Effects of sodium hypochlorite on gutta-percha and Resiloncones: An atomic force microscopy and scanning electronmicroscopy studyÖzgür Topuz, DDS, PhD,a Baran Can Saglam, DDS, PhD,a Fatih Sen, BSc, MSc,b

Selda Sen, BSc, MSc,b Gülsün Gökagaç, DDS, PhD,b and Güliz Görgül, DDS, PhD,a Ankara, TurkeyGAZI UNIVERSITY AND MIDDLE EAST TECHNICAL UNIVERSITY

The aim of this study was to evaluate the effects of 6% sodium hypochlorite (NaOCl) on gutta-percha (GP)and Resilon cones. Six standardized GP and Resilon cones were selected and cut 3mm from their tip. One GP and 1Resilon cone were used as control samples. Cones were immersed in 6% NaOCl for 1, 5, 10, 20, and 30 minutes,thoroughly rinsed with nanopure water, and dried with filter paper. Then, surface topography was analyzed by atomicforce microscopy and scanning electron microscopy coupled to an energy-dispersive x-ray (EDX) spectrometer.According to the root mean square and depth analysis values obtained from atomic force microscopic evalution, therewere no significant differences found among the GP groups. However significant differences were found amongResilon cones (P � .05). SEM images and EDX graphics showed that there were no prominent differences between GPand Resilon groups. These results showed that 6% NaOCl solution can be used in the disinfection of GP and Resiloncones. No alterations were observed on the GP cones, but it can change the surface of Resilon cones. (Oral Surg Oral

Med Oral Pathol Oral Radiol Endod 2011;112:e21-e26)

One of the major objectives of root canal treatment iselimination of the microorganisms from the root canalsystem.1 A positive correlation has been establishedbetween bacteria and endodontic disease.2 Aseptictechniques are paramount in the prevention of contam-ination of the root canal system.3

At present, gutta-percha (GP) cones are the most com-monly used material for obturation of the root canal sys-tem.4 Resilon (Pentron Clinical Technologies) is a ther-moplastic synthetic polymer–based root canal fillingmaterial that performs like GP in handling properties andis similar to GP in size.5 Even though the cones aremanufactured under aseptic conditions, they can becomecontaminated during handling, by aerosols, and by phys-ical sources during the storage process.6 Because of thethermoplastic characteristics of these materials, they can-not be sterilized using the conventional techniques (auto-clave or dry heat), which may cause alterations in the GPstructure7 and subsequent dimensional changes, represent-ing a great potential for failure of the endodontic obtura-tion.9

Sodium hypochlorite (NaOCl) is used a disinfectingagent most frequently in endodontic therapy for irrigationand also for cone disinfection. Increase in the concentra-

aRestorative Treatment and Endodontology, Faculty of Dentistry,Gazi University.bChemistry, Middle East Technical University.1079-2104/$ - see front matter© 2011 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2011.03.002

tion of NaOCl is directly proportional to its antimicrobialeffect.8

A member of the scanning probe microscopy (SPM)family, high-resolution atomic force microscopy (AFM)has made possible several applications in observing thesurface characteristics of different materials. The principleof the AFM is the probing of the sample surface with asmall tip attached to a flexible cantilever. The detection ofseveral parameters between the tip and sample interac-tions provides qualitative and quantitative informationabout the sample structure.9 In AFM, the sample surface isprobed with a sharp tip attached to a flexible cantileverthat deflects in z-direction as a result of the surface topog-raphy during tip scanning over the sample surface.10 AFMoffers the opportunity to view the 3-dimensional surfacetopography of specimens with high spatial resolution un-der a variety of conditions.11

The aim of the present study was to investigate theeffects of NaOCl solution at different time periods onGP and Resilon cones by AFM and scanning electronmicroscopy (SEM).

MATERIALS AND METHODS

Preparation of samples (experimental design)Six standardized ISO size 30 GP (Sure Endo) and

Resilon cones (Pentron Clinical Technologies) were usedfor this study. GP and Resilon cones were cut 3 mm fromtheir tip. One untreated GP cone and 1 untreated Resiloncone were used as control specimens. Cones were im-

mersed in 6% NaOCl for 1, 5, 10, 20, and 30 minutes,

e21

OOOOEe22 Topuz et al. October 2011

thoroughly rinsed with 5 mL nanopure water, and thendried with filter paper.

Atomic force microscopyAFM images were recorded on a multimode scanning

probe microscopy (SPM) Nanoscope IVa (Veeco, SantaBarbara, CA) AFM under ambient conditions in order toevaluate the topographic deviation of these samples. AFMprobes (curvature radius �10 nm; model TAP 300 Al)mounted on 3-dimensional motion cantilevers (125 �m)with force constant of 40 N/m were used. Samples wereattached to a metal base with double-sided tape for AFMevaluation. The analyses in AFM were performed on 15different regions located between 1 and 3 mm from the tip(0.502-Hz scan rate). Scanned areas were uniform squares5 �m � 5 �m. Tapping mode imaging (TMI) profileswere obtained for the first time for these type of materials.

AFM images (512 � 512 lines) were processed andanalyzed with Nanoscope version 6.13 software with

Fig. 1. Tapping mode atomic force microscopy 3-dimensioncones according to time of immersion.

Table I. Root mean square (RMS) and depth analysisMaterial Time RMS SE

Gutta-percha Control 41.6491 8.143961 min 49.8154 3.758205 min 46.9628 4.30053

10 min 55.5072 6.5101320 min 63.6215 7.3371530 min 70.3450 9.88014

Resilon Control 44.15600 5.3452491 min 19.90454 2.1096825 min 15.73569 0.989846

10 min 22.58792 2.26356820 min 15.88127 1.24342430 min 24.29133 2.242379

only background slopes corrected. Three-dimensional

images of GP and Resilon cones were recorded. Forcomparison purposes, the root mean square (RMS), astatistical measure of the magnitude of a varying quan-tity, and depth analysis were chosen to investigate thestructure of GP and Resilon cones.

SEM and energy-dispersive x-ray analysisSame control and treated GP and Resilon samples

were used for SEM and energy-dispersive x-ray (EDX)analysis. Samples were investigated and compared bySEM (Quanta 400F Field Emission SEM) coupled to anEDX spectrometer. Samples were examined under�800, �5,000, �10,000, �20,000, and �40,000 mag-nifications. The EDX spectrum was collected from dif-ferent regions on each sample.

Statistical analysisAll RMS and depth values were evaluated statisti-

cally with the SPSS version 17.0 statistical program.

ges of untreated and treated gutta-percha (GP) and Resilon

s of gutta-percha and Resilon conesepth (nm) SE Statistics

275.3256 67.48806 No statistical differences weredetermined; P � .05270.8808 24.57208

211.8245 15.14941244.8538 33.72477262.4849 28.16440291.2413 33.24336239.5815 26.09098 Significant differences were

determined; P � .05145.5540 22.4392693.9184 6.7252382.8944 4.5726487.9059 10.98355

133.2545 17.58742

al ima

valueD

Means and SEs of the RMS and depth values achieved

nalysis

OOOOEVolume 112, Number 4 Topuz et al. e23

from TMI measurements were calculated. Normal dis-tribution was assumed and checked with profiles, whichwere also used to detect possible outliers. For all data,after outliers were eliminated, Shapiro-Wilks test wasapplied to determine normality of the group distribu-tion. If these values were normal, then 1-way analysisof variance (ANOVA) test was used for the evaluationof data. Kruskal-Wallis test was used if the distribu-tion of the groups was not normal. The differenceamong the studied groups was tested by ANOVA withFisher protected post hoc tests or Mann-Whitney pair-wise multiple comparisons. Here, a P value of �.05was used to indicate a significance level.

RESULTSThree-dimensional AFM images of GP and Resi-

lon cones are shown in Fig. 1. The RMS values of GPcones were increased at all time periods when com-

Fig. 2. Comparison of root mean square (RMS) and depth a

pared with the control sample, but there were no

statistically significant differences. The highest RMSvalue was determined in the 30 minutes group(70.3450 � 9.88014). The depth analysis valueswere decreased at 1, 5, 10, and 20 minutes, but nostatistically significant differences were detected(P � .05). Depth analysis value was higher in the 30minutes group in GP cones compared with the othertime periods (291.2413 � 33.24336). Both RMS anddepth analysis values for Resilon cones were de-creased compared with the control sample, and sta-tistically significant values were obtained (P � .05).The highest RMS and depth analysis values wereobserved in the control group (44.15600 � 5.345249,239.5815 � 26.09098; Table I; Fig. 2).

Figure 3 represents some typical SEM imagesshowing that there were no prominent differences onGP and Resilon surfaces at all magnifications (�800,

of gutta-percha and Resilon cones.

�5000, �10,000, �20,000, and �40,000).

OOOOEe24 Topuz et al. October 2011

Figure 4 shows EDX graphics of GP and Resilon cones.There were no differences in element ratios between thedifferent time periods on both GP and Resilon cones.

DISCUSSIONThe major causative role of microorganisms in the

pathogenesis of pulp and periapical diseases has beendemonstrated clearly. The elimination of microorgan-isms from infected root canal systems is a complicatedprocess involving the use of various instrumentationtechniques, irrigation regimens and intracanal medica-ments.12 When endodontic treatment is performed un-der aseptic conditions and according to accepted clini-cal principles, the success rate is generally high.13

Sodium hypochlorite (NaOCl) is recommended andused by the majority of dentists because of its importantproperties of antimicrobial effect and tissue dissolution

Fig. 3. Scanning electron microsopic images of gutta-perchResilon control; D, Resilon 30 minutes.

capacity with acceptable biologic compatibility.14

Core-filling materials, such as GP and Resilon cones,are thermolabile and therefore ideally should be steril-ized by the manufacturer. If not sterilized or if contam-inated, they should be disinfected chairside throughchemical procedures before their usage.15 GP conescannot be sterilized by heat. Therefore, a chairsidedecontamination using a chemical agent should be ad-opted in routine endodontic practice.13 In this study,6% NaOCl was used as a disinfecting agent, and theeffects of NaOCl on the surface structure of GP andResilon cones were investigated according to differenttimes of immersion. Gomes et al found that 5.25%NaOCl is an effective agent for a rapid disinfection ofgutta-percha cones.16 Retamozo et al. (2010)18 evalu-ated minimum contact time and concentration ofNaOCl to eliminate Enterococcus faecalis from bovinedentin, and reported that the most effective irrigation

) and Resilon cones. A, GP control; B, GP 30 minutes; C,

a (GP

regimen was 5.25% at 40 minutes, whereas irrigation

OOOOEVolume 112, Number 4 Topuz et al. e25

with 1.3% and 2.5% NaOCl for this same time intervalwas ineffective in removing E. faecalis from the infecteddentin cylinders. They concluded that high concentrationand long exposure to NaOCl are needed for elimination ofE. faecalis-contaminated dentin. The commercially avail-able product with 6% NaOCl concentration was used inthe present study. The solution was used in 1-, 5-, 10-, 20-,and 30-minute immersion time periods to analyze thepossible structural changes that may occur on the GP andResilon cones over time. Although 1 or 5 minutes wereenough for rapid disinfection, 10-, 20-, and 30-minuteimmersion time periods in 6% NaOCl solution showedsurface alteration of GP and Resilon cones.

There have been several studies investigating surfacealterations of GP and Resilon cones by using SEM andAFM methods. However, the tapping mode AFM,which provides high-resolution images, was used in thepresent study for the first time for surface characteriza-tion of GP and Resilon cones.17 The advantages of

Fig. 4. Energy-dispersive x-ray graphics. A, gutta-percha (Gminutes.

tapping-mode operation are elimination of the lateral

force, which damages the surface in the contact mode,and the improvement in lateral resolution.

Both RMS and depth analysis values were obtainedfor more accurate calculations and quantitative mea-sures about surface alterations.

In the GP groups, the RMS values increased inconjunction with increased immersion time, but therewere no statistically significant differences in eitherRMS or depth analysis. As a result, we think thatalthough NaOCl affects the surface topography of GPcones, no statistically significant differences occurred.Valois, Silva, and Azevedo (2005)8 investigated byAFM the effects of 0.5%%, and 5.25% NaOCl solu-tions on the surface of GP points. They found that 2.5%and 5.25% NaOCl caused topographic changes on GPcones. In another study, Valois, Silva, and Azevedo(2005)11 used 5.25% NaOCl on GP points for 1-, 5-,10-, 20-, and 30-minute time periods and examined thecones by the contact mode facility of AFM. Their

ntrol; B, GP 30 minutes; C, Resilon control; D, Resilon 30

P) co

results showed that 5.25% NaOCl caused statistically

OOOOEe26 Topuz et al. October 2011

significant surface alterations on GP cones. At 20- and30-minute time periods, they were unable to measurethe deterioration, owing to exceeding the limits of theAFM scanner used. In the present study, we were ableto successfully measure the deteriorations at 1-, 5-, 10-,20-, and 30 minute time periods.

There were statistically significant differences inRMS results and depth analysis values in the Resilongroup. Isci, Yoldas, and Dumani (2006)19 evaluated thesurface changes on Resilon cones treated with 5.25%NaOCl by AFM at 1- and 5-minute periods. The RMSand depth analysis values of 1-, 5-, 10-, 20-, and 30-minute groups were lower than in the control group.Isci, Yoldas, and Dumani19 reported that RMS valuesof the 5-minute immersion time for Resilon cones werelower than in the control group. Their study emphasizesthat NaOCl affected the surfaces of Resilon cones sig-nificantly, consistent with the results of the presentstudy. According to these results, it can be suggestedthat NaOCl has a smoothing effect on Resilon cones. Inroot canal obturation, Resilon adheres to dentin withepiphany sealer. The smooth Resilon cones may causean adverse effect in this adhesive technique, and this isanother matter of concern that requires further study.

In addition to these findings, the surface alterationsof the GP and Resilon cones were analyzed with SEMin an effort to explain the AFM results. It is thought thatthe surface modifications seen in SEM examinationsemanate from the production processes of the cones,regardless of the solution and the immersion times thatwere selected. Gomes et al. (2007)7 used SEM andinvestigated surface alterations on GP and Resiloncones treated with 5.25% NaOCl solution for up to 30minutes. They reported that there were no differencesbetween the surface observations of the samples of GPand Resilon cones immersed in the solutions at thedifferent time periods. These results are also compati-ble with the present study. EDX was used in the presentstudy for evaluating possible chemical changes; how-ever, EDX results showed that there were no differ-ences in GP and Resilon cones at the different timeperiods. The main element rates constituting the GPand Resilon cones did not change.

In conclusion, 6% NaOCl solution can be used in thedisinfection of GP and Resilon cones. No alterationswere observed on the GP cones, but it can change thesurface of Resilon cones.

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