Abstract
Objectives: To compare the antibacterial effect of AH-26, white-coloured mineral trioxide aggregate (WMTA) and CPM sealer against Staphylococcus aureus and Streptococcus mutans after 24 h and 1 week. Materials and methods: All materials were prepared according to the manufacturers’ directions immediately before testing. The antibacterial effect of each agent was determined by measuring the diameter of the zone of inhibition in millimetres after incubation at 37 °C for 24 h and 7 days in a humid atmosphere. Each test was repeated three times. Results: In the 24-h and 7-day samples, the antibacterial activity of AH-26 was significantly greater than that of the two other materials tested against both bacteria (P < 0.05). The antibacterial activities of CPM sealer and WMTA against S. aureus were not significantly different (P > 0.05) in 24-h or 7-day samples, but CPM sealer demonstrated a significantly greater effect than WMTA against S. mutans (P < 0.05). Conclusion: All products studied exhibited antibacterial activity. However, in 24-h and 7-day samples, AH-26 exerted the greatest activity against both organisms tested.
Key words: Agar diffusion test, AH-26 sealer, CPM sealer, MTA, Staphylococcus aureus, Streptococcus mutans
INTRODUCTION
The main purposes of root canal therapy include cleaning, shaping and three-dimensional obturation of the root canal system1. Microorganisms and their byproducts play a major role in the induction, progression and perpetuation of pulpal and periapical pathosis2. Although considerable microbial reduction can be achieved by biomechanical instrumentation, irrigation and intracanal medication, studies reported in the literature have demonstrated the presence of bacteria in dentinal tubules and cementum after treatment3., 4.. For this reason, and particularly when pulpal necrosis and apical periodontitis are present, the choice of a sealer with antimicrobial activity might help to decrease or avoid the growth of the remaining microorganisms5.
In most root canal filling techniques, core materials associated with endodontic sealers are used. Core obturating materials, such as gutta percha, usually occupy space, whereas the endodontic sealers enhance the possible attainment of an impervious seal by serving as a filler for canal irregularities and minor discrepancies between the root canal wall and the core material. Several properties are important for an adequate endodontic sealer. Among them are sealing ability, biocompatibility and antimicrobial activity, which can influence the success of root canal treatment6. Furthermore, sealers that possess both optimum flow ability and antimicrobial properties might theoretically eliminate residual microorganisms located in confined areas of the root canal system6., 7..
Studies have reported that several endodontic sealers have antimicrobial effects8., 9., 10.. Sealers having antimicrobial effects may help to eliminate residual microorganisms unaffected by the influence of both chemomechanical preparation and intracanal medication. In addition, they may limit the ingress of microorganisms from saliva, impeding or, at a minimum, retarding the complete recontamination of the root canal after saliva challenge6. To date, no study has been conducted on the antimicrobial activity of a new mineral trioxide aggregate (MTA)-based sealer (Endo CPM Sealer; CPM Sealer; EGEO SRL, Buenos Aires, Argentina). Therefore, it was decided to evaluate its antibacterial activity and to compare it with AH-26 (a resin-based root canal sealer) and conventional white-coloured MTA after 24 h and 1 week.
MATERIALS AND METHODS
The materials used in the present study were AH-26 (Dentsply, De Trey, Konstanz, Germany), Endo CPM Sealer (EGEO SRL, Buenos Aires, Argentina) and white-coloured MTA (Dentsply, Tulsa Dental, Tulsa, OK, USA). All materials were prepared according to the manufacturers’ directions immediately before testing. The microorganisms used in this study included Staphylococcus aureus and Streptococcus mutans.
Overnight cultures of the microorganisms were used. The bacteria were grown in tryptic–soy broth and adjusted to the turbidity of a 0.5 McFarland BaSO4 standard (∼1.5 × 108 colony-forming units/mL). Forty-four Petri dishes containing tryptic–soy agar enriched with 5% defibrinated sheep blood and supplemented with haemin and vitamin K were seeded with bacteria. For the collection of sheep blood, action was taken to minimise pain and the procedures were performed in accordance with local laws and regulations. Three Petri dishes were used for both 24-h evaluation and 7-day examination. Seeding was performed using sterile cotton-tipped applicators that were brushed across the agar surface. Three wells (depth, 5 mm; diameter, 6 mm) were punched in each agar plate and filled with freshly mixed materials. The plates were then maintained at room temperature for 2 h for pre-diffusion of the material.
Later, the antimicrobial effect of each material was determined by measuring the diameter of the zone of inhibition in millimetres after incubation at 37 °C for 24 h and 7 days in a humid atmosphere. Each test was repeated three times.
Data were analysed using analysis of variance (ANOVA) and Tukey’s test. Differences at the 5% level (P < 0.05) were considered to be statistically significant.
RESULTS
All materials tested demonstrated some antibacterial activity. In the 24-h and 7-day samples, the antibacterial activity of AH-Plus was significantly greater than that of the two other materials tested on both bacteria (P < 0.05). At both time periods, the antibacterial activities of CPM sealer and GuttaFlow on S. aureus were not significantly different (P > 0.05), but RS had a significantly greater effect than white MTA on S. mutans (P < 0.05). The means of the diameters of the zones of microbial inhibition for each sealer against each microorganism are shown in Tables 1 and 2.
Table 1.
Means of the diameters (mm) of the zones of bacterial growth inhibition provided by the tested materials in 24-h samples
| Bacterial species | AH-26 sealer | CPM sealer | White MTA | |||
|---|---|---|---|---|---|---|
| Mean | SD | Mean | SD | Mean | SD | |
| Staphylococcus aureus | 25 | 3.81 | 13.8 | 2.75 | 14.59 | 1.87 |
| Streptococcus mutans | 35.20 | 3.29 | 22.50 | 1.96 | 17.80 | 2.92 |
MTA, mineral trioxide aggregate; SD, standard deviation.
Table 2.
Means of the diameters (mm) of the zones of bacterial growth inhibition provided by the tested materials in 7-day samples
| Bacterial species | AH-26 sealer | CPM sealer | White MTA | |||
|---|---|---|---|---|---|---|
| Mean | SD | Mean | SD | Mean | SD | |
| Staphylococcus aureus | 17 | 1.29 | 13.20 | 2.57 | 13.87 | 0.95 |
| Streptococcus mutans | 22.50 | 2.95 | 21.80 | 1.89 | 12.20 | 1.50 |
MTA, mineral trioxide aggregate; SD, standard deviation.
DISCUSSION
Microorganisms are considered to be the primary aetiological agents in endodontic diseases8. The agar diffusion test has been widely used to evaluate the antibacterial activity of dental materials5., 7., 8., 9. Several factors that are relevant for the diffusion capacity of materials in agar must be considered, such as the contact between the experimental material and agar, molecular weight, size and shape of the antimicrobial agent, load and concentration of the test material, agar gel viscosity and ionic concentration in relation to the medium. Furthermore, the control and standardisation of the inoculation density, evaluation of results, selection of agar medium, selection of microorganisms, depth of agar medium, incubation temperature of plates and reading point of inhibition haloes are restricting factors affecting the dynamics and variability of diffusion tests in an agar medium10. Nevertheless, if most of these variables are carefully controlled, consistent and reproducible results may be obtained5. As a result of the obvious limitations of in vitro studies, clinical inferences should be drawn with strict caution.
Although aerobic and facultative bacteria are usually minor constituents of primary infections, they have been found in cases in which the treatment has been protracted, in flare-ups and associated with endodontic failures. Therefore, in addition to anaerobic bacteria, it is also important to evaluate the antimicrobial activity of endodontic materials against these organisms11. Furthermore, the test microorganisms used in the present study have been employed in several studies to evaluate the antimicrobial effects of root canal sealers8., 10.. Most endodontic sealers possess antimicrobial components that need to be released from the sealer matrix to be effective12.
In the present study, the antibacterial activities of freshly mixed and set materials were assessed. In 24-h and 7-day samples, AH-26 showed significantly greater antibacterial activity relative to the other materials tested (P < 0.05). The antibacterial effect of this resin-based sealer may be related to hexamethylenetetramine. According to Eldeniz et al.13, both freshly mixed and set root-end filling materials, including MTA, amalgam, intermediate restorative material (IRM), Super bond C&B, Geristore, Dyract and Clearfil APX composite with SE bond, demonstrated antibacterial activity against S. aureus, Enterococcus faecalis and Pseudomonas aeruginosa, which is in accordance with the findings of the present study.
MTA consists of 50–75% (by weight) of calcium oxide and 15–25% of silicon dioxide. These two components together comprise 70–95% of the cement. When these raw materials are blended, they produce tricalcium silicate, dicalcium silicate, tricalcium aluminate and tetracalcium aluminoferrite. On addition of water, the cement hydrates to form silicate hydrate gel. It has been shown that, on hydration, MTA produces calcium hydroxide. Thus, it can be concluded that both MTA and calcium hydroxide may have a similar mechanism of action14.
CPM sealer consists of a powder or liquid. The powder consists of fine hydrophilic particles that form a colloidal gel in the presence of moisture. It becomes solid and forms a hard sealer in 1 h. The main components are tricalcium silicate, tricalcium oxide, tricalcium aluminate and other oxides. The liquid consists of saline solution and calcium chloride15.
It should be noted that the antibacterial activity of set materials is decreased relative to that of freshly mixed ones. This can be attributed to a decrease in the release of antibacterial components from the tested materials.
Our findings demonstrated that the decrease in the antibacterial activity of set CPM sealer was less than that of the two other materials. The liquid CPM sealer consists of saline solution and calcium chloride. Antunes Bortoluzzi et al.16 revealed that the addition of calcium chloride to white MTA caused a significant increase in calcium release. Therefore, a smaller decrease in the antibacterial activity of CPM sealer, relative to AH-26 sealer and MTA, is justified.
The antimicrobial activity of Ca(OH)2 is related to the release of hydroxyl ions in an aqueous environment17. Hydroxyl ions are highly oxidant free radicals that show extreme reactivity with several biomolecules. This reactivity is high and indiscriminate, so that this free radical rarely diffuses away from its site of generation18.
Using an agar diffusion method, Sipert et al.19 showed that MTA and Portland cement inhibited the growth of several microorganisms, including Candida albicans, S. aureus, E. faecalis, Staphylococcus epidermidis and P. aeruginosa, which is in accordance with the findings of the present study. In another study, Ribeiro et al.20 demonstrated that the antibacterial activity of MTA against Escherichia coli under aerobic conditions was promoted by the induction of reactive oxygen species. In another study, Ribeiro et al.21 found that E. faecalis was susceptible to grey MTA, as well as calcium hydroxide, after incubation in aerobic conditions. They proposed that an oxygen-enriched medium favours the antimicrobial activity of MTA. Estrela et al.22 showed no antibacterial activity of MTA and Portland cement against S. aureus, E. faecalis, P. aeruginosa, Bacillus subtilis and C. albicans. The antibacterial effect of MTA has been attributed to its high pH or to substances that are released from MTA into the medium.
The results of the agar diffusion test do not depend solely on the toxicity of the material for the particular microorganism, but are strongly influenced by the diffusing ability of the material across the medium.
Bodrumlu et al.23 evaluated the antibacterial activity of six root canal sealers (Diaket, Endomethasone, AH-26, Sealapex, Sultan and Epiphany) on E. faecalis using the agar diffusion method. Their results showed that all of the sealers caused bacterial growth inhibition in descending order as follows: Endomethasone > Sultan > Sealapex > Diaket > Epiphany > AH-26. They also found that the effectiveness of the root canal sealers decreased with increasing duration of incubation, which is in agreement with the results of the present study. Tabrizizadeh and Mohammadi24 evaluated the antibacterial activity of AH-26, Dorifill (a zinc oxide eugenol [ZOE]-based sealer) and pure ZOE on S. aureus and S. mutans after 24 h and 7 days. Their findings revealed that, in 24-h samples, AH-26 showed greater antibacterial activity. However, in 7-day samples, pure ZOE showed greater antibacterial activity. They also found that the effectiveness of the root canal sealers decreased with increasing duration of incubation, which is in agreement with the results of the present study. However, the results of the present study contrast with the findings of Cobankara et al.9, who found that time had no effect on the antibacterial activity of sealers. Within the limitations of the present study, the antibacterial activity of the AH-26 sealer was significantly greater than that of the two other materials.
Acknowledgement
The authors wish to thank research officials of the Hamedan University of Medical Sciences, Hamedan, Iran.
Conflict of interest
The authors report no conflict of interest.
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