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Introduction

Bacteria and their products are major etiologic factors in pulpal and periapical pathosis (1,2). It has also been shown that periapical inflammation does not occur in the absence of microorganisms, regardless of the quality of the root canal filling (3). Enterococci are commensal microorganisms that are found in the mucosal tissues of the oral cavity, gastrointestinal tract and genital tract in humans (4). Enterococci are of clinical importance since they are the third most common nosocomial pathogens (5) and the cause of refractory apical periodontitis (6). They are often resistant to germicides and antiseptics as they can persist under harsh conditions, such as high alkalinity, due to their efficient use of proton pumps (7). To date, 12 enterococci species have been identified, and ~90% of the Enterococcus clinical isolates are of the Enterococcus faecalis species(8).

E. faecalis can survive various extreme environmental conditions and for long periods of time under nutritional deprivation. In addition, it has been shown to be resistant to the calcium hydroxide treatment that is commonly used in the course of endodontic therapy (9). Endodontic therapy mainly attempts to eliminate bacteria from the root canal. The use of chemical irrigation and mechanical instrumentation along with medication of the root canal system between treatment sessions can significantly reduce the population of bacteria inside the infected root canal (10). However, eradication of all bacteria from the root canal system is difficult (11,12).

Plasmas have been used for a number years for the disinfection of medical equipment, implants, blood coagulation and tooth bleaching (13–20). Chen et al reported that He/O2 plasma more effectively killed E. faecalis than pure He plasma (21). Recently, our group designed a special low-temperature plasma device that generates plasma plumes in open space (surrounding air), rather than only in a confined discharge gap (22). This low-temperature plasma device has been used in previous studies to efficiently kill E. faecalis bacteria (19,20,22,23). The aim of the present study was to assess the antimicrobial activity of the plasma jet device with He flowing through 3% hydrogen peroxide in root canals infected with E. faecalis.

Materials and methods

Experimental setup

Fig. 1 shows the plasma jet device with the working gas flowing through 3% hydrogen peroxide, which is used in the treatment of a root canal infected with E. faecalis. The inner diameter of the syringe nozzle was ~1.0 mm. When the root canals were treated, the syringe nozzle was vertically put on the root-canal orifice. The working gas used in all the experiments was He with a flow rate of 2 l/min. All the experiments in the present study used the same frequency of 8 kHz, applied voltage of 8 kV and pulse width of 1,600 ns.

Figure 1. Plasma jet device with helium flowing through 3% hydrogen peroxide, used in the treatment of a root canal infected with Enterococcus faecalis.

Microbial preparation. E. faecalis

(ATCC 29212; American Type Culture Collection, Manassas, VA, USA) were grown in Müller-Hinton (M-H) agar (Difco Laboratories, Detroit, MI, USA), and incubated overnight at 37°C. The microorganisms were then inoculated into a test tube containing 2 ml sterile physiological saline. The suspension was adjusted to a turbidity of 6.0×108 colony forming units (CFU)/ml. Subsequently, the same amount of brain heart infusion (BHI) broth (Difco Laboratories) was added to the test tube. At this point, the concentration of bacteria was 3.0×108 CFU/ml (equivalent to 1.0 McFarland unit).

Preparation of teeth

The study protocol was reviewed and approved by the Ethical Review Board of Investigation at the Tongji Hospital of Huazhong University of Science and Technology (Wuhan, China). Informed consent was obtained from the patients. A total of 42 extracted, human single-rooted anterior teeth with curvatures between 0 and 10 degrees were obtained and stored in physiological saline. The external root surface was cleaned with curettes to remove periodontal soft tissues and calculus. All root canals were prepared in a crown-down technique with K-file (Dentsply Maillefer, Ballaigues, Switzerland) and ProTaper instruments (Dentsply Maillefer) by an experienced endodontist and the size of ProTaper files included sizes 10/15/SX/S1/S2/F1/F2 and F3. Next, the teeth were placed in an ultrasonic bath in 17% ethylenediaminetetraacetic acid for 5 min, followed by 5.25% sodium hypochlorite for a further 5 min in order to remove the smear layer. Root canals were then rinsed with sterile water and placed in an ultrasonic cleaner for 20 min. Next, all teeth were autoclaved for 30 min at 121°C, and parafilm was used to seal all the apical foramina of root canals. A sterile micropipette was used to introduce 10 µl of the bacterial suspension into each root canal, and root canal orifices were sealed with parafilm. Subsequently, the teeth were incubated aerobically at 37°C for 7 days. After 3.5 days of incubation, 10 µl BHI broth was introduced into each root canal. Following the incubation, the root canals were blotted dry with size 40 sterile paper points.

The teeth were randomly divided into one control and six experimental groups (6 teeth in each group), according to the various times and methods of plasma sterilization. The groups were as follows: Plasma jet sterilization with He flowing through 3% hydrogen peroxide for 1 min (Group 1), 2 min (Group 2) or 4 min (Group 3); plasma jet sterilization without He gas flowing through 3% hydrogen peroxide for 1 min (Group 4), 2 min (Group 5) or 4 min (Group 6); and He gas flowing at 2 l/min for 4 min with the plasma off (Group 7; control group).

After the treatment, an experienced endodontist used a new sterile ProTaper file with size F3 to manually file the root canal of each tooth, with each root canal filed 20 times. The parafilm was removed from the root canal orifice, and the tooth and debris in each file were irrigated with 1 ml sterile physiological saline from the apical foramen to the root canal orifice. Subsequently, the debris was collected in a test tube and agitated with adding sterile physiological saline to 5 ml. Serial dilutions (×10) of the samples were plated onto M-H agar and the plates were incubated at 37°C for 48 h. The number of CFU/ml was then obtained for each sample by an observer in a blinded manner.

Optical emission spectra

To identify the various reactive species generated, the optical emission spectrum of plasma jet without or with He flowing through 3% hydrogen peroxide is measured in the 200–1,100 nm wavelength range. A spectrometer (Acton SP-2500i; Princeton Instruments, Acton, MA, USA) was used to measure the emission spectra of the plasma jet with or without the He gas flowing through 3% hydrogen peroxide.

Scanning electron microscopy

A total of 100 µl of the bacterial suspension were seeded on two sterile cellulose nitrate membrane filters (Merck Millipore, Billerica, CA, USA), which were placed in BHI broth and incubated at 37°C for 72 h in an aerobic atmosphere. The experimental group membrane was treated by plasma jet sterilization with He gas flowing through 3% hydrogen peroxide at 2 l/min for 2 min. The control group membrane was treated with He gas flowing at 2 l/min for 4 min with the plasma off. Subsequently, the two membranes were fixed with 2.5% glutaraldehyde, rinsed with distilled water and dehydrated in a series of ethanol. The two dried specimens were coated with gold and then observed under scanning electron microscopy (JEOL JSM-6700F system; JEOL, Tokyo, Japan).

Statistical analysis

Data are expressed as the mean ± standard deviation. SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. Cell counts were logarithmically converted prior to statistical comparison. Kruskal-Wallis test and Mann-Whitney analysis were used to determine the differences in bactericidal efficacy. Statistically significant differences among the groups were considered at P-values of <0.01.

Results

Antibacterial effects of the plasma jet with and without He flowing through 3% hydrogen peroxide

Fig. 2 demonstrates the results of the antibacterial effects of the plasma jet with and without He flowing through 3% hydrogen peroxide. Overall, the viable count of E. faecalis was significantly decreased (P=0.001) in all plasma treatment groups relative to the control group. However, the antibacterial effects of the plasma jet with He flowing through 3% hydrogen peroxide were better when compared with the effect of the plasma jet without He. In addition, a longer sterilization time resulted in improved bactericidal effects of the treatment as observed by the decreased viable count of bacteria after treatment for 4 min (Fig. 2). The greatest reduction in CFU/ml was observed in Group 2 (plasma jet with He for 2 min) and Group 3 (plasma jet with He for 4 min), which presented a reduction by 6.237 and 7.027 log units, respectively (Fig. 2). The reduction in the bacterial count observed in Groups 1, 2 and 3 (treated by plasma jet with He) was significantly greater compared with that in Groups 4, 5 and 6, which were treated by plasma jet without He (P<0.005). In addition, Group 3 showed a significant CFU/ml reduction compared with Groups 1 and 2, which were treated by plasma jet with He for a shorter time length (P=0.0015, P=0.002).

Figure 2. Survival of Enterococcus faecalis following treatment by plasma jet sterilization without or with He flowing through 3% hydrogen peroxide. *P<0.005. He, helium.

Optical emission spectra of plasma jet without or with He flowing through 3% hydrogen peroxide

Fig. 3 demonstrates the optical emission spectra of plasma jet without or with He flowing through 3% hydrogen peroxide at wavelengths between 200 and 1,100 nm. The spectra indicated that plasma jet with He flowing through 3% hydrogen peroxide presented stronger emission lines of atomic oxygen (777.2 nm) and hydroxyl radical (309 nm) compared with that in the plasma jet without He flowing through 3% hydrogen peroxide (Fig. 3). The results indicated that reactive species, including atomic oxygen and hydroxyl radical, serve a dominant role in the plasma jet sterilization, whereas N2 and He are not expected to serve a significant direct role.

Figure 3. Optical emission spectra of plasma jet (A) without He and (B) with He flowing through 3% hydrogen peroxide. He, helium.

E. faecalis cells in Group 2 and the control group were investigated by scanning electron microscopy. As shown in Fig. 4, the images captured following plasma jet treatment demonstrated reduced size, rupture and death of E. faecalis cells, compared with the control cells. The results of scanning electron microscopy clearly explained the reduced viability of bacterial cells observed following treatment by plasma jet with He flowing through 3% hydrogen peroxide.

Figure 4. Scanning electron microscope images of Enterococcus faecalis cells, obtained (A) prior to plasma treatment and (B) after 2-min treatment by plasma jet with He flowing through 3% hydrogen peroxide (magnification, ×20,000).

Discussion

E. faecalis was selected as the test microorganism in the present study owing to its high frequency of isolation from cases of failed endodontic treatment. E. faecalis had been shown to have a high affinity for biofilm formation, and bacteria in the biofilm form were up to 1,000-fold more resistant to phagocytosis, antibodies and other antibiotics compared with those in the planktonic form (24).

In the present study, Groups 3 and 2 treated by plasma jet with He flowing through 3% hydrogen peroxide produced a 7.027 and 6.237 log reduction, respectively, in the bacterial CFU, when compared with the control group. This result suggests that the E. faecalis biofilms were eradicated in Groups 3 and 2. The other experimental groups obtained reductions of at least 2.909 log units. Therefore, the results in all groups were promising.

The bactericidal effect of the plasma jet with or without He flowing through 3% hydrogen peroxide resulted mainly from the atomic and molecular radicals. Reactive oxygen species (ROS) are generally considered to serve a key role in the bactericidal process. Short-time plasma treatment can induce DNA fragmentation (25). The ROS are able to penetrate the cells and, therefore, may induce high levels of DNA damage, which then causes the induction of apoptosis (26,27). Hydrogen peroxide is an oxidizing agent that leads to microbial death by protein denaturation. It is widely applied for disinfection processes in the food, water treatment, healthcare and contact lens industries (28). Hydrogen peroxide decomposition and the recombination of radicals can lead to the formation of molecular oxygen, atomic oxygen and hydroxyl radical (29,30). In the present study, stronger emission lines of atomic oxygen and hydroxyl radical were detected in the plasma jet with He flowing through 3% hydrogen peroxide using a spectrometer, when compared with plasma jet without He flowing through 3% hydrogen peroxide. The atomic oxygen and hydroxyl radical are considered to be responsible for the improved antibacterial effects of the plasma jet with He flowing through 3% hydrogen peroxide when compared with the plasma jet without He. The results indicate that reactive species, including atomic oxygen and hydroxyl radical, serve a dominant role in the plasma jet sterilization, whereas N2 and He are not expected to serve a significant direct role, and these findings are consistent with the results of a previous study (31).

In the current study, the UV intensity emitted by the plasma jet with or without the working gas flowing through 3% hydrogen peroxide was detected, and it was found that the UV intensity was approximately 0.05–0.1 mW/cm2. Therefore, the UV radiation served a minor role in the sterilization of the bacteria (19,32). Heat was not responsible for the bactericidal effect, since the plasma jet with or without the working gas flowing through 3% hydrogen peroxide was at room temperature. Scanning electron microscopy was also used in the present study to reveal damage to E. faecalis by plasma jet with He flowing through 3% hydrogen peroxide. The obtained images of E. faecalis cells demonstrated reduction in size, rupture and death, when compared with the normal cells.

Single-rooted anterior teeth with curvatures between 0 and 10 degrees used in the present study were easily standardized to exclude interference of the dental anatomic complexity. Thus, further studies should examine the sterilizing effect of the plasma jet with He flowing through 3% hydrogen peroxide in complexity root canal systems.

In conclusion, the results of the present study suggested that the antibacterial effects of the plasma jet with He flowing through 3% hydrogen peroxide were better compared with the antibacterial effects of the plasma jet without He flowing through 3% hydrogen peroxide. The atomic oxygen and hydroxyl radical were considered to be responsible for the improved antibacterial effects of the plasma jet with He when compared with the plasma jet without He. The results also suggested that the plasma jet device with or without He flowing through 3% hydrogen peroxide is a valuable tool for root canal disinfection of E. faecalis, and that plasma treatment could be considered as an alternative method for root canal disinfection of E. faecalis in endodontic treatments.

Acknowledgements

The present study was supported by a grant from the Natural Science Foundation of China (grant no. 10875048).

References