International Dental Journal 2012; 62: 70–73

doi: 10.1111/j.1875-595X.2011.00090.x

Antibacterial activity of a new mineral trioxideaggregate-based root canal sealer

Zahed Mohammadi1,2, Luciano Giardino3, Flavio Palazzi4 and Sousan Shalavi5

1Department of Endodontics, Hamedan University of Medical Sciences, Hamedan, Iran; 2Iranian Center for Endodontic Research (ICER), Tehran,Iran; 3Private Practice, Crotone, Italy; 4Department of Odontostomatological and Maxillofacial Sciences, Federico II University of Naples, Naples,Italy; 5Hamedan University of Medical Sciences, Hamedan, Iran.

Objectives: To compare the antibacterial effect of AH-26, white-coloured mineral trioxide aggregate (WMTA) and CPMsealer against Staphylococcus aureus and Streptococcus mutans after 24 h and 1 week. Materials and methods: All materialswere prepared according to the manufacturers’ directions immediately before testing. The antibacterial effect of each agentwas determined by measuring the diameter of the zone of inhibition in millimetres after incubation at 37 �C for 24 h and7 days in a humid atmosphere. Each test was repeated three times. Results: In the 24-h and 7-day samples, the antibacterialactivity of AH-26 was significantly greater than that of the two other materials tested against both bacteria (P < 0.05). Theantibacterial 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 thegreatest activity against both organisms tested.

Key words: Agar diffusion test, AH-26 sealer, CPM sealer, MTA, Staphylococcus aureus, Streptococcus mutans

The main purposes of root canal therapy includecleaning, shaping and three-dimensional obturation ofthe root canal system1. Microorganisms and theirbyproducts play a major role in the induction, progres-sion and perpetuation of pulpal and periapical patho-sis2. Although considerable microbial reduction can beachieved by biomechanical instrumentation, irrigationand intracanal medication, studies reported in theliterature have demonstrated the presence of bacteriain dentinal tubules and cementum after treatment3,4.For this reason, and particularly when pulpal necrosisand apical periodontitis are present, the choice of asealer with antimicrobial activity might help to decreaseor avoid the growth of the remaining microorganisms5.

In most root canal filling techniques, core materialsassociated with endodontic sealers are used. Coreobturating materials, such as gutta percha, usuallyoccupy space, whereas the endodontic sealers enhancethe possible attainment of an impervious seal by servingas a filler for canal irregularities and minor discrepan-cies between the root canal wall and the core material.Several properties are important for an adequateendodontic sealer. Among them are sealing ability,biocompatibility and antimicrobial activity, which caninfluence the success of root canal treatment6. Further-

more, sealers that possess both optimum flow abilityand antimicrobial properties might theoretically elim-inate residual microorganisms located in confined areasof the root canal system6,7.

Studies have reported that several endodonticsealers have antimicrobial effects8–10. Sealers havingantimicrobial effects may help to eliminate residualmicroorganisms unaffected by the influence of bothchemomechanical preparation and intracanal medica-tion. In addition, they may limit the ingress ofmicroorganisms from saliva, impeding or, at a mini-mum, retarding the complete recontamination of theroot canal after saliva challenge6. To date, no study hasbeen conducted on the antimicrobial activity of a newmineral trioxide aggregate (MTA)-based sealer (EndoCPM Sealer; CPM Sealer; EGEO SRL, Buenos Aires,Argentina). Therefore, it was decided to evaluate itsantibacterial activity and to compare it with AH-26 (aresin-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

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Sealer (EGEO SRL, Buenos Aires, Argentina) andwhite-coloured MTA (Dentsply, Tulsa Dental, Tulsa,OK, USA). All materials were prepared according to themanufacturers’ directions immediately before testing.The microorganisms used in this study included Staph-ylococcus aureus and Streptococcus mutans.

Overnight cultures of the microorganisms were used.The bacteria were grown in tryptic–soy broth andadjusted 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 enrichedwith 5% defibrinated sheep blood and supplementedwith haemin and vitamin K were seeded with bacteria.For the collection of sheep blood, action was taken tominimise pain and the procedures were performed inaccordance with local laws and regulations. Three Petridishes were used for both 24-h evaluation and 7-dayexamination. Seeding was performed using sterilecotton-tipped applicators that were brushed across theagar surface. Three wells (depth, 5 mm; diameter,6 mm) were punched in each agar plate and filled withfreshly mixed materials. The plates were then main-tained at room temperature for 2 h for pre-diffusion ofthe material.

Later, the antimicrobial effect of each material wasdetermined by measuring the diameter of the zone ofinhibition in millimetres after incubation at 37 �C for24 h and 7 days in a humid atmosphere. Each test wasrepeated 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 statisticallysignificant.

RESULTS

All materials tested demonstrated some antibacterialactivity. In the 24-h and 7-day samples, the antibacte-rial activity of AH-Plus was significantly greater thanthat of the two other materials tested on both bacteria(P < 0.05). At both time periods, the antibacterialactivities of CPM sealer and GuttaFlow on S. aureuswere not significantly different (P > 0.05), but RS had asignificantly greater effect than white MTA on S. mu-

tans (P < 0.05). The means of the diameters of thezones of microbial inhibition for each sealer againsteach microorganism are shown in Tables 1 and 2.

DISCUSSION

Microorganisms are considered to be the primaryaetiological agents in endodontic diseases8. The agardiffusion test has been widely used to evaluate theantibacterial activity of dental materials5,7–9 Severalfactors that are relevant for the diffusion capacity ofmaterials in agar must be considered, such as thecontact between the experimental material and agar,molecular weight, size and shape of the antimicrobialagent, load and concentration of the test material, agargel viscosity and ionic concentration in relation to themedium. Furthermore, the control and standardisationof the inoculation density, evaluation of results, selec-tion of agar medium, selection of microorganisms,depth of agar medium, incubation temperature of platesand reading point of inhibition haloes are restrictingfactors affecting the dynamics and variability ofdiffusion tests in an agar medium10. Nevertheless, ifmost of these variables are carefully controlled, consis-tent and reproducible results may be obtained5. As aresult of the obvious limitations of in vitro studies,clinical inferences should be drawn with strict caution.

Although aerobic and facultative bacteria are usuallyminor constituents of primary infections, they havebeen found in cases in which the treatment has beenprotracted, in flare-ups and associated with endodonticfailures. Therefore, in addition to anaerobic bacteria, itis also important to evaluate the antimicrobial activityof endodontic materials against these organisms11.Furthermore, the test microorganisms used in thepresent study have been employed in several studiesto evaluate the antimicrobial effects of root canalsealers8,10. Most endodontic sealers possess antimicro-bial components that need to be released from thesealer matrix to be effective12.

In the present study, the antibacterial activities offreshly mixed and set materials were assessed. In 24-hand 7-day samples, AH-26 showed significantly greaterantibacterial activity relative to the other materials

Table 1 Means of the diameters (mm) of the zones ofbacterial growth inhibition provided by the testedmaterials in 24-h samples

Bacterial species AH-26 sealer CPM sealer White MTA

Mean SD Mean SD Mean SD

Staphylococcusaureus

25 3.81 13.8 2.75 14.59 1.87

Streptococcusmutans

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 ofbacterial growth inhibition provided by the testedmaterials in 7-day samples

Bacterial species AH-26 sealer CPM sealer White MTA

Mean SD Mean SD Mean SD

Staphylococcusaureus

17 1.29 13.20 2.57 13.87 0.95

Streptococcusmutans

22.50 2.95 21.80 1.89 12.20 1.50

MTA, mineral trioxide aggregate; SD, standard deviation.

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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 andset root-end filling materials, including MTA, amalgam,intermediate restorative material (IRM), Super bondC&B, Geristore, Dyract and Clearfil APX compositewith SE bond, demonstrated antibacterial activityagainst S. aureus, Enterococcus faecalis and Pseudo-monas aeruginosa, which is in accordance with thefindings of the present study.

MTA consists of 50–75% (by weight) of calciumoxide and 15–25% of silicon dioxide. These twocomponents together comprise 70–95% of the cement.When these raw materials are blended, they producetricalcium silicate, dicalcium silicate, tricalcium alumi-nate and tetracalcium aluminoferrite. On addition ofwater, the cement hydrates to form silicate hydrate gel.It has been shown that, on hydration, MTA producescalcium hydroxide. Thus, it can be concluded that bothMTA and calcium hydroxide may have a similarmechanism of action14.

CPM sealer consists of a powder or liquid. Thepowder consists of fine hydrophilic particles that form acolloidal gel in the presence of moisture. It becomessolid and forms a hard sealer in 1 h. The maincomponents are tricalcium silicate, tricalcium oxide,tricalcium aluminate and other oxides. The liquidconsists of saline solution and calcium chloride15.

It should be noted that the antibacterial activity of setmaterials is decreased relative to that of freshly mixedones. This can be attributed to a decrease in the releaseof antibacterial components from the tested materials.

Our findings demonstrated that the decrease in theantibacterial activity of set CPM sealer was less thanthat of the two other materials. The liquid CPM sealerconsists of saline solution and calcium chloride.Antunes Bortoluzzi et al.16 revealed that the additionof calcium chloride to white MTA caused a significantincrease in calcium release. Therefore, a smallerdecrease 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 tothe release of hydroxyl ions in an aqueous environ-ment17. Hydroxyl ions are highly oxidant free radicalsthat show extreme reactivity with several biomolecules.This reactivity is high and indiscriminate, so that thisfree radical rarely diffuses away from its site ofgeneration18.

Using an agar diffusion method, Sipert et al.19 showedthat MTA and Portland cement inhibited the growth ofseveral microorganisms, including Candida albicans,S. aureus, E. faecalis, Staphylococcus epidermidis andP. aeruginosa, which is in accordance with the findingsof the present study. In another study, Ribeiro et al.20

demonstrated that the antibacterial activity of MTAagainst Escherichia coli under aerobic conditions was

promoted by the induction of reactive oxygen species. Inanother study, Ribeiro et al.21 found that E. faecalis wassusceptible to grey MTA, as well as calcium hydroxide,after incubation in aerobic conditions. They proposedthat an oxygen-enriched medium favours the antimicro-bial activity of MTA. Estrela et al.22 showed noantibacterial activity of MTA and Portland cementagainst S. aureus, E. faecalis, P. aeruginosa, Bacillussubtilis and C. albicans. The antibacterial effect of MTAhas been attributed to its high pH or to substances thatare released from MTA into the medium.

The results of the agar diffusion test do not dependsolely on the toxicity of the material for the particularmicroorganism, but are strongly influenced by thediffusing ability of the material across the medium.

Bodrumlu et al.23 evaluated the antibacterial activityof six root canal sealers (Diaket, Endomethasone,AH-26, Sealapex, Sultan and Epiphany) on E. faecalisusing the agar diffusion method. Their results showedthat all of the sealers caused bacterial growth inhibitionin descending order as follows: Endomethasone > Sul-tan > Sealapex > Diaket > Epiphany > AH-26. Theyalso found that the effectiveness of the root canal sealersdecreased with increasing duration of incubation, whichis in agreement with the results of the present study.Tabrizizadeh and Mohammadi24 evaluated the antibac-terial activity of AH-26, Dorifill (a zinc oxide eugenol[ZOE]-based sealer) and pure ZOE on S. aureus andS. mutans after 24 h and 7 days. Their findings revealedthat, in 24-h samples, AH-26 showed greater antibac-terial activity. However, in 7-day samples, pure ZOEshowed greater antibacterial activity. They also foundthat the effectiveness of the root canal sealers decreasedwith increasing duration of incubation, which is inagreement with the results of the present study. How-ever, the results of the present study contrast with thefindings of Cobankara et al.9, who found that time hadno effect on the antibacterial activity of sealers. Withinthe limitations of the present study, the antibacterialactivity of the AH-26 sealer was significantly greaterthan that of the two other materials.

Acknowledgement

The authors wish to thank research officials of theHamedan University of Medical Sciences, Hamedan,Iran.

Conflict of interest

The authors report no conflict of interest.

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20. Ribeiro CS, Scelza MF, Junior RH et al. The antimicrobialactivity of grey-colored mineral trioxide aggregate (GMTA) andwhite-colored MTA (WMTA) under aerobic and anaerobic con-ditions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod2010 109: e109–e112.

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Correspondence to:Zahed Mohammadi,

Department of Endodontics,Hamedan University of Medical Sciences,

Hamedan,Iran.

Email: zahed_mohammadi@yahoo.com

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