
beams that were reinforced with chitosan. The
improved tensile strength was attributed to the
inherent toughness of chitosan because of
REFERENCES
1. Kishen A. Mechanisms and risk factors for fracture predilection in endodontically treated teeth.
Endod Topics 2006;13:57–83.
2. Ferrari M, Mason PN, Goracci C, et al. Collagen degradation in endodontically treated teeth after
clinical function. J Dent Res 2004;83:414–9.
3. Gu LS, Huang XQ, Griffin B, et al. Primum non nocere - the effects of sodium hypochlorite on
dentin as used in endodontics. Acta Biomater 2017;61:144–56.
6 Hashmi et al.
interfibrillar hydrogen bonding, its hydrophilic
nature facilitating collagen adsorption, and
dentin remineralization12,17,28.
The depth of sealer penetration was
evaluated as an assessment measure of
physicochemical dentin surface modification.
The experimental design was adapted from
earlier established protocols15,29. The choice
of fluorescent imaging technique compared
with SEM is regarded to be superior. The use
of SEM poses difficulties in obtaining an overall
low-resolution image and subsequent analysis
along with concern for artifacts during sample
processing7. Possible factors affecting
penetration were considered; therefore, sonic
activation was used to ensure adequate smear
removal. Sealer placement and obturation
technique were optimized between the
groups. To avoid the presence of sclerotic
dentin/anatomic complexities in the apical
third, the first root section was chosen at the 4-
mm level from the apex30. Slurry of C-HA
nanocomplexes was left in the canal over 30
minutes to standardize for the canal drying
process among samples. Clinically, this
extended drying time may be avoided by using
an alternative method of canal drying. The
percentage of area penetration and other
parameters were measured to account for the
heterogeneity in the structure/ dimensions of
the tested samples. The results are in
accordance with earlier studies that show
increasing sealer penetration toward coronal
sections29.
An earlier study concluded poor TCS
penetration depth in a chitosan nanoparticle–
irrigated group31. However, in the current
study, all penetration parameters assessed
were noted to be higher in C-HA
nanocomplex–conditioned root canals. The
mean and maximum sealer penetration at the
6-mm level was not statistically significant in
the conditioned group unlike at 4 mm. This
might be because of the closer adaptation of
the gutta-percha cone at the 4-mm level that
resulted in generation of higher shear stress on
canal walls, allowing a denser and deeper
nanocomplex coating with the use of manual
dynamics. In contrast to another study, near
zero contact angle measurements were
recorded for C-HA nanocomplex–conditioned
dentin32. Both the earlier studies used a
nonengineered chitosan dispersed in acidic
solution, which has a hydrophobic character
because of the high molecular weight and the
presence of hydrophobic acetyl groups that
tend to aggregate in solution28. Conversely, in
this study, the 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 the hydrophobic
collagen matrix33, 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 C-HA precursor
nanocomplexes formed along the root canal
walls presented carboxyl groups from chitosan
and interstitial water from ACP. This
contributed to the increased dentin wettability
by the TCS19 although 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, this study highlighted the
dentin substrate modification potential of C-HA
nanocomplex conditioning of previously
irrigated root canal dentin by restoring the
surface wettability with synchronous collagen
reinforcement. These findings may find
potential application in establishing improved
TCS sealer-dentin interaction, resulting in
increased TCS sealer penetration and UTS of
dentin. These features would contribute
toward enhancing interfacial integrity in root-
filled teeth.
ACKNOWLEDGMENTS
Supported by the Ontario Centres of
Excellence (grant number 29388) and the
University of Toronto (grant number 206760).
The authors deny any conflicts of
interest related to this study.
JOE  Volume -, Number -, - 2019
 4. 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.

5. Dogan Buzoglu H, Calt S, Gu €mu€sderelioglu 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.

6. Topçuog € Demirbuga S, et al. The effect of different final irrigant activation

lu HS, Tuncay O,
techniques on the bond strength of an epoxy resin-based endodontic sealer: a preliminary study.
J Endod 2014;40:862–6.

7. Mamootil K, Messer HH. Penetration of dentinal tubules by endodontic sealer cements in

extracted teeth and in vivo. Int Endod J 2007;40:873–81.
8. Shao C, Zhao R, Jiang S, et al. Citrate improves collagen mineralization via interface wetting: a
physicochemical understanding of biomineralization control. Adv Mater 2018;30:1–7.

9. Gandhi B, Bollineni S, Janga RK, et al. 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:ZC53–6.

10. Tay FR, Pashley DH. Guided tissue remineralisation of partially demineralised human dentine.
Biomaterials 2008;29:1127–37.

11. Chen Z, Cao S, Wang H, 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.

12. Shrestha A, Friedman S, Kishen A. Photodynamically crosslinked and chitosan-incorporated

dentin collagen. J Dent Res 2011;90:1346–51.
13. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials
2006;27:2907–15.

14. Park JH, Choi YS. Microtensile bond strength and micromorphologic analysis of surface-treated
resin nanoceramics. J Adv Prosthodont 2016;8:275–84.

15. Al-Haddad A, Abu Kasim NH, Che Ab Aziz ZA. Interfacial adaptation and thickness of bioceramic-
based root canal sealers. Dent Mater J 2015;34:516–21.
16. Menezes M, Prado M, Gomes B, et al. 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.
17. Kalliola S, Repo E, Srivastava V, et al. The pH sensitive properties of carboxymethyl chitosan
nanoparticles cross-linked with calcium ions. Colloids Surf B Biointerfaces 2017;153:229–36.

18. Drouet C. Apatite formation: why it may not work as planned, and how to conclusively identify
apatite compounds. Biomed Res Int 2013;2013:12.

19. Sun J, Chen C, Pan H, et al. Biomimetic promotion of dentin remineralization using L-glutamic
acid: inspiration from biomineralization proteins. J Mater Chem B 2014;2:4544–53.
20. Wang Y, Van Manh N, Wang H, et al. Synergistic intrafibrillar/extrafibrillar mineralization of collagen
scaffolds based on a biomimetic strategy to promote the regeneration of bone defects. Int J
Nanomed 2016;11:2053–67.
21. Li X, Lan J, Ai M, et al. Biomineralization on polymer-coated multi-walled carbon nanotubes with
different surface functional groups. Colloids Surf B Biointerfaces 2014;123:753–61.

22. Andrew CA, Khor E, Hastings GW. The influence of anionic chitin derivatives on calcium
phosphate crystallization. Biomaterials 1998;19:1309–16.

23. Gao H, Ji B, Jager IL, et al. Materials become insensitive to flaws at nanoscale: lessons from
nature. Proc Natl Acad Sci U S A 2003;100:5597–600.
24. Inoue S, Pereira PN, Kawamoto C, 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, et al. 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, et al. Tensile strength and microhardness of treated human
dentin. Dent Mater 2004;20:522–9.
27. Smith LJ, Deymier AC, Boyle JJ, et al. Tunability of collagen matrix mechanical properties via
multiple modes of mineralization. Interface Focus 2016;6:20150070.

JOE  Volume -, Number -, - 2019 Dentin Substrate Modification 7

28. Yu Z, Lau D. Flexibility of backbone fibrils in a-chitin crystals with different degree of acetylation.
Carbohydr Polym 2017;174:941–7.

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, et al. Age-related transparent root dentin: mineral concentration,
crystallite size, and mechanical properties. Biomaterials 2005;26:3363–76.
31. € u
Aydın ZU, Ozy €rek 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 2019;107:64–71.

32. Ururahy MS, Curylofo-Zotti FA, Galo R, et al. Wettability and surface morphology of eroded
dentin treated with chitosan. Arch Oral Biol 2017;75:68–73.

33. Ballal NV, Tweeny A, Khechen K, et al. Wettability of root canal sealers on intraradicular dentine
treated with different irrigating solutions. J Dent 2013;41:556–60.

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