Quantitative evaluation of apical extrusion of intracanal bacteria using K3, Mtwo, RaCe and protaper rotary systems: An in vitro study.

AIM: This aim of this study was to evaluate the number of bacteria extruded apically during root canal preparation using Four Rotary instrumentation techniques.
MATERIALS AND METHODS: A total of 50 extracted mandibular premolars were selected, access cavities prepared and inserted in to rubber stoppers of 10 mL glass vial. Root canals were contaminated with a suspension of Enterococcus faecalis (E. faecalis) American Type Culture Collection (ATCC) 29212 and incubated for 24 h at 37°C. Debris extruded from the apical foramen during instrumentation was collected into vials. The numbers of Colony-forming units were determined for each sample. STATISTICAL ANALYSIS: The data obtained were analyzed using the one-way ANOVA followed by post-hoc tukey's test with a P = 0.05 as the level for statistical significance.
RESULTS: Results suggested a statistically significant difference in the number of colony forming units between four experimental rotary Nickel-Titanium instrumentation groups (P < 0.001).
CONCLUSION: Although, extrusion of bacteria was found in all the experimental groups. There was less bacterial extrusion in K3 Group while more bacterial extrusion was seen in Mtwo Group.

INTRODUCTION

Successful cleaning and shaping of root canal is dependent on adherence to specific biologic and mechanical objectives. Biologically, all irritants must be removed from the canal, while at the same time creating sufficient space for adequate flushing and debridement without causing injury to the periradicular tissues.[1] The root canal infection is a dynamic process where different bacterial species apparently dominate at different stages. Microbiological studies on chronically infected root canals state that organisms that infect root canals are mainly anaerobic and gram-negative.[2]

In primary endodontic infections Enterococcus faecalis is occasionally isolated but several studies on endodontically treated teeth requiring retreatment have shown prevalence to facultative anaerobes, especially, E. faecalis is frequently isolated from treatment failure cases.[345]

Apical extrusion of infected debris in to the periradicular tissues is possibly one of the principal causes of post-operative pain. During root canal instrumentation if the infected debris is propelled into periradicular tissues, it may cause or increase the severity of periradicular inflammation. This inflammation is due to virulent bacteria in the root canal system or in the infected debris.[67]

Almost, all the instrumentation techniques have reported apical extrusion of debris, even when the root canal preparation is maintained short of the apical terminus.[8] Reddy and Hicks reported that more apical extrusion of debris was noticed with step-back technique when compared to balanced-force and engine-driven techniques.[9] Though there are few studies, which compared apical extrusion of intra canal bacteria using K3, RaCe, and Protaper Rotary Nickel-Titanium (Ni-Ti) instruments with conventional hand instrumentation technique, there has been no published study to date on Mtwo Rotary Ni-Ti instruments concerning apical extrusion of intra canal bacteria in extracted human teeth.

MATERIALS AND METHODS

Fifty normal, freshly extracted human, single rooted mandibular premolar teeth with mature apices and root curvatures between 0° and 10° were selected. Digital radiographs from proximal and buccal directions were taken to ensure there is a single canal. Endodontic access cavities were prepared and teeth were inserted under pressure into perforated rubber stoppers of 10 mL glass vials. Two coats of nail varnish were applied to the external surface of all roots to prevent bacterial micro-leakage.

The vial was vented with a 27-guage hollow needle alongside the tooth through the rubber stopper to equalize the air pressure inside the vial with outside the vial and it is also used as an electrode for the electronic working length determination. The length of the canal was measured with #10 K-file. When the tip of the file was visible at the apex, 1 mm short of the file penetration length was considered to be the working length, which was also confirmed with an electronic apex locator (Propex-II).

A suspension of pure culture of E. faecalis-ATCC 29212 was prepared by adding 1 mL pure culture of E. faecalis, to freshly prepared Brain Heart Infusion Broth. After 24 h of incubation of E. faecalis in Brain Heart Infusion Broth, changes bacterial growth in turbidity was compared with 0.5 McFarland standard against a ruled paper, which can be comparable to a bacterial suspension of 1.5 CFU/mL × 108 CFU/mL.

Before contamination of root canals a sterilized #15 K-File was placed 1 mm beyond the foramen to ensure patency of apical foramen and clear any clogging due to externally applied nail varnish. Followed by this procedure 10 μL of the prepared bacterial suspension was deposited in to the root canals of all specimens. #10 K-file was used to carry the bacteria down the length of the root canals. The contaminated roots were then placed in an incubator at 37°C for 24 h for bacterial multiplication. Sterilized glass vials were entirely filled with 10 mL of 0.9% NaCl solution. The tooth-rubber stopper-needle unit was then fixed in to the mouth of the vials. Single operator, using aseptic techniques, carried out the preparation and sampling procedures on each specimen under a class II vertical laminar air-flow cabinet to prevent airborne bacterial contamination. Samples were equally divided in to five groups for instrumentation with different techniques.

Group 1 (K3)

K3 instruments were used in a crown-down manner with a gentle in and out motion. File sequences used were: #25, 0.06 taper until half of the working length followed by #25, 0.06 taper between half and 2/3rd of working length. Later instruments #20, #25 and #30 of 0.04 taper were used respectively to the working length.

Group 2 (Mtwo)

Mtwo instruments were used in Standardized technique (Single-length technique) with small stroking, brushing movement. Patency of the canal was checked and glide path to the working length was created with #10 K-file. File sequences used were: #10 with 0.04 taper, #15 with 0.05 taper, #20 with 0.06 taper, #25 with 0.06 taper, and #30 with 0.05 taper all to their working lengths.

Group 3 (RaCe)

RaCe instruments were used in a crown-down manner with a gentle in and out motion. File sequences used were: #25, 0.06 taper until half of the working length followed by #25, 0.04 taper used between half and 2/3rd of working length and instruments of #20, 0.04 taper, #25,0.04 taper, and #30, 0.04 taper simultaneously used to their working length.

Group 4 (Protaper)

Rotary Protaper instruments were used with a gentle in and out motion. Patency of the canal and glide path to the working length was created with #10 K-file. File Sequences used were: S1 file used to one-third of the working length, followed by Sx file used to one-half of the working length, S1 file used to one-half to two-thirds of the working length and then S2 file was used to two-thirds of the working length. F1, F2, and F3 files were later used successively to the full working length.

Group 5 (Control)

No instrumentation was attempted in this group. Glass vials – teeth model contaminated with E. faecalis were kept in the Laminar Air-flow Cabinet for a period of 1 min and taken out from the Cabinet.

During root canal instrumentation 1 mL of NaCl solution was used after every change of instrument. Prior to and at the end of canal preparation, 0.01 mL of solution was collected from all the fifty experimental vials to count the bacteria and this bacterial suspension was placed on Brain Heart agar plates using a bacterial inoculation loop. All the 100 agar plates were placed in an incubator at 37°C for 24 h. After 24 h, colonies of bacteria were counted using classical bacterial counting technique (Collins et al. 2004) and they were counted as number of colony forming units (CFU).[10]

RESULTS

The Statistical package SPSS (Statistical package for social science, version 4) was used for statistical analysis. Mean and standard deviation were estimated from the sample for each Study group. The mean values were compared by one-way ANOVA appropriately followed by post hoc tukey test. Post hoc tukey test was employed to identify the significant groups. In the present study, the level of significance was set at P = 0.05.

Table 1 shows the mean counts, standard deviation and post hoc tukey test of extruded bacteria (E. faecalis) between different experimental groups. Mean CFU was compared in all the groups and there was statistically significant difference among the groups (P < 0.001). [Graph 1] shows the mean amount of extruded bacteria ranged between 0 CFU/mL and 13.46 CFU/mL. Least amount of bacterial extrusion was observed with K3 Rotary Ni-Ti Instruments in comparison with Mtwo, RaCe, and Protaper Rotary Ni-Ti Instruments. Mtwo Rotary Ni-Ti Instruments extruded more bacteria in comparison to other groups.

Table 1

Mean counts, standard deviation and Post-hoc tukey test of extruded bacteria between different experimental groups

Graph 1

Graphical representation of mean number of extruded bacteria

Post-hoc test suggested that Group 5 (Control) had significantly lower mean CFU than all the groups and there was no bacterial extrusion observed in control group where no instrumentation was attempted.

DISCUSSION

Siqueira and Barnett concluded that physical or chemical injury to periradicular tissues during cleaning and shaping of the root canal system can cause degranulation of mast cells, with consequent release of histamine in to the periradicular tissues. Normally, during chemo-mechanical preparation in root canal treatment, apical extrusion of infected debris into the periradicular tissues is the principal cause of post-operative pain. In infected cases over-instrumentation of root canal leads to apical extrusion of infected debris, which may give rise to inter-appointment pain.[11] Furthermore, apical extrusion of debris during root canal preparation causes mid-treatment flare ups and persistent periapical inflammation. These Mid-treatment flare ups are the frequent problems encountered in day to day practice of root canal therapy by practitioners.[12]

In the present study, a standardized protocol was followed-up to decrease the number of variables in root canal instrumentation to reduce the amount of apical extrusion of bacteria. E. faecalis (ATCC 29212) was used as a bacteriological marker because it is the most frequently isolated species and was usually the predominant isolate in the root canal. Microbiological findings from filled root canals with persistent periapical disease have shown a high-proportion of enterococci. During root canal preparation many factors may affect the amount of extruded intra canal bacteria such as instrumentation technique, instrument type, instrument size, preparation end point, and irrigating solution. The apical diameter of master apical instruments in all the groups was standardized at ISO Size 30 to avoid any variations in the amount of extruded bacteria due to the size of apical enlargement.[1314] Therefore, apically extruded debris from root canal specimens could be attributed to the design and technique of the respective instrument used in that particular group. The results of this ex vivo study demonstrated that while all the instruments extruded E. faecalis from the apical foramina, there was statistically significant difference between the four Ni-Ti instrumentation techniques in terms of amount of E. faecalis extrusion.

In the present study, least amount of bacteria was extruded with K3 Rotary Ni-Ti Instruments in comparison with Mtwo, RaCe, and Protaper Rotary Ni-Ti Instruments. Kustarci et al. (2008) in their study found out that bacterial extrusion was comparatively less when instrumented with K3 Rotary Ni-Ti instruments.[15] Ghivari et al. also concluded that there was lesser amount of irrigant and debris extrusion using K3 Rotary Ni-Ti instruments.[1617] Instrumentation in K3 Group was performed by Crown-down technique where dentin chips resulting from the cutting action are easily dislodged from the working area and carried coronally to the orifice via its unique helical angle.[1819] This could be the probable reason for minimal extrusion of E. faecalis in K3 when compared to Mtwo, RaCe, and Protaper.

Tasdemir et al. (2010) showed that RaCe Rotary Ni-Ti instruments extruded less debris than Protaper and Mtwo Rotary Ni-Ti instruments.[20] In the current study, RaCe showed significantly lesser apical extrusion of bacteria than with Mtwo and Protaper instruments. Tanalp et al. (2006) compared Protaper systems with other continuous rotary techniques and found significantly greater amounts of extruded debris when using the Protaper technique.[21] The taper of the Protaper instruments favor the preparation of the apical third in early stages, thus, wear occurs early throughout the whole root canal because the instruments reach the working length in the beginning of the preparation, causing greater apical extrusion of bacteria.

Greater apical extrusion of bacteria is seen with Mtwo Rotary Ni-Ti instruments in comparison with K3, RaCe, and Protaper. This may be attributed to its Standardized length preparation technique (Single-length technique) where all the Ni-Ti instruments are taken to full working length and also to the double cutting-edge geometry of Mtwo instruments whose cross-section resembles an S-Instrument, an instrument with a similar geometry and a smaller cross-section surface than a triple cutting edge instrument with convex geometry.[2223] Because of its specific design characteristics, cutting efficiency, and its standardized length preparation technique Mtwo Rotary Ni-Ti instruments remove adequate amount of dentin in a short period of time thereby unable to displace the debris coronally, leading to significantly greater amount of apical extrusion of bacteria.

In the present study, crown-down technique was used with K3, RaCe, and Protaper Ni-Ti Rotary Instruments. As the greatest number of microorganisms in the root canal lies in the coronal third, initial preparation of this section of the root canal system helps to reduce the number of microorganisms that may be pushed apically.[24] Early flaring of the coronal part of the root canal preparation may also improve instrument control for preparation of apical third of the root canal.[25] The advantages of the early cervical flaring are: Deeper penetration of irrigating solutions, easy removal and clearance of debris from the apical area, reduced possibility of ledging and reduced possibility of debris packing, greatly reducing the number of contaminants that can be extruded from canal. The unique design features of Ni-Ti instruments encourage coronal rather than apical displacement of debris during preparation process. The flat outer edges help keeping the file centered in the canal and inter-flute distance tends to accumulate dentinal debris and direct it coronally.[26]

CONCLUSION

K3 rotary instruments extruded less number of bacteria where instrumentation was performed by crown-down technique. Mtwo rotary instruments extruded more number of bacteria where instrumentation was performed by single-length technique. The direction of instrumentation, whether coronal-apical or apical-coronal or instrumentation to the single length seems to be a more determinant factor of apical debris or bacterial extrusion than the design of a specific rotary instrument.