22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Effective decontamination of soft reline-based oral cancer shutters by means of
non-thermal atmospheric plasma
V. Colombo1,2, M. Gherardi1, R. Laurita1, A. Liguori1, A. Stancampiano1, C. Azzimonti3, A. Cochis4, M. Petri5,
R. Sorrentino3 and L. Rimondini3
1
2
Alma Mater Studiorum-Università di Bologna, Department of Industrial Engineering (D.I.N.), Bologna, Italy
Alma Mater Studiorum-Università di Bologna, Industrial Research Centre for Advanced Mechanics and Materials
(C.I.R.I.-M.A.M.), Bolgna, Italy
3
University of Piemonte Orientale, Laboratory of Applied Microbiology, Novara, Italy
4
University of Piemonte Orientale, Laboratory of Biomedical Materials, Novara, Italy
5
Department of Oral Oncology and Pathology, San Luigi Hospital, Orbassano, Italy
Abstract: The effectiveness of direct cold atmospheric plasma (CAP) treatment in
bacterial decontamination of soft reline-based oral cancer shutters is reported. No
evaluable cytotoxic effect turns out to be induced by plasma treatment to the above reported
substrates.
Keywords: cold atmospheric plasma, oral biofilm, oral cancer, soft reline, oral shutter, in
vitro cyto-compatibility
1. Introduction
Severe oral cancer might require patients to undergo
surgical removal of soft tissues of the oral cavity; in order
to replace these tissues, patients are required to wear
removable shutters, generally patient custom-made, as it
is necessary to mold as much as possible the prosthesis to
the lacking tissue. Among the large number of materials
suitable for this purpose, soft reline holds a relevant
position since it is easily moldable and possesses, thanks
to its sponge-like return, the mechanical characteristics
required to sustain the typical values of the compressive
oral forces. Unfortunately, because of the sophistication
of the raw material and the custom-made production
process, soft reline based oral shutters are very expensive;
therefore, an eventual implant failure might be severely
resource- (because of the implants cost) and time(because of the long production process) consuming,
prompting the desire for novel means for limiting shutter
implant failures, which are primarily caused by severe
bacteria biofilm contaminations [1].
Cold atmospheric plasma (CAP) represents a very
promising strategy to decontaminate soft reline shutters,
given its strong antibacterial potential, related to the
killing activity of the several and synergic biologically
active agents produced by CAP [2-5].
Here we report the results obtained by directly treating,
with two different plasma sources, the surfaces of reline
shutters previously contaminated with a 24 hours-old
mature biofilm of Streptococcus mutans and
Aggregatibacter actinomycetemcomitans, two pathogens
strongly related with biofilm formation in the oral cavity.
The plasma treatment was strongly effective in biofilm
decontamination, determining a bacteria viability decrease
of about 50% and a loss of viable colonies number of
about 2 logs after 30 seconds (p<0.05).
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Finally, with the aim to get some information on the
possible cytotoxic effects of plasma treated samples, we
evaluated the viability of human cells directly cultivated
onto the surface of plasma treated specimens. The
obtained results reported that CAP determined a very
limited toxicity (90% viability with respect to untreated
control, p>0.05).
2. Materials and Methods
2.1. Soft Reline
Soft reline square specimens of 0.5 cm side and 2 mm
thickness width were realized following Manufacturer’s
instructions (GC Reline Soft, CG Corporate, IL, USA).
Specimens were sterilized by 70% ethanol immersion
overnight and washed carefully with phosphate buffered
saline (PBS, pH 7.4) prior to experiments.
2.2. Plasma sources
Two Dielectric Barrier Discharge (DBD) plasma
sources were used. The first configuration, named DBDA is composed by a cylindrical brass electrode (10mm
diameter) with a semispherical end (5mm curvature
radius). Plasma was generated by means of a micropulsed
generator with a peak voltage of 15.4 kV, a burst
repetition frequency of 1 kHz and an internal frequency of
40 kHz. The second configuration, named DBD-B, is
composed by two parallel aluminium-plate electrodes
(one connected to the generator and the other grounded)
covered by a POM-C plate used as dielectric. In this case
the plasma source was driven by a micropulsed generator,
producing high voltage bursts (duration 4 ms) with a
repetition frequency of 100 Hz; during the bursts, a 20
kHz sinusoidal waveform with 12 kV peak voltage is
produced.
1
2.3. Evaluation of plasma antibacterial activity
The ability of plasma to reduce bacteria viability was
evaluated towards two strong biofilm former:
Streptococcus mutans (DSMZ 20523, German DSMZ
Collection of Microorganisms) and Aggregatibacter
actinomycetemcomitans (DSMZ, 11123).
Soft reline specimens were placed into 24 multiwell
plates and their surfaces were submerged for 90 minutes
with in a suspension of 1x107 bacteria/ml to allow biofilm
adhesion (1ml/specimen). Surnatants were removed, fresh
medium (1 ml) added and plates incubated 24 hours at
37°C. Following plasma treatment (30, 60 and 120
seconds), bacteria viability was evaluated by the
colorimetric XTT (Sigma) assay, while the number of
viable colonies was counted by the CFU method.
Untreated samples were used as control and considered as
100% viability.
2.4. Evaluation of cyotocompatibility of plasma treated
soft reline specimens
To evaluate plasma toxicity of plasma treated reline
specimens, human primary gingival fibroblasts were
extracted from patients gingival biopsies by tissue
enzymatic, cultivated in α-MEM (Sigma) 10% serum and
1% antibiotics.
3x104 cells were seeded onto both untreated and plasma
treated reline semples and cultivated for 24 hours at 37°C
in α-MEM. Then, cells viability was evaluated with the
MTT (Sigma) assay. Untreated cells were considered
100% viability.
2.5. Statistical analysis of data
Data were analyzed using the Statistical Package for the
Social Sciences (SPSS v20.0, IBM, USA). Results were
compared by the one-way ANOVA followed by Sheffé’s
test for post-hoc; significance was set at p<0.05.
3. Results
3.1. Soft reline decontamination
Results of biofilm contaminated reline specimens
subjected to the plasma treatment are reported in Fig. 1
and Fig. 2.
In general, both plasma sources were effective in
reducing bacteria number and viability.
Treatment with DBD-A determined a statistically
significant (p<0.05) reduction of bacteria number (Fig.
1A, indicated by the #) and viability (Fig. 1B, indicated
by the *) against A. actinomycetemcomitans biofilm in
comparison with untreated control. Interestingly, the
efficacy was comparable between all the three time points
(30-60-120 sec) suggesting a very fast plasma killing
activity; no statistically significant difference was noticed
between the three time points (p>0.05). Similar results
were obtained by applying the DBD-A source against S.
mutans biofilm; viability ratio was decreased of about
50% in comparison with control (p<0.05, indicated by the
#) and the CFU number reduced of about 2 logs (p<0.05,
indicated by the *). Again, no difference was reported
between the different time-points.
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For the DBD-B source, similar results to DBD-A were
obtained, as reported in Fig. 2. Indeed, the direct
application
of
the
DBD-B
source
to
A.
actinomycetemcomitans biofilm determined a significant
reduction of bacteria number (Fig. 2A) and viability (Fig.
2B) in comparison with controls (indicated by the # and *
symbols respectively). However, a treatment of 30 sec
resulted less effective than the 60 and 120 sec time-points.
The same was observed in the S. mutans biofilm
treatment: the bacteria number was reduced of about 2
logs (Fig. 2C) and the viability of about 50% (Fig. 2D);
better results were achieved for 60 and 120 seconds time
points.
Fig. 1 A-D. DBD-A plasma treatment of contaminated
soft reline specimens.
Fig. 2 A-D. DBD-B plasma treatment of contaminated
soft reline specimens.
3.2. Cytocompatibility of plasma treated reline
specimens y
The cytocompatibility of plasma treated specimens was
evaluated by directly cultivating human primary cells
onto both untreated and treated samples. Results are
reported in Fig. 3. No significant reduction of cells
viability was observed neither on the DBD-A or DBD-B
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treated samples (p>0.05). In particular, cell viability was
reported in a range of 95-97% for samples treated with
DBD-A (Fig. 3A) and 90-97% for those treated with
DBD-B (Fig. 3B), in comparison of control cells
cultivated onto both untreated reline surfaces (polymer
control) and into polystyrene plates (polystyrene control).
Fig. 3 A-B. Human primary gingival fibroblast viability.
4. Discussion
The decontamination of soft reline shutters for patients
presenting severe oral cancer represents a challenge for
clinicians because in most cases it is necessary to remove
and substitute contaminated implants; this procedure is
expensive and time consuming because each shutter
requires a custom-made molding procedure in order to fit
with patients tissue defect. The most recognized reason of
implants failure is represented by biofilm contamination.
Non-thermal atmospheric plasma consists of various
active agents, namely UV photons, and particles as
neutral or excited atoms and molecules, negative and
positive ions, free radicals and free electrons, which were
demonstrated to enable biofilm decontamination [6, 7].
The majority of reactive species generated by CAP are the
following: i) electronically and vibrationally excited
oxygen O 2 and nitrogen N 2 , ii) active form of oxygen
molecules and atoms (reactive oxygen species, ROS) such
as atomic oxygen O, singlet oxygen 1O 2 , superoxide
anion O2 − and ozone O 3 , iii) reactive nitrogen species
(RNS) such as atomic nitrogen N, excited nitrogen N2(A),
nitric oxide NO•, iv) if humidity is present H2O+, OH−
anion, OH radical or hydrogen peroxide (H 2 O 2 ) are also
generated [8]. The effect of non-thermal atmospheric
plasma is not influenced by mechanisms of microbial
resistance to antibiotics (innate or acquired) [8]. This is
reasonable, since as discussed below, plasma consists of a
mixture of various reactive species that contribute to the
inactivation process of microorganisms. At atmospheric
pressure, the most harmful UV components, such as
vacuum UV or UVC (λ<280 nm), which could cause
intrinsic photodesorption or DNA damage, are missing or
are generated to an only small extent, respectively. The
UV light of non-thermal atmospheric plasma is mainly
emitted from N 2 molecules with power densities below 50
μW/cm2, which is not expected to directly affect
inactivation. Plasma discharges create charged particles
and an electrical field. It is proposed that electrical forces
affect the cell membrane, which might cause electrostatic
disruption or at least permeabilization for a very short
time. As a consequence, plasma-derived ROS/RNS
molecules, like reactive free radicals (NO, OH, and
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superoxide) or strong oxidizing agents (H 2 O 2 and O 3 ),
might penetrate into the microorganism. Further chemical
reactions can take place inside the cytoplasm. Then, all
agents oxidize cellular proteins or microbial DNA.
ROS/RNS can violate the integrity of the microbial cell
structure by lipid peroxidation, resulting in membrane
damage.
Our data confirmed the plasma high killing ability for
both A. actinomycetemcomitans and S. mutans biofilm. In
fact, the number and the viability of the bacteria were
significantly reduced by plasma direct treatment.
Moreover, the plasma efficacy was evident even in a very
short time of 30-120 seconds treatment, suggesting that
the production of reactive species is very efficient in the
biofilm polysaccharides matrix penetration. These
findings represent a further step towards solving the
drug-resistance problem.
Furthermore, plasma treatment of soft reline specimens
did not affect the viability of cells cultivated on them. In
fact, no difference in cells viability or morphology was
noticed between cells cultivated onto untreated and
treated specimens.
5. Conclusions
The decontamination of soft reline shutters represents
nowadays a challenge for clinicians in order to avoid
implants replacement. In the present work, we have
shown as the direct appliance of non-thermal atmospheric
plasma represents an effective procedure able to
significantly reduce bacterial contamination even in a
single short time appliance. Moreover, considering these
preliminary in vitro data, the treated samples turn out not
to be toxic for human cells. Therefore, even if more in
vitro and in vivo data are still necessary, it is possible to
state that plasma can be considered a very promising
solution for soft reline decontamination.
6. Acknowledgements
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”.
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