materials

Article
Innovative Curved-Tip Reactor for Non-Thermal Plasma
and Plasma-Treated Water Generation: Synergistic Impact
Comparison with Sodium Hypochlorite in Dental Root
Canal Disinfection
Raúl Arguello-Sánchez 1 , Régulo López-Callejas 2 , Benjamín Gonzalo Rodríguez-Méndez 2, * ,
Rogelio Scougall-Vilchis 1 , Ulises Velázquez-Enríquez 1 , Antonio Mercado-Cabrera 2 , Rosendo Peña-Eguiluz 2 ,
Raúl Valencia-Alvarado 2 and Carlo Eduardo Medina-Solís 1,3

1 Dental Reseach Center and Advanced Studies “Dr. Keisaburo Miyata”, School of Dentistry, Autonomous
University of Mexico State, Av. Paseo Tollocan, 13 Universidad, Toluca de Lerdo 50130, Mexico;
rarguellos@uaemex.mx (R.A.-S.); rscougallv@uaemex.mx (R.S.-V.); uvelazqueze@uaemex.mx (U.V.-E.);
cemedinas@uaeh.edu.mx (C.E.M.-S.)
2 Department of Physics, National Institute for Nuclear Research, Carretera Mexico-Toluca S/N,
Ocoyoacac 52750, Mexico; regulo.lopez@inin.gob.mx (R.L.-C.); antonio.mercado@inin.gob.mx (A.M.-C.);
rosendo.eguiluz@inin.gob.mx (R.P.-E.); raul.valencia@inin.gob.mx (R.V.-A.)
3 Dentistry Academic Area of the Health Sciences Institute, Autonomous University of Hidalgo State,
Exhacienda de la Concepción S/N Carretera Actopan-Tilcuautla, San Agustin Tlaxiaca 42160, Mexico
* Correspondence: benjamin.rodriguez@inin.gob.mx

Citation: Arguello-Sánchez, R.;
López-Callejas, R.; Rodríguez-
Méndez, B.G.; Scougall-Vilchis, R.;
Velázquez-Enríquez, U.; Mercado-
Cabrera, A.; Peña-Eguiluz, R.;
Valencia-Alvarado, R.; Medina-Solís,
C.E. Innovative Curved-Tip Reactor
for Non-Thermal Plasma and
Plasma-Treated Water Generation:
Synergistic Impact Comparison with
Sodium Hypochlorite in Dental Root
Canal Disinfection. Materials 2023, 16,
7204. https://doi.org/10.3390/
ma16227204
Academic Editors: Edgar Schäfer,
Cosmin Sinescu and Mihai Rominu
Received: 16 October 2023
Revised: 13 November 2023
Accepted: 15 November 2023
Published: 17 November 2023
Abstract: Non-thermal plasmas (NTPs), known as cold atmospheric plasmas (CAPs), hold great
potential for diverse medical applications, including dentistry. However, traditional linear and rigid
dielectric barrier discharge reactors used for NTP generation encounter limitations in accessing
oral cavities and root canals. To address this issue, we have developed an innovative NTP reactor
featuring an angled end for improved accessibility. The central copper electrode, with a 0.59 mm
diameter and adjustable length for desired angulation, is coated with zircon powder (ZrSiO4 ) to
ensure stable NTP generation. This central electrode is housed within a stainless steel tube (3 mm
internal diameter, 8 mm external diameter, and 100 mm length) with a 27◦ angle at one end, making
it ergonomically suitable for oral applications. NTP generation involves polarizing the reactor
electrodes with 13.56 MHz radio frequency signals, using helium gas as a working medium. We
introduce plasma-treated water (PTW) as an adjunctive therapy to enhance biofilm eradication within
root canals. A synergistic approach combining NTP and PTW is employed and compared to the gold
standard (sodium hypochlorite, NaOCl), effectively neutralizing Enterococcus faecalis bacteria, even
in scenarios involving biofilms. Moreover, applying NTP in both gaseous and liquid environments
successfully achieves bacterial inactivation at varying treatment durations, demonstrating the device’s
suitability for medical use in treating root canal biofilms. The proposed NTP reactor, characterized
by its innovative design, offers a practical and specific approach to plasma treatment in dental
applications. It holds promise in combatting bacterial infections in root canals and oral cavities.
Keywords: non-thermal plasma; plasma-treated water; veneering; ceramic; bacteria; root canals

Copyright: © 2023 by the authors. 1. Introduction

Licensee MDPI, Basel, Switzerland.
Bacteria lodged in the root canal cause pulpitis and apical periodontitis. Once these
This article is an open access article
bacteria organize themselves into a biofilm, dental debridement becomes difficult due to
distributed under the terms and
their ability to adhere to and colonize the root canal, where they develop and secrete an
conditions of the Creative Commons
exopolysaccharide that acts as an adhesion and protection matrix for the bacteria, making
Attribution (CC BY) license (https://
them more resistant to antimicrobial agents than in planktonic bacteria [1–3]. The purpose
creativecommons.org/licenses/by/
4.0/).
of endodontics is to eliminate the presence of intraradicular biofilm since it is considered

Materials 2023, 16, 7204. https://doi.org/10.3390/ma16227204 https://www.mdpi.com/journal/materials

Materials 2023, 16, 7204 2 of 19

an important factor in the pathogenesis of endodontic infections, and its total elimination is
essential for the success of endodontic treatment; this can be achieved through mechanical
instrumentation of the root canal and irrigation with antiseptic solutions [4,5].
The gold standard for dental disinfection is chemomechanical treatment, which is
carried out with manual or assisted rotary instruments and using chemical solutions such
as sodium hypochlorite (NaOCl) [6–8]. The leading causes of endodontic failure are the
infection of the dentinal tubules and/or lateral canals, anatomical complexity, fractures or
fissures, and extraradicular biofilm [9]. This has led to the search for alternative disinfection
methods to avoid endodontic failure. One of these alternatives could be the application of
non-thermal plasma (NTP) or cold atmospheric plasma (CAP).
NTP is excellent for wound disinfection, sterilization, and healing in biomedical
applications. This is possible because when generating the NTP and depending on the gas
used, reactive species, charged particles, and UV photons can be produced, which have
a high oxidative potential and, therefore, can attack and disrupt the molecular structures
of microorganisms [10]. Likewise, the surfaces of medical devices can be optimized to
improve their biocompatibility and biofunctionality [11,12].
The NTP is generated by providing energy to a gas using an electric field, which
achieves its ionization. An NTP is composed of ions, free electrons, and atoms in their
elementary or excited state. The electric field causes the dissociation of the gas atoms or
molecules into ions and electrons. Subsequently, the atoms or molecules in the excited state
lose their energy by colliding with other particles or by the emission of photons [13]. The
NTP is formed at atmospheric pressure in frequency ranges of the order of units from kHz
to MHz or in microwaves (GHz), with voltages up to 100 kV [14].
Another way to achieve bacterial inactivation is by utilizing water interacting with
NTP, a process known as Plasma-Treated Water (PTW). The application of PTW has gar-
nered significant interest due to its potential environmental, medical, and even food tech-
nology applications, among others. The primary technique for producing PTW involves
generating plasma either outside or within the water to produce ions that result in various
highly reactive oxygen species (ROS) and nitrogen species (RNS), which are essential for
bacterial inactivation. One advantage of PTW is its ability to be applied rapidly and easily,
regardless of the surface’s form. Furthermore, this innovative approach opens new avenues
for enhancing disinfection and treatment strategies, contributing to various fields, including
public health, agriculture, and industrial processes [15].
Plasma can be generated through various methods, with some requiring high vacuum
systems. Examples include glow discharge, microwave discharge, inductively coupled
discharge, and capacitively coupled discharge. However, when operating at atmospheric
pressure, NTP can be generated using corona discharge and dielectric barrier discharge
(DBD). Notably, these methods eliminate the need for high-vacuum systems [16,17].
In the setup of a DBD-type plasma between two electrodes where the discharge
occurs, one or both can be coated with a dielectric material [18,19]. The DBD forms when an
alternating voltage or pulsed voltage is applied across these electrically insulated electrodes,
leading to an accumulation of charge on the surface of the dielectric. This sequence of
events results in multiple discharges in intervals of microseconds, which supplies the
necessary ionization level to sustain a plasma discharge. The plasma thus created can be
maintained as long as the energy supplied is adequate to counteract the recombination
process; otherwise, the plasma discharge will cease to exist [20–22].
An electric field is also established between electrically insulated electrodes, and
if the field strength is sufficient, an electric discharge occurs. However, the insulation
prevents the generation of significant current flow, preventing the gas from heating. Instead,
the electrons migrate toward the positive electrode but cannot escape due to insulation.
In a minimal time, the resulting charge compensates for the high-voltage field created,
extinguishing the discharge [19,20].
Various DBD reactor designs have been described [19]. The characteristics of DBD
plasma reactors mainly depend on the electrical parameters of the power supply, the chosen
Materials 2023, 16, 7204 3 of 19

geometry, and the properties of the dielectric barrier material. Studies have concluded that
three dielectric characteristics are of great importance, the type of dielectric material, the
thickness of the dielectric barrier, and the surface roughness of the dielectric barrier, as they
impact the discharge mode and its emission spectrum [23,24].
Indeed, some reviews focus on dielectric materials and their influence on the perfor-
mance of the DBD plasma reactor, considering factors like thickness [18,24,25]. Researchers
have also investigated the relationship between the concentration of discharge filaments
and the subsequent dielectric breakdown. Additionally, studies have explored the surface
temperature of the dielectric and its effect on the transition from glow discharge to fila-
mentary discharge. Notably, it has been demonstrated that certain ceramics, known for
their corrosion resistance in both high- and low-temperature conditions, exhibit excellent
dielectric properties and heat conduction [24].
Ceramic materials serve specific purposes based on their application. Multifunctional
ceramics consist of different materials, each contributing a distinct function. Some of the
most studied ceramics include aluminium oxide doped with MgO [26], calcium zirconate
oxide doped with MgO [24], and yttrium-stabilized zirconia [27]. These compositions have
been extensively investigated for their unique properties and potential applications.
The reactors that generate NTP for biomedical applications need specific characteris-
tics, including being manageable and ergonomic. However, conventional NTP reactors are
often rigid and straight, making them unsuitable for direct use in oral cavity root canals.
A jet-type reactor with a curved terminal has been developed to overcome this limita-
tion [28,29]. These reactors can operate with peak voltages of up to 18 kV and working
frequencies between 20 and 50 kHz. The curved design allows for easier insertion and
application of NTP in root canals, making it more practical for dental treatments.
Another type of reactor used for NTP generation is the Thorns plasma reactor [30],
which creates a plasma in pre-established radial directions. It operates with voltages around
16 kV and frequencies of 10 kHz, and the diameter of the tip is 6 mm. Although it can
be inserted into the root canal for treatment, the tip size may make it impractical in some
instances where the canal’s dimensions are too narrow. This limitation could restrict its
applicability in specific dental procedures.
This study introduces an innovative approach for addressing biofilms within root
canals. The primary objective is the efficient elimination of these biofilms by utiliz-
ing a novel curved-tip reactor designed for NTP generation. This unique reactor was
also employed for PTW production. Both processes were compared to the conventional
gold standard in root canal treatments involving sodium hypochlorite. In vitro assess-
ments demonstrated the effectiveness of bacterial and biofilm removal from dentin sam-
ples. The curved reactor design enhances manoeuvrability and facilitates application in
challenging-to-access regions, potentially offering an improved treatment modality for
endodontic infections.

2. Experimental Setup
2.1. Non-Thermal Plasma Reactor Design with a Curved Terminal
The NTP-generating reactor proposed in this study features a coaxial design with a
27◦ angle at one end. This configuration allows for convenient insertion into the oral cavity
and precise positioning to apply NTP from the incisors to the lower and upper molars
(refer to Figure 1). Consequently, the plasma beam is directed perpendicular to the target
area, ensuring accurate and targeted treatment for optimal therapeutic outcomes.
The reactor has specific characteristics, including an internal electrode length of 99 mm
and an external electrode length of 100 mm. Both electrodes have an angle of 27◦ at one
end, enabling easy access to the molars, as depicted in Figure 1. The inner electrode is
made of copper and has a diameter of 0.59 mm. On the other hand, the outer electrode is a
stainless-steel tube with an internal diameter of 3 mm and an external diameter of 8 mm.
 The reactor has specific characteristics, including an internal electrode length of 99
mm and an external electrode length of 100 mm. Both electrodes have an angle of 27° at
one end, enabling easy access to the molars, as depicted in Figure 1. The inner electrode is
Materials 2023, 16, 7204 made of copper and has a diameter of 0.59 mm. On the other hand, the outer electrode 4 of 19
is
a stainless-steel tube with an internal diameter of 3 mm and an external diameter of 8
mm.
The inner
The inner electrode
electrode isis coated
coated with
with aa dielectric
dielectric material
material toto create
create the
the dielectric
dielectric barrier
barrier
discharge. For
discharge. For this
this purpose,
purpose, commercial
commercial zircon
zircon(ZrSiO
(ZrSiO44)) powder
powder (zircon
(zircon potting
potting cement
cement
no. 13,
no. 13, Sauereisen,
Sauereisen, Inc.,
Inc.,Pittsburgh,
Pittsburgh,PA,PA,USA)
USA) was
was chosen.
chosen. This
This dielectric
dielectric material
material exhibits
exhibits
highelectrical
high electricalinsulation,
insulation, thermal
thermal conductivity,
conductivity, and anticorrosive
and anticorrosive properties.
properties. When
When mixed,
itmixed,
forms ita chemical
forms a chemical settingcapable
setting cement cement ofcapable of withstanding
withstanding temperaturestemperatures
above 1400 ◦ C,
above
making
1400 °C,itmaking
suitable for the plasma
it suitable conditions
for the plasma and potential
conditions temperature
and potential gradients
temperature and
gradients
thermal shocks
and thermal within
shocks the reactor.
within the reactor.

(a) (b)
Figure1.1.Placement
Figure Placement of
ofthe
theNTP-generating
NTP-generating reactor
reactor inside
inside the
themouth,
mouth,illustrating
illustrating its
itsapplication
application in
in
themost
the mostprofound
profoundareas:
areas:(a)
(a)upper
uppermolar
molarand
and(b)
(b)lower
lowermolar.
molar.

As
As mentioned
mentioned earlier,
earlier,the
thecentral
centralcopper
copper electrode
electrodeisiscoated
coated by by mixing
mixing 10 10 gg of
of zircon
zircon
powder
powderwith with1 1mL mLofof distilled water.
distilled water.TheThe
mixture
mixtureis degassed
is degassed using an electric
using vibrator
an electric (Ray
vibrator
Foster DentalDental
(Ray Foster Equipment, Huntington
Equipment, Beach, CA,
Huntington Beach,USA)CA,forUSA)
2 min, foras2shown
min, asinshown
Figure in 2a.Fig-
To
improve adhesion, any linear irregularities on the copper electrode
ure 2a. To improve adhesion, any linear irregularities on the copper electrode are recti- are rectified by rolling
itfied
on by
sandpaper
rolling itbetween two flatbetween
on sandpaper crystals,twothusflat
providing
crystals,surface roughness.
thus providing surface rough-
ness.After injecting the mixture to cover the copper electrode, it is inserted into a silicone
hose with
Afteran internalthe
injecting diameter
mixtureofto2.1 mmthe
cover andcopper
an external diameter
electrode, of 3 mm.into
it is inserted Theamixture
silicone
is carefully
hose with an injected
internalinside the hose
diameter until
of 2.1 mm theand
entire electrodediameter
an external volume is of covered.
3 mm. The Before
mixturethe
mixture sets, the silicone hose with the covered electrode is introduced
is carefully injected inside the hose until the entire electrode volume is covered. Before into an 80 mm long
carbon fibre sets,
the mixture tube the
with an internal
silicone diameter
hose with of 3.1 mm
the covered and anisexternal
electrode introduced diameter
into an of 80
5 mm.
mm
The ◦
long carbon fibre tube with an internal diameter of 3.1 mm and an external diameter 27
silicone hose and carbon fibre tube are placed in a mould to create the required of 5
curvature for the reactor
mm. The silicone hose and design
carbon(Figure
fibre2b).
tube are placed in a mould to create the required
After the zircon mixture has been
27° curvature for the reactor design (Figure 2b). injected and set around the copper electrode inside
the silicone hose and carbon fibre tube,
After the zircon mixture has been injected and the central electrode,
set around nowthe coated
copperwith the zircon
electrode in-
material,
side the silicone hose and carbon fibre tube, the central electrode, now coated awith
is carefully retrieved from the hose by making a transverse cut using scalpelthe
blade.
zirconItmaterial,
is then adjusted to the
is carefully requiredfrom
retrieved length
thetohose
fit inside the reactor
by making (Figure 2c).
a transverse cut using a
The schematic diagram of the entire process is depicted
scalpel blade. It is then adjusted to the required length to fit inside the reactorin Figure 2d. Finally, Figure2c).
(Figure 2e
shows the NTP reactor applied to a root canal model, and in the enlarged area of the model,
The schematic diagram of the entire process is depicted in Figure 2d. Finally, Figure
plasma penetration is observed throughout the canal. This demonstrates the successful
2e shows the NTP reactor applied to a root canal model, and in the enlarged area of the
application of the NTP reactor for dental purposes.
model, plasma penetration is observed throughout the canal. This demonstrates the
successful application of the NTP reactor for dental purposes.
Materials 2023,
Materials 16, x7204
2023, 16, FOR PEER REVIEW 5 5of
of 20
19

(a) (b) (c)

BNC connector Anode Cathode

RF source
Gas inlet
Dielectric Plasma

(d) (e)
Figure
Figure 2.
2. (a)
(a)Preparation
Preparationofofthe
themixture
mixtureforfor
thethe
coating
coatingof the central
of the electrode.
central (b) The
electrode. mixture
(b) The and
mixture
corresponding angle cover the central electrode. (c) Non-thermal plasma reactor. (d)
and corresponding angle cover the central electrode. (c) Non-thermal plasma reactor. (d) Schematic Schematic
diagram.
diagram. (e)
(e) The
The NTP
NTP reactor
reactor test
test device
device was
was applied
applied toto aa root
root canal model.
canal model.

2.2. Materials
Materials for
for the
the Experimentation In Vitro
Several
Several publications
publications havehave discussed
discussed using
using NTP
NTP reactors
reactors for
for treating
treating biofilms
biofilms[12,31].
[12,31].
Various in vitro tests were conducted to verify the efficiency efficiency of the designed device for
applying itit to
applying topatients
patientswith
withroot
rootcanal
canalproblems
problemsinfected
infectedwithwith biofilms.
biofilms. AsAs documented
documented in
in previous studies, different sampling techniques and identification
previous studies, different sampling techniques and identification methods have methods have been
employed to to examine
examinemicrobial
microbialpopulations
populations within
withinrootroot
canals [32,33].
canals These
[32,33]. studies
These have
studies
revealed
have the presence
revealed of a polymicrobial
the presence community
of a polymicrobial communitywithinwithin
endodontic infections.
endodontic infections.
The literature reports the presence of various bacteria and microorganisms microorganisms in root
canals [32,34], including
canals including Gram-positive
Gram-positive bacteria
bacteria such
such as Peptostreptococcus spp.,
as Peptostreptococcus Strepto-
spp., Strepto-
coccus spp., Enterococcus faecalis, Staphylococcus salivarius, Lactobacillus spp.,
coccus spp., Enterococcus faecalis, Staphylococcus salivarius, Lactobacillus spp., Actinomyces Actinomyces
spp., Candida albicans,
spp., Candida albicans, Eubacterium
Eubacterium spp., Bacillus spp.,
spp., Bacillus spp., and
and Gram-negative
Gram-negative bacteria
bacteria suchsuch as
as
Porphyromonas spp., Prevotella spp., Veillonella spp., and Escherichia coli [35],
Porphyromonas spp., Prevotella spp., Veillonella spp., and Escherichia coli [35], among others. among others.
Some species
Some species are
are less
less prevalent
prevalent in in root
root canals.
canals.
The enduring
The enduring presence
presence of of microorganisms
microorganisms in in the
the apical
apical region
region ofof the
the root
root canal
canal can
can
potentially result in endodontic diseases, even following the completion
potentially result in endodontic diseases, even following the completion of root canal of root canal
treatment. Enterococcus
treatment. Enterococcusfaecalis
faecalisholds
holdssignificant
significantrelevance
relevance in the
in field, givengiven
the field, its widespread
its wide-
spread presence in cases of endodontic failure. It has been detected in necroticbefore
presence in cases of endodontic failure. It has been detected in necrotic pulps pulpsandbe-
afterand
fore unsuccessful treatments
after unsuccessful [34]. This[34].
treatments bacterium is inherently
This bacterium highly resistant
is inherently highlyto antimi-
resistant
crobial
to drugs compared
antimicrobial to other Gram-positive
drugs compared bacteria, making
to other Gram-positive it clinically
bacteria, makingsignificant in
it clinically
dentistry. Therefore, for this in vitro study, Enterococcus faecalis ATCC 29212
significant in dentistry. Therefore, for this in vitro study, Enterococcus faecalis ATCC 29212 was selected as
the bacterium to be investigated.
was selected as the bacterium to be investigated.
Materials 2023, 16, x FOR PEER REVIEW 6 of 20

Materials 2023, 16, 7204 6 of 19

2.3. Construction of Transparent Root Canal Models with Inserted Dentin

This investigation
2.3. Construction employed
of Transparent RootaCanal
lowerModels
secondwithpremolar,
Insertedwhich
Dentinhad been extracted for
orthodontic purposes. Access to the pulp chamber
This investigation employed a lower second premolar, which had was established, and a type
been K file was
extracted for
orthodontic purposes. Access to the pulp chamber was established, and a type length
meticulously inserted until it was visible through the apical foramen. The working K file
was meticulously
was determined by deducting
inserted until0.5 mm,visible
it was and chemomechanical
through the apical instrumentation
foramen. The workingwas per-
length was determined by deducting 0.5 mm, and chemomechanical instrumentation wasa
formed using a solution of 5.25% NaOCl and rotary instruments, ultimately achieving
final diameter
performed using ofa0.45 mm with
solution a taper
of 5.25% NaOClof 0.06%.
and rotary instruments, ultimately achieving a
To obtainof3D
final diameter 0.45images
mm with of the premolar,
a taper of 0.06%.high-definition Planmeca OP300 5 × 5 FOV
coneTobeam,
obtaincomputed
3D images tomography
of the premolar, (Planmeca, Helsinki,
high-definition Finland)
Planmeca OP300produced
5 × 5 FOV DICOM
cone
beam, computed tomography (Planmeca, Helsinki, Finland) produced DICOM formata
format images. Using the inVivo5 3D design program (Anatomage, San Jose, CA, USA),
three-dimensional
images. recreation
Using the inVivo5 3D of the premolar
design program was generatedSan
(Anatomage, in Jose,
STL format.
CA, USA), Theadesign
three-
included a 3recreation
dimensional mm slot above of thethe apex inwas
premolar its vestibular
generatedportion, with dimensions
in STL format. The designofincluded
3 mm in
adepth
3 mmand 3.8 mm
slot above theinapex
height, ensuring
in its vestibular it occupied
portion, withthe dimensions
entire root canal
of 3 mm space. Onceand
in depth the
final
3.8 mmdesign was completed,
in height, ensuring it the modelthe
occupied was 3D printed
entire root canalusing standard
space. Once thetransparent
final designbio-
compatible
was completed,resin,
the specifically
model was 3D theprinted
3D AnyCubic 8k with
using standard LCD technology
transparent biocompatible(AnyCubic,
resin,
specifically the 3D AnyCubic
Shenzhen, China) (see Figure 3a,b). 8k with LCD technology (AnyCubic, Shenzhen, China) (see
FigureA3a,b).
0.45/0.06 rotary file was introduced into the 3D-printed models along their final
A 0.45/0.06
length to preparerotary file wasAdditionally,
the samples. introduced into the15
a size 3D-printed
K file wasmodels
passed along
0.5 mm their final
through
length to prepare
the foramen the samples.
to ensure Additionally,
the patency a size 15
of the apical K file was
foramen andpassed
to check0.5 for
mmany through
resin the
ob-
foramen
structionsto in
ensure the patency
the duct. of the apical
The samples foramen
underwent a 10and to check
min for any
wash with resin obstructions
isopropyl alcohol in
in
anthe duct. The
ultrasonic samples
bath, and an underwent
additional a 10
10minminwash
rinse with
withisopropyl alcohol
distilled water inin ansame
the ultrasonic
ultra-
bath, and
sonic bath.an additional 10 min rinse with distilled water in the same ultrasonic bath.

(a) (b) (c) (d) (e) (f)

Figure3.3.AA3D
Figure 3Dprinted
printedtransparent
transparent tooth
tooth model
model with
with thethe following
following images:
images: (a) top
(a) top viewview showing
showing the
the simulated root canal, (b) lateral view, (c) presentation of the cast and dentin, (d)
simulated root canal, (b) lateral view, (c) presentation of the cast and dentin, (d) dentin mounted dentin
on
mounted on the model, (e) dentin and cast showing the stamp, and (f) biofilm growth.
the model, (e) dentin and cast showing the stamp, and (f) biofilm growth.

Humanmandibular
Human mandibularpremolars
premolarswere were carefully
carefully selected
selected forfor
thisthis study
study based
based on spe-
on specific
cific criteria, including having a fully formed apex, no root curvature,
criteria, including having a fully formed apex, no root curvature, no fractures, and no signs no fractures, and
no signs of resorption. Each tooth had a single root canal. Dentin segments
of resorption. Each tooth had a single root canal. Dentin segments were obtained from these were obtained
from theseusing
premolars premolars
a diamond usingdisc,a diamond
and theirdisc, and their were
measurements measurements were precisely
precisely matched to the
matched
groove to the
in the groove in
3D-printed the 3D-printed
models. To simulate models.
the root Tocanal
simulate
space,the root canal space,
a standardized rounda
standardized
groove round groove
with dimensions of 1.0with
mm in dimensions
depth and of 1.31.0
mm mm in depth
in width and 3c)
(Figure 1.3was
mmcreated
in widthat
(Figure 3c) was created at the centre of each dentin specimen using
the centre of each dentin specimen using a number 1157 tungsten carbide high-speed bur a number 1157 tung-
stenWhite,
(SS carbide high-speed
Lakewood, NJ,bur
USA. (SS White, Lakewood, NJ, USA.
Toverify
To verifythethe structural
structural integrity
integrity of samples,
of the the samples, they scrutinized
they were were scrutinized
under an under an
operat-
operating
ing microscope
microscope at a 20×at a 20× magnification
magnification level (Opmi level (Opmi
Pico, Carl Pico,
Zeiss,Carl Zeiss, Oberkochen,
Oberkochen, Germany).
Germany).this
Following Following
preparation,this preparation,
the dentin samplesthe dentin
were samples were17%
treated with treated with 17% eth-
ethylenediaminete-
ylenediaminetetraacetic
traacetic acid for 1 min toacid remove for 1themin to remove
smear the smear
layer, ensuring layer,and
a clean ensuring a clean
consistent and
surface.
consistent surface.
Subsequently, Subsequently,
the prepared dentin the prepared
segments were dentin
insertedsegments
into thewere insertedspaces
designated into the
in
the 3D-printed
designated models
spaces (Figure
in the 3d). Gingival
3D-printed models locking
(Figure resin
3d).(Ultradent Productsresin
Gingival locking Inc., (Ultra-
South
Jordan, UT, USA),
dent Products Inc.,was
South then used UT,
Jordan, to seal thewas
USA), dentin
thenfragments
used to sealin place securely
the dentin without
fragments in
causing any damage
place securely without to the samples
causing any (Figure
damage3e). to the samples (Figure 3e).
Before
Before inoculating
inoculating and and preparing
preparing bacterial
bacterial biofilms,
biofilms, thethe models
models shown
shown in in Figure
Figure 3f 3f
underwent
underwentgammagammairradiation
irradiationto tocreate
createaasterile
sterileenvironment.
environment. The The sterilization
sterilizationprocedure
procedure
involved exposing the model to a dose of gamma rays of 15 kGy, which was emitted from
Materials 2023, 16, 7204 7 of 19

a cobalt-60 radioactive source. This ensured the creation of a sterile environment while
preserving the physical integrity of the conduits in the model.

2.4. Bacterium Selection and Preparation

The Enterococcus faecalis bacterium was cultured in 5 mL of Luria–Bertani (LB) nutrient
medium for 20 h at 37 ◦ C. After incubation, the nutrient medium was removed by cen-
trifugation using a Dynamica-brand centrifuge (Dynamica, Livingstone, UK) operating at
5000 rpm for 10 min. This centrifugation process was repeated twice, and, each time, sterile
phosphate-buffered saline (PBS) with a pH of 7.2 was used. The supernatants obtained after
centrifugation were discarded, and the residues were washed three times with distilled
water to eliminate any potential interference from the culture media. Subsequently, the
washed residues were resuspended in a PBS solution.
Bacterial counting was conducted using a Carl Zeiss phase-contrast microscope (Carl
Zeiss, Oberkochen, Germany) and a Neubauer chamber (Marienfeld GmbH & Co. KG,
Lauda-Königshofen, Germany). To guarantee the precision of the bacterial count, serial
dilutions at a 1:10 ratio were meticulously prepared and subsequently incubated for 24 h
at 37 ◦ C under aerobic conditions. The bacterial cell count in the suspension was deter-
mined through the conventional plate count method, and the bacterial cell density was
adjusted to 1 × 106 colony-forming units per mL (CFU/mL) and 1 × 105 CFU/mL for
subsequent experiments.

2.5. Intracanal Biofilm Formation

A single representative colony was chosen to assess the susceptibility of Enterococcus
faecalis biofilms to NTP, and it was then diluted in 1 mL of sterile PBS. A total of 20 µL of
Enterococcus faecalis culture was injected into each root canal model containing inserted
dentin. Then, 15 mL of Luria–Bertani agar was placed into an 80 mL beaker to introduce
the root canal models with inserted dentin until the complete coverage of the specimens
with the bacterial suspension up to the dentin of the model was achieved. Each group of
specimens consisted of 10 individual models. These groups of specimens were carefully
placed in a circular plate made of stainless steel schedule 316L. The circular plate had
perforations with a diameter of 0.296 mm, which allowed each model to be securely
positioned (Figure 3f).
After the specimens were positioned on the circular plate, they were incubated at
37 ◦ C for three weeks, and this incubation period facilitated the formation and maturation
of the biofilm on the dentin surfaces.
The bacterial culture medium was regularly replaced throughout the three-week incu-
bation period. This regular replacement ensured the biofilm had access to a fresh nutritional
environment, promoting its continuous growth and development during the study.

2.6. Application of Non-Thermal Plasma in the Root Canal

NTP was generated in the plasma reactor, as described in Section 2.1, by utilizing an
RF source that operated at a frequency of 13.56 MHz and a power level of 20 W. Helium
was employed as the working gas, and it was introduced into the reactor at a flow rate
of 0.5 LPM. These parameters resulted in an energy density of 0.50 W/cm2 at the tip of
the reactor, which is an order of magnitude lower than the recommended exposure limit
for individuals according to the International Commission on Non-Ionizing Radiation
Protection [36], which is 4 W/cm2 .
The 3D root canal models from the experimental and control groups were randomly
divided for microbiological and SEM analysis. Dentin sections were disassembled from the
3D models after the respective treatments for microbial analysis and transferred to 1 mL
tubes. They were then sonicated in a water bath to remove biofilms. Subsequently, samples
were diluted from 107 to 103 , with 100 µL aliquots from each dilution step of every sample
being plated in triplicate on LB agar. Afterward, the plates were left to incubate at 37 ◦ C for
48 h, during which time the resulting colonies were enumerated as CFU/mL.
 samples were diluted from 107 to 103, with 100 µL aliquots from each dilution step of
every sample being plated in triplicate on LB agar. Afterward, the plates were left to in-
Materials 2023, 16, 7204 cubate at 37 °C for 48 h, during which time the resulting colonies were enumerated as19
8 of
CFU/mL.

2.7. Generation of Plasma-Treated Water (PTW)

2.7. Generation of Plasma-Treated Water (PTW)
Using the curved-tip reactor, we generated PTW on the surface of sterile distilled
Using the curved-tip reactor, we generated PTW on the surface of sterile distilled
water (SDW). The SDW container was positioned on a shaker device to ensure a con-
water (SDW). The SDW container was positioned on a shaker device to ensure a consistent
sistent dispersion of the reactive components within the water. The operational parame-
dispersion of the reactive components within the water. The operational parameters of
ters of the NTP reactor remained consistent with those detailed in Section 2.6. The reac-
the NTP reactor remained consistent with those detailed in Section 2.6. The reactor’s tip
tor’s tip was positioned at a 3 mm distance from the surface of the SDW within a 5 mL
was positioned at a 3 mm distance from the surface of the SDW within a 5 mL volume,
volume, which underwent plasma activation for 10 min. The primary objective of this
which underwent plasma activation for 10 min. The primary objective of this process was
process was to produce the PTW utilized in the current study.
to produce the PTW utilized in the current study.
2.8.
2.8.Experimental
ExperimentalDescription
Description
Within
Within the scopeofofthis
the scope thisresearch,
research,we wecultivated
cultivatedbiofilms
biofilmsonon3D3Droot
rootcanal
canalmodels
models
containing
containing embedded dentin, as depicted in Figure 3. These biofilms were formedover
embedded dentin, as depicted in Figure 3. These biofilms were formed over
three
threeweeks,
weeks,creating
creatingaaconducive
conduciveenvironment
environmentfor fordeveloping
developingand andestablishing
establishingbiofilms
biofilms
within
withinthe
thedentin
dentinroot
rootmodels.
models.This Thisbiofilm
biofilmformation
formationprocess
processwas
wasdeliberately
deliberatelydesigned
designed
totofaithfully
faithfullyemulate
emulateconditions
conditionsobserved
observedininreal-world
real-worldclinical
clinicalsettings,
settings,thus
thusproviding
providing
ananauthentic
authenticframework
frameworkfor forthe
thesubsequent
subsequentinvestigation
investigationand andassessment
assessmentofoftreatment
treatment
outcomes. As illustrated
outcomes. As illustrated in in Figure 4, various combinations were
4, various combinations were conductedconductedtoto compre-
comprehen-
hensively explore
sively explore and and evaluate
evaluate thethe effects
effects of multiple
of multiple treatments
treatments on biofilm
on biofilm formation
formation and
and elimination
elimination within
within environments
environments thatthat faithfully
faithfully replicate
replicate actual
actual clinical
clinical conditions.
conditions. In In
the
the experimental
experimental phase
phase (refer
(refer to Figure
to Figure 4), concentration
4), the the concentration of Enterococcus
of Enterococcus faecalis
faecalis bac-in
bacteria
teria
the biofilms was 107was
in the biofilms 107 CFU/mL,
CFU/mL, establishing
establishing this as this as our control
our control condition;
condition; in this
in this case, the
case, the samples
samples did not undergo
did not undergo any treatment
any treatment after inoculation,
after inoculation, and their
and their initial CFUinitial
countsCFUwere
used as
counts a reference
were used as for comparison
a reference with the experimental
for comparison group.
with the experimental group.

Experimentalconditions
Figure4.4.Experimental
Figure conditionsapplied
appliedtotothe
the3D
3Dmodels
modelsofofroot
rootcanals.
canals.

Theααcondition
The conditiondenoted
denotedthe
theapplication
applicationofofNTP
NTPininthe
theroot
rootcanal
canalfor
for55min,
min,with
withthe
the
reactor’s tip positioned at a 5 mm distance. The γ condition entailed the introduction
reactor’s tip positioned at a 5 mm distance. The γ condition entailed the introduction of
of PTW into the root canal for 5 min. In another condition, the reactor’s tip was placed
PTW into the root canal for 5 min. In another condition, the reactor’s tip was placed di-
directly onto the model, in contact with the dentin, at a distance of 0 mm, designated as the
ε condition. Furthermore, we employed NaOCl in 0.6% and 5.25% concentrations. The first
concentration was defined as the ζ condition and was applied to the root canal for 5 min,
while the second concentration of NaOCl was used for 1 min (β condition) and for 5 min (η
condition). All these conditions were considered baseline reference conditions.
Materials 2023, 16, 7204 9 of 19

In the subsequent stage of experimentation, we replicated the baseline procedures, i.e.,

α + α, γ + γ, and ε + ε. In each instance, the initial condition was administered, the model
with the dentin was refrigerated, and the same procedure was repeated one hour later.
Moreover, we explored specific combinations of the baseline conditions. For instance,
we applied NTP treatment (α), immediately followed by the application of PTW (γ) for an
additional 5 min, which was referred to as α + γ. Similarly, the combinations were carried
out as γ + ε, and, finally, η + γ.
Another phase of the experimentation entailed the combination of the NaOCl at 5.25%
(η) and NTP at 0 mm (ε). After applying the NaOCl solution for 5 min, the model with
the dentin was refrigerated for 60 min, followed by NTP; we referred to this procedure
as η + ε. Lastly, the experiment identified as γ + ε + ε was performed, in which PTW
was administered to the model with the dentin for 5 min (γ) without removing the PTW
residues. Subsequently, the ε condition was applied, followed by 60 min of refrigeration,
and then the ε condition was reapplied for 5 min more.

2.9. Scanning Electron Microscopy

We utilized scanning electron microscopy (SEM) and energy-dispersive X-ray micro-
analysis (EDX) to examine zircon powder during this study. Additionally, SEM was used
independently to investigate the ultrastructural and physicochemical characteristics of the
formed biofilm, and the morphology of both the biofilms and microorganisms within the
dentin treatment areas.
Dentin samples were collected from the treated and control root canal models and
prepared for SEM analysis using the following described methodology. The samples
were harvested, washed, and fixed with glutaraldehyde in solution at 6% in 0.1 M of PBS
and placed under refrigeration at 4 ◦ C for 24 h. Samples were then postfixed in osmium
tetroxide (solution at 4%) in 0.1 M of PBS and refrigerated at 4 ◦ C for 2 h. Finally, the
samples were rinsed with PBS and dehydrated in ascending graded ethanol series (20, 30,
40, 60, 70, 80, 90, and 100%). The samples were subsequently affixed to be mounted on a
stub and then subjected to an ion sputter process to apply an ultra-thin layer of gold.
To perform the SEM analysis, we utilized a scanning electron microscope model JEOL
JSM 5900 (JEOL Ltd., Tokyo, Japan), with the following radiation parameters: high voltage
of 20 kV and a probe current of 25 pA. These settings provided the necessary resolution and
sensitivity to examine the surface morphology and composition of the biofilm, enabling us
to gain valuable insights into its structure and interactions with the dentin surfaces.

2.10. Statistical Analysis

The data analysis was conducted using two software tools: a spreadsheet program
(Excel 97, Microsoft Corp., Richmond, VA, USA) and Origin-Pro software ver. 8.0 SR2
(OriginLab Corporation, Northampton, MA, USA). Statistical tests, including the ANOVA
with Bonferroni and Tukey’s post hoc tests, were performed at a significance level of 5%. We
rigorously analysed and interpreted the experimental results by applying these statistical
methods, deriving meaningful conclusions from the data.

3. Results
3.1. Results of the Non-Thermal Plasma Reactor with a Curved Terminal
The analysis of the zircon powder was conducted to evaluate the composition of the
dielectric using a scanning electron microscope JEOL JSM 5900, operating at an electron
energy of 20 kV. Figure 5a displays SEM micrographs of the zircon-based powder, revealing
a dispersed crystal form, the characteristics of which are detailed in Table 1. In Figure 5b,
the solid structure of the central electrodes of the DBD reactor covered with the zircon
coating is presented, and its features are detailed in Table 1. The uniformity of the powder
used across the entire surface can be observed after being heated at 1200 ◦ C. This uniform
coating is crucial to ensure the desired effect of the DBD in the plasma reactor.
 The analysis of the zircon powder was conducted to evaluate the composition of the
dielectric using a scanning electron microscope JEOL JSM 5900, operating at an electron
energy of 20 kV. Figure 5a displays SEM micrographs of the zircon-based powder, re-
vealing a dispersed crystal form, the characteristics of which are detailed in Table 1. In
Figure 5b, the solid structure of the central electrodes of the DBD reactor covered with the
Materials 2023, 16, 7204 10 of 19
zircon coating is presented, and its features are detailed in Table 1. The uniformity of the
powder used across the entire surface can be observed after being heated at 1200 °C. This
uniform coating is crucial to ensure the desired effect of the DBD in the plasma reactor.
A ® CuKa diffractometer (Siemens Corp., New York, NY, USA) was
A Siemens
Siemens D5000
D5000® CuKa diffractometer (Siemens Corp., New York, NY, USA) was
utilized to determine
utilized to determine thethe crystalline phases in
crystalline phases in the
the zircon
zircon powder used to
powder used to coat
coat the
the central
central
electrode. The diffractometer was operated with an acceleration set at 35 kV
electrode. The diffractometer was operated with an acceleration set at 35 kV and a current and a current
of ◦ (2θ) range using a step size of 0.02◦ ,
of 25
25 mA.
mA.Diffractograms
Diffractogramswere wereobtained
obtained in in
thethe
15–80
15–80° (2θ) range using a step size of
and
0.02°,each
andstep
eachtook
stepapproximately 38.9 s. 38.9 s.
took approximately
The
The X-ray diffraction (XRD)
X-ray diffraction (XRD) results
results were
were analysed
analysed by by comparing
comparing them them with
with refer-
refer-
ence
ence standards
standards from
from the
the ICDD
ICDD (International
(International Centre
Centre for
for Diffraction
Diffraction Data)
Data) database
database using
using
DIFFRAC.EVA
DIFFRAC.EVA V4.1.1 software (Bruker, Billerica, MA, USA). Figure 6 displaysthe
V4.1.1 software (Bruker, Billerica, MA, USA). Figure 6 displays thecorre-
cor-
sponding
responding XRD
XRDdiffractogram,
diffractogram,which indicates
which the presence
indicates of zircon
the presence and magnesium
of zircon and magnesiumoxide
in the in
oxide powder.
the powder.
The
The thermal
thermaltreatment
treatmentofofthe
thezircon powder
zircon powder during
duringthethe
coating of the
coating of central electrode
the central elec-
did not lead to substantial changes in peak widths, indicating no significant alterations in
trode did not lead to substantial changes in peak widths, indicating no significant altera-
the crystal lattice structure.
tions in the crystal lattice structure.

(a) (b)
Figure 5.
Figure 5. (a)
(a) SEM
SEM micrograph of the
micrograph of the raw
raw powder.
powder. (b)
(b) SEM
SEM micrograph
micrograph of
of the
the processed
processed powder.
powder.

Table 1. Composition characteristics of zircon before and after processes.

Table 1. Composition characteristics of zircon before and after processes.
Element Raw Powder [at.%] Processed Powder [at.%]
Element Raw Powder [at.%] Processed Powder [at.%]
C 63.70 40.71
C 63.70 40.71
O 25.10 39.31
Mg O 0.39 25.10
6.22 39.31
Si Mg 5.16 0.39 3.82 6.22
Zr Si 5.25 5.16 3.28 3.82
Al Zr 0.31 5.25 - 3.28
P Al - 0.31 6.66 -
P - 6.66

The NTP generated by the curved-tip reactor produced a significant concentration of

reactive species. In particular, the generated NTP provides an optical emission spectrum
(OES) identical to that observed in previous works [37,38], where primary reactive oxygen
species (ROS) were identified, such as the hydroxyl radical (OH), the nitric oxide (NO),
and singlet oxygen (1 O2 ), which give rise to the formation of secondary species, includ-
ing molecular oxygen (O2 ), hydrogen peroxide (H2 O2 ), atomic oxygen (O), and ozone
(O3 ). Some of these primary species have a half-life of the order of microseconds [39].
Furthermore, various species were identified because NTP is generated with helium gas,
such as the molecular nitrogen ion (N2 + ) and helium atomic radicals. The presence of the
NOγ(A-X) system was also determined in the spectrum, which results from the dissociation
of the surrounding air and the subsequent formation of NO molecules from oxygen (O)
and nitrogen (N) atoms.
Materials2023,
Materials 2023,16,
16,7204
x FOR PEER REVIEW 11 of 19
11 of 20

110
^2 0 0
100
90
80
70

^3 1 2
Dielectric
60
∗=MgO

^1 0 1
50

^1 1 2

^3 0*11 2 0
40 ^=Zr(SiO4)
30

^4 0 0

^4 2 *0 1 3 0
^2 2 0

^3 2 1
20

^1 0 3
^2 1 1

^4 2 0
^2 0 2

^4 3 1

* 222
∗020

^4 1 1

^4 1 3
10
0
20 30 40 50 60 70 80
2Θ
Figure6.6.Diffractogram
Figure Diffractogramofofthe
thepowder
powderused
usedfor
forthe
thecoating
coatingofofthe
thecentral
centralelectrode.
electrode.

Secondary reactive species

The NTP generated can persistreactor
by the curved-tip from produced
milliseconds to severalconcentration
a significant days [40]. In of
summary, the generation
reactive species. In particular,and evolution
the generated of reactive species an
NTP provides produced by NTP represent
optical emission spectrum
a(OES)
complex and dynamic
identical process that
to that observed involvesworks
in previous multiple stages,
[37,38], transitioning
where from gasoxy-
primary reactive to
liquid phases. This profound understanding of the underlying
gen species (ROS) were identified, such as the hydroxyl radical (OH), the nitric oxide chemistry carries significant
implications
(NO), and singlet for microbial
oxygen inactivation.
(1O2), which give rise to the formation of secondary species, in-
cluding molecular oxygen (O2), hydrogen peroxide (H2O2), atomic oxygen (O), and ozone
3.2.
(O3).NTPSome Temperature
of these Measurement
primary species have a half-life of the order of microseconds [39].
In biomedicine,
Furthermore, various species the temperature generatedbecause
were identified in the NTP NTP is critical and should
is generated not exceed
with helium gas,
40 ◦ C to avoid the thermal damage and+destruction of living tissue [41]. In this study, the
such as the molecular nitrogen ion (N2 ) and helium atomic radicals. The presence of the
NTP beam was
NOγ(A-X) systemdirected
was also ontodetermined
a glass plate in where a type Kwhich
the spectrum, thermocouple wasthe
results from placed for
dissocia-
temperature measurements. The outlet nozzle of the
tion of the surrounding air and the subsequent formation of NO molecules from oxygenreactor was varied perpendicular
to
(O)theandglass plate between
nitrogen (N) atoms. 1 and 10 mm, with the specific power of the RF generator set
between 16 and 24 W and
Secondary reactive species a constant canhelium
persistgasfromflowmilliseconds
rate of 0.5 LPM. to several days [40]. In
Subsequently, at a fixed distance of 5 mm,
summary, the generation and evolution of reactive species produced the plasma application bytime
NTPwas varieda
represent
between 2 and 7 min (see Figure 7). These conditions
complex and dynamic process that involves multiple stages, transitioning from gas towere tested for their effectiveness
in eliminating
liquid phases. bacteria,
This profoundand theunderstanding
results are presented in Figures chemistry
of the underlying 6 and 7a. For each
carries data
signifi-
point, five samples were averaged,
cant implications for microbial inactivation. and the average of these measurements was reported as
the result. Figure 7a,b exemplify that with 20 W of power and a five-minute application
Materials 2023, 16, x FOR PEER REVIEW
time, the sample’s
3.2. NTP Temperature temperature
Measurement is maintained at a reasonable 28 ◦ C, making it suitable 12 offor
20
application to patients and ensuring no thermal damage to biological tissue.
In biomedicine, the temperature generated in the NTP is critical and should not ex-
ceed32
40 °C to avoid the thermal damage and destruction of living tissue [41]. In this
24 W
study,
31 the NTP beam was directed onto a glass plate 22 W where a type K thermocouple was
31 20 W
30
placed for temperature measurements. The outlet 18 W nozzle of the reactor was varied per-
29
Temperature [°C]

16 W
pendicular
28 to the glass plate between 1 and 30 10 mm, with the specific power of the RF
Temperature [°C]

generator
27 set between 16 24and W 24 W and a constant 29 helium gas flow rate of 0.5 LPM.
26 22 W
Subsequently, at a fixed 20 W distance of 5 mm, the plasma application time was varied
25 28
between
24 2 and 7 min (see18 W
Figure
16 W
7). These conditions were tested for their effectiveness in
23
eliminating bacteria, and the results are presented 27 in Figures 6 and 7a. For each data
22
point,
21
five samples were averaged, and the average
26 of these measurements was reported
as 20
the result. Figure 7a,b exemplify that with 20 W of power and a five-minute applica-
2 3 4 5
1
tion time, 2 the3 sample’s
4 5 6 temperature
Distance [mm]
7 8 9 10 is maintained at a reasonable Time [min] 286°C, making
7
it suita-
ble for application to patients and ensuring no thermal damage to biological tissue.
(a) (b)
Figure 7.7. Temperature
Figure Temperature measurements
measurements with
with two
two graphs:
graphs: (a)
(a) effect
effect of
of applied
applied power
power concerning
concerning
distance; (b) effect of applied power concerning time.
distance; (b) effect of applied power concerning time.

3.3. Experimentation with the Reactor with Bacteria and Biofilms In Vitro
Experiments were conducted initially in Petri dishes and subsequently on root canal
models with embedded dentin to assess the time required for Enterococcus faecalis bacte-
rial inactivation. Figure 8 illustrates the outcomes of in vitro bacterial inactivation at 106
and 105 CFU/mL concentrations, employing a 5 min NTP application while maintaining a
5 mm distance between the reactor tip and the plate. In Figure 8, the region where the
plasma was applied is delineated by a circle, covering an approximate area of 1 cm².
 22

T
21 26
20
2 3 4 5 6 7
1 2 3 4 5 6 7 8 9 10 Time [min]
Distance [mm]
(a) (b)
Materials 2023, 16, 7204 12 of 19
Figure 7. Temperature measurements with two graphs: (a) effect of applied power concerning
distance; (b) effect of applied power concerning time.

3.3. Experimentation with

3.3. Experimentation with the
the Reactor
Reactor with
with Bacteria
Bacteria and
and Biofilms
Biofilms InIn Vitro
Vitro
Experiments
Experiments were
were conducted
conducted initially
initially in
in Petri
Petri dishes
dishes and
and subsequently
subsequently on on root
root canal
canal
models
models with
with embedded
embeddeddentin
dentintotoassess
assessthe
thetime
timerequired
required Enterococcus
forfor Enterococcus faecalis bacterial
faecalis bacte-
inactivation. Figure 8 illustrates the outcomes of inof
vitro bacterial inactivation 6 and
at 10at
rial inactivation. Figure 8 illustrates the outcomes in vitro bacterial inactivation 106
10 5
and 10 CFU/mL concentrations, employing a 5 min NTP application while maintaining aa
CFU/mL
5 concentrations, employing a 5 min NTP application while maintaining
55 mm
mm distance
distance between
between the
the reactor
reactor tip
tip and
and the
the plate. In Figure
plate. In Figure 8, 8, the
the region
region where
where thethe
plasma 2
plasma was applied is delineated by a circle, covering an approximate area of 1 cm
was applied is delineated by a circle, covering an approximate area of 1 cm²..

(a) (b) (c) (d)

Figure 8.
Figure 8. The
The inactivation
inactivation of
of Enterococcus
Enterococcus faecalis
faecalis bacteria
bacteria under
under various
various conditions.
conditions. (a) Control at
(a) Control at
10 6 CFU/mL. This image illustrates bacterial growth without any treatment. (b) Concentration of
6
10 CFU/mL. This image illustrates bacterial growth without any treatment. (b) Concentration of
106 CFU/mL treated with NTP. The treated area is highlighted by a circle, demonstrating bacterial
106 CFU/mL treated with NTP. The treated area is highlighted by a circle, demonstrating bacterial
inactivation. (c) Control at 105 CFU/mL. This image displays bacterial growth without NTP treat-
inactivation. (c) Control at 105 CFU/mL. This image displays bacterial growth without NTP treatment.
ment. (d) Concentration5 of 105 CFU/mL treated with NTP. Once again, the treated area is marked
(d)
with Concentration of 10 CFU/mL
a circle, showcasing bacterial treated with at
inactivation NTP.
thisOnce again, the treated area is marked with a
concentration.
circle, showcasing bacterial inactivation at this concentration.
We conducted bacterial inactivation using PTW on the same strain in the subse-
We conducted bacterial inactivation using PTW on the same strain in the subsequent
quent phase. Water distilled with a volume of 5 mL was utilized, and the treatment du-
phase. Water distilled with a volume of 5 mL was utilized, and the treatment duration
ration was set to be 5 min. Water characterization revealed a pH of 4.3, ozone concentra-
was set to be 5 min. Water characterization revealed a pH of 4.3, ozone concentration of
tion of 0.8 ppm, and a hydrogen peroxide concentration of 3 ppm. Employing PTW, En-
0.8 ppm, and a hydrogen peroxide concentration of 3 6ppm. Employing PTW, Enterococcus
terococcus faecalis was exposed to the bacteria at a 10 CFU/mL concentration, leading to
faecalis was exposed to the bacteria at a 106 CFU/mL concentration, leading to complete
completeinactivation
bacterial bacterial inactivation within 5 min.
within 5 min.
3.4. Experimentation
3.4. of the
Experimentation of the Reactor
Reactor with
with Bacteria
Bacteria and
and Biofilm
Biofilm Root
Root Canals
Canals
The experimental
The experimental phase
phase aimed
aimed at at removing
removing biofilms
biofilms fromfrom root
root canals
canals followed
followed the
the
protocol outlined in Section 2.8 and depicted in Figure 4. Biofilms were cultivated
protocol outlined in Section 2.8 and depicted in Figure 4. Biofilms were cultivated within within
root canal
canal models
modelscontaining
containingembedded
embeddeddentin,
dentin,asasillustrated
illustratedinin Figure
Figure 3. 3. The
The formation
formation of
of these
these biofilms
biofilms spanned
spanned three
three weeks
weeks (see(see Section
Section 2.5),2.5), enabling
enabling bacterial
bacterial proliferation
proliferation and
andestablishment
the the establishment of biofilms
of biofilms withinwithin the dentin.
the dentin. This biofilm
This biofilm formation
formation processprocess rep-
replicates
licates conditions
conditions encountered
encountered in practice,
in clinical clinical practice,
providing providing
a realistica framework
realistic framework for
for research
research
and and subsequent
subsequent treatmenttreatment
evaluation.evaluation.
The concentration of Enterococcus faecalis bacteria in the control model was consistent
at approximately 1.52 × 107 CFU/mL, as depicted in Figure 9 and Table 2. A comparative
analysis was conducted among the NTP, PTW, and NaOCl at 5.25% procedures, along with
the gold standard NaOCl at 0.6%, as detailed in Table 2. This table delineates the conditions
under which the processes exhibited statistical significance (value 1, p < 0.001) and those
where significance was not observed (value 0, p = 0.999).
These findings emphasize the importance of treatment combinations and their sequenc-
ing to achieve effective inactivation of Enterococcus faecalis within root canals. Additionally,
they provide valuable insights into the effectiveness of different approaches and potential
limitations in this specific application. These results contribute to advancing therapeutic
strategies in biofilm removal in endodontic procedures.
 The concentration of Enterococcus faecalis bacteria in the control model was consistent
at approximately 1.52 × 107 CFU/mL, as depicted in Figure 9 and Table 2. A comparative
analysis was conducted among the NTP, PTW, and NaOCl at 5.25% procedures, along
with the gold standard NaOCl at 0.6%, as detailed in Table 2. This table delineates the
Materials 2023, 16, 7204
conditions under which the processes exhibited statistical significance (value 1, p <13 of 19
0.001)
and those where significance was not observed (value 0, p = 0.999).

7
10

6
10

5
10

CFU/mL
4
10

3
10

2
10

1
10

0
10
Control

γ+ε+ε
γ

γ+ε
ε
α

γ+γ

η+γ
α+γ

η+ε
α+α

ε+ε
Condition
Figure 9.
Figure 9. Survival
Survival graph
graph of
of biofilms
biofilms in
in the
the biomodels
biomodels under
under different
different conditions.
conditions.

Table2.
Table 2. Comparison
Comparison with
with NaOCl
NaOCl “the
“thegold
goldstandard”.
standard”.

Treatment
Treatment loglog
10 CFU/mL
10 CFU/mL
p pValue
Valueversus
versus ζζ Significance
Significance * *
ζζ 5.59 ± 0.03
5.59 ± 0.03
Without value
Without value
Without value
Without value
Control 7.17 ± 0.11 0.999 0
Control 7.17 ± 0.11 0.999 0
α 6.16 ± 0.01 0.999 0
α 6.16 ± 0.01 0.999 0
α+α 6.02 ± 0.25 0.999 0
αγ+ α 6.02± ±
6.09 0.25
0.05 0.999
0.999 00
γ +γγ 6.09± ±
5.87 0.05
0.48 0.999
0.999 00
αγ++γγ 5.56
5.87± ±
0.07
0.48 0.999
0.999 00
αε+ γ 2.88 ± 0.16
5.56 ± 0.07 <0.001
0.999 01
ε +εε 2.15
2.88± ±
0.21
0.16 <0.001
<0.001 11
β 2.47 ± 0.06 <0.001 1
ε+ε 2.15 ± 0.21 <0.001 1
η 2.03 ± 1.36 <0.001 1
β 2.47 ± 0.06 <0.001 1
η+γ 1.26 ± 0.10 <0.001 1
η 2.03 ± 1.36 <0.001 1
η+ε 0.97 ± 0.06 <0.001 1
γη++εγ 1.26± ±
1.60 0.10
0.08 <0.001
<0.001 11
γ +ηε++ε ε 0.97± ±
0.87 0.06
0.15 <0.001
<0.001 11
* The significance
γ+ε level is 1 if the
1.60difference
± 0.08 to NaOCl 0.6% is significant at 0.05 and1 0 if it is not
<0.001
significant at the same level.
γ+ε+ε 0.87 ± 0.15 <0.001 1
* The significance level is 1 if the difference to NaOCl 0.6% is significant at 0.05 and 0 if it is not significant at the
These findings emphasize the importance of treatment combinations and their se-
same level.
quencing to achieve effective inactivation of Enterococcus faecalis within root canals. Ad-
ditionally, they provide
We utilized SEM to valuable
investigateinsights into the effectiveness
how different of different
treatments affected approaches
the morphology
and
of potential
bacteria limitations
attached to theindentin
this specific
surfaceapplication.
(Figure 10).These results contribute
The Enterococcus faecalis to advanc-
biofilm in
ing therapeutic
Figure 10a appearsstrategies in to
to adhere biofilm removalthe
and colonize in sample’s
endodontic procedures.
surface, with generally intact cell
membranes. NTP treatment led to some Enterococcus faecalis cells displaying a deflated and
ruptured morphology, accompanied by debris, as indicated by arrows in Figure 10b. The
cell membranes became considerably rough and unstructured in samples where the NTP
was applied at a 0 mm distance from the nozzle (Figure 10c). Similar results were observed
when the NTP was used for 5 min and then again after a 60 min interval (Figure 10d). The
treatment with PTW (Figure 10e) reduced the presence of these microbes in the sample;
however, some cells were still visible, and, among them, specific cells exhibited ruptured or
damaged cell walls, as indicated by the arrows. In the case of NaOCl treatment (Figure 10f),
 flated and ruptured morphology, accompanied by debris, as indicated by arrows in Fig-
ure 10b. The cell membranes became considerably rough and unstructured in samples
where the NTP was applied at a 0 mm distance from the nozzle (Figure 10c). Similar re-
sults were observed when the NTP was used for 5 min and then again after a 60 min in-
Materials 2023, 16, 7204
terval (Figure 10d). The treatment with PTW (Figure 10e) reduced the presence of14theseof 19
microbes in the sample; however, some cells were still visible, and, among them, specific
cells exhibited ruptured or damaged cell walls, as indicated by the arrows. In the case of
NaOCl treatment (Figure 10f), the canal walls appeared clean, and the presence of cells
the
wascanal wallsWhen
minimal. appeared clean,treatments
combined and the presence of cellssuch
were applied, wasas
minimal.
PTW andWhen
NTPcombined
at a 0 mm
treatments were applied, such as PTW and NTP at a 0 mm distance from the nozzle
distance from the nozzle (Figure 10g) and NaOCl and PTW (Figure 10h), the number of
(Figure 10g) and NaOCl and PTW (Figure 10h), the number of attached cells on the surface
attached cells on the surface rapidly decreased, and ruptured cell membranes were ob-
rapidly decreased, and ruptured cell membranes were observed.
served.

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 10. Longitudinal section of root canal analysed by SEM: (a) Dentin fully colonized by En-
Figure 10. Longitudinal section of root canal analysed by SEM: (a) Dentin fully colonized by Entero-
terococcus faecalis. Biofilms under various treatment conditions: (b) α, (c) ε, (d) ε + ε, (e) γ, (f) η, (g) γ
coccus faecalis. Biofilms under various treatment conditions: (b) α, (c) ε, (d) ε + ε, (e) γ, (f) η, (g) γ + ε,
+ ε, (h) η + γ, and (i) Treatment η + ε. Arrows indicate changes in cellular morphology, alterations,
(h) + γ, and (i) Treatment η + ε. Arrows indicate changes in cellular morphology, alterations,
andηdebris.
and debris.

4. Discussion
Emerging microbial control technologies offer numerous advantages in the scientific
field, including the preservation of oral health. Microbial load and the virulence of various
oral microorganisms significantly impact microbial contamination assessment, with specific
pathogens posing risks in root canals. Over the decades, innovative techniques have been
developed to address oral health challenges. However, it is essential to note that some
decontamination methods may adversely affect oral properties.
Combining multiple treatment methods, especially for root canals, has shown promise
by synergistically combating oral health-threatening microorganisms, providing effective
alternatives for achieving desired outcomes. Although this multidisciplinary approach
creates an inhospitable environment for microorganisms, a definitive solution still needs to
be discovered. These approaches aim to optimize therapeutic interventions and enhance
Materials 2023, 16, 7204 15 of 19

patients’ quality of life, contributing to ongoing dental advancements and promoting

optimal oral health.
This study aimed to develop a non-thermal plasma generator reactor with a curved
terminal and assess its performance in the inactivation of biofilms in Petri dishes and root
canals. Furthermore, the coating exhibited no detectable porosities, ensuring electrical
insulation and the effective operation of the plasma reactor. The absence of porosities is
critical to maintaining electrical integrity and preventing any potential plasma leakage,
thereby contributing to the overall success of the plasma reactor in its intended appli-
cations. Traditional plasma generators typically possess rigid and straight macroscopic
characteristics. To initiate the discharge, the polarization of the two electrodes is achieved
using a high-voltage source in the order of kilovolts, employing alternating or pulsed
current with an operating frequency in the tens of kilohertz range. The gas flow rate during
the experimentation was consistently maintained at approximately 10 LPM [42–48]. The
electrical properties resemble those of curved-type reactors, such as plasma jets [28,29],
or the Thorns plasma reactor [30]. In our previous work, we designed and constructed a
reactor with a straight output, which has found applications in clinical settings for treating
patients with burns [38], addressing oral health issues [49–52], managing diabetic foot
conditions [53,54], and performing neck surgeries [55].
The power supply characteristics used in these applications included a voltage range
of ±200 V, a frequency of 13.56 MHz, and a gas flow rate of 0.5 LPM [56], significantly
reducing the electrical characteristics at which the reactor operates. Bacterial inactivation
studies conducted with the curved-output NTP reactor demonstrated excellent bacterial
inactivation. These disinfection studies highlight the reactor’s potential for root canal
treatment. Furthermore, during its use, the proposed curved-output NTP reactor does not
significantly increase the heat on the surface where the plasma is applied, making it very
useful in preventing heat damage to dental pulp [57].
Many research endeavours have been dedicated to unveiling the bacterial species
inhabiting root canals [58–61]. A substantial body of literature has documented the effec-
tive inactivation of bacteria through NTP treatment, underscoring its potent antibacterial
efficacy [44,60,62]. The results consistently demonstrate complete bacterial inactivation
within the treated area, primarily attributed to the influence of reactive oxygen and nitrogen
species, a phenomenon that has been observed by other researchers as well [41]. These
seminal findings provided a solid foundation for the subsequent application of NTP in
root canals. Furthermore, encouraging outcomes have been reported regarding the in vitro
eradication of the Gram-positive Enterococcus faecalis using reactors featuring curved
outlets [28,29]. These reactors typically operate under high voltages and frequencies in the
tenths of kilohertz range. In contrast, our proposed reactor sets itself apart by employing
lower voltages and frequencies in the megahertz range, representing a significant departure
from established methodologies in the field.
Chronic periradicular lesions, both before and after root canal therapy, have garnered
significant attention in the literature [12,35]. Biofilms have proven to be more resistant to
antimicrobial agents than planktonic bacteria. Given this challenge, Enterococcus faecalis was
chosen as the model organism for our in vitro studies, representing endodontic disinfection
investigations. Preclinical and clinical studies have reported that the separate application of
NTP and PTW promotes anti-inflammatory and microbicidal functions, facilitating tissue
repair and contributing to the restoration of periodontal tissue. The extent of this recovery
is contingent on the number of applications, as supported by research such as [49–52,63,64].
The outcomes of our experiments involving Enterococcus faecalis and its treatment with
NTP and PTW using an innovative reactor with a curved terminal underscore a remarkable
level of success. This achievement can be attributed to our meticulous examination of the
distinct phases involved in the inactivation process of Enterococcus faecalis. These findings
emphasize the importance of treatment combinations and their sequencing in achieving
Enterococcus faecalis inactivation within root canals, offering valuable insights into the
effectiveness of different approaches for this specific application.
Materials 2023, 16, 7204 16 of 19

Enterococcus faecalis, a bacterium known for its resistance to conventional sterilization

methods, including heat, acid, calcium hydroxide, and UV irradiation [65], has posed a
significant challenge in root canal treatments. The combined experiments conducted in
this study unveil several effective strategies for enhancing the inactivation of Enterococcus
faecalis within root canals. These experiments clearly illustrate the efficacy of treatment
combinations involving NTP, PTW, and NaOCl applied under various sequences and
conditions. In our study, the microbiological results strongly indicate the susceptibility
of Enterococcus faecalis to the NTP and PTW generated by our proposed reactor, with
noticeable effects on Enterococcus faecalis biofilms becoming apparent after just 5 min
of treatment. Plasma exposure rapidly disrupts the cell wall, as visually depicted in
Figure 10, leading us to infer that the cell membrane incurred damage from the NTP [66].
This compromised membrane renders microorganisms highly vulnerable to the reactive
environment, facilitating the penetration of reactive oxygen and nitrogen species, photons,
and charged particles, ultimately reaching the DNA material [67]. It is this mechanism
that underscores the effectiveness of our NTP-based approach in inactivating Enterococcus
faecalis, providing a promising avenue for improved root canal sterilization.
The colonization of Enterococcus faecalis bacteria was also studied using SEM. These
results revealed distinct differences in root canals, with significant effects observed when
using NTP and PTW treatments, especially when combined, compared to the individual
use of sodium hypochlorite. Notably, the combined treatment of NaOCl and NTP at
a 0 mm distance from the nozzle (Figure 10i) demonstrated more effective microbial
detachment. These findings and microbiological analysis demonstrate the promising
potential of these mixed methods in oral treatments, particularly for root canals. These
approaches synergistically combat oral health-threatening microorganisms, providing
adequate alternatives. While a definitive solution remains elusive, these multidisciplinary
approaches aim to enhance therapeutic interventions and improve patients’ quality of life,
contributing to dental advancements and optimal oral health promotion. In the studies
reported by Kerlikowski and collaborators [68], they suggested the design and construction
of a reactor that would facilitate the application of the NTP, and we consider that compliance
is achieved with the proposed reactor. It is worth noting that treatments with our innovative
curved-tip reactor involve perpendicular application to the 3D model, achieving a similar
effect to that offered by the researchers mentioned above.

5. Conclusions
In this study, we introduce an innovative reactor design with a curved termination
for generating non-thermal plasma (NTP) and plasma-treated water (PTW) to inactivate
biofilms in root canals. The reactor utilizes zircon (ZrSiO4 ) powder coating on the central
electrode, ensuring stable NTP production suitable for dental applications, particularly
in root canal treatments. The curved-terminal reactor offers an ergonomic and efficient
solution for accessing root canals, applying NTP, and inactivating biofilms of Enterococcus
faecalis. The combined treatments involving NaOCl, NTP, and PTW exposure demonstrated
significantly heightened antimicrobial efficacy compared to the control group. The unique
antimicrobial efficacy of NTP and PTW is attributed to their ability to disrupt microbial
structures, including those within biofilms. This breakthrough can revolutionize root
canal disinfection, providing a superior and optimized approach. In summary, our study
represents a substantial advancement in dentistry, presenting an innovative method for
biofilm inactivation in root canals using NTP generated by a curved-terminal reactor.
Implementing this technology can improve the prevention and management of endodontic
infections. Further research with this reactor design is essential for complete bacterial
inactivation in root canals.

Author Contributions: Conceptualization, R.A.-S., R.L.-C. and B.G.R.-M.; Methodology, R.A.-S.,

R.L.-C. and B.G.R.-M.; Software, A.M.-C. and R.V.-A.; Validation, R.S.-V., U.V.-E. and C.E.M.-S.;
Formal analysis, A.M.-C.; Investigation, R.A.-S., R.L.-C., B.G.R.-M., U.V.-E. and C.E.M.-S.; Resources,
R.A.-S.; Data curation, A.M.-C., R.P.-E. and R.V.-A.; Writing—original draft, R.A.-S., R.L.-C., B.G.R.-M.,
Materials 2023, 16, 7204 17 of 19

R.S.-V., U.V.-E., A.M.-C., R.P.-E., R.V.-A. and C.E.M.-S.; Writing—review & editing, R.A.-S., R.L.-C.
and B.G.R.-M.; Supervision, R.L.-C. and R.S.-V.; Project administration, R.L.-C. All authors have read
and agreed to the published version of the manuscript.
Funding: The CB-302 research project from the National Institute of Nuclear Research and the Faculty
of Dentistry of Autonomous University of Mexico State funded this research.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: We want to acknowledge the contributions of technicians M.T. Torres-Martínez,
P. Ángeles-Espinosa, and I. Contreras-Villa in constructing the device and conducting the in vitro tests.
Conflicts of Interest: The authors declare no conflict of interest.

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