The Journal of the Australian Society of Endodontology Inc.

and
the Australian and New Zealand Academy of Endodo ntists

Aust Endod J 2009; 35: 5258

ORIGINAL RESEARCH

Effectiveness of propolis and calcium hydroxide as a short-term

intracanal medicament against Enterococcus faecalis:
A laboratory study
Lama Awawdeh, BDS, MSC, PhD1; Maha AL-Beitawi, BDS, MSC1; and Mohammad Hammad, BDS, PhD2
1 Department of Restorative Dentistry, Jordan University of Science and Technology, Irbid, Jordan
2 Department of Preventive Dentistry, Jordan University of Science and Technology, Irbid, Jordan

Keywords
calcium hydroxide, Enterococcus faecalis,
intracanal medicaments, propolis.
Correspondence
Lama Awawdeh, Department of Restorative
Dentistry, Faculty of Dentistry, Jordan
University of Science and Technology,
PO Box 3030, Irbid 22110, Jordan.
Email: lawawdeh@just.edu.jo
doi:10.1111/j.1747-4477.2008.00125.x

Abstract
The aim of this study was to investigate the antimicrobial activity of propolisbased intracanal medicament against Enterococcus faecalis using infected dentine
models, and to compare its antimicrobial efficacy with that of the non-setting
calcium hydroxide paste when used as a short-term medication for 1 and 2
days. A total of 50 dentine discs of 7-mm length was obtained from extracted
human teeth. Five dentine discs were kept sterile to serve as a negative control.
The remaining 45 were contaminated with E. faecalis and divided into two
groups (n = 20) in addition to five discs that served as a positive control. The
discs were treated as follow: 20 discs were filled with propolis, while the other
20 discs were filled with non-setting calcium hydroxide. Microbiological sampling was performed utilising sterile paper point, headstrom file and disc
immersion. Results showed that propolis was significantly more effective than
non-setting calcium hydroxide against E. faecalis after short-term application,
which made comparison from this prospect unlevelled. The most effective
microbiological sampling technique was abrading the lumen with headstrom
file. Propolis is very effective as intracanal medicament in rapidly eliminating
E. faecalis ex vivo.

Introduction
A favourable outcome of the endodontic treatment of
teeth with apical periodontitis depends on effective
control of the root canal infection (1,2). Chemomechanical cleaning and shaping of the root canal can greatly
reduce the number of microorganisms but not completely
eliminate them (3). Therefore, an effective antimicrobial
treatment protocol should be used to reduce bacterial
insult to minimum allowing hosts defence system to take
over and provide a favourable environment for healing
(4,5).
Enterococcus faecalis is part of normal oral flora, and is
found in small numbers in uninstrumented infected root
canals. Its role in endodontic infections was and still is a
big issue in root canal therapy. E. faecalis is the most
prevalent species isolated from root canals of previously
root-filled teeth with chronic apical periodontitis (6).
52

The activity of different intracanal medicaments against

E. faecalis was investigated widely and mostly calcium
hydroxide was the standard against which other medicaments were compared (79).
It was demonstrated that the use of calcium hydroxide
as a root canal medicament has limitations because it does
not eliminate the whole spectrum of microorganisms (10)
and it needs long time to provide its antimicrobial effect
(11).
Propolis is a natural flavanoid-rich resinous product of
honeybees, which is known for its biological properties,
including antibacterial, antifungal and healing properties
(12). A number of studies have been conducted, mainly
on animals and to a lesser extent on humans, to investigate the use of propolis in different dental fields (13,14).
Recently, the response of rat dental pulp capping with
propolis was assessed. It was suggested that flavonoids
from propolis may stimulate reparative dentine formation

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L. Awawdeh et al.

and may delay pulp inflammation by stimulating production of transforming growth factor (TGF)-b1 and synthesis of collagen by dental pulp cells (15).
At present, sodium hypochlorite as an irrigant and
calcium hydroxide as an intracanal medication is the
most used approach for root canal disinfection. Unfortunately, a number of studies have demonstrated that
despite the use of such antimicrobial agents, microorganisms such as E. faecalis may still persist (16,17). This may
allow further bacterial colonisation of the root apex and
surrounding periapical tissues, and consequently prevent
healing which would have a negative influence on the
prognosis of the treatment (18).
Therefore, many recent studies have focused on an
alternative intracanal disinfection protocol which is able
to eradicate such resistant microorganisms more rapidly.
There is mounting evidence, however, that obtaining
sterility of the infected root canal by presently available
treatment methods might be more difficult than once
thought (3,19).
This ex vivo study was conducted mainly to investigate
the antimicrobial properties of a Jordanian propolis-based
intracanal medicament against E. faecalis, to find the
minimum time needed to achieve its optimal antibacterial
effect using infected dentine models, and to compare
its antimicrobial efficacy with that of the non-setting
calcium hydroxide paste when used as a short-term
medication for 1 and 2 days. Three microbiological sampling methods: paper point, headstrom file and immersion of the dentine disc, were compared as well.

Materials and methods

Preparation of ex vivo model of root canal infection
Microorganism
Enterococcus faecalis (ATCC 29212, CULTI-LOOPS, OXOID
Ltd., Hampshire, England), used in this study, was
obtained from 24-h cultures and suspended in sterile
Tryptic Soy broth (OXOID Ltd., Hampshire, England) to
obtain concentrations of approximately 1.5 108 cells.

Root dentine specimens

Fifty human sound single-rooted teeth extracted for
orthodontic reasons were collected and stored in 10%
formalin. All teeth were sectioned at the cementoenamel
junction using low-speed Edenta diamond discs (Hauptstrasse, Switzerland) with water cooling. Each root canal
was prepared using ProTaper (DENTSPLY, MAILLEFER,
Switzerland) rotary system according to manufacturer
instructions (files S1 and S2 followed by F1 and finally

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Effectiveness of Propolis against E. Faecalis

F2). Then root dentine discs of 7-mm length each were

obtained from each root by transverse sectioning using
a low-speed diamond edge-coated disc mounted on a
milling machine (Paraskop M, BEGO, Germany) with
water cooling. The dentine discs were placed in ultrasonic
bath of 18% EDTA (Ultradent, South Jordan, UT, USA),
followed by a bath of 5.25% NaOCl each for 5 min to
open dentinal tubules for liquids and microorganisms to
penetrate. The root specimens were placed in 5 mL test
tubes (three discs per tube) containing distilled water and
autoclaved (SES little sister autoclave, UK) for 30 min at
121C (7).

Inoculation of root specimens

Five dentine discs were selected randomly and kept sterile
to serve as negative control. The remaining 45 discs were
placed in test tubes containing Tryptic Soy broth inoculated with E. faecalis. Every three discs were placed in
5 mL test tube. Each tube was filled with 3 mL inoculated
broth. All incubated at 37C for 21 days. The medium was
renewed every 3 days. At the day 21, two specimens were
selected randomly from the 50 (one sterile and one
infected with E. faecalis), and sent for examination under
scanning electron microscope to confirm contamination
of the root canal leaving a total of 48 discs.

Preparation for scanning electron microscope

The two dentine discs used for scanning electron microscope (SEM) examination were sectioned longitudinally
to obtain two halves so that the inner surface of the canal
can be viewed. Specimens were fixed in 2.5% glutaraldehyde for 2 h at room temperature. Then each specimen
was washed in 0.1 M sodium phosphate buffer three
times for 5 min each. Dehydration was performed using
ascending concentrations of ethyl alcohol (30%, 50%,
70%, 90% and 100%) for 10 min each. The 100% concentration was used three times. After dehydration of
the samples, they were dried using critical point dryer
employing liquid CO2 replacement (BALZERS critical
point dryer CPD 030, UK). Then specimens were goldcoated using 1200 V and 20 mA (BIO-RAD, Polaron
Division E5350, UK) and examined under scanning
electron microscope (FEI Quanta 200, the Netherlands).

Disinfection of the infected root discs

After contaminating the root discs they were divided into
two equal experimental subgroups (n = 20 discs): subgroups I (A1 & A2) and II (B1 & B2), and positive and
negative control groups (n = 4) (Fig. 1). Each disc was
treated as follows: rinsed with sterile saline, blotted dry
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Effectiveness of Propolis against E. Faecalis

L. Awawdeh et al.

Figure 1 Flowchart summarising the study design. SEM, scanning electron microscope.

with sterile gauze and sterile paper points (Excel Dental

Supplies Ltd., Hong Kong). Then the external surfaces
were coated with two layers of nail varnish (Diamond
Hard Max Factor, Protector & Gamble, Weybridge, UK).
The discs in each subgroup were treated as follows:
20 discs received a solution of Jordanian propolis (30%
propolis, Nature Home, Amman, Jordan) as intracanal
medicament, 20 received non-setting calcium hydroxide
(UltraCal XS, Ultradent, South Jordan, UT, USA) and
four discs as positive control. The positive control specimens served to provide data on bacterial growth over
time and consisted of inoculated root discs treated in the
same manner as the other experimental groups except
that they received sterile saline rather than a test medicament. In addition, a negative control group consisted of
54

four specimens treated in the same manner as the positive control group with the exception that they were
incubated with sterile uninoculted broth. Then discs were
mounted in an individual 22-mm diameter tissue wells
(greiner bio-one, cellstar, Maybachstr, Germany) on
bases of utility wax approximately 5 mm high. Two millilitres of sterile saline (El Nasr Pharmaceutical Chemicals,
Abu Zaabal, Egypt) were added to each tissue well (surrounding the wax but out of contact with the root discs)
to ensure a humid environment. The lumen of each disc
was filled with the medicament and sealed coronally and
apically with CavitTM G (3M ESPE, Seefeld, Germany).
The plates were labelled and re-incubated at 37C for 1
and 2 days (Fig. 2). Samples were taken after day one
medicament for A1 & B1 and after day two medicament

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L. Awawdeh et al.

Descriptive statistics including means, standard deviations and frequency distribution were calculated for each
subgroup. The results of CFU mL-1 from all test groups
were submitted to analysis of variance and the post-hoc
Tukey test to demonstrate differences between pairs of
groups. Probability values of P 0.05 were set as the
reference for statistically significant results.

Results

Figure 2 Tissue culture plate showing system set-up, which consists of

tissue culture well, utility wax and dentine discs coated with nail varnish
and grey cavity covering the disc lumen.

for A2 & B2. At the end of the medication period, each

canal was washed with 5 mL sterile saline. Microbiological samples were collected by three different methods:
paper points, headstrom files and immersion of the
dentine disc. The fluid in the lumen was absorbed by
sterile paper points and transferred to a sterile Eppendorf
tube containing 1 mL fresh broth, shaken for 30 s on a
Vortex (v-1 BOECO, Germany). A 10-fold dilution was
made and 0.1 mL aliquots was seeded on Agar plates in
duplicates and incubated at 37C for 24 h to observe
any microbial growth. Moreover, after the paper point
sampling, headsrtom files sized 40 (Thomas, BouRGES
CEDEX, France) were used to obtain dentine samples
from the lumen, and then the same procedure described
for paper points was applied to the files (the methodology
is a modification to that of Baker et al. (20) and Evanov
et al. (21)). Finally, the dentine disc itself was transferred
to a sterile Eppendorf tube containing 1 mL fresh broth,
vortex for 30 s. Agar plates were seeded with 0.1 mL
aliquots from the Eppendorf tubes and incubated at 37C
for 24 h.

Estimating CFU mL-1

After the incubation period, the colonies on the agar
plates were counted. The agar plate was divided into
four quadrants and the colonies on one quadrant were
counted. Then the number gained was multiplied by four
to get the whole number of colonies on the agar plate
(minimal colonies appearing in some plates were counted
all without dividing the plate into quadrants).

Statistical analysis
The SPSS computer software version 11.0 (SPSS Inc.,
Chicago, IL, USA) was used to conduct data analysis.

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Journal compilation 2008 Australian Society of Endodontology

In group I, the mean of CFU mL-1 of E. faecalis for dentine

discs treated with propolis was zero after both 1 and 2
days of application. The means of CFU mL-1 for dentine
discs treated with non-setting calcium hydroxide after 1
and 2 days of application are shown in Table 1.
A significant difference was found between propolis
and calcium hydroxide groups (P < 0.05) with the later
being ineffective against E. faecalis after such short duration. Also, a significant difference was found between
samples collected after 1 and 2 days of medication application (P < 0.05) (Table 1).
The difference between the three sampling methods
was significant (P < 0.05). Post-hoc Tukey test was performed to determine the differences within sampling
groups. A significant difference was found between paper
point and headstrom file sampling methods (P < 0.001)
and paper point and disc immersion sampling methods
(P < 0.001), but no significant difference was found
between headstrom file and disc immersion sampling
methods (P = 0.111).
Table 1 shows that the disc immersion method yielded
the highest number of colonies followed by the headstrom file and the least was the paper point sampling
method.
Figure 3 shows SEM of the sterile dentine disc with
dentinal tubule opened and no smear layer. Figures 4 and
5 show SEM of contaminated sample with E. faecalis
colonising dentine surface and dentinal tubules invaded
by E. faecalis.

Discussion
This study is the first to investigate the beneficial use of a
Jordanian propolis as an endodontic intracanal medicament. A 30% solution of propolis was very effective in
eliminating the tested microorganism after both 1 and 2
days of application. Our results are similar to some of
those published studies in which propolis was effective
against E. faecalis (12,22,23).
Various methodologies, ex vivo and in vivo, can be
used to assess the antimicrobial activities of intracanal
medicaments. However, antimicrobial evaluations ex vivo,
although difficult to correlate to clinical results, are
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Effectiveness of Propolis against E. Faecalis

L. Awawdeh et al.

Table 1 Effect of non-setting calcium hydroxide on root canals contaminated with Enterococcus faecalis after 1 and 2 days of application using different
sampling methods
E. faecalis (CFU mL-1)
n
Sampling method
Paper point
Headstrom les
Disc immersion

Day 1
10
10
10

Mean
Day 2
10
10
10

Day 1

SD

Day 2
3

2.3 10
7.3 103
1.0 104

Day 1
2

1.3 10
4.4 103
4.7 103

594.8
3761.8
3512.0

Minimum
Day 2
157.8
3467.8
3967.6

Day 1

Day 2
3

1.5 10
2.0 103
3.5 103

0.0
6.0 10
3.0 10

Maximum
Day 1

Day 2
3

3.3 10
1.2 104
1.6 104

4.1 102
9.0 103
1.0 104

n, number of samples; SD, standard deviation.

Figure 3 Scanning electron microscope of sterile root dentine disc

(negative control) (Original magnication 5000).

Figure 4 Scanning electron microscope of Enterococcus faecalis colonising root dentine disc (positive control) (Original magnication 20 000).

justified for simple comparisons and screening of materials and application techniques. Indeed, the methodology
can be a possible explanation for the dissimilarities of the
results of different studies.
Because of the large numbers of variables that may
affect the results, rigorous quality control is important for
antimicrobial susceptibility testing. The main control is
provided by a series of reference microorganisms strain
including E. faecalis (ATCC 29212). The ideal control
strains have susceptibility end points in the mid range of
antimicrobial concentrations tested and have minimal
tendencies to change susceptibility patterns over time
(24). This explains the choice of this specific microbial
strain in this study.
The contamination period of dentine discs was 21 days
and the microbial broth was renewed every 3 days. At the
day 21, two specimens were selected randomly from each
group (sterile and infected) and sent for examination
under scanning electron microscope to confirm con-

tamination of the root canal (Figs 35). Haapasalo and

rstavik reported that after 3 weeks of incubation of
dentine discs with E. faecalis, a dense infection from the
canal side reached up to 300400 mm (25).
In the current study, microbiological sampling was
accomplished using three methods: sterile paper point
that absorbed the root canal contents, headstrom file that
was used to abrade the disc lumen producing dentine
debris, and immersion of the disc in the broth. The first
two methods have the advantage that both can be used in
ex vivo and in vivo studies. However, paper point sampling
method suffers from an obvious limitation as only microorganisms in the root canal space can be sampled, while
those located within the dentinal tubules could not be
reached. This does not seem to apply to the other two
methods of sampling. Therefore, a wide variation among
the CFU mL-1 was observed between the three methods
of sampling with the paper point sampling yielding the
least CFU mL-1.

56

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Effectiveness of Propolis against E. Faecalis

L. Awawdeh et al.

Figure 5 Scanning electron microscope of longitudinal section of the dentine showing dentinal tubules invaded by Enterococcus faecalis (original
magnication 20 000).

In this study, calcium hydroxide showed minimal antimicrobial effect compared with propolis. One might argue
that the basis for comparison with calcium hydroxide in
the present study was an unlevelled field as the time of
application was not sufficient for the calcium hydroxide
to act against the microorganisms. This could be partly
true; however, it is not clear what minimum time might
be needed for a calcium hydroxide dressing to achieve an
optimal antibacterial effect (11). Safavi et al. have shown
E. faecalis to be eliminated from infected dentine specimens by 24-h exposure (26). On the other hand, according to Sjgren et al. (11), calcium hydroxide must be
applied for 7 days in the canal to act as antimicrobial
agent. In this study the main aim was to test the
effectiveness of propolis as a short-term intracanal medicament and to find the minimum time needed to achieve
its optimal antibacterial effect using infected dentine
models, therefore medications were applied for 1 and
2 days.
It is also important to recognise that the buffering effect
of dentine could be another contributing factor to explain
why calcium hydroxide showed minimal antimicrobial
effect compared with propolis. It is reported that ex vivo,
dentine powder had an inhibitory effect on calcium
hydroxide (27). Clinically, calcium hydroxide is routinely
used as an intracanal medicament. However, several published studies showed that E. faecalis (9) is resistant to
calcium hydroxide, which are in agreement with this
study. Moreover, E. faecalis can survive in the dentinal
tubules despite long periods of calcium hydroxide therapy
(8). E. faecalis has the ability to survive in root canal as
a single organism without support from other bacterial
species. It also has the ability to remain viable within the
dentinal tubules and maintain the capability to invade the
tubules and adhere to collagen in the presence of human
serum (28). In the present study, E. faecalis used was in

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Journal compilation 2008 Australian Society of Endodontology

the starvation phase, and this is the most resistant phase

to medication. Also it is reported that E. faecalis in the
starvation phase produce organic products and release
them intercellularly; this may play a role in the inactivation of the antibacterial activity of the medicament (29).
Moreover, removal of the smear layer enables the E.
faecalis to penetrate the dentinal tubules deeply (30).
Within the limitation of the current study, it became
apparent that the natural bee product propolis is very
effective ex vivo in eliminating E. faecalis within 1 day
and its effectiveness is not weakened by dentine.
However, using calcium hydroxide alone in resistant
endodontic cases where E. faecalis play a major role seems
to be questionable. Among the sampling methods used,
paper point sampling technique was the least effective,
while the headstrom file sampling was the most effective
one.
Further laboratory and clinical investigations should be
carried out to validate findings of beneficial use of propolis as intracanal medicament or as any other endodontic material.

Acknowledgement
The authors wish to thank Jordan University of Science
and Technology for supporting this project financially
(Grant number 1/2006). The authors express their
sincere appreciation to Dr F. Lundy (Oral Science
Research Center, School of Medicine and Dentistry,
Queens University Belfast) for her helpful suggestions.

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