doi:10.1111/j.1365-2591.2010.01696.

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. faecalis. 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 hydroxide). 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 statistically 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 signicant in all groups compared to the control group (Saline). Group V (chlorhexidine gluconate) (100%) produced better antimicrobial efcacy followed by 2% POV-I (87%), propolis (71%), MCJ (69%), and calcium hydroxide (55%). There was no signicant difference between propolis and MCJ and no signicant 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 disinfection of the root canal system (Lee et al. 2008). Chemomechanical instrumentation removes the major-

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: babu_endo@yahoo.com).

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 endodontic 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 chemomechanical instrumentation and can provide an environment conductive to periapical tissue repair (Lee et al. 2008).

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Enterococcus faecalis is more likely to be found in n et al. 1997). cases with post-treatment infection (Sire 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 intracanal 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 grampositive and gram-negative microbes and biocompatible (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-inammatory agent. The main chemical elements present in propolis are avonoids, phenolics, and various aromatic compounds. Flavonoids are wellknown plant compounds that have antioxidant, antibacterial, antifungal, antiviral, and anti-inammatory 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-inammatory, 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 an endodontic irrigant in combination with EDTA followed by a nal ush with 6% MCJ (Murray et al. 2008). The American Heart Association (AHA) recommend the use of topical antimicrobials such as povidoneiodine (POV-I) as an adjunct to systemic antibiotic cover to enhance the efciency 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 (polyvinylpyrrolidone), a dextran-like molecule (Fleischer & Reimer 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). This study was undertaken to evaluate the disinfection 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 modied. One hundred and eighty single-rooted human 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
Mean N Propolis Morinda 2% POV-I CHX Ca(OH)2 10 10 10 10 10 200 lm 3.3 3.2 3.1 0 3.3 400 lm 3.2 3.4 3.2 0 3.3 Standard deviation 200 lm 0.48 0.20 0.17 0.00 0.15 400 lm 0.42 0.51 0.42 0.00 0.48 Median 200 lm 3 3 3 0 3 400 lm 3 3 3 0 3 Interquartile ranges 200 lm 1.0 1.0 0.25 0 1.0 400 lm 0.25 1.0 0.25 0 1.0

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

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

400 lm
Mean N Propolis Morinda 2% POV-I CHX Ca(OH)2 10 10 10 10 10 200 lm 2.7 3 1.4 0 3.3 400 lm 2.7 2.8 2.2 0 3.3 Standard deviation 200 lm 0.67 0.66 0.51 0.00 0.48 400 lm 0.67 0.63 0.42 0.00 0.48 Median 200 lm 3 3 1 0 3 400 lm 3 3 2 0 3 Interquartile ranges 200 lm 1.0 0.5 1.0 0 1.0 400 lm 1.0 1.0 0.25 0 1.0

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

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

400 lm
Mean N Propolis Morinda 2% POV-I CHX Ca(OH)2 10 10 10 10 10 200 lm 2.4 2.3 1.1 0 2.7 400 lm 2.1 2.6 1.6 0 2.5 Standard deviation 200 lm 0.60 0.48 0.31 0.00 0.48 400 lm 0.56 0.51 0.51 0.00 0.52 Median 200 lm 2.5 2 1 0 3 400 lm 2 3 2 0 2.5 Interquartile ranges 200 lm 1.0 1.0 0 0 1.0 400 lm 0.25 1.0 1.0 0 1.0

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

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 rst 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.

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 = 30 dentine 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 parafn 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

Contamination of the specimens

The test organism used for this study was E. faecalis, which is a gram-positive facultative anaerobic bacterium that is common in root lled teeth with posttreatment 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 ow. Purity of the culture was checked

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diluted, 100 lL of the broth in 100 lL of sterile saline ve 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 comparison 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 signicantly lower in comparison with the control group (Saline). Group V (CHX) (100%) demonstrated better antimicrobial efcacy followed by 2% POV-I (87.07%), propolis (71%), MCJ (69%), and calcium hydroxide (55%). No statistical 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 appointments may reduce or eliminate bacteria in the root canal system and signicantly increase the success of root canal treatment (Bystro m et al. 1985). The in vitro model developed by Haapasalo & rstavik (1987) has been used to assess the efcacy of endodontic medicaments in the disinfection of dentinal tubules. Lynne et al. (2003) modied this model to include quantitative analysis of bacteria in the dentine tubules to dene a percentage of reduction in CFU in infected dentine before and after the application of intracanal medicaments. The model has clear limitations because it does not reect 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 efciently 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 of E. 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% chlorhexidine. 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. faecalis 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 afnity 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. faecalis 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 avanoid content (Grange & Davey 1990). Grange & Davey (1990) assessed the bacteriocidal ability of propolis against a wide range of gram-positive and gram-negative organisms. 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 antimicrobial 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 signicant inhibition against E. faecalis followed by POV-I, propolis, MCJ, and Ca(OH)2

References
Awawdeh L, AL-Beitawi M, Hammad M (2008) Effectiveness of propolis and calcium hydroxide as a short-term intracanal

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medicament against Enterococcus faecalis: a laboratory study. Australian Endodontic Journal 35, 5258. Basrani B, Tjaderhane L, Santos M et al. (2003) Efcacy of chlorhexidine- and calcium hydroxide containing medicaments against Enterococcus faecalis in vitro. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 96, 61824. Bystro m A, Claesson R, Sundqvist G (1985) The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol, and calcium hydroxide in the treatment of infected root canals. Endodontics and Dental Traumatology 1, 170 5. Carlo CG, Bergammante V, Calabrese V, Biserni S, Ronchi C, Fini A (2006) A design and evaluation on vitro of controlled release mucoadhesive tablets containing chlorhexidine. Drug Development and Industrial Pharmacy 32, 5361. Cherry M, Daly CG, Mitchell D (2007) Effect of rinsing with povidone iodine on bacteraemia due to scaling: a randomizedcontrolled trial. Journal of Clinical Periodontology 34, 14855. Dajani AS et al. (1997) Prevention of bacterial endocarditis: recommendations by the American Heart Association. Journal of the American Dental Association 128, 114251. Delany GM, Patterson SS, Miller CH, Newton CW (1982) The effect of chlorhexidine gluconate irrigation on the root canal ora of freshly extracted necrotic teeth. Oral Surgery, Oral Medicine, and Oral Pathology 53, 51823. Fleischer W, Reimer K (1997) Povidone-iodine in antisepsis: state of the art. Dermatology 195, 39. Froeman PC, Barnes IE (1990) A review of calcium hydroxide. International Endodontic Journal 23, 28397. Gomes BPFA, Souza SFC, Ferraz CCR, Teixeira AA, Valdrighi L, Filho FJS (2003) Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentin in vitro. International Endodontic Journal 36, 267 75. Gopikrishna V, Baweja PS, Venkateshbabu N, Kandaswamy D (2008) Comparison of coconut water, propolis, HBSS, and milk on PDL cell survival. Journal of Endodontics 34, 5879. Grange JM, Davey RW (1990) Antibacterial properties of Propolis (Bee Glue). Journal of the Royal Society of Medicine 83, 15960. Haapasalo M, rstavik D (1987) In vitro infection and disinfection of dentinal tubules. Journal of Dental Research 66, 13759. Hancock HH, Sigurdsson A, Trope M, Moiseiwitsch J (2001) Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 91, 57986. Krithikadatta J, Indria R, Dorothykalyani AL (2007) Disinfection of dentinal tubules with 2% Chlorhexidine, 2% Metronidazole, Bioactive Glass when compared with Calcium Hydroxide as intracanal medicaments. Journal of Endodontics 33, 14736. Lee Y, Han SH, Hong SH, Lee JK, Ji H, Kum KY (2008) Antimicrobial efcacy of a polymeric chlorhexidine release

device using in vitro model of Enterococcus faecalis dentinal tubule infection. Journal of Endodontics 34, 8557. Lynne RE, Lieweher FR, West LA, Patton WR, Buxton TB, McPherson JC (2003) In vitro antimicrobial activity of various medication preparations on E. faecalis in root canal dentin. Journal of Endodontics 29, 18790. lez AM, Pozos A, Herzog DS, Frierman S Manzur A, Gonza (2007) Bacterial quantication in teeth with apical periodontitis related to instrumentation and different intracanal medications: a randomized clinical trail. Journal of Endodontics 33, 1148. Murray PE, Farber RM, Namerow KN, Kuttler S, Godoy FG (2008) Evaluation of Morinda citrifolia as an endodontic irrigant. Journal of Endodontics 34, 6670. rstavik D, Haapasalo M (1990) Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endodontics and Dental Traumatology 6, 1429. F, Luder HU, Senner B, Zehnder M (2006) Tubular Paque sclerosis rather than the smear layer impedes dye penetration into the dentine of endodontically instrumented root canals. International Endodontic Journal 39, 1825. Portenier I, Waltimo T, rstavik D, Haapasalo M (2005) The susceptibility of starved stationary phase, and growing cells of Enterococcus faecalis to endodontic medicaments. Journal of Endodontics 31, 3805. ngberg SW, Langeland K (1990) Root canal Safavi KE, Spa dentinal tubule disinfection. Journal of Endodontics 16, 207 10. Schreier H, Erdos G, Reimer K (1997) Molecular effects of povidone-iodine on relevant micro-organisms: an electro microscopic and biochemical study. Dermatology 195, 111 6. Siqueria JF, Uzeda M (1997) Intracanal medicaments: evaluation of the antibacterial effects of Chlorhexidine, Metronidazole, and Calcium Hydroxide associated with three vehicles. Journal of Endodontics 23, 1679. n EK, Haapasalo MPP, Ranta K, Salmi P, Kerosuo ENJ Sire (1997) Microbiological ndings and clinical treatment procedures in endodontic cases selected for microbiological investigation. International Endodontic Journal 30, 905. Spangberg LSW (1994) Intracanal medication. In: Ingle JI, Bakland LK, eds. Endodontics, 4th edn. Baltimore: Williams & Wilkins, pp. 62740. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB (2005) Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. Journal of Endodontics 32, 938. nc , C , Salih B Uzel A, Sorkun K, O ag O ogulu D, Genc ay O (2005) Chemical compositions and antimicrobial activities of four different Anatolian propolis samples. Microbiological Research 160, 18995. Yesilsoy C, Whitaker E, Cleveland D, Phillips E, Trope M (1995) Antimicrobial and toxic effects of established and potential root canal irrigants. Journal of Endodontics 21, 5135.

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