Effects of sodium hypochlorite on gutta-percha and Resilon cones: An atomic force microscopy and scanning electron microscopy study

Effects of sodium hypochlorite on gutta-percha and Resilon cones: An atomic force microscopy and scanning electron microscopy study Özgür Topuz, DDS, PhD, a Baran Can Sağlam, DDS, PhD, a Fatih Şen, BSc, MSc, b Selda Şen, BSc, MSc, b Gülsün Gökağaç, DDS, PhD, b and Güliz Görgül, DDS, PhD, a Ankara, Turkey GAZI 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 1 Resilon 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 atomic force 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, there were no significant differences found among the GP groups. However significant differences were found among Resilon cones (P.05). SEM images and EDX graphics showed that there were no prominent differences between GP and Resilon groups. These results showed that 6% NaOCl solution can be used in the disinfection of GP and Resilon cones. 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) a Restorative Treatment and Endodontology, Faculty of Dentistry, Gazi University. b Chemistry, Middle East Technical University. 1079-2104/$ - see front matter 2011 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2011.03.002 One of the major objectives of root canal treatment is elimination of the microorganisms from the root canal system. 1 A positive correlation has been established between bacteria and endodontic disease. 2 Aseptic techniques are paramount in the prevention of contamination of the root canal system. 3 At present, gutta-percha (GP) cones are the most commonly used material for obturation of the root canal system. 4 Resilon (Pentron Clinical Technologies) is a thermoplastic synthetic polymer based root canal filling material that performs like GP in handling properties and is similar to GP in size. 5 Even though the cones are manufactured under aseptic conditions, they can become contaminated during handling, by aerosols, and by physical sources during the storage process. 6 Because of the thermoplastic characteristics of these materials, they cannot be sterilized using the conventional techniques (autoclave or dry heat), which may cause alterations in the GP structure 7 and subsequent dimensional changes, representing a great potential for failure of the endodontic obturation. 9 Sodium hypochlorite (NaOCl) is used a disinfecting agent most frequently in endodontic therapy for irrigation and also for cone disinfection. Increase in the concentration of NaOCl is directly proportional to its antimicrobial effect. 8 A member of the scanning probe microscopy (SPM) family, high-resolution atomic force microscopy (AFM) has made possible several applications in observing the surface characteristics of different materials. The principle of the AFM is the probing of the sample surface with a small tip attached to a flexible cantilever. The detection of several parameters between the tip and sample interactions provides qualitative and quantitative information about the sample structure. 9 In AFM, the sample surface is probed with a sharp tip attached to a flexible cantilever that deflects in z-direction as a result of the surface topography during tip scanning over the sample surface. 10 AFM offers the opportunity to view the 3-dimensional surface topography of specimens with high spatial resolution under a variety of conditions. 11 The aim of the present study was to investigate the effects of NaOCl solution at different time periods on GP and Resilon cones by AFM and scanning electron microscopy (SEM). MATERIALS AND METHODS Preparation of samples (experimental design) Six standardized ISO size 30 GP (Sure Endo) and Resilon cones (Pentron Clinical Technologies) were used for this study. GP and Resilon cones were cut 3 mm from their tip. One untreated GP cone and 1 untreated Resilon cone were used as control specimens. Cones were immersed in 6% NaOCl for 1, 5, 10, 20, and 30 minutes, e21

e22 Topuz et al. October 2011 Fig. 1. Tapping mode atomic force microscopy 3-dimensional images of untreated and treated gutta-percha (GP) and Resilon cones according to time of immersion. Table I. Root mean square (RMS) and depth analysis values of gutta-percha and Resilon cones Material Time RMS SE Depth (nm) SE Statistics Gutta-percha Control 41.6491 8.14396 275.3256 67.48806 No statistical differences were 1 min 49.8154 3.75820 270.8808 24.57208 determined; P.05 5 min 46.9628 4.30053 211.8245 15.14941 10 min 55.5072 6.51013 244.8538 33.72477 20 min 63.6215 7.33715 262.4849 28.16440 30 min 70.3450 9.88014 291.2413 33.24336 Resilon Control 44.15600 5.345249 239.5815 26.09098 Significant differences were 1 min 19.90454 2.109682 145.5540 22.43926 determined; P.05 5 min 15.73569 0.989846 93.9184 6.72523 10 min 22.58792 2.263568 82.8944 4.57264 20 min 15.88127 1.243424 87.9059 10.98355 30 min 24.29133 2.242379 133.2545 17.58742 thoroughly rinsed with 5 ml nanopure water, and then dried with filter paper. Atomic force microscopy AFM images were recorded on a multimode scanning probe microscopy (SPM) Nanoscope IVa (Veeco, Santa Barbara, CA) AFM under ambient conditions in order to evaluate the topographic deviation of these samples. AFM probes (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 were attached to a metal base with double-sided tape for AFM evaluation. The analyses in AFM were performed on 15 different regions located between 1 and 3 mm from the tip (0.502-Hz scan rate). Scanned areas were uniform squares 5 m 5 m. Tapping mode imaging (TMI) profiles were obtained for the first time for these type of materials. AFM images (512 512 lines) were processed and analyzed with Nanoscope version 6.13 software with only background slopes corrected. Three-dimensional images of GP and Resilon cones were recorded. For comparison purposes, the root mean square (RMS), a statistical measure of the magnitude of a varying quantity, and depth analysis were chosen to investigate the structure of GP and Resilon cones. SEM and energy-dispersive x-ray analysis Same control and treated GP and Resilon samples were used for SEM and energy-dispersive x-ray (EDX) analysis. Samples were investigated and compared by SEM (Quanta 400F Field Emission SEM) coupled to an EDX spectrometer. Samples were examined under 800, 5,000, 10,000, 20,000, and 40,000 magnifications. The EDX spectrum was collected from different regions on each sample. Statistical analysis All RMS and depth values were evaluated statistically with the SPSS version 17.0 statistical program. Means and SEs of the RMS and depth values achieved

Volume 112, Number 4 Topuz et al. e23 Fig. 2. Comparison of root mean square (RMS) and depth analysis of gutta-percha and Resilon cones. from TMI measurements were calculated. Normal distribution was assumed and checked with profiles, which were also used to detect possible outliers. For all data, after outliers were eliminated, Shapiro-Wilks test was applied to determine normality of the group distribution. If these values were normal, then 1-way analysis of variance (ANOVA) test was used for the evaluation of data. Kruskal-Wallis test was used if the distribution of the groups was not normal. The difference among the studied groups was tested by ANOVA with Fisher protected post hoc tests or Mann-Whitney pairwise multiple comparisons. Here, a P value of.05 was used to indicate a significance level. RESULTS Three-dimensional AFM images of GP and Resilon cones are shown in Fig. 1. The RMS values of GP cones were increased at all time periods when compared with the control sample, but there were no statistically significant differences. The highest RMS value was determined in the 30 minutes group (70.3450 9.88014). The depth analysis values were decreased at 1, 5, 10, and 20 minutes, but no statistically significant differences were detected (P.05). Depth analysis value was higher in the 30 minutes group in GP cones compared with the other time periods (291.2413 33.24336). Both RMS and depth analysis values for Resilon cones were decreased compared with the control sample, and statistically significant values were obtained (P.05). The highest RMS and depth analysis values were observed in the control group (44.15600 5.345249, 239.5815 26.09098; Table I; Fig. 2). Figure 3 represents some typical SEM images showing that there were no prominent differences on GP and Resilon surfaces at all magnifications ( 800, 5000, 10,000, 20,000, and 40,000).

e24 Topuz et al. October 2011 Fig. 3. Scanning electron microsopic images of gutta-percha (GP) and Resilon cones. A, GP control; B, GP 30 minutes; C, Resilon control; D, Resilon 30 minutes. Figure 4 shows EDX graphics of GP and Resilon cones. There were no differences in element ratios between the different time periods on both GP and Resilon cones. DISCUSSION The major causative role of microorganisms in the pathogenesis of pulp and periapical diseases has been demonstrated clearly. The elimination of microorganisms from infected root canal systems is a complicated process involving the use of various instrumentation techniques, irrigation regimens and intracanal medicaments. 12 When endodontic treatment is performed under aseptic conditions and according to accepted clinical principles, the success rate is generally high. 13 Sodium hypochlorite (NaOCl) is recommended and used by the majority of dentists because of its important properties of antimicrobial effect and tissue dissolution capacity with acceptable biologic compatibility. 14 Core-filling materials, such as GP and Resilon cones, are thermolabile and therefore ideally should be sterilized by the manufacturer. If not sterilized or if contaminated, they should be disinfected chairside through chemical procedures before their usage. 15 GP cones cannot be sterilized by heat. Therefore, a chairside decontamination using a chemical agent should be adopted in routine endodontic practice. 13 In this study, 6% NaOCl was used as a disinfecting agent, and the effects of NaOCl on the surface structure of GP and Resilon cones were investigated according to different times of immersion. Gomes et al found that 5.25% NaOCl is an effective agent for a rapid disinfection of gutta-percha cones. 16 Retamozo et al. (2010) 18 evaluated minimum contact time and concentration of NaOCl to eliminate Enterococcus faecalis from bovine dentin, and reported that the most effective irrigation regimen was 5.25% at 40 minutes, whereas irrigation

Volume 112, Number 4 Topuz et al. e25 Fig. 4. Energy-dispersive x-ray graphics. A, gutta-percha (GP) control; B, GP 30 minutes; C, Resilon control; D, Resilon 30 minutes. with 1.3% and 2.5% NaOCl for this same time interval was ineffective in removing E. faecalis from the infected dentin cylinders. They concluded that high concentration and long exposure to NaOCl are needed for elimination of E. faecalis-contaminated dentin. The commercially available product with 6% NaOCl concentration was used in the present study. The solution was used in 1-, 5-, 10-, 20-, and 30-minute immersion time periods to analyze the possible structural changes that may occur on the GP and Resilon cones over time. Although 1 or 5 minutes were enough for rapid disinfection, 10-, 20-, and 30-minute immersion time periods in 6% NaOCl solution showed surface alteration of GP and Resilon cones. There have been several studies investigating surface alterations of GP and Resilon cones by using SEM and AFM methods. However, the tapping mode AFM, which provides high-resolution images, was used in the present study for the first time for surface characterization of GP and Resilon cones. 17 The advantages of 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 obtained for more accurate calculations and quantitative measures about surface alterations. In the GP groups, the RMS values increased in conjunction with increased immersion time, but there were no statistically significant differences in either RMS or depth analysis. As a result, we think that although NaOCl affects the surface topography of GP cones, no statistically significant differences occurred. Valois, Silva, and Azevedo (2005) 8 investigated by AFM the effects of 0.5%%, and 5.25% NaOCl solutions on the surface of GP points. They found that 2.5% and 5.25% NaOCl caused topographic changes on GP cones. 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 the cones by the contact mode facility of AFM. Their results showed that 5.25% NaOCl caused statistically

e26 Topuz et al. October 2011 significant surface alterations on GP cones. At 20- and 30-minute time periods, they were unable to measure the deterioration, owing to exceeding the limits of the AFM scanner used. In the present study, we were able to successfully measure the deteriorations at 1-, 5-, 10-, 20-, and 30 minute time periods. There were statistically significant differences in RMS results and depth analysis values in the Resilon group. Isci, Yoldas, and Dumani (2006) 19 evaluated the surface changes on Resilon cones treated with 5.25% NaOCl by AFM at 1- and 5-minute periods. The RMS and depth analysis values of 1-, 5-, 10-, 20-, and 30- minute groups were lower than in the control group. Isci, Yoldas, and Dumani 19 reported that RMS values of the 5-minute immersion time for Resilon cones were lower than in the control group. Their study emphasizes that NaOCl affected the surfaces of Resilon cones significantly, consistent with the results of the present study. According to these results, it can be suggested that NaOCl has a smoothing effect on Resilon cones. In root canal obturation, Resilon adheres to dentin with epiphany sealer. The smooth Resilon cones may cause an adverse effect in this adhesive technique, and this is another matter of concern that requires further study. In addition to these findings, the surface alterations of the GP and Resilon cones were analyzed with SEM in an effort to explain the AFM results. It is thought that the surface modifications seen in SEM examinations emanate from the production processes of the cones, regardless of the solution and the immersion times that were selected. Gomes et al. (2007) 7 used SEM and investigated surface alterations on GP and Resilon cones treated with 5.25% NaOCl solution for up to 30 minutes. They reported that there were no differences between the surface observations of the samples of GP and Resilon cones immersed in the solutions at the different time periods. These results are also compatible with the present study. EDX was used in the present study for evaluating possible chemical changes; however, EDX results showed that there were no differences in GP and Resilon cones at the different time periods. The main element rates constituting the GP and Resilon cones did not change. In conclusion, 6% NaOCl solution can be used in the disinfection of GP and Resilon cones. No alterations were observed on the GP cones, but it can change the surface of Resilon cones. REFERENCES 1. Kakehashi S, Stanley HP, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. J Oral Surg 1965;20:340-9. 2. Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1994;78:522-30. 3. Royal MJ, Williamson AE, Drake DR. 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