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ENDODONTOLOGY Editor: Larz S. W. Sp!
angberg
Effectiveness of a calcium hydroxide and chlorhexidine digluconate mixture
as disinfectant during retreatment of failed endodontic cases
Joseph A. Zerella, DMD, MDS,
a
Ashraf F. Fouad, BDS, DDS, MS,
b
and
Larz S. W. Sp
!
angberg, DDS, PhD,
c
Farmington, Conn, and Baltimore, Md
UNIVERSITY OF CONNECTICUT AND UNIVERSITY OF MARYLAND
Objective. The purpose of this in vivo investigation is to compare the effect of a slurry of Ca(OH)
2
mixed in aqueous 2%
chlorhexidine (CHX) versus aqueous Ca(OH)
2
slurry alone on the disinfection of the pulp space of failed root-filled teeth during
endodontic retreatment.
Study design. Forty single-rooted previously root-filled teeth with associated periradicular lesions were included. The teeth
were nonsurgically retreated and medicated over 3 treatment visits with 7-10-day intervals with either Ca(OH)
2
in water
or Ca(OH)
2
in 2% aqueous CHX. Root canal cultures were collected in fluid thioglycollate, and bacterial growth was assessed
by turbidity daily for 1 week, then weekly for an additional 3 weeks. The presence of enterococci in the root canals at the initial
treatment session was determined.
Results. Of the total sample population, 12 of 40 (30%) were positive for bacteria before root filling. The control
medication disinfected 12 of 20 (60%) teeth including 2 of 4 teeth originally diagnosed with enterococci. The experimental
medication resulted in disinfected 16 of 20 (80%) teeth at the beginning of the third appointment. None of the teeth originally
containing enterococci showed remaining growth. This difference between the overall positive cultures was not statistically
significant (P[.05).
Conclusions. Canal dressing with a mixture of 2% CHX and Ca(OH)
2
slurry is as efficacious as aqueous Ca(OH)
2
on the
disinfection of failed root-filled teeth.
(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:756-61)
The success of endodontic treatment is negatively influ-
enced by the presence of microorganisms within the root
canal system.
1-2
Studies have shown the relationship
between the development of apical periodontitis and
the colonization of the pulp space by bacteria.
3-6
The
necrotic pulp harbors a delicate ecology of resident
microbes and microbial by-products.
7
The necrotic tis-
sue remnants and dentin serve as a sufficient environment
for the establishment of bacterial growth. Apical peri-
odontitis is an inflammatory process of the periradicular
region and is initiated and sustained by endodontic
infection.
3,7
Endodontic treatment of teeth with apical periodon-
titis consists of the elimination of microorganisms from
the root canal. Thus, the success of endodontic treatment
is directly contingent on the eradication of the infection
before root filling.
1,8,9
The flora of untreated necrotic
teeth consist of polymicrobial infection, often 8-11
different species, dominated by obligate anaerobes.
Studies have found that the microbial flora of failed cases
is significantly different from those of necrotic pulps.
Previously root-filled teeth with chronic periradicular
lesions often contain an isolated microflora, dominated
by gram-positive facultative anaerobes. Enterococcus
faecalis, which is not normally recovered in high
quantities of initially infected teeth, has been isolated
more often from failed root-filled teeth.
10-12
Therefore,
a
Former Resident and Postgraduate Student in Endodontology,
Department of Endodontology, School of Dental Medicine, Univer-
sity of Connecticut.
b
Associate Professor and Chairman, Department of Endodontics,
Prosthodontics and Operative Dentistry, Baltimore College of Dental
Surgery, University of Maryland.
c
Professor of Endodontology, Department of Endodontology, School
of Dental Medicine, University of Connecticut.
Received for publication Oct 10, 2003; returned for revision Jan 15,
2004; accepted for publication May 17, 2005.
1079-2104/$ - see front matter
!2005 Mosby, Inc. All rights reserved.
doi:10.1016/j.tripleo.2005.05.072
756
Vol. 100 No. 6 December 2005
a very selective environment may exist within the root
canal system of previously root-filled teeth that favors
the survival of microorganisms like E faecalis and
Candida albicans.
13
These microorganisms appear to
have an ability to utilize opportunities created by the
removal of other microorganisms and to survive and
multiply in the low-nutrient environment of the treated
root canal. E faecalis and C albicans are also less
susceptible to common deposit medicaments such as
calcium hydroxide.
14-17
Efforts have been made to enhance the antimicrobial
effectiveness of Ca(OH)
2
using the addition of suitable
antimicrobial mixing vehicles. Chlorhexidine (CHX)
is an antimicrobial agent currently under investigation
as an endodontic irrigant.
18,19
CHX possesses broad-
spectrum antibacterial activity, biocompatibility with
periodontal tissues, and substantivity.
20-22
However, the
effectiveness of aqueous CHX as a mixing vehicle on
enhancing the antimicrobial efficacy of Ca(OH)
2
slurry
has not been fully examined in vivo.
CHX is a synthetic cationic bis-biguanide (pKa 2.3
and 10.3) that remains stable at pH 5-7. As the pH is
increased, ionization will decrease.
23
Thus, at higher
pH values, there are a greater proportion of unionized
CHX molecules present. At high pH the CHX precipi-
tates and may be unavailable as an antimicrobial agent.
In preparation for this investigation, pilot studies were
performed to explore the chemical interaction between
Ca(OH)
2
and CHX and the CHX concentration needed
for clinical effectiveness. Therefore, the pH of mixtures
of various concentrations of CHX with Ca(OH)
2
were
recorded. The average pH of an aqueous solute of CHX
as a mixing vehicle for Ca(OH)
2
was 12.7, which is
similar to the pH value when using sterile water as the
mixing vehicle. This suggests that high concentrations
of hydroxide ion are generated when aqueous CHX is
used as a mixing vehicle.
CHX precipitates when mixed with Ca(OH)
2
.Theresult
of this precipitation on CHX was investigated. When a
slurry of Ca(OH)
2
in 0.5%, 0.4%, 0.3%, 0.2%, and 0.1%
CHX was centrifuged to separate the insoluble Ca(OH)
2
particulate from the fluid, a supernatant containing a
saturated solution of Ca(OH)
2
ions in aqueous CHX
resulted. The absorbance of ultraviolet light at 254 nm,
which is the peak absorbance for CHX, showed that
there was a significant loss of CHX ([99%) when mixed
with Ca(OH)
2
at this span of concentrations. Some
residual CHX is still available in this aqueous mixture
but in very low concentrations and dependent on the
original CHX concentration. When the Ca(OH)
2
pre-
cipitate was suspended again in water, the supernatant
was devoid of free CHX. Thus, after being mixed with
Ca(OH)
2
the CHX remains as a base, with little or no
solubility in water. Because of this observed loss of
CHX when mixed with Ca(OH)
2
, the bactericidal efficacy
of mixtures of various concentrations of Ca(OH)
2
in
aqueous CHX on cell suspensions of E faecalis
(ATCC19433) was studied. Despite the potential loss of
CHX ([99%) when mixed with Ca(OH)
2
, the anti-
microbial efficiency of this mixture was as effective as
CHX alone on E faecalis (ATCC19433) in agar inhibi-
tion tests in vitro. Thus, despite the remarkable high loss
of CHX when mixed with Ca(OH)
2
the combined
resulting effect may have clinical value. Other recent
studies in vitro have demonstrated a reduced effective-
ness of CHX after mixing with Ca(OH)
2
powder.
24,25
Because there is significant loss of CHX when
mixed with Ca(OH)
2
powder a 2% aqueous solution
was chosen as the mixing vehicle for the clinical trial.
The purpose of this in vivo investigation was to
compare the effect of a mixture of aqueous 2% CHX and
Ca(OH)
2
powder with an aqueous mixture of Ca(OH)
2
alone on the disinfection of the pulp space of failed
root-filled teeth during endodontic retreatment.
MATERIALS AND METHODS
Preparation of test medicament
A 2% solution of CHX was prepared by diluting a
20% stock solution (Sigma Chemical, St Louis, Mo) in
sterile deionized water. The CHX solutions were mixed
with sterile Ca(OH)
2
powder (Sigma Chemical; 0.5 g
Ca(OH)
2
to 1 mL CHX in water) until smooth slurry
was formed. Aqueous calcium hydroxide slurry was
prepared with sterile water in the same proportions.
Patient/sample selection
The subjects for this study were selected from
patients referred to the Department of Endodontology,
University of Connecticut School of Dental Medicine.
Forty single-rooted previously root-filled teeth with asso-
ciated apical periodontitis were included. The patients
were 20 years of age or older. Exclusion criteria included
teeth that could not be easily isolated with a rubber dam,
crowns with leaky cervical margins, and teeth with large
intraradicular posts. Informed consent was obtained
from all patients who participated in this investigation
in accordance with protocol obtained from the institu-
tional review board (IRB #03-041). A nonparticipating
dental assistant prepared the intracanal dressing on a
case-by-case basis. The operator was blinded to all inter-
visit medications. After complete instrumentation of the
root canal, and just before the time of initial placement
of intracanal medication, a lot was drawn from an initial
sample of 40, marked with either A or B, representing
control and experimental. The medication was coded as
A or B. This determined which medication was to be
used on that tooth for all treatment visits.
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Tooth preparation
The surface of the tooth was isolated with a rubber
dam and old restorations were removed and caries ex-
cavated. Before accessing the root filling, the operating
field was disinfected and then neutralized according
to the protocol by Mo
¨ller (1966)
26
Briefly, the rubber
dam, retainer, tooth, and surroundings were disinfected
for 2 minutes each with 30% hydrogen peroxide and
5% tincture of iodine. The disinfectants were neutralized
with 5% sodium thiosulfate, and then a bacteriologic
sample (CR
1
) of the tooth surface was obtained with
sterile paper points.
Endodontic treatment
The root filling was mechanically removed with
Gates-Glidden burs and hand files. An operating
microscope (Leica Microsystems, Bannockburn, Ill)
was used to aid in the removal of residual root filling
and sealer. A sterile endodontic file was placed into the
canal and the length was adjusted to within 1 mm of the
apex with the aid of a Root ZX electronic apex locator
(J Morita Corp, Irvine, Calif). A digital radiograph (Schick
CDR, Schick Technologies, Long Island City, NY) was
exposed to confirm the working length. Sterile saline
was then added to the root canal. A sterile #20 file,
advanced to the working length, was used to agitate the
canal contents for 1 minute. The entire canal contents
were absorbed onto sterile paper points and transfered
to prereduced thioglycolate broth (Becton-Dickinson
Microbiology, Cockeysville, Md), and the culture was
labeled (C1). The root canal was then cleaned and
shaped with endodontic files using conventional end-
odontic technique. A copious amount of 1.0% NaOCl
solution was used for irrigation. The canal was dried
with sterile paper points, and 5% sodium thiosulfate was
used to neutralize the sodium hypochlorite. The canal
was again dried and saline was added to the root canal,
and the entire contents were cultured as described
above (C2).
Next, the root canal was irrigated with 5 mL of 1%
NaOCl and agitated with the master apical file. The
canal was dried with sterile paper points and packed
with either aqueous Ca(OH)
2
slurry or 2%CHX 1
Ca(OH)
2
slurry and temporized with a thick layer of
Cavit (3M ESPE, St Paul, Minn). The next appointment
was scheduled for 7-10 days thereafter.
At the second appointment, the tooth was isolated
with a rubber dam, disinfected, and again neutralized,
as outlined earlier. A microbial sample of the operating
field was obtained (CR
2
). The temporary filling was
removed with a sterile round bur and the dressing was
flushed with copious sterile saline. The canal was dried
with sterile paper points. All root canals were filled with
a mixture of 0.3% lecithin (Sigma Chemical), 3%
Tween 80 (Sigma Chemical), and 5% sodium thiosulfate
(American Regent Laboratories, Shirley, NY) to neu-
tralize any residual CHX and were agitated with the
master apical file at working length. The canal was
dried and then filled with saline, and a culture sample
was obtained and labeled (C3). The root canal was
instrumented with the master apical file to working
length and irrigated with 1% NaOCl. The root canal was
dried with sterile paper points. The sodium hypochlorite
was neutralized with 5% sodium thiosulfate and the root
canal again dried. Saline was added to the root canal and
the entire contents were cultured (C4). The tooth was
remedicated with a fresh mixture of the same medica-
ment that was used during the first visit and temporized
with Cavit. The third visit was scheduled for 7-10 days
thereafter.
At the third appointment, the tooth was treated
similarly to the preceding appointments and cultured
twice again (CR
3
and C5). The root canals were finally
filled with gutta percha and AH26 sealer using cold
lateral compaction. The tooth was temporized with
Cavit and a permanent restoration planned.
All cultures were collected in prereduced fluid
thioglycolate.
27
The thioglycolate broth cultures were
incubated at 378C and inspected daily for turbidity
for the first 7 days, then weekly for another 3 weeks
to assure that slow-growing microorganisms were in-
cluded. The initial cultures (C1) were also analyzed
with molecular technique using PCR amplification and
molecular sequencing for identification of enterococci.
The results of this analysis were previously reported.
28
Statistical analysis
The material from the culture data was distributed
into 2 groups: experimental and control. The cultures
were recorded according to turbidity as positive or
negative and were compared using the chi-square test
(significance level set at P\.05).
RESULTS
Clinical experiment
The control cultures (CR
1
, CR
2
, and CR
3
) were all
negative, indicating successful disinfection of the opera-
ting field before the root canal was accessed. All teeth
contained cultivable microorganisms when first sam-
pled (C1). Four teeth in each group harbored E faecalis
as determined by molecular technique.
28
Bacterial cultures obtained after root canal medica-
tion at the beginning of the second appointment resulted
in 10 positive cultures (50%) for the control group, of
which 3 belonged to the group that initially contained
enterococci. In the experimental group there were 7
positive cultures (35%), of which 2 belonged to the
group that initially contained enterococci (Table I).
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These differences were not statistically significant
(P[.05).
Bacterial cultures obtained after root canal medica-
tion and at the beginning of the third appointment
resulted in 8 positive cultures (40%) for the control
group, of which 2 belonged to the group that initially
contained enterococci. In the experimental group
there were 4 positive cultures (20%), of which none be-
longed to the group that initially contained enterococci
(Table I). These differences between the overall positive
cultures were not statistically significant (P[.05).
Of the total sample population, 12 of 40 (30%) were
positive for bacteria before root filling. The control
medication disinfected 12 of 20 (60%) teeth, including
2 of 4 teeth originally diagnosed with enterococci. The
experimental medication resulted in disinfected 16 of 20
(80%) teeth at the beginning of the third appointment.
None of the teeth originally containing enterococci
showed remaining growth. This difference between
the overall positive cultures was not statistically signif-
icant (P[.05).
DISCUSSION
Numerous treatment strategies and regimens have
been suggested for the treatment of teeth with failed
endodontic therapy. Thorough chemomechanical de-
bridement is still the mainstay of therapy and the
placement of an intracanal antimicrobial dressing is
usually recommended. However, owing to the special
microflora present in retreatment cases, the traditional
intracanal antimicrobial dressing with Ca(OH)
2
may
under certain circumstances be ineffective.
16,29
Ca(OH)
2
is poorly soluble in water but dissociates
into OH
!
ions, which creates a highly alkaline solution
(pH [12.0 at 378C). The hydroxide ion concentration
remains constant in solution as long as the presence of
undissolved Ca(OH)
2
is in contact with the saturated
solution.
30,31
Sukawat and Srisuwan
32
tested the antimicrobial
efficacy of 3 Ca(OH)
2
-slurry formulations on human
dentin infected with E faecalis. After exposure to the 3
Ca(OH)
2
mixtures (distilled water, 0.2% chlorhexidine,
or camphorated paramonochlorophenol (CMCP)) for
7 days, the CMCP mixture was completely effective
against dentinal tubule infection, and distilled water
and CHX mixed with Ca(OH)
2
were ineffective. The
low effectiveness of the CHX and Ca(OH)
2
mixture was
attributed to a possible decrease in the resulting pH. Our
results, however, indicate that the high pH is maintained
in a mixture. Therefore, the substantial reduction of
CHX concentration, due to the precipitation at high pH
of an already low concentration of CHX is most likely
responsible for the lack of effect in the study by Sukawat
and Srisuwan. Podbielski et al
33
used a combination
of Ca(OH)
2
and CHX and tested the effectiveness of
this mixture in disinfecting dentin. They concluded that
an antibacterial synergism exists between the Ca(OH)
2
suspension and CHX when used against E faecalis.
They also found that Ca(OH)
2
did not adversely affect
the solubility and activity of CHX but rather exhibited
an additive effect on common endodontic pathogens.
In contrast, our preliminary studies showed that most
CHX precipitates out of solution when mixed with
Ca(OH)
2
. However, at higher concentrations of CHX it
appears that a small residue of active CHX may still be
present. The reduced efficacy of the CHX and Ca(OH)
2
mixtures, in addition to precipitation, may be due to
the deprotonation of the biguanide at pH [8.0 and
therefore to a reduced solubility, which may hinder
the interaction with the negatively charged bacterial cell
membrane. Therefore, a mixture of CHX and Ca(OH)
2
may not provide a sufficient reservoir of free CHX
molecules.
The present study shows that sodium hypochlorite
in conjunction with mechanical instrumentation is only
partially effective as a method to reduce microbial con-
tent of previously treated root canals that have failed.
Table I. Positive culture results from the root canals during retreatment of failed endodontic cases in forty single-
rooted teeth
Dressing: CHX1Ca(OH)
2
Dressing: Ca(OH)
2
Sample # All samples C contained Enterococcus All samples C contained Enterococcus Total positive
C1 20 4 20 4 40
C2 2 — 7 — 9
C3 7 2 10 3 17
C4 2 — 3 — 5
C5 4 0 8 2 12
Ca(OH)
2
,calcium hydroxide slurry.
All teeth were treated in similar fashion except for the intervisit dressing.
Cultures C1, C3, and C5 were obtained at the beginning of visits 1, 2, and 3, respectively. C2 was obtained at the end of visits 1 and 3. C4was taken at the end of visit 2.
Chi-square analysis. The distribution is not significant at the 0.05 level.
No difference between 1- and 4-week observations.
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Our findings correspond to results reported by others
when treating teeth with necrotic pulp.
34,35
Although
there were fewer samples with growth in the CHX1
Ca(OH)
2
group compared to the Ca(OH)
2
group, these
findings did not reach statistical significance. The sam-
ple size, however, does not allow the conclusion that
there is no significant difference. Further research is
needed to explore whether the difference between the
experimental and control groups in residual cultivable
bacteria of 20% and 40%, respectively, remains consis-
tent in larger samples of cases.
An analysis of the original samples from the infected
root canals showed an even distribution of Enterococcus
species between the 2 groups. Four out of 20 teeth in
both the experimental as well as the control group
initially contained Enterococcus species. The Ca(OH)
2
and CHX slurry was effective in completely eliminating
bacteria in these specimens but a Ca(OH)
2
slurry was
only partially effective. If the presence of Enterococcus
species in therapy resistance is important these trends
may be of value. Therefore, to further explore the
observed enhancement in disinfection when using a
slurry of Ca(OH)
2
in 2% CHX, larger size clinical
experiments must be undertaken to verify or reject the
trend.
There were noticeable differences in the rate of
growth in cultures taken at the end of each appointment
compared with the culture obtained at the beginning of
the subsequent visit. There is always the risk of leakage
and contamination of the pulp space between visits but
great care was taken to maintain reliable temporary
fillings and asepsis. The difference observed is more
likely associated with a significant decrease in number
of bacterial cells after thorough instrumentation and
irrigation during each treatment session. If the cell
numbers are too low the cultures obtained at the end of
the treatment session may fail to grow in this specific
culture medium.
Myers et al
36
determined the incidence of microor-
ganisms present in the root canals of teeth scheduled to
be filled following 1 or 2 consecutive negative cultures.
They found that root canals to be filled following
1 negative culture yielded 25.9% positive cultures at the
time of root filling and root canals filled following
2 successive negative cultures produced a reversal rate
of 13.2%. Engstro
¨m and Lundberg
37
found a reversal
rate of 16.2% after 2 successive negative cultures.
The length of time elapsed between the last treatment
and the final filling appointment was regarded as the most
important factor differentiating the culture results between
canals filled after 1 negative culture and those filled
after 2 negative cultures.
Another reason for the difficulty to more successfully
eliminate microorganisms from these root canals may
be the effect of remaining root filling materials inhibi-
ting effective disinfection of the root canal walls. Despite
the intracanal dressing used, all microorganisms
may not have been reached by the antiseptics, which
allowed bacterial repopulation during the intervisit
period. This observation is not unique, but difficult to
explain, because the root canals were filled with some
form of calcium hydroxide slurry between visits.
38
Our results suggest that 2 sessions of intracanal
dressing may be of value when retreating teeth with
failed endodontic treatment. No cultures were obtained
after irrigation and instrumentation during the third
visit. Judging from the remarkable decrease in numbers
of root canals with microorganisms between the begin-
ning and end of the first and second visit, an equivalent
decrease might be expected immediately before the
placement of the root filling. All teeth where enterococci
were found at the initial sampling were successfully
disinfected when Ca(OH)
2
was mixed with CHX. Only
2 of the 4 teeth with initial content of enterococci were
successfully disinfected when a Ca(OH)
2
slurry was
used as intracanal dressing. These observations are
similar to the observations by Evans et al,
39
who found
dentin cylinders infected with E faecalis (ATCC 29212)
were more effectively disinfected with 2% CHX 1
Ca(OH)
2
compared with Ca(OH)
2
alone.
CONCLUSION
Root canal dressing with a mixture of 2% CHX
and Ca(OH)
2
slurry is at least as efficacious as aqueous
Ca(OH)
2
on the disinfection of root canal dentin of
failed root-filled teeth. The difference, however did
not reach statistical significance in this study. When
using an alkaline canal dressing for the retreatment of
failed endodontic cases, a 3-visit retreatment procedure
resulted in fewer cases of root canals with residual in-
fection than a 2-visit treatment routine. Complete dis-
infection of all cases was not achieved but all cases that
initially harbored Enterococcus species were success-
fully disinfected with the Ca(OH)
2
and CHX combina-
tion. To elucidate if the slurry of Ca(OH)
2
in CHX has an
enhanced clinical disinfection efficacy over a Ca(OH)
2
slurry a larger clinical trial must be undertaken.
This study was funded by Endodontic Alumni Association at
the University of Connecticut Health Center School of Dental
Medicine, University of Connecticut.
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Reprint requests:
Dr Joseph A. Zerella, DMD, MDS
1275 Post Road
Fairfield, CT 06824
josephzerella@yahoo.com
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Volume 100, Number 6 Zerella, Fouad, and Sp
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angberg 761