The DCT used in the present study is a quantitative and reproducible method designed to simulate the contact of the microorganism with retrograde filling materials [15]. This procedure allows us to assess the antimicrobial effect of test materials at different stages of the setting reaction. In 2009, Zhang et al. reported a modified DCT [16], in which the suspension of MTA was obtained to contact the bacteria suspension. However, the retrograde filling materials were in direct contact with the microorganisms inside the root canals, the DCT applied in the present study might better mimic the clinical situation. The agar diffusion test (ADT) is another method to evaluate the antimicrobial activity of root-end filling materials. Since the outcome of ADT depend on the material diffusibility in the medium [4], the solid root-end filling materials may not be diffusible, which could be a possible explanation for the negative outcome of ADT in the present study. Therefore, DCT seems more appropriate in evaluating the antimicrobial activity of solidified materials.
MTA was introduced innovatively as a root-filling material by Dr. Torabinejad in 1995 [17]. According to previous studies [18, 19], the antibacterial and antifungal properties of MTA were associated with elevated pH value. In the present study, no inhibition zone was observed in agar diffusion test, while direct contact test revealed antimicrobial effect of MTA against both E. faecalis and P. gingivalis. An in vitro study [3] showed that MTA exerts antibacterial effects against some facultative bacteria but not on any species of absolute anaerobes, however, another study by Kim et al. [20] found that freshly mixed ProRoot MTA formed a bacterial growth inhibition zone against P. gingivalis in disk diffusion test. Since MTA has been tested in many researches but with contradictory results [18] such difference may be attributed to the usage of different methodologies, bacterial strains, aerobic and anaerobic conditions.
Biodentine was developed as dentin replacement material. In addition to shorter setting time and satisfactory strength, it was also reported to be less porosity and less leakage [21], less tooth discolors [22–24] and excellent biocompatibility [25] compared with MTA. In the present study, the antimicrobial effect of Biodentine against E. faecalis was similar to that of MTA, and the effect was lower when tested 7 days after setting, which is in accordance with a previous study by Koruyucu et al [15].
iRoot FS (Innovative Bioceramix, Vancouver, BC, Canada) was introduced as a root canal repair material. As a premixed material, iRoot FS solidifies only when exposed to a moist environment. Previous studies have reported that iRoot FS has similar apical sealing ability and mechanical properties to MTA [26] and that iRoot FS has a shorter setting time (initial 18 min and final 57 min) than MTA. There are great potentials for the clinical application of iRoot FS as the material is cytocompatible while facilitating cell adhesion, proliferation, differentiation and maintenance of normal cell function [27]. However, the antimicrobial effect of iRoot FS is unknown. In the present study, iRoot FS showed satisfactory antimicrobial effect when tested 20 min or 1 day after setting, and the effect became relatively lower than MTA and Biodentine when tested 7 days after setting, which might be attribute to its shorter setting time.
The pH values measured in this study were between 11 and 12, all the three materials showed strong alkaline pH, which is in accordance with previous studies [18, 28]. However, though Biodentine exhibited the highest pH value at all time intervals, which might explain its superior antimicrobial effect 7 days after setting, it did not show the strongest antibacterial activity against E. faecalis. Therefore, as Zhang et al. mentioned in a previous study [29], the antibacterial action cannot be rationally explained by pH alone. Moreover, in clinical situations, a desirable high pH after MTA application cannot be maintained due to the buffering capacity of dentin [20].
The selection of the used bacterial species in this in vitro study was intended to represent the poly-micro flora in the periapical lesions. However, the real situation in vivo is far more complex and hard to simulate in vitro. Further in vivo studies are required to better understand the various properties of the retrograde filling materials.