JOURNALOF ENDODONTICS
Copyright © 1999 by The American Association of Endodontlsts
Antimicrobial Evaluation of Calcium Hydroxide in
Infected Dentinal Tubules
Carlos Estrela, DDS, MSc, PhD, Fabiana Cristina Pimenta, MSc, Izabel Yoko Ito, MSc, PhD, and Lili
Luschke Bammann, MSc, PhD
The objective of this study was to evaluate the
antimicrobial activity of calcium hydroxide in in-
fected dentinal tubules. Four microorganisms,
strains of ATCC (Streptococcus faecalis (ATCC-
29212), Staphylococcus aureus (ATCC-6538), Ba-
cillus subtilis (ATCC-6633), and Pseudomonas
aeruginosa (ATCC-27853)) and one mixture of
these were used. These strains were inoculated in
brain heart infusion (BHI) and incubated at 37°C for
24 h. Sixty-three human maxillary central incisors
were prepared and sterilized by autoclaving. Five
groups of 12 teeth each were contaminated for 28
days using new 24-h cultures every 72 h, prepared
and adjusted to tube 2 of the MacFarland scale (6 x
108 cells/ml). Root canals were then irrigated with
5 ml of saline, dried, and completely filled with
calcium hydroxide paste. At intervals of 0, 48, and
72 h, and 7 days, dressings were removed and
teeth were immersed in 5 ml of BHI and incubated
at 37°C for 48 h to observe the growth and multi-
plication of the microorganisms. Three uninocu-
lated teeth were maintained in a humid environ-
ment as an aseptic control. These teeth were
immersed in BHI and maintained at 37°C for 7 days
to determine microbial growth. Bacterial growth
was shown by turbidity of the culture medium and
confirmed by seeding these broths on BHI agar at
37°C for 24 h. The positive BHI tubes were se-
lected, and inoculum was spread on the surface of
BHI agar, followed by the same incubation condi-
tions. Gram stain was conducted from BHI growth
and from colonies growing on solid medium. Cal-
cium hydroxide in infected dentinal tubules
showed no antimicrobial effect on S. faecalis, S.
aureus, B. subtilis, P. aeruginosa, or on the bacte-
rial mixture used throughout the experiment.
The control of microorganisms in infected root canals with apical
periodontitis is a frequent concern, as shown by research to eval-
uate the efficacy of mechanical instrumentation, irrigation, and
416
Printed in U.S.A.
VOL. 25, NO. 6, JUNE 1999
intracanal and systemic dressings, and to find alternatives for
antimicrobial treatment of this pathosis (1-3).
Associations between microbial species in root canal infections,
related to availability of nutrients and low oxygen tensions, lead to
polymicrobial infections with elevated virulence (4).
The establishment of in vitro experimental models for root canal
infection has allowed observation of the depth of penetration of the
various microorganisms in dentinal tubules as a function of the
time of incubation, as well as the efficacy of endodontic dressings
and irrigants (5-9).
The difficulty in eliminating microorganisms in dentinal tubules
after cleaning and shaping demonstrates the necessity of using an
intracanal dressing. Calcium hydroxide is the antimicrobial agent
recommended for different clinical situations, such as root canal
infections, root closure, and root resorption (10-12). The antimi-
crobial action of calcium hydroxide, given the high pH, is deter-
mined by the liberation of hydroxyl ions, which requires an ideal
time for effective destruction of microorganisms, acting in direct or
indirect contact in dentinal tubules (13). However, the liberation of
hydroxyl ions to attain a high pH that may completely eliminate the
microorganisms can be delayed (14-16). The time required for
calcium hydroxide, alone or in association with endodontic irrig-
ants, to have an effect on microorganisms considered resistant to
antimicrobial agents has been questioned and has not yet been
determined. Sjogren et al. (17), studying the antimicrobial effect of
calcium hydroxide as a short-term intracanal dressing in teeth with
periapical lesions for 10 rain and 7 days, respectively, reported that
the effective destruction of microorganisms was complete after 1
week. Estrela et al. (13) determined, in vitro, the direct antimicro-
bial effect of calcium hydroxide on different microorganisms,
observing positive results after 72 h.
The purpose of this study was to evaluate the antimicrobial
effect of calcium hydroxide, in infected dentinal tubules after 0, 48,
and 72 h, and 7 days.
M A T E R I A L S AND M E T H O D S
Four bacterial strains of the American Type Cultures Collection
(ATTC) were selected for this study: Streptococcus faecalis
(ATCC-29212), Staphylococcus aureus (ATCC-6538), Bacillus
subtilis (ATCC-6633), and Psetuhmumas aeruginosa (ATCC-
27853). These strains were inoculated in brain heart infusion (BHI:
Difco Laboratories, Detroit, MI) and incubated at 37°C for 24 h.
The microorganism suspensions were adjusted to tube 2 of the
Vol. 25, No. 6, June 1999 Antimicrobial Evaluation of Calcium Hydroxide 417

TABLE 1. Antimicrobial evaluation of calcium hydroxide in antimicrobial effect on S. Jaecalis (ATCC-29212), S. aureus
infected dentinal tubules (ATCC-65381, B. subtilis (ATCC-6633), P. aeruginosa (ATCC-
27853), or on a mixture of these.
Microorganisms 0 h 48 h 72 h 7 clays
S. faecalis +++ +++ +++ +++
(AGPC)
DISCUSSION
S. aureus +++ +++ +++ +++
(AGPC)
P. aeruginosa + + + ++ + + ++ + + +
The microbial composition of infected root canals is an impor-
(AGNR) tant factor in the selection of intracanal dressings. The antimicro-
B. subtilis +++ +++ +++ +++ bial action of these dressings must reach different types of micro-
(AGPR) organisms, inhibit the osteoclastic activity in root resorption, and
Mixture +++ +++ +++ +++ favor tissue repair. Significant results have been shown with cal-
+++, presence of growth, AGPC, aerobic Gram-positive coccus; AGNR, aerobm cium hydroxide in these situations (3, 10-131.
Gram-negative rods; AGPR, aerobic Gram-positive rods, spore-forming; mixture, S. fae- The time necessary for this dressing to manifest action on
oa#s + S. aureus + P. aeruginosa * B. subtihs.
microorganisms present in endodontic infections has been dis-
cussed (13, 17). Estrela et al. (18) reported that the determining
factor in the speed of action of this dressing is the maintenance of
MacFarland scale (6 × l0 s cells/ml). From each experimental
a high concentration of hydroxyl ions that inactivate bacterial
suspension, 1 ml was removed and a mixture of the four selected
enzymes of the cytoplasmic membrane, influence chemical trans-
microorganisms was prepared.
port, and alter the availability of nutrients, thus causing toxic
Sixty-three human maxillary central incisors, extracted for dif-
effects on bacterial cells. This bacterial enzymatic inactivation is
ferent reasons, were used. The apical foramen was closed with
reflected in the growth, cellular division, and metabolic activity
composite resin to reduce possible contamination of the external
that occur in the cytoplasmic membrane. The chemical disintegra-
surface. Teeth were prepared up to size 1SO 60 (K-file, Maillefer,
tion of the membrane is related to the destruction of unsaturated
Switzerland) 1 mm before the apical foramen, using a step-back
fatty acids or phospholipids, due to a high concentration of hy-
preparation technique. The cervical third was enlarged with #3 and
droxyl ions that interferes with the lipidic peroxidation process and
#4 Gates-Glidden burs. Three milliliters of 1% sodium hypochlo-
saponification reaction. Safavi and Nichols (19) demonstrated that
rite were used for irrigation after each file, while instrumenting the
the hydroxyl ions of calcium hydroxide can hydrolyze the lipo-
root canals. Root canals were dried and filled with 17% E D T A / p H
polysaccharide present in bacterial cell walls, causing the degra-
7.2) and held for 3 min. After cleaning and shaping, root canals
dation of lipid A and neutralizing its toxic effect. Estrela and Pesce
were steriiized by autoclaving (30 rain at 120°C).
(20) studied the liberation of calcium and hydroxyl ions of calcium
Five groups of 12 teeth each were inoculated with the micro-
hydroxide pastes in the connective tissue of the dog. They reported
organisms or a mixture of these, using syringes of sufficient
that, in 1 mol of dissociated calcium hydroxide, 45.89% is hy-
volume to fill the root canal. This procedure was repeated every
droxyl ions and 54.11% is calcium ions, and the speed of the ionic
72 h for 28 days, always using 24-h cultures prepared and adjusted
dissociation is influenced by the vehicle, being more effective
to tube 2 of the MacFarland scale. Teeth were maintained in a
when hydrosoluble vehicles are used.
humid environment at 37°C. Root canals were irrigated with 5 ml
The direct contact of calcium hydroxide on different microor-
of saline, dried with sterilized absorbent paper, and completely
ganisms (Streptococcus sp., P. aeruginosa, S. aureus, Escherichia
filled with calcium hydroxide paste. The paste was prepared using
coil, Fusobacterium nucleatum, and Micrococcus luteus) showed
calcium hydroxide P.A. (Quimis Mallinkrodt, Inc.) plus saline as
positive effects after 3 days (131. However, it is necessary for the
vehicle, with a viscosity of 350 l cP at 0.1 rpm (Brookfield Digital
dressing to have activity over distance and time to destroy micro-
Reometer, model DV-III-IV), corresponding to the consistency of
organisms in the dentinal tubules. Perez et al. (9) observed, in vitro,
toothpaste, with a pH 12.6 determined using a pH meter/Analion,
that the depth of penetration into the dentinal tubules of Strepto-
pH digital, PM 605).
coccus sanguis was 479 /xm after 28 days of incubation, with a
At intervals of 0, 48, and 72 h, and at 7 days, calcium hydroxide
maximum value of 737 /xm. Orstavik and Haapasalo (8) found
paste was removed from three teeth of each group using saline and
Enterococcus faecalis and S. sanguis at depths of 300 to 400 p,m
mechanical stirring with a file. Root canals were dried and teeth
after 2 to 3 weeks, whereas P. aeruginosa infected the dentin
were immersed in 5 ml of BHI and incubated for 48 h.
quickly (after 3 daysJ. However, examination of the specimens
Bacterial growth was shown by turbidity of the culture medium.
with a scanning electron microscope showed few microorganisms.
The positive BHI tubes were selected, and inoculum was spread on
Data reported in Table 1 show that calcium hydroxide was
the surface o f B H I agar (Difco Laboratories), followed by the same
ineffective at a distance (indirect action) after 7 days on the
incubation conditions. Gram stain was conducted from BHI growth
microorganisms (S. faecalis, S. aureus, B. subtilis, P. aeruginosa,
and from colonies growing on BHI agar.
and a mixture of these); therefore, under the present conditions,
During the 28 days of contamination of the root canals, three
these microorganisms {in pure or mixed suspensions) can survive
teeth not inoculated were maintained in a humid environment and
in high pH. The contamination period of the root canals in this
then transferred to BHI, followed by incubation at 37°C for 7 days,
study was 28 days, enough time for deep infection of the dentinal
as an aseptic control.
tubules, as described previously (8, 9). Sjogren et al. (171 reported
that, in 7 days, calcium hydroxide eliminated microorganisms that
RESULTS survive biomechanical preparation. Their endodontic irrigant was
sodium hypochlorite, an excellent antimicrobial agent. In the
The results are shown in Table 1. During the 7-day period, present investigation, saline was used as an irrigant to avoid
calcium hydroxide in infected dentinal tubules showed no positive masking the antimicrobial potential of calcium hydroxide.
418 Estrela et al. Journal of Endodontics

The amount of hydroxyl ions liberated from calcium hydroxide
in 7 days did not alter the integrity of the cytoplasmic membrane
of these microorganisms to cause their destruction. These results
confirm the hypothesis of Estrela et al. (18), who reported the
possibility of calcium hydroxide producing reversible and irrevers-
ible bacterial enzymatic inactivation. Irreversible inactivation can
be observed in extreme pH conditions after an extended period,
with the loss of biological activity of the cytoplasmic membrane,
as has been shown to occur in pools of microorganisms after 3 days
of exposure (13). Reversible inactivation can be found when the
pH does not reach high enough levels or returns to ideal pH.
Further research must be done to determine the ideal time to
maintain the antimicrobial activity of calcium hydroxide in in-
fected root canals with apical periodontitis.
Dr. Estrela is chairman and professor of Endodontics. Federal University of
Go=~ts, Goifinia, Brazil. Dr. Pimenta is professor of Microbiology, Institute of
Tropical Pathology and Public Health, Federal University of Goias, Goifinia,
Brazil. Dr. Ito is chairman and professor of M~crobiology, Unwersity of SAo
Paulo, RibeirAo Preto, Brazil. Dr. Bammann is chairman and professor of
Microbiology, Federal University of Petotas, Pelotas, Brazil. Address requests
for reprints to Dr. Carlos Estrela, Rua B-l, Quadra 6, Lote 2, Setor Bueno,
74,530-180 Goi&nia, GO, Brazil.
References
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results of treatment of extraradicular endodontic infection. Endod Dent Trau-
matol 1990;6:129-36.
3. Holland R, Soares IJ, Soares IM Influence of irrigation and intracanal
dressing on the healing process of dog's teeth with apical periodontitis.
Endod Dent Traumatol 1992;8:223-9.
4. Sundqvist G. Associations between microbial species in dental root
canal infections. Oral Microbiol Immunol 1992;7:257-62.
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tubules. Acta Qdontol Scand 1974;32:61-70.
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Endodon 1981 ;7:17-21.
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