Basic ResearchBiology

The Effect of Detergents on the Antibacterial Activity of Disinfecting Solutions in Dentin

Zhejun Wang, DDS,* Ya Shen, DDS, PhD,* Jingzhi Ma, DDS, PhD, and Markus Haapasalo, DDS, PhD*
Abstract
Introduction: Detergents have been added into different disinfecting solutions to lower their surface tension and to enhance their antibacterial effects. The purpose of this study was to evaluate the effectiveness of dentin disinfection by different antibacterial solutions in the presence and absence of detergents using a novel dentin infection model and confocal laser scanning microscopy (CLSM). Methods: Semicylindrical dentin specimens were infected with Enterococcus faecalis by centrifugation according to a previously described protocol. After 1 day of incubation, the infected dentin specimens were subjected to 1 and 3 minutes of exposure to sterile water, 0.1% cetrimide (CTR), 2% sodium hypochlorite (NaOCl), 2% NaOCl + 0.1% CTR, 6% NaOCl, 6% NaOCl + 0.1% CTR, Chlor-Xtra (Vista Dental, Racine, WI), 2% chlorhexidine (CHX), CHX-Plus (Vista Dental, Racine, WI), 2/4% iodine potassium iodide (IPI), and IPI + 0.1% CTR. The specimens were then stained for bacterial viability and examined by CLSM to analyze the proportions of dead and live bacteria inside dentinal tubules. Results: More bacteria in dentin were killed after 3 minutes of exposure than after 1 minute of exposure to the disinfecting solutions in all experimental groups (P < .05). The antibacterial solutions with detergents (0.1% CTR, 2% NaOCl + 0.1% CTR, CHX-Plus, and IPI + 0.1% CTR) showed a statistically higher proportion of dead bacteria than the corresponding solutions without detergents (sterile water, 2% NaOCl, 2% CHX, and IPI) (P < .05) except for the 6% NaOCl group (6% NaOCl, 6% NaOCl + 0.1% CTR, and Chlor-Xtra) (P > .05). Six percent NaOCl, 6% NaOCl + 0.1% CTR, and Chlor-Xtra were the most effective solutions, killing over 45% and 65% of the bacteria after 1 and 3 minutes of exposure, respectively. Only 3% to 4% of the bacteria were dead in the sterile water group, whereas 0.1% CTR alone was able to kill 24% to 36% of the E. faecalis cells. Conclusions: The addition of detergents in the disinfecting solutions used in the present study increased their antibacterial effects against E. faecalis in the dentinal tubules. When used alone as a single agent, CTR showed antibacterial effectiveness comparable to 2% NaOCl, 2% CHX, and 2/4% IPI. (J Endod 2012;38:948953)

Key Words
confocal laser scanning microscopy, dentin, detergent, disinfection, Enterococcus faecalis

he prime function of endodontic medicaments in root canal treatment is to kill the infecting organisms in the whole root canal system (1, 2). One of the possible ways to improve the bactericidal efcacy of the disinfecting solutions is to incorporate different detergents as surface active agents that could help to reduce the surface tension and increase the wettability of the solutions (3). High wettability is speculated to enable the disinfecting solutions a better adaption to dentin and penetration into the dentinal tubules (4, 5). Previous studies have shown that different antibacterial agents with detergents have lower surface tension values and eradicate bacteria in direct contact faster than agents without a detergent (68). The reason for better killing of bacteria by the addition of detergents may be related to the weakening of the cohesive forces in extracellular polymeric substance and bacterial membranes (9). However, in the clinical situation, the challenge of removing and/or killing all bacteria still remains (10). The survival of bacteria may be partly attributed to their invasion into dentinal tubules, where they are better protected from endodontic medicaments than in the main root canal (11, 12). There are several studies where killing of bacteria in dentin has been examined (10, 13). However, there is no evidence so far showing that detergents can help antibacterial solutions to kill bacteria in dentin more effectively. There may be several reasons for the lack of such studies, one of them being the lack of a standardized dentin infection model and sensitive detection method, which together could give reliable results of the antibacterial effectiveness of the medicaments in dentin. Recently, a new dentin infection model was introduced to establish bacterial presence in the dentinal tubules in vitro (13). Compared with some previous bacterial invasion models based on culturing methods (1416), the new model allowed a standardized, dense, and deep penetration of bacteria into dentinal tubules using the power of centrifugation. Hence, the application of this new model was suggested to provide a more predictable platform than previously to measure the effectiveness of disinfecting agents in infected dentin. In the present study, we aimed to compare the performance of different antibacterial solutions with and without detergents

From the *Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada; The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; and Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Address requests for reprints to Dr Markus Haapasalo, Division of Endodontics, Oral Biological and Medical Sciences, UBC Faculty of Dentistry, 2199 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3. E-mail address: markush@dentistry.ubc.ca 0099-2399/$ - see front matter Copyright 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.03.007

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against bacteria in dentin canals using a new dentin infection model and the noninvasive detection method by confocal laser scanning microscopy (CLSM). iodide (18) was used for staining the 44 fractured dentin specimens following the manufacturers instruction. The specimens were then rinsed with phosphate-buffered saline for 1 minute. Fluorescence from the stained cell was viewed using a CLSM (Nikon Eclipse C1; Nikon Canada, Mississauga, ON). The mounted specimens were observed using a 20 lens with an additional zoom of 2. CLSM images were acquired by the software EZ-C1 v.3.40 build 691 (Nikon) at a 512 512 pixel scan area. Five randomly selected places on the border of the root canal were chosen for CLSM scanning on each specimen. A total of 220 scans were performed for all 11 groups. A stack of 20 slices (0.5mm step size) were scanned at the 5 areas (0.30 0.30 mm for each area) in each sample. Live/dead ratios of the infected dentinal tubules were analyzed using the Imaris 7.2 software (Bitplane Inc, St Paul, MN). The volume ratio of red uorescence to green and red uorescence indicated the proportion of killed cells for each medicament. The proportions of dead cell volume after exposure to different solutions were subjected to univariate analysis of variance using SPSS 16.0 (SPSS Inc, Chicago, IL). Post hoc multiple comparisons were used to isolate and compare the results at a signicance level of P < .05.

Materials and Methods

Sample Preparation Eleven extracted caries-free single-rooted teeth were collected under a protocol approved by the local ethics committee of the School and Hospital of Stomatology Wuhan University, Wuhan, China. The teeth were kept in 0.01% NaOCl solution before use. According to a previously described protocol (13), a cylindrical root dentin block was horizontally sectioned from each tooth, and the root canals were enlarged to 1.5 mm with a Gates Glidden drill (Dentsply Tulsa Dental, Tulsa, OK) at 300 rpm under water cooling. Twenty-two semicylindrical halves were vertically fractured from the 11 dentin blocks followed by shaping and rening to a size of 4 4 2 mm. The smear layer on both sides of the specimen was removed by immersion in 5.25% NaOCl and 6% citric acid (pH = 4.0), each for 4 minutes. The prepared dentin specimens, with the canal side up, were placed on the bottom (lter) of the upper chamber of a microltration tube (Pall Corporation, Ann Arbor, MI), and the gaps between the inner tube and the dentin specimen were sealed by a composite lling material (Kerr Co, Orange, CA) light cured for 20 seconds. Dentin Infection Enterococcus faecalis VP3-181 isolated from a case of persistent apical periodontitis was used as the test organism (17) and grown in air at 37 C overnight on brain-heart infusion (BHI) agar (Becton-Dickinson, Sparks, MD) plates. The bacteria were suspended in BHI broth (Becton-Dickinson) and standardized spectrophotometrically to 3 106 colony-forming units/mL. In accordance with the previously described detailed protocol (13), E. faecalis suspension was sequentially centrifuged into the dentinal tubules. All microltration tubes with infected dentin specimens inside were then incubated at 37 C in BHI broth in air for 24 hours to allow recovery of the bacteria after centrifugation. Disinfection of Dentin The dentin specimens were detached from the composite and removed from the lter tubes and rinsed in sterile water for 1 minute followed by air drying. The cemental (outer) sides of the specimens were closed by nail varnish. The 22 specimens were randomly divided into 11 groups with 2 specimens each: sterile water (control) (pH = 6.8), 0.1% cetrimide (CTR) (Sigma Chemical Co, St Louis, MO) (pH = 4.6), 2% sodium hypochlorite (NaOCl) (EMD Chemicals Inc, Darmstadt, Germany) (pH = 12.0), 2% NaOCl + 0.1% CTR (pH = 11.8), 6% NaOCl (pH = 12.4), 6% NaOCl + 0.1% CTR (pH = 12.2), Chlor-Xtra (Vista Dental, Racine, WI) (pH = 9.3), 2% CHX (Sigma Chemical Co) (pH = 6.1), CHX-Plus (Vista Dental) (pH = 5.2), 2/4% iodine potassium iodide (IPI, Sigma Chemical Co) (pH = 8.0), and IPI + 0.1% CTR (pH = 6.2). Fifty microliters of each medicament was placed on the root canal side of each dentin specimen for 1 or 3 minutes. The specimens were then rinsed in sterile water for 1 minute and vertically fractured through the root canal into 2 halves to expose a fresh surface of longitudinally fractured dentin canals for CLSM examination as previously described (13). CLSM Examination The uorescent LIVE/DEAD BacLight Bacterial Viability stain (Molecular Probes, Eugene, OR) containing SYTO 9 and propidium
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Results
A homogenous penetration of E. faecalis deep into the dentinal tubules of the dentin specimens from the root canal side was observed by CLSM (Figs. 1 and 2). With no antibacterial treatment (control group with sterile water), 3% to 4% of the bacteria inside dentin were dead (Table 1). In the experimental groups, the proportion of dead bacteria was between 24% and 70% depending on the disinfecting agent and the time of exposure (Table 1). After 1 minute of treatment, 6% NaOCl + 0.1% CTR was the most effective but with a nonsignicant difference to regular 6% NaOCl and Chlor-Xtra. These 3 were followed by IPI + 0.1% CTR, 2% NaOCl + 0.1% CTR, CHX-Plus, IPI, 2% NaOCl, 0.1% CTR, and 2% CHX. After 3 minutes of exposure, the order of effectiveness was the same with the exception that 0.1% CTR was slightly better than 2% NaOCl and 2% CHX, but the difference was not statistically signicant. The solutions with detergent (except for 6% NaOCl) were equally effective in killing bacteria in dentin in 1 minute as the same solutions without detergent in 3 minutes (P > .05). In general, a higher percentage of dead bacteria was detected in the detergent-containing groups (Table 1). In the 6% NaOCl, 6% NaOCl+0.1% CTR, and Chlor-Xtra groups, dead bacteria could be seen throughout around the 200-mm-deep scanned dentin area in the 3-minute specimens (Fig. 1E2, F2, and G2). Surfactants (unknown in CHX-Plus and Chlor-Xtra, cetrimide in others) lowered the pH by 0.2 to 3.1 pH units depending on the original pH and concentration of the main disinfectant. There was no obvious link between the pH effect of the surfactant and killing effectiveness of the solutions.

Discussion
Wettability has been shown to play an important role in the penetration of disinfecting solutions into small spaces of the root canal system (5). The wettability of a solution depends on its surface tension, which is dened as the force between molecules tending to reduce the surface area of a liquid (19). The decrease of surface tension may be accomplished by the addition of chemicals known as detergents (20), which can destabilize cohesive forces and facilitate the destruction of extracellular polymeric substance matrix and bacterial cell membranes (9, 21). In order to extend the antibacterial capability of disinfecting agents in the endodontic treatment, detergents were introduced to facilitate the medicaments entry into places of difcult access (21). However, the advantage of detergents in disinfecting agents only has been veried by the direct contact of medicaments with biolm or 949

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Figure 1. CLSM of E. faecalisinfected dentinal tubules after exposure to different disinfecting solutions and viability staining. A1, sterile water 1 minute; A2, sterile water 3 minutes; B1, 0.1% CTR 1 minute; B2, 0.1% CTR 3 minutes; C1, 2% NaOCl 1 minute; C2, 2% NaOCl 3 minutes; D1, 2% NaOCl + 0.1% CTR 1 minute; D2, 2% NaOCl + 0.1% CTR 3 minutes; E1, 6% NaOCl 1 minute; E2, 6% NaOCl 3 minutes; F1, 6% NaOCl + 0.1% CTR 1 minute; F2, 6% NaOCl + 0.1% CTR 3 minutes; G1, Chlor-Xtra 1 minute; G2, Chlor-Xtra 3 minutes. P, pulpal side; D, dentin.

planktonic bacteria in vitro (7, 22). Little information exists of the antibacterial efcacy with and without detergents against bacteria invaded into dentinal tubules. Previous studies have shown that dentin has an inhibitory effect on the antibacterial effectiveness of different disinfecting agents (23). It has also been reported that the antibacterial effect of NaOCl is reduced by the various components of dentin (11). Traditionally, attempts to establish the invasion of bacteria into dentin have been predominantly based on culturing methods in which bacteria are grown in liquid media in the root canals of extracted teeth. Haapasalo and rstavik (14) developed a dentin block model for the 950

evaluation of the antibacterial effect in dentin for more than 2 decades ago. More recent studies have observed the presence of live and dead bacteria in the dentinal tubules by CLSM (9, 15). However, creating comparable and strong enough dentin infections has been difcult using culturing methods, making it challenging to quantitatively measure bacterial viability after exposure to disinfecting agents. To improve the predictability of obtaining a strong presence of bacteria deep in dentin, a recently presented technique using the power of centrifugation was applied in this study, resulting in the dense and heavy presence of bacteria deep in the dentinal tubules as veried by CLSM scanning after uorescent staining (13). Despite using the natural
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Figure 2. CLSM of E. faecalisinfected dentinal tubules after exposure to different disinfecting solutions and viability staining. A1, 2% CHX 1 minute; A2, 2% CHX 3 minutes; B1, CHX-Plus 1 minute; B2, CHX-Plus 3 minutes; C1, IPI 1 minute; C2, IPI 3 minutes; D1, IPI + 0.1% CTR 1 minute; D2, IPI + 0.1% CTR 3 minutes. P, pulpal side; D, dentin.
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TABLE 1. Proportion of Dead E. faecalis Cell Volume in a Dentin Infection Model Treated by Different Disinfection Solutions Medicaments
Sterile water 0.1% CTR 2% NaOCl 2% NaOCl + 0.1% CTR 6% NaOCl 6% NaOCl + 0.1% CTR Chlor-Xtra 2% CHX CHX-Plus IPI IPI+0.1% CTR

1 min
0.03 0.01 0.24 0.13b 0.26 0.11bc 0.38 0.13de 0.45 0.13dg 0.48 0.14fg 0.46 0.14dg 0.24 0.10b 0.36 0.14de 0.29 0.10b 0.41 0.13dg
a

3 min
0.04 0.02a 0.36 0.13de 0.34 0.14e 0.47 0.12fg 0.65 0.08h 0.70 0.07h 0.68 0.13h 0.33 0.09ce 0.45 0.12g 0.39 0.15de 0.55 0.12f

Different superscript letters indicate statistically signicant differences between groups (P < .05).

environment (dentin canals) in the present study, it is important to understand that experimental models also have limitations. In our study, only young, fresh cultures were examined. Also, centrifugation packed so many bacteria in the tubules that the resulting infection may be even a greater challenge to disinfecting agents than many in vivo situations. However, the experimental design used in the present study allowed a high level of standardization of the conditions, which is difcult or impossible to achieve in in vivo studies. Detergents have been used in endodontic irrigants such as CHXPlus and MTAD to reduce the surface tension of the solution (24). The results of the present study clearly indicate that the addition of a surfactant increases the antibacterial effect of a variety of disinfecting solutions against bacteria in dentin. However, the mechanism by which cetrimide (and other surfactants in CHX-Plus and Chlor-Xtra) increased the antibacterial effect in dentin is not clear. Cetrimide used alone was equally effective as 2% NaOCl, 2% CHX, and IPI. Therefore, it is possible that the surfactant merely added its own antibacterial effect to the disinfecting solution in the combined solutions. Another possibility is that the surfactants improved the penetration of the solutions into the dentinal tubules. In the main root canal, low surface tension is likely to help the spreading of a solution. However, in the narrow and long dentinal tubules, penetration can occur either by capillary forces or by diffusion/ow into the dentin canals. The mechanisms by which solutions penetrate into dentin tubules is not fully understood, but if capillary forces play a role, low surface tension may in fact even limit the trend of liquid penetration into the tubules (25, 26). Although the relative amount of viable and killed bacteria at various depths in dentin was not measured separately, it seemed that the better performance by surfactant-containing solutions was seen in all layers and not just in the deeper areas of dentin. CTR is a cationic surfactant that reduces the surface tension of liquids and weakens the cohesive forces of bacteria (7). It has usually been used together with other irrigants. The combination of CHX with CTR has been reported to be more efcient against E. faecalis than CHX alone (21). The bactericidal effect of CTR is possibly caused by the fact that CTR targets the cell wall and may have an additive or synergistic effect with other medicaments such as CHX (7). The effectiveness of CTR was also shown with 2% NaOCl and IPI in this study. Iodine compounds are known for their effectiveness against a variety of resistant microbes in in vitro models (27, 28). In the dentin model of the present study, IPI alone killed 29% and 39% E. faecalis cells in 1 and 3 minutes, respectively, whereas over 10% more bacteria were killed with the addition of 0.1% CTR in IPI. The 2 commercial products, CHX-Plus and Chlor-Xtra, were used in the present study as surfactant-containing products to compare their antibacterial effects with CHX and NaOCl at the same concentrations but without surfactant. The exact formulations of these 2 products are not 952

known. According to the manufacturers declaration, CHX-Plus contains proprietary surface modiers to reduce viscosity, and ChlorXtra contains a wetting agent, proprietary surface modiers, and alkylating agents. The results show that better killing by CHX-Plus as compared with regular 2% CHX was comparable to the difference between surfactant-added formulations of 2% NaOCl and IPI and their counterparts without surfactant. CHX-Plus has been reported to kill in vitro biolm by plaque bacteria much faster than regular 2% CHX (8), whereas another study claimed no statistically signicant difference between 2% CHX and CHX-Plus against 48-hour E. faecalis in planktonic culture (29). In accordance with a previous study using the same experimental design (13), high-concentration NaOCl (6%) showed a signicantly stronger antibacterial effect against E. faecalis than low-concentration NaOCl (2%) and any other solution tested. Interestingly, there was no difference between the 3 6% NaOCl solutions regarding their ability to kill E. faecalis in dentin (Table 1). This result supports the ndings by Williamson et al (29) who reported no statistically signicant difference between 6% NaOCl and Chlor-Xtra in killing E. faecalis biolm. One possible explanation for this is that at this high concentration NaOCl even without surfactants reaches maximal killing within the timeframes selected for disinfectant exposure, and, therefore, the added surfactants cannot contribute to any increase in antibacterial activity. A recent study conrmed that the surface tension of 3 NaOCl-based irrigants including Chlor-Xtra was signicantly lower than that of regular 5.25% NaOCl (4). Other studies have indicated that the addition of surfactants enhances the ability of NaOCl to dissolve tissue (3, 6, 30). In conclusion, using the new dentin infection model, viability staining, and confocal microscopy, the results showed that with the exception of 6% NaOCl, the addition of a detergent (such as cetrimide) in the disinfecting solutions can increase their antibacterial effect against bacteria in infected dentin. Besides the combined mode of application, the use of cetrimide as a single agent showed a good antibacterial effect against E. faecalis.

Acknowledgments
The authors deny any conicts of interest related to this study.

References
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