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EGYPTIANDENTAL JOURNAL

Vol. 60, 4739:4747, October, 2014

* Associate Professor, Department of Oral Biology, Faculty of Dentistry, Mansoura University, Egypt.** Associate Professor, Department of Dental Biomaterials, Faculty of Dentistry, Mansoura University, Egypt.*** Associate Professor, Department of Endodonditcs, Faculty of Dentistry, Tanta University, Egypt.

SURFACE ROUGHNESS OF APICAL ROOT CANAL WALLS PREPARED BY ROTARY VS HAND INSTRUMENTATION WITH TWO IRRIGATING

PROTOCOLS; ATOMIC FORCE MICROSCOPIC STUDY

Heba M. Elsabaa* ; Salwa Abd El-Raof El-Negoly** and Ahmed Hussien Labib***

ABSTRACTAim The aim of root canal treatment is to achieve a canal free of micro organisms, residual pulp

remnants and smear layer. This study aimed to compare ex vivo the surface roughness of apical root canal walls prepared by rotary Vs hand instrumentation with two irrigating protocols using atomic force microscopy.

Methodology Seventy-five freshly extracted central incisors sound teeth were selected. Their roots were allocated to four experimental groups and a negative control one (n=15). The roots of group 1 were prepared using St St K-type hand file and irrigated with 15 ml of 2.5% NaOCl and 5 ml of 17% EDTA. In between each instrument change, the canal was filled with EDTA for 1 min. While the roots of group 2 were prepared as group 1 and irrigated with 15 ml of 2.5% NaOCl and after complete preparation, the canal was irrigated with 9% HEBP for 5 minutes. In addition, the roots of group 3 were prepared using Protaper Next NiTi rotary file and irrigated using the same protocol as group 1. The roots of group 4 were prepared as group 3 and irrigated using the same protocol as group 2. The root canals of the negative control were left unprepared. The roots of all groups were split longitudinally in a labiolingual direction. The apical thirds’ root dentin of the negative control were serially polished and placed in an ultrasonic cleaner with distilled water to remove polishing debris. Two halves of each tooth was subjected to AFM surface analyses and each half was analyzed at five different points. The photomicrographs of each specimen were scored for smear layer remnants and the results of the scoring system for the four experimental groups were evaluated statistically and in addition, the results of roughness average (Ra) for all five groups were expressed as means±SD.

Results The values of surface roughness varied significantly depending on the type of instrumentation (St St hand K-type file Vs Protaper Next NiTi rotary file) and type of irrigating protocol (NaOCl with EDTA Vs NaOCl associated with HEBP chelating agent) (P> 0.001). The highest surface roughness with large amount of smear layer was found after St St hand K-type file instrumentation and NaOCl with EDTA irrigation. A slightly rough surface due to the presence of a small amount of smear layer was found with Protaper Next NiTi rotary file instrumentation followed by NaOCl associated with HEBP chelating agent.

Conclusions Based on the results of atomic force microscopy and compared with the other groups, root canals prepared using Protaper Next NiTi rotary file and irrigated with NaOCl associated with HEBP had the least amount of smear layer and surface roughness.

Keywords: Surface roughness; smear layer; root dentin; rotary instrumentation; hand instrumentation; irrigating solutions; atomic force microscopy.

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INTRODUCTION

Cleaning and shaping are the main goals of root canal instrumentation [1]. In the last 20 years, several new instrumentation systems based on rotary nickel-titanium (NiTi) have been developed for root canal preparation. The advantages of rotary NiTi instruments over hand ones include facilitating canal preparation, preserving the shape of curved canals and producing smooth surfaces in lesser time [2]. The traditional stainless steel (St St) K-type hand files are triangular in cross-section with sharp cutting edges. In contrary, most of rotary files were designed to have a broad radial lands with non-cutting tips [3].

Various intracanal irrigants can be used to minimize the bacterial load in the infected teeth. Irrigation serves as a physical flush to remove debris as well as serves as an antimicrobial agent, tissue solvent and lubricant. There is no single irrigating solution that alone sufficiently covers all of the functions required from an irrigant [4]. Sodium hypochlorite solution (NaOCl) is the most common irrigating agent used in biomechanical preparation based on its excellent antimicrobial activity and tissue-dissolving capabilities of pulpal remnants and collagen [5]. In spite of its unpleasant taste, toxicity, and inability to completely remove the smear layer, NaOCl remains the recommended irrigant of choice [6]. The hydroxyethylidene bisphosphonate (HEBP), also known as etidronate, is a decalcifying agent that shows only little short-term interference with sodium hypochlorite. It has recently been suggested as a possible alternative to citric acid or ethylenediaminetetraacetic acid (EDTA) [7].

Many investigators [8, 9] demonstrated that a smear layer is created by endodontic instruments and may be altered by irrigant solutions used during the endodontic procedures. Different endodontic systems and procedures may produce a different amount of debris and a different morphology of smear layer [8, 10]. The presence of such a layer

compromises the penetration of root canal irrigants and the obturating materials into dentinal tubules, which increases the risk of bacterial infection and microleakage [11]. Thus, the removal of smear layer can improve the adaptation of root canal filling materials [12] and the apical microleakage is less in the absence of the smear layer [13].

Atomic force microscopy (AFM) offers the opportunity to image the three-dimensional surface topography of biological specimens with high spatial resolution under a wide variety of conditions. These include exposure to air, water and other storage solutions at elevated or reduced temperatures [14]. Up to date, most of the previous studies evaluated the nanostructural alterations of rotary instruments after their immersion in irrigating solutions [15, 16]. Also, there is a scarcity of information regarding the potential role of AFM for the examination of the root canal dentinal walls changes after using different endodontic instrumentation techniques and irrigation protocols. This study aimed to investigate the surface roughness for the apical one-third of the root canal dentinal walls prepared by rotary Vs hand instrumentation with two irrigating protocols using atomic force microscopy.

MATERIALS AND METHODS

Seventy-five periodontally involved freshly extracted central incisors sound teeth from patients aged 50 to 65 years were selected to be used in the present study. The teeth were obtained according to a protocol that was approved by our Institution Committee for Ethics of Research. The inclusion criteria for the study were single rooted teeth with complete formed apices and straight canals (checked with periapical radiographs from mesial and distal surfaces), almost equal dentinal walls proportions and teeth having no calcifications. Teeth of tortuous canals, open apex, curved canals, thin dentinal walls were excluded.

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All the collected teeth were subjected to thorough scaling (Varios 550, NSK Nakanishi, Japan) and ultrasonic cleaning (Pro-sonic 300 MTH, Sultan Chemists Inc, Englewood, NJ) to get rid of both hard and soft deposits. The teeth were stored in artificial saliva described by Arvidson and Johansson [17] that was prepared by Chemistry Department, Faculty of Pharmacy, Mansoura University. The roots were separated from their crowns at the cemento-enamel junction using a low speed water-cooled diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). The roots were allocated to four experimental groups and a negative control according to the type of instruments and the irrigation protocols used (n=15). The pulp tissues were extirpated using a barbed broach (Dentsply Maillefer, Ballaigues, Switzerland). The working lengths were measured by reducing 1mm from lengths recorded when tips of # 10 St St K-type hand file (Dentsply Maillefer, Ballaigues, Switzerland) were visible at the apical foramina.

The roots of group 1 were prepared using step back technique up to size 30/0.02 St St K-type hand file (Dentsply Maillefer, Ballaigues, Switzerland) at the end point apically and irrigated alternately with 15 ml of 2.5% NaOCl (Sigma-Aldrich, St Louis, MO, USA) and 5 mL of 17% EDTA (Sigma Aldrich, St. Louis, MO, USA). During instrumentation, the canal was filled with NaOCl. In between each instrument change, the canal was filled with EDTA for 1 min. While the roots of group 2 were prepared as group 1 and irrigated with 15 ml of 2.5% NaOCl and after complete preparation, the canal was irrigated with 9% HEBP (Zschimmer & Schwarz Mohsdorf GmbH & Co KG, Burgstädt, SN, Germany) for 5 minutes [18]. In addition, the roots of group 3 were prepared using Protaper Next NiTi rotary file up to size 30/0.07 file (Dentsply Maillefer, Ballaigues, switzerland ) at the end point apically and irrigated using the same protocol as group 1. The roots of group 4 were prepared as

group 3 and irrigated using the same protocol as group 2. All canals were dried with sterile adsorbent paper points. The negative control group was left unprepared.

The roots of all groups were split using a tapering fissure diamond bur cutting a groove longitudinally in a labiolingual direction. To avoid contamination of the canals by the separation process, the last part of the separation was performed by splitting the root with a chisel. The root halves were cleaned from grinding material and dried using water and air-blasting for three seconds [19]. The apical root thirds of the negative control group were fixed with stick wax in acrylic resin cylinders, and the root canal surfaces of the specimens were then flattened and serially polished up to #2500-grit roughness with silicon carbide abrasive papers. Specimens were placed in an ultrasonic cleaner (T1440D, Odontobra´ s Ltda., Ribeira˜o Preto, SP, Brazil) with distilled water for 10 min to remove polishing debris.

A stereomicroscope (Nikon 88286, Tokyo, Japan) at 40x magnification was employed to select the samples without cracks or structural defects, which would compromise the results of the study. Two halves of each tooth was subjected to AFM (Shimadzu Co, Kyoto, Japan) surface analysis for the apical one-third of the root canal. Each specimen was fixed to the AFM microscope holder with a cyanoacrylate adhesive and the surface morphology of the prepared root canal was probed in ‘contact’ mode; physical contact between the surface and the AFM tip was maintained at all times with constant force. Each half was analyzed at five different points; four at the corner and one in the center of each specimen. The surface roughness of each point was scored from Ra of all particles included in the area (Fig. 1). Imaging was performed with standard geometry silicon nitride probes. AFM images were collected at a very low scan rate of 1 Hz to obtain details of the dentin structure and

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to avoid damaging the tip. An area of 2.0 μm2 was captured for the AFM image using a scanning speed of 80 μms in air [20]. To assess the degree of smear layer removal, scoring criteria were recorded for the photomicrographs of each specimen for the four experimental groups according to the rating system of none, mild, moderate and heavy smear layer amount.

The scoring was performed individually in a blinded fashion by 2 calibrated examiners to determine the inter- and intra-examiner reliability of a standardized scoring method. The results of the scoring system for the four experimental groups were evaluated statistically using Kruskal-Wallis test/ Mann-Whitney U-test. In addition, the results of roughness average (Ra) for all five groups were expressed as the means±SD. A two-way Analysis of Variance (ANOVA) followed by least significant difference (LSD) post hoc test were conducted for Ra to determine if there was a significant difference among the five groups. A P value <0.05 was considered statically significant. The factors for ANOVA were 1) type of instrumentation and 2) type of irrigation protocol. All analyses were performed with SPSS, for windows statistical package version 17 (SPSS Inc., Chicago, IL, USA).

RESULTS

In group 1, a uniform and homogenous heavy smear layer was found completely covering the root canal dentinal surface. Group 2 showed inhomogeneous and irregular moderate smear layer covering the root canal dentinal wall. Specimens of group 3 revealed moderate amount of the smear layer covering the openings of the dentinal wall (Fig. 2A). While, group 4 showed a slightly rough surface due to the presence of a small amount of smear layer (Fig. 2B). The micromorphological observation of the dentin obtained from negative control group revealed the least amount of the smear layer.

There were no statistically significant differences between the intraexaminer (P= 0.519) or interexaminer (P= 0.724) readings regarding the scoring features that were observed for the four experimental groups (Tab. 1). Application of Kruskal-Wallis test showed that there were statistically significant differences in the amount of smear layer removal between the four experimental groups (P> 0.001) (Tab. 2). Using the Mann-Whitney U for two groups comparison revealed a significant differences between group 1 and groups 2,3 & 4, group 2 and groups 3 & 4 and between group 3 &4 (P> 0.001) (Tab. 3).

The results of two-way ANOVA for the five groups indicated that surface roughness values var-ied significantly depending on the type of instru-mentation (St St hand K-type file Vs Protaper Next NiTi rotary file) and type of irrigating protocol (Na-OCl with EDTA Vs NaOCl associated with HEBP chelating agent) (P> 0.001). Meanwhile, significant interaction was found between type of instrumenta-tion and type of irrigating protocol (P>0.001) (Tab. 4). The mean Ra values and SDs of the groups are presented in Table 4 & Figure 3. The LSD post hoc test showed that the lowest mean surface roughness value for particle size was obtained in the nega-tive control group (222.63±06) and the highest one was for group1 (1190.65±08) followed by group 2 (950.68±02), group 3 (700.00±05) and group 4 (354.64±06). There were significant differences among the five groups (Tab. 4).

FIG. (1) AFM contact mode images of root canal dentinal walls at the apical one third for the root canals analyzed for surface roughness for a point of 2µm x 2µm.

SURFACE ROUGHNESS OF APICAL ROOT CANAL WALLS PREPARED (4743)

FIG. (2) Three-dimensional presentation of the AFM contact mode images of root canal dentinal walls at the apical one third for the root canals prepared by Protaper Next NiTi with NaOCl and EDTA (A), Protaper Next NiTi with NaOCl associated with HEBP (B).

TABLE (1) Statistical results for intraexaminer and interexaminer variability attributed to the scoring system used in the present study.

Intraexaminer variability Interexaminer variability

First reading (mean ± SD)

Second reading (mean ± SD)

P valueFirst examiner (mean ± SD)

Second examiner (mean ± SD)

P value

1.7 ± 0.9 1.9 ± 0.8 0.519 0.3 ± 0.4 0.3 ± 0.7 0.724

*. The mean difference is significant at the 0.05 level. SD, Standard deviation.

TABLE (2) Kruskal-Wallis statistical results for the scoring system and their significance.

Groups N Mean Rank Chi-Square df P value

Scoring K-type file+ NaOCl with EDTA 30 104.83 109.380 3 0.0001

K-type file+ NaOCL with HEBP 30 75.80

Protaper Next NiTi+ NaOCl with EDTA 30 42.82

Protaper Next NiTi+ NaOCl with HEBP 30 18.55

*. The mean difference is significant at the 0.05 level.

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DISCUSSION

Thorough instrumentation of the apical re-gion has long been considered to be an essential component in the cleaning and shaping process. Spangberg [21] concluded that the last few milli-meters that approach the apical foramen are criti-cal in the instrumentation process. Thus, one aim of the present study was investigating the surface roughness of apical root canal with different instru-mentation techniques and irrigation protocols. Up to date, most of the previous studies had scanned the root canal dentinal walls with the convention-al scanning electron microscopy [21-24]. The greater

TABLE (3) Mann-Whitney U statistical results for the scoring system and their significance.

Groups Mann-Whitney U Z P value

Group 1 x group 2 20.000 -6.650 0.0001

Group 1 x group 3 0.000 -6.932 0.0001

Group 1 x group 4 0.000 -6.957 0.0001

Group2 x group3 11.000 -6.872 0.0001

Group 2 x group 4 0.000 -7.042 0.0001

Group 3 x group 4 91.500 -5.783 0.0001

*. The mean difference is significant at the 0.05 level.

TABLE (4) Two-way ANOVA and LSD post-hoc test for instruments and irrigant effects on Ra and their statistical significance.

Two-way ANOVA (F ratio and P-value) LSD post-hoc test

Instruments IrrigantsInstruments * Irrigants

Instrumentation Irrigating solutions Mean± SD

116104.027 (0.0001)

19944.587 (0.0001)

372.493 (0.0001)

Negative control Negative control 222.63±06*

K-type fileNaOCl with EDTA 1190.65±08*NaOCl with HEBP 950.68±02*

Protaper Next NiTi

NaOCl with EDTA 700.00±05*NaOCl with HEBP 354.64±06*

*. The mean difference is significant at the 0.05 level.

FIG. (3) Line chart histogram showing the mean Ra values (nm) regarding the type of instrumentation and the different irrigation protocols used.

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advantages of AFM over the conventional ones pro-vided a second rational for performing the present study. Among these, the high resolution pattern, avoidance of special sample preparation and the real-time detection of samples under nearly physi-ological environment [25].

Using a negative control group was to measure the normal value for surface roughness of root dentin at the apical one third. In addition, the present study compared two irrigating protocols (NaOCl with EDTA Vs NaOCl associated with HEBP). Selecting NaOCl as the standard irrigating protocol comes from its ability to dissolve both vital and necrotic tissues as well as its broad-spectrum antimicrobial property. It also removes the organic portion of the smear layer created during instrumentation of the root canal space [26]. However, the main disadvantage of NaOCl is its inability to remove the inorganic portion of the smear layer from the root canal system [27]. To overcome its disadvantage NaOCl with EDTA or associated with HEBP was used as possible alternative for NaOCl in the present study. This in compliance with Zehnder et al [28]

who assessed the interaction of citric acid, sodium triphosphate, amino tris methylenephosphonic acid, EDTA and HEBP with NaOCl, the indispensable endodontic irrigant. They concluded EDTA and citric acid negatively interfered with NaOCl, while HEBP did not.

The smear layer of dentin debris was formed in the other four experimental groups. The amount of smear layer varied according to the instrumentation systems and irrigation protocols used. The hand St St K-file step-back technique with NaOCl and EDTA produced a uniform and homogenous heavy smear layer and this in compliance with Tinaz et al, [29]

who evaluate the smear layer removal effectiveness of EDTA using two techniques through scanning electron microscope and they concluded that all groups had significantly higher smear layer scores at apical compared to coronal sections.

Using hand St St K-file step-back technique with NaOCl irrigant associated with HEBP as an adjuvant

chelating agent resulted in irregular inhomogeneous moderate smear layer covering the root canal dentinal wall. These results were in accordance with Zehnder et al [30] who considered HEBP as a unique chelator that can be mixed with NaOCl without interfering with its antimicrobial property and suggested it as a substitute for traditional chelators due to fewer effects observed on dentin structure. Also, Lottanti et al [31] reported that HEBP appear to have a minimal effect on dentine walls, yet can still reduce smear layer.

The roots that were prepared using Protaper Next NiTi rotary file and irrigated using NaOCl with EDTA revealed moderate amount of the smear layer and this comes with Yang et al [32] who concluded that ProTaper and Hero Shaper instruments in combination with NaOCl and EDTA irrigation produced a clean and debris-free canal surface in the coronal and middle thirds, but were unable to produce a canal surface free from debris and smear layer in the apical third. However, the canals prepared with ProTaper instruments showed smaller amounts of debris and smear layer remaining in the apical region.

Mean while, the roots that were prepared using Protaper Next NiTi rotary file and irrigated using NaOCl associated with HEBP showed a slightly rough surface due to the presence of a small amount of smear layer. This in agreement with Paqué et al [33] who investigated the amount of hard-tissue debris accumulation and reduction during rotary root canal instrumentation by etidronic acid in a sodium hypochlorite irrigant and concluded that a hypochlorite-compatible chelator can reduce but not completely prevent hard-tissue debris accumulation during rotary root canal instrumentation.

AFM results of our study reveled that a higher surface roughness and large amount of smear layer were for the St St K-file step-back technique with NaOCl irrigant and EDTA chelator and the lowest surface roughness values were for Protaper Next

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NiTi rotary file with NaOCl and HEBP irrigant. This was inagreement with, Baumgartner and Mader [34] who found that the combination of 17% EDTA and 5% NaOCl is an effective irrigating solution in removing the smear layer in the apical third of instrumented canals. In contrast with our results Mancini and Cianconi [35] concluded that the use of NaOCl with chelating agent failed to clean the root canal system and left remnants of the smear layer in the apical third. These different results may be explained by the different volume of irrigants used.

In Endodontics, an increase in surface roughness could be clinically beneficial because it may enhance the micromechanical bonding of root canal sealers, which requires irregularities on the surface of the adherent for penetration [36]. However, too much roughness can facilitate bacterial adhesion, which might lead to plaque formation [37]. Tartari et al [38]

found a result that the smallest changes in surface roughness after the use of a chelating agent were observed in regimens that employed 9% HEBP for 5 min after NaOCl treatment. These findings confirm that HEBP is a weak chelating agent that attacks less dentin surface than other commonly used chelators, such as EDTA, but the HEBP solutions need 300 seconds to completely remove the smear layer.

CONCLUSION

Based on the results of this study, compared with the other groups, root canals prepared using Protaper Next NiTi rotary file and irrigated with NaOCl associated with HEBP had the least amount of smear layer and surface roughness.

ACKNOWLEDGEMENT

The authors deny any conflicts of interest

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