er:yag laser modification of root canal dentine: influence of pulse duration, repetitive irradiation...

Lasers Med Sci 2002, 17:198–207Ownership and Copyright© 2002 Springer-Verlag London Limited

Er:YAG Laser Modification of Root Canal Dentine:Influence of Pulse Duration, Repetitive Irradiationand Water Spray

A. Ebihara1,2, B. Majaron1,3, L-H.L. Liaw1, T.B. Krasieva1 and P. Wilder-Smith1

1Beckman Laser Institute, University of California at Irvine, Irvine, CA, USA; 2Pulp Biology and Endodontics, Department ofRestorative Sciences, Tokyo Medical and Dental University, Tokyo, Japan; 3Joef Stefan Institute, Jamova, Ljubljana, Slovenia

Abstract. The aim of this study was to determine the e#ects of varying parameters of Er:YAG laserirradiation with and without water spray cooling on root canal dentine in vitro. After horizontally removingtooth crowns from extracted human teeth, roots were axially sectioned into thin slices, exposing the root canalsurface. An Er:YAG laser delivered 10–30 J/cm2 into a 0.4-mm diameter laser spot on the root canal surface.Single pulses of di#erent lengths (80–280 �s) were applied with and without water spray cooling/irrigation,and sequences of three pulses at a repetition rate of 30 Hz were applied at selected pulse parameters. Theirradiated samples were investigated using both confocal laser scanning microscopy (CLSM) and scanningelectron microscopy (SEM). At most irradiation conditions, the root canal dentine surface was ablated.Three-dimensional images from CLSM revealed that the cavity walls were not smooth. Depths of the cavitiesrevealed significant di#erences between the cavities. No debris was observed at the surface of cavities at anyirradiation condition. Strong melting and recrystallisation, or unusually flat surfaces with open dentinaltubules were obtained with sequences of three pulses without water cooling. CLSM is an e#ective tool forinvestigation of laser e#ects on root canal dentine. By varying the irradiation conditions, the Er:YAG lasercan induce di#erent modifications of root canal surface, which may be very interesting for root canalpreparation.

Keywords: Ablation; Confocal laser scanning microscopy; Dentistry; Endodontics; Hard tissue;Morphological change; Scanning electron microscopy

INTRODUCTION

Root canal preparation is usually performedusing hand instruments and/or rotary instru-ments [1]. However, preparing root canalsusing those instruments is not easy, and oftenleaves a smear layer and debris on the surfaceof the root canal. The smear layer is composedof debris, composed of fractured bits of dentineand soft tissue from the canal. In clinicalpractice, chemical irrigation is used to removethe smear layer, but it is very di$cult toachieve a complete removal. Moreover, ordi-nary root canal preparation methods are notable to disinfect the root canal surface.

As a possible solution, novel modalities forroot canal therapy utilising laser irradiationhave recently been investigated. Two studiesof root canal disinfection using lasers havebeen performed [2,3]. Le Go# et al. reported thee#ect of CO2 laser on animal teeth contami-nated with an endodontic bacterial strain [2].Berkiten et al. investigated the disinfectionability of Nd:YAG laser on the root canal walls[3]. Harashima et al. [4] evaluated the e$cacyof Nd:YAG laser for removing smear layer andsuggested that it was useful and that it causedmelting of internal structures on the instru-mented root canal walls. Blum et al. [5] usedthe Nd:YAP laser and reported that laser has apotential in ensuring optimal canal cleanli-ness. Machida et al. [6] applied the KTP:YAGlaser to remove the smear layer and debrisfrom the root canal surface, and indicatedits e#ectiveness and thermal safety. One

Correspondence to: Petra Wilder-Smith, Beckman LaserInstitute and Medical Clinic, University of California atIrvine, 1002 Health Sciences Road East, Irvine, CA 92612,USA. e-mail: [email protected]

possibility is ablation of the root canal dentinewith the Er:YAG laser (wavelength 2.94 �m),which is used clinically for cavity preparation.However, only a few studies thus far haveinvestigated the feasibility of root canaltreatment using this laser [7–12].

The main purpose of the present study wasto determine the surface and subsurface modi-fications of root canal dentine after Er:YAGlaser irradiation under variable conditions.The exposed surface of human root canal den-tine was irradiated in vitro by an Er:YAG laserwith various pulse durations and fluences,with and without water spray cooling/irrigation. Water cooling is clinically advis-able to reduce the risk of thermal damage tothe adjacent root canal and periodontal struc-tures [11]. The irradiated surface was investi-gated using both confocal laser scanningmicroscopy (CLSM) [13,14] and scanning elec-tron microscopy (SEM). In contrast with thelatter, more customary method, CLSM doesnot require the samples to be dehydrated, andtherefore does not produce artefactual cracks.Furthermore, CLSM has the potential to ana-lyse the subsurface layer of irradiated dentine,and produce three-dimensional images of theablation craters.

MATERIALS AND METHODS

Sample Preparation

Extracted human teeth were stored indemineralised water with 0.01% (w/v) thymol.After removing the tooth crown horizontally,the roots were sectioned axially using a low-speed saw with water coolant (Isomet, Buchler,IL, USA). In this manner, approximately600 �m thick slices were prepared, exposingthe surface of the root canal for laserirradiation.

Laser Irradiation

This study was performed using the ‘Fidelis’Er:YAG laser (Fotona, Ljubljana, Slovenia),which permits the user to choose betweenfour laser ‘modes’, featuring various pulsedurations at nearly square temporal profiles,and repetition rates from 2 to 50 Hz. Inaddition, a manufacturer-provided custom-isation of the system enabled us to externallycontrol the laser to generate repeatable pulse

sequences at high repetition rates. Beam deliv-ery via the instrument’s articulated arm and anon-contact focusing handpiece (RO-8, Fotona,Ljubljana, Slovenia) was used to irradiate per-pendicularly nominally 0.4-mm-diameter spotson the root canal surface.

Laser pulses generated in three di#erentlaser ‘modes’ were used in the study: VSP(‘very short pulse’, pulse duration tp=80 �s),LP (‘long pulse’, tp=180 �s), and VLP (‘verylong pulse’, tp=280 �s). In each laser mode, theoutput energy was set at around 35 mJ toproduce an estimated energy density of28–29 J/cm2 on the dentine surface. The tem-poral dependence of laser intensity in thesethree laser ‘modes’, as detected with an InSbphotodetector, is presented in Fig. 1. One ortwo external attenuators were used to reducethe sample irradiation fluence by 31% (oneattenuator) or 52%, to 13–14 J/cm2 (with twoattenuators), while preserving the spatial andtemporal structure of the laser pulse. Inaddition, using the custom external control,we programmed the laser to emit sequences ofthree pulses with the temporal profile resem-bling the LP laser mode, at a repetition rate of30 Hz (see Table 1 for an overview of theirradiation conditions). All fluence valuesused were lower than the manufacturer-recommended settings for e$cient ablation ofcaries and dentine in cavity preparation appli-cation (45–100 J/cm2, repetition rates up to12 Hz).

Fig. 1. Temporal dependence of Er:YAG laser intensity inthe three pulsed modes used in the study: (a) VSP, (b) LPand (c) VLP.

Modification of Root Canal Surface with Variable Er:YAG Laser Irradiation 199

At each of the above laser settings, two siteson the exposed root canal surface were irradi-ated without water cooling. Then, the com-plete experimental protocol was repeatedusing a dental water spray cooling/irrigationsystem at a water flux of 4 ml/min, for a total of44 irradiated sites on 15 tooth sections.

Confocal Laser Scanning Microscopy(CLSM)

Laser-irradiated samples were stained usingRhodamine 123 (Sigma, MO, USA) at a con-centration of 10�5 in phosphate bu#eredsaline (PBS, Sigma, MO, USA) for 24 h. Afterstaining, they were rinsed two to three timeswith PBS, and fixed on slide glasses withcyanoacrylate glue.

Stained samples were examined with aZeiss LSM 410 inverted laser scanning con-focal microscope (Carl Zeiss, Oberkochen,Germany). The objective lense used was thePlan-Neofluar 20� bright field (Carl Zeiss,Oberkochen, Germany). The 488-nm line of acontinuous argon laser was used for sampleexcitation, and the fluorescence emission wasisolated with a long-pass filter at 590 nm. Anumber of optical sections parallel to thesample surface (i.e. horizontal) were obtainedfor each sample. The distance between individ-ual optical sections was 2–10 �m, depending onthe depth of the cavity. The manufacturer-supplied software enabled generation of verti-cal cross sections, as well as three-dimensional(3-D) images through the cavity, from stacks ofacquired horizontal sections.

Scanning Electron Microscopy (SEM)

SEM was performed to identify the microstruc-tural e#ects of laser irradiation on the rootcanal dentine surface. After being imaged byCLSM, the samples were dehydrated in a

graded series of aqueous ethanol (50, 70, 90 and100% ethanol) for 10 min at each concen-tration. Then, they were mounted on stubsusing colloidal silver liquid (Ted Pella, CA,USA), and gold coated on a PAC-1 Pelcoadvanced coater 9500 (Ted Pella). Micrographsof the root canal dentine surface were takenwith a Philips 515 (Mohawk, NJ, USA) SEM.

RESULTS

Confocal Laser Scanning Microscopy

At all irradiation conditions tested in thisstudy, root canal dentine was ablated andsmall cavities were prepared by laserirradiation when no water spray cooling/irrigation was used. Fluorescence from thesurface and subsurface of the resulting cav-ities was detected by the CLSM in well-definedplanes, perpendicular to the axis of the micro-scope objective. Figure 2(a–d) presents a set ofsuch optical sections, acquired from ablationcavity caused by a single LP pulse (tp=180 �s)at the energy density of F=28 J/cm2, and nowater spray. The images were acquired withthe 20� bright-field objective from horizontalplanes separated by 4 �m, and roughly corre-spond to cross sections of the ablation cavity.The size bars in the images mark the lateraldimension of 100 �m. Figure 2(f) shows a 3-Drepresentation of the same cavity, viewedalong the cavity axis, composed from theacquired CLSM images.

The manufacturer-supplied software enablesalso reconstruction of cross sections throughthe cavity, perpendicular to the root canalsurface. Figure 3(a–g) presents such verticalsections (of the same ablation cavity as shownin Fig. 2), positioned in equidistant distancesof 20 �m from its centre towards the periphery.In addition, a 3-D lateral view of one half of thecavity is presented in Fig. 3(h). The imagesreveal the uneven depth and rather rough

Table 1. Overview of the laser irradiation parameters

Laser modetp

(�s)Elaser

(mJ)F0

(J/cm2)F1

(J/cm2)F2

(J/cm2)

VSP (very short pulse) 80 35 28 20 13LP (long pulse) 180 34 28 19 13VLP (very long pulse) 280 37 29 20 14Sequence (3�LP, 30 Hz) 180 30 — 18 12

200 A. Ebihara et al.

Fig. 3. Vertical cross sections of the laser ablation cavity from Fig. 2, from the centre (a), towards its periphery (g), asreconstructed from 48 horizontal optical sections. The presented sections are equidistant at 20 �m, the size bars are 100 �m long.(h) The composed 3-D lateral view of one-half of the same ablation cavity (LP, 28 J/cm2, no water spray).

Fig. 2. CLSM images of an ablation cavity (LP, 28 J/cm2, no water spray), as acquired at depths of (a) 0 �m, (b) 40 �m, (c)80 �m, (d) 120 �m and (e) 160 �m. (f) shows a 3-D presentation of the same cavity, composed from 48 optical sections, separatedby 4 �m. The size bars in the images are 100 �m long (objective magnification: 20×).


surface of this particular cavity (LP, 28 J/cm2,no water spray).

Figure 4 shows an overview of CLSM imagesof another ablation cavity (VSP, F=28 J/cm2),where water spray cooling was applied duringthe laser irradiation. 3-D views of the cavity,reconstructed from 50 horizontal CLSM scans,(a) separated by 4 �m, (b) in vertical crosssection through the centre and (c) in a 3-Dlateral view of one half of the same ablationcavity are shown. Although the general shapeof the cavity is similar to that seen in Figs 2and 3 (reflecting the intensity profile ofthe laser), it features significantly rounder,smoother cavity walls.

In Fig. 5, no significant di#erence can beobserved between the cavities produced withsequential Er:YAG laser irradiation (3�LP,

12 J/cm2, 30 Hz), without (a) and with (b)application of water spray cooling.

The depths of all ablation cavities, asassessed using CLSM, are collected in Fig. 6.The symbols mark the average values deter-mined from two samples, and error bars markthe standard error of the mean. The end pointsof each bar, therefore, correspond to individ-ual values for the two craters, and only onecrater was available where no error bar isplotted. For the sequential irradiation, one-third of the cavity depth is plotted, to yield theablation depth per pulse. The plotted curvesare not based on theory and serve only as avisual guide.

The cavities resulting from single laserpulses of three di#erent durations (VSP, LP, orVLP) and sequences of three LP pulses without

Fig. 4. (a) 3-D presentation of a laser ablation cavity (VSP, 28 J/cm2, with water spray), as composed from 50 horizontal CLSMimages, separated by 4 �m. (b) Vertical cross section through the centre, and (c) 3-D lateral view of one half of the same ablationcavity. The size bars are 100 �m long.

Fig. 5. 3-D presentation of laser ablation cavities produced with sequences of three laser pulses (LP, 12 J/cm2, 30 Hz), without(a) and with (b) application of dental water spray. These images were composed from 46 and 20 CLSM scans (for a and b,respectively), obtained with the –0× objective from horizontal planes separated by 6 �m. The size bars are 100 �m long.


water spray cooling (Fig. 6a) display similarfluence dependences. However, the VSP data(pulsewidth tp=80 ms) indicate a greaterablation e#ect at moderate pulse fluences(F�20 J/cm2), but also the strongest satur-ation (i.e. diminution of ablation e$ciency,causing a deviation from linear dependence)with increasing fluence values, as compared to

the LP and VLP pulses. Within the experimen-tal margin of error, there is no di#erencebetween the two latter data sets. The greatestvariability of the results is observed with thesequential irradiation.

Very similar observations can be made fromresults obtained when water spray cooling wasapplied during the irradiation (Fig. 6b). The

Fig. 6. Ablation depth per pulse as a function of single-pulse fluence for irradiation with individual pulses in three laser ‘modes’(VSP, LP, VLP), and sequences of three LP pulses at 30 Hz repetition rate, without (a) and with (b) application of the water spray.

Fig. 7. SEM images of the bottom of ablation cavities, produced by irradiation with individual Er:YAG laser pulses in three‘modes’: (a), (b) VSP, 28 J/cm2; (c), (d) LP, 28 J/cm2; (e), (f) VLP, 29 J/cm2. (a, c, e) no water cooling; (b, d, f) with application ofwater spray cooling. (Original magnification: 1000×)


ablation e#ect at moderate pulse fluences isagain greatest in the VSP mode, which dis-plays a significant saturation at higher fluencevalues, whereas the LP and VLP results arecomparable within the experimental error. Bycomparing the results in Fig. 6(a) and (b), wesee that the application of water spray cooling/irrigation increased the ablation threshold forall laser operation modes. Nevertheless, thedi#erential ablation e$ciency (i.e. increasein ablation depth per unit increase of pulsefluence) seems somewhat higher when thewater spray is used, in particular for the VSPlaser mode.

A comparison of the cavity diameters hasrevealed that, in addition to increasing theablation threshold, water spray cooling/irrigation also reduces the cavity diameter,regardless of the laser mode [14].

Scanning Electron Microscopy

No debris was observed in any of the ablationcavities obtained with single-pulse irradiationat 28–29 J/cm2, regardless of the pulse durationor application of the water spray (Fig. 7). Inall presented examples, the root canal den-tine is cleanly ablated, with dentinal tubules

open, and no evidence of melting orrecrystallisation.

In contrast, a large variety of surface mor-phologies resulted when sequences of lower-fluence pulses were applied with or withoutwater spray cooling (Fig. 8). Very strong melt-ing of dentine and recrystallisation is evidentat single-pulse fluence of 18 J/cm2 and no waterspray (Fig. 8a). In contrast, just moderatelyannealed, but very flat surface with open den-tinal tubules is observed at 12 J/cm2 (Fig. 8c).With aplication of water spray cooling, thethermal e#ect is strongly reduced at both flu-ence values. At 18 J/cm2, a nice flat surfacewith just slightly annealed ‘edges’, but opendentinal tubules results (Fig. 8b), whereas at12 J/cm2, the micromorphology of the surfaceis essentially identical to that observed aftersingle-pulse irradiation (Fig. 8d).

DISCUSSION

During routine root canal treatment usinghand and rotary instruments and chemicalirrigation, debris may remain on the root canalwalls [15]. In this study, no smear layer wasobserved on the surface of the root canaldentine after Er:YAG laser irradiation, just as

Fig. 8. SEM images of the root canal dentine at the bottom of ablation cavities, resulting from irradiation with 30-Hz sequencesof three LP pulses with single-pulse fluence of (a), (b) 18 J/cm2; (c), (d) 12 J/cm2. (a, c) no water cooling, (b, d) with applicationof water spray cooling. (Original magnification: 1000×)


reported by Takeda et al. [8]. On the otherhand, it has been reported that irrigation withwater followed by Er:YAG laser irradiationincreases dentine permeability, which leads togreater cleanliness and more open tubules [10].This finding may be an important factor inachieving disinfection of the root canals anda higher mechanical bonding between theroot canal sealer and dentine [10]. Since theEr:YAG laser was reported to ablate cariousdentine more readily than sound dentine [16],it might be particularly suitable for removal ofcaries from root canal dentine.

In addition, the Er:YAG laser has a highbactericidal potential at a low energy level[17,18]. Mehl et al. [18] reported that Er:YAGlaser radiation (pulse energy 50 mJ; repetitionrate 15/s) for 15 or 60 s could e#ectively reduceStaphylococcus aureus and Escherichia coli,and concluded that Er:YAG laser radiationexerted very e#ective antimicrobial propertiesin dental root canals, depending on the dur-ation of irradiation. The antibacterial e#ec-tiveness of the Nd:YAG, Ho:YAG and Er:YAGlaser in infected root canals were compared,and the most favourable results were obtainedby the Er:YAG laser – achieving a meanbacterial elimination of 99.64% [19]. Thus,the Er:YAG laser has the potential to achievemultiple e#ects of shaping, cleaning anddisinfecting the root canal.

Matsuoka et al. [9] evaluated the e$cacy ofa fibrescope for the assessment of remnantdebris on the root canal walls, and suggestedthat it was e#ective for evaluation of rootcanals of intact teeth. In this study CLSM wasused to evaluate the surface of the irradiatedroot canal. Since this method does not requirethe samples to be dehydrated, it avoids produc-ing artefactual cracks. Depth of cavitiescould be determined from CLSM 3-D recon-structions of the cavity shape and crosssections. Moreover, CLSM can visualise sub-surface dentine. These properties of CLSMrender this modality ideal for the investigationof dental structures, particularly for the non-invasive visualisation of surface and subsur-face microstrucures in laser-irradiated hardtissues.

The depths of cavities resulting from threesingle-pulse laser modes (VSP, LP, VLP) arequite similar between themselves, both withand without the application of water spraycooling/irrigation. However, in both cases,VSP laser pulses (tp�80 ms) display thehighest ablation e$ciency at moderate pulse

fluences (�20 J/cm2), but also the strongestsaturation at higher fluence values. Suchbehaviour is in perfect agreement with earliersystematic measurements on human dentine,as well as theoretical considerations of theinvolved mechanisms [20,21]. On one hand, itresults from di#usion of heat from the laser–tissue interaction layer during the laser pulse.This e#ect, which is more pronounced withlonger laser pulses, reduces the amountof energy available for explosive dentineablation, and therefore diminishes more theablation e$ciency of the longer laser pulses(LP, VLP). On the other hand, scattering andabsorption of laser radiation on the ejecteddebris is strongest with the shortest laserpulses; however, it diminishes the ablatione$ciency primarily at larger pulse fluences,where the deeper ablation cavities trap theejected debris more e#ectively [20,21].

Our results demonstrate that the applicationof water spray cooling/irrigation increasesthe single-pulse ablation threshold for allpulsewidths under test (from 5–10 J/cm2 to>13 J/cm2, see Fig. 6a, b). This matches nicelyto earlier observations in ablation of toothdentine with sequences of ten 200–300 �s longEr:YAG laser pulses [22], and can be attributedto the energy necessary to ablate the stronglyabsorbing water film on the sample surface[23,24]. On the other hand, the water spraycan substantially increase di#erential ablatione$ciency (i.e. increase of ablation e#ect perincrease of pulse fluence). This e#ect has beenobserved in previous studies on laser ablationof enamel, and was tentatively attributed toreduced enamel desiccation [20,22]. However,although this explanation is viable for experi-ments involving repetitive laser irradiation,we would expect the influence of this factorto be minimal in single-pulse ablationexperiments, such as presented in this study.Therefore, one or several of the earlier sug-gested non-thermal ablation mechanisms, suchas intense acoustic transients, or fast waterjets associated with the collapse of laser-induced water bubbles, might indeed be atwork here [22,24–26]. In an extensive study,Ashouri et al. [27] have recently investigatedthe influence of the water layer on ablationwith free-running and Q-switched Er:YAG andEr:YSGG lasers, as well as Ho:YAG and 9.6-�mTEA CO2 laser. The authors have concludedthat ‘water augments the ablation of dentalenamel by aiding in the removal of looselyattached deposits of non-apatite mineral


phases from the crater surface, thus producinga more desirable surface morphology’.

With sequences of three laser pulses, theablation depth per pulse is observed to beessentially the same as with single pulses,when no water spray was used (Fig. 6a). Thismight be incidental, however, since one wouldexpect the incomplete thermal relaxation ofthe sample between successive pulses toreduce the ablation threshold for subsequentlaser pulses, whereas the possible desiccationof the root canal dentine would certainly tendto increase it. Since such additional e#ects areintroduced when the water spray is used, therelation between the single-pulse and repeti-tive ablation e#ect can depend on numerousparameters, such as the number of pulses inthe sequence, repetition rate, water spraycharacteristics, etc. For example, the reducedablation threshold for the repetitive, as com-pared to single-pulse, Er:YAG irradiation (Fig.6b), might indicate that the water film on thedentine surface, which is removed by the firstlaser pulse, does not reconstitute before thenext pulse arrives, thus diminishing the e#ectof ablation threshold increase with the waterspray.

High-resolution SEM images of the dentinesurface structure reveal a clean ablation andno debris on the cavity walls after single-pulseirradiation, regardless of the pulse durationand water spray application (at 28–29 J/cm2).In contrast, a very smooth, possibly slightlyannealed, surface was observed at somesequence-irradiated sites, with and withoutapplication of the water spray. It might be veryinteresting to further explore this regime forroot canal preparation. Pecora et al. [28]reported that dentine treated with Er:YAGlaser displayed greater adhesion of sealersthan dentine treated with EDTAC. The latter,in turn, was greater than dentine whichreceived no treatment. Therefore, the sealingEr:YAG laser modification of the dentinesurface ability of root canal fillings mightbe considerably a#ected by Er:YAG lasermodification of the dentine surface [28].

A non-contact hand piece was used in thisstudy, and only a few investigations have beenreported thus far using fibre tips in contactmode [7,29]. As reported earlier, it is verydi$cult to treat the canal walls with standardfibre tips, which emit primarily in the forwarddirection [7,30]. Therefore, it seems imperativethat a suitable side-firing fibre tip should bedeveloped before Er:YAG or similar lasers can

be applied clinically for treatment of rootcanals.

CONCLUSION

In conclusion, confocal laser scanningmicroscopy is an e#ective tool for investi-gation of laser e#ects on root canal dentine.The cavities resulting from Er:YAG laserirradiation at di#erent pulse durations (80–280 �s) without water spray cooling displaysimilar fluence dependences. However, theshortest pulses feature a larger ablation e#ectat moderate pulse fluences (�20 J/cm2), butalso the most pronounced saturation. This iseven more pronounced when water sprayis applied during the irradiation, whichmarkedly increases the ablation threshold.Scanning electron microscopy indicates cleanablation and no debris on the cavity walls aftersingle-pulse irradiation at �30 J/cm2, regard-less of the pulse duration or water spray appli-cation. However, a very smooth surface withno debris and open dentinal tubules isobserved at some repetitively irradiated sites(at 30 Hz), with and without application of thewater spray. It might be very interesting tofurther explore such regimes for root canalpreparation.

ACKNOWLEDGEMENTS

This work was supported by: Ministry of Education,Culture, Sports, Science and Technology, Japan, NIH(LAMMP) RR01192 and the Slovenian Ministry of Scienceand Technology (BM).

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Paper received 23 November 2001;accepted after revision 3 January 2002.


er:yag laser modification of root canal dentine: influence of pulse duration, repetitive irradiation...

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