Reduction of Enterococcus faecalis in curved root canals after various sizes and tapers of canal preparation.

AIMS: The aim of this study was to evaluate the reduction of Enterococcus faecalis in curved root canals after various sizes and tapers of the canal preparation.
MATERIALS AND METHODS: Mandibular first molars (n = 103) with curved mesiobuccal canals were divided into one control (n = 5) and 7 experimental (n = 14) groups, were inoculated with E. faecalis (ATTC 29212) and prepared with the following RaCe files (FKG Dentaire) as master apical file: Groups: 25.04, 25.06, 30.04, 30.06, 35.04, 35.06 and 40.06. All the experimental groups were irrigated with 2 mL of 1% sodium hypochlorite during instrumentation and finally rinsed with 17% ethylenediaminetetraacetic acid (EDTA) (2 mL) followed by 5.25% NaOCl (2 mL) and sterile distilled water. Colony counting was performed after incubation. STATISTICAL ANALYSIS USED: Resulting data were analyzed using one-way ANOVA and Tukey's post-hoc test, (P < 0.05). RESULTS AND
CONCLUSIONS: All the experimental groups showed significant bacterial reduction (P < 0.001). Although the greater the size/taper or both led to more decreased amount of bacteria, differences between the groups with the identical size and different tapers, and among the groups with the same taper and different sizes were not significant. Based on this study, 25.04 along with using 2 mL of 1% NaOCl during instrumentation, and using 17% EDTA and 5.25% NaOCl as final rinse successively after the termination of preparation, can effectively reduce intra-canal bacteria and preserve root structure.

INTRODUCTION

Endodontic literatures have well established the role of bacteria and their byproducts in pathogenesis of endodontic diseases.[1] Therefore, prevention or reduction of root canal bacterial contamination is the cornerstone of endodontic treatment.[23] This will be achieved through proper chemo-mechanical preparation[4] along with ensuring maximum conservation of tooth structure and maintaining the original canal anatomy.[5] Therefore minimal mechanical preparation with proper chemical irrigation may significantly reduce intra-canal bacteria, and would be desirable.

Irrigation has been shown to be more efficacious with increased canal preparation size and taper.[367] Different studies suggested different amount of apical enlargement.[678] Some studies found that apical preparation up to size 30 could effectively clean root canals.[89] Preparation to sizes larger than 30 or 35 was found to be necessary for NaOCl to be effective as an antimicrobial agent in another study.[10] One study suggested apical enlargement to 40.04 as a good balance between preservation of tooth structure and maximum volume of irrigation using negative pressure irrigation system in apical third.[11] Although ElAyouti et al.[12] stressed on keeping the apical enlargement to minimum size required for effective irrigation, Akhlaghi et al.[13] concluded that apical preparation even to 30 (0.10) left minimum required root wall thickness. No study reported the simultaneous effect of size and taper of master apical file (MAF) from the microbiological point of view. The purpose of this study was to evaluate the reduction of E. faecalis in curved root canals after various sizes and tapers of canal preparation.

MATERIALS AND METHODS

Sample collection

In this experimental study fully developed mandibular first molars extracted for periodontal reasons were collected and disinfected (immersion in 5.25% NaOCl 1 h). After providing the periapical radiographs, teeth with external or internal root resorption, visible cracks, fracture, caries, calcification and previous root canal treatments were excluded. After preparing the access cavity, presence of the two separate mesial canals and the patency of mesiobuccal (MB) canals were confirmed, and the roots with apical constriction diameter wider than a size 15 file were excluded. A total of 103 teeth were selected. Working length (WL) was determined by inserting a size 10 K-File (Dentsply Maillefer, Ballaigues, Switzerland) until the tip emerged from the apical foramen and subtracting 1 mm from this length. Degree of curvature was determined for MB canal according to Schneider[14] technique in bucco-lingual and mesio-distal directions, using parallel radiographs. Canals with curvatures of 20-35° were included. Then the teeth were numbered and prepared to no. 20 K-File.

Sterilization

Each tooth was autoclaved in 121°C, 25 psi for 30 min in a separate vial. Six teeth were randomly selected to confirm sterilization. Three teeth were immersed in three separate vials containing brain heart infusion (BHI) broth and three in separate vials of Thioglycollate culture medium. Each vial was incubated for 14 days at 37°C. A clear culture medium after incubation confirmed sterility.

Bacterial inoculation

Each vial was opened in a laminar air flow cabinet — under sterile conditions — and a fresh suspension of E. faecalis (ATCC 29212) with the darkness equal to the standard of McFarland was introduced into each MB canal using no. 15 K-File. Then each tooth was immersed in a presterilized vial containing sterile BHI. The presence of turbidity confirmed bacterial contamination in all samples.

Experimental and control groups

The teeth were randomly (simple randomization method) divided into 7 experimental groups (n = 14) and one control group (n = 5) without rotary instrumentation.

Rotary preparation was performed under sterile conditions in a laminar air flow cabinet, using sterilized RaCe instruments (FKG Dentaire — La-Cheaux-de Fonds — Switzerland) and a motor controller device (XSMART, Dentsply Maillefer, Ballaigues, Switzerland) according to the manufacturer. Coronal and apical preparation in all groups were performed with a crown-down technique. Coronal part of the root canal in all the samples was prepared with 40.10, 35.08, 30.06, respectively (the first number refers to the ISO size and the latter is the representative for the instrument taper ex. 40.10 means ISO size no. 40 with 10% taper). The apical and middle parts of the canals were prepared in each group as follows:

Group 1: 25.04

Group 2: 25.04, 25.06

Group 3: 25.04, 30.04

Group 4: 25.04, 30.04, 30.06

Group 5: 25.04, 30.04, 35.04

Group 6: 25.04, 30.04, 35.04, 35.06

Group 7: 25.04, 30.04, 35.04, 35.06, 40.06

The last bold size/taper in each group were considered as the final apical file.

A postgraduate student of endodontics prepared all the canals. Each rotary instrument was used for preparation of five canals. Each instrument was applied for 5 s to the WL with an anticurvature filing method. Subsequent to each rotary file, canal was irrigated with 2 ml 1% NaOCl using 28 gauge needles (Dentsply Rinn, Elgin, IL) and canal patency checked using no. 10 K-File. The needle was passively penetrated from coronal to middle third at the end of coronal preflaring. Needle was placed within the apical 5 mm during apical preparation sequence. In all experimental groups final irrigation was performed using 2 ml of 17% ethylenediaminetetraacetic acid (EDTA) solution (Roth International Ltd., Chicago, IL) and 2 ml NaOCl 5.25%, each for 1 min, followed by final flushing using 5 ml sterilized distilled water to eliminate the irrigation solutions from the root canal. In the control group, only 5 ml of normal saline was used.

Sampling procedure

After preparation, all MB canals were filled with sterilized BHI broth using sterile 18 gauge needle, and each tooth immersed in a vial containing 5 ml sterilized BHI broth, then incubated for 24 h at 37°C. After that, vials opened under sterile conditions in laminar air flow cabinet. Sampling procedure was performed using three sterile paper points no. 20, each left for 10 s in canal length to absorb canal contents. All three paper points of each tooth were placed in a separate vial containing 10 ml sterilized BHI broth. Vials were placed on vortex with power for 30 s with a setting of four. 1 ml from each vial content transported to KF (KF streptococcus Agar [7610]) with a micro-pipette. Colony counting was done after 48 h incubation at 37°C.

STATISTICS

Data analysis was performed using one-way ANOVA with Tukey's post-hoc test. Significance was set at P < 0.05 (statistical analysis performed using SPSS #16 software, SPSS Inc., Chicago, IL, USA).

RESULTS

All the experimental groups showed significant bacterial reduction while control group still contained 100% of initial contamination [Table 1].

Table 1

CFU/mL (mean ± SD) and percentage of bacterial reduction after preparation in control and each of the experimental groups

Although the greater size/taper or both led to more decreased amount of bacteria, differences between the groups with identical size and different tapers and between the groups with the same taper and different sizes were not significant (P > 0.05). There was no file deformity, broken instrument, and apical perforation occurrence during instrumentation.

DISCUSSION

The present ex vivo study compared different sizes and tapers of RaCe rotary files in decreasing the bacterial amount of mandibular first molar curved MB canals. There was no significant difference among the experimental groups with the same sizes and different tapers. Furthermore, the difference between the groups having identical tapers and different sizes was not significant (P < 0.05).

This study and some others were performed on curved MB canals of mandibular first molars.[813]

Shuping et al.[10] and Siqueira et al.[15] studied premolar teeth with one straight canal, thus did not assess the challenge of bacterial reduction in curved and narrow canals. The former was an in vivo study. Some other similar studies did not mention the degree of curvature.[61115] Since canal curvature can affect depth of needle and irrigant penetration in the root canal and also canal preparation, the results of above mentioned studies could not be generalized to most clinical situations.[16] Likewise, some other studies we considered curved (20-35°)canals.[1213] According to several previous studies, debriding ability of RaCe was superior to some other NiTi rotary files.[1417]

Irrigant type and method of irrigation in our study were similar to some other studies.[678] However, compared to Khademi et al.[8] we used a greater volume of irrigating solution. In some studies, using lower concentrations of NaOCl resulted in acceptable results.[310] According to the results of Soares et al.[18] the best irrigating solution for eliminating bacterial biofilm from root canal is 5.25% NaOCl. In addition, the best regimen for acceptable elimination of smear layer is using 17% EDTA, followed by a rinse with 5.25% NaOCl.

Intra-canal bacterial sampling procedure was similar some other studies.[1920] Unlike some other studies we considered similar time, size and number of absorbent points for absorbing fluid from the canal.[2310]

In this study, there was a significant difference between pre- and post-instrumentation bacterial amount.

Furthermore, increasing the size/taper of MAF did not result in a significant decrease of remaining bacteria. The control group had significantly more bacteria than all other experimental groups.

Card et al.[3] used 2 ml of 1% NaOCl as irrigant and showed that increasing apical preparation size leads to more bacterial reduction. This was more obvious in teeth with single straight canal. Shuping et al.[10] used 1.25% NaOCl without reporting the volume. They confirmed the results of Card et al.[3] and stated that bacterial reduction would be much more effective when NaOCl is used as irrigating solution. Results of Siqueira et al.[2] showed that increasing the size of apical file to no. 40 could reduce bacterial count significantly more than smaller sizes. They used 7 ml of 0.85% normal saline as irrigating solution to eliminate the chemical aspect of chemo-mechanical preparation, thus assessed the effect of mechanical instrumentation per se.

In the present study increasing size of apical file resulted in less remaining bacteria, but because of using NaOCl, reduction was not significant. Another study concluded that there were no additional benefits to apical enlargements more than three sizes after the first file, which binds in the WL.[21] Some other studies showed that there was no significant difference in bacterial reduction even with different instruments and preparation techniques.[222324] In contrast with the present study, Wu and Wesselink[25] concluded that preparation of canals in molars with no. 40 hand files leaves significantly less bacteria than smaller files. Dalton et al.[1] concluded that preparing the canal with larger sizes lead to more disinfection, but even larger sizes could not render the root canal bacteria free. In this study, MAF 25 (0.04) did not show significant difference comparing to the other experimental groups. Elayouti et al.[12] as well suggested keeping the apical size of curved canals as the minimum size required for irrigation sufficiency.

Based on this study, regarding nonsignificant difference of remaining intra-canal bacteria in canals prepared with different sizes/tapers of MAF, and respecting that less canal enlargement leads to more tooth structure preservation, 25 (0.04) may be suggested as the MAF in curved canals. However, further studies on cleaning effectiveness, tooth structure preservation and canal shaping ability with evaluation of bacterial reduction considering different size/taper of MAF is recommended.