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Year : 2014  |  Volume : 3  |  Issue : 1  |  Page : 19-22

Stability of antibacterial activity of Chlorhexidine and Doxycycline in bovine root dentine

1 Department of Endodontics, Torabinejad Dental Research Center, Iranian Academy of Medical Science, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Endodontics, Islamic Azad University of Khorasgan, Isfahan, Iran
3 Department of Endodontics, Dental Implant Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Web Publication19-May-2014

Correspondence Address:
Sanaz Jahadi
Department of Endodontics, Dental Implant Research Center, Isfahan University of Medical Sciences, Isfahan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2279-042X.132705

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Objective: The aim of this study was to compare the substantivity of chlorhexidine (CHX) and doxycycline bond to the dentin in diffusion disk method.
Methods: A total of 92 dentin disks were prepared from Bovine's teeth root. After removing cementum and standardizing disks in weight and outer diameter, they were irrigated with sodium hypochlorite 2.5%, ethylenediaminetetraacetic acid 17% and sterile saline and sterilled in autoclave. Then, the first group (n = 36) were irrigated with chlrohexine 2% and the second group (n = 36) with doxycycline 100 mg/mL, each for a period of 10 min. The third group (n = 20) was considered as the control group. Samples were divided into four subgroups, which after 1 day, 3, 6, and 12 weeks were incubated in plates containing Muller Hinton agar broth and Enterococcus faecalis for a period of 48 h at 37°C temperature; then the diameter of the zone of inhibition was measured. The antimicrobial effect of irrigating solutions without binding to the dentin was also studied using the plate well method. One-way and univariate variance tests as well as Post-hoc Tukey were used for data analyses.
Findings: The diameter of the zone of inhibition of doxycycline group was significantly more than the CHX group from the beginning to the 3 rd week (P < 0.005). After 3 rd week, mean diameter in doxycycline group declined as compared with the CHX group up to 12 th week (P < 0.005). The difference between means in two groups was not significant in the 3 rd week (P = 0.87).
Conclusion: The results of this study show that CHX and doxycycline show antibacterial activity for 12 weeks; although after 3 rd week, the substantivity of CHX was significantly greater than doxycycline. Both of these irrigants can be used in other pharmacological and medical fields whenever a long-lasting antibacterial action is needed.

Keywords: Chlorhexidine; Doxycycline; enterococcus faecalis; substantivity

How to cite this article:
Khademi AA, Saleh M, Khabiri M, Jahadi S. Stability of antibacterial activity of Chlorhexidine and Doxycycline in bovine root dentine. J Res Pharm Pract 2014;3:19-22

How to cite this URL:
Khademi AA, Saleh M, Khabiri M, Jahadi S. Stability of antibacterial activity of Chlorhexidine and Doxycycline in bovine root dentine. J Res Pharm Pract [serial online] 2014 [cited 2023 Mar 27];3:19-22. Available from: https://www.jrpp.net/text.asp?2014/3/1/19/132705

  Introduction Top

Microorganisms play a significant role in development of pulp and periapical diseases, which has been demonstrated in previous studies. [1],[2] Disinfection of the root canal system to reduce the microbial load present in the root canal is one of the main goals of endodontic therapy. Although, mechanical instrumentation removes a portion of the microbes, but irrigation and intracanal medicaments help more microorganisms and necrotic pulp tissues to be removed. [3] Calcium hydroxide (Ca[OH]) is commonly used as an intracanal medicament and can predictably disinfect the root canal, but after mechanical removal of Ca (OH), no residual antimicrobial effect could remain. [4] To obtain more reductions in the microbial load some antimicrobial irrigants such as chlorhexidine (CHX) and Tetracyclines have been used. Unlike sodium hypochlorite (NaOCl), [5] these irrigants are capable of adsorbing on to the dentin walls, which imparts long-lasting antimicrobial effects, termed substantivity. [6] CHX is a cationic biguanide that adsorbs on to the cell wall of microorganisms and cause leakage of intracellular components. CHX is effective against both Gram-positive and Gram-negative bacteria. [7] Some studies have shown 0.02-2% CHX kills the Enterococcus faecalis cells in 30 s or less. [8] In-vivo studies found that antimicrobial effect of CHX remains for up to 48 h or even longer period of time. [9] Tetracyclines are effective against a wide range of microorganisms and other than substantivity show antiresorptive activity and may remove the smear layer from instrumented root canal walls. [10],[11] Due to residual alive micro-organisms in root canal system, which may cause failure of root canal therapy [12] and since leakage of temporary or final restorations can lead to reinfection of root canal irrigation, [13],[14],[15] using these irrigants seems to be necessary. The aim of this study was to evaluate the antimicrobial efficacy and residual activity (substantivity) of 2% CHX and doxycycline against E. faecalis in the bovine root dentin disks in an ex-vivo model. Long lasting antibacterial action of such irrigants can prevent the reinfection and may be useful in other pharmacological and medical fields, especially in oral medicine.

  Methods Top

Ninety-two dentin disks of bovine incisor root were used in this study. First, teeth having complete roots and enough dentine thickness were selected. Crowns were sectioned off from cementoenamel junction using a diamond saw and tap water irrigation, cementom was removed using polish paper (Matador factory, softlex 240, Germany) and dentin disks were obtained from 1/3 coronal portion of each tooth using a slow speed diamond saw. By caliper (Varnier, caliper 0.02%, China) and analytical balance (R and D Scaltex, [0.001] GR, USA) disks were measured and samples with 7 mm outer diameter and 150-200 mg weight were selected. All samples were preserved in vials containing tap water to avoid dehydration. Disks were individually treated with 5.25% NaOCl (Merck, Germany) and 17% ethylenediaminetetraacetic acid (EDTA) (pH = 7.2) (Meta Diomed, MD-Cleaner, Korea) to remove the smear layer. Samples were divided into three groups: Group A (n = 36) were treated by immersion in 2% CHX (Merk, Germany) for 10 min, Group B (n = 36) were immersed in doxycycline solution 100 mg/mL and Group C (n = 20), as the control group, were treated by immersion in sterile saline for 10 min. All of the disks were then blotted dry paper and after removal of extra liquid they were put in a sterile dish in 37°C. A single colony of E. faecalis (ATCC 29272) was selected from blood agar plates and inoculated in brain-heart infusion until it turned turbid. The broth was adjusted to 0.5 McFarland standard equal to 1.5 × 10 8 bacteria/mm. Muller-Hinton agar plates (Antec diagnosis) were swabbed with E. faecalis. Experimental and control disks were placed on the plates and incubated for 48 h in 37°C. Each of main groups divided to four subgroups. The zones of inhibitions were measured by the average of two perpendicular diameters after 1 day and 3, 6, and 12 weeks.

  Results Top

The zones of inhibitions in two used irrigants were measured and analyzed using two-way analysis of variance. Type of irrigant and the time, both had a significant effect on diameter of zones of inhibitions (P < 0.001). Tukey's Post-hoc test showed significant differences between CHX and doxycycline. [Table 1] demonstrates results of mean diameter of bacterial inhibition zone in two irrigants and control group at four periods of time. The control group revealed inhibition zones and the differences with experimental ones were significant (P < 0.001). Except 6 th and 12 th weeks, there were significant differences between all time periods. The diameter of the zone of inhibition of doxycycline was significantly more than the CHX group from the beginning to the 3 rd week (P < 0.005). The difference between means in two groups was not significant in the 3 rd week (P = 0.87) and the mean diameter in doxycycline group declined from the 3 rd to 12 th week as compared to the CHX group (P < 0.005).
Table 1: Means of zone of inhibition of E. faecalis in experimental and control groups

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  Discussion Top

After cleaning and shaping of the root canal, some of the areas may be untouched and remained bacteria may cause failure of treatment; thus a suitable irrigant is needed to aid in debridement of the canals and killing of bacteria. In this study, E. faecalis was chosen as the test organism because it is well-recognized as a pathogen associated with persistent apical periodontitis in endodontically treated teeth and is resistant to intracanal medication with Ca (OH). [16] Antibacterial substantivity of CHX and doxycycline is compared for 12 weeks in this study. Although doxycycline has shown more effective antibacterial action than CHX in 1 st day, its effect suddenly drops off after 3 weeks and show constant level, which is lower than CHX at 6 th up to 12 th weeks. CHX shows less antibacterial effect at 1 st day than doxycycline, but its effect increases in next periods. Hence, it seems that CHX has more substantiative antimicrobial activity that lasts up to 12 th week. High level of antibacterial activity of doxycycline disks at the beginning which continues up to 3 rd week shows that doxycycline substantivity decreases after 3 weeks and hence that less antibacterial activity of the disk is shown up to 12 th week. In contrast, CHX disks show low level antibacterial activity at 1 st day, which increases up to 12 th week. Zone of inhibition in control groups (disks without use of irrigants) may be a result of residual hypochlorite and EDTA used in previous steps. The finding of the present study showed 2% CHX and doxycycline both have substantiative antimicrobial activity that lasts up for 12 weeks. These results are similar to the results of Mohamadi [17] and Rosenthal [18] study which showed CHX substantivity for 12 weeks, and complete the result of White study [6] or in-vivo study of Leonardo and Shahani, which had shown antibacterial activity of CHX just up to 72 h and 48 h. [19],[20] Although, Lenet and komorowski have reported that for induction of substantivity, dentin should be treated for 7 days, [21],[22] but in the present study, 10 min contact of dentin with doxycycline or CHX has been shown to be enough to make substantivity. Our results showed that doxycycline up to 3 rd week is more potent antibacterial irrigant than CHX; however, Stabholz found that antimicrobial substantivity of tetracycline was significantly greater than CHX for 14 days and CHX didn't show antibacterial effect even as subgingival irrigant. [23],[24] After 6 weeks, CHX antibacterial effect increases and becomes more than doxycycline. Rasmik showed that half-life of CHX is more than doxycycline (14 weeks against 3 weeks). [25] In other studies, substantivity of CHX has been continued up to 28 days and in all periods, CHX has been more potent than doxycycline. [26] The point to make is the effect of different methods used in these two studies. For instance, in the latter study canals were initially infected by bacteria and then treated with irrigants and antibacterial activity was measured by culturing and colony forming unit counting, but when dentin is exposed to irrigants before bacterial infection, as used in the recent study, it removes errors of sampling and culturing. In addition, this method resembles secondary infections after leakage of final or temporary restorations. Because antibacterial effect of CHX and doxycycline is affected by factors like local pH and endodontic sealers can change the pH, so clinical relevance of the results is limited, autoclaving might denature dentin collagens and reduce the retention of CXH and doxycycline.

Finally, results of this study show that CHX and doxycycline are suitable irrigants against E. faecalis with substantivity, but CHX show more antibacterial effect than doxycycline after 3 weeks and it means that CHX is more substantiantive antimicrobial factor.

  Authors' Contribution Top

All authors contributed the idea of research, design of study, data analysis and manuscript preparation.

  References Top

1.Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.  Back to cited text no. 1
2.Möller AJ, Fabricius L, Dahlén G, Ohman AE, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand J Dsent Res 1981;89:475-84.  Back to cited text no. 2
3.Matos Neto M, Santos SS, Leão MV, Habitante SM, Rodrigues JR, Jorge AO. Effectiveness of three instrumentation systems to remove Enterococcus faecalis from root canals. Int Endod J 2012;45:435-8.  Back to cited text no. 3
4.Lin YH, Mickel AK, Chogle S. Effectiveness of selected materials against Enterococcus faecalis: Part 3. The antibacterial effect of calcium hydroxide and chlorhexidine on Enterococcus faecalis. J Endod 2003;29:565-6.  Back to cited text no. 4
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7.Basrani B, Lemonie C. Chlorhexidine gluconate. Aust Endod J 2005;31:48-52.  Back to cited text no. 7
8.Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza-Filho FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424-8.  Back to cited text no. 8
9.Baca P, Junco P, Arias-Moliz MT, Castillo F, Rodríguez-Archilla A, Ferrer-Luque CM. Antimicrobial substantivity over time of chlorhexidine and cetrimide. J Endod 2012;38:927-30.  Back to cited text no. 9
10.Mohammadi Z, Giardino L, Palazzi F, Shalavi S, Farahani MF. Substantivity of three concentrations of tetraclean in bovine root dentin. Chonnam Med J 2012;48:155-8.  Back to cited text no. 10
11.Rasimick BJ, Shah RP, Musikant BL, Deutsch AS. Bacterial colonisation of root canal dentine previously treated with endodontic irrigants. Aust Endod J 2010;36:489-92.  Back to cited text no. 11
12.Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997;30:297-306.  Back to cited text no. 12
13.Friedman S, Komorowski R, Maillet W, Klimaite R, Nguyen HQ, Torneck CD. In vivo resistance of coronally induced bacterial ingress by an experimental glass ionomer cement root canal sealer. J Endod 2000;26:1-5.  Back to cited text no. 13
14.Deveaux E, Hildelbert P, Neut C, Romond C. Bacterial microleakage of Cavit, IRM, TERM, and Fermit: A 21-day in vitro study. J Endod 1999;25:653-9.  Back to cited text no. 14
15.Kazemi RB, Safavi KE, Spångberg LS. Assessment of marginal stability and permeability of an interim restorative endodontic material. Oral Surg Oral Med Oral Pathol 1994;78:788-96.  Back to cited text no. 15
16.Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93.  Back to cited text no. 16
17.Mohammadi Z, Abbott PV. On the local applications of antibiotics and antibiotic-based agents in endodontics and dental traumatology. Int Endod J 2009;42:555-67.  Back to cited text no. 17
18.Rosenthal S, Spångberg L, Safavi K. Chlorhexidine substantivity in root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488-92.  Back to cited text no. 18
19.Shahani MN, Subba Reddy VV. Comparison of antimicrobial substantivity of root canal irrigants in instrumented root canals up to 72 h: An in vitro study. J Indian Soc Pedod Prev Dent 2011;29:28-33.  Back to cited text no. 19
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20.Leonardo MR, Tanomaru Filho M, Silva LA, Nelson Filho P, Bonifácio KC, Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. J Endod 1999;25:167-71.  Back to cited text no. 20
21.Lenet BJ, Komorowski R, Wu XY, Huang J, Grad H, Lawrence HP, et al. Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles. J Endod 2000;26:652-5.  Back to cited text no. 21
22.Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315-7.  Back to cited text no. 22
23.Stabholz A, Kettering J, Aprecio R, Zimmerman G, Baker PJ, Wikesjö UM. Antimicrobial properties of human dentin impregnated with tetracycline HCl or chlorhexidine. An in vitro study. J Clin Periodontol 1993;20:557-62.  Back to cited text no. 23
24.Stabholz A, Kettering J, Aprecio R, Zimmerman G, Baker PJ, Wikesjö UM. Retention of antimicrobial activity by human root surfaces after in situ subgingival irrigation with tetracycline HCl or chlorhexidine. J Periodontol 1993;64:137-41.  Back to cited text no. 24
25.Rasimick BJ, Wan J, Musikant BL, Deutsch AS. Stability of doxycycline and chlorhexidine absorbed on root canal dentin. J Endod 2010;36:489-92.  Back to cited text no. 25
26.Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J 2006;32:112-5.  Back to cited text no. 26


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