Non-Thermal Atmospheric Plasma for Endodontic Treatment

Avinash S. Bansode

, Aumir Beg

, Swanandi Pote

, Bushra Khan

, Rama Bhadekar

, Alok Patel

S. V. Bhoraskar

, V. L. Mathe

Department of Physics, University of Pune, 411007 Pune, India

Department of Microbial Biotechnology, Bharati Vidyapeeth University, 411046 Pune, India

Department of Pedodontics and Preventive Dentistry, Bharati Vidyapeeth University, 411046 Pune, India

Keyword: Atmospheric Non-Thermal Plasma, Plasma for Endodontic Treatment, Plasma Treatment on E. Faecalis.

Abstract: Gas discharge plasma is being explored nowadays for its application as an alternative to the conventional

sterilization and disinfection techniques in medical sciences. We have developed the non-thermal

atmospheric plasma torch to study the effect of plasma treatment on the growth rate of E. faecalis culture

and biofilms. E. faecalis treated with plasma was then compared with helium gas exposed and

chlorohexidine treated cultures and biofilms. All the results are analysed for significance (P < 0.001) using

ANOVA and TUCKEY’S test. Optical emission spectroscopy technique has been employed in. situ to

identify the species interacting with the samples. It is found that atmospheric non-thermal plasma proves to

be a promising alternative to traditional disinfectants for disinfection during endodontic treatment.

1 INTRODUCTION

Recently, cold plasma is being explored for its

application as an alternative to the conventional

sterilization and disinfection techniques in medical

field. Amongst the different types of plasmas,

studies on atmospheric non-thermal plasma has

gained importance in the medical field due to its

property of being functional at room temperature

and atmospheric pressure; unlike low pressure cold

plasmas. The antimicrobial property of atmospheric

plasma has been demonstrated against several

bacteria for example Escherichia coli, Candida

albicans, Streptococcus mutans, Bacillus subtilis etc

(

Hong, 2009).

In the field of dentistry, microorganisms and

their by-products are considered to be the major

cause of pulp and periradicular pathosis. The

objective of endodontic therapy is to i] remove

diseased tissue, ii] eliminate bacteria present in the

canals and dentinal tubules and iii] prevent post

endodontic recontamination. Hence, a major

objective in root canal treatment is to disinfect the

entire root canal system and to eliminate all the

possible sources of infection. This can be

accomplished by using mechanical instrumentation

and chemical agents, in conjunction with medication

of the root canal system between treatment sessions.

Inspite of these treatments, the survival of

microorganisms in the apical portion of root filled

teeth is still the major cause of endodontic failure.

Studies have revealed that this increased resistance

is due to the formation of biofilms by the

microorganisms. Microorganisms like Enterococcus

faecalis, Streptococcus mutans, Candida albicans

can adhere to the root canal walls and form

communities organized in biofilm which makes

them more resistant to phagocytosis, antibodies, and

antimicrobials. This is due to the presence of

exopolysaccharides in comparison with non-biofilm

producing organisms (

Sabeena, 2010). Also, due to

the presence of dentin tubules; the disinfection of

dentin posses special challenges during caries

therapy. Conventionally disinfection is achieved by

i] invasive removal, ii] use of chemicals like

chlorhexidine. But these treatments do not disinfect

the dental tubules completely. Contact dermatitis is a

common adverse reaction to chlorhexidine.

Chlorhexidine is also liable to cause desquamative

gingivitis, discoloration of tongue and teeth or

dysgeusia (distorted taste). Plasma being in the

gaseous form would have a better reach in the

confined and tortuous root canal system & may

prove to be a better adjunct to root canal

instrumentation for canal sterilization. Hence,

Bansode A., Beg A., Pote S., Khan B., Bhadekar R., Patel A., Bhoraskar S. and Mathe V..

Non-Thermal Atmospheric Plasma for Endodontic Treatment.

DOI: 10.5220/0004246200730077

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2013), pages 73-77

ISBN: 978-989-8565-34-1

 2013 SCITEPRESS (Science and Technology Publications, Lda.)

inactivation of microorganisms using plasma has

attracted much attention recently.

Numerous in vitro studies have been conducted

on the effects of atmospheric plasma on pathogens

like Streptococcus mutans, Candida albicnas,

Chromobacterium violaceum etc. Amongst the

various oral pathogens Enterococcus faecalis is one

of the primary organisms in patients with post

treatment endodontic infection (

Mohammadi, 2009).

E. faecalis is known to form intracanal biofilms,

periapical biofilms and biomaterial centered

infection. Inspite of being one of the important

infection causing organism, relatively few reports

are available that describe the efficacy of

atmospheric plasma against E. faecalis. Hence, this

study was aimed at evaluating the inhibitory effect

of atmospheric plasma against E. faecalis culture

and biofilms.

The paper reports the use of He (atmospheric

pressure) as a plasma generating gas which enables

the formation of active reactive species useful for

bactericidal properties.

2 MATERIALS AND METHODS

Atmospheric non-thermal plasma torch is found to

be useful for varieties of applications. Its use for

dental applications is an emerging field of research.

In the present article we report on generation of

miniaturize plasma torch using helium as plasma

forming gas. Fig 1 shows the schematic of the

atmospheric pressure non thermal plasma torch

which consisted of a tungsten cathode in the form of

thin wire of axially fitted into a cylindrical glass

tube with a fine nozzle. The anode was a stainless

steel plate which formed the base below the

microtitre plate. Helium was made to flow at a flow

rate of 1 lpm through the torch system so as to reach

the bacterial culture placed inside the microtitre

plate. Pulsed dc voltage of 24 kV was used to

operate the torch. The plasma plume 1-2 mm in

diameter extended outside the nozzle upto a distance

of 2-3 cm and reached the E. faecalis culture.

Helium having a low ionization potential serves as a

suitable ionizing medium and helps in extracting the

plasma plume to larger distances. Compared to other

gas plasmas it is easy to obtain plume of few

centimeter by using helium gas as plasma forming

gas (

Sladek, 2004). Figure 2 shows the photograph of

the plasma torch in operation, treating culture and

biofilms in microtitre plate



Figure 1: Shows the schematic of the atmospheric pressure

non thermal plasma torch.

The glow in the plasma is due to electron

excitation de-excitation and ionization of gas atoms,

so that it serves as a visual indicator of the presence

of energetic electrons and photons. These energetic

electrons generate radicals by dissociating gas

molecules such as H

O and O

, or by generating

metastable He ions that probably dissociate H

molecules. The air mixed with water molecules help

in generating the reactive OH and O radicals which

helps in killing the E. faecalis bacteria (

J. Goree,

2006).

Figure 2: Photograph of actual atmospheric non-thermal

plasma torch while treating the samples.

Enterococcus faecalis (NCIM 5025) was procured

from National Collection of Industrial

Microorganisms, Pune. The strain was maintained

on MRS (de Man, Rogosa and Sharpe) medium

(composition per liter: peptone 10 g, meat extract 10

g, yeast extract 5 g, sodium acetate 5 g, dipotassium

phosphate 2 g, ammonium citrate 2 g, magnesium

sulfate 0.2 g, manganese sulfate 0.05 g, glucose 20

He gas

Plasma

Plume

Plasma

Torch

E.Faecalis

culture

High

Voltage

BIODEVICES2013-InternationalConferenceonBiomedicalElectronicsandDevices

g, tween 80 1 g) slants and stored at 4°C.The effect

of atmospheric plasma against E. faecalis culture

was monitored as follows. 100 µl of culture was

placed in each well of 96 well microtitre plate. The

culture was then treated with atmospheric plasma for

2 minutes. The wells were grouped into 4 groups

viz. group A-helium treated, group B, plasma treated

and group C- chlorohexidine treated D- control

group with group size of 30 wells per group. E.

faecalis culture was treated with chlorohexidine for

2 minutes by adding 100 µl 0.2 % chlorohexidine

solution to the wells and subsequent removal of the

solution after 2 minutes. The viability of the cells

was checked by

Triphenyl tetrazolium chloride

(TTC) assay.

Preparation of bio-films: E. faecalis was

inoculated in MRS broth and incubated at 37°C at

120 rpm for 24 hours. 100 µl of culture (OD 1.0

according to McFarland’s scale, colony forming unit

(CFU) 4× 108 / ml) was added in each well of 96-

well flat base microtitre plate. The plate was

incubated at 37°C for 1 hour in order to allow the

organisms to adhere to the surface of the wells. After

1 hour, remaining culture form the wells was

replaced by 100 µl of fresh MRS broth and the plate

was further incubated at 37°C for 24 hours. After

incubation, the excess medium form the wells was

removed and the biofilms then treated with

atmospheric plasma for 2 minutes. The experiment

was divided into 4 groups as mentioned above.

2.1 Viability Assay

The viability of the organisms was determined using

the TTC viability assay described by C E

Nwanyanwu with some modifications (Nwanyanwu

et al, 2011). 100 µl of 1 % (w/v) solution of TTC

prepared in sterile distilled water was added in the

wells along with 100 µl of MRS broth following the

treatments. The plates were incubated at 37°C

overnight. The color change was measured at 490

nm using Elisa plate reader (Bio Rad model no:

IMark)

All the results are analysed for significance (P <

0.001) using ANOVA and TUCKEY’S test. Optical

emission spectrometer (OES) model HR-4000

(manufactured by Ocean Optics) was employed for

identification of the active ionic species present in

the plasma. This spectrometer is having detector

(model TCD1304AP) with linear CCD array and the

range of detection 200-1100 nm. Other

specifications of the spectrometer are availability of

shutter mode, fiber optical input and the optical

resolution is ~0.03nm (FWHM).

3 RESULTS AND DISCUSSION

E. faecalis culture was exposed to atmospheric non-

thermal helium plasma for 2 min. Similarly, culture

was also treated with chlorohexidine for 2 minutes.

The results of the viability assay were then

compared for the two processes as shown in figure

3. Figure shows the mean bacterial count in terms of

optical density in all the four groups. The wells in

the control group and the wells treated with helium

exhibited no change in the optical density (OD 1.0)

indicating 100 % survival of the culture. However

significant reduction in optical density was observed

in the wells exposed to chlorohexidine (OD 0.7) and

plasma (OD 0.6). Inhibitory effects of atmospheric

plasma have also been reported on E. faecalis

culture by

Cao et al (2011). There the authors had

exposed the culture to helium -oxygen plasma for 5,

10 and 15 min. The post exposure viability was

determined by measuring the zone of inhibition.

Effects of atmospheric plasma, helium and

chlorohexidine on E. fecalis biofilms adhered on

wells of the microtitre plates were determined by

TTC viability assay. The results were similar to

those observed for the culture. Chlorohexidine and

plasma treated biofilms showed decrease in optical

density.

Figure 3: Viability Assay of effect of plasma treatment

and chlorohexidine treatment on E. faecalis culture.

It was 0.5 for chlorohexidne, 0.47 for plasma as

compared to 0.8 of control and helium treated

biofilms. This clearly indicated reduction in viability

of bacteria in biofilms (fig3).

In order to confirm the presence of the reactive

species we have carried out the spectral

identification using optical emission spectroscopy.

Fig 4 shows the OES spectrum recorded during the

plasma exposure of the E. faecalis culture. Optical

Non-ThermalAtmosphericPlasmaforEndodonticTreatment

emission spectrum shows that the species present in

the plasma include helium, atomic oxygen (O),

Ozone (O

), and OH radicals.

Figure 4: Optical emission spectrum of helium plasma

generated by atmospheric non-thermal plasma torch.

Fig 5 shows helium gas did not show inhibitory

effect on bio films. Inhibitory effects of atmospheric

plasma have also been examined on

Chromobacterium violaceum (

Jonathan, 2009) and on

Streptococcus mutans biofilms (Sladek, 2007). Du et.

al. (2011) have also reported reduction in viability of

E. faecalis biofilms after treatment with atmospheric

plasma. The authors determined viability using

confocal laser scanning microscopy. Cao et al

(2011) have reported antibacterial effects of

atmospheric plasma on E. faecalis biofilms prepared

on nitrocellulose membrane using SEM. Our results

of 2 min exposure leading to significant reduction in

the viability suggest that this technique can be used

as an adjunct for endodontic therapy in dentistry.

Further research in this line could prove its potential

as an alternative for traditional procedures of

disinfection.

Figure 5: Shows inhibitory effect on E. faecalis biofilms.

Oxygen radical and OH radicals are known to be

bactericidal. It reacts with the cell wall of the

bacteria due to which it gets ruptured and cytoplasm

comes outside cause the death of the bacteria.

4 CONCLUSIONS

Atmospheric plasma proves to be a promising

alternative to traditional disinfectants for

disinfection during endodontic treatment. Using

plasma will be beneficial mainly due to its gaseous

nature, improved dispersion of disinfectant in the

dentinal tubules, better reach in the tortuous canals

and no dysgeusia (distorted taste). Hence,

application of atmospheric non-thermal plasma can

be a time saving method since post disinfection

clean up procedures can be minimized during

endodontic treatments.

ACKNOWLEDGEMENTS

ASB is thankful to UGC for the award of Ph. D.

fellowship. SVB is thankful to DST and CSIR, New

Delhi for the financial support and award of ES

fellowship.

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Non-ThermalAtmosphericPlasmaforEndodonticTreatment