Introduction
Oral bacteria are major causative agents of pulpal and periapical diseases. The treatment of these is performed through dental root canal enlargement, cleansing, and disinfection and the main purpose of root canal therapy is to remove bacteria present in the root canal and to prevent reinfection [1-3]. The persistent or secondary infection of the root canal is the main cause of root canal treatment failure. Staphylococcus aureus and Enterococcus faecalis are the pathogenic bacteria of endondontic infection [4]. S. aureus is a representative purulent bacterial species and is also isolated from endodontic infection lesions, so it has been used as a target bacterium for evaluation of antimicrobial property of dental materials used for various root canal treatments [5,6]. E. faecalis has been reported as a major causative agent of root canal failure [7,8].
Recently, chlorhexidine or Mixture of Tetracycline, an Acid, and a Detergent (MTAD) has been used as antimicrobial agent mixed with root canal sealer for root canal treatment [3,9]. However, chlorhexidine has toxicity to oral tissue cells and MTAD has possibility of inducing new tetracycline-resistant bacteria or has not effect tetracycline-sensitive bacteria that are existed in root canal [10,11]. Therefore, they are used as root canal cleaners rather than as sealers for root canals [10,12]. Therefore, studies are being conducted to find an antimicrobial substance derived from natural products that can be used in combination with an endodontic treatment material to overcome these problems [13]. The previous study, the ethanol extract of Garcinia mangostana L. (magosteen) had strong antimicrobial activity against S. aureus and E. faecalis [14,15].
Since mineral trioxide aggregate (MTA) was introduced by Torabinejad and Chivian [16], it has been widely used in the field of endodontic treatment. Recently, MTA-based root sealer has been developed and delivered via a syringe. This sealer was reported to have better biocompatibility than conventional resin sealers for root canal filling [17]. However, it was reported that the antimicrobial effect of MTA was very low [18]. Therefore, in this study, the antimicrobial activity of Endoseal TCS® (MARUCHI Co., Ltd., Wonju, Korea), an MTA-based root canal sealer, mixed with the water-soluble mangostin derivatives (WsMD) of the ethanol extract of magosteen against E. faecalis and S. aureus was evaluated.
Materials and Methods
1. Bacteria and bacterial culture
The E. faecalis (KCOM 1083, KCOM 1161, KCOM 2823) and S. aureus (KCOM 1335, KCOM 1395, KCOM 1491) strains used in this study were obtained from the Korean Collection for Oral Microbiology (Gwangju, Korea). These strains were grown on brain heart infusion (BHI, BD Difco Laboratories, Franklin Lakes, NJ, USA) broth or BHI agar medium in an incubator at 37℃ for 24 hours.
2. Preparation of water-soluble mangostin derivative powder
Mangosteen ethanol extract was purchased from Maypro Industries® (Purchase, NY, USA). To impart water solubility to the mangosteen ethanol extract, a certain amount of mangosteen ethanol extract powder was added to purified water and stirred at 50℃ and 1 atmosphere (atm) for 1 hour. Thereafter, L-arginine was added to the solution and stirred at 50℃ and 1 atm for 1 hour. The pH at this time was maintained at 9–10. L-arginine (1%, w/w) and KOH, an alkaline metal salt used as a catalyst, were added to the solution and stirred at 70℃ and 1 atm for 2 hours to form WsMD. The pH was maintained at 9–10. After the color of the solution changed from ocher to black, it was cooled to 4℃ and left at room temperature for 12 hours to increase the stability of the WsMD. The solution was vacuum-dried at 0.4 atm and 60℃ to prepare WsMD powder (Fig. 1).
3. Preparation of mixed sample of WsMD powder and Endoseal TCS®
Endoseal TCS® was mixed with a WsMD at weight proportions of 0.023%, 0.07%, or 0.115% to prepare premixed injectable pastes. These were provided by MARUCHI Co., Ltd.
4. Antibacterial test
Agar diffusion tests were performed by a modification of the method of Hasheminia et al. [19]. Briefly, 10 mL of the sterilized BHI agar medium was poured into a sterilized 90-mm Petri dish and then a paper disk (8 × 0.5 mm) was placed into the agar medium. The bacterial strain was grown in BHI broth at 37℃ for 16 hours in an incubator. It was diluted to 106 CFU/ mL with BHI agar solution cooled to 40℃ and then 10 mL was poured onto the previously solidified BHI agar medium. After the BHI agar solution mixed with the bacterial strain was solidified, the paper disc was carefully removed. Two mL of Endoseal TCS® (control), or Endoseal TCS® mixed with 0.023% (w/ w), 0.07% (w/w), or 0.115% (w/w) of WsMD by injecting using a syringe with a 24G needle. The plates were incubated for 24 hours in an incubator at 37℃ and the zone of inhibition was measured. The length from the edge of the Endoseal TCS® to the edge of the inhibition zone was measured and recorded. This experiment was repeated three times and the antibacterial activity is presented as the mean ± standard error of the length of the zone of inhibition.
Results
The antimicrobial activity of the WsMD mixed with Endoseal TCS® against E. faecalis and S. aureus strains was evaluated by agar diffusion tests. The data showed that Endoseal TCS® mixed with 0.115% WsMD created a zone of inhibition that ranged from 0.7 ± 0.2 to 2.4 ± 0.1 mm (Fig. 2 and Table 1). Among the strains used in this study, E. faecalis KCOM 2823 was more resistant to Endoseal TCS® containing the most WsMD compared to the other strains (Fig. 2 and Table 1). In contrast, the S. aureus KCOM 1491 strain was the most susceptible (Fig. 2 and Table 1). In the case of Endoseal TCS®, which was used as a control, antibacterial activity was not observed (Fig. 2 and Table 1).
Discussion
In this study, WsMD were mixed with Endoseal TCS®, an MTA-based root canal sealer, to impart antibacterial properties, which were investigated by agar diffusion tests. The zone of inhibition was increased by adding higher amounts of WsMD (Fig. 1 and Table 1). According to previous studies, the minimum bactericidal concentration (MBC) values of the ethanol extract of mangosteen containing about 40% alpha-mangostin against E. faecalis (KCOM 1083, KCOM 1161, KCOM 2823) and S. aureus (KCOM 1335, KCOM 1395, KCOM 1491) were between 2 and 8 μg/mL [14,15]. To investigate the antimicrobial activity of the WsMD compared to the mangosteen ethanol extract, the MBC value of WsMD against S. aureus KCOM 1491 was measured. The MBC value of WsMD against S. aureus KCOM 1491 was the same, 2 μg/mL, as that of mangosteen ethanol extract (data not shown). These results indicate that the antimicrobial activity of the WsMD was maintained and might be released when mixed with Endoseal TCS®. However, further study is needed to investigate how the WsMD were produced and released from the Endoseal TCS® mixture.
Little antimicrobial activity of MTA Fillapex, another MTAbased root canal sealer developed in Brazil, against E. faecalis was seen in the agar diffusion tests in previous studies [18,19]. However, the antimicrobial activity of MTA Fillapex that was cured and pulverized against E. faecalis was excellent [19]. The antimicrobial mechanisms of MTA are explained in two ways. The first is that MTA produces reactive oxygen species in the atmosphere, with antibacterial effects [20]. Second, MTA contains calcium oxide. Therefore, when it comes into contact with water, calcium hydroxide is formed, which increases the pH and exhibits an antibacterial effect [21-23]. It is known that E. faecalis cannot survive at a pH of 11.5 or higher. Therefore, water is essential for MTA to have an antibacterial effect on E. faecalis. This means that the MTA-based root canal sealer loses its antimicrobial activity after curing. Thus, the addition of a water-soluble antibacterial agent is considered necessary to impart antibacterial activity to the MTA-based root canal sealer, as in this study.
As a result of studies comparing the antimicrobial activity of various types of root canal sealer currently in use, the antibacterial effect of the MTA-based sealer against E. faecalis was lower than that of the other types of root canal sealers [18,24- 26]. When using only MTA, the antibacterial effect was lower than that of the conventional sealer. Therefore, there have been attempts to increase the antibacterial effect by adding chlorhexidine to MTA [9,27]. However, when chlorhexidine was added, the antibacterial effect was increased, but the physical properties of the MTA itself were deteriorated [9]. Based on these results, we intend to conduct future studies on whether Endoseal TCS® containing the WsMD used in this study has clinically usable properties.
Another study on the antimicrobial effect of MTA-based sealers against E. faecalis showed some antimicrobial effects of MTA Fillapex and Endofill against E. faecalis but did not display complete inhibition and the high pH of the MTA-based sealer was increased after seven days [28]. In this study, the Endoseal TCS® component spread to the inhibitory zone from the second day and began to become opaque, and from the third day, the agar medium dried, making it difficult to identify the antibacterial activity (data not shown). Therefore, animal experiments will be used to evaluate the duration of the antimicrobial activity of Endoseal TCS® containing WsMD in the future.
According to the above results, the higher the content of WsMD mixed with Endoseal TCS®, the higher the antibacterial effect. With more than 0.115% WsMD mixed with Endoseal TCS®, the color tone of the sealer changed and the flowability was impaired, resulting in the loss of function as a sealer for root canal filling. Therefore, the proper concentration of WsMD for mixing with Endoseal TCS® was 0.115%.
In summary, Endoseal TCS® mixed with the WsMD of the ethanol extract of Magosteen might be useful as a root canal sealer with antibacterial activity against E. faecalis and S. aureus.
