2. Chlorhexidine gel was the most effective at inhibiting the growth of Enterococcus faecalis, eliminating bacterial growth at both 200 and 400 micrometer depths over 5 days. Metronidazole gel showed moderate inhibition of 86.5%, followed by bioactive glass at 62.8% and calcium hydroxide at 58.5%. Chlorhexidine gel completely inhibited bacterial growth at all time intervals tested, demonstrating its strong antibacterial properties compared to the other medicaments.

1. Basic Research—Technology
Disinfection of Dentinal Tubules with 2% Chlorhexidine,
2% Metronidazole, Bioactive Glass when Compared with
Calcium Hydroxide as Intracanal Medicaments
Jogikalmat Krithikadatta, MDS,* Rajamani Indira, MDS,† and Alfred Leo Dorothykalyani, MSc‡
Abstract
Thorough disinfection of the root canal system is es-
sential for the success of root canal therapy. This re-
quires the use of an intra-canal medicament. Entero-
M icroorganisms play a fundamental role in the etiology of pulp and periapical
diseases. Their control and elimination are important during endodontic treat-
ment (1). Aerobic and facultative anaerobic microorganisms are usually minor con-
coccus faecalis is the most frequently found species in stituents of primary infections and are found in higher frequency in endodontic flare-
persistent/secondary intracanal infection associated ups and in failed cases. Enterococcus faecalis is more likely to be found in failed cases
endodontic treatment failure. This study evaluates the than in primary infection (2), and starvation increases the resistance of E. faecalis
disinfection of dentinal tubules using 2% chlorhexidine 1000-fold to 10,000-fold (3). It is probable that the physiologic state of the cells,
gel, 2% metronidazole gel, bioactive glass (S53P4) in particularly in retreatment cases, is closest to the starvation phase (4).
comparison with calcium hydroxide. The antibacterial Cleaning and shaping of the root canal reduce the bacterial population but do not
efficacy of the four medicaments against Enterococcus completely eliminate them. One possible reason for persistent endodontic infection
faecalis was assessed in vitro using extracted premolar might be due to the retention of microorganisms in the dentinal tubules of the root canal
teeth at the depths of 200 m and 400 m. The overall (5). Hence, the use of an intracanal medicament helps in the elimination of bacteria that
percentage inhibition of bacterial growth (at 200 m remain even after cleaning and shaping, thereby providing an environment conducive
and 400 m depth) was 100% with 2% chlorhexidine for periapical tissue repair (6).
gel. The inhibition of growth was moderate with 2%
Calcium hydroxide is the most widely used intracanal medicament, requiring a
metronidazole gel (86.5%), followed by bioactive glass
disinfection period of 7 days (7). The high pH of calcium hydroxide formulations alters
(62.8%) and calcium hydroxide (58.5%). It can be
the biologic properties of bacterial lipopolysaccharides in the cell walls of gram-neg-
concluded from the present study that 2% chlorhexi-
ative species and inactivates membrane transport mechanisms, resulting in bacterial
dine gel alone was most effective against E. faecalis
cell toxicity (8). However, E. faecalis has been reported to be resistant to this effect as
when compared to other medicaments tested. (J Endod
a result of its ability to penetrate the dentinal tubules and adapt to changing environment
2007;33:1473–1476)
(9). The search for a better alternative has lead to the introduction of newer antimi-
Key Words crobial agents like chlorhexidine, metronidazole, and particulate bioactive glass.
Bioactive glass, calcium hydroxide, chlorhexidine, den- Hence, this study was undertaken to evaluate the disinfection of dentinal tubules con-
tin tubule disinfection, Enterococcus faecalis, metroni- taminated with E. faecalis by using 2% chlorhexidine gel, 2% metronidazole gel, and
dazole bioactive glass (S53P4) when compared with calcium hydroxide as intracanal medica-
ments.
From the *Department of Conservative Dentistry & End- Materials and Methods
odontics, Meenakshi Ammal Dental College, Maduravoyal; The model proposed by Haapasalo and Orstavik (10) was modified for this study,
†
Department of Conservative Dentistry & Endodontics, Ragas
Dental College & Hospitals, Uthandi; and ‡Mohan Diabetes and freshly extracted single rooted human premolar teeth were selected.
Specialty Center, Gopalapuram, Chennai, Tamilnadu, India.
Address requests for reprints to Dr Jogikalmat Krithika-
datta, No. 32, Arumuga Nainar Street, Thirunagar, Valasara- Preparation of the Blocks
vakkam, Chennai 600087, Tamilnadu, India. E-mail address: A rotary diamond disk was used to decoronate the teeth 5 mm below the cemen-
drkrithikadatta@hotmail.com. toenamel junction. The remaining root was then sectioned such that 6 mm of the middle
0099-2399/$0 - see front matter
Copyright © 2007 by the American Association of
third of the root was obtained. Cementum was removed from the root surface to stan-
Endodontists. dardize the external diameter to 4 mm (11). The internal diameter was standardized to
doi:10.1016/j.joen.2007.08.016 Gates Glidden drill no.3 (Mani Inc, Tachigi-ken, Japan) in a slow speed handpiece
(NSK, Tokyo, Japan). Organic and inorganic debris was removed by treating the blocks
in an ultrasonic bath of 17% ethylenediaminetetraacetic acid for 5 minutes followed by
3% sodium hypochlorite for 5 minutes. The blocks were immersed in an ultrasonic bath
of distilled water for 5 minutes to remove all traces of the chemicals used and sterilized
in an autoclave at 121°C. The blocks were subjected to a second cycle of sterilization,
with the blocks immersed in 1 mL of tryptone soya (TS) broth (HiMedia, Mumbai,
India) in individual microcentrifuge tubes. This allows better penetration of the broth
into the dentinal tubules (12).
JOE — Volume 33, Number 12, December 2007 Disinfection of Dentinal Tubules 1473

2. Basic Research—Technology
TABLE 1. Mean Colony Counts for Different Intracanal Medicaments at 200 and 400 m Depths at Different Time Intervals
Mean (Standard Deviation) Colony Count ( 105)
GROUPS Day 1 Day 3 Day 5
200 m 400 m 200 m 400 m 200 m 400 m
Saline 3.26 (0.26) 3.46 (0.26) 3.12 (0.56) 3.38 (0.48) 2.8 (0.53) 3.16 (0.42)
Calcium hydroxide 0.85 (0.31) 0.98 (0.36) 1.73 (0.77) 1.86 (0.78) 1.2 (0.26) 1.30 (0.17)
Bioactive glass 1.31 (0.22) 1.38 (0.21) 1.06 (0.42) 1.34 (0.65) 1.0 (0.20) 1.07 (0.20)
Chlorhexidine 2% gel 0.00 0.00 0.00 0.00 0.00 0.00
Metronidazole 2% gel 0.39 (0.05) 0.44 (0.08) 0.36 (0.15) 0.41 (0.13) 0.38 (0.09) 0.54 (0.19)
Contamination of the Blocks and 400 m) by using Gates Glidden drills nos. 4 and 5 (Mani Inc),
E. faecalis was used as the test organism in this study. This gram- respectively, and collected in 1 mL of sterile TS broth and incubated in
positive facultative anaerobic bacterium is the most common isolate found an anaerobic environment at 37°C for 24 hours. After the incubation
in endodontically failed cases. Isolated 24-hour colonies of pure culture of period, the content of each microcentrifuge tube was serially diluted,
E. faecalis (ATCC 29212) grown on tryptone soya agar were suspended in 100 L of broth in 100 L of normal saline for 5 times. Five microliters
5 mL of TS broth and incubated for 4 hours at 37°C. The culture suspen- of this diluted sample was plated on TS agar plates and incubated for 24
sion was adjusted to match the turbidity equivalent to 0.5 McFarland hours. Colonies were counted, and readings were tabulated.
standard. Fifty microliters of the inoculum was transferred to presteril-
ized individual microcentrifuge tubes containing 1 mL of the TS broth
and dentin block. The dentin blocks were transferred to fresh broth Statistical Analysis
containing E. faecalis every second day. All the procedures were carried The data were statistically analyzed with one-way analysis of vari-
out under laminar flow (Clean Air, Mumbai, India).The purity of the ance followed by Tukey multiple comparison means to check the dif-
culture was checked by subculturing 5 L of broth from the incubated ferences in bacterial inhibition between groups (P .01). The paired
dentin block in TS broth on tryptone soya agar plates (HiMedia). The t test was used to check for differences in growth at different time
dentin blocks were contaminated during a period of 21 days. Five intervals within groups and for differences at the 2 depths (P .01).
blocks were picked randomly and assessed for the depth of penetration
of E. faecalis by using light microscopy and scanning electron micros-
copy.
Results
Antimicrobial Assessment The current study showed that all 4 medicaments studied exerted
After the incubation period, the blocks were irrigated with 5 mL of antibacterial activity. The scanning electron microscopy and light mi-
sterile saline to remove the incubation broth. The dentin blocks were as- croscopy evaluation of 5 dentin blocks showed invasion of the bacteria
signed to the following groups (n 45 dentin blocks): group 1, saline within the dentinal tubules. Infection of the dentin blocks was confirmed
(negative control); group 2, calcium hydroxide (Ref no.23,923-2; Sigma- when debris samples harvested from the saline group (negative con-
Aldrich, Mumbai, India); group 3, bioactive glass S53P4 (AbminDent; Ab- trol) yielded positive growth. Table 1 shows the antibacterial activity,
min Technologies Ltd, Turku, Finland); group 4, 2% chlorhexidine gel (Ref measured at 2 depths (200 and 400 m) and at 3 time intervals (1, 3,
no.24800; Sigma-Aldrich); and group 5, 2% metronidazole gel (Ref and 5 days). The inhibition of growth in all the groups was statistically
no.M3761-5G; Sigma-Aldrich). significant in comparison to the control group (saline). Group 4 (2%
According to Fava and Saunders (13), the antibacterial activity of chlorhexidine gel) was the most effective against E. faecalis to the depth
intracanal medicaments is enhanced by the vehicle used. Hence, appro- of 400 m on all days of incubation. Intergroup comparison showed
priate vehicles were chosen for the individual medicament as described that the inhibition in group 5 (2% metronidazole gel) was statistically
below. significant compared with groups 2 and 3 (calcium hydroxide and
Bioactive glass S53P4 with a particle size of less than 45 m bioactive glass, respectively). Intergroup comparison of inhibition be-
enables the obtainment of a paste-like consistency when mixed with tween groups 2 and 3 showed no statistical difference.
saline. The bioactive glass S53P4 powder used in this study is composed The inhibition of growth of E. faecalis at 200 and 400 m was
of 53% SiO2 (wt/wt), 23% Na2O, 20% CaO, and 4%P2O5 (12). Group1 uniform with no statistical difference.
received no medicament. In groups 2 and 3, the powder was mixed with
The inhibition of growth of E. faecalis at the end of day 1, day 3,
sterile saline in the ratio of 1.5:1 (wt/vol) to obtain a paste-like consis-
and day 5 was varied with different medicaments. Calcium hydroxide
tency. This was carried into the canal by using a plastic instrument and
condensed with a hand plugger. Polyethylene glycol 400 (HiMedia) was showed an increased antibacterial activity on day 3, which declined by
used as a vehicle in group 4. In a pilot study, metronidazole was found day 5. Bioactive glass showed a gradual increase in antibacterial activity
to dissolve better in glycerin than in polyethylene glycol. Hence, purified at the end of day 5. Two percent metronidazole gel showed a decrease
glycerin (HiMedia) was used as the vehicle in group 5. Methyl cellulose in antibacterial activity at the end of day 5. However, the differences in
was used as a thickening agent in both groups. All blocks after medica- antibacterial potential of the medicaments during different time inter-
tion were sealed above and below with paraffin wax and incubated in an vals were not statistically significant. Two percent chlorhexidine gel
anaerobic environment at 37°C. showed complete inhibition of E. faecalis on all time intervals.
Antibacterial assessment was performed at the end of 1, 3, and 5 To summarize the results, the overall percentage inhibition at 2
days, with 15 blocks from each group for every time interval. The blocks depths and different time intervals was 100% with 2% chlorhexidine gel,
were washed with 5 mL of sterile saline combined with ultrasonics to 86.5% with 2% metronidazole gel, followed by 62.8% with bioactive
remove the medicament. Dentin debris was harvested at 2 depths (200 glass and 58.5% with calcium hydroxide (Figure 1).
1474 Krithikadatta et al. JOE — Volume 33, Number 12, December 2007

3. Basic Research—Technology
when compared with gram-positive bacteria (21). On the contrary, it
was found in the present study that E. faecalis, a gram-positive bacte-
rium, was found to be resistant to the effects of bioactive glass. This
strange phenomenon calls for further exploration. Antibacterial activity
of bioactive glass increases with time (12), and similar observations
were seen in this study.
Metronidazole is known to be more effective against obligate an-
aerobic bacteria than on aerobic and facultative anaerobic bacteria
(22). But the present study showed it to have antibacterial effect even on
E. faecalis, a facultative anaerobic bacterium. Anaerobic bacteria con-
tain electron transport components such as ferrodoxin that have a suf-
ficiently negative redox potential to donate electrons to metronidazole.
This single electron transfer forms a highly reactive nitro radical anion
that kills susceptible organisms by radical-mediated mechanism that
targets DNA and other biomolecules (22). A complete elimination of
E. faecalis might be achieved by increasing the concentration of met-
Figure 1. Percentage reduction in bacterial growth at 200 and 400 m for ronidazole (23).
different medicaments. In the present study, 2% chlorhexidine gel provided 100% inhibi-
tion of E. faecalis at the depths of 200 m as well as 400 m from day
1 to day 5. The plausible reason could be the bactericidal dosage of 2%
Discussion and increased diffusion of the medicament into the dentinal tubules.
This article reports on the disinfection potential of 4 intracanal Basrani et al (11) observed that 2% chlorhexidine gel produced a better
medicaments. The model proposed by Haapasalo and Orstavik (10) has antimicrobial action when compared with 0.2% chlorhexidine gel or
been modified for this study. Human permanent teeth were used instead calcium hydroxide mixed with 0.2% chlorhexidine. In another study, a
of the bovine teeth as suggested by Basrani et al (11, 14). The canal 10-minute irrigation with 2% chlorhexidine before obturation of the
lumens of the bovine blocks were 3 times larger than those of human root canal resulted in complete elimination of E. faecalis (24). Two
blocks, thus influencing the antimicrobial activity of certain medica- percent chlorhexidine gel has been reported to have sustained antibac-
ments (14). In addition, studies with human dentin blocks would def- terial action beginning from the first day of medication (25). Similar
initely be more suitable to simulate the clinical scenario. results were found in this study. Basrani et al (26) found lower contact
The results of the present study revealed significant information on angle in preparations containing chlorhexidine, enabling better diffu-
the newer intracanal medicaments against E. faecalis. On average, com- sion into the tubules.
plete inhibition of E. faecalis at both depths (200 and 400 m) was Portenier et al (27) and Haapasalo et al (16) have shown in
observed with 2% chlorhexidine gel, followed by 86.5 % reduction with independent studies that dentin matrix and collagen type I have inhibi-
2% metronidazole gel. However, bioactive glass and calcium hydroxide tory effect on chlorhexidine. But both studies tested a concentration of
had an overall reduction of only 62.8% and 58.5%, respectively. 0.2% chlorhexidine, which is much lower than the concentration used
There was no increase in antimicrobial effect of calcium hydroxide in the present study. The inhibitory effect of dentin on chlorhexidine can
when left for longer periods in the root canal because the hydroxyl ions be overcome by increasing the concentration (16). This appears to be
do not pass through patent dentinal tubules to alkalize the medium true, because the present study used a higher concentration and
surrounding the teeth (15). Calcium hydroxide has a pH of 12, but achieved complete inhibition. However, caution must be exercised
because of the buffering effect of dentin, it is unlikely that this high pH when drawing conclusions to in vivo situations. There is plausibility of
would be maintained within the dentinal tubules, enabling E. faecalis to negative interactions between endodontic disinfecting agents and the
survive and replicate (16, 17). The results of our study also show no various compounds present in the root canal environment. This might
increase in the inhibition over time and thus corroborate these findings. have a vital role in deciding the clinical effectiveness of antibacterial
Evans et al (18) demonstrated that the proton pump activity of E. fae- agents (28, 29).
calis offers resistance to high pH of calcium hydroxide. The ability of E. Under the limitations of the present study it can be concluded that
faecalis to penetrate deep into the tubules is attributed to Ace, a bacte- 2% chlorhexidine was most effective against E. faecalis.
rial adhesin (19).
In this study, the results of antimicrobial activity of bioactive glass
against E. faecalis were moderate. However, its mechanism of action is Acknowledgments
not completely understood. An increase in pH of the aqueous bioactive The authors thank Dr V. Mohan, Professor and Chairman, Ma-
glass has been the principle behind the observed effects (20, 21), and dras Diabetes Research Foundation, for financial help for the study;
this increase was found to be brought about by the surface deterioration Dr. Deepa Raj and Ms. M. Deepa, Research scientist, Madras Diabetes
of glass, which releases sodium oxide (12). The smaller the particle size Research Foundation, for assistance with the statistics; and Dr Mat-
of the glass, the greater is the surface deterioration (21). thais Zehnder, Professor, Division of Endodontology, Department of
According to Zehnder et al (12), a mixture of bioactive glass and Preventive Dentistry, Periodontology and Cariology, University of
dentin increased the antibacterial efficacy of bioactive glass. They hy- Zurich Center for Dental Medicine, for providing the bioactive glass.
pothesized that dentin might serve as a source of Ca and P ions, which
allows bioactive glass to mineralize the bacterial cell wall. However,
even though the dentin block model was used in the present study, the References
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1476 Krithikadatta et al. JOE — Volume 33, Number 12, December 2007