Afterward, the microbiologist adjusted each broth culture suspension of bacteria and C. albicans to No. 1 MacFarland standard (approximately 3 x 10⁸ cells/mL). He dispersed 100-microliter aliquots of each microbial suspension (except P. endodontalis) on the surface of Mueller-Hinton agar medium (Merck, Darmstadt, Germany) until the surface was covered. The inoculated plates were dried for 15 minutes at 37 C. The microbiologist dispersed aliquots of the suspension containing P. endodontalis (100 µL) on 100-millimeter petri dishes containing anaerobe basal agar medium (Oxoid, Unipath, Hampshire, England).

The microbiologist aseptically transferred three Resilon cones (0.02 taper points) and three conventional gutta-percha cones (number 35 size) (ML.029, DiaDent Group, Chongju, South Korea) into separate halves of each previously inoculated plate.

Subsequently, the microbiologist placed the agar plates with the aerobic bacteria in an incubator and incubated them aerobically for 24, 48 and 72 hours at 37 C.

The microbiologist placed the agar plates with the anaerobic bacterium (P. endodontalis) into an anaerobic culture jar (Oxoid AnaeroJar AG025A, Unipath) with a GasPak (Oxoid Anaerogen System AN025A, Unipath) anaerobic atmosphere and incubated them for 24, 48 and 72 hours at 37 C.

We maintained the positive and negative controls—namely, the inoculated plates and the uninoculated plates, respectively—for the same time and under the same laboratory conditions.

After each incubation interval, we measured the diameters of zones of microbial inhibition in millimeters. For each strain, we performed experiments in triplicate and determined the mean value.

We compared the means of the diameters via one-way analysis of variance and then used Duncan’s method to make the necessary pairwise comparisons. We established statistical significance at P < .05.

	RESULTS

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The table shows the means of the diameters of the inhibition zones for the tested materials at different periods. Trials in triplicate yielded consistent results, and uniform growth was evident on all control plates.

The results showed no significant difference between the Resilon and conventional gutta-percha groups.

The Resilon cones exhibited no antimicrobial effect against any of the bacteria tested, except for S. aureus. The cones showed antimicrobial efficacy against S. aureus during the first 24-hour period (P < .05). However, after 48 and 72 hours, the cones no longer inhibited the growth of S. aureus. In addition, the Resilon cones demonstrated no antifungal activity in any of the three periods.

The gutta-percha cones did not exhibit any antibacterial or antifungal effects against any of the tested microorganisms for all three periods.

Thus, the results showed no significant difference between the Resilon and conventional gutta-percha groups (P > .05).

	DISCUSSION

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The antimicrobial activity of root canal filling material and sealer may help eliminate residual microorganisms unaffected by chemomechanical preparation or intracanal medication, and it also may discourage infection.^1,7 Therefore, to avoid the growth of microbial attachments, the root canal filling materials should have an antibacterial effect, if possible.

Several researchers have investigated the antimicrobial effects of different endodontic materials on various bacteria by using different techniques. The agar diffusion test we used in this study is one of the methods most frequently used for assessing the antimicrobial activity of endodontic materials.^4,8–10 However, the environment in the dentinal tubule certainly is dissimilar to the in vitro conditions of an agar plate. Therefore, the size of the inhibition zone does not show definitively the antimicrobial efficacy of the material. Solubility and diffusibility can influence the antimicrobial efficacy of the material.¹¹

Al-Khatib and colleagues¹² reported that the antimicrobial activity of a material may differ depending on the microorganisms tested and the penetrating ability of the tested material in agar. However, the agar diffusion method permits direct comparisons between materials, and it indicates which core material is likely to exhibit antimicrobial activity within the root canal system. Because of the obvious limitations of in vitro studies, clinicians should use strict caution when drawing clinical inferences. In addition, we were unable to identify any research related to the buffering effect of agar on Resilon. Buffering may reduce the antimicrobial activity of core materials.

Bacteria tested. We tested the antimicrobial effects of Resilon by using the bacteria that are found most frequently in the root canal flora.^7,13,14 We tested the cones against the five most common species of pathogens. However, an infected root canal usually contains many species of pathogens. Also, root canals contain necrotic and/or viable tissues and tissue fluids, which may reduce the antimicrobial activity of the root canal core material and sealer. In addition, the antibacterial effects of endodontic materials depend on the quantity and virulence of the pathogens.¹ We might interpret this as microbial selection, which may lead to a quantitative or qualitative shift in the composition of the endodontic microflora.

Secondary infections. Aerobic and facultative microorganisms (bacteria and fungi), usually minor constituents of primary infections, have been found in cases of protracted endodontic treatment and in flare-ups, and they are associated with endodontic failures.^7,14 These microorganisms can enter the root canal system during endodontic therapy and cause secondary infections by exploiting opportunities created by the removal of the primary infection and by surviving in the low-nutrient environment of the treated root canal.⁷

In addition to evaluating anaerobic bacteria, investigators need to evaluate the antimicrobial activity of endodontic materials against aerobic and facultative microorganisms. Clinicians commonly find E. faecalis, P. aeruginosa and S. aureus in infected root canals, and these microorganisms are resistant to the calcium hydroxide–based intracanal medications used routinely in endodontic treatment.¹⁵ In view of the prevalence of facultative anaerobes and aerobes such as E. faecalis, S. aureus and P. aeruginosa in cases of unsuccessful endodontic treatment,^3,7,16 clinicians should direct all stages of root canal treatment toward eliminating endodontic microbial flora.

Test organism. We chose C. albicans as a test organism in this study because it has been found in infected root canals and in periradicular tissue.¹⁷ It may enter the pulp through dentinal tubules or deep carious lesions or as a contaminant from the oral microflora during endodontic therapy. The results of our study showed the root canal filling cone to have no antifungal efficacy against C. albicans.

Gutta-percha. Conventional gutta-percha also exhibited no antimicrobial efficacy against tested microorganisms in this study. Therefore, our findings do not support those of Moorer and Genet.⁵ The discrepancies between studies may be due to differences in methodology, such as inoculum density, agar viscosity or type of agar medium.

Melker and colleagues⁴ detected no antibacterial properties of Resilon against Actinomyces israelii, Actinomyces naeslundIi, E. faecalis and Fusobacterium nucleatum. However, our results regarding E. faecalis support those of Melker and colleagues.⁴ In addition, Shipper and colleagues¹⁸ analyzed Resilon core material and Epiphany sealer (Pentron Clinical Technologies, Wallingford, Conn.), as well as gutta-percha/AH 26 sealer (Dentsply Maillefer, Tulsa, Okla.) by using a bacterial penetration test; the results showed less penetration for Streptococcus mutans and E. faecalis than for other bacteria.

This good performance of Resilon in bacterial leakage studies may be explained by the effective attachment of the Epiphany sealer to the root canal walls and to the Resilon core material, or perhaps the sealer has antibacterial efficacy with regard to these bacteria.¹⁹ Although Resilon exhibited antimicrobial efficacy against one microorganism in our study, its antimicrobial and antifungal efficiency was not superior to that of conventional gutta-percha. However, the antimicrobial activity of various components of sealers plays a role that is as important as the antimicrobial activity of the filling materials in the success of endodontic treatment. Thus, if endodontic treatment requires an antimicrobial and anti-fungal effect, the clinician should use a root canal filling material or sealer that is composed of something other than Resilon.

Further research is needed to determine the antimicrobial effectiveness of Resilon with different assaying methods, as well as to evaluate potential bacterial penetration through gutta-percha and Resilon cones and the bacterial growth within these cones.

	CONCLUSION

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Results obtained from in vitro antimicrobial efficacy studies do not have direct clinical applicability, but they do permit comparisons. In vitro tests can identify only those materials that have the potential to inhibit microbial growth in the local microenvironment of the root canal. The results of our study indicate that the only antimicrobial activity of Resilon cones was against S. aureus in the first 24 hours. Therefore, these cones do not appear to be superior to conventional gutta-percha cones with regard to antibacterial efficacy.