EFFECT OF SODIUM HYPOCHLORITE

International Journal of Clinical Dentistry ISSN: 1939-5833 Volume 6, Number 2 Nova Science Publishers, Inc. EFFECT OF SODIUM HYPOCHLORITE ON THE SUBSTANTIVITY OF CHLORHEXIDINE Zahed Mohammadi 1, Luciano Giardino 2, Flavio Palazzi 3, Sousan Shalavi 4, Mohammad Yousof Alikhani 5, Giuseppe Lo Giudice 6, and Noloofar Davoodpour 7 1 Department of Endodontics, Hamedan University of Medical Sciences, Hamedan, Iran Iranian Center for Endodontic Research (ICER), Tehran, Iran 2 Department of Endodontology, Dental School, University of Torino, Italy 3 Department of Odontostomatological and Maxillofacial Sciences, Federico II University of Naples, Italy 4 Hamedan University of Medical Sciences, Hamedan, Iran 5 Department of Bacteriology, Hamedan University of Medical Sciences, Hamedan, Iran 6 Department of Odontostomatoly, University of Messina, Italy 7 General Dentist, Hamedan, Iran ABSTRACT The aim of this in vitro study was to evaluate the effect of sodium hypochlorite on the substantivity of chlorhexidine (CHX). Forty dentin tubes prepared from human maxillary central and lateral incisor teeth were used. Specimens were contaminated with Enterococcus faecalis for 14 days and were divided into five groups as follows: 2% CHX; 1.3% NaOCl; 2% CHX+1.3% NaOCl; infected dentin tubes (positive control); and sterile dentin tubes (negative control). Dentin chips were collected with round burs into brain heart infusion broth and after culturing, the number of colony-forming units (CFU) was counted. Data were analyzed using analysis of variance and covariance with repeated measures. Significance level was set at p< 0.05. The number of CFU was minimum in the first cultures in all experimental groups, and the results obtained were significantly different from each other at any time period (P < 0.05). At the first and second cultures the number of CFU in the CHX+NaOCl group was lower than two other groups. At the 14 day and 21 day experimental periods the CHX group showed the most effective antibacterial action. NaOCl group showed the worst result at third and fourth periods (P < 0.05). In each group, the number of CFU increased significantly by time-lapse (P < 0.05). In conclusion, NaOCl had no additive effect the residual antibacterial activity of CHX. Keywords: Chlorhexidine, Enterococcus faecalis, residual antibacterial activity, sodium hypochlorite

174 Zahed Mohammadi, Luciano Giardino, Flavio Palazzi et al. INTRODUCTION It has been demonstrated that bacteria and their products are the main etiologic factors in the initiation and perpetuation of pulpal and periapical diseases [1]. Although mechanical instrumentation reduces the microbial load up to 100 to 1000 times, due to the complexity of the root canal system, it has been demonstrated that at least 35% of the root canal surfaces remained untouched by the instruments [2]. Therefore, an appropriate antibacterial irrigation solution is required to improve disinfection of the root canal system. Various irrigation solutions have been suggested for canal disinfection. Sodium hypochlorite is the most commonly used irrigation solution in concentrations from 0.5% to 6%. NaOCl possesses excellent antimicrobial and tissue dissolving abilities. In high concentrations it is toxic and may cause damage to the oral and periapical tissues. On the other hand, in low concentrations its antimicrobial activity may reduce. Furthermore, NaOCl has little to none substantivity. In addition, NaOCl may cause corrosion of endodontic instruments and discoloration of clothes and has an unpleasant odor [3]. Another irrigation solution is chlorhexidine (CHX). CHX has a wide range of antimicrobial activity against endodontic pathogens. Its antibacterial activity is comparable to that of NaOCl. Substantivity of CHX has been revealed in various studies. However, it has no tissue dissolving ability [4]. In a protocol proposed by Zehnder [5] for root canal irrigation, NaOCl is used during cleaning and shaping followed by irrigation with EDTA. Thereafter, CHX is used as final rinse. Kuruvilla and Kamath [6] found that combining NaOCl with CHX increased their antimicrobial activity. However, there is no study on the effect of combining with NaOCl on the substantivity of CHX. Therefore, the purpose of this study was to assess the effect of NaOCl on the substantivity of CHX. MATERIALS AND METHODS Human maxillary central and lateral incisor teeth were used. Up to reaching the needed number teeth were stored in sterile saline solution. Thereafter, in order to remove remaining hard and soft tissues and to surface disinfection, the teeth were kept in 0.5% NaOCl solution for up to 7 days. The clinical crown and apical third were removed from each tooth with a rotary diamond saw at 1000 rpm (Isomet Plus precision saw, Buehler, IL, USA) under watercooling. Cementum was removed by using polish paper (Ecomet 3, variable-speed grinderpolisher, Buehler, IL, USA), which resulted in a center-holed piece of root dentin with a 6- mm outer diameter. The remained piece of each tooth was then cut into 4-mm thick slices with a diamond saw as above. The canals of the 4-mm blocks were enlarged (standardized) with an ISO 023 slow speed round bur (Figure 1). In order to prevent dehydration, all teeth and dentin slices were preserved in vials containing tap water during the procedures. Each dentin block (n = 40) was individually treated with 5.25% NaOCl and 17% EDTA (with ph 7.2) to remove the smear layer. The specimens were then placed in BHI broth (Oxoid, Basingstoke, UK) and autoclaved. To monitor the efficacy of the sterilization they were then kept in an incubator at 37 C for 24 h. A total of 75 specimens were randomly divided into five groups as follows: Group 1 (10 specimens): 2% CHX; Group 2 (10 specimens): 5.25%

Effect of Sodium Hypochlorite on the Substantivity of Chlorhexidine 175 NaOCl; Group 3 (10 specimens): 2% CHX+5.25% NaOCl; Group 4 (5 specimens): positive control (infected dentin tubes); and Group 5 (5 specimens): negative control (sterile dentin tubes). Isolated 24-h colonies of pure cultures of E. faecalis (ATCC 29212) were suspended in 5 ml of BHI. The bottles containing each specimen in Groups 1, 2, 3 and 4 were opened under laminar flow. Figure 1. Schematic view of used dentin tubes. Two milliliters of sterile BHI was removed with sterile pipettes and replaced with 2 ml of bacterial inoculum. The bottles were closed and kept at 37 C for 14 days, with the replacement of 1 ml of contaminated BHI for 1 ml of freshly prepared BHI every 2 days, to avoid medium saturation. After the contamination period, each specimen was removed from its bottle under aseptic conditions, and the canal was irrigated with 5 ml of sterile saline and dried with sterile paper points. In order to prevent contact of the medicament with the external surface, the outer surface of the specimens was covered with two layers of nail varnish. Thereafter, using decontaminated sticky wax, specimens were fixed at the bottom of wells of 24-well cell culture plates which also obliterated the apical surface of the root canal. Finally, the irrigating solutions were inserted into the canal lumen with sterile 3-ml plastic syringes and 27-gauge needles until the dentin tubes were totally filled. Solutions were removed using sterile paper points ten minutes after placement into the lumen. The specimens were then incubated at 37 C for 21 days to maintain humidity. At experimental times of 0, 7, 14, and 21 days, dentin chips were removed from the canals with sequential sterile low-speed round burs with increasing diameters of ISO sizes: 025, 027, 029, and 031, respectively. Each bur removed approximately 0.1 mm of dentin around the canal. The powder dentin samples obtained with each bur were immediately collected in separate test tubes containing 3 ml of freshly prepared BHI. Thereafter, l00 μl from each test tube was cultured on blood agar. Growing colonies were counted and recorded as CFU. Analysis of variance and covariance with repeated measures was used (ANOVA) to indicate differences between the experimental groups and the positive control. In addition, One-way ANOVA (Tukey's method) was used to indicate differences within each layer. Significance level was set at P<0.05.

176 Zahed Mohammadi, Luciano Giardino, Flavio Palazzi et al. RESULTS The number of CFU obtained from four consecutive dentinal layers was presented in Table 1. The number of CFU in all three experimental groups was minimum after treatment. The positive control group showed viable bacteria at all experimental times, which indicated the efficiency of the method. In contrast, the negative control group showed no viable bacteria at all experimental times. At the first and second cultures, the CHX+ NaOCl group showed the most effective antibacterial action (P < 0.05). However, at days 14 and 21 CHX group demonstrated more effective antibacterial action than two other experimental groups (P < 0.05). In addition, the difference between CHX and CHX+ NaOCl was not significant at 7 and 14 days (P > 0.05). Furthermore, in all groups, the number of CFU increased significantly by time-lapsed (P< 0.05). Table 1. The number of CFU in three experimental groups at four intervals Time Day 0 Day 7 Day 14 Day 21 Periods Irrigants NaOCl 6.818±7,055 9.182±7.55 61.909±7.55 222.369±7.867 CHX 11.397±7.448 14.097±7.448 35.947±7.448 121.836±7.882 NaOCl+CHX 6.193±7.442 8.773±7.442 48.993±7.442 203.970±9.775 DISCUSSION Current root canal instrumentation techniques leave many areas of the root canal system completely untouched by the instruments [7]. Therefore, an appropriate irrigation solution is needed to improve debridement of the root canal system. For improvement of their efficacy, the root canal irrigants must be in contact with the dentin walls and debris [8, 9]. It is well known that microorganisms penetrate into dentinal tubules to varying depths [10]. Therefore, if paper point be used to taking sample from the root canal system, the possibility of false negative culture is significantly increased [11]. Thus, in order to decrease the possibility of false negative result of the culturing, it is advised to cut dentin from root canal walls [11]. There are three ways to achieve this goal: using hand files [12], using Gates-Glidden drills [13], and using burs [11]. Hand files and Gates-Glidden drills can be used both in vitro and in vivo. However, because of the increased risk of perforation, burs should be used in vitro only. In the present study both the antibacterial substantivity and penetration depth of CHX, NaOCl, and CHX+NaOCl were assessed. The use of synergism between two active agents seems a logical pharmaceutical way to achieve maximal therapeutic effect with minimal side effects. CHX and NaOCl are both potent antibacterial agents, however, these two agents have considerable side effects; CHX is irritating to the skin [14] is unable to dissolve necrotic tissue remnants [15], is less effective on Gram-negative than on Gram-positive bacteria(16) while NaOCl is cytotoxic and tissue toxic [3]. In order to reduce their side effects, we tested the hypothesis that a combination of sub-bactericidal concentrations of these two agents may act synergistically. The use of CHX together with NaOCl has a clinical advantage: it can be

Effect of Sodium Hypochlorite on the Substantivity of Chlorhexidine 177 postulated that the interactions between NaOCl and CHX may reduce side effects such as teeth-staining due to the oxidative properties of NaOCl that may counteract the staining caused by CHX. Findings showed that the number of CFU of the CHX+NaOCl group was lower than the other two groups, which is in accordance to the findings of Kuruvilla and Kamath (6) and confirms the synergistic effect between two agents. Synergistic effect between CHX and NaOCl as also been demonstrated. Heling and Chandler [17] found that at determined concentrations, CHX and NaOCl had synergistic activity. The method of their study was very similar to the method of present study. Steinberg et al. [18] showed the additive antibacterial effect of CHX and hydrogen peroxide as well. Recently, Shahriari et al. [19] demonstrated that hydrogen peroxide increased the antibacterial activity of CHX only at the first culture, however did not increase its long-term (residual) antibacterial activity. The burs used for removing dentin from the lumen of the dentin tubes were selected consecutively and each bur removed a thin layer of 0.1mm thickness. Additionally, the irrigation solutions used (CHX and NaOCl) can kill E. faecalis only in direct contact. Therefore, it can be stated that besides antibacterial substantivity, the penetration depth of the irrigants into dentinal tubules was assessed. CONLUSION Within the limitations of the present study, NaOCl increased the antibacterial activity of CHX only at the first culture, however did not increase its residual antibacterial activity. REFERENCES [1] Sundqvist G. Ecology of the root canal flora. J. Endod. 1992; 18: 427-30. [2] Mohammadi Z. Chemomechanical strategies to manage endodontic infections. Dent. Today 2010; 29: 91-2, 94, 96. [3] Mohammadi Z. Sodium hypochlorite in endodontics: an update review. Int. Dent. J. 2008; 58: 329-341. [4] Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int. Endod. J. 2009; 42: 288-302. [5] Zehnder M. Root canal irrigants. J. Endod. 2006; 32: 382-96. [6] Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J. Endod. 1998; 24: 472-6. [7] Peters OA, Laib A, Gohring TN, Barbakow F. Changes in root canal geometry after preparation assessed by high resolution computed tomography. J. Endod. 2001; 27: 1-6. [8] Pecora JD, Guirnaraes LF, Savioli RN. Surface tension of several drugs used in endodontics. Braz. Dent. J. 1991; 2: 123-7. [9] Tasman F, Cehreli ZC, Ogan C, Etikan I. Surface tension of root canal irrigants. J. Endod. 2000; 26: 586-7. [10] Love RM, Jenkinson HF. Invasion of dentinal tubules by oral bacteria. Crit. Rev. Oral. Biol. Med. 2002; 13: 171-83.

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