TITLE: 

Ex vivo microbial leakage after using different final irrigation
regimens with chlorhexidine



ABSTRACT.OBJECTIVE:
To assess the influence of final irrigation protocols with chlorhexidine in the
coronal leakage of Enterococcus faecalis in filled root
canals.

ABSTRACT.MATERIAL AND METHODS:
Seventy single-root canals from extracted teeth were prepared using ProTaper
instruments. The irrigation protocol accomplished an alternating irrigation with 5
mL of 2.5% sodium hypochlorite (NaOCl) and 17% EDTA between each file. The teeth
were randomly divided into four experimental groups (n=15) according to the final
irrigation regimen: group 1, without final irrigation; group 2, irrigation with 10
mL 2.0% chlorhexidine (CHX); group 3, with a final application of
EC40TM; and group 4, irrigation with the combination (1:1) of 0.2%
CHX + 0.1% cetrimide (CTR). All the teeth were mounted in a two-chamber apparatus
and the coronal access was exposed to E. faecalis. The presence
of turbidity in the BHI broth over a period of 180 days was observed. The Friedman
test was used for statistical analysis.

ABSTRACT.RESULTS:
EC40TM varnish showed the least leakage at 180 days, and was
statistically similar to 2% CHX. No significant differences were observed between
the group without final irrigation and the 2% CHX group or 0.2% CHX + 0.1%
CTR.

ABSTRACT.CONCLUSIONS:
In this ex vivo study, EC40TM showed the longest
delayed coronal leakage of E. faecalis, although without
significant differences from 2% CHX.

BODY.INTRODUCTION:
The use of irrigating solutions in root canal treatments is essential to dissolve
organic tissue, to eliminate bacteria and to remove the smear layer formed on root canal
surfaces by the action of endodontic instruments40. The alternating use of sodium hypochlorite (NaOCl) with
chelating agents, like ethylenediaminetetraacetic acid (EDTA) or citric acid, has been
recommended during root canal preparation to this end25. Chlorhexidine (CHX) has also been recommended as a root
canal irrigant because of its antimicrobial activity, its capacity to adhere to dental
hard tissues, its gradual release (substantivity), and its low grade of
toxicity22. Kishen, et
al.21 (2008) showed that the
bacterial adherence to dentin was significantly influenced by the last irrigant used on
the root canal dentin; thus, 2% CHX used as the final solution, alone or after 17%
EDTA-5.25% NaOCl, reduced the adherence of Enterococcus faecalis.
Vivacqua-Gomes, et al.35 (2002)
reported a decrease in coronal leakage in root canals irrigated with a combination of 1%
NaOCl and 17% EDTA, or with 2% CHX gel, than for those irrigated with only 1% NaOCl or
with 1% NaOCl followed by 2% CHX gel. When applied in the gel form, a concentration of
2% CHX may exert antimicrobial activity15,29 and delay
recontamination of the root canals6,18. CHX varnishes have mainly been used for
the control of dental caries. The EC40TM varnish has 35% CHX, 27% sandarac
resin and 38% ethanol and is able to effectively reduce plaque acidogenicity17. EC40TM can penetrate and
seal tubules in dentin2, reducing the
level of mutans streptococci in exposed root surfaces38. Nonetheless, we found no studies assessing the efficacy
of EC40TM in root canals against E. faecalis.
CHX can be used alone or combined with surfactant agents such as cetrimide
(CTR)9; in turn, CTR is a
cationic surfactant largely used in endodontics in conjunction with other irrigants.
When combined with CHX, it is capable of eliminating E. faecalis
planktonic cultures24 as well
as biofilms3. Considering the
antimicrobial efficacy of CHX in root canals, the objective of the present study was to
assess the influence of different final irrigation protocols, using diverse
concentrations and CHX vehicles, in the coronal leakage of E. faecalis
in filled root canals.

BODY.MATERIAL AND METHODS:
Seventy freshly extracted mandibular incisor teeth with single, straight root canals and
fully developed apices were collected. Any remaining tissue was mechanically removed
using a curette, without producing damage to the root surface. The teeth were cleaned
with 0.5% NaOCl and then stored in 0.9% sterile normal saline solution at room
temperature until use.
The crowns were removed and the coronal surfaces of the root were sectioned
perpendicular to the long axis of the root using a turbine handpiece and a diamond bur.
To ensure uniformity in the samples, the root length was set at approximately 15 mm from
the coronal surface to the apex of the root. The protocol was approved by the ethics
Committee of the University of Granada.
BODY.ROOT CANAL PREPARATION:
All the teeth were prepared by the same operator. The patency of each canal was
confirmed by inserting an ISO size 10 K file (Dentsply Maillefer, Ballaigues,
Switzerland) until the tip was just visible at the apical foramen. The working length
was determined by subtracting 1 mm from the length. The root canals were prepared
using ProTaper instruments (Dentsply Maillefer) with the crown-down technique
recommended by the manufacturer; all were enlarged with a F3 file (finishing file
number 3; taper 0.09-0.05; size 30). A size 10 K file was used between each ProTaper
instrument to verify the patency of the canal.

BODY.IRRIGATION PROTOCOLS:
Next, the irrigation was carried out using a 3 mL Luer-Loc syringe coupled to a
30-gauge needle tip placed passively into the canal up to 2 mm from the apical
foramen without binding. Alternating irrigation of 5 mL of 2.5% NaOCl and 5 mL of 17%
EDTA solutions was performed between each file. After the root canal preparation, the
teeth were flushed with 10 mL of 2.5% NaOCl and then randomly divided into four
experimental groups (n=15) according to the final irrigation
protocol. Group I was without final irrigation. The other three groups, before the
final irrigation regimen, were flushed with 5 mL distilled water and dried with F3
paper points (Dentsply Maillefer). Group II received a final flush with 10 mL of 2.0%
CHX solution; Group III a final application of EC40TM varnish (Biodent,
Nijmegen, The Netherlands) using a microbrush at an established length of 15 mm from
the canal orifice; and Group IV, a final flush with 10 mL of 0.2% CHX + 0.1% CTR. To
prepare this combination, both solutions were prepared at double concentration and
mixed 1:1 to obtain the desired concentrations in the mixture. All the root canals
were dried after the final irrigation protocols with F3 absorbent paper points.
Ten teeth served as controls: 5 positive (roots that were not obturated) and 5
negative (teeth with intact crowns).
The root canals were filled using a F3 master gutta-percha point (Dentsply Maillefer)
and AH Plus® sealer (Dentsply Maillefer). Lateral condensation was
performed using a #25 spreader (Dentsply Maillefer). The gap left by the spreader was
filled with #25 gutta-percha points. The excess guttapercha was cut off using a hot
instrument, and the coronal portion of the warm gutta-percha was firmly condensed
vertically. All these samples were stored at 37ºC for 72 hours to allow the sealer to
set.

BODY.MICROBIAL LEAKAGE TEST:
The external surfaces of all teeth, except 2 mm around the apical foramen, were
covered with two layers of nail varnish in order to prevent bacterial leakage through
the lateral canals. The negative controls were fully covered by two layers of
varnish.
The microbial test consisted of a 2-chamber method10. The upper chamber contained 1.5 mL eppendorf plastic
tubes (elkay, Shrewbury, MA, USA) tapered and cut to receive the root end. The
interface between the tooth and the eppendorf tube was put into the rubber cork of a
penicillin bottle previously cut to fit inside the lower chamber. The junctions
between the root, the eppendorf tube and the rubber cork were sealed with
cyanoacrylate adhesive (Super Glue-3, Henkel Ibérica, S.A., Barcelona). The mounts
were sterilized for 45 minutes in hydrogen peroxide gas plasma (Sterrad-50, Advanced
Sterilization Products, Johnson & Johnson, Irvine, CA, USA).
After sterilization, the apparatus was placed in a glass flask (the lower chamber)
containing sterile Brain Heart Infusion broth (BHI, Scharlau Chemie S.A., Barcelona,
Spain). The root tip had 2-3 mm immersed in the broth. The junctions between the
Eppendorf tubes and the glass flasks were tightly sealed with Parafilm MTM
(Pechiney Plastic Packaging, Chicago, IL, USA) and cyanoacrylate adhesive.
An initial bacterial suspension containing 6 x 108 CFU/mL of E.
faecalis (ATCC 29212) was obtained in a turbidimeter (bioMérieux S.A.,
Densichek Plus, NC, USA). The Eppendorf reservoirs were filled with 1 mL of the
initial suspension keeping the bacterial suspension in contact with the coronal
portion of the filled roots. The mounts were always handled in sterile conditions
under a laminar flow hood (Nuaire, Plymouth, MN, USA) to avoid bacterial
contamination. They were placed in an oven at 37C during a period of 180 days. The
culture medium in the upper chamber was replaced with freshly grown broth twice a
week. The viability and purity of the bacteria were checked every week by seeding
some in blood agar plates and observing the colony morphology.
The lower chambers of all mounts were observed daily, the turbidity time was recorded
for each specimen as an indicator of entire root canal contamination. Once turbidity
was present, a sample of the turbid broth was streaked onto blood agar plates and the
bacteria were identified to ensure that there was no contamination other than
E. faecalis. The presence of E. faecalis in the
root canal walls was confirmed under scanning electron microscopy (Figure 1).
Figure 1Scanning electron microscopy (SEM) micrograph. E. faecalis
biofilm attached to the root canal wall and entering the dentinal tubule

BODY.STATISTICAL ANALYSIS:
The number and percentage of specimens not leaking were determined for each group in
the five evaluation periods (45, 60, 90, 120 and 180 days). Since the assessment of
the days of bacterial leakage did not show a normal distribution, the Friedman test
was used to evaluate the significance of the differences over time for the four
irrigating protocols, as well as for the paired comparisons. Moreover, the
significance and sign of the slope regression over time served to study the
intragroup behaviour of the irrigating protocol. The level of statistical
significance was set at 5%. All statistical analyses were performed with the SPSS
15.0 software (SPSS Inc, Chicago, IL, USA).


BODY.RESULTS:
Table 1 shows the percentage of specimens with
no leakage at the different periods of evaluation, and the comparisons among the groups.
No leakage was observed in the negative control group at any time period of study. The
positive control group leaked on the second day.
Table 1Specimens not leaking in the different time periods (initial sample n=15 in each
group)
 
Evaluated times (days)
 
45
60
90
120
180
FINAL PROTOCOL
n (%)
n (%)
n (%)
n (%)
n (%)
2.5% NaOCl*
13 (86.7)
12 (80.0)
11 (73.3)
9 (40.0)
3 (20.0)
2% CHX*,**
15 (100.0)
15 (100.0)
10 (66.7)
7 (46.7)
3 (20.0)
EC40TM varnish**
15 (100.0)
15 (100.0)
14 (93.3)
9 (60.0)
5 (33.3)
0.2% CHX + 0.1% CTR*
12 (80.0)
12 (80.0)
9 (60.0)
8 (53.3)
1 (6.7)
The same superscript small asterisks show statistically similar groups
according to the Friedman test. In all cases p<0.05 is considered a
statistically significant difference.
The global comparison among the groups, simultaneously taking into account the five
evaluation periods using the Friedman global model, gave significant differences with a
value of p=0.021. The results of the pair-by-pair comparisons between
protocols accounting for the five evaluation periods can be viewed in the Table.
EC40TM exhibited a similar behaviour to 2% CHX. Yet, in the 90 day period,
the EC40TM group had only one leaked sample, in contrast with the five
samples showing leakage in the 2% CHX group. EC40TM had significant
differences when compared to the 2.5% NaOCl group and the group 0.2% CHX + 0.1% CTR.
No significant differences were observed between the 2.5% NaOCl group and the 2% CHX
group or 0.2% CHX + 0.1% CTR. The slopes of the regression models relating to the
percentage of non-leaked specimens with the time period were statistically significant
and negative for all the irrigating protocols, thereby showing an intra-group reduction
of non-leaking over time.

BODY.DISCUSSION:
Obtaining hermetic tri-dimensional sealing of a root canal is essential for isolating
the microorganisms that may remain after biomechanical preparation and preventing their
entry from the oral cavity30,32. In this sense, coronal restoration is
an important requisite for long-term endodontic success, and restorative materials
should provide a permanent, leak-proof seal. Defective temporary or permanent
restoration, during or after root canal treatment, is known to be a main cause of
coronal leakage8.
Several studies have shown that coronal micro-leakage can occur with any of the
different obturation techniques used10,26. However, the irrigation procedure seems
to play a key role in the success of endodontic treatment, as it can delay or decrease
bacterial contamination of the treated canal21,35. Therefore, in order to
enhance efficient antimicrobial action over time, the use of irrigating solutions with a
residual antiseptic capacity, such as CHX, is recommended22, although it is not effective for smear layer or debris
removal33.
Diverse in vitro methods for evaluating the sealing ability of root
canal filling materials have involved the use of dyes, scanning electron microscopy,
fluid filtration techniques, electrochemical methods, radioisotopes, and
bacteria34. Evaluation of
coronal leakage by bacteria provides data that are more biologically significant and
clinically relevant than other methods34. In our study, the 2-chamber model described by Imura, et
al.19 (1997) and modified by
De-Deus, et al.10 (2005) was applied.
It is a static model that does not exactly simulate clinical conditions, requiring a
long period of observation, and not allowing for quantification of the number of
penetrating bacteria30. In addition,
the presence of leakage evaluated as positive turbidity is a binary variable that is not
very accurate. The bacteria selected for our study was E. faecalis,
often isolated from necrotic or improperly filled root canal systems, as well as from
properly filled ones16. E. faecalis
ATCC 29212 is a strain of reference widely used in antimicrobial susceptibility studies,
and it exhibits a great capacity for forming biofilms20.
Mandibular incisors with single oval canals were used in this study. It is known that
the prevalence of long oval root canals in the apical third of mandibular incisors is
> 50%39, representing a great
challenge for proper cleaning and disinfection of the root canal1,27. In canals with these anatomical conditions, hand and rotary
instruments work in reaming motion and would leave some un-instrumented recesses, which
have the potential to harbour persistent bacteria7,36. In this sense,
instruments with greater taper, like the ProTaper, are more efficient than NiTi- hand
files, but in some cases, at the expense of remaining dentin-wall thickness13. Moreover, the quality of the root
fillings in oval canal-mandibular incisors may be compromised31; however, different obturation techniques in oval-shaped
canals have not shown statistical differences10,11.
Preparation of root canals was similar in all the groups of our study and included
sequential use of 2.5% NaOCl and 17% EDTA with a final flush of 2.5% NaOCl. However, all
groups were not treated with the same amount of final irrigating solution, which may
have influenced the results. The regimen of final irrigation has been shown to influence
the capacity of adherence of E. faecalis21. Alternating use of NaOCl and EDTA can prove to be
beneficial. NaOCl can dissolve organic matter and necrotic tissue as well as exert
strong antimicrobial action14. In
turn, EDTA is able to remove the smear layer40 formed during preparation of the root canal, although its
antimicrobial activity is still a matter of debate. Using NaOCl as the final irrigating
solution is widely recommended40, but
it has not demonstrated any residual antibacterial activity23.
A 2% CHX solution is recommended as the final irrigant in the root canal
preparation22. Nevertheless, the
use of a CHX varnish such as EC40TM has not been tested in endodontics. Our
results indicate that the delay of leakage was greater in the EC40TM group,
while the 2% CHX group showed an intermediate effectiveness. Vivacqua-Gomes, et
al.35 (2002) obtained better
results with a 2% CHX gel than with a 2% CHX solution, suggesting the importance of the
vehicle of application. Moreover, the antimicrobial substantivity of CHX depends on the
number of CHX molecules available to interact with the dentin22, and EC40TM has a very high concentration of
this active agent. White, et al.37
(1997) evaluated substantial antimicrobial activity in instrumented root canals using
2.0% and 0.12% CHX as irrigants; their results indicated that the antimicrobial activity
remaining in the 2% CHX-treated teeth was significantly greater than in the 0.12%
CHX-treated teeth.
Another factor to bear in mind is the vehicle used. EC40TM can be considered
a slow release device, and varnishes were formulated with the objective of prolonging
the delivery of the active agent. Attin, et al.5 (2008) observed a greater release of CHX on bovine dentin fissures
treated with CHX varnishes as compared to dentin treated with CHX preparations, in gel
or in solution. Their study found the varnish providing the best release of CHX to be
EC40TM. Attin, et al.4
(2003) and Derks, et al.12 (2008)
compared the efficacy of different concentrations of CHX in varnish (EC40TM,
Cervitec) in reducing the levels of mutans streptococci, lactobacilli and in plaque
formation in interproximal plaque and saliva. According to their findings, application
of the highly concentrated varnish EC40TM results in a higher decrease of
mutans streptococci in plaque sites and saliva. Furthermore, it should be emphasized
that the 2% CHX was a solution, and since the root canals were dried with paper points,
it would have been better removed than the varnish.
Although it was recently demonstrated that the association of CHX and CTR enhances the
in vitro capacity to eradicate E. faecalis
biofilm3, our results
for the group 0.2% CHX + 0.1% CTR showed no differences with respect to the control
group. This may be due to the relatively low concentration of CHX, promoting a less
intense substantive action.
Notwithstanding, high concentrations of CHX in the form of a varnish (EC40TM)
would appear to ensure some delay in coronal leakage. Further investigations are needed
to evaluate the efficacy of different vehicles and concentrations of CHX and to assess
how they might affect the complete sealing of the root canal.

BODY.CONCLUSION:
In this ex vivo study, high concentrations of CHX in the form of a
varnish (EC40TM) would appear to ensure some delay in coronal leakage.

Question: Compared to irrigation with 10 mL 2.0% chlorhexidine (CHX) what was the result of irrigation with a final application of EC40TM on Specimens not leaking?
Evidence: EC40TM exhibited a similar behaviour to 2% CHX.
Answer: no significant difference