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Aust Endod J 2011; 37: 128–133

ORIGINAL RESEARCH

A comparison of three rotary systems and hand


instrumentation technique for the elimination of Enterococcus
faecalis from the root canal aej_239 128..133

Melahat Gorduysus, DDS, PhD ; Emre Nagas, DDS ; Ozgur Yildirim Torun, DDS, PhD2; and
1 1

Omer Gorduysus, DDS, PhD1


1 Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey
2 Department of Clinical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Keywords Abstract
bacteria, hand instrumentation, HeroShaper,
K3, ProTaper, rotary instrument. The aim of this study was to compare the in vitro reduction of a bacterial
population in a root canal by mechanical instrumentation using three rotary
Correspondence systems and hand instrumentation technique. The root canals contaminated
Dr Melahat Gorduysus, Department of
with a suspension of Enterococcus faecalis were instrumented using ProTaper, K3,
Endodontics, Faculty of Dentistry, Hacettepe
HeroShaper and K-file hand instrumentation technique. Later the root canals
University, 06100 Sıhhiye, Ankara, Turkey.
Email: mgorduysus@yahoo.com were sampled. After serial dilutions, samples were incubated in culture media
for 24 h. Bacterial colonies were counted and the results were given as number
doi:10.1111/j.1747-4477.2010.00239.x of colony-forming units per millilitre. The results showed that all the canal
instrumentation systems reduced the number of bacterial cells in the root
canals. Statistically, ProTaper instruments were more effective in reducing the
number of bacteria than the other rotary files or hand instruments.

support the treatment objectives (11). However, bacterial


Introduction
penetration is deeper coronally and at mid-root levels than
Microorganisms play a fundamental role in the aetiology in the apical part (12,13). As a result, root canals instru-
of pulp and periapical disease (1,2), and their control and mented with greater taper files may contain fewer bacteria
elimination are important during endodontic treatment than those instrumented with smaller taper files (14).
(3). Reducing the bacterial count in infected root canals is Siqueira et al. (15) compared conventional taper files
accomplished by a combination of mechanical instru- (0.02 mm/mm) with greater taper files (0.06), and found
mentation, various irrigation solutions and antibacterial no significant difference in bacterial reduction. Dalton
medicaments placed into the canal. et al. (10) found no difference in bacterial reduction using
Mechanical instrumentation is the core method either 0.04 taper rotary or stainless-steel K-file hand
for bacterial reduction during endodontic treatment of instrumentation with sterile saline as the irrigant. Sub-
infected root canals (4). Irrigation and dressing with anti- stantial bacterial reduction was achieved with progressive
microbial agents further reduce or eliminate the number filing, regardless of file type and fewer bacteria remained
of intracanal bacteria (5–7). The effect of canal instru- when larger file sizes were used. Neither of these tech-
mentation using non-antimicrobial irrigation is reported niques could predictably render canals free of bacteria.
in the previous studies (8,9). It has been demon- The ProTaper (Dentsply/Maillefer; Ballagigues, Switzer-
strated that mechanical instrumentation with a non- land), the K3 (SybronEndo; Orange, CA, USA) and
antimicrobial irrigant reduces the intracanal bacterial the HeroShaper (Micro-Mega; Besançon, France) have
count enough to detect a quantifiable difference using become popular rotary systems, particularly among
appropriate sampling techniques (10). general practitioners. Although the earlier developed
Bacteria in the canal can penetrate the dentinal tubules. rotary nickel-titanium (Ni-Ti) systems (e.g. ProFile) have
Increasing the size of the apical preparation reduces the published research evaluating their ability to remove
bacterial count and yields cleaner canals, which may bacteria, there is only one currently published data in

128 © 2010 The Authors


Australian Endodontic Journal © 2010 Australian Society of Endodontology
M. Gorduysus et al. Elimination of Bacteria from the Root Canal

peer-reviewed journals comparing the recently developed MI, USA) with 5% sheep blood agar plates incubated at
rotary systems (ProTaper, HeroShaper) for ability to 37°C for 48 h in a CO2 incubator (Sanyo MCO-18AIC,
remove bacteria (14). Japan), were inoculated in BHI broth and left for 24 h.
The aim of this study was to compare the in vitro reduc- A bacterial suspension was prepared in a fresh broth
tion of a bacterial population in root canals by mechanical from a culture of E. faecalis, grown in BHI broth for 24 h.
instrumentation using three rotary systems (ProTaper, McFarland standard no. 4 was then used to evaluate the
K3, HeroShaper) and K-file hand instrumentation. broth in order to ensure that the bacterial count was
approximately 1.2 ¥ 109 colony-forming units per millili-
tre. Each root canal was completely filled with 20 mL of
Materials and methods
E. faecalis suspension through their cervical apertures
Selection and preparation of teeth using sterile automatic micropipettes. The root canals of
negative control were filled with 20 mL of sterile BHI
Experimental protocols were performed on freshly broth. The cervical access was sealed with temporary
extracted human lower premolars with a single-root cement (Nucavfil PSP Dental Co. Ltd. Belvedere, Kent,
canal (n = 135). All experiments were conducted under UK). The roots were incubated at 37°C for 7 days with the
aseptic conditions in a laminar air flow cabinet to prevent replacement of 20 mL of fresh BHI broth at the third day
airborne bacterial contamination. Teeth were initially after incubation.
stored in 10% formalin for 24 h, to provide disinfection
and organic tissue fixation. After this procedure, bone,
calculus and soft tissues on the root surface were Verification of specimen contamination
removed with curettes. The crowns were then sectioned with E. faecalis
with a diamond disk and the root lengths were standar-
dised to 15 mm. They were then stored in physiological To confirm the contamination by E. faecalis, the negative
saline solutions until use. control roots were analysed by scanning electron micros-
Canals were evaluated for apical patency with a size 10 copy (SEM) (JEOL JSM-6400, Japan). The specimens
file. Root canals were instrumented to a size 25 K-file were first fractured, fixed, completely dehydrated and
1 mm from the apex of the root (determined working attached to slides. Then they were sputter-coated with
length is 14 mm) and irrigated with 2 mL of saline solu- gold before SEM examination.
tion. The root canals were then filled with 17% ethylene
diamine tetraacetic acid for 3 min to remove the smear
layer. All the canals were finally rinsed with 5 mL of Root canal instrumentation
distilled water. To prevent bacterial leakage, the apical All procedures were carried out by the same researcher to
foramen was sealed with an epoxy resin and the surfaces rule out personal variations.
of all roots were varnished with nail polish. To improve Group 1: Twenty-five root canals were enlarged using
handling, the roots were mounted vertically in acrylic the ProTaper system (Dentsply/Maillefer; Ballagigues,
blocks. Switzerland). The ProTaper instruments were used in a
crown-down manner according to the manufacturer’s
Sterilisation of specimens instructions.
Group 2: Twenty-five root canals were enlarged using
All specimens were sterilised in an autoclave at 121°C for the K3 system (Sybron Endo; Orange, CA, USA). The K3
15 min. The sterilised roots were randomly divided into instruments were used in a crown-down manner accord-
five groups with 27 teeth in each group and one of the ing to the manufacturer’s instructions
groups served as the positive control group. Before the Group 3: Twenty-five root canals were enlarged using
contamination procedure, a negative control group (total the HeroShaper system (Micro-Mega; Besançon, France).
of five roots) were collected by randomly selecting one The preparation was in a crown-down manner. The blue
root from each group in order to confirm the sterilisation HeroShaper sequence was employed.
procedure. Group 4: Twenty-five root canals were instrumented
using 0.02 tapered K-file hand instrument (Dentsply;
Maillefer; Ballagigues, Switzerland). The standard tech-
Contamination with Enterococcus faecalis
nique was used as described by Kerekes and Tronstad
A pure culture of Enterococcus faecalis (ATCC 29212) was (16). For coronal enlargement, #2, #3 and #4 Gates-
used to contaminate the root canals. Colonies of E. faeca- Glidden drills were used for the two-thirds of the coronal
lis, grown on brain heart infusion (BHI, Difco; Detroit, part.

© 2010 The Authors 129


Australian Endodontic Journal © 2010 Australian Society of Endodontology
Elimination of Bacteria from the Root Canal M. Gorduysus et al.

Group 5 (Control group): Twenty-five root canals There was no bacterial growth in the negative control
were used as the control group. In this group, no further roots.
instrumentation or irrigation was done. All the canal instrumentation systems appreciably
Each root canal was irrigated with a total volume of reduced the number of bacterial cells in the root canals
10 mL sterile saline solution. Irrigation was delivered in (Fig. 2). Comparisons with the control group showed
the canals using a 2.5 mL plastic syringe with a 23-gauge that the ProTaper system was more effective (P < 0.001)
needle. in reducing the number of bacteria than the K3 system
(P < 0.001), HeroShaper system (P < 0.001) and the
K-file hand instrumentation technique (P < 0.05). The
Post-preparation specimen sampling method reduction of bacterial numbers was also significantly
greater in the ProTaper system when compared with the
Bacterial samples were acquired with #25 paper points.
K3 system (P < 0.05), HeroShaper system (P < 0.05) and
The paper point was placed into the canal and kept there
the K-file hand instrumentation technique (P < 0.001).
for about 1 min and then transferred into tubes contain-
There were no other statistically significant differences.
ing 1 mL saline for 1 min. After 10-fold serial dilutions
in saline, 100 mL aliquots were plated onto BHI agar
and incubated at 37°C for 24 h. Bacterial colonies were Discussion
counted and the results were expressed as the number of
One of the most important objectives during root canal
colony-forming units per millilitre.
instrumentation is the removal of vital and/or necrotic
pulp tissue, infected dentin and dentin debris in order to
Statistical analysis eliminate most of the microorganisms from the root canal
system (17).
Data were expressed as mean ⫾ SEM. Kruskal–Wallis Several methods have been described to evaluate endo-
variance analysis was used for determining the differ- dontic instrumentation techniques, including microscopic
ences among groups and for multiple comparisons observation of the remaining debris, morphometric
Dunn’s post hoc test was used. A P-value less than 0.05 analysis and a bacteriological assessment. A bacteriologi-
was considered statistically significant. cal assessment was chosen for the present study because
of the importance of canal disinfection in successful treat-
ment (10).
Results Mechanical instrumentation is the core method for
In all of the specimens studied by SEM, the presence of bacterial reduction in the infected root canal (8,9). It is
bacteria in the root canals as well as within the dentinal
tubules was demonstrated (Fig. 1).

Figure 2 The reduction of bacterial numbers in root canals by ProTaper,


Figure 1 Sample scanning electron microscopy picture of a root canal K3, HeroShaper and K-file systems. CFU, colony-forming unit. All data are
contaminated with Enterococcus faecalis. expressed as mean ⫾ SEM. *P < 0.05, ***P < 0.001.

130 © 2010 The Authors


Australian Endodontic Journal © 2010 Australian Society of Endodontology
M. Gorduysus et al. Elimination of Bacteria from the Root Canal

well established that mechanical preparation with saline Table 1 Mean of colony counts (CFU mL-1)
as the irrigating solution, cannot predictably eliminate Control ProTaper K3 HeroShaper K-file
the bacteria from infected root canals. Possible bacterial
Mean 870 400 29 544 133 840 183 776 212 080
invasion into dentinal tubules and the lateral canals
SEM 135 927 9 468.083 23 711.02 49 583.0405 32 803.35
from the main root canal, the complexity of the root
SD 679 635.2 47 340.42 118 555.1 247 915.203 164 016.7
canal system in most teeth, the physical limitations of 25 25 25 25 25
instruments and the insignificant antibacterial activity of
CFU, colony-forming unit.
saline may all play a part in bacterial regrowth (9).
Hence, the use of irrigation solutions is an important
part of effective chemomechanical preparation. As our
aim was to assess the effects of the instrumentation that the specimens were successfully reinfected with the
techniques alone, an antibacterial irrigation solution organism (Fig. 1).
was not used in this study. In this study, all three rotary systems were used with
All techniques reduced the number of bacteria recov- the crown-down technique. Effective cleaning of root
ered from infected canals (8,9,18). Standard instrumen- canals may also be influenced by the apical size of a
tation with stainless-steel K-files was performed as a preparation (25). In the present study, the apical pre-
comparative technique. However, other clinical effects of paration was standardised in four techniques to a size
large instrumentation, including compromised restor- 30.
ability, fracture susceptibility and canal path alterations, It has been shown that the bacterial count is decreased
should also be considered when using any instrumenta- with larger instrument sizes (10). The bacterial numbers
tion technique (10). Lam et al. (19) showed that greater continue to decrease with progressive filing to larger sized
apical enlargement (Lightspeed) or increased canal taper files. This study showed that the ProTaper system was
(Greater Taper files) did not increase fracture suscep- more effective in reducing the number of bacteria than
tibility of tooth roots. the HeroShaper system, K3 and K-file hand instrumen-
NiTi rotary instrumentation is faster and its lower tation technique (Table 1 and Fig. 2). It was expected that
rate of procedural errors has popularised its use (4). Such the more aggressive removal of dentin (ProTaper) would
features are biologically important if the practitioner eliminate more bacteria and lead to lower bacterial counts
is to safely instrument to larger file sizes, thereby fur- in the final test sample.
ther reducing intracanal bacteria. However, Rollison Siqueira et al. (15) compared conventional taper files
et al. (20)showed that a standardised preparation with (0.02 mm/mm) with greater taper files (0.06), and found
increased apical enlargement of root canals appears to no significant difference in bacterial reduction. In the
result in significantly greater bacterial removal than mentioned study, further apical instrumentation with the
tapered instrumentation preparations. These findings are conventional files to a larger size was significantly more
in agreement with those of Ørstavik et al. (9) and Dalton effective in eliminating bacteria from the root canal than
et al. (10). Although manual instrumentation is still the greater taper files that stopped at a smaller apical size,
most popular means of root canal preparation, many supporting the concept that the size of the apical prepa-
specialists and an increasing number of general practitio- ration is important in intracanal bacteria reduction
ners are using rotary Ni-Ti instruments. However, rotary (9,11).
instruments have not always been found to be superior to The reason for the differences in these studies could be
hand instruments, when the various aspects of prepara- the test sample. Infection of the canals in vitro may have
tion were compared (21). Ahlquist et al. (22)and Schafer affected the results (14), or the anatomy of the root canals
and Lohmann (23)showed that hand instrumentation may have been a contributory factor. Mandibular premo-
produced cleaner canals than preparation with rotary lars, in horizontal section, have an oval-shaped root
instruments. canal, which is broad in the buccolingual direction
E. faecalis was chosen as the bacteriological marker in until the middle-third of the root canal (26). Surfaces
this study. It is a non-fastidious, easy-to-grow facultative untouched by the instruments may remain.
anaerobic bacterium of significant clinical importance In this study, the bacteriological sampling was accom-
that can be used in a study applying a bacteriological plished with a sterile paper point to observe the root canal
assessment method. Other bacteria commonly asso- contents. The paper point was then transferred to tubes
ciated with endodontic infections may require symbiotic containing saline solution. The use of paper points has
support from other bacteria, but E. faecalis has been the advantage that it can be performed in vitro and in vivo.
reported to survive and successfully increase alone (24). On the other hand, bacteriological sampling with paper
The SEM evaluation used in the present study confirmed points is limited because only the microorganisms that

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Australian Endodontic Journal © 2010 Australian Society of Endodontology
Elimination of Bacteria from the Root Canal M. Gorduysus et al.

are in the root canal can be sampled, while those located rial infection during treatment of apical periodontitis: a
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It is suggested that further studies be performed with 10. Dalton BC, Ørstavik D, Philips C, Pettiette M, Trope M.
irrigation solutions and intracanal medicaments in in vitro Bacterial reduction with nickel-titanium rotary instru-
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13. Matsuo T, Shirakami T, Ozaki K, Nakanishi T, Yumoto
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