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Dentinal tubule disinfection with 2% chlorhexidine

gel, propolis, morinda citrifolia juice, 2% povidone

iodine, and calcium hydroxide

D. Kandaswamy1, N. Venkateshbabu1, D. Gogulnath1 & A. J. Kindo2

Departments of 1Conservative Dentistry and Endodontics; and 2Microbiology, Sri Ramachandra Dental College, Porur, Chennai,

India

Abstract

Kandaswamy D, Venkateshbabu N, Gogulnath D, Kindo

AJ. Dentinal tubule disinfection with 2% chlorhexidine gel,

propolis, morinda citrifolia juice, 2% povidone iodine, and

calcium hydroxide. International Endodontic Journal, 43, 419

423, 2010.

Aim To investigate the antimicrobial activity of 2%

chlorhexidine gel, propolis, Morinda citrifolia juice

(MCJ), 2% povidone Iodine (POV-I), and calcium

hydroxide on Enterococcus faecalis-infected root canal

dentine at two different depths (200 lm and 400 lm)

and three time intervals (day 1, 3 & 5).

Methodology One hundred and eighty extracted

human teeth were infected for 21 days with E. fae-

calis. Samples were divided into six groups. Group I

(Saline) (Negative control), Group II (Propolis), Group

III (MCJ), Group IV (2% Povidone Iodine), Group V

(2% Chlorhexidine Gel), Group VI (Calcium hydrox-

ide). At the end of 1, 3, and 5 days, the remaining

vital bacterial population was assessed. Dentine

shavings were collected at two depths (200 lm and

400 lm), and total numbers of colony forming units

were determined. The values were analysed statisti-

cally with one-way analysis of variance followed by

Tukey multiple comparison test. The paired t-test was

used to check for differences in growth at different

time intervals within groups and for differences at the

two depths (P < 0.01)Results The number of colony-forming units was

statistically significant in all groups compared to the

control group (Saline). Group V (chlorhexidine gluco-

nate) (100%) produced better antimicrobial efficacy

followed by 2% POV-I (87%), propolis (71%), MCJ

(69%), and calcium hydroxide (55%). There was no

significant difference between propolis and MCJ and no

significant difference between data at 200 lm and

400 lm.

Conclusion Propolis and MCJ were effective against

E. faecalis in dentine of extracted teeth.

Keywords: calcium hydroxide, chlorhexidine gel,E. faecalis, morinda citrifolia juice, povidone iodine,

propolis.

Received 30 March 2009; accepted 5 January 2010

Introduction

Achieving predictable long-term success of root canal

treatment requires effective debridement and disinfec-

tion of the root canal system (Lee et al. 2008).

Chemomechanical instrumentation removes the major-

ity of infecting bacteria, together with necrotic pulp

debris (Manzur et al. 2007). However, this is not

always achieved completely because of anatomical

complexity and the limitation in accessing the canal

system by instruments and irrigants. Persistent end-

odontic infection might be attributed to the retention of

microorganisms in dentinal tubules (Safavi et al.

1990). Intracanal medicaments can help in reducing

the remaining bacteria remaining even after chemo-

mechanical instrumentation and can provide an

environment conductive to periapical tissue repair

(Lee et al. 2008).

Correspondence: Dr Nagendrababu Venkateshbabu, M.D.S,

Senior Lecturer, Department of Conservative Dentistry and

Endodontics, Sri Ramachandra Dental College, Porur, Chennai

600116, Tamilnadu, India (Tel.: +9144 24768403; e-mail:

[email protected]).

doi:10.1111/j.1365-2591.2010.01696.x

2010 International Endodontic Journal International Endodontic Journal, 43, 419423, 2010 419


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Enterococcus faecalis is more likely to be found in

cases with post-treatment infection (Siren et al. 1997).

Starvation conditions appear to increase the resistance

of E. faecalis substantially (Portenier et al. 2005). It is

probable that the physiological state of the cells

particularly in retreatment cases approximates to the

starvation phase (Stuart et al. 2005).Calcium hydroxide has been advocated as an intra-

canal medicament because of its bactericidal properties

(Froeman & Barnes 1990). Its high pH (of about 12.5)

has a destructive effect on bacterial cell membranes and

protein structure (Spangberg 1994). However, calcium

hydroxide is not effective in eliminating bacteria from

dentinal tubules. Gomes et al. (2003) reported that

E. faecalis present in the dentinal tubules was resistant

to calcium hydroxide over 10 days.

Two per cent chlorhexidine gluconate (CHX) has

been used as an irrigant and intracanal medicament in

endodontics. CHX is a bis-bis-guanide (Carlo et al.

2006) that acts by adsorbing onto the cell wall of

microorganism resulting in leakage of intracellular

components. CHX has a broad-spectrum antimicrobial

activity (Delany et al. 1982), targeting both gram-

positive and gram-negative microbes and biocompati-

ble (Yesilsoy et al. 1995).

Propolis, also known as bee glue and bee propolis, is

a brownish resinous substance collected by bees,

mainly from plants. It is used to reinforce the combs

and to keep the hive environment aseptic (Uzel et al.

2005). It is a potent antimicrobial, antioxidant, and

anti-inflammatory agent. The main chemical elements

present in propolis are flavonoids, phenolics, andvarious aromatic compounds. Flavonoids are well-

known plant compounds that have antioxidant, anti-

bacterial, antifungal, antiviral, and anti-inflammatory

properties (Gopikrishna et al. 2008).

Morinda citrifolia (Rubiaceae) (MCJ), commercially

known as noni, is indigenous to tropical countries and

is considered as an important folk medicine. Its juice has

a broad range of therapeutic effects including antibac-

terial, antifungal, antiviral, antitumor, antihelminthic,

analgesic, hypotensive, anti-inflammatory, and immune

enhancing effects (Murray et al. 2008). Murray et al.

(2008) compared the effectiveness of MCJ with sodium

hypochlorite and chlorhexidine gluconate to remove the

smear layer from the walls of instrumented root canals.

They concluded that 6% MCJ could be used as anendodontic irrigant in combination with EDTA followed

by a final flush with 6% MCJ (Murray et al. 2008).

The American Heart Association (AHA) recommend

the use of topical antimicrobials such as povidone-

iodine (POV-I) as an adjunct to systemic antibiotic

cover to enhance the efficiency of current prophylactic

measures (Dajani et al. 1997). POV-I, which is used

widely as a topical antiseptic agent, is an iodophore in

which iodine is linked to povidone (polyvinylpyrroli-

done), a dextran-like molecule (Fleischer & Reimer

1997). POV-I appears to be active against all microor-

ganisms, including gram-positive and gram-negative

bacteria, spores, mycobacteria, fungi, viruses, and

protozoa (Cherry et al. 2007).

This study was undertaken to evaluate the disinfec-

tion of dentinal tubules when contaminated with

E. faecalis using propolis, MCJ, POV-I, 2% CHX gel

when compared to calcium hydroxide.

Materials and methods

Preparation of dentine specimens

The model proposed by Haapasalo & rstavik (1987)

was modified. One hundred and eighty single-rootedhuman mandibular premolar teeth freshly extracted for

orthodontic reasons from 90 individuals were selected.

A rotary diamond disc was used to decoronate the teeth

below the cementoenamel junction and the apical part

of the root to obtain 6 mm of the middle third of the

root. Cementum was removed from the root surface.

Gates Glidden drills no. 3 (Mani Inc, Tachigi-ken,

Japan) in a slow-speed handpiece was used to stan-

Table 1 Mean, standard deviation, median, and interquartile ranges for various intracanal medicaments at Log Day 1 at 200 &

400 lm

N

Mean Standard deviation Median Interquartile ranges

200 lm 400 lm 200 lm 400 lm 200 lm 400 lm 200 lm 400 lm

Propolis 10 3.3 3.2 0.48 0.42 3 3 1.0 0.25

Morinda 10 3.2 3.4 0.20 0.51 3 3 1.0 1.0

2% POV-I 10 3.1 3.2 0.17 0.42 3 3 0.25 0.25

CHX 10 0 0 0.00 0.00 0 0 0 0

Ca(OH)2 10 3.3 3.3 0.15 0.48 3 3 1.0 1.0

POV-I, povidone Iodine; CHX, chlorhexidine gluconate.

Dentinal tubule disinfection Kandaswamy et al.

International Endodontic Journal, 43, 419423, 2010 2010 International Endodontic Journal420


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dardize the internal diameter of the root canals. The

specimens were placed in an ultrasonic bath of 17%

ethylenediaminetetraacetic acid for 5 min followed by

3% NaOCl for 5 min to remove organic and inorganic

debris. The traces of chemicals used were removed by

immersing the dentine specimens in an ultrasonic bath

containing distiled water for 5 min. All the specimens

were sterilized in an autoclave for two cycles. The first

cycle was at 121 C and the second was with the

specimens immersed in 1 mL of tryptone soya (TS)broth in individual microcentrifuge tubes.

Contamination of the specimens

The test organism used for this study was E. faecalis,

which is a gram-positive facultative anaerobic bacte-

rium that is common in root filled teeth with post-

treatment infection. E. faecalis (ATCC 29212) was

grown in tryptone soya agar for 24 h. The culture

was suspended in 5 mL of TS broth and incubated for

4 h at 37 C and its turbidity adjusted to 0.5

McFarland standard. Each dentine block was placed

in pre-sterilized microcentrifuge tubes containing 1 mL

of the TS broth. Fifty microlitres of the inoculum

containing the E. faecalis was transferred into each of

the microcentrifuge tubes. At the end of 24 h, the

dentine specimens were transferred into fresh broth

containing E. faecalis. All procedures were carried out

under laminar flow. Purity of the culture was checked

by subculturing 5 lL of the broth from the incubated

dentine specimens in TS broth on tryptone soya agar

plates. Contamination of the dentine specimens was

carried out for a period of 21 days.

Antimicrobial assessment

At the end of 21 days, the specimens were irrigated

with 5 mL of sterile saline to remove the incubation

broth. They were assigned into seven groups (n = 30dentine blocks). Group 1, saline (negative control);

Group 2, propolis; Group 3, MCJ; Group 4, 2% POV-I;

Group 5, 2% CHX gel; Group 6, calcium hydroxide.

Calcium hydroxide (Sigma-Aldrich, Mumbai, India)

was mixed with sterile saline in a ratio of 1.5 : 1(wt/

vol) to obtain a paste-like consistency (Krithikadatta

et al. 2007). Methyl cellulose was used as a thickening

agent for Groups 2, 3, and 4.

The medicaments were placed inside the canals and

sealed at both ends with paraffin wax. They were

incubated in an anaerobic environment for 37 C. At

the end of 1, 3, and 5 days an assessment of microbial

cells was carried out with 10 specimens at each time

interval. Harvesting of dentine was carried out at two

depths (200 and 400 lm) with Gates Glidden drills no

4 and 5, respectively. The collected dentine shavings

were transferred into 1 mL of sterile TS broth and

incubated in an anaerobic environment at 37 C for

24 h. After 24 h, the contents of each tube was serially

Table 3 Mean, standard deviation, median, and interquartile range for various intracanal medicaments at Log Day 5 at 200 &

400 lm

N



Propolis 10 2.4 2.1 0.60 0.56 2.5 2 1.0 0.25

Morinda 10 2.3 2.6 0.48 0.51 2 3 1.0 1.0

2% POV-I 10 1.1 1.6 0.31 0.51 1 2 0 1.0

CHX 10 0 0 0.00 0.00 0 0 0 0Ca(OH)2 10 2.7 2.5 0.48 0.52 3 2.5 1.0 1.0


Table 2 Mean, standard deviation, median, and interquartile range for various intracanal medicaments at Log Day 3 at 200 &

400 lm

N



Propolis 10 2.7 2.7 0.67 0.67 3 3 1.0 1.0

Morinda 10 3 2.8 0.66 0.63 3 3 0.5 1.0

2% POV-I 10 1.4 2.2 0.51 0.42 1 2 1.0 0.25

CHX 10 0 0 0.00 0.00 0 0 0 0

Ca(OH)2 10 3.3 3.3 0.48 0.48 3 3 1.0 1.0


Kandaswamy et al. Dentinal tubule disinfection



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diluted, 100 lL of the broth in 100 lL of sterile saline

five times. Fifty microlitres of the dilution was then

plated on TS agar plates and incubated for 24 h.

Colonies were counted and readings were tabulated.

Statistical analysis

The data were statistically analysed with one-way

analysis of variance followed by Tukey multiple com-

parison means to check the difference in bacterial

inhibition between groups (P < 0.01). The paired t-test

was used to check for differences in growth at different

time intervals within groups and for differences at the

two depths (P < 0.01).

Results

The number of colony-forming units in all the

experimental groups was significantly lower in

comparison with the control group (Saline). Group V

(CHX) (100%) demonstrated better antimicrobial effi-

cacy followed by 2% POV-I (87.07%), propolis (71%),

MCJ (69%), and calcium hydroxide (55%). No statis-

tical difference was seen between propolis and MCJ.

There was no difference in the data between 200 lm

and 400 lm (Tables 1,2,3).

Discussion

The use of a biocompatible intracanal medicament

possessing antimicrobial properties between appoint-

ments may reduce or eliminate bacteria in the rootcanal system and significantly increase the success of

root canal treatment (Bystrom et al. 1985). The in vitro

model developed by Haapasalo & rstavik (1987) has

been used to assess the efficacy of endodontic medica-

ments in the disinfection of dentinal tubules. Lynne

et al. (2003) modified this model to include quantitative

analysis of bacteria in the dentine tubules to define a

percentage of reduction in CFU in infected dentine

before and after the application of intracanal medica-

ments. The model has clear limitations because it does

not reflect the situation in apical dentine, which is

mostly sclerotic (Paque et al. 2006). E. faecalis was

chosen as a test organism because it is a facultative

organism that is non-fastidious, easy-to-grow, and

efficiently and rapidly colonizes tubules (rstavik &

Haapasalo 1990). It has been used extensively in

endodontic research because it has been found to be

present in 63% of teeth with post-treatment disease

(Hancock et al. 2001).

In the present study, 2% CHX gel provided 100%

inhibition ofE. faecalis at depths of 200 lm and 400 lm

from day 1 to day 5. The possible reason could be the

bactericidal dosage of 2% and increased diffusion of the

medicament into the dentinal tubules (Krithikadatta

et al. 2007). Basrani et al. (2003) observed that 2% CHX

gel was a better antimicrobial when compared to 0.2%CHX gel or calcium hydroxide mixed with 0.2% chlorh-

exidine. The result of the present study was similar to

that of Krithikadatta et al. (2007), Gomes et al. (2003),

and Siqueria & Uzeda (1997).

POV-I produced 68% and 72% inhibition of E. fae-

calis at depths of 200 lm and 400 lm from day 1 to

day 5. The possible reason might be attributed to the

povidone molecule, by virtue of its affinity for cell

membranes, delivers diatomic free iodine directly to the

bacterial cell surface where it exerts its antibacterial

effects (Schreier et al. 1997). POV-I appears to be active

against all microorganisms, including gram-positive

and gram-negative bacteria, spores, mycobacteria,

fungi, viruses, and protozoa (Cherry et al. 2007).

Propolis produced 66% and 70% inhibition of E. fae-

calis at depths of 200 lm and 400 lm from day 1 to day

5. The possible reason for the antimicrobial action of

propolis might be attributed to its flavanoid content

(Grange & Davey 1990). Grange & Davey (1990)

assessed the bacteriocidal ability of propolis against a

wide range of gram-positive and gram-negative organ-

isms. They reported complete inhibition of cultures of

Staphylococcus aureus, including MRSA strains. The

results of the present study was similar to the study of

Awawdeh et al. (2008) who compared the antimicro-bial activity of propolis with calcium hydroxide as

intracanal medicament against E. faecalis and that

propolis was effective in eliminating the microorganism.

MCJ showed 41% inhibition of E. faecalis at depths of

200 lm and 400 lm from day 1 to day 5. MCJ

contains the antibacterial components L-asperuloside

and alizarin which might be the reason for the

antibacterial property (Murray et al. 2008).

Conclusion

Two per cent chlorhexidine demonstrated significant

inhibition against E. faecalis followed by POV-I, propo-

lis, MCJ, and Ca(OH)2

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