JOURNAL OF ENDODONTICS Copyright 2003 by The American Association of Endodontists

Printed in U.S.A. VOL. 29, NO. 4, APRIL 2003

Effectiveness of Stannous Fluoride and Calcium Hydroxide Against Enterococcus faecalis

Andre K. Mickel, DDS, MSD, Priya Sharma, BDS, and Sami Chogle, BDS, MSD

The objective of this study was to evaluate the antimicrobial activity of stannous fluoride, calcium hydroxide, or the two in combination against Enterococcus faecalis. Fifteen Mueller-Hinton agar plates were used to conduct a disc diffusion susceptibility test. The plates were inoculated with E. faecalis. The groups tested were: (a) stannous fluoride (Stanimax); (b) calcium hydroxide; and (c) a combination of stannous fluoride and calcium hydroxide. Three filter paper discs, each soaked with one of the groups, were placed along with an antibiotic control (ampicillin) on each agar plate. The plates were then incubated at 37C. The zones of inhibition were measured at 24 and 48 h. One-way ANOVA revealed a statistically significant difference (p < 0.05) between the three groups. Stannous fluoride displayed the widest zone (1.7 mm), followed by the combination of stannous fluoride and calcium hydroxide (1.1 mm), whereas calcium hydroxide by itself displayed a minimal zone of inhibition (0.05 mm).

asserted that all biological properties of calcium hydroxide are due to the ionic dissociation into Ca2 and OH ions (9). Viscous and oily vehicles promote continuous slow diffusion of ions with low solubility (10). Various authors have suggested mixing calcium hydroxide with rinses like chlorhexidine and antibiotics like metronidazole and ciprofloxacin (1113). Stanimax (SDI Labs, Glenview, IL) is a relatively new periodontal rinse with 0.63% stannous fluoride in a nonaqueous base. Studies on antimicrobial effects of various concentrations of stannous fluoride have reported positive results (14, 15). Oosterwaal et al. (16) demonstrated bacterial reductions comparable to those by chlorhexidine. The purpose of this study was to evaluate the antibacterial activity of stannous fluoride separately and in combination with calcium hydroxide against E. faecalis. MATERIALS AND METHODS The three solutions tested were Stanimax in sterile water (0.3% stannous fluoride), calcium hydroxide, and a combination of calcium hydroxide with Stanimax. Ampicillin was used as a positive control. Antibacterial activity was evaluated against the Grampositive facultative anaerobic microorganism E. faecalis (ATTCC 29212). The bacteria were incubated in brain-heart infusion (BHI) broth. The E. faecalis culture medium turned turbid after 24 h. The turbidity was assessed using a Colorimeter. The paper disk method to determine the susceptibility of the microorganism to a particular chemotherapeutic agent was used. Ten Mueller-Hinton plates were streaked with the E. faecalis inoculum. The chemotherapeutic (test solution) saturated paper disks (6 mm in diameter) were placed on the plates. Measurement of zones of inhibition was performed after 24 and 48 h of incubation at 37C for optimum growth of the bacteria. The zones of inhibition were measured from the edge of the paper disk with a transparent ruler and recorded. To allow a general view of the antibacterial effectiveness of the test solutions, data were statistically analyzed using ANOVA and t tests. Significance was established at p 0.05. RESULTS Stanimax created the largest mean zone of inhibition for E. faecalis (1.65 mm) with the largest single zone being 3 mm. The mean zones of inhibition along with variance and maximum and minimum zones are presented in Table 1. Calcium hydroxide alone
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Although a multitude of instrumentation and irrigation techniques exist, debris is often left behind (1, 2). Persistent intraradicular infection contributes to failure of root canal treatment (3). In a study by Sundqvist et al. (4) a high frequency of Enterococcus faecalis, a Gram-positive facultative anaerobe, was observed from the root canals of teeth in which the previous treatment had failed. In addition only 33% of teeth that were positive for E. faecalis at the time of reobturation were successful. E. faecalis seems to be highly resistant to medications used during treatment and is one of the few microorganisms that has been shown to resist the antibacterial effect of calcium hydroxide (5). Calcium hydroxide, a white, odorless powder with a high pH and low solubility in water, was introduced into dentistry by Hermann (6) in the 1930s to promote healing in many clinical situations. The medication is widely used as an intraappointment dressing in endodontic therapy and possesses significant antibacterial properties (7). Sjo gren et al. (8) suggested an intracanal dressing of calcium hydroxide for a week to counter bacteria remaining after initial instrumentation and irrigation. Studies have

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Mickel et al. TABLE 1. Descriptive statistics Experimental Groups Stanimax Calcium hydroxide Stanimax with calcium hydroxide n 10 10 10 Mean 1.650 0.450 1.050 Variance 0.5583 0.1361 0.2472 Minimum 0.50 0.00 0.50

Journal of Endodontics

Maximum 3.00 1.00 1.50

p 0.05.

created an average zone of inhibition of 0.45 mm. The combination of stannous fluoride with calcium hydroxide produced an average zone of 1.05 mm with the largest zone being 1.5 mm. The positive control displayed a mean inhibition zone of 10.4 mm. There was no statistical difference between the 24- and 48-h samples. The differences between the three experimental groups were statistically significant. The stannous fluoride group produced the largest mean zone of inhibition, which was statistically significant (p 0.05). DISCUSSION Based on the averages of the zones of bacterial growth inhibition, the antibacterial effects of the solutions could be ranked from strongest to weakest as follows: stannous fluoride 0.3%, stannous fluoride and calcium hydroxide, and calcium hydroxide. In addition to the toxicity, zones of inhibition may be altered by diffusibility of the irrigant through the agar, interaction with media components, and microenvironmental conditions in an in vivo environment. The plaque inhibitory effects of 0.3% stannous fluoride were reported to be comparable to that of 0.5% chlorhexidine (17). Shapira et al. (18) reported that stannous fluoride enhances the oxygen-dependent antibacterial activity of neutrophils, which may contribute to more efficient elimination of bacteria from the periodontal environment. Andres et al. (14) suggested that the bacterial effects resulted from the combined action of tin and fluoride ions in stannous fluoride. Hughes et al. (19) suggested that the ions from stannous fluoride may bind with phosphate groups of lipoteichoic acid (LTA) by electrostatic interaction and interfere with membrane stability and bacterial metabolism. Despite an aqueous vehicle (sterile water) used for calcium hydroxide, which causes maximum dissociation of ions (20), this group still showed the least zone of bacterial inhibition. Our study is in agreement with the study by Safavi et al. (5), which reported E. faecalis to resist the antibacterial effect of calcium hydroxide. The combination of stannous fluoride and calcium hydroxide yielded a moderate zone of bacterial inhibition. This could be because of the nature of the vehicle for calcium hydroxide and possible interaction between stannous fluoride and calcium hydroxide. Further studies are required for any form of clinical application of the results of this in vitro study.
Dr. Mickel is director, Postgraduate Endodontics Program; Dr. Sharma is a second-year postdoctoral resident; and Dr. Chogle is assistant professor, Department of Endodontics, Case Western Reserve University School of

Dentistry, Cleveland, OH. Address requests for reprints to Dr. Andre K. Mickel, CWRU School of Dentistry, Department of Endodontics, 10900 Euclid Avenue, Cleveland, OH 44106-4905.

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