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.

(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).

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.

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.

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).

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.

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).

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.

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.