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 »  Home  »  Endodontic Articles 5  »  Reduction in intracanal bacteria during root canal preparation with and without apical enlargement
Reduction in intracanal bacteria during root canal preparation with and without apical enlargement
Introduction - Materials and methods.



L. G. Coldero, S. McHugh, D. MacKenzie & W. P. Saunders
Dental School, Department of Statistics, University of Glasgow, Glasgow, UK.
University of Dundee Dental School, Dundee, UK.

Introduction.
The role of bacteria and their products in the initiation, propagation and persistence of periradicular periodontitis has been established (Kakehashi et al. 1965, Sundqvist 1976, Moller et al. 1981). These microorganisms obtain their nutritional supply from vital, degenerating and necrotic pulpal tissue, saliva from the mouth, serum protein from the periradicular tissues and metabolites from other bacteria. The number of microorganisms within an infected root canal system may vary anywhere from 102 to more than 108 (Sjogren et al. 1991). Microbes are present in all parts of the root canal system, including fins, and anastomoses and may be found at varying depths of up to 300 m within the dentinal tubules, from the pulpal end (Horiba et al. 1990). There also appears to be a regional variation in the extent to which dentine is invaded; cervical tubules are invaded to a greater extent than the midroot tubules, which are, in turn invaded more than those in the apical region (Love 1996). Any method used to disinfect the root canal should be capable of accessing and eliminating microbes as much as possible from all parts of the system.
The aims of root canal treatment are to disinfect the root canal system thoroughly, completely obturate the space created so as to entomb any microbes that have escaped elimination and to prevent reinfection; without causing any iatrogenic damage (Saunders & Saunders 1997, Sundqvist & Figdor 1998). A chemomechanical preparation technique is advocated to disinfect root canals (Bystrom et al. 1985) because it allows a greater number of root canals to be rendered bacteria free; between 20% (without the use of an antimicrobial irrigant) to around 80% (with the use of an antimicrobial irrigant) of canals can be disinfected (Bystrom & Sundqvist 1981, Bystrom & Sundqvist 1983).
The latest generation of root canal instruments include engine-driven nickel–titanium (Ni–Ti) instruments. These have been found to produce good shaping (Glossen et al. 1995) by virtue of their increased flexibility (Walia et al. 1988). This superelasticity together with the development of specially configured cutting blades means that these engine-driven instruments can be efficiently and safely used in even narrow, curved root canals (Short et al. 1997).
Investigators have evaluated the efficacy of different preparation techniques in cleaning the apical region of the root canal (Wu & Wesselink 1995, Siqueira et al. 1997). Wu & Wesselink (1995) compared the efficiency of three techniques, step-back, crown-down pressureless and the balanced-forced techniques using K-files, in cleaning the apical portion of curved root canals. The remaining surface debris was used as the evaluation criteria and they concluded that the apical portion of the canal was cleaned less than the middle and the coronal portions, regardless of the technique used. The balancedforce technique produced a cleaner apical portion of the canal when compared to the other two techniques. Siqueira et al. (1997) compared the effectiveness of five instrumentation techniques for cleaning the apical third of root canals using a histological evaluation method. Canals were prepared with the step-back technique using stainless steel or Ni–Ti files, an ultrasonic technique using a ISO size 15 ultrasonic file, balanced-force using Flex-R-files and Canal Master U instruments. They concluded that none of the five instrumentation techniques tested were effective at completely debriding the root canal system. Apical enlargement during canal cleaning and shaping procedures has the potential to eliminate more bacteria from the root canal system (Parris et al. 1994, Yared & Bou Dagher 1994, Siqueira et al. 1999). However, there still remains some controversy as to whether apical enlargement is necessary. Buchanan (1993a,b, 1996, 1998) has advocated minimal apical preparation on the grounds that it minimizes the potential for the creation of apical zips and provides better control of filling materials.
The aim of this study was to compare the intracanal bacterial reduction during root canal preparation using Ni–Ti GT rotary files and an antimicrobial irrigant with and without apical enlargement to test the null hypothesis that there was no difference between the two techniques.

Materials and methods.
The method used was based on previously published protocols (Orstavik & Haapasalo 1990, Dalton et al. 1998, Siqueira et al. 1999). Forty-five extracted permanent human maxillary first and second molar teeth were obtained so that their palatal roots could be used in the study. These teeth were stored in normal saline with a few crystals of thymol. In order to be included in the study the palatal root had to satisfy the following criteria:

  1. Long, minimally curved, narrow and caries free.
  2. Mature root apex with no evidence of external root resorption.
  3. Sufficiently separated from the buccal roots to allow easy palatal root resection.

A slow speed diamond disc under saline irrigation was used to separate the palatal root from each tooth. A barbed broach (ISO size 20) was used to remove any pulp remnants from the root canal ensuring that the canal wall was not instrumented. The patency of each canal was established by gently inserting an ISO size 10 KFlexofile (Dentsply Maillefer, Ballaigues, Switzerland) until the tip emerged from the apical foramen. This length was noted and the working length (WL) of each root specimen was calculated by subtracting 1 mm. After WL determination, the apical 3 mm, including the foramen, of each specimen was covered with Dyract AP (DeTrey, Dentsply, UK) to prevent any extrusion or leakage of material during root canal preparation and sample collection.

Sterilization of specimens.
Aliquots of 5 mL of sterile Tryptic Soy Broth (TSB) solution (Bioconnections, Leeds, UK) were added to each universal container with the specimen. Agitation for about 30 s on a vortex mixer (McQuilkin, Glasgow, UK) was performed to aid the penetration of TSB into the root canal of the specimen. The 45 universal containers with the specimens were placed on an aluminium stack tray and autoclaved at 121C for 15 min. The specimens were then stored in an aerobic incubator at 37C. Frequent visual checks were made to determine if the TSB in any of the containers became turbid, indicating that bacteria had survived the autoclaving. Each container and specimen were vortexed for 30 s after each observation and before returning to the incubator. The TSB in all the containers remained clear.

Maintenance of viable Enterococcus faecalis, preparation of infecting broth, test for viability of infectious TSB and infection of samples.
A vial of frozen Enterococcus faecalis NCTC 29212 was the source of bacteria and a sample on a sterile wire loop was used to inoculate a blood agar plate. The plate was incubated aerobically at 37C overnight. A viable growth of E. faecalis was maintained by subculturing two colonies from the previous plate to blood agar, every other day. Every 3–4 days, four bottles containing 100 mL sterile TSB were each inoculated with two colonies of E. faecalis and after overnight incubation 0.5 mL aliquots from these were used to infect and maintain infection of the samples for the duration of the experiment. At around 10 am on the days following the TSB inoculation (i.e. on every Tuesday and Friday for the duration of the experiment), the liquid content of each specimen container was decanted and replaced with 5 mL of infected TSB. These were vortexed for 30–40 s to enhance the penetration of the infected TSB into the root canal of each specimen. The purity of the E. faecalis that each specimen was being exposed to was checked on a regular basis by plating samples taken randomly from several universal containers. Initially, an infecting period of 2 weeks was designated for infecting the specimens, based on the study by Orstavik & Haapasalo (1990). After this initial 2 weeks, the infected TSB was replaced for the remaining samples at the same frequency for the total duration of the experiment.

Verification of specimen reinfection.
Evidence proving that the specimens were successfully reinfected with E. faecalis was obtained in two ways. First, visual evidence using scanning electron microscopy was performed on two randomly selected specimens. The two additional specimens were prepared for SEM examination (Philips SEM 500, Eindhoven, The Netherlands). The specimens were first freeze-fractured in liquid nitrogen, fixed, completely dehydrated, attached to glass slides and sputter coated with gold before being examined in the SEM (Fig. 1). Secondly, the specimens used in the experimental groups were each tested for the presence of viable E. faecalis within their canals. A sample was obtained from the canal of each specimen before they were prepared and the concentration in CFUmL–1 was later determined (vide infra).

Specimen grouping.
Five specimens from the initial 45 were used for a pilot study of the experiment protocol. Two specimens were randomly chosen for SEM examination.
The 38 remaining specimens were first grouped according to their working lengths (WL). Based on this WL grouping, specimens were then randomly assigned to three study groups; two experimental groups contained 16 specimens each and the third, the control group, contained six specimens. A preliminary statistical evaluation was performed to verify that the specimens were evenly distributed with respect to their working lengths.

Determination of E. faecalis sensitivity to materials used in study.
The sensitivity of E. faecalis to the materials (at their specific concentration or state) used in the study was investigated. This was deemed necessary as a result of the pilot study. The materials tested were Dyract AP (DeTrey Dentsply, UK), 4.4% NaOCl (Tesco, Cheshunt, UK), 15% EDTA (McQuilkin, Glasgow, UK), sodium thiosulphate (McQuilkin, Glasgow, UK), 0.85% NaCl (McQuilkin, Glasgow, UK), paper points (Dentsply Maillefer, Ballaigues, Switzerland). Seven labelled blood agar plates were inoculated with infected tryptic soya broth (TSB) using a sterile cotton swab for each plate. Absorbent paper disks were impregnated with each liquid, the paper points were presoaked in sterile TSB and the Dyract AP was light cured with its primer on a clean glass slide before placing on the surface of the inoculated plates. Two samples of each material were used on each plate. On the final plate the disks were first dipped in NaOCl and then in sodium thiosulphate (Na2S2O3). This was to determine if there was complete neutralization of the NaOCl by the Na2S2O3 concentration used. All plates were incubated over night at 37C and were then examined for the presence or absence of zones of inhibited E. faecalis growth.

Prepreparation sampling and root canal preparation sequence applied to experimental groups.
A single operator, using aseptic techniques, carried out the preparation and sampling procedures on each specimen under a class I laminar air flow cabinet (Fischer et al. 1998) to prevent airborne bacterial contamination. The following procedures were applied to each specimen regardless of their grouping. The infected broth was carefully decanted from the universal container and the specimen removed with sterile cotton pliers. An ISO size 20 sterile paper point was used to obtain a broth sample from the root canal of the specimen. The paper point was kept in the canal for about 30 s and then transferred to a prelabelled bijou bottle (Inverclyde Biological, Bellshill, UK) containing 1 mL of sterile TSB. This sample was called the prepreparation (preprep.) sample and the concentration of bacteria was determined. The external surface of the root specimen was washed with 3 mL of sodium hypochlorite (NaOCl). The entire specimen, except for the coronal 2–3 mm, was then carefully wrapped in a presterilized 5 cm2 sheet of aluminium foil. The specimen and foil were then held firmly in the jaws of a mini-vice; the jaws of the vice were first lined with a sheet of barrier plastic.

Group A: Crown-down chemomechanical preparation with apical enlargement to a GT rotary file size 35/.04 taper file to the WL.
The nickel–titanium GT rotary and 04 Profile file series (Dentsply Maillefer) were used for root canal preparation. In group A, this was initiated with a GT rotary file size 20/.12 taper used in a crown-down manner. The file was rotated at 350 r.p.m. in an 18 : 1 handpiece driven by an electric motor (Analytic Endodontics, Orange, CA, USA). The GT rotary files, sizes 20/.10, 20/.08 and 20/.06, were all used serially in a crown-down technique toward the apex. Each file went deeper into the canal but was not allowed to go further than 1 mm short of the WL. Irrigation was performed between each instrument using 1 mL of 4.4% NaOCl delivered in a luer-lock syringe with a 27- gauge endodontic needle (QED, Peterborough, UK). The apical preparation for specimens in group A was initiated by placing the Profile size 20/.04 taper to the WL. This was followed sequentially by the size 25/.04 file, size 30/ .04 file and then the 35/.04 file, all to the WL. Again, the root canal was irrigated with 1 mL of 4.4% NaOCl after each instrument. Finally, the GT rotary file size 20/.10 taper was placed to the WL followed by 1 mL of NaOCl irrigation. The root canal system was irrigated with 1 mL of 15% EDTA solution to remove the smear layer. This solution was kept in the canal for 3 min before being washed out with 1 mL of NaOCl. A total of 10 mL of 4.4% NaOCl was used for irrigation in each specimen. On average, the NaOCl was kept in contact with the root canal contents for about 15 min (Senia et al. 1971, Bystrom & Sundqvist 1983).

Group B: Crown-down chemomechanical preparation with 0.25 mm step-back apical preparation to a size 35/.04 taper 1 mm short of the WL, using GT rotary files.
In this group, the same preparation regimen that was applied in group A with the first four GT rotary files (i.e. sizes 20/.12–20/.06 tapers). However, the Profile .04 tapered files were applied using a 0.25-mm incremental step-back technique, starting with the size 20/.04 file at the WL and ending with the size 35/.04 file at 1 mm short of the WL. The root canal was irrigated with 1 mL of 4.4% NaOCl after each file. As with the preparation regimen for group A, the final GT file used was the size 20/.10 taper to the WL and this was followed with another 1 mL 4.4% NaOCl irrigation. The specimens in group B were also each irrigated with 1 mL of 15% EDTA solution, which was then left in the root canal for 3 min before being irrigated with another 1 mL of 4.4% NaOCl solution. Each specimen was again exposed to a total of 10 mL of 4.4% NaOCl for about 15 min.

Group C: Positive control group with irrigation without mechanical preparation.
In this group, each of the six specimens were set up the same as those in groups A and B. However, no mechanical preparation with GT rotary files was performed. Instead, irrigation with 4.4% NaOCl and 15% EDTA solutions was performed so that the specimens were exposed to the same volume of irrigants for the same length of time. The tip of the needle was able to penetrate only to a depth of approximately 5 mm into each root canal.
Following the irrigation regime for each group, root canals were then irrigated with 3 mL sterile NaCl solution to dilute the NaOCl within the canal. The NaOCl was then neutralized with 3 mL sodium thiosulphate (Na2S2O3) solution. A sterile syringe with luer-lock connection and 27-gauge endodontic needle was used with each irrigant. The same syringe that was used to deliver the Na2S2O3 was used to aspirate the majority of the solution from the canal and each specimen was then ready for sampling.

Postpreparation specimen sampling method.
A sample was collected from each specimen after preparation and this was used to calculate the numbers of bacteria surviving the preparation process. After the Na2S2O3 solution was aspirated from the canal of each specimen, 1 mL of sterile TSB was used to flush the canal. With the canal filled with TSB, a sterile cotton roll was used to remove excess TSB from the coronal surface of each specimen. The contents within the canal were then mixed using a sterile file (Dalton et al. 1998). The files used were GT rotary files 35/.04, 20/.04 and K-Flexofile size 15/.02 for groups A, B and C, respectively. Each sterile instrument was inserted manually into the root canal of each specimen to the WL and pumped five times vertically with minimal reaming action. A sterile paper point was then used to absorb the fluid content from the canal with care being taken not to touch the outer surface of the specimen. In groups B and C, ISO size 20 paper points were used and size 35 points were used in group A. The points were left in each canal for about 30 s before they were each transferred to a sterile bijou bottle with a 1-mL aliquot of TSB. This constituted the postpreparation (postprep.) sample. The bijou bottle with the postprep. paper point sample and the other with the preprep. sample taken earlier, were vortexed for 30 s. Serial 10-fold dilutions were produced from each sample and were used to inoculate blood agar plates using an automatic spiral inoculator. The labelled plates were incubated for 24 h at 37C and the concentration of each preprep. and postprep. sample was determined in CFU mL−1; these were called the preprep. and the postprep. concentrations, respectively. The bijou bottle with the neat sample was also incubated for 24 h and was examined the following day for any sign of turbidity.

Statistical evaluation.
A one-way analysis of variance was used to compare the preprep. concentrations and the working lengths for the groups. Generalized linear model and stepwise logistic regression tests were used to determine if there were differences amongst the methods of preparation. A Bonferroni adjustment, with a 95% confidence interval, was used to make paired comparisons between the three groups.