22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Investigation of antibacterial efficacy of a plasma gun source for endodontic
applications
D. Barbieri1, M. Boselli2, V. Colombo1,2, M. Gherardi1, R. Laurita1, A. Liguori1, E. Simoncelli1, A. Stancampiano1 and
L. Viola1
1
Alma Mater Studiorum-Università di Bologna, Department of Industrial Engineering (D.I.N.), Via Saragozza 8, IT40123 Bologna, Italy
2
Alma Mater Studiorum-Università di Bologna, Interdepartmental Center for Industrial Research – Advanced
Applications in Mechanical Engineering and Materials Technology (I.C.I.R.-A.M.M.), Via Saragozza 8, IT-40123
Bologna, Italy
Abstract: The contribution focuses on the evaluation of the antibacterial efficacy of
plasma treatments with properly designed Plasma Guns (PG) for endodontic applications.
Firstly, bacterial inactivation experiments on contaminated tryptone soy agar plates were
carried out in order to identify the best PG source configuration and its operating
conditions. The PG was tested on a liquid suspension contaminated with Enterococcus
faecalis to prove the antibacterial properties of plasma treatment in a liquid environment.
Bacterial decontamination tests on contaminated dental root canal models are also
envisaged, to investigate the effects of plasma treatment on bacteria, both in planktonic and
biofilm state.
Keywords:
applications
non-equilibrium plasma, plasma gun, bacterial inactivation, endodontic
1. Introduction
Atmospheric pressure non-equilibrium plasma sources
able of propagating plasma through dielectric capillaries
over distances of tens of centimeters, Plasma Guns (PG)
[1-3], are raising great interest for their potential in a wide
range of biomedical applications, such as cancer cells
treatment and endoscopic therapies [4-5].
In particular, the air plasma plume generates a great
amount of reactive species of oxygen (ROS) and nitrogen
(RNS) and UV radiation that play a key role in the
bacterial decontamination [6].
Oral cavity is a perfect ecosystem for the bacterial flora
of the mouth where all internal surfaces are coated with a
plethora of bacteria biofilms. A relevant challenge in
dental clinics is represented by the complete
decontamination of tooth root canal that could evolve in
an undesirable granuloma in the apical region [7].
Unfortunately, the complete disinfection of dental canal
roots is an important unresolved challenge for endodontic
treatments and fully efficient decontamination methods do
not yet exist [8-9]. This research is focused on the
valuation of efficacy of non-equilibrium atmospheric
pressure plasmas in the bacterial decontamination of
standardized models of root canal (endodontic training
blocks) contaminated with the most common bacteria in
dental clinics such as Enterococcus faecalis and
Streptococcus mutans, both in planktonic and biofilm
state.
At first, the antibacterial potential of different PG
configurations developed in our laboratories, properly
designed for endodontic treatments, was comparatively
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analysed on tryptone soy agar (TSA) plates contaminated
with E. faecalis, for the purpose of selecting the best PG
configurations and operating conditions by measuring the
respective inactivation areas.
Since nowadays the importance of plasma-induced
liquid chemistry acting on cells is well established in
many fields of Plasma Medicine, such as in the
decontamination of living tissue [10-11], the antibacterial
efficacy of the previously selected PG treatment was also
evaluated on E. faecalis bacteria suspended in a smallvolume liquid solution, in order to simulate an oral
environment. In this frame, the antibacterial efficacy of
PG treatments can be even more realistically investigated
on root canal models contaminated with E. faecalis and S.
mutans in planktonic and biofilm form, varying the
exposure time.
Furthermore, an Optical Emission Spectroscopy (OES)
analysis of the produced plasma plume was performed to
investigate the chemical composition of the effluent, both
in experiments on the liquid bacterial suspension and on
the contaminated endodontic training blocks.
For reasons of space limitation results will be here
presented and discussed only for the treatment of
contaminated agar dishes and liquid bacterial suspension,
detailing only materials and methods for the experiments
on root canal models.
2. Preliminary tests on contaminated TSA plates
Qualitative information about the antibacterial efficacy
of different plasma treatments were achieved with an
inactivation area analysis on TSA plates contaminated
1
with E. faecalis. One millilitre of bacterial suspension at
2,8·107 CFU (Colony Forming Units)/ml was uniformly
spread over the agar surface and subjected to 3 minute of
plasma exposure.
Two different configurations of PG source were tested
in different operating conditions, investigating the role of
the peak Voltage (V) and the helium flow rate (Q). PG
sources are characterized by a high voltage electrode
made of a tungsten wire with a diameter of 1 mm and a
length of 50 mm; as the outer electrode, a 25 mm width
grounded aluminium foil wrapped around the dielectric
channel was used (Fig. 1). The working gas was 99.999%
pure helium and the plasma source was driven by a
micropulsed generator producing high voltage sinusoidal
pulses with a rise time of 40-50 µs; in the experiments,
the pulse width was set at 600 µs, the duty cycle at 6%
and the frequency at 22 kHz, while the applied voltage
varied from 7 to 15 kV. In all tests the distance between
the dielectric channel outlet and the agar surface was kept
constant at 5 mm.
The bacterial suspension was prepared from an
overnight culture and adjusted to approximately 1.5·108
CFU/ml based on McFarland turbidity standards (0.5
McFarland).
Fig. 1. Sample configuration of PG developed for
endodontic treatments.
The best source configuration and operating conditions
were selected evaluating inactivation areas and the
neatness (absence or a lower number of bacterial colonies
identify a neater inactivation area) after 24 h of incubation
at 37°C.
The results, presented in Fig. 2, have shown an increase
of dimension and neatness of the inactivation area for
increasing peak voltage. Otherwise, an increase of helium
flow rate causes a lower level of neatness on a bigger
inactivation area.
3. Plasma Gun decontamination of bacterial liquid
suspension
The plasma source which has been consequently
adopted in the following experiments is a wire electrode
PG with 4 mm of inner diameter, 75° of inclination angle
and with necking at the channel outlet. The operating
conditions were set at 15 kV of peak voltage, 2 slpm of
helium flow rate, 22 kHz of frequency, 6% of duty cycle.
In order to simulate a thin liquid layer that covers the
inner surfaces of the oral cavity, a 20 µl-volume of
physiological saline solution (NaCl 0.9%) contaminated
with E. faecalis was chosen for the quantitative
assessment of the PG antibacterial activity.
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Fig. 2. Inactivation area results in TSA plates (above) and
in mm2 (below) for different PG configurations.
A working suspension at 107 CFU/ml was prepared and
20 µl were placed in different wells of a 96-wells plate
(about 2·105 CFU/well). Immediately after treatment,
samples were recovered and diluted in 480 µl of saline
solution; ten-fold serial dilutions were prepared, plated on
TSA plates and incubated for 24 h at 37°C to quantify the
number of viable bacterial cells by colony counting (drop
counting method), as shown in Fig.3. The number of
bacterial cells/ml recovered after plasma treatment is then
compared to that of untreated samples (control).
Plasma Gun treatment of contaminated suspensions was
performed in triplicates varying the treatment time (1-3-5
min), keeping the source outlet at 5 mm from the well
upper surface (each well is deep about 7 mm). The results
reported in Tab.1 have shown a strong decontamination
for all tested conditions.
Tabel1. Bacterial load reduction results for untreated (control)
and plasma treated samples in different operating conditions.
*after sample recovering
**method sensitivity
Test
Starting*
CFU/ml
After treatment
CFU/ml
Log
Reduction
Control
1,7·105
###
###
All treatments
1,7·105
< 101 **
4
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Fig. 3. Representative photos of TSA plates for dropcolony counting of untreated (control) and treated
samples.
These experimental evidences have proved the
antibacterial efficacy of PG source on contaminated
solution, advancing plasma treatment on biological targets
living in a liquid environment such as teeth and root
canals.
4. Plasma Gun treatment on contaminated root canal
models
Having proved the antibacterial efficacy of a PG
treatment of contaminated agar dishes and of
contaminated liquid suspensions, an important step
forward is the study of decontamination on root canal
models with a PG suitably designed for endodontic
applications.
The plasma source used in these experiments will be the
PG in the prearranged configuration (4 mm of inner
diameter, 75° of inclination, with necking) at the previous
operating condition (15 kV of applied voltage, 22 kHz of
frequency, 2 slpm of helium flow).
The standardized endodontic training blocks have been
contaminated with 20 µl of E. faecalis and S. mutans
suspension in planktonic form (104-105 CFU/root canal).
The experiments on planktonic form have been
performed for three different plasma exposure time (1, 3
and 5 minutes) with a constant distance of 5 mm between
the source outlet and the root canal inlet, as in Fig.5.
3
Fig. 5. Optical emission spectrum of the plasma plume
produced by PG with He flow rate 3 slpm and peak
voltage 9.5 kV.
Fig. 4. Plasma Gun treatment on an endodontic training
block simulating a contaminated root canal.
Otherwise, in the biofilm decontamination tests, the
endodontic training blocks, contaminated with 104-105
CFU of E. faecalis and S. mutans, are incubated up to 7
days in anaerobic or 5% CO 2 conditions, respectively, to
create a biofilm layer in the inner surface of root canal. In
these cases, since biofilms are complex communities of
bacteria embedded in a polysaccharide matrix more
resistant than the planktonic form, longer treatment times
(5, 8 and 11 minutes) have been investigated.
Immediately after treatment, samples have been
recovered in saline solution by sonication, serially diluted,
plated on agar plates suitable for E. faecalis and S. mutans
and incubated for 24-48 h at 37°C to quantify the number
of viable bacterial cells. Each test is performed in
triplicate comparing the number of bacterial cells
recovered on the treated sample, on control and on
samples treated with only gas flow (gas control).
5. Optical Emission Spectroscopy of plasma plume
produced by Plasma Gun
In order to get some qualitative information on the
reactive species produced in the plume between the outlet
of the PG and the biointerphase of the contaminated
substrates, optical emission spectra in the UV, visible
(VIS) and near infrared (NIR) regions have been
collected. The UV-VIS spectrum for the flexible PG
source operated with a peak voltage of 9.5 kV is shown in
Fig. 5. The OES analysis shows that the main components
of optical radiation are in the wavelength region between
280-450 nm, where the bands of excited molecular
nitrogen and OH radicals are detected. Future studies will
focus on the analysis of the chemical species forming in
the liquid suspension during plasma treatment, in order to
better understand the physical and chemical mechanisms
behind the inactivation processes induced by the PG.
4
6. Acknowledgement
Work partially supported by COST Action MP1101
“Biomedical Applications of Atmospheric Pressure
Plasma Technology” and COST Action TD1208
“Electrical discharges with liquids for future applications”
7. References
[1] E. Robert, Plasma Sources Science Technology, 21,
034017 (2012).
[2] E. Robert, Plasma Processes and Polymers, 6, 795
(2009).
[3] Z. Xiong, Journal of Physics D: Applied Physics, 45,
27 (2012).
[4] E. Robert, Clinical Plasma Medicine, 1, 2 (2012).
[5] E. Robert, Plasma Processes and Polymers, 6, 795
(2009).
[6] V. Boxhammer, New Journal of Physics, 11, 115013
(2009).
[7] R. Gendrom, Microbes and Infection, 2, 897 (2000).
[8] V. Arora, International Dental Journal, 4, 1 (2014).
[9] G. C. Kim, Plasma Processes and Polymers, 10, 199
(2013).
[10] V. Boxhammer, New Journal of Physics,14, 113042
(2012).
[11] B. K. H. L. Boekema, Journal of Physics D: Applied
Physics, 46, 422001 (2013).
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