JOURNAL OF ENDODONTICS Printed in U.S.A.

Copyright © 2004 by The American Association of Endodontists VOL. 30, NO. 9, SEPTEMBER 2004

Antibacterial Efficacy of Calcium Hydroxide and

Chlorhexidine Gluconate Irrigants at 37°C and 46°C
Chris Evanov, DDS, Frederick Liewehr, DDS, MS, Thomas B. Buxton, PhD, and Anthony P. Joyce, DDS

This study investigated the ability of two endodon- irrigant meets all of these requirements, and the most commonly
tic irrigants to eliminate Enterococcus faecalis used irrigant, sodium hypochlorite (NaOCl), has been found to
from dentinal tubules, and whether their antimicro- cause severe inflammatory reactions if extruded into the periapical
bial action was enhanced by heat. The lumens of tissues (5). Chlorhexidine gluconate (CHX) (6) has been suggested
as an irrigant because of its biocompatibility and its substantivity,
disks prepared from extracted bovine roots were
a prolonged antibacterial effect due to its adherence to dentin (7).
infected with E. faecalis and incubated for 72 h.
A 10% calcium hydroxide solution (Ca(OH)2) also has been rec-
Specimens were then filled with saline, 10% cal- ommended as a biocompatible irrigant (8). Ca(OH)2 has long been
cium hydroxide (Ca(OH)2), or 0.12% chlorhexidine used in endodontics as an intracanal medication and irrigant, with
gluconate (CHX) at 24°C or 46°C and incubated at its antibacterial effects attributed to its high pH (approximately
37°C or 46°C. The samples were then pulverized 12.5), which has a destructive effect on cell membranes and protein
and plated to quantify residual bacteria. No statis- structure (9). Ca(OH)2 owes its biocompatibility to its low solu-
tical difference (p > 0.05) in bacterial growth was bility in water and limited diffusion, which effectively limits its
seen between the two saline groups, or between cytotoxicity to the tissues with which it is in contact. The low
the two medication groups at a given temperature. solubility and diffusibility of calcium hydroxide, along with the
CHX and Ca(OH)2 at either temperature produced buffering capacity of dentin, make it difficult to rapidly reach and
significantly less growth than either saline group, sustain the high pH necessary for effective antimicrobial action
and CHX or Ca(OH)2 at 46°C produced significantly against E. faecalis sequestered in dentinal tubules. Barbosa et al.
(10) found that a saturated calcium hydroxide irrigant had limited
less growth than either group at 37°C. Heat en-
success against most aerobic microorganisms with a 60-min ex-
hanced the antibacterial action of both experimen-
posure. Therefore, Ca(OH)2 may require prolonged exposure for
tal irrigants against E. faecalis, but heating saline effective action.
produced no increase in bactericidal effect. Several studies have demonstrated that increasing the temper-
ature of NaOCl can enhance its antibacterial action. Cunningham
and Joseph (11) demonstrated that increasing the temperature of
NaOCl from room temperature to body temperature (37°C) in-
The goal of endodontics is the complete debridement of the root- creases its antibacterial effectiveness. Abou-Rass and Oglesby (12)
canal system to eliminate all bacteria, bacterial byproducts, and demonstrated that heating NaOCl to 60°C increased its effective-
tissue debris. Byström and Sundqvist (1) demonstrated that me- ness as a tissue solvent. However, Raphael et al. (13) found no
chanical instrumentation with nonantibacterial irrigants was effec- direct relationship between the temperature of NaOCl and its
tive in significantly reducing the number of bacteria within the root antibacterial efficacy. The purpose of this study was to investigate
canal, but Ingle and Zeldow (2) observed that irrigation with sterile the ability of two endodontic irrigants to eliminate E. faecalis from
water alone yielded positive cultures in 80% of initially infected dentinal tubules, and whether their antimicrobial action was en-
root canals. Furthermore, these authors observed that by the time hanced by heat.
of the second appointment 24 h later, the number of canals that
yielded positive cultures had increased to 95.4%, referred to as a
reversal, which suggests that approximately 75% of the canals MATERIALS AND METHODS
presumed to have been mechanically sterilized actually contained
undetected residual bacteria. Siqueira et al. (3) demonstrated that A preliminary study was conducted to determine the time
irrigants with antibacterial properties were more effective than needed for a dentin disk specimen to reach a specific temperature
saline at eliminating bacteria. Therefore, thorough chemomechani- after being returned to the incubator. A T-type thermocouple
cal preparation with an antibacterial irrigant is recommended. (Model No. MLT1401, PowerLab, ADInstruments, Colorado
An ideal irrigant should be nontoxic and capable of dissolving Springs, CO) was attached to a 5-mm dentin disk, using a 1⁄8-inch
both vital and necrotic pulp tissue. It should kill bacteria, lubricate orthodontic band, to measure the disk temperature. The thermo-
the canal, and remove the smear layer (4). Currently, no single couple was connected to a PowerLab 400 peripheral device spe-

653
654 Evanov et al. Journal of Endodontics

FIG 1. Dentin disk mounted within a tissue well.

cifically designed to perform data acquisition and preprocessing. Everett, MA). The specimens were then mounted in individual
The PowerLab communicated with a personal computer (AMD 22-mm diameter tissue wells (Corning Cell Wells, Corning Glass
800 mH), which stored the digital signal in real time using graph- Works, Corning, NY) on bases of sticky wax approximately 5-mm
ing software (Chart v4.2, PowerLab, ADInstruments). The dentin tall. The lumens of each specimen were completely filled (approx-
disk was then placed in an incubator at 37°C or 46°C. When the imately 25 ␮l) with brain-heart infusion (BHI) broth (Difco, De-
computer indicated that the incubator had reached the desired troit, MI) containing 1.0 ⫻ 107 colony-forming units/ml (approx-
temperature, the trial experiment was begun. The doors of the imately 2.5 ⫻ 105 colony-forming units/dentin disk) of E. faecalis
incubator were opened, and this exposure to ambient temperature (ATCC 29212). Each tissue well received 2 ml of sterile water
caused a decrease in incubator temperature. The time for the (surrounding the wax base but not contacting the specimen) to
incubator to reestablish the desired temperature was measured. maintain humidity (Fig. 1). All manipulations of the specimens
Temperature readings were repeated five times for accuracy. Re- were performed under a laminar flow hood (NUAIRE, Plymouth,
sults indicated that 5 min were necessary for the specimens to MN) to avoid contamination by outside organisms. The specimens
return to their desired temperature. This time was added to 30 min were then incubated at 37°C in 5% CO2 (6000 Incubator, Napco,
(chosen to represent the approximate time of irrigant exposure Winchester, VA) for 72 h. Sterile BHI was added to replenish each
during clinical instrumentation) for a total of 35 min of irrigant lumen every 24 h as needed to compensate for any evaporation.
exposure at a specified temperature. Then the BHI broth was flushed from each specimen using a 10-ml
The in vitro model for preparation of dentin disk specimens syringe containing 2 ml of sterile saline at ambient temperature
originally described by Haapasalo and Ørstavik (14) was used in (24°C). The excess fluid from the 2-ml flush, and the saline in the
the present study with some modifications. Ninety-five, intact, lumen, was suctioned away.
bovine, mandibular, central and lateral incisors (Shapiro Packing Two temperatures for each irrigant were tested: 24°C irrigants
Co, Augusta, GA) were extracted from frozen jaws and stored (ambient temperature) were placed in 37°C incubators (oral tem-
overnight in 5.25% sodium hypochlorite for surface disinfection perature) to simulate clinical conditions; 46°C irrigants (experi-
and soft-tissue dissolution, and then stored in tap water to prevent mental temperature) were placed in 46°C incubators (to maintain
dehydration. The crowns and apical 5-mm were removed with a the experimental temperature) to test the antimicrobial effect of
diamond saw (Isomet, Buehler LTD, Evanston, IL) at slow speed increased temperature. Ninety teeth were divided into six groups
(⬍100 rpm) with water coolant. The roots were prepared to cy- (two temperatures for each of the three irrigants) consisting of 15
lindrical test specimens 5 mm in height. The pulpal lumens were teeth each. The irrigant for groups 1 and 2 was saline. The irrigant
standardized to 2.5-mm diameter with an ISO 025 bur (Brasseler for groups 3 and 4 was prepared by mixing 10 g of Ca(OH)2 USP
USA, Savannah, GA) in a MCS Heavy Duty Drill Press (Model per 100 ml of sterile water. The irrigant for groups 5 and 6 was
951220, Manhattan Supply Company, Central Islip, NY). Disk CHX. The negative control group consisted of five teeth treated in
thicknesses were measured with calipers (Mahr, 16ES, Esslingen, the same manner as the experimental teeth with the exception that
Germany). The smear layer was removed by using an ultrasonic they were incubated with sterile, uninoculated BHI broth.
bath with 17% EDTA for 4 min, followed by an ultrasonic bath in Each specimen was then filled with one of the test irrigants at a
5.25% NaOCl for 4 min. The specimens were then placed in glass specified temperature. Group 1 additionally served as a positive
containers with tap water and sterilized in a steam autoclave for 15 control group to provide baseline data on bacterial growth over
min at 121°C and 15 psi (Market Forge, Sterilmatic Model STM-E, time. The specimens were returned to the incubator at 37°C or
Vol. 30, No. 9, September 2004 Heating Irrigants 655

FIG 3.

Recently, E. faecalis has been cultivated from a high percentage

of teeth that have been unsuccessfully treated endodontically.
FIG 2. E. faecalis cultured after application of the experimental Hancock et al. (15) found that 63% of failed root canals were
irrigants. infected with E. faecalis, the most frequently recovered species.
Although calcium hydroxide has been shown to reduce or elimi-
nate bacteria from the root canal when used as an interim dressing
46°C, as appropriate, for 35 min. Then, the saline or test irrigant (16), E. faecalis has demonstrated resistance to this medication
was flushed from each specimen using 2 ml of 24°C sterile saline, (14). The mechanism of action of calcium hydroxide is considered
which was then suctioned away. The specimens were then frozen to be caused by the high pH (pH 12.5), which is related to the
in a ⫺70°C freezer, weighed, pulverized in liquid nitrogen with a release of hydroxyl ions in an aqueous environment (17). Various
mortar and pestle, placed in 2 ml of phosphate buffered saline, and mechanisms have been postulated for the resistance of E. faecalis
vortexed for 5 min. Serial dilutions (1:100, 1:1000, 1:10,000, and to this medication, but it is considered to be primarily due to the
1:100,000) were made of the samples, and 100-␮l aliquots of each ability of the bacteria to sequester themselves in the dentinal
dilution were plated on BHI plates in triplicate. The BHI plates tubules of the infected tooth (18).
were incubated in 5% CO2 at 37°C, and the number of bacterial One approach that has been suggested to increase the efficacy of
colony forming units was recorded 24 h later. The dilutions that Ca(OH)2 against E. faecalis is to combine it with another medi-
yielded an average number between 30 and 300 bacterial colonies cation, which could produce an additive or synergistic effect.
on the plates were used for data analysis. Following Frank’s classic technique for apexification (19), Leo-
A one-way ANOVA was performed, followed by the Student- nardo et al. (20) suggested adding camphorated p-monochlorophe-
Newman-Keuls multiple comparison test. The level of significance nol (CMCP) to Ca(OH)2, and found that it produced better apical
was set at p ⬍ 0.05 (n ⫽ 95). bridging than either medication alone when used alone as a dress-
ing in infected dog teeth. Siqueira and Uzeda (21) infected bovine
RESULTS dentin cylinders with E. faecalis and found that a paste of Ca(OH)2
plus CMCP killed all bacteria within 1 day. The problem posed by
There was no statistical difference (p ⬎ 0.05) in bacterial CMCP is its toxicity. Testing the biocompatibility of seven com-
growth between the two saline groups. CHX and Ca(OH)2 at either monly used intracanal medications and irrigants in cultures of
temperature produced significantly (p ⬍ 0.05) less growth than human lung fibroblasts, Masillamoni et al. (22) found CMCP to be
either saline group. There was no difference in growth between the the least biocompatible of the materials tested. Spångberg et al.
two medication groups when considered at the same temperature, (23) also found CMCP to be highly cytotoxic, and Fager and
and CHX or Ca(OH)2 at 46°C produced significantly (p ⬍ 0.05) Messer (24) noted that CMCP passed rapidly and extensively
less growth than either group at 37°C (Fig. 2). beyond the apical foramen and into the bloodstream where it could
potentially cause systemic toxicity. Although the likelihood and
extent of tissue damage by this medication are unknown, it seems
DISCUSSION prudent to continue the search for an efficacious but less toxic
material.
When endodontic therapy is unsuccessful, continued infection Mickel et al. (25) compared the efficacy of Ca(OH)2, stannous
or reinfection of the root-canal system can create or perpetuate fluoride, and a combination of the two against E. faecalis, and
periapical inflammation caused by the escape of these bacteria or found that stannous fluoride displayed the widest zone of inhibition
their byproducts from the apical foramen. To maximize the prob- in vitro (1.7 mm), followed by the combination (1.1 mm) and the
ability of retreatment being successful, thorough chemomechanical Ca(OH)2 group. The biocompatibility of stannous fluoride in the
preparation with an antibacterial irrigant is essential (4). periapical tissues is unknown, however, and the authors caution
656 Evanov et al. Journal of Endodontics

that further studies are required before clinical use could be mediate microbial analysis eliminates the possibility of culture
advocated. reversals.
Another suggested approach is to change the consistency of the The results of our study are at variance with Lin et al. (33), who
Ca(OH)2. Behnen et al. (26) and Lynne et al. (27) demonstrated found Peridex to produce larger zones of inhibition than Ca(OH)2,
that a thin mix of Ca(OH)2 penetrated dentinal tubules better than whereas we found the two medications to produce equal killing at
a thick mix and was effective against E. faecalis. We investigated a given temperature. Lin et al. placed the experimental mixtures in
the use of 10% Ca(OH)2 in this study, because it is effective in a E. faecalis colonies cultured on blood agar plates, whereas we used
thin mix and because of its known biocompatibility. dentin disks, because a suspected reason for the resistance of E.
Still another strategy is to change the irrigant or intracanal faecalis to intracanal medications is its ability to sequester itself in
medication used. Heling and Chandler (28) found chlorhexidine dentinal tubules in which it is more difficult for intracanal medi-
gluconate and sodium hypochlorite were similarly effective at cations to penetrate in effective dosage. Portenier et al. (34) found
eradicating E. faecalis. CHX is a broad-spectrum antibacterial that dentin matrix exerted a strong inhibitory effect on the antimi-
agent effective against Gram-positive and Gram-negative bacteria. crobial properties of CHX, which may have been operative in our
CHX acts by absorbing into the cell wall of the microorganism and study and not in the agar plates of Lin et al. It is possible that our
causing leakage of cytoplasmic substances. White et al. (29) dem- thin solution of Ca(OH)2, which has already been found more
onstrated that 0.12% CHX instills substantive activity for 6 to 24 h. effective when used on dentin than a thick mixture (25), was able
We used 0.12% CHX in this study because it is efficacious, to compensate for its lower antimicrobial efficacy by resistance to
biocompatible, and readily available at this concentration as Peri- the attenuation produced by dentin on CHX.
dex (Zila Pharmaceuticals Inc, Phoenix, AZ). Our results demonstrated that heat (46°C) enhanced the antimi-
The use of bovine teeth was based on their availability and their crobial action of both 10% Ca(OH)2 and 0.12% CHX relative to
similar morphology (size and density of dentinal tubules) to human saline (the positive control) and to either experimental medication
teeth (30). The in vitro model developed by Haapasalo and Ørsta- at 37°C. Increasing the temperature of saline, however, produced
vik was used in this study with modification to include quantitative no increased bacterial efficacy. Heating 10% Ca(OH)2 and 0.12%
analysis of the bacteria in the dentin tubules after the application of CHX to 46°C produced approximately a 1.8-log reduction of E.
irrigants. In addition, both lumen preparation and smear layer faecalis relative to saline at 37°C.
Currently, there are several systems and devices available to the
removal were accomplished before sterilization to eliminate the
endodontic community that provide the delivery of warm irrigating
effect of two confounding variables, mechanical instrumentation,
solutions. Our results suggest that delivering 10% Ca(OH)2 or
and the antimicrobial effect of smear layer removal, in reducing
0.12% CHX at 46°C would increase its antibacterial efficacy
bacterial counts. The removal of the smear layer also allowed
without the addition of more cytotoxic materials. Further studies
deeper penetration of both the microorganisms and the irrigant.
are needed to test these devices for their effectiveness in main-
Results are given in logarithmic numbers of microbes rather than
taining the prescribed level of heat.
arithmetic numbers to allow a linear assessment of the results of
the statistical analysis of exponential growth/reduction of bacteria This study is the work of the United States government and may be
(e.g. one-log decrease equals 90% of microbial population killed). reprinted without permission. Opinions expressed herein, unless otherwise
specifically indicated, are those of the authors. They do not represent the
A preliminary study was conducted to determine the time views of the Department of the Army or any other Department or Agency of the
needed for a 5-mm, dentin disk specimen to return to incubation United States Government.
temperature after opening the incubator door to flush the inoculum
The authors thank Ms. Jordan T. Mastrodonato for her expertise in ren-
from the lumens and place the irrigant. This procedure took ap- dering the figures for this publication, and Mr. Royce R. Runner and Ms.
proximately 2 min, during which time the temperature of both the Phyllis D. Brewer for their technical investigative expertise.
incubator and disk specimens would decrease. After repeated trials, Dr. Evanov is endodontist, U.S. Army Dental Corps, and former resident,
we discovered that the disk specimen and incubator would take 5 U.S. Army Endodontic Residency Program, Fort Gordon, GA. Dr. Liewehr is
min to return to the original temperature, so that time was added to former program director, and Dr. Joyce is program director, U.S. Army End-
odontic Residency Program, Fort Gordon, GA. Dr. Buxton is a microbiologist,
the 30-min incubation period considered to represent experimen- Department of Clinical Investigation, Dwight D. Eisenhower Army Medical
tally the time the material would be in contact with the dentin in Center, Fort Gordon, GA.
clinical use. Address correspondence to Frederick Liewehr, DDS, MS; E-mail:
We used 46°C as our experimental temperature because Eriks- doctorendo@comcast.net.
son and Albrektsson (31) found that the threshold temperature for
bone survival was 47°C for 1 min. Cunningham and Joseph (11)
and Abou-Rass and Oglesby (12) both found that the effectiveness
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