JOURNALOF ENCODONTICS
Copyright 0 2001 by The American Association of Endodontists
Antimicrobial Activity of Several Calcium Hydroxide
Preparations in Root Canal Dentin
Michael J. Behnen, DDS, Lesley A. West, DDS, MS, Frederick R. Liewehr, DDS, MS,
Thomas B. Buxton, PhD, and James C. McPherson, 111, PhD
The purpose of this study was to evaluate the an-
timicrobial activity of several calcium hydroxide
(Ca(0H)dpreparations in root canal dentin infected
with Enterococcusfaecalis. Roots of extracted bo-
vine incisors were prepared to standardized cylin-
drical test specimens of 5 mm in height; the smear
layer was removed, and the specimens were incu-
bated for 24 h at 37°C in bacteriological culture
medium that contained 7.0 x lo4 colony forming
units per milliliter of E. faecalis. The specimens
were mounted in individual 4-mm diameter culture
wells, and the test material was applied to fill the
canal lumen. There were five treatment groups:
group 1, a thick mixture of Ca(OH), USP (1.0 g/ml
H,O); group 2, a thin mixture of Ca(OH), USP (0.1
g/ml H20); group 3, Pulpdent TempCanalTMpaste;
group 4, sterile H20(positive control); and group 5,
25 dentin specimens in sterile, uninoculated brain-
heart infusion broth that were included as negative
controls. Quantitative microbiological analysis of
dentin at various depths was completed after 24 h.
All groups showed a significant (p e 0.001) de-
crease in numbers of E. faecalis in all depths of
dentin compared with the control. Groups 2 and 3
demonstrated significantly greater antimicrobial
activity (73%-86% reduction) at all depths of den-
tin tested compared with group 1 (13%-26%) (p c
0.05). These results suggest that Ca(OH), can de-
crease the numbers of E. faecalis at all depths of
dentinal tubules within 24 h and that thin prepara-
tions of Ca(OH), may be more effective in the elim-
ination of E. faecalis from dentinal tubules than
thick preparations.
Bacteria are the primary etiological agents in pulpal and periapical
inflammation; successful endodontic therapy depends on their re-
duction or elimination (1). Sjogren et al. (2) reported complete
radiographic healing in only 68% of root canals that yielded a
positive culture before obturation compared with 94% of root
785
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VOL. 27. No. 12, DECEMBER 2001
canals that yielded a negative culture. These results emphasize the
importance of completely eliminating bacteria from the root canal
system. Bystrom et al. (3) demonstrated that instrumentation and
irrigation will eliminate bacteria in only approximately 50% of root
canals. However, when biomechanical instrumentation is com-
bined with placement of an antimicrobial dressing for an appro-
priate length of time before root canal obturation, bacteria can be
more effectively eliminated (3, 4).
Calcium hydroxide (Ca(OH),) has been widely used as an
intracanal medication to eliminate bacteria that survive instrumen-
tation and is currently acknowledged to be one of the most effec-
tive antimicrobial dressings used during endodontic therapy (5).
Although the exact mechanism of action of Ca(OH), is still un-
known, its antimicrobial activity is generally considered to be
related to the release of hydroxyl ions in an aqueous environment,
producing a pH of approximately 12.5, even in very dilute mix-
tures. Most endodontic pathogens are unable to survive in this
alkaline environment (5). The mechanisms that Ca(OH), uses to
eliminate bacteria may include damage to the bacterial cytoplasmic
membrane by inducing lipid peroxidation, protein denaturation,
and damage to bacterial DNA and by serving as a physical barrier
that withholds nutrients for bacterial growth and limits space for
bacterial multiplication (6).
Previous studies have shown Ca(OH), to be a very effective
intracanal medication. Bystrom et al. (3) reported that a 30-day
application of Ca(OH), effectively eliminated bacteria in root
canals, and Sjogren et al. (4) found that a 7-day application
eliminated the bacteria in root canals that survived instrumentation.
Because of its low solubility, a relatively large amount of Ca(OH),
can be packed into the root canals with little risk of periapical
irritation. The slow dissociation of hydroxyl ions over time results
in a long duration of the antimicrobial effect of Ca(OH), in the root
canal, in contrast to other antiseptic solutions (7).
Dentin tubules may act as an important reservoir for bacteria
that can result in re-infection of the root canal and ultimately the
failure of endodontic treatment. Sundqvist et al. (8) found that
Enterococcus faecalis was the bacterium most commonly isolated
from teeth after failed root canal therapy, even though it is rarely
found in the untreated canal. Haapasalo and 0rstavik (9)found that
a commercial Ca(OH), paste (CalaseptB, Swedia, Knivsta, Swe-
den) failed to eliminate, even superficially, E. faecalis in the dentin
tubules of bovine incisors. In 1990,the same authors reported that
it could take more than 1 week for Ca(OH), to disinfect dentin
tubules infected by facultative bacteria (10) and that E. faecalis
766 Behnen et al.
could survive at least 10 days in the dentin tubules after withdrawal
of nutrient support. Additional studies by Safavi et al.( 11) and
Siqueira and Uzeda (12) also suggested that Ca(OH), preparations
placed in root canals for an extended period were unable to
eliminate E. faecalis.
Previous studies have reported resistance of E. faecalis to
Ca(OH), but have not included a quantitative analysis of the
bacteria remaining at various depths of root canal dentin after the
placement of Ca(OH), preparations. The purpose of this study was
to evaluate the antimicrobial activity of several Ca(OH), prepara-
tions after 24 h in various depths of bovine root canal dentin
infected with E. faecalis.
MATERIALS AND METHODS
In this study we modified the in vitro model for testing the
antibacterial activity of medications in dentin tubules of bovine
incisors developed by Haapasalo and 0rstavik in 1987 (9) to
include the quantitative analysis of bacteria present in dentin.
Intact bovine central incisors, extracted from frozen jaws, were
kept in 0.5% sodium hypochlorite (NaOCl) overnight for surface
disinfection. The crowns and apical 5 mm were removed with a
diamond saw (Isomet, Buehler LTD,Evanston, IL) at slow speed
(<lo0 rpm) with water-cooling. The roots were prepared to cy-
lindrical test specimens 5 mm thick with the pulpal lumen stan-
dardized to an I S 0 025 bur (Brasseler, Savannah, GA). The smear
layer was removed by an ultrasonic bath in 17% EDTA for 4 min,
followed by an ultrasonic bath in 5.25% NaOCl for 4 min. The
specimens were then placed in water and sterilized in a steam
autoclave for 15 min at 121°C.
The dentin specimens were placed in brain-heart infusion (BHI)
broth (Difco, Detroit, MI) containing 7 X lo4colony-forming units
(cfu)/ml of E. faecalis (ATCC 29212) and incubated at 37°C in 5%
CO, for 24 h with inversion. The dentin cylinders were then
mounted in individual 22-mm diameter tissue culture wells (Com-
ing Cell Wellsm, Coming Glass Works, Coming, NY) on a base
of sticky wax approximately 5 mm tall. Each test medication was
applied to fill the canal lumen to the top of the dentin cylinder.
Approximately 150 p l of sterile water was added to each well to
ensure a humid environment. The culture plates were sealed with
tape, and the medicated cylinders were incubated under humid
conditions at 37°C in 5% CO, for 24 h. All manipulations of the
specimens were performed under a laminar flow hood
( W A R E T M ,Plymouth, MN) to avoid contamination from outside
organisms.
The experimental groups were chosen to represent a range of
concentrations of Ca(OH),. Twenty-five dentin specimens for each
of the five medication groups were tested. Group 1 was a thick
paste of Ca(OH),, consisting of 1.0 g of Ca(OH), USP per 1.0 ml
of sterile water, mixed to approximate the consistency of Pulp-
dentTMas it is used clinically. Group 2 was a thin 10% preparation
consisting of 0.1 g of Ca(OH), USP per 1.0 ml of sterile water.
Group 3 was the commercial product Pulpdent TempCanalTM
(Pulpdent Corporation, Watertown, MA), which is approximately
52.5% Ca(OH), in an aqueous suspension of methylcellulose.
Group 4, which served both as a positive control and to provide
baseline data on bacterial growth over time, consisted of inoculated
dentin specimens that received sterile water only. Group 5 con-
sisted of 25 dentin specimens in sterile, uninoculated BHI broth
that were included as negative controls.
Journal of Endodontics
Bur 1 Bur 2 Bur 3
IS0 029 I S 0 035 IS0 042
FIG 1. f. faecalis versus medications at increasing dentin depths.
Following the 24-h incubation period, excess medication was
removed from the canal lumen with a sterile IS0 025 (2.5 mm
diameter) bur that was placed passively through the canal lumen.
Three consecutive bacterial samples were taken from each of the
dentin cylinders with sterile round burs (bur 1: IS0 029; bur 2: IS0
035; bur 3: I S 0 042) that were mounted in a straight handpiece and
operated at low speed. The specimens were prepared directly over
tubes containing 10 ml of BHI broth. The dentin shavings collected
in the tubes were then vortexed (Vortex Genie, Scientific Indus-
tries, Bohemia, NY) for 10 s. Serial dilutions (l:lO, 1:100, and
1:lOOO) were made, and a 100-pl aliquot of each dilution was
plated on trypticase soy agar (TSA) plates in triplicate. In most
experiments, the 1 X dilution yielded a reproducible number
(between 30 and 300) of bacterial colonies on the plates. The TSA
plates were incubated in 5% CO, at 37"C, and the number of
bacterial colony forming units were counted at 24 h. A two-way
analysis of variance followed by the Student-Newman-Keuls mul-
tiple comparison procedure for means were used for statistical
evaluation of the data. Statistical significance was set at p 5 0.05.
RESULTS
No growth was seen in the negative control specimens (group
5). The quantity of bacteria that was present in various depths of
dentin that was produced by the experimental and positive control
groups is shown in Fig. 1. E. faecalis was present in all depths of
all dentin specimens that were tested after 24 h. As expected, their
numbers were lower in the more peripheral (deeper) dentin spec-
imens. The sterile water group (group 4) had a mean bacterial
count of 2.72 X 10' cfu/ml in the dentin that was collected using
bur 1, which was closest to the canal lumen, and a mean of 1.58 X
lo5 cfdml from the most peripheral level of dentin specimen that
was obtained using bur 3. For each of the test medications, there
was a, significant decrease in bacteria at all bur levels compared
with the sterile water control (p = 0.001). The thick Ca(OH), paste
(group 1) had a larger number of bacteria present at all bur levels
than group 2 or group 3. There was no significant difference
between the thin 10% Ca(OH), preparation and the PulpdentTM
Tempcanal group (groups 2 and 3) (p > 0.05).
Table 1 illustrates the reduction of E. fueculis by each medica-
tion, which is indicated in t e r n of percent killing of the bacteria
at different bur levels. The positive control (group 4) count was
considered 0% killing. The thick paste SOUP eliminated only 13%
Vol. 27, No. 12, December 2001
1. Percent (YO)
TABLE reduction of E. faecalis at increasing
dentin depths compared with control
Group Bur 1 Bur 2 Bur 3
Thick paste 26 13 23
10% paste 73 76 86
PulpdentTM 78 76 86
to 26% of the bacteria, demonstrating a considerably lower anti-
microbial activity than either the PulpdentTMor the 10% Ca(OH),
group, which produced 73% to 86% killing of the bacteria at all bur
levels tested.
DISCUSSION
In our study, E. faecalis demonstrated the ability to penetrate
into the dentinal tubules of bovine teeth after a 24-h incubation.
Waltimo et al. (13) also observed that very short incubation periods
were sufficient for growth of E. faecalis in dentinal tubules. The
results of our study are in agreement with studies by Siqueira and
Uzeda (12) and Haapasalo and 0rstavik (9, lo), which reported
that Ca(OH), was unable to completely eliminate E. faecalis from
dentinal tubules after 10 days. Our results, however, suggest that
Ca(OH), does produce a significant decrease in bacteria at all
depths of dentin in as little as 24 h.
Siqueira and Uzeda (12) suggested that the agglomeration of
bacterial cells colonizing root canal walls could protect those
bacteria more deeply located in the dentin tubules. Studies includ-
ing those by Haapasalo and 0rstavik (9, 10) used large numbers of
bacteria, which may have mechanically blocked the dentin tubules,
preventing the Ca(OH), from entering them and may not accu-
rately represent the clinical situation.
Since Frank (14) published the original article advocating the
use of Ca(OH), as an agent to promote apexification, mixing a
thick paste has generally been recommended. Our results, how-
ever, suggest that thick mixtures of Ca(OH), may not be ideal for
use as an interappointment antimicrobial agent. The antimicrobial
action of Ca(OH), is related to the release of hydroxyl ions in an
aqueous environment and therefore depends on the availability of
hydroxyl ions in the solution and the ability of these ions to diffuse
through dentin and pulpal tissue remnants to reach sequestered
bacteria. To do so, the ionic diffusion of the Ca(OH), should
exceed the buffering ability of the dentin. Ca(OH), owes its bio-
compatibility to its low water solubility, but this low solubility and
diffusibility mediates against its ability to produce the rapid in-
crease in pH necessary to eliminate bacteria from dentinal tubules
and root canal intricacies. Differences in the velocity of hydroxyl
ion dissociation are seen when various vehicles are employed to
produce the Ca(OH), paste (15). The lower the viscosity of the
paste, the higher will be the ionic dissociation. Aqueous vehicles,
including water, saline, dental anesthetics, and methylcellulose,
promote a rapid release of hydroxyl ions.
Our results additionally suggest that, although water and car-
boxymethylcellulose are both considered aqueous vehicles (15).
carboxymethylcellulose may possess additional qualities that allow
the Ca(OH), to diffuse more readily than an aqueous solution of
equal viscosity. Further research is required to elucidate those
Calcium Hydroxide in Dentin 767
qualities, but our research suggests that using a more dilute aque-
ous solution of Ca(OH), may produce equivalent results.
The use of an antimicrobial agent as an intracanal medication
between appointments may significantly increase the chances for
successful endodontic treatment by reducing residual bacteria in
the root canal system. After longstanding endodontic infection,
bacteria may infiltrate into root canal isthmuses, apical deltas,
lateral canals, and dentinal tubules where they cannot be elimi-
nated by chemomechanical preparation alone. Therefore, use of a
Ca(OH), preparation with maximum ability to diffuse into these
areas may be of critical importance. Our results suggest that
Ca(OH), produces a significant antimicrobial effect in the dentin
tubules and that a thin mix or the commercial product hlpdentTM
has a greater effect than a thick mixture.
The opinions and assertions contained herein are the private ones of the
authors and are not to be construed as official or as reflectingthe views of the
United States Army or the Department of Defense.
Dr. Behnen is a major in the U.S. Army Dental Corps and a former resident
in the US. Army Endodontic Residency Program, Fort Gordon, Georgia. He is
currently the assistant chief of endodontics, Fort Lewis, Washington. Dr. West
is the director, U.S. Army Endodontic Residency Program, Fort Gordon,
Georgia. Dr. Liewehr is the assistant director, U.S. Army Endodontic Resi-
dency Program, Fort Gordon, Georgia. Dr. Buxton is a microbiologist, De-
partment of Clinical Investigation, Dwight D. Eisenhower Army Medical Cen-
ter, Fort Gordon, Georgia. Dr. McPherson is a biochemist, Department of
Clinical Investigation, Dwight D. Eisenhower Army Medical Center, Fort Gor-
don, Georgia. Address requests for reprints to COL Frederick R. Liewehr, US.
Army Dental Activity, Fort Gordon, GA 30905-5650.
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