An In-vivo Study Comparing Antimicrobial Activity of Chlorhexidine 0.2% to Sodium Hypochlorite 0.5% as Canal Irrigant.

Vahid A, et al Journal of Dentistry, Tehran University of Medical Sciences An In-vivo Study Comparing Antimicrobial Activity of Chlorhexidine 0.2% to Sodium Hypochlorite 0.5% as Canal Irrigant. Vahid A 1, Aligholi M 2, Namazi HR 3 1 Assistant Professor, Dept. of Endodontic, Faculty of Dentistry, Tehran University of Medical Sciences, Tehran, Iran 2 Assistant Professor, Dept. of Microbiology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3 Dentist M Statement of Problem: Bacterial agents are the primary cause of pulpoperiapical diseases. Therefore, an irrigating solution with proper antibacterial ability should be used to eliminate the bacterial agents during canal instrumentation. Purpose: The aim of present study was to compare the antibacterial effect of 0.2% chlorhexidine to 0.5% sodium hypochlorite as canal irrigating solutions. Materials and Methods: In this study 20 single canal teeth with necrotic pulps were selected by vitality tests and divided in two groups each one consists of ten. Each canal irrigated with 12 ml of 0.2% chlorhexidine in the first group and 0.5% sodium hypochlorite in the second one. Each canal was instrumented by 6 k- type files by step back procedure (two minutes for each file). After canal instrumentation, sterile cotton pellet was placed on the pulp chamber and the canals were filled temporarily by cavit. Samples were selected by two strile paper points both before the canal instrumentation (after accessing the canal) and 48 hours later. Samples were cultured on Mitis salivarius agar and anaerobic blood agar and then, the colony counts were reported in terms of CFU/ml. Results: Elimination percent mean of streptococcus mutans and anaerobics with 0.2% chlorhexidine were 99.9 and 99.02 respectively and for 0.5% hypochlorite were 99.7, and 92.7 respectively. Conclusion: There was no significant difference between these two irrigating solutions by non parameteric Mann-Whitney test. Key words: Chlorhexidine- Sodium Hypochlorite- Canal Irrigant. Journal of Dentistry, Tehran University of Medical Sciences, Tehran, Iran (2004; Vol: 1, No. 1) echanical debridement and irrigation of the canal play a fundamental role in achieving endodontic success. In spite of all attempts, attaining complete sterility of the canal system is still extremely difficult. Microorganisms can penetrate deep into accessory canals, apical ramifications, isthmi, tubules and smear layers. Hence, finding ways to overcome this issue is essential. The ideal irrigant should be strongly antibacterial but not toxic to the periapical tissue (selective toxicity). (1) It should be tissue solving and lubricate the file during instrumentation. (1) Also, it has been shown that surface tension of an irrigating solution and its wettability on solid dentin are important factors in penetration into dentin and lateral canals. (2) Sodium hypochlorite solution has been widely 2004; Vol. 1, No. 1 43

Journal of Dentistry, Tehran University of Medical Sciences Vahid A, et al recommended as an irrigant to aid in chemomechanical debridement of the root canal, because it has many of the above mentioned advantages, except its toxic effect for periapical tissue and wettability. (1) Also, its smell and discoloring action are other disadvantages. Spangberg et al (3) found that all medicaments are toxic compared to their antibacterial effect. They said on the basis of a balance between the cytotoxicity, the antibcaterial effect and the specific needs, a recommendation for an adequate irrigating solution is being made. They believed that sodium hypochlorite at 5% concentrations is more stronger than it is needed to kill bacteria. (3) Huggers et al (4) in their investigation on antimicrobial activity and tissue toxicity of different concentrations (0.25%, 0.025% and 0.0125%) of sodium hypochlorite, found that even 0.25% concentration could be tissue toxic. Chlorhexidine gluconate has been recognized as an effective antimicrobial agent in periodontics, and its use as a potential endodontic irrigating solution has been demonstrated in the last decade. (5) It seems chlorhexidine has most of the mentioned criteria. It has a long term effect and the reservoir of this agent forms on the teeth and on the oral mucosa after mouth rinse. Antimicrobial activity is released from these locations as the concentration of chlorhexidine decreases in the environment. (6) The absorption of chlorhexidine by hydroxyapatite, teeth and protein demonstrated previously by a chemical method. (6) This characteristic can also be useful inside of the canal. Many in vitro studies have been performed comparing these two irrigating solutions while few clinical trials have been done. In vivo studies are more natural and closer to the actual specifics of the human. Therefore, the aim of this study was to compare the antimicrobial effect of sodium hypochlorite 0.5% and chlorhexidine 0.2% during root canal therapy of twenty infected canals in patients. Materials and Methods Twenty single rooted teeth with necrotic pulps were chosen. Pulpal necrosis was determined either by radiographic presence of apical rarefaction or by the lack of response to pulp vitality tests. Ages of patients were between 15 to 50 years old. Apices were obviously formed and pulp chambers and canals were free of pulp stones, calcifications, or sharp dilacerations. Teeth were randomly divided into two groups. Each of the groups received one of the irrigating solutions, sodium hypochlorite (SH) 0.5% or chlorhexidine gluconate (CHG) 0.2%. SH had the concentration that could have antimicrobial activity with least hazard to periapical tissue. (1) Irrigating solutions used in this study were chlorhexidine gluconate %0.2 from Shahredaru and sodium hypochlorite 5.25% from Taj Co which diluted to 0.5%. Study had two phases which will be described: Clinical phase: After selecting teeth (taking primary X-ray and making diagnosis by vitality tests) informed consent was taken. The decay was removed by sharp excavator without opening the pulp chamber. Tooth isolated with rubber dam and access cavity prepared. Before entering the canal, pulp chamber was cleaned with sterile distilled water to clean debris resulted from access preparation. Pulp chamber was dried with sterile cotton pellets and two paper points were put into the canal. After one minute, paper points were withdrawn and dropped in screw top test tubes containing transfer media and was sent to microbiologic laboratory. Taking samples and preparing dilutions took less than 10 minutes. This was referred as first sample. Then, after length measurement, canals prepared by step back technique for cleaning and shaping. Between the procedures, canals were rinsed by 2 cc of any of irrigating solutions for each file. Finally, canals were irrigated by sterile distilled water, then, access cavity closed with ZOE. Two days later, after isolation, the area surrounding the tooth and 44 2004; Vol. 1, No. 1

Vahid A, et al Journal of Dentistry, Tehran University of Medical Sciences clamp was irrigated with SH, and temporary filling was removed. Two papers were put in the canal and then dropped into transfer media and were sent for culture. Sterility of the process and instruments during study was axiomatic. After irrigation, master point with X-ray was prepared and canals were filled. Temporary filling was placed back. Microbiologic phase: For microbiologic phase, all media autoclaved and distributed into plates. Thioglicholate (Difco) transferring medium was poured into vials and autoclaved. Vials containing paper points agitated on a vortex mixer until a homogeneous suspension of microorganism were obtained. (7,8) For ease of colony counting, 1 ml. of the homogenized solution transferred to five test tubes of ringer solution with 1/10 to 1/100000 dilutions. (9,10) In first sampling (before instrumentation), 0.1 ml from dilutions 1/10, 1/1000, 1/100000 was inoculated to plates of blood agar (Merk) for anaerobic organisms and three plates of Mitis salivarius agar (Difco) for sterptococcus mutans and spread with sterile bent glass rod. (9) Plates with gas pack, catalyzer and indicator were put in anaerobic jar (Oxoid) and incubated under 35 degree centigrade for 7 days. In second sampling, culture of undiluted sample added to previous dilutions and culture was performed as before. After 7 days colony-forming units (CFU) were counted. The results were analyzed by nonparametric Mann- Whitney test. Results Tables I-IV indicated the CFU results of the effect of CHG and SH as irrigating solutions on streptococcus mutans and anaerobe bacteria. A generalized decrease of flora of the canal from culture one to culture two was present. (For streptococcus mutans exact P=0.089 and for anaerobe bacteria exact P=0.315). But none of the teeth were totally free of cultivable microorganisms. According to table V, the results showed no significant difference between groups. As shown on tables I-IV, the elimination rates for streptococcus mutans and anaerobe bacteria in the presence of C.H.G. were 99.9% and 99.02%; and in presence of S.H. were 99.7% and 92.7% respectively. Discussion An important adjunct for chemomechanical preparation of the canal system is irrigation which aids in debridement and flushing debries from the canal. (11) At present study antimicrobial activity of sodium hypochlorite (SH) 0.5% and chlorhexidine gluconate (CHG) 0.2% compared as canal irrigants. SH as the most common proteolytic irrigating solution has most of the required characteristics, except for its surface tension, (2) its unpleasant odor and taste, corrosion of endodontic instruments, ineffectiveness on some microorganisms when used at low concentration, and SH accident. It doesn t differentiate between necrotic and vital tissue in contact with apical and periapical tissue, and is unable to be dressed within the canal. Studies indicate that SH dissolves tissue but experiments in clinical usage are less impressive. (11) Histologic and scanning electron microscope studies do not show that tissue dissolution or sterilization properties are consistently effective. (12) It seems that in clinical conditions irrigating solutions have limited surface contact and are neutralized in the canal. (11) In present research, SH 0.5% (Dakin solution) was used because of its lower toxicity for periapical tissue despite being effective in the canal. (4) CHG as a mouthwash is well known for few decades and it is available at 0.12% in the market at USA. The concentration chosen was 0.2% which is used in many studies (7,10) and now is available in Iran. CHG has most of the advantages needed except tissue solving which hasn t been proven yet. It is active against a wide range of microorganisms, such as Gram positive, Gram-negative bacteria, bacterial spores, lipophilic viruses and yeasts at ph of 2004; Vol. 1, No. 1 45

Journal of Dentistry, Tehran University of Medical Sciences Vahid A, et al 5.5-7. However, compared to NaOCl, it seems to have fewer advantages during instrumenttation. (11) Table I- Antimicrobial activity of 0.2% chlorhexidin solution on Streptococcus mutants with respect to No. of CFUs/ml. Case Before 48 hour after Percentage No. instrumentation instrumentation of reduction 1 22500 0 100 2 10000 10 99/9 3 0 0-4 700000 0 100 5 100 0 100 6 241900 0 100 7 100 0 100 8 0 0-9 800 0 100 10 100 0 100 Mean elimination percent =99..9% Table II- Antimicrobial activity of 0.2% chlorhexidine solution on anaerobe microorganisms (CFU/ml). Mean elimination percentage= 99.02% Case Before 48 hour after Percentage No. instrumentation instrumentation of reduction 1 67550 0 100 2 8250 265 96.8 3 103000000 40 ~100 4 1300000 0 100 5 16500 50 99.7 6 100000000 13700 99.99 7 30000000 720 ~100 8 50145000 26600 99.95 9 2475000 2860 99.88 10 490000 30100 93.9 Table III- Antibacteial activity of 0.5% sodium hypochlorite solution on Streptococcus mutans (CFU/ml). Mean elimination percentage=%99.7. Case No. Before 48 hour after instrumentation instrumentati on 1 0 0-2 0 0-3 14950 180 98/8 4 0 0-5 8400 0 100 6 700 0 100 7 1500 0 100 8 0 0-9 0 0-10 0 0 - Percentage of reduction Table IV- Antimicrobial activity of 0.5% sodium hypochlorite solution on anaerobe microorganisms (CFU/ml). Elimination mean percent =92.71% Case No. Before 48 hour after Reduction instrumentation instrumentation percentage 1 915000 110000 87.98 2 3195000 30000 99.07 3 580000 50400 99.32 4 2400000000 1000000 99.59 5 7220000 50 ~100 6 6580000 600 ~100 7 4455000 3100 99.94 8 670000 2600 99.7 9 11000 52 99.52 10 63250 2 ~100 Table V- Mann- Whitney test (A): Ranks Group No. Mean Sum of Rank Ranks DPANA Soduime 10 9.05 90.50 hypochlorite 0.5% Chlorhexdine 0.2% 10 11.95 119.50 Total 20 DPSM Soduime hyochlorite 10 8.25 82.50 0.5% Chlorhexdine 0.2% 10 12.75 127.50 Total 20 (B): Test statistics Statistical tests DPANA DPSM Mann- Whitney U 35.500 27.500 Wilcoxon W 90.500 82.500 Z -1.120-1.887 Asymp. Sig (2-tailed).263.059 Exact Sig. [2* (1- tailed Sig.].280 (a).089 (a) a Not corrected for ties. b Grouping Variable: Group Concerning more than one decade that CHG is recommended as canal irrigant, its antibacterial effect is not well evaluated clinically. At present study its antibacterial activity evaluated on anaerobic bacteria and Streptococcus mutans. Although anaerobics are the most microbial flora of necrotic canals (obligatory and facultative), since studies still aren t very clear about a specific species of anaerobic bacteria of mix canal flora, the anaerobic bacteria of the canal at present research was studied nonspecifically. 46 2004; Vol. 1, No. 1

Vahid A, et al Journal of Dentistry, Tehran University of Medical Sciences Streptococcus Mutans is a facultative anaerobe, which prefers anaerobic condition and is not always presents in the canal. Since its susceptibility to CHG is like other pathogens in endodontic (Porphyromonas Gingivalis, Prevotella Intermedia, and Fusobacterium Nucleatum), and also it has been used for other irrigants anti-microbial studies, (13) it was used at present study. This microorganism can enter dentinal tubules of open apices and continues its life. However in this study at first culture 12 out of 20 canals (60%), rendered positive results. The results of this research showed that antimicrobial activity of CHG 0.2% is more effective than SH 0.5% in-vivo, although the difference is not significant. Vahdaty et al (14) compared 2% and 0.2% of SH to 2% and 0.2% CHG, and showed that in equal concentration, CHG is more effective. Ringel et al (7) Similarly, used 2.5% SH with 0.2% CHG and found out that SH 2.0% is more effective. It seems that lower concentration of CHG is less effective compared to higher concentration of SH. By the way, comparing 0.2% concentration of CHG to 0.5% SH, the results were like the study of Yesiloy et al. (1) Regarding the growth of microorganisms in the canal system between appointments, lots of studies comparing different antimicrobial agents as dressing have been performed.( 15,16,17) According to Barthel and others, CHG gel and calcium hydroxide paste both had good results in preventing microbial colonization. (17) Apparently using CHG solution as dressing after cleaning the canal is easier and takes less time. According to Delany et al, when CHG is used as irrigant and interappointment dressing, its 0.2% solution can reduce 70% bacteria in multiradicular and 80% in uniradicular teeth. (10) The study of Leonardo et al (18) resulted in aerobe bacteria elimination equal to 100% and for anaerobes 77.75%. Mean elimination percent in the present study in chemomechanical preparation of the canal and by benefiting of 12 ml of CHG 0.2% were 99.9 for Streptoccocus mutans and 99.02 for anaerobes. These results for 0.5% SH were 99.7 and 92.7, respectively. Almost in none of the studies the elimination percent, particularly for anaerobes rendered 100%. Also dressing the canal with CHG, as mentioned previously, could have positive results in present study that for similarity in methods for none of the irrigating solutions was considered. Besides all, the issue of SH accident should be mentioned. It is a complication that some times can happen. In the first case reported by Becking, the accident happened through a perforation at CEJ of buccal wall and it seems as much more the concentration the accident would be worse. (19) Thus with everything discussed, it seems that in spite of SH effectiveness, CHG can be a more effective irrigating and antimicrobial agent with much fewer side effects for irrigating the canal. More studies for higher concentrations are recommended. Conclusions In clinical experiment antimicrobial effect of Chlorhexidine Gluconate 0.2% was similar to Sodium Hypochlorite 0.5% and no significant statistical difference existed. Thereby it can be concluded that CHG, an effective mouth wash for treating oral infections can be used as irrigation in endodontic therapy and can easily replace Sodium Hypochlorite that is caustic, malodorous, toxic for periapical tissue with low remaining antimicrobial activity and can cause corrosion on dental instruments. CHG 0.2% with high antimicrobial effect can be used for patients sensitive to SH also in open apices teeth without hazard to periapecal tissues. CHG can be dressed in the canal between appointments and it can keep its antimicrobial activity during this period of time and thus prohibits bacterial re-growth. Every 2004; Vol. 1, No. 1 47

Journal of Dentistry, Tehran University of Medical Sciences Vahid A, et al References: unwanted entering of irrigating solution (CHG) in the mouth can t cause gagging and bothering the patient. Since it seems, higher concentrations of CHG can be more effective comparable to higher concentrations of SH. 1-Yesiloy C, Whitaker E, Cleveland D, Phillips E, Trope M. Antimicrobial and toxic effects of established and potential root canal irrigants. J Endod 1995; 21(10): 513-15. 2-Tasman F, Cehrely Z, Ogan C, Etikan I. Surface tension of root canal irrigants. J Endod 2000; 26(10) 586-87. 3-Spangberg L, Engstrom B, Langland K. Biologic effects of dental material. Oral Surg 1973; 36: 856-71. 4-Huggers JP, Sazy AJ, Stenberg BD. Bacterial and wound-healing properties of sodium hypochlorite solution. J Burn Care Rehab 1991;12: 420-24. 5-Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endodon 1998; 24(7): 472-76. 6-Rolla G, Loe H, Schiott RC. Retention of chlorhexidine in the human oral cavity. Archs Oral Biol 1971; 16: 1109. 7-Ringel AM, Patterson SS, Newton CW, Miller CH, Mulhern JM. In- vivo evaluation of chlorehexidine gluconate solution and sodium hypochlorite solution as root canal irrigants. J Endod 1982; 8(5): 200-204. 8-Gold G, Olga Jordan HV, Van Houte J. A selective medium for Streptococcus mutans. Arch Oral Biology 1973; 18: 1357-64. 9-Bailley, Scott s. Diagnostic Microbiology. Betty A. Forbes, Daniel F. Sahm, Alice S. Weissfeld. 10 th ed. St. Louis: Mosby; 1998. 10-Delany GM, Patterson SS., Miller CH, Newton CW. The Effect of chlorhexidine gluconate irrigation on the root canal flora of freshly extracted necrotic teeth. Oral Surg 1982 May; 53(5): 518-23. 11-Torabinejad M, Walton RE. Principles and Practice of Endodontics. 3 rd ed. Philadelphia: Saunders; 2002. 12-Modnick RM, Darn SO, Feldman MJ. Efficacy of biomechanical instrumentation: An electron microscope study. J Oral Endod 1976; 2: 261. 13-White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Oral Endod 1997; 23(4): 229-31. 14-Vahdaty A, Pitt Ford TR, Wilson RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro. Endod Dent Traumatol 1993; 9: 243-48. 15-Yang Shue F, Rivera EM, Walton RE, Baumgardner KR. Canal debridement: effectiveness of sodium hypochlorite and calcium hydroxide as medicaments. J Oral Endod 1996; 22(10): 521-26. 16-Orstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990; 6: 142-49. 17-Barthel CR, Zimmer S, Tanomaru M. In situ antimicrobial effectiveness of chlorhexidine and calcium hydroxide: gel and paste versus gutta-percha points. J Oral Endod 2002; 28(6): 427. 18-Leonardo MR, Filho Tanomaru M, Silva LA, Filho Nelson P, Bonifacio KC, Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. J Oral Endod 1999; 25(3): 167-71. 19-Becking AG. Complications in the use of sodium hypochlorite during endodontic treatment. Oral Surg Oral Med Oral Pathol 1991; 71(3); 346-48. 48 2004; Vol. 1, No. 1