Sealing ability of MTA, CPM, and MBPc as root-end filling materials: a bacterial leakage study

ABSTRACT Objectives To evaluate the sealing ability of three root-end filling materials (white MTA, CPM, and MBPc) using an Enterococcus faecalis leakage model. Material and Methods Seventy single-root extracted human teeth were instrumented and root-ends were resected to prepare 3 mm depth cavities. Root-end preparations were filled with white MTA, CPM, and MBPc cements. Enterococcus faecalis was coronally introduced and the apical portion was immersed in BHI culture medium with phenol red indicator. The bacterial leakage was monitored every 24 h for 4 weeks. The statistical analysis was performed using the Wilcoxon-Gehan test (p<0.05). Results All cements showed bacterial leakage after 24 hours, except for the negative control group. The MBPc showed significantly less bacterial leakage compared with the MTA group (p<0.05). No significant differences were found between the CPM and the other groups. Conclusions The epoxy resin-based cement MBPc had lower bacterial leakage compared with the calcium silicate-based cements MTA and CPM.


INTRODUCTION
Persistent microorganisms are the main factors associated with chronic apical periodontitis and consequent Endodontic failures 10 . One of the indications for periradicular surgery is when the conventional treatment is not able to eliminate the microorganisms of the apical portion 7 . These prevent bacterial leakage and the reinfection of the root canal system 27 . Therefore, it is crucial for the cement used to obturate root-end cavities to be Several materials were suggested for retrograde and MTA 2,32 . The Mineral Trioxide Aggregate (MTA) satisfactory physicochemical 15,29 and biological properties 28,30 . Other cements are studied as alternatives for the MTA. In 2004, a novel root-SA, Buenos Aires, Argentina) is composed of a powder mainly made of Portland cement and a liquid with distilled water in its composition. The CPM cement presents antimicrobial activity 23 , satisfactory biological response 18,20 , and adequate physicochemical properties 4,24 .
Epoxy-resin based cements have been widely studied for Endodontic procedures since 1957 8,19 . In 1984, Moraes and Berbert developed an epoxy resin-based cement containing calcium hydroxide to 13 . The MBPc is consisted of hydrophobic base paste/catalyst paste cement 14 . Promissory results regarding its physical 34 , chemical 14,33 , and biological 3,9 properties were found. Nonetheless, this cement requires more investigation to be considered clinically useful.
There are several methods to evaluate the 34 , radioisotope 22 , dye penetration 6,14 , and bacterial leakage 21 . Radioisotope and dye penetration methods present the disadvantage of having the molecular size of radioisotopes tracers 2016;24(2):148-52 and dye particles smaller than bacteria 31 . On the other hand, bacterial leakage is considered more clinically relevant 31 . This method is widely used in Endodontic research to evaluate the sealing ability of root canal sealers 12,26 materials 1,11 . A previous study has compared MTA, CPM, and MBPc cements using a dye penetration model 14 . Considering the limitations of this method, it is indispensable for us to investigate if there is cements. Thus, the aim of the study was to evaluate (white MTA, CPM, and MBPc) using an Enterococcus faecalis leakage model.

MATERIAL AND METHODS
Seventy intact, caries-free, single-rooted permanent human teeth were selected. The ethics committee of the Bauru School of Dentistry approved the use of teeth for research purposes (CEP 049-2007). All teeth were autoclaved. The coronal portions were sectioned 1.0 mm below the dental-enamel junction using a 0.3 mm Isomet saw (Buehler, Lake Bluff, Illinois, USA). Roots were standardized at 15 mm and the working length (WL) was established 1.0 mm from the apical foramen. Root canals were prepared using the crown-Ballaigues, Switzerland). Apical preparation was The shaping procedure was completed with 2 Ballaigues, Switzerland). After the use of each instrument, we irrigated the canal with 2 mL of 2.5% sodium hypochlorite. The smear layer was removed with 2 mL of 17% EDTA (Biodinâmica, Ibiporã, Paraná, Brazil) for three minutes, then each root was resected with a cylindrical diamond bur, perpendicularly to the long axis of the root. A root-end cavity with 3.0 mm of depth and 1.0 mm of diameter was prepared. A gutta-percha cone of size 40.02 was inserted into the root canal 3 mm The specimens were randomly divided in three groups (n=20): The composition of the evaluated cements is present in Figure 1. Cements were manipulated by following the manufacturer's instructions. They were incrementally inserted into the rootend cavities and vertically compacted using a hand plugger. Specimens were stored in 100% of humidity at 37°C for one week to allow the cements to set. For negative control (n=5), the entire root surface was coated with two layers of nail varnish (Colorama, L'Oréal, São Paulo, São Paulo, Brazil)

Bacterial leakage
The apparatus used to evaluate the leakage was prepared as previously described 21 with rubber stoppers were adjusted to use. Using a high-speed handpiece, a hole was made through the centre of each rubber stopper in which each tooth was inserted under a pressure up to 4 mm from the apical portion. An epoxy resin-based varnish (Araldite, Brascola, São Paulo, São Paulo, Brazil) was used to seal the interface between tooth and rubber stopper. Cylinders prepared with 10 mL plastic syringes were adapted to the outer surface of the stoppers to create a chamber around the crown of the tooth. The apparatus was sterilized in ethylene oxide gas for a 4 h cycle at 56°C (Figure 2).
The standard bacterial strains of Enterococcus faecalis (ATCC 29212) were used in the study.   Sterile pipettes were used to place 400 μl of bacterial inoculum in each access cavity found in the syringe apparatuses (in the upper region, removing the gauze stop). The 4 mm of root-ends were immersed in sterile BHI. The apparatus was incubated at 37°C for 120 days and checked daily for turbidity in the BHI broth. Bacterial leakage was considered when turbidity was observed (Table 1).

Statistical analysis
Statistical analysis was performed using the groups (p<0.05).

RESULTS
The criteria evaluated with experimental periods are shown in Figure 3. All cements showed bacterial leakage after 24 hours, except for the negative control group. The MTA showed a higher number of specimens that leaked compared with the MBPc group (p<0.05). Statistical similarities for bacterial leakage were found between the CPM and the MBPc and also the MTA (p>0.05). After 31 days until the

DISCUSSION
properties of the cement used 17 . The aim of root-end conditions to periapical healing 32 . The sealing ability of a cement can be determined using leakage methods 12,14 , confocal microscopy 32 , scanning electron microscopy 25 , and more recently microcomputed tomography analysis 35 . The bacterial leakage method has the advantage of providing a clinically relevant adaptability date of materials for root canal walls 31 . The Enterococcus faecalis was chosen for this study model because it is commonly present in secondary infections from Endodontic treatment failures. This microorganism is a gram-positive coccus and is highly resistant to alkaline pH such as the one present in the calcium hydroxide canals and their drug resistance, the E. faecalis has been proposed as an Endodontic pathogen 16 . Furthermore, this microorganism is easily arranged and interpreted from the study data 11 . Under the conditions of this study, this microorganism was able to leak in all experimental cements. On the other hand, Jacobovitz, et al. 5 (2009), using another model of in vitro microleakage analysis with the same microorganism, found that the MTA showed no microbial growth after 30 days.
The results found in the present study showed that the bacterial leakage occurred after 24 hours in at least one specimen for MTA, CPM, and MBPc cements. During the experimental period, all groups showed decrease in the number of leaked specimens. A possible explanation is that the cements expanded into the cavities during the in the present study are according to that previously found by Orosco, et al. 35 (2008), although they have used the dye penetration method.
Although no statistical differences were found between MBPc and CPM cements (p>0.05), the with the MTA (p<0.05). In a previous study, the MBPc showed satisfactory results for marginal adaptation and leakage 14 . Vasconcelos, et al. 34 (2011) reported low leakage ranges for epoxyresin sealers MBPc and AH Plus compared with the presence of epoxy-resin improved the sealing of MBPc and AH Plus in dentin walls. calcium carbonate, silicon dioxide, bismuth trioxide, and barium sulphate 33 . Probably, the presence of calcium carbonate could be the responsible for offering a great calcium ions release, which may promote the adhesion to dentinal canal walls, thus improving sealing properties 33 . According to the results, this cement has not been able to inhibit bacterial leakage completely. The absence of studies regarding its sealing ability.
Further studies should use the confocal laser scanning (CLSM) microscopy to detect and quantify bacterial viability in void spaces or gaps between cavity walls. Moreover, prospective clinical studies evaluating the success rate of Endodontic surgeries using the tested sealers might prove to be informative.

CONCLUSION
The epoxy resin-based cement MBPc had lower bacterial leakage compared with the calcium silicate-based cements MTA and CPM.