Bactericidal Activity of Stabilized Chlorine Dioxide as an Endodontic Irrigant in a Polymicrobial Biofilm Tooth Model System John R. Lundstrom, DDS, MS, Anne E. Williamson, DDS, MS, Alissa L. Villhauer, Deborah.V. Dawson, PhD, and David R. Drake, PhD Abstract Introduction: The purpose of this study was to deter- mine bactericidal efficacy of 0.04% stabilized chlorine dioxide, 3% sodium hypochlorite, 2% chlorhexidine gluconate, and sterile distilled water in a polymicrobial biofilm model. Methods: Roots of 35 permanent bovine incisors had pulps extirpated, and their apical size and root length were standardized. Teeth were coated with mucin, inoculated with standardized suspensions of Streptococcus sanguinis, Actinomyces viscosus, Fusobacterium nucleatum, Peptostreptococcus micros, and Prevotella nigrescens and incubated anaerobically. Teeth were randomly divided into four groups and rinsed for 3 minutes with 15 mL of irrigant. Biofilms were harvested and spiral-plated on selective media. Numbers of bacteria in the harvested biofilms was determined via the standard spiral-plating method- ology. Treatment groups were evaluated using the nonparametric Kruskal-Wallis procedure. Pair-wise comparisons among the four groups and five organisms were made using the Wilcoxon-Mann-Whitney proce- dure. Adjustments for multiple comparisons were made using the Holm method with p < 0.05. Results: Results provide strong evidence of a significant difference in levels of bactericidal activity associated with the type of irrigant for all five bacterial species tested. Levels of bactericidal activity were significantly higher for the NaOCl group than for the stabilized chlorine dioxide (ClO 2 ) group for S. sanguinis, A. viscosus, and P. ni- grescens. Results for F. nucleatum and P. micros were not significant after the adjustment for multiple comparisons. Conclusions: The triple-inoculation bovine tooth model system is a robust, consistent, and reproduc- ible model system to study polymicrobial biofilms. It should be used with the knowledge expansion of biofilm structure and function as well as the development of anti- microbial protocols. (J Endod 2010;36:1874–1878) Key Words Bovine tooth, chlorine dioxide, endodontic irrigant, polymicrobial biofilm P erhaps the most important step in endodontic treatment is the disinfection of the root canal system. This is accomplished by the chemomechanical debridement of the canal space. The mechanical portion of this debridement is accomplished by a variety of instruments that attempt to clean and shape the canal system in order to have access for irrigation and smooth taper for efficient obturation. The chemical portion of this debridement is accomplished with a variety of endodontic irrigants. These irrigants are flushed into and out of the canal system as they remove debris and microbes via fluid motion. Other desirable qualities of endodontic irrigants are well known. These include bactericidal efficacy, dissolution of necrotic tissue, low- surface tension, substantivity, lubrication, and a harmless effect on the microhardness and roughness of root canal dentin (1). Undesirable qualities include the formation of precipitate, tissue toxicity upon extrusion, and corrosion of dental instruments (1). To date, no irrigant or concentration of irrigant has been formulated that can accomplish all the desirable qualities while avoiding the undesirable ones while at the same time showing a high rate of bactericidal efficacy on known endodontic pathogens existing in polymicrobial biofilms. Therefore, the purpose of this study was to determine bacte- ricidal efficacy of 0.04% stabilized chlorine dioxide, 3% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) gluconate, and sterile distilled water in a polymicrobial biofilm bovine tooth model. Materials and Methods Preoperative Preparation of Teeth Thirty-nine permanent bovine incisors were selected. Inclusion/exclusion criteria included no root curvatures and no root fractures. The teeth were extracted with eleva- tors and forceps and placed in a 1.5% solution of NaOCl for 24 hours. The teeth were subsequently stored in 0.1% thymol solution until use, which was approximately 1 week. External root surfaces were debrided of bone, calculus, and soft tissue using a Gracey curette. The apical 3 mm was resected with a fissure bur and high-speed hand- piece with water spray. The teeth were then decoronated to retain exactly 25 mm of the root. A number 6 Gates Glidden was used in a retropreparation fashion to create a fixed apical size. Care was taken to not extend the Gates Glidden coronal to the apical 2 mm. The pulps were extirpated with a barbed broach. Bovine pulps were always removed in total using this procedure. The canals were then rinsed with 3% NaOCl and 17% EDTA (Vista Dental, Racine, WI) for 60 seconds followed by a final rinse with NaOCl. EDTA was used to remove the minimal smear layer produced by the Gates Glidden drills in the apical 2 mm. The apical portions were then etched with 37% phosphoric acid and restored with Optibond (Kerr Dental, Orange, CA) and Filtek composite (3M ESPE, St Paul, MN), creating 25-mm ‘‘test tubes’’ with a fixed apical size. The entire outside surfaces were then twice coated with clear fingernail polish. The teeth were thoroughly rinsed with distilled H 2 O and placed in a glass jar with screw cap filled with dH 2 O and steam sterilized for 20 minutes. From the College of Dentistry, University of Iowa, Iowa City, IA, USA. Address requests for reprints to Dr Anne E. Williamson, 435 Dental Science Bldg South, Iowa City, IA 52242-1001. E-mail address: anne-williamson@uiowa.edu. 0099-2399/$ - see front matter Copyright ª 2010 American Association of Endodontists. All rights reserved. doi:10.1016/j.joen.2010.08.032 Basic Research—Technology 1874 Lundstrom et al. JOE — Volume 36, Number 11, November 2010