Keywords
bacterial reduction -
Enterococcus faecalis
- WaveOne Gold - ProTaper Gold
Introduction
One of the major objectives of root canal treatment is the reduction of bacteria flora
in the root canal system. Chemomechanical instrumentation achieves a major part of
this task.[1]
[2] Therefore, the development of chemomechanical strategies that maximize root canal
disinfection before obturation is of prime importance. Many rotary nickel-titanium
systems have emerged to facilitate the cleaning and shaping procedure; however, anatomic
complexities might represent physical constraints that pose a severe challenge to
adequate root canal disinfection. The introduction of single-file systems in comparison
to multiple file systems presents another challenge among endodontists to select the
system that can provide a superior bacterial reduction in the root canal.
The WaveOne Gold system (Dentsply Maillefer, Ballaigues, Switzerland), with its reciprocating
motion, was shown to have a favorable performance in root canal preparation[3] and bacterial reduction.[4] However, the WaveOne system has also been shown to produce a higher amount of smear
layer in the canal.[5] ProTaper Gold (Dentsply Tulsa Dental Specialties, Tulsa, Oklahoma, United States)
was recently introduced as a multiple file system that is manufactured with advanced
metallurgy through heat treatment technology. ProTaper Gold has two-stage specific
transformation behavior and high austenite finish temperatures, which is around 55°C.[6] It consists of three shaping files (SX, S1, and S2) and five finishing files (F1,
F2, F3, F4, and F5). It has triangular cross-sections and progressive tapers as ProTaper
Universal (Dentsply Tulsa Dental Specialties).[7] In addition, it uses the same rotary motion and settings. However, it was found
that it provides less transportation,[8] higher flexibility, and higher cyclic fatigue resistance.[6]
[9]
Therefore, due to the increasing interest in single-file systems, the aim of the present
study was to compare the ability of a single (WaveOne) and a multiple file (ProTaper
Gold) systems to reduce the bacterial flora in the root canal system. The null hypothesis
tested in this study was that there is no difference in the bacterial reduction between
the rotary file systems under investigation.
Materials and Methods
Sample Selection and Classification
The proposed study was approved by the institutional review board (IRB # 16–00287).
Maxillary central incisors (n = 62) were collected from 30 to 50 years old patients attending the outpatient clinic
of MetroHealth Medical Center Oral Health Department, Ohio, United States. The teeth
were sectioned at the cementoenamel junction using a fine steel disc (NTI diamond
disc, Axis Dental, United States) mounted on a high-speed handpiece with water coolant.
All samples were standardized to 15 mm and instrumented to a working length (WL) of
14 mm up to 15 K-file (Dentsply Maillefer) under irrigation with sterile saline. The
root canals were filled with 17% ethylenediaminetetraacetic acid for 2 minutes then
washed with 5 mL sterile saline. The apex was covered with composite filling material
(3M, Saint Paul, Minnesota, United States), and the root surface was covered using
a bonding agent (3M). All samples were fixed onto polystyrene microtiter plates using
self-acrylic resin and were sterilized using a steam autoclave (MELAG, Medizintechnik
Geneststrase, Berlin, Germany) at 134°C for 15 minutes. Samples were divided into
two groups: the WaveOne Gold group (n = 30) and the ProTaper Gold group (n = 30). Each group was further subdivided into subgroup A (n = 15), where no activation of the irrigant was performed, and subgroup B (n = 15) where passive ultrasonic activation (PUI) was applied. Two samples served as
the negative controls to assess the sterility of teeth.
Bacterial Culture and Inoculation of Specimens for Biofilm Formation
Enterococcus faecalis (ATCC 51299) was cultured overnight in thioglycollate broth (Remel, Lenexa, Kansas,
United States) under the aerobic condition at 37°C. A 0.5 McFarland Standard Solution
(MSS; ~1 × 108 colony-forming unit [CFU]/mL) was made from this fresh overnight culture. A subsequent
1:10 dilution of 0.5 MSS with thioglycollate broth was prepared to inoculate the teeth
specimens by immersing them in 3 mL of the solution and incubated at the aerobic condition
at 37°C for 4 weeks. The culture medium was replaced with a fresh thioglycollate broth
every other day.[10]
[11] Negative control samples (n = 2) were incubated in phosphate-buffered saline (PBS; pH 7.4; Life Technologies,
Grand Island, New York, United States).
After incubation, positive control samples (n = 4) were randomly selected from each subgroup for scanning electron microscopy (SEM)
evaluation (magnification 5000×) for biofilm formation ([Fig. 1]) and for Enterococcus genus-specific quantitative real-time polymerase chain reaction (qPCR).
Fig. 1 (A, B) Untreated positive controls (5000×); (C) sample from ProTaper Gold group with passive ultrasonic activation (PUI) (8000×)
showing no microorganisms; (D) sample from ProTaper Gold group without PUI (5000×) showing few microorganisms;
(E) sample from WaveOne Gold without PUI (2500×) showing few microorganisms; (F) sample from WaveOne Gold with PUI (2500×) showing few microorganisms.
Root Canal Instrumentation
The WL was determined by passing a 10 K-type file through the apical foramen and withdrawing
it 0.5 mm. Samples of the ProTaper Gold group were instrumented following the directions
of the manufacturer with the following sequence: S1, S2, F1, F2, and F3. Each canal
preparation was performed with a new set of instruments mounted on endodontic 6:1
reduction handpiece (Dentsply Maillefer) powered by an electric motor (Dentsply Maillefer)
at 300 revolutions per minute and 2.5 Ncm. Samples of the WaveOne Gold group were
instrumented following the directions of the manufacturer in a pecking motion until
reaching the full WL. The WaveOne Gold files were mounted on a 6:1 reduction handpiece
connected to a reciprocating motor (Dentsply Maillefer).
In subgroups, irrigation was accomplished with a 10-mL syringe and a 30-gauge needle
(NaviTip). In subgroup B, irrigation was performed with an ultrasonic device (Piezon
Master 400) and a stainless steel K-type file size 15 (Endosonore; Dentsply Maillefer),
with its power set at the ¼ of the scale. In all subgroups, a total volume of 20 mL
NaOCl (1%) was delivered per canal. The flow rate in subgroup A was approximately
5 mL/min. In subgroup B, the delivery rate during the PUI with a continuous flush
of the irrigant was 10 mL/min. The insertion depth of the needle and the ultrasonic
file was 1 mm short of WL in all subgroups.
Microbiological Assessment
All samples were split mesio-distal into two parts. Two samples from each subgroup
were selected for SEM magnification of 10,000× and 20,000×. SEM examination of positive
controls showed excellent biofilm formation by E. faecalis (ATCC 51299) in the root canal walls after 4 weeks of incubation, and the test organism
could clearly be identified based on cocci morphology in clusters ([Figs. 1A] and [B] ).
The remaining samples were sonicated for CFUs and qPCR.[12] Sonication (Branson 8800, model CPX8800H, Branson Ultrasonics, Danbury, Connecticut,
United States) was performed for each sample for 5 minutes in 1 mL of PBS. A 10-µL
aliquot of the sonicated fluid was plated in triplicates with blood agar (Remel) and
incubated overnight at 37°C; then, the CFUs per mL were counted. The remaining sonicated
fluid was stored at –70°C for qPCR assay to determine the bacterial load. Total nucleic
acid was extracted from 200 µL of sonication fluid from each teeth specimen using
the NucliSENS easyMAG automated extraction system (bioMerieux Inc., Durham, North
Carolina, United States). The Enterococcus genus-specific primers targeting tuf gene (Ent1: 5′-TAC TGA CAA ACC ATT CT GAT G-3′ and Ent2: 5′-AAC TTC GTC ACC AAC GCG
AAC-3′) were used at 1 µM concentration.[3] The qPCR was performed with a LightCycler 1.5 instruments using LightCycler FastStart
DNA Master SYBR Green 1 Hot start reaction mix (Roche Molecular, Indianapolis, Indiana,
United States) per manufacturer’s instructions. The following thermal cycling protocol
was used: 95°C for 10 minutes (preincubation), 45 cycles of 95°C for 10 seconds (denaturation),
55°C for 5 seconds (annealing), and 72°C for 25 seconds (extension), 1 cycle of melt
curve with 95°C for 0 second (denaturation), 65°C for 15 seconds (annealing), and
95°C for 0 second (ramp rate = 0.1°C/s for melting), and 1 cycle of 40°C for 30 seconds
(cooling). The quantification of bacterial copies was determined from the standard
curve generated by testing a known number of bacterial cells using the Roche Molecular
quantification procedure.
Statistical Analysis
Statistical analysis was performed using statistical analysis software SPSS (Statistical
Packages for the Social Sciences 20.0; IBM, Armonk, New York, United States). One-way
analysis of variance followed by Tukey’s post hoc test was performed. Significance
level was set at 0.05.
Results
Scanning Electron Microscopy
Among representative treated teeth specimens, two showed no viable organisms at all
(one treated with ProTaper Gold without prior ultrasonic treatment, [Fig. 1C], [a]nd another treated with WaveOne Gold with prior ultrasonic treatment, [Fig. 1F]). In general, less organism burden was observed in experimental teeth specimens
compared with untreated controls ([Figs. 1D] and [E] ).
Colony Formed Units
Data collected from the CFU evaluation method is presented in [Table 1]. The ProTaper Gold group showed statistically superior bacterial reduction than
WaveOne Gold with or without the use of PUI (p < 0.001). Compared with the control group, both groups under investigation showed
a statistically significant reduction in CFU with or without the use of PUI (p < 0.001).
Table 1
Bacterial count (Log10 CFU/mL) for ProTaper Gold and WaveOne Gold with or without
ultrasonic
|
ProTaper Gold group
|
WaveOne Gold group
|
Positive control
|
p-Value
|
|
Abbreviations: CFU, colony-forming unit; PUI, passive ultrasonic activation.
Note: Different capital letters represent statistically significant differences (p < 0.05) in the same row, whereas different small letters represent significant differences
in the same column (p < 0.05).
|
|
With no PUI
|
95 ± 72Aa
|
976 ± 821Ba
|
5,010 ± 1,345Ca
|
< 0.001
|
|
Using PUI
|
103 ± 39Aa
|
436 ± 469Ba
|
4,353 ± 1,290Ca
|
< 0.001
|
|
p-Value
|
0.7609
|
0.087
|
0.27
|
|
Quantitative Real-Time PCR
Data collected from the qPCR evaluation method is presented in [Table 2]. The ProTaper Gold group showed statistically superior bacterial reduction than
WaveOne Gold without the use of PUI (p < 0.001). However, there was no significant difference between both groups upon using
PUI. Compared with the control group, both groups under investigation showed a statistically
significant reduction in CFU with or without the use of PUI (p < 0.001).
Table 2
Quantitative PCR (PCR E+6) for ProTaper Gold and WaveOne Gold with or without ultrasonic
|
ProTaper Gold group
|
WaveOne Gold group
|
Positive control
|
p-Value
|
|
Abbreviations: PCR, polymerase chain reaction; PUI, passive ultrasonic activation.
Note: Different capital letters represent statistically significant differences (p < 0.05) in the same row, whereas different small letters represent significant differences
in the same column (p < 0.05).
|
|
With no PUI
|
3.06 ± 2.37Aa
|
7.18 ± 4.33Ba
|
50.1 ± 17.4Ca
|
< 0.001
|
|
Using PUI
|
3.45 ± 2.38Aa
|
5.44 ± 3.31Aa
|
44.34 ± 5.8Ba
|
< 0.001
|
|
p-Value
|
0.713
|
0.326
|
0.334
|
|
Discussion
Evaluation of endodontic instrumentation includes morphometric analysis, microscopic
observation of remaining debris,[13] and bacteriological assessment.[14]
[15] The bacterial reduction was selected for this study because of the utmost importance
of canal disinfection in the treatment and prevention of apical periodontitis.
A. faecalis, facultative anaerobic Gram-positive cocci, was selected for this study because it
is always involved in resistant endodontic infections, having the ability to survive
under extreme environmental conditions,[16]
[17] which may contribute to bacterial resistance to intracanal antimicrobial procedures.[18] The specimens were infected with E. faecalis for 4 weeks to ensure deeper bacterial penetration into dentinal tubules,[19]
[20] which makes it harder to eliminate them from the infected root canal.[21]
Single-file systems have become recently available for root canal instrumentation,
but evidence as to their cleaning and disinfecting abilities is still questionable.[3]
[22]
The present quantitative results showed that chemomechanical preparation promoted
a highly significant reduction in intracanal bacterial count irrespective of the system
used for preparation.[14]
[15]
[23]
[24]
Real-time PCR was used as a quantification method in this study, because of its high
sensitivity and the ability to detect and quantitate not only cultivable bacteria
but also culture-difficult species, and noncultivable.[24]
The use of single-file instrumentation is thought to compromise the disinfection ability.
This is because of the claimed simplification of the preparation process hence a decrease
in the amount of antibacterial irrigant being used, combined with a shorter time in
the canal.[25] This was confirmed in this study, in which significant reduction was observed in
favor of the multifile system, ProTaper Gold without the use of activation method.
However, the use of PUI as an activation method resulted in a significant reduction
in the case of the single-file WaveOne Gold, and hence there was no significant difference
between the single and multifile system in that case. These findings were consistent
with the conventional CFU counting method. The minor conflict was noticed between
both quantification methods in case of absence of activation method as there was no
difference in bacterial reduction between the single and multifile systems in CFU
count while there was a significant difference in the PCR quantification method. This
conflict might be attributed to the fact that dead bacteria that remained in the canal
with their deoxyribonucleic acid made it detectable in PCR but not cultivable using
the CFU counting method.
Under the conditions of this study, the two instrumentation systems used, single and
multifile techniques, significantly reduced the bacterial counts. However, multifile
techniques showed better antibacterial results compared with a single-file technique
without the use of any activation methods. The use of PUI dramatically improved the
antibacterial performance of single files. The use of activation methods is recommended
with single-file techniques.
