Basic Research—Technology

Evaluation of Cold Plasma Treatment and Safety in

Disinfecting 3-week Root Canal Enterococcus faecalis
Biofilm In Vitro
Yinglong Li, PhD,* Ke Sun, MDS,* Guopin Ye, MDS,* Yongdong Liang, PhD,† Hong Pan, MDS,*
Guomin Wang, MS,* Yijiao Zhao, MS,‡ Jie Pan, DDS,*§ Jue Zhang, PhD,*† and Jing Fang, PhD*†

Abstract
Introduction: Although endodontic infection is caused
by multi-bacteria species, Enterococcus faecalis is
usually isolated in chronic apical periodontitis. The aim
of this study was to evaluate the effectiveness and me-
chanical safety of cold plasma therapy in disinfecting
3-week E. faecalis biofilms. Methods: Teeth with
3-week E. faecalis biofilm were treated with AC
argon/oxygen (Ar/O2) cold plasma for various treatment
times and compared with those treated with Ca(OH)2,
2% chlorhexidine gel, and Ca(OH)2/chlorhexidine for a
week. Antimicrobial efficacy was assessed by colony-
forming unit method. Scanning electron microscopy
was used to assess the morphologic changes of E.
faecalis biofilm by plasma. Confocal laser scanning
microscopy was used to confirm the viability of the bio-
film after the plasma treatment. Microhardness and
roughness changes of root canal dentin caused by
plasma were verified with Vickers Hardness Tester and
3D Profile Measurement Laser Microscope, respectively.
Results: There were no detectable live bacteria after 12
minutes of cold plasma treatment. This was further
confirmed by scanning electron microscopy and confocal
laser scanning microscopy results. Microhardness and
roughness of root canal dentin showed no significant
difference after plasma treatment. Conclusions: Atmo-
spheric pressure cold plasma is an effective therapy in
endodontics for its strong sterilization effect on fully
matured biofilm within a few minutes. Meanwhile, it
has an accepted mechanical safety for its low tempera-
ture and not affecting the microhardness and roughness
of root canal dentin significantly. (J Endod 2015;-:1–6)
Key Words
Cold plasma, Enterococcus faecalis biofilm, me-
chanical safety, microhardness, roughness
G enerally, endodontic infection is a polymicrobial infection of dental root canal sys-
tem. When treating persistent root canal infection, the main task to increase success
rate is to effectively eradicate infection during the therapy. However, traditional treat-
ments including mechanical instrumentation techniques and syringe or ultrasound irri-
gation cannot achieve a sufficient effect (1) because of the complex root canal system
and the innate resistance capacity of biofilms (2, 3). Many studies have demonstrated
that Enterococcus faecalis are commonly isolated bacteria associated with persistent
periapical lesions and often exist in the form of biofilms (4–6). Biofilms are complex
microbial communities attached to an interface and typically embedded with
extracellular polymeric substance (EPS) (7). Usually biofilms exhibit more resistance
to phagocytosis, antibodies, and antimicrobials than planktonic bacteria (8, 9).
Interappointment intracanal medication strategy has been recommended to
enhance disinfection after chemomechanical preparation. Ca(OH)2 paste has been
widely applied as an interappointment medication for its ability to produce extremely
alkaline environments. However, E. faecalis are resistant to Ca(OH)2 (10–12). The
2% chlorhexidine (CHX) could maintain an antibacterial effect for a prolonged
duration when adhered to the root canal walls with a better antimicrobial effect than
Ca(OH)2, although it still cannot kill E. faecalis biofilm entirely (13, 14). The
combinatorial usage of Ca(OH)2 and 2% CHX gel has shown better antimicrobial
properties than Ca(OH)2 alone (15), but this combination also is not completely
able to eliminate the E. faecalis biofilms (16).
Atmospheric pressure cold plasma, a partially ionized gas containing molecules,
charged particles with negative ions, positive ions, electrons, photons, and free radicals,
is a novel antimicrobial intervention. Cold plasma can be obtained at low pressure, and
the temperature is at or near room temperature. Its applications in the biological and
biomedical field such as tooth whitening (17), promoting blood clotting and wound
healing (18), cell detachment and migration (19), and inducing apoptosis of tumor
cells (20) have all been proved. Compared with regular antibiotic and intracanal dress-
ings to disinfect E. faecalis biofilm, cold plasma therapy has been increasingly high-
lighted in recent years. It has been witnessed that cold plasma technology has
outstanding sterilization effects in endodontic treatment in recent years (21, 22).
Although cold plasma is effective for young biofilms (23, 24), few studies have been
focused on mature E. faecalis biofilms, which have a more complex structure and
internal environment. To the best knowledge of the authors, the mechanical safety of
dentin has never been demonstrated in dental applications.

From the *Academy for Advanced Interdisciplinary Studies, Peking University; †College of Engineering, Peking University; ‡National Engineering Labo-
ratory for Digital and Material Technology of Stomatology; and §Department of General Dentistry, Peking University School and Hospital of Stomatology,
Beijing, China.
Address requests for reprints to Dr Jie Pan, 22 Zhongguancun South Street, Haidian District, Department of General Dentistry, Peking University School and Hospital
of Stomatology, Beijing, China 100081. E-mail address: panjie72@sina.com
0099-2399/$ - see front matter
Copyright ª 2015 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.10.020

JOE — Volume -, Number -, - 2015 Evaluation of Cold Plasma Treatment and Safety on E. faecalis 1
Basic Research—Technology
In this study, we investigated the disinfection effect of a cold plasma
jet for 3-week E. faecalis biofilm in root canals and compared the re-
sults with the effects of commonly adopted medications in root canal
treatment. Meanwhile, the microhardness and roughness changes
were tested.

Materials and Methods

Sample Preparation
One hundred human caries-free single-rooted teeth with mature
apices were used for cultivation of E. faecalis biofilm in the study.
The teeth were selected, cleaned, and stored in 0.1% thymol solution
at 4 C. The crown was removed at 2–3 mm below the cement-
enamel junction to obtain a standard length of 10 mm by using a dia-
mond bur. Then the pulp was extirpated with a barbed broach (Mani
Inc, Takanezawa, Japan). All canals were cleaned and shaped by rotary
nickel-titanium ProTaper instruments (Dentsply Maillefer, Ballaigues,
Switzerland) until F3 with a special torque-testing device (rotational
speed, 250 rpm). The canals were irrigated with 1 mL 5.25% sodium
hypochlorite (NaOCl) after each instrument size (25). Subsequently,
the smear layer was removed by an ultrasonic bath of 17% EDTA, fol-
lowed by an ultrasonic bath of 5.25% NaOCl for 2 minutes, respectively.
Figure 1. Picture of cold plasma jet during root canal treatment in vitro, and
All root apical foramina were sealed with composite resin. Finally, the
(a) is a thermal imaging of temperature distribution.
root canals were autoclaved for 20 minutes at 121 C to ensure no bac-
teria contamination and kept at 4 C (23).

Assessment of Antibacterial Activity of AC

Cultivation of E. Faecalis Biofilm
E. faecalis (ATCC 29212) was grown on brain-heart infusion agar
(BHI) (Land Bridge Technology Co, Ltd, Beijing, China) plates in an
aerobic environment at 37 C. A single colony was inoculated in 1 mL
sterile BHI broth at 37 C. The root canals were placed into 1.5-mL Ep-
pendorf tubes with 1 mL BHI broth containing 108 colony-forming units
(CFU)/mL E. faecalis and then incubated anaerobically at 37 C for 3
weeks. The sterile BHI broth was refreshed every other day to ensure
bacteria viability.
Cold Plasma Device
A non-thermal atmospheric pressure plasma device was used in
this study. Figure 1 shows a photograph of the in vitro plasma treatment
of a single-root tooth. A detailed description of the device can be found in
our previous article (23). A quartz tube jet with inner and outer diam-
eters of 7 mm and 10 mm was used in this study, which was different from
the previous study where a Teflon tube was used. The working gas in the
current study was Ar (98%) and O2 (2%) admixture, with a gas flow rate
of 5 L/min. During each treatment, the working distance from the tip of
the plasma jet to the sample was 10 mm. To measure the temperature
values during the plasma processing, we used a thermal imaging camera
(FLIR E50, Danderyd, Sweden), which can provide high-quality thermal
imaging at 19,200 pixels (160  120) with a thermal sensitivity of
0.07 C. The mean temperature of the cold plasma near the top of the
root canal and the room temperature were 30.1 C and 27.6 C, respec-
tively (Fig. 1A). The results indicate that the cold plasma treatments are
performed at an acceptable biocompatible temperature range.
Cold Plasma and Intracanal Dressings
Fifty root canals were used to evaluate the effectiveness of the
cold plasma disinfection of E. faecalis biofilms in root canals. After
incubation for 3 weeks, the biofilms were formed inside the pulpal
wall. The root canals were randomly divided into 5 groups of 10 teeth.
The root canals in group 1 (negative control group) did not receive
any treatment. The groups 2–5 were treated by the cold plasma for
3, 6, 9, and 12 minutes, respectively (Table 1).
Another 30 root canals, the positive control groups (G6–G8),
were used to evaluate the effectiveness of endodontic medicaments
including Ca(OH)2 paste (Meta Biomed Co, Ltd, Cheongju City,
Chungbuk, Korea), 2% CHX gluconate gel (Cerkamed Group, Nisko,
Poland), and a combination of both (Ca(OH)2/CHX). Each root canal
was completely filled with endodontic medicaments, with the orifices
sealed by Coltosol F (Colt
ene AG, Altst€atten, Switzerland), and incu-
bated for 7 days at 37 C (Table 1).
The antimicrobial efficacy was determined by using the CFU
counting method. After cold plasma treatment, 15 mL sterile
normal saline was injected into the root canal. The total biofilm
volume (TBV) was collected with a smooth broach and churned
for 1 minute, and then two #30 sterile paper points were inserted
into the canal to collect the bacteria for 1 minute. The paper points
were transferred into 1 mL normal saline, and the process was
repeated 3 times as described. The centrifuge tubes were shaken
vigorously for 1 minute. The number of E. faecalis was determined
by an EasySpiral instrument (Interscience, Saint Nom la Bret^eche,
France).

TABLE 1. Experimental Groups of Different Treating Methods

Treatment Treatment
Group 1 Cold plasma 0 min Group 5 Cold plasma 12 min
Group 2 Cold plasma 3 min Group 6 Ca(OH)2 7 days
Group 3 Cold plasma 6 min Group 7 2% CHX 7 days
Group 4 Cold plasma 9 min Group 8 Ca(OH)2/CHX 7 days

2 Li et al. JOE — Volume -, Number -, - 2015

 Basic Research—Technology
Scanning Electron Microscopy
Scanning electron microscopy (SEM) (S-4800; Hitachi, Tokyo,
Japan) was introduced to study structural changes of the biofilms before
and after cold plasma treatment. Ten of the processed root canals were
immersed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate
(pH = 7.4) for 2 days at 4 C and a gradient of dehydration in ethanol.
Subsequently, they were split longitudinally, dried naturally, fixed, and
sputter-coated with gold-palladium for examining.

Confocal Laser Scanning Microscopy Analysis

The viability of the biofilms after plasma treatment was determined
by confocal laser scanning microscopy (CLSM) (A1R-si; Nikon, Tokyo,
Japan) with the excitation/emission wavelengths of argon ion laser
488/543 nm. Ten root canals were prepared for CLSM analysis, which
were split longitudinally into 2 parts. Both halves were infected with E.
faecalis for 3 weeks. Then, one of the halves received plasma treatment
for 12 minutes; the other half served as a control without any treatment.
According to the manufacturer’s instructions, the biofilms were stained
by using LIVE/DEAD BackLight Bacterial Viability Kit (Invitrogen, Carls-
bad, CA) for 15 minutes and then rinsed slightly with phosphate-
buffered saline. The mounted samples were observed and imaged by
using 10 lens with the Nikon A1R-si confocal microscope system.
Mechanical Safety Tests
Mechanical safety tests including microhardness and roughness
changes of pulpal dentin caused by plasma were tested. Ten single-
root canals were split longitudinally, and the middle third of the pulpal
dentin area was chosen. Subsequently, the samples were cleaned in an
ultrasonic bath for 15 minutes and finally embedded and kept for 24
hours in epoxy resin at room temperature. The pulpal wall of the
root canal was ground and polished by wet grinding paper with a grain
size of up to #4000. Afterwards, the samples were cleaned in an ultra-
sonic bath for 15 minutes. All 20 samples were divided into 2 groups.
Ten samples were used for roughness (mm) test by 3D Profile Measure-
ment Laser Microscope VK-X200 (Keyence Corp, Osaka, Japan) with its
software at an interval of 3 minutes. Another 10 samples were prepared
for microhardness test with Vickers Hardness Tester (Shimadzu Corp,
Tokyo, Japan). The indentations were made on the cut surface of each
sample. Three different areas of each sample were measured by using a
300-g load and a dwell time of 20 seconds. The mean value of 3 areas
represents the hardness value for each sample.
Measurement of Major Reactive Species
Generated by Plasma
To measure the major reactive species generated by the Ar/O2
plasma equipment, conventional optical emission spectroscopy
(OES) was applied with the Fibre Optic Spectrometer (Avantes, Broom-
field, CO). The dispersed emission spectra were recorded by a 2048-
pixel charge-coupled device detector array. To acquire the light signals,
a fiberoptics cable was used at the bottom of the plasma jet. Detailed
OES detection method of the cold plasma can be found in our earlier
article (26).
Statistical Analysis
The statistical analysis was determined by using one-way analysis
of variance, followed by Duncan multiple range post hoc tests. Statistical
significance was considered when P < .05.
Figure 2. Disinfection results of Ar/O2 plasma and intracanal medicaments.
E. faecalis biofilm was treated with Ca(OH)2, 2% CHX, and Ca(OH)2 plus 2%
CHX (CH/CHX) for 7 days and plasma treatment for 12 minutes (P < .05).
(a) In the plasma treatment groups, there is significant decrease of TBV
when prolonging the treatment time.
Results
Effectiveness of Plasma Disinfection
CFU counting method was adopted to evaluate the antimicrobial
effectiveness (Fig. 2). After 3-week incubation, the total bacteria vol-
umes (TBV) of biofilms all reached 107–108 CFU/mL. After 7 days of
intracanal dressing and plasma therapy for 12 minutes, all groups ac-
quired a CFU reduction compared with the control group, but only the
plasma group was able to kill E. faecalis biofilm completely. In all medi-
cation groups, the Ca(OH)2 group and CH/CHX group decreased about
4 and 2 logs, respectively. The 2% CHX gel group behaved most effec-
tively in all intracanal dressing groups.
For all groups of plasma treatment, the bactericidal effect
improved enormously as exposure time prolonged. When exposure
time extended from 3 minutes to 12 minutes, CFU reduced from 5 to
0, which had a significant reduction compared with the control.
SEM
SEM images were taken at the apical one third of the split root ca-
nals (white box area) with different magnifications. In the control group
(without cold plasma), a thick, matured biofilm embedded through EPS
and covered the entire pulpal canal surface (Fig. 3A). A highly organized
biofilm structure consisting of E. faecalis within the EPS was observed
(Fig. 3C–E). However, after plasma treatment for 12 minutes, the 3-
dimensional (3D) biofilm architectures were destroyed, and the biofilm
was separated from the pulpal dentin wall (Fig. 3B) and replaced with
ruptured bacteria (Fig. 3F–H).
CLSM Analysis
The CLSM 3D and 2-dimensional (2D) images show the efficacy of
cold plasma treatment (Fig. 4). For one half of the root canal after 3
weeks of incubation, nearly the whole layer exhibited green fluores-
cence, which indicated that all bacteria were alive (Fig. 4A). The other
half of the root canals was treated with plasma for the duration of 12
minutes, which resulted in almost all of the bacteria being killed by
plasma (red fluorescence, Fig. 4C). The thickness of the biofilm of
both groups was similar (as shown in Fig. 4B and Fig. 4D), which indi-
cated that cold plasma could even kill the bacteria located at the bottom
of the biofilm.

JOE — Volume -, Number -, - 2015 Evaluation of Cold Plasma Treatment and Safety on E. faecalis 3
Basic Research—Technology
Figure 3. SEM images of control group and plasma-treated group at low resolution and high resolution. (A) Biofilms adhered to the pulpal wall. (B) Biofilms are
separated from the pulpal wall after 12-minute plasma treatment. The arrow shows the E. faecalis biofilm before and after plasma treatment. (C–E) The 3D biofilm
architectures are embedded with EPS and linked with each other (as the arrow shows in E). (F–H) The architectures are destroyed after plasma treatment for 12
minutes, with debris and fused cell bodies left (as the arrow shows in H).
Mechanical Safety Evaluation
The values of pulpal dentin microhardness (HV) and roughness
(Ra, mm) are shown in Figure 5. The plasma treatment does not signif-
icantly change the microhardness and roughness of pulpal dentin after
plasma treatment with different durations (P > .05), indicating that cold
plasma treatment did not change the mechanical properties of pulpal
dentin.
OES Analysis
As shown in Figure 6, the emission spectra are dominated by Ar
emission lines, because Ar is the major component (98%) of the work-
ing gas. Strong atomic oxygen emission at 777 nm and 844 nm can also
be identified. However, the OH emission at 308 nm was not detected in
this study.
Discussion
Bacteria are a common cause of root canal infection and endodon-
tic treatment failure. Thus, efforts in treatment/retreatment should be
focused on maximal bacterial elimination from the root canal systems.
However, chemomechanical procedures and even intracanal medica-
tion strategies cannot achieve this goal because of the complexity of
the root canal system and the antibiotic resistance of biofilms (1–3).
Although monospecies infection is unlikely to happen in real life, the
E. faecalis species is closely related to persistent periapical infection
(2, 6), and the E. faecalis biofilm is a commonly adopted model to
investigate the disinfection methods (5). One of the reasons for this
is that the antibacterial effects of biofilm can be up to 1000-fold higher
than planktonic bacteria (8). Moreover, 3-week E. faecalis biofilms
become more resistant than the young biofilms less than a week
(27), which is hard to eliminate with traditional treatment (1, 13).
Thus, we used 3-week E. faecalis biofilm in this study.
Although TBV has been reduced by traditional intracanal dress-
ings, it is not as effective and stable as plasma therapy. Because
4 Li et al.
Ca(OH)2 has the property of a high pH value to disinfect infection, it
has been used in clinical applications for a long time (28, 29).
However, it has limited effect on E. faecalis in the biofilms because
E. faecalis are resistant to Ca(OH)2 (30). Therefore, 2% CHX has
been suggested as an irrigation solution and intracanal medication
(31). When used as intracanal medicament, 2% CHX has a better effect
than Ca(OH)2 in disinfecting E. faecalis and has a wide antibacterial
spectrum to eliminate microorganisms associated with persistent infec-
tions (13). High concentrations of CHX (2%) cause precipitation of
intracellular constituents, especially phosphate entities such as adeno-
sine triphosphate and nucleic acids, which leads to the precipitation of
the cytoplasmic contents and results in cell death. The combination of
2% CHX gel and Ca(OH)2 seems to have a similar bactericidal effect as
with Ca(OH)2 (32). In other words, 2% CHX gel is the most effective
intracanal dressing in killing E. faecalis biofilm, which is in accordance
with our present research. A possible explanation is that optimal anti-
microbial activity of CHX occurs at pH of 5.5–7.0 (33). Additional
Ca(OH)2 might change pH value for both CHX and Ca(OH)2, which
leads to a relatively poor antimicrobial effect. However, results showed
that 2% CHX gel still could not completely disinfect biofilms. Because of
the complex anatomy of the root canal system, medications in the forms
of liquid, gel, or paste are difficult to penetrate into the whole root canal
system; thus the disinfection effect is limited. One of the advantages in
cold plasma is being able to reach these areas more easily than tradi-
tional methods, which is more likely to achieve a better disinfection ef-
ficacy.
In our study, non-thermal atmospheric pressure plasma equip-
ment was used to kill E. faecalis biofilm incubated for 3 weeks. No
cultured bacteria were recovered from the BHI agar plate after 12 mi-
nutes of treatment, which indicates that the E. faecalis biofilm was killed
completely. SEM analysis showed that the normal spherical structure of
E. faecalis disappeared, and the 3D structure of biofilm was destroyed.
The vitality of E. faecalis in the biofilm was further confirmed by CLSM
analysis; the whole layer of biofilm was inactivated. Before plasma
JOE — Volume -, Number -, - 2015
 Basic Research—Technology

Figure 4. CSLM 3D images of E. faecalis biofilm before and after plasma treatment for 12 minutes. (A) Living bacteria (green fluorescence). (C) Dead bacteria
(red fluorescence). (B and D) The space between 2 arrows shows the thickness of biofilm.

treatment, bacteria of biofilms were almost green, but after 12 minutes
of plasma treatment, biofilm expressed red fluorescence, which means
almost all of the bacteria had died, which is in accordance with CFU
counting and SEM results. Meanwhile, there is no significant change
of microhardness and roughness of the dentin after 12-minute expo-
sure to plasma.
To investigate the major inactivation mechanisms of cold plasma,
OES was used in this study. Plasma induced UV intensities and thermal
impact on the bacteria can be neglected. Thus, the major cause of cell
death in this study is due to the membrane damage induced by cold
plasma treatment (34). The results of OES indicated that the reactive
oxygen species, especially oxygen, was generated and led to oxidative
stress and cell damage, which are the key inactivation agents in cold
plasma (35). Other possible mechanisms of reactive oxygen species,
including damage to the redox state of antioxidants, damage to the
structure of the cell membrane, and degradation of DNA, have been re-
vealed in our previous study (36). Nevertheless, the current study has
some limitations. For example, it is unknown whether the remnants of
bacteria on the root canal wall are a possible promoter of inflammatory
processes, and further studies will be carried out.

Figure 5. Microhardness (HV) and roughness (Ra, mm) analysis. There are
no significant changes before and after plasma treatment with different treat- Figure 6. Optical emission spectra of Ar/O2 cold plasma. (A) O (777 nm)
ment times (P > .05). and (B) O (844 nm) emission intensities of cold plasma.

JOE — Volume -, Number -, - 2015 Evaluation of Cold Plasma Treatment and Safety on E. faecalis 5
Basic Research—Technology
In conclusion, atmospheric pressure cold plasma has been
demonstrated to have a promising application in root canal treatment
for its effectiveness and safety in an accepted level, with a potential to
serve as an alternative solution in the near future.
Acknowledgments
The authors express their sincere appreciation to the Dental
Material Laboratory, Peking University School and Hospital of
Stomatology for its valuable technical assistance.
This study was supported by the 985 program of Peking
University.
The authors deny any conflicts of interest related to this study.
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