Effect of Ethylenediaminetetraacetic Acid and Sodium Hypochlorite Irrigation on Enterococcus faecalis Biofilm Colonization in Young and Old Human Root Canal Dentin: In Vitro Study Hu ¨seyin Ozgur Ozdemir, DDS, PhD,* Hatice Dogan Buzoglu, DDS, PhD,* Semra Calt, DDS, PhD,* Adam Stabholz, DMD, † and Doron Steinberg, MD, PhD ‡ Abstract Introduction: The alterations in dentin tissue depend- ing on increasing age might cause different adhesion capability of bacteria, yielding differences in clinical approaches regarding root canal irrigation. This study, therefore, aimed to evaluate the effects of ethylenedia- minetetraacetic acid (EDTA) and sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilm growth in root canal dentin of young and old individuals. Methods: The root canals of extracted young (<30 years) and old (>60 years) single-rooted human teeth were sectioned at the crown and the apical parts. The root canals of the mid-root sections were enlarged with #2 Gates-Glidden burs. After treatment with 17% EDTA + 2.5% NaOCl, 17% EDTA alone, 2.5% NaOCl alone, or saline, the samples were incubated in E. faecalis suspension for 24 hours. Thereafter, root canal samples were enlarged again with #3 Gates- Glidden burs, and the removed dentin chips were collected. Bacteria were dispersed by using sonication, serially diluted, and then plated for counting on agar plates as colony-forming units. Scanning electron microscopy and confocal laser scanning microscopy investigations were also carried out to examine the bio- film formation on the dentin. Data were analyzed with Kruskal-Wallis test and Mann-Whitney U test with Bon- ferroni adjustment. Results: Combination of EDTA and NaOCl significantly reduced the amount of intracanal bi- ofilm in both age groups (P < .01). However, the bacterial counts of E. faecalis in the old group were still higher (P < .05). Conclusions: It might be suggested that root canals from elderly population are more susceptible to canal infection. However, combined application of EDTA and NaOCl significantly reduces the amount of intracanal biofilm. (J Endod 2010;36:842–846) Key Words Aging, biofilm, dentin, EDTA, Enterococcus faecalis B acteria are the primary etiologic agents of periradicular diseases (1). Root canal treatment aims to eliminate the bacteria from the infected canal and to prevent rein- fection. Although chemomechanical preparation and the use of antimicrobial medica- ments are effective in reducing bacterial colonization in root canal systems, some bacteria might survive despite the treatment, leading to reinfection of the root canal (2). Enterococcus faecalis, a facultative anaerobic, gram-positive coccus, is frequently isolated from endodontically treated teeth with persistent periradicular disease (3, 4). It is commonly found in monoinfections, but it is also observed in mixed infections of the root canal system and has the potential of forming a biofilm structure on root canal walls (5, 6). Several stages are critical in biofilm formation. One of the important stages is the initial adhesion of the bacteria onto the tooth surfaces. The primary adhesion of bacteria depends on surface characteristics of dentin as well as specific adhesion characteristics of the bacteria (7, 8). A smear layer forms on the dentin surface during root canal instrumentation, which might affect the adhesion of bacteria to the root canal wall (9–13). It has been reported that removing the smear layer decreased the adhesion of E. faecalis (12, 13). On the other hand, bacterial invasion of dentinal tubules might be responsible for persistent root canal infection (2). The exposure time of dentin to bacteria and tubule diameter might play an important role in bacterial penetration into the tubuli. Tubules that are sclerotic or obliterated can physically impede bacterial invasion. Recently, Ka- koli et al (14) revealed that the depth of E. faecalis penetration into the dentin tubules was lower in aged dentin samples. With increasing age, several changes occur in the dentin-pulp complex. Dentin sclerosis occurs as a result of an increase in peritubular dentin. Dentinal tubules become obliterated, resulting in narrowing of the tubule to approximately 2.5 mm in diameter near the pulp and 0.9 mm in diameter near the enamel/cement. Thus, a tubule is normally larger in diameter than the average E. faecalis cell diameter of approxi- mately 0.8–1 mm (15–19). Recent studies showed that collagen, which forms the organic matrix of dentin, plays a key role in the adhesion capability of E. faecalis to the dentin surface (2, 6). However, Yang et al (12) suggested that E. faecalis adhesion might be related to nonspecific interaction on the basis of surface properties rather than specific binding to collagen. From the *Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey; and † Department of Endodontics and ‡ Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University-Hadassah, Jerusalem, Israel. Address requests for reprints to Dr Hatice Dogan Buzoglu, Department of Endodontics, Faculty of Dentistry, Hacettepe University, 06100 Sihhiye, Ankara, Turkey. E-mail address: hdogan67@hotmail.com. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.01.008 Basic Research—Technology 842 Ozdemir et al. JOE — Volume 36, Number 5, May 2010