In vitro antimicrobial effect of chlorhexidine-

impregnated gutta percha points on

Enterococcus faecalis

J. N. Lui1, V. Sae-Lim1, K. P. Song2 & N. N. Chen3

1
Department of Restorative Dentistry, Facultyof Dentistry, 2Department of Microbiology, Facultyof Medicine, National Universityof
Singapore, and 3Restorative Department, National Dental Centre, Singapore

Abstract and the colony-forming units (CFU) of E. faecalis were

then plate-counted after culturing. Statistical analysis
Lui JN, Sae-Lim V, Song KP, Chen NN. In vitro antimicrobial
was completed using the paired t-test.
effect of chlorhexidine-impregnated gutta percha points on Enterococcus
Results In comparison to the positive control, treat-
faecalis. International Endodontic Journal, 37, 105^113, 2004.
ment with calcium hydroxide (P ˆ 0.000 and 0.000) or
Aim To evaluate the in vitro antimicrobial e¡ect of activ points (P ˆ 0.000 and 0.002) produced signi¢-
chlorhexidine-impregnated gutta percha points, Roeko cantly lower colony counts of E. faecalis at dentine
activ point (Roeko, Langenau, Germany) on Enterococcus depths of 100 and 250 mm, respectively. Calcium hydro-
faecalis. xide (2.10  102 CFU mL 1) was signi¢cantly more e¡ec-
Methodology Human maxillary premolar roots were tive than activ points (1.58  103 CFU mL 1) at 100 mm
prepared with .04 rotary ProFile instruments to a master (P ˆ 0.013), but not at 250 mm (P ˆ 0.353). Neither of
apical ¢le size 40, autoclave-sterilized and then infected these two medications was able to eliminate E. faecalis
with E. faecalis (ATCC 29212) for 3 weeks. Baseline con- completely.
trols were carried out verifying negligible e¡ects of plain Conclusions Chlorhexidine-impregnated activ
gutta percha cones on E. faecalis. Subsequent to intraca- points did not possess an in vitro inhibitory activity
nal placement of calcium hydroxide, `activ points' or strong enough to eliminate E. faecalis completely from
saline (positive control) and the 2-week incubation in 54 infected dentinal tubules.
root specimens, dentine sampling at depths of 100 and
Keywords: activ point, antimicrobial e¡ect, E. faeca-
250 mm was carried out using .04 rotary ProFile instru-
lis, intracanal medicament.
ments at sizes 60 and 90 to assess the quantityof bacteria
present. Inactivating agents were used prior to sampling Received 9 April 2003; accepted13 August 2003

the main cause of root-canal treatment failure is the per-

Introduction
sistence of microorganisms after therapy (Molander
Bacteria and their by-products are considered the pri- et al. 1998, Sundqvist et al. 1998) or the reinfection of
mary aetiologic agents of necrotic pulps and apical peri- the root-canal system because of inadequate coronal
odontitis (Kakehashi et al. 1965, Sundqvist 1976). seal (Ray & Trope 1995).
Accordingly, the aim of root-canal treatment is the elim- Infections of untreated root canals with necrotic
ination of infection from the canal system. Similarly, pulps and apical periodontitis are typically poly-
microbial, with approximately equal proportions of
Gram-positive and Gram-negative anaerobic bacteria
(Sundqvist 1976). The microbial £ora of the retreatment
Correspondence: Dr Varawan Sae-Lim, Department of Restora- cases, however, has been characterized as monoinfec-
tive Dentistry, Faculty of Dentistry, National University of Singa-
pore, 5 Lower Kent Ridge Road, Singapore 119074, Republic of tions of predominantly Gram-positive microorganisms,
Singapore (Tel.: ‡65 6 7724954; fax: ‡65 6 7732603; e-mail: with approximately equal proportions of facultative
rsdvsl@nus.edu.sg). and obligate anaerobes (Molander et al. 1998, Hancock

ß 2004 Blackwell Publishing Ltd International Endodontic Journal, 37, 105^113, 2004 105
 Chlorhexidine-impregnated gutta percha Lui et al.

et al. 2001). Enterococcus faecalis, a facultative Gram- were collected and stored in physiological saline. These
positive coccus, is the most frequently isolated species teeth were included based on the criteria of root length
and may also sometimes be the only isolate (Molander ranging between 11 and 13 mm measured from the low-
et al. 1998, Sundqvist et al. 1998, Peciuliene et al. 2000, est level of the cemento-enamel junction (CEJ) to the
Hancock et al. 2001). mature root apices and root curvature less than 158, as
The use of intracanal calcium hydroxide as an adjunct well as uniform dentine thickness veri¢ed radiographi-
to chemo-mechanical instrumentation (Bystrom & cally in the buccolingual and mesiodistal dimensions.
Sundqvist 1981, Bystrom & Sundqvist1983), and preven- Adherent soft tissue was mechanically removed without
tion of culture reversal (Bystrom & Sundqvist 1985) in damaging the root surface. Inspection was further car-
teeth with primary apical periodontitis has been well ried out to ensure that there were no cracks, fractures
established. However, several studies have shown that or areas of root resorption.
E. faecalis is relatively resistant to calcium hydroxide
(Bystrom et al. 1985, Haapasalo & Orstavik 1987), which
Teeth preparation
may partially explain the lower prognosis of retreatment
cases with secondary apical periodontitis (Sjogren et al. Decoronation and separation of the two roots was per-
1990). formed using a high-speed diamond bur. All roots were
Chlorhexidine, a cationic bisguanide with the ability adjusted to10 mm length and con¢rmed to have a minor
to adsorb onto dentine (Parsons et al.1980), is considered apical foramen of size 20. The root canals were instru-
a broad-spectrum antimicrobial agent. It acts by adsorb- mented to 9 mm using rotary .04 ProFiles (Dentsply
ing onto the microorganism cell wall and causing intra- Maillefer, Ballaigues, Switzerland) in conjunction with
cellular component leakage. Chlorhexidine has been 6 mL of 1% sodium hypochlorite irrigation (Ricochlor,
suggested as an e¡ective irrigant (Delany et al.1982, Jean- Orion Laboratories, Welshpool, Western Australia,
sonne & White 1994) and an intracanal medicament Australia). At the completion of canal preparation to a
because of its ability to disinfect dentinal tubules against master apical ¢le of .04 ProFile size 40, the teeth were
E. faecalis (Heling et al. 1992,Vahdaty et al. 1993). treated in an ultrasonic bath with 17% EDTA (Pulpdent
In addition to the gel form of chlorhexidine suggested Corporation, Watertown, USA) for 4 min and 5.25%
for use as an intracanal medication (Siqueira & Uzeda sodium hypochlorite for 4 minto remove the smear layer
1997), a new formulation of a chlorhexidine-impreg- (Haapasalo & Orstavik1987). Copious irrigation with dis-
nated gutta percha point,`activ point' (Roeko, Langenau, tilled water was carried out to remove any remaining
Germany), has recently been marketed. According to EDTA and sodium hypochlorite.
the manufacturer, `activ points' contain gutta percha
matrix embedded with 5% chlorhexidine diacetate. This
Sterility and asepsis control
innovation allows ease of introduction and retrieval
from the root canal. However, previous studies on `activ Each root specimen was placed in an individual Bijou
points' only evaluated the e¡ect of direct exposure to a bottle containing 2 mL of Brain Heart Infusion (BHI)
suspension broth of bacteria (Podbielski et al. 2000, broth (Becton Dickonson, Franklin Lakes, USA) and
Petschelt et al. 2002). As there has been no study estab- autoclaved for 20 min at 121 8C. They were then incu-
lishing the antimicrobial e¡ect of `activ points' against bated for 48 h at 37 8C and inspected daily to ensure that
bacteria within the infected dentinal tubules, this study the BHI broth showed no signs of turbidity as a sterility
was undertaken to investigate, using an in vitro infected control prior to proceeding with the subsequent stages
dentine model, the antimicrobial e¡ect of `activ points' of the experiment.
against E. faecalis when used as an intracanal medica- As a further step to assess the asepsis protocol used
ment. throughout the experimental procedures, ¢ve additional
instrumented and sterilized roots in BHI broth were
incubated for 3 weeks at 37 8C. The roots were washed
Materials and methods
with saline in a vortex mixer for10 s and the root surface
was disinfected with 70% ethanol and dried with sterile
Teeth selection
gauze. Five microlitres of sterile saline was placed in
Human permanent maxillary premolars extracted for each root canal prior to 2-week incubation of the
orthodontic reasons with no existing caries or restora- root specimen wrapped with saline-dampened sterile
tions, and the buccal and palatal roots clearly separated gauze within an Eppendorf tube at 37 8C under humid

106 International Endodontic Journal, 37, 105^113, 2004 ß 2004 Blackwell Publishing Ltd
 Lui et al. Chlorhexidine-impregnated gutta percha

Table 1 Statistical analysis of the number of CFU for the baseline control experimental groups

Dentine depths
100 mm 250 mm Mann^Whitney (dentine depths)

Treatment (n ˆ 6) Mean  SD Median Mean  SD Median Pair-wise comparison 100 mm 250 mm

Activ points 1205  1154 778 1027  669 1282 Activ points and saline 0.004 0.004
Saline (positive control) 31650  34377 21225 16083  16992 10600 Saline and gutta percha 1.000 0.337
Gutta percha 21158  4689 20225 14408  4872 14175 Gutta percha and activ points 0.004 0.004

Kruskal^Wallis test P ˆ 0.003.

conditions. At the end of the incubation, bacterial sam- compared (Table 1). Statistical analysis using Kruskal^
pling from the dentinal walls performed in accordance Wallis and Mann^Whitney tests showed negligible
to the prescribed protocol in the experiment showed no e¡ects (P ˆ 1.000 and 0.337) of the 9-mm gutta percha
bacterial growth. cones size 40 (Top color, Roeko, Langenau, Germany)
on E. faecalis (2.02  104 and 1.42  104 CFU mL 1),
and similar e¡ects to that of the positive control (saline
Inoculation control of E. faecalis
group; 2.12  104 and 1.06  104 CFU mL 1).
A pure culture of the test strain, E. faecalis (ATCC 29212;
Global Bioresource CenterTM, Manassas, Virginia, USA)
Experimental proper and treatment groups
was prepared in sterile BHI broth and adjusted using a
spectrophotometer (Spectronic Genesys 5; Milton Roy, Twenty-seven teeth were selected for the experiment
Ivyland, USA) to an optical density of 1.0 at 600 nm cor- proper with each pair of roots randomly assigned to
responding to1.15108 colony-forming units (CFU) mL 1. two of the three treatment groups. The three treatment
The prepared root specimen in each Bijou bottle was groups, each of a sample size of 18 roots, were prepared
incubated at 37 8C with the E. faecalis broth changed according to the following protocols prior to the 2-week
every 3 days for the total inoculation period of 3 weeks humid incubation at 37 8C.
(Akpata & Blechman 1982, Haapasalo & Orstavik 1987).  Group A (calcium hydroxide):10 mg of calcium hydro-
Random sampling and plating of the inoculum to con- xide powder (USP; Pulpdent Corporation, Watertown,
¢rm the viability and purity of the E. faecalis broth was USA) mixed with 10 mL of distilled water into a creamy
done weekly using a bacterial identi¢cation kit (API 20 paste was placed into the root canal of each root speci-
strep; bioMerieux, MO, USA). In addition, the root-canal men with a lentulo spiral.
dentine of another ¢ve infected roots was sampled  Group B (`activ points'): An `activ point' size 40
according to the dentine sampling protocol after the trimmed to a 9 mm length was inserted into the root
3-week inoculation period to validate the e¡ectiveness canals according to the manufacturer's instructions
of the dentine inoculation protocol. Mean counts of (Roeko 1999) in the presence of 5 mL distilled water.
1.33  105 and 6.85  104 CFU mL 1 were recovered at  Group C (positive control): 5 mL of sterile saline was
dentine depths of 100 and 250 mm, respectively. All pro- syringed into the root canals of the specimens.
cedures were carried out under strict aseptic conditions
in a laminar air£ow chamber (Gelman Sciences, Ann
Preparation for bacterial sampling
Arbor, Michigan, USA).
The specimens were retrieved from the Eppendorf tubes,
and intracanal materials were removed according to
Baseline e¡ect of gutta percha
the following protocol prior to the dentine sampling.
In order to ascertain the extent of the speculated anti-  Group A (calcium hydroxide): Calcium hydroxide paste
bacterial e¡ect of gutta percha cones (Moorer & Genet was removed from the canals by rotating a rotary ProFile
1982), which form the inner core of the `activ point' .04 taper size 40 instrument for 20 s (Han et al. 2001).
coated with chlorhexidine diacetate, a baseline control The canals were then £ushed with 3 mL of 0.5% citric acid
experiment was carried out. Three groups of six inocu- (Moller 1966) followed by 3 mL of sterile saline.
lated root samples each were incubated for 2 weeks with  Group B (`activ point'): The `activ point' was removed
intracanal `activ points', gutta percha cones and saline using a pair of sterile tweezers. The canal was then
(positive control), and the e¡ect on E. faecalis was £ushed with 3 mL of 3% Tween 80 (Sigma Chemical,

ß 2004 Blackwell Publishing Ltd International Endodontic Journal, 37, 105^113, 2004 107
 Chlorhexidine-impregnated gutta percha Lui et al.

St Louis, USA) and 0.3% L -a-lecithin (Sigma Chemical, St LOGCOUNT

Louis, USA), and then with 3 mL of sterile saline 14
(Zamany & Spangberg 2002). 12
 Group C (positive control): The canals were £ushed
.

with 6 mL of sterile saline. 10

.
8
Dentinal sampling and bacteriological evaluation
6
New sterile rotary ProFile .04 tapers sizes 60 and 90
4
were used at 300 r.p.m. to remove the dentine from the
root-canal wall in the presence of the irrigated BHI 2
broth. The dentine sample was collected within 5 mL
0 .

of BHI broth in a test tube. The suspension was then vor-

texed for 10 s and serially diluted. Aliquots of 100 mL -2
N= 18 18 18
were subsequently plated onto Tryptic Soy Agar (TSA) CALCIUM CHLORHEXIDINE POSITIVE
blood agar plates (Biomedia Laboratories, Melaka, HYDROXIDE

Malaysia) in duplicates and incubated at 37 8C for 24 h.

Figure 2 Box plot representing the number of CFU of
The quantity of CFU grown was counted and the identity E. faecalis present in groups A^C at100 mm into dentine after
was evaluated using the API 20 strep (bio Merieux) log transformation.
bacterial identi¢cation kit.

COLONY FORMING UNITS

Statistical analysis 60000
The results obtained from groups A^C were subjected to 2
50000
logarithmic transformation to obtain a near-normal dis-
tribution (Figs 1^4). Thereafter, the paired t-test was 40000
8
used to investigate the di¡erences in the CFU of E. faecalis
obtained betweentreatment groups at each depth of den- 30000
tine as well as the di¡erences at the two di¡erent depths
20000
within each treatment group. The level of signi¢cance
was set at 5%. 10000
9
0

COLONY FORMING UNITS -10000

N= 18 18 18
120000 CALCIUM CHLORHEXIDINE POSITIVE
HYDROXIDE
100000 .

Figure 3 Box plot representing the number of CFU of

80000 E. faecalis present in groups A^C at 250 mm into dentine.

60000

40000
Results
20000
All experimental samples inoculated showed puri¢ed
.

0 . inoculum of E. faecalis in all ¢nal plated samples. There

was a trend to recover higher mean counts in the ino-
-20000
N= 18 18 18 culation controls at dentine depths of 100 (1.33 
CALCIUM CHLORHEXIDINE POSITIVE 105 CFU mL 1) and 250 mm (6.85  104 CFU mL 1),
HYDROXIDE
respectively, compared to the positive controls (group
Figure 1 Box plot representing the number of CFU of C) at the same dentine depths (2.92  104 and
E. faecalis present in groups A^C at100 mm into dentine. 1.28  104 CFU mL 1).

108 International Endodontic Journal, 37, 105^113, 2004 ß 2004 Blackwell Publishing Ltd
 Lui et al. Chlorhexidine-impregnated gutta percha

LOGCOUNT
12

250 mm

0.353

0.000
0.002
2

Dentine depths

^
10

100 mm

0.000

0.000
0.013
8

^
6

between groups
Paired t-test
4

A and B

C and A
B and C
^
2
N= 18 18 18

between depths
CALCIUM CHLORHEXIDINE POSITIVE
HYDROXIDE

Paired t-test
Figure 4 Box plot representing the number of CFU of

0.000

0.000
0.101
E. faecalis present in groups A^C at 250 mm into dentine

^
after log transformation.

Table 2 Statistical analysis of the number of CFU for the three treatment groups and between the two dentine depths

25000^127500

2800^50000
20^1460

65^4115
The results of the E. faecalis counts (CFU) recovered

Range
from the dentine samples of groups A^C is presented in
Table 2. At the 100 and 250-mm dentine depths, there
were signi¢cantly fewer colonies isolated in group A
Median

treated with calcium hydroxide (2.1 102, 5.11 102)

1055
305

9325
43000

or group B with chlorhexidine `activ points' (1.58  103,

1.11 103) as compared to the positive control group
(P < 0.05). There was also a signi¢cant di¡erence
68500  46544

12830  11504
1105  1037

between the two treatment groups at a depth of

511 452
Mean  SD

A, calcium hydroxide; B,`activ points'; C, saline (positive control); I, inoculation control.

100 mm, with calcium hydroxide being signi¢cantly
250 mm

more e¡ective than the chlorhexidine points in reducing

the number of colonies of E. faecalis recovered
(P ˆ 0.013). This di¡erence did not reach statistical sig-
5600^100000

ni¢cance for samples obtained at the depth of 250 mm

66000^217500

125^7850
0^675

(P ˆ 0.353).
In the calcium hydroxide group, CFU detected was sig-
Range

ni¢cantly less in super¢cial dentine (100 mm) compared

to deeper layers (250 mm; P ˆ 0.000). The reverse was
noted for the positive control (P ˆ 0.000), whereas there
Median

175

23925
1202
94000

was no signi¢cant di¡erence in the CFU detected at

either depth of dentine for `activ points' (P ˆ 0.101;
Table 2).
132800  69958

Level of significance set at 0.05.

29210  21081
Dentine depths

1581 1836
210  183
Mean  SD

Discussion
100 mm

The in vitro infected dentine model used in this experi-

ment was modi¢ed from that developed by Haapasalo &
Orstavik (1987) without the removal of cementum
(n ˆ 18)

(n ˆ 18)

(n ˆ 18)
(n ˆ 5)

(Almyroudi et al. 2002). Human two-rooted maxillary

Group

premolars were selected with root lengths and diameters

A

C
I

ß 2004 Blackwell Publishing Ltd International Endodontic Journal, 37, 105^113, 2004 109
 Chlorhexidine-impregnated gutta percha Lui et al.

appropriate for dentine sampling using rotary nickel^ layers at 250 mm (5.11 102). These results appeared to
titanium ¢les (Han et al. 2001). In this study, ProFile .04 suggest that microorganisms were exposed to lethal
series sizes 60 and 90 were able to sample dentine to levels of hydroxyl ions only at the tubule ori¢ce (Siqueira
depths of 100 and 250 mm, where a mean count of & Lopes 1999) and that the hydroxyl ions were increas-
1.33  105 and 6.85  104 CFU mL 1 of E. faecalis were ingly bu¡ered and impeded by di¡usion with increasing
recovered, respectively, under the inoculation controls. dentine depth (Wang & Hume 1988, Nerwich et al. 1993)
Scanning electron microscopic evaluation had earlier and, consequently, too overwhelmed to exert their lethal
demonstrated a correlation between culturing from den- e¡ect on E. faecalis.
tine samples and dentine tubule infection (Orstavik & Chlorhexidine has been shown to be e¡ective against
Haapasalo 1990). The latter was facilitated by the E. faecalis in vitro (Hennessey 1973). In this study, the
removal of smear layer (Safavi et al. 1989, Love et al. antimicrobial e¡ect of `activ points'was similarly demon-
1996) using 17% EDTA and 5.25% sodium hypochlorite strated (Table 2). As veri¢ed by the baseline control of
for 4 min each (Haapasalo & Orstavik 1987, Love 1996). gutta percha prior to the experiment proper, this was
Enterococcus faecalis, ATCC 29212, was chosen as the unlikely to be because of the high content of zinc oxide
test species because of its implication as a possible micro- in the gutta percha exerting an antimicrobial e¡ect
bial factor in therapy-resistant apical periodontitis (Moorer & Genet 1982, Podbielski et al. 2000). `Activ
(Molander et al. 1998, Sundqvist et al. 1998, Hancock points' were found to result in a 10-fold less count of
et al. 2001). It is a nonfastidious microbe that is relatively E. faecalis (1.58  103 and 1.11 103 CFU mL 1) com-
easy to culture and has been shown in vitro to predicta- pared to the positive controls (2.92  104 and
bly penetrate deeply into human dentinal tubules 1.28  104 CFU mL 1). These signi¢cant di¡erences
(Akpata & Blechman1982). It is also suggested to be con- (P ˆ 0.000 and 0.002) were possibly attributed to the
siderably resistant to calcium hydroxide (Stevens & large quantities of embedded chlorhexidine diacetate
Grossman1983, Bystrom et al.1985, Orstavik &Haapasalo that initially dissociated and was gradually released
1990), which is the most commonly used intracanal from the surface of the `activ points' upon contact with
medicament. This study compared the antimicrobial moisture. Presumably, the rate of release is dependent
e¡ect on infected dentinal tubules of intracanal chlor- on the amount of £uid present in the canal available for
hexidine-impregnated gutta percha points (`activ dissociation. In this study, 5 mL of distilled water was
points') and calcium hydroxide. Precautions to prevent added to the `activ points' in the root canal, and the root
dehydration of the latter, speculated to reduce its alkali- specimens were thereafter kept moist to promote the dis-
nity (Almyroudi et al. 2002), was achieved by surround- sociation kinetics. The fact that there was no signi¢cant
ing the root specimens with sterile saline-dampened di¡erence (P ˆ 0.101) in the CFU detected at the two
gauze in individually closed Eppendorf tubes. depths of dentine sampled seemed to indicate that the
The results of this study indicated that calcium hydro- antimicrobial e¡ect of chlorhexidine, though present at
xide was not able to completely eliminate E. faecalis, as a deeper depth of 250 mm, might be at too low an inhibi-
evidenced by the counts recovered (Table 2). The relative tory concentration to achieve complete elimination of
ine¤cacy of calcium hydroxide against E. faecalis found E. faecalis. The limited volume of £uid in the root canal
in this study was in accordance with other studies on might have hindered further drug release because of
both bovine (Orstavik & Haapasalo 1990, Siqueira & drug accumulation and the associated saturation
Uzeda 1996) and human teeth (Safavi et al. 1990, Estrela (Huang et al. 2000). Gomes et al. (2001) found that 1%
et al. 1999). It was reported that intracanal calcium and 2% chlorhexidine gluconate took signi¢cantly less
hydroxide reached inner peak dentine pH as high as time to kill E. faecalis compared to 0.2% chlorhexidine,
12.3 cervically (Ho et al. 2003) and 9.7 apically (Nerwich although all concentrations were eventually e¡ective.
et al. 1993) within hours, whereas outer peak dentine It is not known if the antibacterial e¡ect of `activ points'
pH of 9.3 cervicallyand 9.00 apically were achieved after found in this study would be improved if they had been
2^3 weeks (Nerwich et al. 1993). While most bacteria left in the root specimens for a longer period of time.
are readily susceptible to a pH of 9 (Harper & Loesche As shown in Table 2, chlorhexidine `activ points' were
1984), E. faecalis has been shown to survive at pH 11.5 found to be signi¢cantly less e¡ective compared to cal-
or less (Bystrom et al. 1985). It was also noted in the cal- cium hydroxide paste at a depth of 100 mm into dentine
cium hydroxide group that there was signi¢cantly less (P ˆ 0.013), but at the deeper depth of 250 mm, the di¡er-
CFU mL 1 of E. faecalis detected in the super¢cial den- ence was not distinct (P ˆ 0.353). It is not known if the
tine at100-mm depth (2.10  102) compared to the deeper better antimicrobial e¡ect of calcium hydroxide was

110 International Endodontic Journal, 37, 105^113, 2004 ß 2004 Blackwell Publishing Ltd
 Lui et al. Chlorhexidine-impregnated gutta percha

because of its speculated ability to act as a physicochem- seemed to imply that the withdrawal of nutrient support
ical barrier depriving bacteria of nutrients and space to is detrimental to the survival of E. faecalis (Orstavik &
multiply or the di¡erence in the formulations or both. Haapasalo1990). However, at least1.28  10 4 CFU mL 1
Previous studies reported more favourable results with was still detected at a dentine depth of 250 mm. Entero-
a gel formulation of chlorhexidine gluconate compared cocci have been shown to possess virulence factors that
to similar concentrations in a sustained release device allow survival under harsh ecological conditions as a
(Lenet et al. 2000, Barthel et al. 2002, Basrani et al. single organism (Fabricius et al. 1982, Love 2001).
2002). The better adaptability of the calcium hydroxide Although the consequences of dentine tubule infection
paste to the intricacies of the root-canal system might and the possible role of the remaining bacteria in the
similarly allow for better di¡usion. In contrast, only dentinal tubules after root-canal treatment leading to
one`activ point'with .02 taper was used in the inoculated long-term failure (Oguntebi1994, Peters et al.1995) have
root canal prepared to .04 taper. It is possible, however, not been ascertained, the mainstay of root-canal treat-
speculative, if better adaptation would improve the anti- ment is still the elimination of bacteria. Therefore, mea-
bacterial e¡ect of `activ points' in addition to the sures should constantly be employed to search for
improved dissociation kinetics in the presence of replen- alternative, more e¡ective intracanal medications.
ished intracanal £uid. Further studies with other clinical isolates of this bacter-
The substantivityof chlorhexidine on hard tissue (Par- ium are warranted.
sons et al. 1980) was a concern in this study, where the
dentinal shavings were cultured to obtain bacterial
Conclusions
counts. In order to avoid false-negative results, a combi-
nation of 0.3% L -a-lecithin and 3% Tween 80 was used Based on the current experimental design, the following
(Zamany & Spangberg 2002). The former contains phos- conclusions could be made:
phatidylcholine that inactivates chlorhexidine, while 1 Chlorhexidine-impregnated points (`activ points') did
the latter maintains the phosphatidylcholine in solution not possess an inhibitory activity strong enough to com-
(Zamany & Spangberg 2002). The use of these chemicals pletely eliminate a moderately large number of a single
has been found to have no detrimental e¡ect on the endodontic pathogen, E. faecalis, from infected human
recovery of test bacteria (Zamany & Spangberg 2002). dentinal tubules.
Likewise, 0.5% citric acid was used as the inactivating 2 The antimicrobial e¡ect of chlorhexidine-impreg-
agent for calcium hydroxide (Moller 1966). It was nated points (`activ points') was less than that of the aqu-
believed that 0.5% citric acid should have no bearing eous calcium hydroxide paste; however, this was not
on the results as its antibacterial e¡ect would be bu¡ered signi¢cant at the dentine depth of 250 mm.
by live bacteria (Shuping et al. 2000). In this study, inac-
tivators were used (Bender & Seltzer 1954) rather than
Acknowledgements
centrifuging and diluting in a large volume of culture
medium (Vahdaty et al. 1993) to predictably eradicate This study was supported by research grants from the
any possible trace of residual antimicrobial agents. National University of Singapore and National Dental
Di¡erent methodologies have been employed as mea- Centre. The authors appreciate the statistical analysis
sures for bacterial growth. These include turbidity provided by Dr WangYou Gan, Biostatistics Department,
assessment of the broth containing incubated dentine National University of Singapore, as well as the technical
shavings by optical density readings from a spectrophot- assistance from Kai Soo and other members of the micro-
ometer (Heling et al.1992, Komorowski et al. 2000, Lenet biology laboratory. The supply of activ points by Roeko,
et al.2000, Han et al.2001, Basrani et al.2002) or the incu- Langenau, Germany, is hereby acknowledged.
bated root specimens by visual inspection (Safavi et al.
1990). The present study adopted a quantitative mea-
surement taking into account the viability of the micro- References
organisms. After obtaining the dentine samples,
Akpata ES, Blechman H (1982) Bacterial invasion of pulpal den-
E. faecalis was cultured and the number of colonies were tin wall in vitro. Journal of Dental Research 61, 435^8.
quanti¢ed using the plate count technique (Vahdaty Almyroudi A, Mackenzie D, McHugh S, SaundersWP (2002) The
et al. 1993, Almyroudi et al. 2002). The observation that e¡ectiveness of various disinfectants used as endodontic
the mean counts obtained for the positive controls were intracanal medications: an in vitro study. Journal of Endo-
lower than that from the 3-week inoculation controls dontics 28, 163^7.

ß 2004 Blackwell Publishing Ltd International Endodontic Journal, 37, 105^113, 2004 111
 Chlorhexidine-impregnated gutta percha Lui et al.

Barthel CR, Zimmer S, Zilliges S, Schiller R, Gobel UB, Roulet JF in a sustained-release device for dentine sterilization. Interna-
(2002) In situ antimicrobial e¡ectiveness of chlorhexidine tional EndodonticJournal 25, 15^9.
and calcium hydroxide: gel and paste versus gutta-percha Hennessey TD (1973) Some antibacterial properties of chlorhex-
points. Journal of Endodontics 28, 427^30. idine. Journal of Periodontal Research 8, 61^7.
Basrani B, SantosJM,Tjaderhane L et al. (2002) Substantive anti- Ho CH, KhooA,Tan R,TehJ, Lim KC, Sae-LimV (2003) pH changes
microbial activity in chlorhexidine-treated human root den- in root dentin after intracanal placement of improved cal-
tin. Oral Surgery, Oral Medicine, Oral Pathology, Oral cium hydroxide containing gutta-percha points. Journal of
Radiolology and Endodontics 94, 240^5. Endodontics 29, 4^8.
Bender IB, Seltzer S (1954) Probability of error of the negative Huang J,Wong HL, ZhouYet al. (2000) In vitro studies and mod-
culture with use of combination of antibiotics in endodontic eling of a controlled-release device for root canal therapy.
treatment. Oral Surgery, Oral Medicine and Oral Pathology 4, Journal of Controlled Release 67, 293^307.
886^90. Jeansonne MJ, White RR (1994) A comparison of 2.0% chlor-
Bystrom A, Sundqvist G (1981) Bacteriologic evaluation of the hexidine gluconate and 5.25% sodium hypochlorite as anti-
e¤cacy of mechanical root canal instrumentation in endo- microbial endodontic irrigants. Journal of Endodontics 20,
dontic therapy. Scandinavian Journal of Dental Research 89, 276^8.
321^8. Kakehashi S, Stanley HR, Fitzgerald RJ (1965) The e¡ects of sur-
Bystrom A, Sundqvist G (1983) Bacteriologic evaluation of the gical exposures of dental pulps in germ-free and conventional
e¡ect of 0.5 percent sodium hypochlorite in endodontic ther- laboratory rats. Oral Surgery, Oral Medicine and Oral Pathology
apy. Oral Surgery, Oral Medicine and Oral Pathology 55,307^12. 20, 340^9.
Bystrom A, Sundqvist G (1985) The antibacterial action of Komorowski R, Grad H,Wu XY, Friedman S (2000) Antimicro-
sodium hypochlorite and EDTA in 60 cases of endodontic bial substantivity of chlorhexidine-treated bovine root den-
therapy. International Endodontic Journal 18, 35^40. tin. Journal of Endodontics 26, 315^7.
Bystrom A, Claesson R, Sundqvist G (1985) The antibacterial Lenet BJ, Komorowski R,Wu XYet al. (2000) Antimicrobial sub-
e¡ect of camphorated paramonochlorophenol, camphorated stantivity of bovine root dentin exposed to di¡erent chlorhex-
phenol and calcium hydroxide in the treatment of infected idine delivery vehicles. Journal of Endodontics 26, 652^5.
root canals. Endodontics and Dental Traumatology 1, 170^5. Love RM (1996) Regional variation in root dentinal tubule infec-
Delany GM, Patterson SS, Miller CH, Newton CW (1982) The tion by Streptococcus gordonii. Journal of Endodontics 22,
e¡ect of chlorhexidine gluconate irrigation on the root canal 290^3.
£ora of freshly extracted necrotic teeth. Oral Surgery, Oral Love RM (2001) Enterococcus faecalis ^ a mechanism for its role
Medicine and Oral Pathology 53, 518^23. in endodontic failure. International Endodontic Journal 34,
Estrela C, Pimenta FC, Ito IY, Bammann LL (1999) Antimicrobial 399^405.
evaluation of calcium hydroxide in infected dentinal tubules. Love RM, Chandler NP, Jenkinson HF (1996) Penetration of
Journal of Endodontics 25, 416^8. smeared or nonsmeared dentine by Streptococcus gordonii.
Fabricius L, Dahlen G, Holm SE, Moller AJ (1982) In£uence of International Endodontic Journal 29, 2^12.
combinations of oral bacteria on periapical tissues of mon- MolanderA, Reit C, Dahlen G, Kvist T (1998) Microbiological sta-
keys. Scandinavian Journal of Dental Research 90, 200^6. tus of root-¢lled teeth with apical periodontitis. International
Gomes BP, Ferraz CC,Vianna ME, BerberVB,Teixeira FB, Souza- Endodontic Journal 31, 1^7.
Filho FJ (2001) In vitro antimicrobial activity of several con- MollerAJ (1966) Microbiological examination of root canals and
centrations of sodium hypochlorite and chlorhexidine gluco- periapical tissues of human teeth. Methodological studies.
nate in the elimination of Enterococcus faecalis. International Odontological Tidskr 20, 74 (5), 1^380 (Suppl).
Endodontic Journal 34, 424^8. Moorer WR, Genet JM (1982) Antibacterial activity of gutta-
Haapasalo M, Orstavik D (1987) In vitro infection and disinfec- percha cones attributed to the zinc oxide component. Oral
tion of dentinal tubules. Journal of Dental Research 66,1375^9. Surgery, Oral Medicine and Oral Pathology 53, 508^17.
Han GY, Park SH, Yoon TC (2001) Antimicrobial activity of Nerwich A, Figdor D, Messer HH (1993) pH changes in root den-
Ca(OH)2 containing pastes with Enterococcus faecalis in vitro. tin over a 4-week period following root canal dressing with
Journal of Endodontics 27, 328^32. calcium hydroxide. Journal of Endodontics 19, 302^6.
Hancock HH III, Sigurdsson A, Trope M, Moiseiwitsch J (2001) Oguntebi BR (1994) Dentine tubule infection and endodontic
Bacteria isolated after unsuccessful endodontic treatment therapy implications. International Endodontic Journal 27,
in a North American population. Oral Surgery, Oral Medicine, 218^22.
Oral Pathology, Oral Radiolology and Endodontics 91, 579^86. Orstavik D, Haapasalo M (1990) Disinfection by endodontic irri-
Harper DS, Loesche WJ (1984) Growth and acid tolerance of gants and dressings of experimentally infected dentinal
human dental plaque bacteria. Archives of Oral Biology 29, tubules. Endodontics and Dental Traumatology 6, 142^9.
843^8. Parsons GJ, Patterson SS, Miller CH, Katz S, KafrawyAH, Newton
Heling I, Sommer M, Steinberg D, Friedman M, Sela MN (1992) CW (1980) Uptake and release of chlorhexidine by bovine pulp
Microbiological evaluation of the e¤cacy of chlorhexidine and dentin specimens and their subsequent acquisition of

112 International Endodontic Journal, 37, 105^113, 2004 ß 2004 Blackwell Publishing Ltd
 Lui et al. Chlorhexidine-impregnated gutta percha

antibacterial properties. Oral Surgery, Oral Medicine and Oral Siqueira Junior JF, de Uzeda M (1996) Disinfection by calcium
Pathology 49, 455^9. hydroxide pastes of dentinal tubules infected with two obli-
Peciuliene V, Balciuniene I, Eriksen HM, Haapasalo M (2000) gate and one facultative anaerobic bacteria. Journal of Endo-
Isolation of Enterococcus faecalis in previously root-¢lled dontics 22, 674^6.
canals in a Lithuanian population. Journal of Endodontics Siqueira Junior JF, de Uzeda M (1997) Intracanal medicaments:
26, 593^5. evaluation of the antibacterial e¡ects of chlorhexidine,
Peters LB,Wesselink PR, MoorerWR (1995) The fate and the role metronidazole, and calcium hydroxide associated with three
of bacteria left in root dentinal tubules. International Endo- vehicles. Journal of Endodontics 23, 167^9.
dontic Journal 28, 95^9. SiqueiraJuniorJF, Lopes HP (1999) Mechanisms of antimicrobial
Petschelt A, Ebert J, Kolowos K (2002) Antimicrobial behaviour activity of calcium hydroxide: a critical review. International
of di¡erent `active' gutta-percha points against Enterococcus EndodonticJournal 32, 361^9.
faecalis in simulated root canals. International Endodontic Sjogren U, Hagglund B, Sundqvist G, Wing K (1990) Factors
Journal 35, 87 (Abstract). a¡ecting the long-term results of endodontic treatment. Jour-
Podbielski A, Boeckh C, Haller B (2000) Growth inhibitory activ- nal of Endodontics 16, 498^504.
ity of gutta-percha points containing root canal medications Stevens RH, Grossman LI (1983) Evaluation of the antimicrobial
on common endodontic bacterial pathogens as determined potential of calcium hydroxide as an intracanal medicament.
byan optimized quantitative in vitro assay. Journal of Endodon- Journal of Endodontics 9, 372^4.
tics 26, 398^403. Sundqvist G (1976) Bacteriological Studies of Necrotic Dental
Ray HA,Trope M (1995) Periapical status of endodontically trea- Pulps. Dissertation. Umea, Sweden: University of Umea.
ted teeth in relation to the technical quality of the root ¢lling Sundqvist G, Figdor D, Persson S, Sjogren U (1998) Microbiologic
and the coronal restoration. International Endodontic Journal analysis of teeth with failed endodontic treatment and the
28, 12^8. outcome of conservative re-treatment. Oral Surgery, OralMed-
Roeko (1999) Activ Point Instructional Guide. Germany: Lan- icine, Oral Pathology, Oral Radiology and Endodontics 85,
genau. 86^93.
Safavi KE, Spangberg LS, Langeland K (1989) Smear layer VahdatyA, Pitt Ford TR,Wilson RF (1993) E¤cacy of chlorhexi-
removal e¡ects on root canal dentin tubule infection. Journal dine in disinfecting dentinal tubules in vitro. Endodontics
of Endodontics 15, 175^6. and Dental Traumatology 9, 243^8.
Safavi KE, Spangberg LS, Langeland K (1990) Root canal dent- Wang JD, HumeWR (1988) Di¡usion of hydrogen ion and hydro-
inal tubule disinfection. Journal of Endodontics 16, 207^10. xyl ion from various sources through dentine. International
Shuping GB, Orstavik D, Sigurdsson A, Trope M (2000) Reduc- EndodonticJournal 21, 17^26.
tion of intracanal bacteria using nickel^titanium rotary Zamany A, Spangberg LS (2002) An e¡ective method of inacti-
instrumentation and various medications. Journal of Endo- vating chlorhexidine. Oral Surgery, Oral Medicine, Oral Pathol-
dontics 26,751^5. ogy, Oral Radiology and Endodontics 93, 617^20.

ß 2004 Blackwell Publishing Ltd International Endodontic Journal, 37, 105^113, 2004 113