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CLINICAL DENTISTRY AND RESEARCH 2012; 36(2): 15-21
BACTERIAL REDUCTION IN INFECTED ROOT CANALS TREATED
WITH CALCIUM HYDROXIDE USING HAND AND ROTARY
INSTRUMENT: AN IN-VIVO STUDY
M. Ömer Görduysus, DDS, PhD
Professor, Department of Endodontics,
Faculty of Dentistry, Hacettepe University,
ABSTRACT
Background and Aim: The aim of this in vivo study was
Ankara, Turkey
to evaluate the microbial flora in infected root canals after
Zeliha Yılmaz, DDS, PhD
the treatment with and without calcium hydroxide (Ca(OH)2)
Associate Professor, Department of Endodontics,
Faculty of Dentistry, Hacettepe University,
Ankara, Turkey
dressing using hand and rotary instruments.
Subjects and Methods: Twenty-eight patients were selected.
After access cavity preparation completed, the first culture
Melahat Görduysus, DDS, PhD
were taken from each root canal with sterile paper points.
Professor, Department of Endodontics,
Four groups were formed. Group I and II were instrumented
Faculty of Dentistry, Hacettepe University,
Ankara, Turkey
Canan Kaya, DDS
Department of Endodontics,
Faculty of Dentistry, Hacettepe University,
Ankara, Turkey
Dolunay Gülmez, MD
Assistant Professor, Department of Medical Microbiology,
Faculty of Medicine, Hacettepe University,
Ankara, Turkey
with ProTaper instruments, group III and IV were instrumented
step-back technique with K files. Group I and III were medicated
with Ca (OH)2 paste. One week after, the second culture was
taken and the canals were filled. All samples were submitted
to microbiological processing to evaluate anaerobe and aerobe
microorganism. The results statistically analyzed with McNemar
and Chi-Square tests at p<0.05.
Results: There was a significant decrease in bacteria
elimination in Group III compared to the other groups (p<0.05).
Lindihana Emini, M.Sc
Groups, in which Ca(OH)2 was applied, were found significantly
Specialist of Dental Pathology and
better to eliminate bacteria in root canals than the groups
Endodontics Dental Clinic, Medident,
Tetovo, Fyrom
Veton Hoxha, DDS, PhD
Professor, Department of Dental Pathology and
Endodontics University Dental Clinic Center,
without Ca(OH)2.
Conclusion: Better root canal disinfection could be obtained
by mechanical preparation with application of Ca(OH)2 for one
week.
Prishtina, Kosova
Correspondence
Zeliha Yılmaz DDS,PhD
Department of Endodontics,
Faculty of Dentistry, Hacettepe University,
06100 Sıhhiye Ankara/Turkey
Phone: +90 312 3052260
Key words: Calcium Hydroxide, Microbiology, Root Canal
Treatment, Rotary Instruments
Fax: +90 312 3052832
Submitted for Publication: 10.05.2011
E-mail: [email protected], [email protected]
Accepted for Publication : 06.21.2012
15
CLINICAL DENTISTRY AND RESEARCH
INTRODUCTION
It is well known that remaining bacteria, either in the root
canal space or in parts of root canal system that are not filled,
is the main cause for root canal treatment failure.1,2 Thus,
removal of vital and necrotic pulp tissue, microorganisms and
their toxins is an important part of endodontic treatment.3
Unfortunately, studies have shown that both stainless
steel file systems which have been used for years and NiTi rotary instruments that have gained popularity recently
have lead to inadequate disinfection of root canal space
because of the complexity of root canal morphology, apical
ramifications, isthmuses, other morphological challenges
and bio-film form of root canal bacteria.4 As a result of
technological developments, Ni-Ti rotary instruments have
gained increased taper and different designs that can
significantly improve the cleaning and shaping procedure.
Many studies have been conducted to compare Ni-Ti
rotary instruments with stainless steel instruments.5,6
However, there have been few in-vivo studies in which NiTi rotary instruments provided better results in reducing or
eliminating intra-canal bacteria, compared with stainless
steel instruments.
During endodontic treatment, both mechanical preparation
and intra-canal dressing are used to reduce or eliminate
bacteria. Some studies have shown that mechanical
preparation significantly decreases bacteria in the root canal.
Yet, in approximately 20%-50% of the canals, remaining
bacteria was observed.7,8 For this reason, in addition to
mechanical preparation, different concentrations and types
of irrigation solutions and various medicaments are used to
remove microorganisms and to achieve disinfection.4 It has
been reported that the use of sodium hypochlorite (NaOCl)
in different concentrations during mechanical preparation
decreases the number of bacteria. Despite the use of this
solution, however, positive culture was obtained in 4060% of the root canals.1,9 In addition to irrigation solutions,
intra-canal medicaments are used to remove bacteria and to
disinfect the root canal systems. Calcium hydroxide (Ca(OH)2)
has been the most frequently used intra-canal medicament
due to its antimicrobial effect, osteoclastic activity and
suitable tissue response.10-12 Whether or not there is an apical
lesion, in cases with necrotic pulp, multiple-visit treatment
and the use of Ca(OH)2 as medicament is recommended for
a week to achieve the desired disinfection.10,13
This in-vivo study takes stock of results obtained in previous
research highlighted above and aims to assess the amount
16
of microbial flora in infected root canals by using Ni-Ti rotary
instruments and stainless steel instruments, once with and
once without the use of Ca(OH)2.
SUBJECTS AND METHODS
Case selection
Patient population consists of 28 people (18 female, 10
male) who frequented Hacettepe University, Faculty of
Dentistry, Department of Endodontics. In total, 28 teeth
(9 maxillary incisors, 2 maxillary premolars, 6 maxillary
molars, 2 mandibular incisors, 1 mandibular premolar and
8 mandibular molar) have been treated. Patient selection
criteria were as follows:
1- The teeth with intact pulp chamber walls,
2- Necrotic pulps, negative response to the pulp
tests (electrical and cold),
3- Clinical and radiographic evidence of chronic apical
periodontitis lesions,
4- Teeth from patients who have not received
antibiotic therapy within the preceding month,
5- Patients without swelling or any symptoms or
periodontal problems.
Before initiation, the study was approved by the Ethics
Committee of the Hacettepe University and informed
consent was obtained from each patient.
Endodontic Treatment and Sampling Procedures
After the clinical and radiographic examination of the
patients, each tooth was cleaned of plaque, calculus and
attachments. All decay and unsupported tooth structure
were removed and canal orifices were localized after access
cavity preparation. Following the rubber-dam isolation, all
tooth surfaces were cleaned with a cotton pellet immersed
in 2.6% NaOCl. A #15 K file was inserted into canals for
apical patency; then, the first root canal sample was taken.
The sterile paper point was placed in each root canal to
a level approximately 1 mm short of the apex, based on
diagnostic radiographs, and was used to soak up the fluid
in the canal. Each paper point was kept in the canal for 3
minutes. Paper points were then placed into aseptically
tubes containing 1 mL tiyoglukonated buyyon.
Four groups were formed, each with 7 teeth. After estimating
the working length, root canals in Groups I and II were
prepared with ProTaper Ni-Ti rotary instruments (DentsplyMaillefer, Ballaigues, Switzerland) to a master apical size of
#30 using an endodontic micromotor (X-Smart, Dentsply,
Infected Root Canals Treated with Calcium Hydroxide
Maillefer). RC-Prep (RC-Prep; Premier Dental, Norristown, PA)
chelating agent was used for lubrication. In Groups III and
IV, root canals were prepared with the step-back technique
using stainless steel files, with the master apical file being
determined according to each tooth’s anatomy. The canals
were irrigated with 0.5 ml 2.5% NaOCl after each file. In all
cases, chemo-mechanical preparation was completed at
the first appointment. After the instrumentation, 5 ml 2.5%
NaOCl was applied as final flush; then each canal was dried
up by using sterile paper point (Spident, Incheon, Korea). In
Groups I and III, root canals were filled with Ca(OH)2 (SUREPaste, Korea). Ca(OH)2 paste was placed by using a lentulo
spiral and packed with a cotton pellet at the level of canal
orifice. A radiograph was taken to ensure proper placement
of the paste in the canal. Then the access cavities were
filled with at least 4 mm of a temporary filling material
(Nucavfil; PSP Dental, Belvedere, Kent, UK).
The second appointment was scheduled for the following
week. At this time, the tooth was isolated with rubber-dam
and the operative field was disinfected. In Groups I and
III where Ca(OH)2 was applied, Ca(OH)2 paste was rinsed
out of the canal by using 2 ml sterile saline solution and
master apical files. In Groups II and IV, after temporary filling
material was removed, each canal was irrigated with only
2 ml sterile saline solution. The second culture was taken
from each canal as outlined above. Subsequently, the canals
were filled with gutta-percha (Diadent, Seoul, Korea) and AH
26 root canal sealer (Dentsply Detrey, Konstanz, Germany)
using cold lateral compaction technique. The tooth was
temporized with glass ionomer cement and a permanent
restoration was planned.
Microbiologic Analysis
Root canal samples were taken with sterile paper points
which were left in the canal for at 3 minutes and placed in 1
ml of thioglycolate broth. Each sample was vortexed and 10μl
of the sample was transferred onto each of the following
media: Sheep blood agar, chocolate agar, MacConkey agar
and Schaedler agar. All media were incubated overnight in
5-10% CO2 at 37 ºC (-/+ 2), except Schaedler agar which
was incubated in anaerobic atmosphere for 48 hours.
Colony counts were determined for each group of bacteria
with different colony morphologies and subcultured for
identification. Aerobic bacteria were identified to genus
level by Gram stain morphology, catalase and coagulase
tests, growth on bile-esculin agar, growth in 6.5% NaCl
and CAMP reaction. Anaerobic bacteria were identified to
species level by catalase test, indole reaction and Crystal
System (Becton Dickinson, USA).
Statistical Analysis
A non-parametric test (McNemar) was used for determining
the differences between first and second measurement
in the intergroup and the Chi-Square test was used for
evaluating the differences between the groups. A p value
<0.05 considered as statistically significant.
RESULTS
Table 1 shows the total distribution of bacterial types at
the beginning of access cavity preparation in the first visit
and at the end of preparation in the second visit. While the
total number Colony Forming Unit (CFU) of the first samples
was 551170 CFU/ml, after instrumentation, irrigation and
applying Ca(OH)2 the CFU count had dropped significantly
to total 391300 CFU/ml. In culture results, 44% of the
microbial flora of root canals was found to be aerobe while
46% of the flora was anaerobe. Most observed aerobe
bacteria species was Alpha hemolitic streptococcus with
a ratio of 28.8%. Most observed anaerobe bacteria species
was Veillonella spp with a ratio of 22.8%.
In all experiment groups, when the final measurements of
difference in the decrease of both aerobe and anaerobe
bacteria were evaluated (Table 2). Statistically significant
difference was only seen in Group III in which Ca(OH)2 was
applied after cleaning and shaping procedures with hand
instrument (p<0.05). There is a significant decrease in
bacteria elimination in Group III compared to other groups.
When initial and final changes in cultures were evaluated,
Group IV registered the highest level of bacteria compared
to other groups in which cleaning and shaping procedures
were carried out with hand instrument without using
Ca(OH)2 (p=0.0036). Groups in which Ca(OH)2 was applied
proved to be significantly better in eliminating bacteria in
root canals compared to groups in which Ca(OH)2 was not
applied (Figure 1). Cleaning and shaping procedures with
hand instrument were significantly better than rotary
instrument groups in eliminating bacteria from root canals
(p=0.03) (Figure 2).
DISCUSSION
An effective endodontic treatment requires the eradication
of bacteria or at least the reduction in bacterial counts in the
root canal space.14 Chemo-mechanical preparation of root
canal using different types and concentrations of irrigation
17
CLINICAL DENTISTRY AND RESEARCH
Table 1. The total distribution of bacterial types in the first visit and second visit
Aerobe Bacteria
Patient
Prevalance
n
CFU
%
Alfa Hem. Strep.
34,5
253500
28,81664
Coagulase-Stap
29,14
420800
47,83449
Neisseria
14,54
10400
1,182221
S. Viridans
5,46
17100
1,943844
Difteroid
5,46
24100
2,73957
Other
10,9
153800
17,48323
Total
100%
879700
100%
Anaerobe Bacteria
Patient
Prevalance
n
CFU
%
Veillonella ssp
34,70
16870
22,86837
Actinomyces
8,69
6000
8,133388
Fusobacterium
13,17
8200
11,11563
Eurobacterium lim
4,34
10000
13,55565
Peptostreptococus
8,69
11500
15,58899
Bacteriodes cap
4,34
5000
6,777823
Prevotella
4,34
5300
7,184492
Other
21,73
10900
14,77565
Total
100%
73770
100%
CFU: Colony Forming Unit
Table 2. Result of one week treatment according to groups
Groups
Result of one
week treatment
Rotary with Ca(OH)2
Rotary w/o Ca(OH)2
Manual with Ca(OH)2
Manual w/o Ca(OH)2
n
%
n
%
n
%
n
%
Elimination
7
53,8
0
0
14
100
2
12,5
Repeated-growth
2
15,4
5
41,7
0
0
2
12,5
Stagnation
0
0
2
16,7
0
0
0
0
Reduction
2
15,4
2
16,7
0
0
2
12,5
New
2
15,4
4
25
0
0
10
62,5
Total 13
100
13
100
14
100
16
100
n: The number of species of microorganism
solution has been shown to be critical step for reduction
of bacterial populations in the root canal but about 40%
to 60% of the canals still yield positive cultures after
instrumentation and irrigation.9,15 Bacteria in the infected
root canal are mostly anaerobic bacteria.16 Our study shows
that 44% of the microbial flora of root canals was aerobe
18
while 46% was anaerobe. According to the results reported
by Sundquist et al.17, Byström & Sundqvist18, Baumgartner
& Falker,19 Siqueira & Lopes,20 and Sassone et al.21, the most
microbial flora in infected root canals is anaerobic bacteria.
These authors concluded that the infection in infected root
canal is mainly polymicrobial, with anaerobic flora being less
Infected Root Canals Treated with Calcium Hydroxide
90
77,8
80
70
60
46,4
50
%
40
25
30
20
10
7,4
7,1
7,1
0
0
Elimination
Repeated
stagnation
Repeated
growth
With Ca(OH)2
4,3
7,4
7,4
Repeated
reduction
New
Without Ca(OH)2
Figure 1. The effect of Ca(OH)2 on bacterial flora
100
80
%
60
Group 1
Group 2
Group 3
Group 4
40
20
0
Elimination
Repeated
growth
Repeated
stagnation
Repeated
reduction
New
Figure 2. Results of one week treatment according to groups
frequent. The results reached in our study conflict with
those reported by the mentioned authors. This may be
explained by the method we applied for the identification
of microbes in root canal.
Although cleaning, shaping and irrigating the canal greatly
reduce the number of bacteria,16 it is generally believed
that the number of bacteria can also be controlled by
placing an inter-appointment dressing such as Ca(OH)2
within the prepared root canal.9 Estrela et al.22 analyzed
the antimicrobial properties of Ca(OH)2 and concluded that
the latter affects the cell membrane of both aerobe and
anaerobe bacteria. Sjögren et al.10 clinically evaluated the
antibacterial effect of Ca(OH)2 as a short-term intra-canal
dressing by applying the medicament for 7 days in root canals
of teeth with periapical lesions. These authors showed that
the 7 day dressing effectively eliminated bacteria which
survived biomechanical instrumentation of the canal. The
present study also shows that groups in which Ca(OH)2 was
applied were significantly better in eliminating bacteria in
root canals compared with groups in which Ca(OH)2 was
not applied. After the final measurement, a statistically
significant difference was observed only in Group III (treated
with Ca(OH)2 and hand instruments). Our results parallel
those of Sakamoto et al.23 showing the success of hand
instrumentation and treatment with Ca(OH)2 in 97% of
cases. De Rossi et al.24 investigated the effect of Ca(OH)2 in
infected root canals using hand and rotary instruments and
showed that, regardless of the instrumentation technique,
the use of intra-canal dressing is important in the endodontic
treatment for the elimination of bacteria in the root canal.
The present study demonstrates that instrumentation,
irrigation and applying Ca(OH)2 significantly reduces the
total number of microorganisms, namely approximately to
16 % of the original number. Peters et al.16 found that the
total number of microorganisms was reduced by 0.18 %
after instrumentation, irrigation and dressing with Ca(OH)2
on infection in pulpless teeth with periapical lesions. They
also concluded that such reduction was not related to the
use of Ca(OH)2 for 4 weeks. The difference in our findings
might relate to the duration of the use of Ca(OH)2 and the
transport media used. In our study, Ca(OH)2 was applied for
one week as opposed to four weeks.
In this study, we analyzed the effectiveness of hand and
rotary instrumentation in the elimination of bacteria from
root canal. Our results show that hand instruments are far
better than rotary instruments in cleaning and shaping
(p=0.03). Even if the crown–down technique was the
basis for the preparation with rotary files, taper and apical
diameter caused changes in the preparation. Similarly, in the
preparation with hand files, the taper and apical diameter
was different. In all Groups, master apical file was determined
according to each tooth’s number and anatomy. This might
have had an impact on dentin elimination and bacterial
reduction. ProTaper instruments have active cutting surface
which allows such instruments to progress easily in the root
canal and remove more infected dentin from the root canal
compared with hand instruments. However, although rotary
instruments appear to be better than hand instruments in
reducing bacteria counts, in this study, the Groups prepared
with hand files turned out to be better than rotary files on
bacterial reduction. The results of this study conflict with
those of Chuste-Guillot et al.25, Limongi et al.26 and Beun et
al.27 who concluded that hand and rotary instruments were
not significantly different in eliminating bacterial flora from
root canal. Evans et al.28 also showed no difference between
the hand-file step-back and NiTi Quantec systems regarding
pulp and pre-dentin elimination.
19
CLINICAL DENTISTRY AND RESEARCH
CONCLUSION
Our findings show that regardless of the technique and the
instrument, a similar quantity of bacterial reduction may be
achieved. A good root canal disinfection may be obtained
by mechanical preparation coupled with the application of
Ca(OH)2 for one week.
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