Regenerative Endodontics

Dentin Conditioning with Bioactive MoleculeReleasing Nanoparticle System EnhancesAdherence, Viability, and Differentiationof Stem Cells from Apical Papilla

Suja Shrestha, MSc, PhD, Calvin D. Torneck, DDS, MS, and Anil Kishen, BDS, MDS, PhD

Abstract

Introduction: Temporal-controlled bioactive molecule(BM) releasing systems allow the delivery of appropriateconcentration of BM to enhance the interaction of stemcells to dentin matrix and subsequent odontogenic dif-ferentiation in regenerative endodontics. Objectives:The goal of this study was to evaluate the effect ofdentin conditioning with 2 variants of dexamethasone(Dex) releasing chitosan nanoparticles (CSnp), (1) Dex-CSnpI (slow releasing) and (2) Dex-CSnpII (rapidreleasing), on adherence, viability, and differentiationof stem cells from apical papilla (SCAP) on root dentinexposed to endodontic irrigants.Methods: Slab-shapeddentin specimens were prepared parallel to the root canaland treated with 5.25% sodium hypochlorite (NaOCl) for10 minutes and/or 17% EDTA for 2 minutes. Dentin wasthen conditioned accordingly by (1) no nanoparticle treat-ment, (2) CSnp, (3) Dex-CSnpI, and (4) Dex-CSnpII. Theeffect of nanoparticle conditioning on SCAP viabilitywas determined by cell count and a circularity index.SCAP adherence and viability on dentin were assessedby fluorescence and scanning electron microscopy andodontogenic differentiation by immunofluorescence. Re-sults: SCAP on dentin treated with NaOCl alone or NaOClas the last irrigant showed the least adherence, minimalcytoplasmic extensions, and higher circularity. SCAPadherence and viability on Dex-CSnpI and Dex-CSnpIIconditioned dentin were increased and had a well-developed cytoplasmic matrix and significantly lowercircularity (P < .05). SCAP cultured in Dex-CSnpII groupexpressed higher levels for DSPP and DMP-1 than inCSnp or Dex-CSnpI groups. Conclusions: Dex-CSnpIand Dex-CSnpII conditioning of dentin enhanced SCAPadherence and viability. Temporal-controlled release ofDex from Dex-CSnpII enhanced odontogenic differentia-tion of SCAP. This study highlighted the ability of dentinconditioning with temporal-controlled BM releasing

From the Discipline of Endodontics, Faculty of Dentistry, UniverAddress requests for reprints to Dr Anil Kishen, Faculty of Dent

akishen@gmail.com0099-2399/$ - see front matter

Copyright ª 2016 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2016.01.026

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nanoparticles to improve the local environment in regenerative endodontics. (J Endod2016;42:717–723)

Key WordsCell adherence, chitosan nanoparticles, dentin, dexamethasone, odontogenic differen-tiation, regenerative endodontic procedures, stem cells from apical papilla

Regenerative endodontic procedures are biologically based procedures designedto replace damaged, diseased, or missing portions of the pulp-dentin complex

(1). Although disinfection of root canal is an essential prerequisite for successfulregeneration of tissue (2), many of the agents used for this purpose are cytotoxicand potentially damaging to the dentin. This, in turn, can alter the bioactivity ofdentin matrix and compromise the survival, adherence, proliferation, and odonto-genic differentiation of the stem cells (3). Conversely, treatment of the dentin withethylenediaminetetraacetic acid (EDTA) appears to improve stem cell adherenceand survival, but when the root canal is irrigated with sodium hypochlorite(NaOCl) after irrigation with EDTA, its positive effect is negated and dentin erosionaccelerated (4). This further compromises the interaction between the dentin andstem cells and impedes neotissue integration.

Chitosan is a cationic natural biocompatible polymer with broad-spectrumantibacterial properties and excellent biodegradable characteristics when used inregenerative endodontic procedures (5). Its molecular structure is similar toextracellular matrix component and contains numerous free hydroxyl and aminogroups that allow it to be easily modified when necessary (5). The incorporationof chitosan nanoparticles (CSnp) with dentin matrix appears to enhance themechanical properties and its resistance to bacterial enzymatic degradation (6).CSnp possesses favorable physicochemical characteristics such as a nanoscalesize, a large surface area/mass ratio, and an increased chemical reactivity, whichmakes it useful in the local delivery of bioactive molecules (BMs) in regenerativeprocedures (7). Temporal-controlled release of bovine serum albumin from CSnp,for example, has been shown to increase the viability and the alkaline phosphataseactivity of stem cells from apical papilla (SCAP) (8). More recently, temporal-controlled release of dexamethasone (Dex) from CSnp enhanced the odontogenicdifferentiation of SCAP in vitro. Furthermore, the rapid releasing variant of CSnp,Dex-CSnpII, has been shown to be more effective in increasing biomineralizationand expression of odontogenic markers in cultured SCAP than a slow-releasingvariant Dex-CSnpI (9).

sity of Toronto, Toronto, Ontario, Canada.istry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada. E-mail address:

Dentin Conditioning with Nanoparticle System 717

Figure 1. Representative images of SCAP adherence on chemically untreated/treated dentin specimens. SCAP were cultured on dentin specimens for 24 hours andstained with calcein-AM (bar: 100 mm) and phalloidin-TRITC/DAPI (bar: 20 mm). Few cells with cytoplasmic extensions were present in NaOCl/EDTA group.Significantly less numbers of cells with rounded morphology and devoid of cytoplasmic extensions were observed in NaOCl and NaOCl/EDTA/NaOCl groups.

Regenerative Endodontics

Strategies to improve the success of tissue engineering shouldallow for (1) favorable interaction between stem cells and dentinmatrix and (2) availability of BMs at an optimal time and concentration.It has been hypothesized that dentin conditioned with a temporal-controlled Dex releasing system can neutralize the detrimental effectof some root canal irrigants on dentin and provide a bioactive extracel-

Figure 2. Representative images of SCAP adherence on NaOCl/EDTA-treated dentdentin specimens for 24 hours and stained with calcein-AM (bar: 100 mm) andunder scanning electron microscope (SEM). Cell adherence and morphology wereNaOCl/EDTA-treated specimens.

718 Shrestha et al.

lular matrix that promotes SCAP adherence, viability, and differentia-tion. The purpose of this study was to evaluate the effects of a slowDex releasing nanoparticle system (Dex-CSnpI) and a rapid Dexreleasing nanoparticle system (Dex-CSnpII) in promoting SCAP adher-ence, viability, and odontogenic potential of SCAP seeded on dentinexposed to endodontic irrigants.

in specimens without/with nanoparticle conditioning. SCAP were cultured onphalloidin-TRITC/DAPI (bar: 10 mm). SCAP ultrastructure was also observedimproved by nanoparticle conditioning on dentin specimens. Unconditioned:

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Regenerative Endodontics

Materials and MethodsChitosan, sodium tripolyphosphate, Dex, a-minimum essential

medium, and EDTA were purchased from Sigma-Aldrich Inc (St Louis,MO). EDTA was used as a 17% solution. Fetal bovine serum, L-gluta-mine solution, and antibiotic:antimycotic solution were obtainedfrom Gibco (Carlsbad, CA). NaOCl was purchased from Lavo Inc (Mon-treal, Quebec, Canada) and used as a 5.25% solution. All the chemicalswere of analytical grade (purity$ 95%).

Teeth Collection and Dentin Specimen PreparationFreshly extracted caries-free human teeth were collected for use in

the study in accordance with University of Toronto Ethical Guidelines.After extraction all soft tissue was removed, and teeth were cleanedbefore storage in deionized water at 4�C. The crown of a tooth selectedfor use was removed at the level of cementoenamel junction, and theroot was shortened by grinding to allow for the creation of uniformroot sections 5 mm in length. Root dentin sections 1 mm in thicknesswere prepared from either side of the root canal by using a slow speeddiamond-wafering blade (Buehler, Coventry, UK) under continuous wa-ter spray (10). Sections were then reduced by using wet emery paper,grit sizes 400, 800, and 4000, under a continuous water spray to pro-duce dentin slabs 5 � 5 � 0.5 mm in size. Specimens were stored infresh deionized water at 4�C until used.

Chemical Treatment ProtocolDentin specimens were autoclaved for 20 minutes at 121�C before

use, and all experimentation was performed under a laminar flow hoodto assure asepsis (11). A total of 30 specimens were treated with NaOCland/or EDTA to simulate clinical condition (12) by using 5 protocols(n = 6/group): group 1, no treatment (control); group 2, 10-minutetreatment with NaOCl (NaOCl); group 3, 2-minute treatment withEDTA (EDTA); group 4, 10-minute treatment with NaOCl followed by2-minute treatment with EDTA (NaOCl/EDTA); and group 5, 10-minute treatment with NaOCl followed by 1-minute treatment withEDTA followed by 1-minute treatment with NaOCl (NaOCl/EDTA/NaOCl). Specimens were thoroughly rinsed with sterile deionized waterafter each treatment protocol (3, 13, 14).

Nanoparticle ConditioningNanoparticles used for dentin conditioning were synthesized as

previously described (8, 9). Physical characterization and releasekinetics of 2 variants Dex-CSnpI (slow releasing) and Dex-CSnpII(rapid releasing) have been previously published (9). Seventy-twoof NaOCl/EDTA-treated dentin specimens were divided into 4 groups(n = 18) and treated accordingly with 300 mg/mL nanoparticles(8, 9) in deionized water (1 mL) for 12 hours. The 4 groups weredesignated as (1) unconditioned, (2) CSnp conditioned, (3) Dex-CSnpI conditioned, and (4) Dex-CSnpII conditioned.

Figure 3. (A) Relative viability of SCAP in NaOCl/EDTA-treated dentin speci-mens without/with nanoparticle conditioning. Groups conditioned with nano-particles demonstrated no significant change in viability relative to theunconditioned group. (B) Circularity of cells cultured in NaOCl/EDTA-treated dentin specimens without/with nanoparticle conditioning. Cells inthe unconditioned group demonstrated 3-fold increase in circularity ascompared with the control group and 2-fold increase in value as comparedwith the CSnp, Dex-CSnpI, or Dex-CSnpII conditioned groups. Data are pre-sented as mean � standard deviation of the mean (n = 3). **P < .05 versusthe control group. ##P < .05 versus unconditioned group.

SCAP CultureA previously characterized SCAP cell line was used in all experi-

ments (15). Cells were cultured and expanded by adding single-cellsuspensions (1� 105 cells) to a-minimum essential medium supple-mented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 100units/mL antibiotic:antimycotic solution. Cells were allowed to expandin culture to 70%–80% confluency and then released with 0.05%trypsin (Gibco, Carlsbad, CA). Cells from the third to fifth passages(15–17) were used in all experiments.

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Characterization of SCAP on Nanoparticle ConditionedDentin

Six dentin specimens from each chemical treatment group wereplaced in a 24-well plate, and 1 mL SCAP at a concentration of 1.0 �105 was introduced into each well. The cells were allowed to grow at37�C in a humidified incubator in an atmosphere of 5% CO2 for24 hours. Specimens were washed with phosphate-buffered saline(PBS) and stained for fluorescence microscopy. Similarly, SCAP wascultured on 9 dentin specimens from each nanoparticle conditionedgroup and stained for fluorescence microscopy and prepared for scan-ning electron microscopy.

SCAP Adherence and Viability on Nanoparticle Condi-tioned Dentin. Three specimens in each group were stained withcalcein-AM (1 mmol/L in PBS; Life Technologies, Grand Island, NY)by directly adding the staining solution to cultured cells (18) and exam-ined under a fluorescence microscope (Carl Zeiss, Gottingen, Ger-many). The number of cells adherent to dentin was calculated from12 microscopic fields at �20 magnification. Images were capturedand processed by using ImageJ software (National Institutes of Health,Bethesda, MD) and then saved as a 32-bit jpeg file. Cell counts were

Dentin Conditioning with Nanoparticle System 719

Figure 4. (A) Scanning electron microscopic micrographs showing morphology and biomineralization of SCAP cultured for 2 weeks on NaOCl/EDTA-treateddentin specimens with/without nanoparticle conditioning. CSnp conditioned specimen without cells was studied as negative control to determine the effect ofCSnp on mineralization. Mineralization was present in the conditioned groups, with the greatest degree present in the Dex-CSnpII conditioned group. Immuno-fluorescence localization of DSPP (B) and DMP-1 (C) during odontogenic differentiation. Fluorescein isothiocyanate (FITC)–conjugated antibody was used todetect the localization of the protein (green signal); all samples were counterstained with DAPI (blue signal). Scale bar = 25 mm.

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made from 8-bit images normalized to 50–500 pixels by using ImageJ.Cell viability was calculated as a percentage relative to the total numberof cells attached to control dentin that was designated as 100% viability.

SCAP Spreading on Nanoparticle Conditioned Dentin.Cell circularity was determined by analyzing cells in the same specimensthat were stained with calcein-AM by using ImageJ. Circularity was as-sessed as 1, a perfect sphere or rounded cell, to 0, representing anon-circular or highly spread cell (19). Ten cells in each microscopicview were analyzed and averaged to assign circularity values.

In addition, SCAP present on 3 specimens from each group werefixed for 5 minutes in 10% normal buffered formalin and thoroughlywashed with PBS. Cells were permeabilized with 0.1% Triton X-100(Sigma-Aldrich) in PBS for 20minutes and washed again with PBS. Cellswere stained with DAPI (Roche, Basel, Switzerland) for 5 minutes andthen fluorescent phalloidin-tetramethylrhodamine B isothiocyanate(Phalloidin-TRITC; Sigma-Aldrich) solution in PBS for 40 minutes atroom temperature. They were washed several times with PBS to removethe unbound phalloidin conjugate. The excitation and emission wave-lengths for Phalloidin-TRITC are 540 nm and 570 nm, respectively.The specimens were examined under a fluorescent microscope to visu-alize cytoskeletal actin.

SCAP Ultrastructure on Nanoparticle Conditioned Dentin.Three specimens from each group were fixed overnight in 2.5%glutaraldehyde. These specimens were then dehydrated in graded so-lutions of ethanol and dried with hexamethylene disiloxane. Afterpalladium coating, specimens were examined under a scanning elec-tron microscope (Hitachi S-2500, Ibaragi, Japan) to assess SCAP ul-trastructure.

SCAP Differentiation on Nanoparticle Conditioned DentinThe remaining 9 specimens in each nanoparticle conditioned

group were placed in an 8-chamber polystyrene treated vessel of a tis-sue culture glass slide (BD Biosciences, Bedford, MA) and seededwith 2 � 104 cells in standard culture medium containing 50 mg/mL ascorbic acid, 10 mmol/L b-glycerolphosphate, and 1.8 mmol/L potassium dihydrogen phosphate (KH2PO4). Cultures were main-tained at 37�C in 5% CO2 humidified incubator for 2 weeks. Compar-isons were made between the 2 variants, Dex-CSnpI (slow releasing)and Dex-CSnpII (rapid releasing). All experiments were performed intriplicate before analysis.

SCAP Biomineralization on Nanoparticle ConditionedDentin. After 2 weeks, 3 specimens from each group were preparedfor scanning electronmicroscopy as described earlier to assess biomin-eralization (20). Ten images were analyzed in a sequential manner foreach sample. The effect of CSnp on biomineralization was studied byincubating CSnp conditioned dentin under the same conditions/dura-tion without cells (negative control).

Immunofluorescence Analysis on Nanoparticle Condi-tioned Dentin. At the end of 2 weeks, specimens were washed inPBS and fixed with 4% paraformaldehyde in PBS containing 0.1% TritonX-100 at 4�C for 30 minutes. After blocking the fixed specimens in 2.5%bovine serum albumin for 30 minutes at room temperature, 3 speci-mens from each group were incubated with the following primary an-tibodies, mouse anti-DMP-1 (Santa Cruz Biotechnologies, Santa Cruz,CA) or mouse anti-DSPP (Santa Cruz Biotechnologies), diluted 1:50in blocking reagent at 37�C for 2 hours. After 3 washes in PBS/Tween20, specimens were incubated with secondary antibody goat anti-mouseimmunoglobulin G fluorescein isothiocyanate conjugate (Santa CruzBiotechnologies), diluted 1:1500 in PBS at 37�C, for 1 hour. Afterrinsing, specimens were counterstained with DAPI and examined by us-

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ing confocal laser scanning microscopy (Leica Microsystems, Rich-mond, IL).

Statistical AnalysisViability and circularity results were subjected to one-way analysis

of variance with Dunnett test. The difference between 2 groups wasconsidered statistically significant if P < .05.

ResultsEffect of Nanoparticle Conditioning on SCAPCharacteristicsSCAP Adherence. Figure 1 shows the typical fluorescence micro-scopic images of SCAP adherence on dentin. Numerous cells with afibroblast-like morphology were homogenously distributed on the un-treated dentin (control group) in a unidirectional manner. The numberand morphology of cells present on EDTA only treated dentin weresimilar to those present in the control group, but cells were more flat-tened. Few cells with cytoplasmic extensions were present on dentintreated with NaOCl/EDTA. Significantly less numbers of cells withrounded morphology and devoid of cytoplasmic extensions wereobserved in NaOCl and NaOCl/EDTA/NaOCl groups. The morphologyof cells after F-actin staining with phalloidin-TRITC was concurrentwith the green fluorescent images stained with calcein-AM. Cells ondentin treated with NaOCl and NaOCl/EDTA/NaOCl displayed less ctyto-plasmic F-actin compared with the rest of the groups.

Nanoparticle conditioning on NaOCl/EDTA treated dentin resultedin significant increase in number of SCAP adherence compared with un-conditioned dentin as shown in Figure 2. Cells in all the conditionedgroups were uniformly distributed and displayed well-developed lamel-lipodia and filopodia.

SCAP Ultrastructure. Cells on unconditioned dentin were roundand devoid of cell extensions (Fig. 2). Cells on dentin conditionedwith nanoparticles were flattened, appeared more numerous andmore attached, and had well-developed cytoplasmic extensions, similarto what was seen in these same groups under fluorescence microscopy.

SCAP Viability and Circularity. The quantitative relative viabilityof cells on dentin varied among groups (Fig. 3A). The viability of cells inthe unconditioned group was decreased by 50% when compared withthe untreated control group. Groups conditioned with nanoparticlesdemonstrated no significant change in viability relative to the uncondi-tioned group. Cells in the unconditioned group demonstrated a 3-foldincrease in circularity as compared with the control group (P < .05)and a 2-fold increase in value as compared with the CSnp, Dex-CSnpI, or Dex-CSnpII conditioned groups (P < .05) (Fig. 3B).

Effect of Nanoparticle Conditioning on SCAPDifferentiationSCAP Biomineralization. No evidence of mineralization could beseen under scanning electron microscopy in the unconditioned andnegative control groups (Fig. 4A). Mineralization was present in thenanoparticle conditioned groups, with the greatest degree present inthe Dex-CSnpII conditioned group.

SCAP Immunofluorescence Localization of Dentin MatrixProtein-1 and Dentin Sialophosphoprotein. As shown inFigure 4B and C, expressions of dentin sialophosphoprotein (DSPP)and dentin matrix protein-1 (DMP-1) were not observed in uncondi-tioned group. Cells in the rest of the groups demonstrated a highersignal for cytoplasmic DSPP expression than for DMP-1 expression.Higher signals for DSPP and DMP-1 expression were seen in the cellsof the Dex-CSnpI and Dex-CSnpII conditioned groups compared with

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the CSnp conditioned group, and signals for DSPP and DMP-1 weremarkedly higher in cells of the Dex-CSnpII group than in cells of theDex-CSnpI group.

DiscussionA treatment protocol that promotes adherence of dental stem cells

to root canal dentin and enhances their odontogenic differentiation isessential in reestablishing dentinogenesis in a pulpless environment(21). Dex has been identified as an odontogenic stimulant for SCAP inregenerative endodontics. Ideally this stimulation works best when thedentin is undamaged (13). Many commonly used root canal irrigants,especially those used in root canal disinfection, have a potential to dam-age dentin and therefore impede cell adherence and new tissue forma-tion (22, 23). NaOCl, a highly effective root canal antimicrobial agent forexample, alters the surface characteristics of dentin and impedes theodontogenic differentiation of pulp stem cells (22, 24, 25).Conditioning with CSnp or Dex-CSnp, as shown in this study, allowedcells to adhere and retain their viability on NaOCl/EDTA-treated dentin.

Of the numerous agents currently used in root canal irrigation,only EDTA demonstrates an ability to promote cell adherence andviability on dentin (22, 23). This has been reported several times inthe literature (4, 13, 22, 23) and was supported by the findings inthis study. It has been suggested that this effect is related to anincrease in dentin surface wettability (26) and the release of dentino-genic BMs from the matrix of chelated dentin (27). Furthermore,EDTA as a final irrigant removes smear layer and exposes organic sub-strate for interaction with the nanoparticles. The adherence of stem cellsto dentin has been attributed to fibronectin, an adhesion protein presentin the dentin matrix and preferentially adsorbed to a hydrophilic surface(28). Nanoparticle conditioning of NaOCl/EDTA-treated dentin appearsto provide that hydrophilic surface (5) and hence enhance the degree ofcell adherence.

In the current study, biomineralization and signals from bio-markers such as DSPP and DMP-1 were distinctly higher in SCAPcultured on Dex-CSnpI and Dex-CSnpII conditioned dentin than inSCAP cultured on CSnp conditioned dentin alone. DSPP is a matrixphosphoprotein strongly expressed in odontoblasts and a marker ofbiomineralization (29). DMP-1 is a non-collagenous matrix protein ex-pressed in odontoblasts and regulates biomineralization during toothdevelopment and repair (30). Because DSPP and DMP-1 are expressedin odontoblasts in the early stages of odontogenesis (31), the elevatedexpression of these 2 odontogenic proteins in Dex-CSnpI and Dex-CSnpII conditioned specimens suggested that Dex has been made avail-able to the cells in an appropriate concentration and at a critical time.Furthermore, because cells conditioned with Dex-CSnpII (rapidrelease) showed higher expression of these odontogenic markersthan cells conditioned with Dex-CSnpI (slow release), a temporal sys-tem of BM release may be advantageous when developing a regenerativeendodontic protocol (9).

The usefulness of temporal-controlled BM release in regenerativeendodontics has been previously discussed (8, 9). The advantage oftemporal-control release over static availability is release of a desiredconcentration of BM and a preservation of BM stability during a prede-termined time. When used as a vehicle to deliver BMs, CSnp adds anantimicrobial and antibiofilm effect and greater hydrophilicity to thedentin, making it an ideal nanoparticle for use in regenerative proce-dures (32). Release of endogenous dentin matrix components, suchas transforming growth factor–b, bone morphogenic protein–2,platelet-derived growth factor, has been shown to be advantageous inpromoting stem cell proliferation and differentiation (27, 33, 34)and can potentially be delivered in a temporal manner. Growth

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factors which regulate specific components of the healing processthat are currently being used as therapeutic agents to enhancerepopulation of root surfaces denuded by periodontal disease (35)have a potential that can be readily applied to endodontic regenerativeprocedures.

In conclusion, results of this investigation demonstrate that condi-tioning of dentin with CSnp, Dex-CSnpI, and Dex-CSnpII has a potentialto reverse the deleterious effect of NaOCl on stem cell viability andadherence in regenerative procedures. As such, it may allow NaOCl tobe used in the disinfection of the root canal when regenerative proce-dures are planned. The study has also demonstrated that by controllingthe concentration and duration of BMs release, the odontogenic differ-entiation of SCAP can be enhanced over static administration. Ultimately,this may lead to a more favorable outcome when regenerative proce-dures are undertaken.

AcknowledgmentsThe authors gratefully acknowledge the support of Dr Anibal

Diogenes in this study.This study was supported by the University Start Up Fund, Uni-

versity of Toronto, Ontario, Canada and a Research Grant from theAmerican Association of Endodontists (AAE) Foundation(2014).

The authors deny any conflicts of interest related to this study.

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