BASIC RESEARCH – TECHNOLOGY

Anam Hashmi, BDS, MSc,*

Rana N.S. Sodhi, PhD,† andAnil Kishen, BDS, MDS, PhD*‡

Interfacial Characterization ofDentin Conditioned withChitosan HydroxyapatitePrecursor NanocomplexesUsing Time-of-flight SecondaryIon Mass Spectrometry

SIGNIFICANCE

Dentin substrate conditioningwith C-HA nanocomplexesmay find potential applicationin enhancing the chemicalinteraction of sealers withdentin for the reinforcement ofinterfacial integrity.

From the *Kishen Lab, Faculty of Dentistryand †Ontario Centre for theCharacterization of Advanced Materials,

ABSTRACT

Introduction: The purpose of this study was to evaluate the effect of chitosan-hydroxyapatite precursor (C-HA) nanocomplex conditioning on the chemical modifications atthe tricalcium silicate sealer-dentin interface using time-of-flight secondary ion mass spec-trometry. Methods: Dentin slabs from human premolar root dentin were prepared,demineralized, and randomly distributed between control and C-HA nanocomplexconditioned groups. Tricalcium silicate sealer was applied, and the slabs were allowed to setin 100% humidity for 10 days. The cross-sectional area was exposed, and the sealer-dentininterface was characterized for chemical/ultrastructural evaluation with time-of-flight sec-ondary ion mass spectrometry and transmission electron microscopy, respectively.Results: Chemical analysis revealed the presence of an ion-rich layer constituted of abun-dant phosphates (PO2

2, PO32, and PO4

2), hydroxide (OH2), and chitosan fragments(C2H4NO

2, C3H4NO22, C2H5O2

1, C2H6NO1, C4H6NO2

1, C5H6NO1, and C5H5O2

1) on thedentin surface at the sealer-dentin interface and subsurface dentin after conditioning withC-HA nanocomplexes. In contrast, a decreased interfacial presence of calcium (Ca1) andcalcium phosphates (CaPO2

1, CaPO31, CaPO4

1, and Ca2PO31) and the absence of

phosphate fragments in the control were noted. Ultrastructural evaluation showed an inter-facial layer (,1 mm) with interrupted mineral aggregates in the controls as opposed to acontinuous (5 mm) mineral layer formation on the conditioned dentin. Conclusions: C-HAnanocomplex conditioning of dentin before tricalcium silicate sealer application resulted in achemically modified dentin substrate with an ion-rich layer consisting of phosphate, calcium,calcium phosphates, and chitosan that chemically modified the dentin surface/subsurface. (J Endod 2019;45:1513–1521.)

KEY WORDS

Chitosan; dentin conditioning; interface; nanocomplexes; sealer-dentin interface

Department of Chemical Engineering andApplied Chemistry, University of Toronto,Ontario, Canada; and ‡Department ofDentistry, Mount Sinai Health System,Mount Sinai Hospital, Toronto, Ontario,Canada

Address requests for reprints to Dr AnilKishen, Faculty of Dentistry, University ofToronto, Toronto M5G 1G6, Canada.E-mail address: anil.kishen@utoronto.ca0099-2399/$ - see front matter

Copyright © 2019 American Associationof Endodontists.https://doi.org/10.1016/j.joen.2019.08.011

Dentin is a biocomposite composed of carbonated hydroxyapatite mineral crystallites, type I collagenfibrils, and noncollagenous macromolecules spanning over several length scales1. However, iatrogenicapplication of chemicals and/or medications during root canal therapy induces ultrastructural andcompositional alterations in dentin that affect its physicochemical and mechanical characteristics2,3. Theuse of EDTA and sodium hypochlorite on dentin as commonly used root canal irrigants results in surface/subsurface demineralization and irreversible, nonspecific dissolution of the organic constituents4. Themineral denuded collagen shows poor surface polarity and poses challenging substrate forremineralization5. The endodontic chemical may also compromise the surface wettability, which furtherlimits the extent of interaction between the root canal sealer and dentin at the interface3,6. Furthermore,dentin possesses an anastomosing network of secondary dentinal tubules that encourages fluidmovement and contaminant ingress even when frank interfacial gaps are lacking7. All of these factors

JOE � Volume 45, Number 12, December 2019 Interfacial Characterization of Nanoparticles Conditioned Dentin 1513

facilitate progressive collagen degradation andincreased fracture predilection in root-filledteeth with time8.

Tricalcium silicate–based materials(TCSs) are hydraulic in nature and interact withdentin through carbonated apatite formationon contact with physiological fluids9,10.Therefore, the physicochemical profile ofdentin plays a crucial role in directing themechanics of this interaction and subsequentmineral formation5,11. Differences in the natureof precipitate formation in the bulk of TCSs andthat formed at the sealer-dentin interface havebeen reported, chiefly because of theinadequacy and lack of accessibility ofphosphorous ions12. The availability of asustained mineral source and subsequentnucleation on a hydrophobic, mineral denudedcollagen surface are current challenges indentin remineralization5. However, to the bestof our knowledge, no studies havecharacterized the TCS sealer–dentin interfaceor developed strategies to improve the sealer-dentin interfacial integrity via concepts ofdentin remineralization. In this line, polymer-based guided tissue mineralization usingchitosan-hydroxyapatite precursor (C-HA)nanocomplexes may serve as an effectivestrategy.

Chitosan is an abundant biopolymerconsisting of b-(1-4) glucosamine units13.Chitosan and its derivatives have been foundto offer different advantages because of theirbiocompatible, bioactive, and antibacterialnature13. C-HA nanocomplexes are anorganic-inorganic composite that arefunctionally inspired by the role ofnoncollagenous proteins. Their bioactivity isfacilitated through a dense polyanionic surfacecharge (227 mV)14 on the water-solublechitosan backbone that allows sequestrationand stabilization of hydroxyapatite precursorphases (amorphous calcium phosphate) withits carboxyl groups14. This facilitatessynergistic intra- and extrafibrillar collagenmineralization to take place15,16, similar to howit occurs in biomineralization. Dentinconditioning with C-HA nanocomplexes hasbeen reported to promote TCS sealer–dentininteraction through increased surfacewettability, greater sealer penetration into thedentinal tubules, and enhanced ultimate tensilestrength of endodontic irrigant–treateddentin14.

Time-of-flight secondary ion massspectrometry (TOF-SIMS) is a well-establishedtechnique in material sciences that allowsanalysis and identification of both organic andinorganic fractions such as hydroxyapatite withmillimass precision and monolayersensitivity17. It provides high-resolution massspectra along with their spatial ion maps18–20.

1514 Hashmi et al.

Recently, TOF-SIMS analysis has been used tostudy precipitate formation on the dentinsurface and in dentinal tubules subsequent tothe application of different irrigants21. Althoughthere is a lack of knowledge on how dentinsurface chemistry affects the interfacialcharacteristics of TCS sealers, TOF-SIMS,because of its inherent advantages, would bean ideal method to characterize the sealer-dentin interface. The aim of the current studywas to chemically characterize the TCS sealer–dentin interface after prior dentin conditioningwith C-HA nanocomplexes.

MATERIALS AND METHODS

Five extracted human premolar teeth werecollected under the university ethical guidelines(protocol identification number: 35073) andstored in 0.9% saline until use. Slabs of rootdentin were prepared by sectioning the rootalong the long axis of the tooth using adiamond wafering blade (4 ! 0.12 ! 1/2inches [Precision Smart Cut; UKAM IndustrialSuperhard Tools, Valencia, CA]) mounted on aslow-speed saw (Isomet Low Speed Saw;Buehler, Lake Bluff, IL) under running water.Each slab (n 5 6) was then shaped andgrinded with carbide paper discs to finaldimensions of 6 ! 4 ! 0.2 mm. Slabs weredemineralized (17% EDTA for 7 days),ultrasonicated (10 minutes with deionizedwater), and randomly distributed between thecontrol and C-HA nanocomplex conditionedgroup.

Carboxymethyl chitosan wassynthesized according to an earlier protocol22.Amorphous calcium phosphate was thengrafted by the addition of K2HPO4 andCaCl2

12. The resultant gel was freeze-driedand processed to a powder16. Fresh slurry ofC-HA nanocomplexes was prepared bydissolving 2 mg C-HA nanocomplex powder indeionized water. Demineralized dentin slabswere conditioned in 1 mL of a 2-mg/mLsolution of C-HA nanocomplexes for 30minutes followed by sealer application in thetest group. Demineralized slabs in the controlgroup remained unexposed to conditioningtreatment before sealer application. In eachgroup, sealer (iRoot SP; Innovative BioCreamixInc, Vancouver, Canada) was allowed tospread into a uniform thickness layer inbetween 2 dentin slabs that were sandwichedunder weight and incubated in 100% humidityat 37�C with 5% CO2 for a 10-day period. Thesamples were stabilized on a customized,polystyrene jig; secured in a mount; andsectioned to expose the sealer-dentin interfaceusing a microtome (Leica EM UC6/FC6 Ultra-cryomicrotome; Leica Microsystems GmbH,Wetzlar, Germany). One sample from each

group was processed for ultrastructuralanalysis with transmission electronmicroscopy.

TOF-SIMS AnalysisDentin surface analysis was performed byTOF-SIMS (TOF-SIMS V; IONTOF GmbH,M€unster, Germany). A Bi3

11 cluster primaryion source was used with a bismuth liquidmetal ion gun operated in a high massresolution bunched mode over an area of 500! 500 mm for 100 seconds. Additionally, ahigh spatial resolution imaging mode (“burstalignment”) was used to obtain spectralimages (256! 256 pixels) from 20 scans overan area of 150 ! 150 mm. A pulsed electronflood gun was used for charge neutralization.The calibration of the mass scale wasperformed using standard, identifiable, andwell-spaced peaks found in all the spectra.Both positive and negative polarity massspectra and spatial chemical maps formolecular fragments of interest weregenerated through regions of interest (ROIs) atthe interface (5 ! 25um) and total dentin (150! 70 mm). Individually run C-HA nanocomplexpowder, set TCS sealer, and demineralizeddentin were used as reference samples inorder to select characteristic fragments.Spectral comparisons were performed afternormalization of the intensity, proportionally tothe total intensity of each spectrum23. Thus,data from both normalized spectral intensitiesand spectral chemical maps are presentedlater.

Transmission Electron MicroscopicEvaluationSpecimens were fixed with Karnovsky fixative(2.5 wt% glutaraldehyde buffered to pH5 7.3)for 3 days at 4�C and postfixed in 1% osmiumtetroxide for 1 hour. The specimens weredehydrated in an ascending ethanol series(30%–100%), immersed in propylene oxide asa transition medium, and ultimately embeddedin pure epoxy resin. Ninety-nanometer-thicksections were prepared. Sections containingthe material-dentin interface were stained with2% aqueous uranyl acetate and Reynolds’lead citrate. The sections were examined alongthe cross section using a JEM-1230transmission electron microscope (JEOL,Tokyo, Japan) at 110 kV.

RESULTS

TOF-SIMS AnalysisTotal ion and CN2 maps are shown inFigure 1A–D. A distinguishable interfacial zone(5 mm) because of differences in pixel intensityis seen lacking in the control (Fig. 1A) incomparison with the conditioned dentin

JOE � Volume 45, Number 12, December 2019

FIGURE 1 – Total ion maps for the (A ) control and (B ) C-HA nanocomplex conditioned dentin. The interfacial layer with differences in pixel intensity can be seen (dashed linemarks theinterfacial boundary). (C ) The CN2 chemical ion map of the control sample showing no signal zones (yellow arrows ) suggestive of degraded collagen. (D ) Intense CN2 signals ofintegrated C-HA nanocomplexes at the interface/subsurface in the conditioned dentin. (E and F ) Red-green overlays of the organic fragment (C2H4NO [red]) with phosphates (PO22,PO3

2 , and PO42 [green]). (E ) A minimal presence of phosphate ions at the sealer-dentin interface (dashed line ) in the control dentin. (F ) A well-defined interfacial band of phosphates

(PO22, PO3

2) in C-HA nanocomplex conditioned dentin. (G ) A red-green overlay of chitosan fragment (C5H6NO1 [red]) with calcium (Ca1 [green]) showing the presence of calciumislands in the conditioned dentin in areas lacking chitosan matrix (yellow arrows ).

(Fig. 1B). Ion maps for CN2 (Fig. 1C) show lowintensity with no signal areas in the control,whereas increased signals are observed inconditioned dentin (Fig. 1D). Figure 1E showsred-green overlays of chemical ion maps. Thepresence of PO3

2 and PO42 fragments was

nonexistent at the interfacial zone in the controlsample, whereas a greater intensity of PO2

2

was found. In contrast, the presence ofphosphate was noted in well-defined interfacialbands of PO2

2 and PO32 in the conditioned

dentin (Fig. 1F). The PO42 map, although less

defined, depicted a dense phosphatepresence. The distribution of Ca1 in islandscan be noticed from Figure 1G in theconditioned dentin.

The results from normalized spectralintensities are shown in Figure 2. Higherintensities for nonspecific (negative polarity)organic fragments of O2, CN2, and CNO2 atboth ROIs (Fig. 2A and B) were observed forthe conditioned dentin, with the exception ofcysteine residue (SH2), in comparison with thecontrol. OH2 intensity was higher in totaldentin of the conditioned group only. Theconditioned dentin (Fig. 2C–E) showed anincreased intensity for specific organicfragments of chitosan with negative polarity(C2H4NO

2 and C3H4NO22 at both ROIs) and

JOE � Volume 45, Number 12, December 2019

positive polarity (C2H5O21, C2H6NO

1,C4H6NO2

1, C5H6NO1, C5H5O2

1 andC5H6NO2

1 fragments at interface) comparedwith the control. C2H4NO

2 was used as acharacteristic marker fragment of thecarboxymethyl group to tag chitosanpresence. CHO2

2 was noted to be higher atthe interface of the conditioned dentin. Othernonspecific organic fragments (NH4

1, CH4N1,

and C4H8N1) were noted to be higher (Fig. 2F)

at the interface in the conditioned dentin.Inorganic fragments of phosphates

such as PO22, PO3

2, and PO42 (Fig. 2G and

H) registered higher intensity at the interfaceand total dentin ROIs in the conditioned group,whereas PO3

2 and PO42 fragments were

absent in the control. An increased intensity forCaO1 and CaOH1 (Fig. 2I) was noted in thecontrol sample with Ca1 and all other highermolecular mass fragments (CaPO2

1, CaPO31,

CaPO41 and Ca2PO3

1) to be greater inintensity in the conditioned dentin (Fig. 2J).

Transmission Electron MicroscopicEvaluationTransmission electron microscopicmicrographs are shown in Figure 3A and B.Isolated mineral aggregates as a thin (1 mm)

Interfacial Characteriz

discontinuous layer with denuded collagenmatrix are noted in the control (Fig. 3A). Frayingof collagen fibril ends as a sign of degradationcan also be noted, whereas a consistentinterfacial layer with appreciably increasedthickness (5 mm) (Fig. 3B) and abundant incoarse mineral aggregates encapsulated in agel-like matrix was observed in the conditioneddentin. The nanometric-sized aggregates werenoted deposited in close adaptation to theunderlying collagen matrix.

DISCUSSION

In this study, chemical modification at the TCSsealer–dentin interface after conditioning with C-HA nanocomplexes was characterized withmonolayer sensitivity. Previously, interfacialcharacterization for mineral trioxide aggregate(MTA)-based cements has been attemptedusing scanning electron microscopy coupledwith energy-dispersive spectroscopy andelectron probe microanalysis, which have limitedresolution requiring peak deconvolution such asthat reported for phosphorus masking byzirconium peaks24,25. Further samplepreparation steps, the inability to detect traceelements, and organic materials are otherreported disadvantages. Techniques like

ation of Nanoparticles Conditioned Dentin 1515

FIGURE 1 – (Continued)

confocal laser scanning microscopy are unableto provide chemical information that warrants theuse of additional analysis26. The use offluorescent dyes has also been reported;nevertheless, rhodamine is preferentially imbibedby MTA-based materials, causing its movementacross the sealer-dentin interface difficult27.

1516 Hashmi et al.

In addition, the small-sized fluorescein particlesare easily taken up by dentin, which furthermakes the investigations on interfacial gradientdifficult27. Hence, an advanced high-resolutionsurface characterization technique was animportant prerequisite for interfacialcharacterization in endodontics.

In TOF-SIMS, a primary ion beamdirected onto the sample causes surfaceionization (1–2 monolayers)28. The time of flightbased on molecular mass is then used forfingerprint identification of these secondaryions29. Specific advantages for using TOF-SIMS in the current study included its

JOE � Volume 45, Number 12, December 2019

0

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A B

Interface Den�n

CO2H- C2H4NO- C3H4NO2-

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nitneDecafretnI

C2H5O2+ C2H6NO+ C4H6NO2+ C5H6NO+ C5H5O2 + C5H6NO2 +

Interface

OH- O- CN- CNO- SH-

Interface

FIGURE 2 – Normalized mass spectral intensities for negative and positive polarity at the interfacial and total dentin ROIs. (A ) Negative polarity nonspecific protein fragments at theinterface and (B ) total dentin. (C ) Negative polarity characteristic chitosan fragments at the interface and (D ) total dentin. (E ) Positive polarity characteristic chitosan fragments and (F )nonspecific organic fragments at the interface. (G ) Phosphate fragments at the interface and (H ) total dentin. (I ) The presence of tricalcium silicate hydration products (Ca1, CaO1,and CaOH1) at the interface. (J ) Higher molecular mass fragments (CaPO21, CaPO31, CaPO41, and Ca2PO31) of calcium phosphates at the interfacial dentin.

sensitivity to identify spatially resolved chemicalcharacteristics of elemental and molecularfragments of the thin modified layer on theconditioned dentin. This is significant becausechemical associations provide a far greaterunderstanding of the nature ofmineralization30. Retrospective analysisgranted complete freedom to choose specific

JOE � Volume 45, Number 12, December 2019

areas with precision for analysis. Operatingparameters are crucial because they dictatethe yield of secondary ions; therefore, thecontrol and conditioned dentin samples wererun under the same experimentalparameters31.

One of the major limitations of usingTOF-SIMS analysis in biological samples is the

Interfacial Characteriz

difficultly in generating quantitative informationfor statistical analysis. This is attributed to thehigh resolution and high sensitivity of thespectra/maps generated by the system andvariations associated with different biologicalsamples. Thus, TOF-SIMS is primarily used forqualitative assessments17–21. In the currentstudy, qualitative analysis was performed to

ation of Nanoparticles Conditioned Dentin 1517

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Interface

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FIGURE 2 – (Continued)

examine the interfacial chemical changes andassociated trends. The ion maps generatedfrom the TOF-SIMS have 256 ! 256 pixelsfrom an average of 20 scans over an area of150 ! 150 mm, which contains more than65,000 spectra. It is challenging to processthese large data sets quantitatively while thesmallest spectral changes are considered.

FIGURE 3 – Transmission electron microscopic micrographsthin, discontinuous interfacial layer with collagen fraying. (B )

1518 Hashmi et al.

Thus, keeping in mind the limitationsassociated with TOF-SIMS analysis based onsmall ROIs from a single window of therastered area along with the limited ion yield ofhigher mass fragments from nonstandardizedbiological interfaces, normalized intensitieswere created for comparison, and statisticalanalysis was avoided.

showing the sealer-dentin (D) interface (IF) with the presenceThe conditioned dentin shows a closely adapted interfacial la

The presence of Ca, P, and Si ion-richdeposits along the calcium silicate cement–dentin interface and dentin subsurface tovarying depths was reported by previousstudies24,25. The variation in interfacial layerthickness (4.8–14.5 um) from scanningelectron microscopic measurements has beenattributed to the decreased length of

of an interfacial layer (IL). (A ) The control dentin shows ayer with markedly increased thickness.

JOE � Volume 45, Number 12, December 2019

interaction between the phosphate from thesupersaturated solution and the calciumleached from the calcium silicate cements12.Along similar lines, a 5-mm-thick layer wasidentified on the conditioned dentin from thetotal ion chemical maps and transmissionelectron microscopic micrographs. However, itwas difficult to measure a consistent layer inthe controls.

The presence of phosphate as a well-delineated band in the conditioned dentingroup was an important finding in this study.TCS was tested as a calcium source based onthe earlier biomineralization studies10,32. Thepresence of a calcium phosphate monobasicphase in the sealer could have been a potentialsource for phosphate fragments. However, inthe control sample, this contribution was verynominal and virtually nonexistent at theinterface for PO3

2 and PO42 fragments. The

same finding was reinforced with a lowerregistration of calcium phosphate intensities.Similarly, as the TCS hydration continued,more calcium hydroxide (Ca[OH]2) wasformed, but because of the limited phosphateavailability in the control sample, it remainedunused and recorded a higher intensity9,10.This is in contrast to earlier studies in which alarge quantity of phosphate-based fluid isprovided25. Such an approach clearlyestablished the source of phosphate to be theC-HA nanocomplexes without anyconfounding factors.

Demineralized dentin allowed mapping ofcalcium gradients from the interface into thedentin. Registration of Ca1 in the conditioneddentin as dense islands may be explained by thelack of chitosan fragment in those areas as isevident on the red-green overlay, whereas in therest of the image, the Ca1 signals were maskedby the near homogenous presence of chitosanof w5 mm, which is way larger than the 1 to 2monolayer sampling depth of TOF-SIMS.Among others, high O2 and OH2 signals werenoted in the conditioned sample, whichrepresented substrate signals and weresuggestive of calcium phosphate precipitation30.

An overlap of signals occurred from thedentin matrix and the chitosan layer. Therefore,the chemical nature of the conditioned layerwas established through the presence of

JOE � Volume 45, Number 12, December 2019

characteristic positive and negative chitosanfragments along with nonspecificfragments23,30. The presence of SH2 as acysteine residue was identified as an intensesignal in the control group in comparison withthe conditioned group wherein its presencewas almost negligible. Because cysteineresidue is not present in the chitosanstructure30, this further supported theadsorption/integration of chitosan as anadditional, modified layer at the sealer-dentininterface. Also, the minor SH2 signals in thenanocomplex group may also point at theclose adaptation of the modified layer to theunderlying dentin.

As reported in an earlier study, theformation of Ca(OH)2 by calcium silicatecement results in caustic etching anddegradation of collagen matrix26. Such afinding may also be supported by the lack ofCN2 signals seen (black zone) in the fewmicrons nearest to the interface in the controlsample, whereas strong CN2 signals can beclearly seen emanating from the integration ofC-HA nanocomplexes in the conditioneddentin. This is supplemented by transmissionelectron microscopic images showing collagenfraying, whereas a very homogenous surface isobserved in the conditioned dentin,highlighting a potential protective effect ondentin collagen.

TCS-based cements have beenreported to interact with dentin through theformation of an ion-rich layer and sealertags24. The use of C-HA nanocomplexes inthis study provided a basis for restoring thechemical characteristics of demineralizeddentin. This may be explained by the factthat as the TCS continued to set, Ca(OH)2was produced and underwent hydrolysis toprovide a continued supply of calcium andOH2 ions32. The hydrophilic nature ofcarboxymethyl chitosan would haveattracted more water molecules close tothe C-HA nanocomplexes at the sealer-dentin interface forming amicroenvironment, allowing amorphouscalcium phosphate dissolution andprecipitation33 that aided in providing aphosphate source. The accumulation ofcalcium, hydroxyl, and phosphate ions

Interfacial Characteriz

inside the chitosan microenvironmentformed the ion-rich layer, which wouldfacilitate subsequent phase transformationtoward the thermodynamically stable phaseassisted by medium alkalinity10. Thepresence of C-HA nanocomplexes couldhave also provided added heterogeneousnucleation sites to sequester Ca1 ions withits carboxyl groups as well and hastenedthe reaction kinetics.

All biomimetic mineralization schemesare composed of fundamental componentsrequiring organic-inorganic interaction toinitiate nucleation and an uninterrupted mineralreplenishment source, which was provided byC-HA nanocomplex conditioning of the dentin.By mimicking the role of noncollagenousproteins, the adsorption of C-HAnanocomplexes on dentin collagen couldproduce a negatively charged surface andtherefore reduced interfacial energy betweenthe aqueous microenvironment and dentin,allowing mineral deposition5. Becausenucleation and growth of minerals isproportional to the concentration of theavailable ions34, enhanced bioactivity of TCSwas observed with C-HA nanocomplexconditioning, which resulted in a modifiedinteraction between the TCS and dentin asestablished in the current study.

The findings from this study highlightedthe possible contributions of C-HAnanocomplex conditioning in chemicallymodifying the dentin surface and subsurfaceby forming an ion-rich layer. This may facilitateenhanced interfacial integrity of sealer-dentininterfaces and improved dentin mechanicalintegrity in root-filled teeth.

ACKNOWLEDGMENTS

Supported by the Ontario Centres ofExcellence (grant number 29388), CanadianFoundation of Innovation-Leading Edge Fund(grant number 30765), and the University ofToronto. The authors would also like to thankInnovative BioCeramix Inc, Vancouver,Canada for graciously supplying iRoot SPsealer for this study.

The authors deny any conflicts ofinterest related to this study.

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