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Page 1
International Journal of
Molecular Sciences
Review
Applications of Plasma-Activated Water in Dentistry: A Review
Noala Vicensoto Moreira Milhan 1, * , William Chiappim 2 , Aline da Graça Sampaio 1 ,
Mariana Raquel da Cruz Vegian 1 , Rodrigo Sávio Pessoa 2 and Cristiane Yumi Koga-Ito 1,3
1 Oral Biopathology Graduate Program, São José dos Campos Institute of Science & Technology,
São Paulo State University, UNESP, São Paulo 12245-000, Brazil; aline.sampaio@unesp.br (A.d.G.S.);
mary.rcv@hotmail.com (M.R.d.C.V.); cristiane.koga-ito@unesp.br (C.Y.K.-I.)
2 Plasma and Processes Laboratory, Department of Physics, Aeronautics Institute of Technology,
Praça Marechal Eduardo Gomes 50, São José dos Campos 12228-900, Brazil; chiappimjr@yahoo.com.br (W.C.);
rspessoa@ita.br (R.S.P.)
3 Department of Environment Engineering, São José dos Campos Institute of Science & Technology,
São Paulo State University, UNESP, São Paulo 12247-016, Brazil
* Correspondence: milhan.noala@gmail.com; Tel.: +55-12-991851206
Abstract: The activation of water by non-thermal plasma creates a liquid with active constituents
referred to as plasma-activated water (PAW). Due to its active constituents, PAW may play an
important role in different fields, such as agriculture, the food industry and healthcare. Plasma
liquid technology has received attention in recent years due to its versatility and good potential,
mainly focused on different health care purposes. This interest has extended to dentistry, since the
use of a plasma–liquid technology could bring clinical advantages, compared to direct application
of non-thermal atmospheric pressure plasmas (NTAPPs). The aim of this paper is to discuss the
applicability of PAW in different areas of dentistry, according to the published literature about
NTAPPs and plasma–liquid technology. The direct and indirect application of NTAPPs are presented
in the introduction. Posteriorly, the main reactors for generating PAW and its active constituents with
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??????? a role in biomedical applications are specified, followed by a section that discusses, in detail, the use
Citation: Milhan, N.V.M.; Chiappim, of PAW as a tool for different oral diseases.
W.; Sampaio, A.d.G.; Vegian,
M.R.d.C.; Pessoa, R.S.; Koga-Ito, C.Y. Keywords: plasma-activated water; plasma-treated water; atmospheric plasma; gliding arc discharge;
Applications of Plasma-Activated DBD; dentistry; decontamination; oral cancer; tooth bleaching
Water in Dentistry: A Review. Int. J.
Mol. Sci. 2022, 23, 4131. https://
doi.org/10.3390/ijms23084131
Academic Editor: Mary Anne Melo 1. Introduction
Received: 5 March 2022
Plasma medicine is a multidisciplinary research field that investigates the uses of
Accepted: 6 April 2022
plasma in the healthcare field. Currently, non-thermal plasma technology is mainly focused
Published: 8 April 2022
on applications at atmospheric pressure, which is commonly referred to as non-thermal
atmospheric pressure plasmas (NTAPPs). Direct applications of NTAPP have been used
Publisher’s Note: MDPI stays neutral
for decontamination of food [1,2] and food contact surfaces [1,3] in the food industry, in
with regard to jurisdictional claims in
air purification [4] and as an antimicrobial agent in the medical and dentistry fields [5–9].
published maps and institutional affil-
In 2021, NTAPP focused on plasma medicine reached a quarter of a century since the first
iations.
published study [10]. Over time, in addition to the antimicrobial effects, other applications
of NTAPP have been discovered in biomedical fields, such as the benefits for wound healing
and cancer treatment [11,12].
Copyright: © 2022 by the authors.
NTAPPs can also be applied indirectly through the activation of water or liquids and
Licensee MDPI, Basel, Switzerland. through the treatment of contaminated or polluted water [13,14]. Their antimicrobial ef-
This article is an open access article fect [15,16] and applicability in the treatment of cancer [17,18] and wound healing [19] have
distributed under the terms and also been observed in the indirect modality of treatment. In recent years, plasma medicine
conditions of the Creative Commons has expanded the frontiers towards plasma dentistry. NTAPP has shown efficacy against
Attribution (CC BY) license (https:// oral microorganisms, in addition to anti-inflammatory properties, with possible application
creativecommons.org/licenses/by/ in cariology, endodontic, periodontics and oral oncology [20]. Besides, plasma-activated
4.0/). water (PAW) has also demonstrated potential application in dentistry fields [21,22].
Int. J. Mol. Sci. 2022, 23, 4131. https://doi.org/10.3390/ijms23084131 https://www.mdpi.com/journal/ijmsPage 2
Int. J. Mol. Sci. 2022, 23, 4131 2 of 26
It is important to emphasize the differences between both modalities of treatment:
direct and indirect. The first implies that plasma is applied directly on a given substrate,
such as wounds, skin, food etc., [23,24]. Conversely, in the indirect modality of NTAPPs,
a given liquid is activated prior to the application to the substrate. To exemplify, Table 1
shows the main review articles published in the last three years on the applications of direct
and indirect NTAPPs, in areas ranging from medicine, biomedicine, dentistry, agriculture
and the food industry. It is worth mentioning that although the present work is focused
on dentistry, the direct and indirect NTAPPs are of great importance for agriculture and
the food industry. This wide range of applications of direct and indirect NTAPPs is
schematically presented in Figure 1. Figure 1a highlights the applicability of direct NTAPPs
that cover different human and food healthcare areas. Figure 1b illustrates the applications
of NTAPPs through the modality of generating plasma-activated liquid (PAL) or PAW,
which are later applied to substrates. As described, PAW and PAL studies are focused on
different purposes, which shows the effort of many scientists to improve and make plasma
technology more accessible.
Figure 1. Schematic illustration of direct (a) and indirect (b) applications of non-thermal atmospheric
pressure plasmas (NTAPPs). Note that due its antimicrobial effect, direct NTAPPs have been used forPage 3
Int. J. Mol. Sci. 2022, 23, 4131 3 of 26
different purposes such as decontamination/preservation of food, air purification and decontamina-
tion of medical and dental devices. Additionally, an improvement in wound healing and anti-cancer
properties has also been observed after direct exposure to NTAPPs. Similarly, the indirect modality
has demonstrated antimicrobial effect against some types of microorganisms, with applicability
in different fields. The reactive oxygen and nitrogen species generated in the liquids after plasma
exposure may favor the seed germination. In addition, good responses have also been observed
in wound healing and cancer treatment, which brings good perspectives for healthcare due to the
clinical advantages of plasma-activated water (PAW) compared to direct NTAPPs. Some figures were
reprinted with permission from Delben et al. [25], CC BY 4.0 license (2016); Whittaker et al. [26],
copyright Elsevier (2004); Weltmann and Woedtke, [7], copyright IOP (2017); Misra and Cheorun, [27],
copyright elsevier (2017); Keidar et al. [28], CC BY 4.0 license (2011); Sivachandiran and Khacef, [29],
CC BY 3.0 license (2017); Guo et al. [15], CC BY 4.0 license (2018); Chen et al. [18], copyright Wiley
(2016); Xu et al. [19], CC BY 4.0 license (2020); Li et al. [22], copyright Wiley (2017); Ma et al. [30],
copyright Elsevier (2015); Chiappim et al. [31], copyright Wiley (2021).
To understand the mechanisms of action related to NTAPPs, it is essential to know that
the plasma is a partially ionized gas consisting of particles as electrons, ions, metastable
species, radiation as ultraviolet (UV), visible (VIS), the electromagnetic field and reactive
species. It is worth highlighting that the term reactive species is commonly used for
free radicals and reactive oxygen species (ROS) and reactive nitrogen species (RNS). It
is highly reactive due to the presence of unpaired valence electrons or non-static bonds
on its structure. Despite the various components produced in plasma, reactive oxygen
and nitrogen species (RONS) play a fundamental role in plasma medicine [32,33]. RONS
generated in NTAPP are divided into short-lived and long-lived species. Hydroxyl radical
(OH− ), delta oxygen singlet (1 O2 ) and superoxide anion (O2 − ) are examples of short-lived
species, which last from a few seconds to minutes [34]. In contrast, hydrogen peroxide
(H2 O2 ), nitrite (NO2 ), nitrate (NO3 − ), nitrous acid (HNO2 ) and ozone (O3 ) belong to the
groups of long-lived species [35,36]. When NTAPP is used in an indirect mode, i.e., in
liquid or water activation, most RONS found in the liquid phase are long-lived ones,
generated from the gas phase of the plasma and the plasma–liquid interface interaction.
The generation of RONS also occurs through the primary transformation of reactive species
generated in the liquid [37]. Therefore, water or liquid exposure to NTAPP induces active
constituents, called RONS, which are useful for biomedical applications [21].
Due to the growing interest of the scientific community in plasma technology applied
to dentistry, a review article is presented here to serve as a quick guide for dentists, physi-
cians, physicists, engineers and health professionals in general. This review aims to discuss
the applicability of PAW in dentistry, which, as can be seen in Table 1, is a topic review
with little or no exploration. Thus, based on the published literature, the findings of PAW
applied to dentistry are presented, as well as the future perspectives for the area considering
the main results related to direct and indirect NTAPP for biomedical applications.
Table 1. Summary of the main review articles published in the last three years on applications of
direct and indirect NTAPPs.
Publication
Approach NTAPP Modalities
Year/Reference
Molecular Mechanisms of the Efficacy of NTAPP in Cancer Treatment Direct 2020 [38]
A Powerful Tool for Modern Medicine Direct 2020 [11]
The New Frontier in Low Temperature Plasma Applications Direct 2020 [39]
Atmospheric Cold Plasma Treatment in Fruit Juices Direct 2020 [40]
Chemical, Physical and Physiological Quality Attributes of Fruit and
Direct 2020 [41]
Vegetables Induced by Cold Plasma TreatmentPage 4
Int. J. Mol. Sci. 2022, 23, 4131 4 of 26
Table 1. Cont.
Publication
Approach NTAPP Modalities
Year/Reference
Cold Plasma as a New Hope in the Field of Virus Inactivation Direct 2020 [42]
The Effects of Plasma on Plant Growth, Development and Sustainability Direct 2020 [43]
Cold Plasma for the Control of Biofilms in Food Industry Direct 2020 [44]
Potential of Cold Plasma Technology in Ensuring the Safety of Foods and
Direct 2020 [45]
Agricultural Produce
Plasma Agriculture from Laboratory to Farm Direct 2020 [46]
Aurora Borealis in Dentistry Direct 2021 [47]
Applications of Cold Atmospheric Pressure Plasma in Dentistry Direct 2021 [20]
Cold Atmospheric Pressure Plasma Technology in Medicine, Agriculture
Direct 2021 [48]
and Food Industry
The Antimicrobial Effect of Cold Atmospheric Plasma against
Direct 2021 [49]
Dental Pathogens
Influence of Atmospheric Cold Plasma on Spore Inactivation Direct 2021 [50]
Plasma-Assisted Agriculture: History, Presence and Prospects Direct 2021 [51]
Improving Seed Germination by Cold Atmospheric Plasma Direct 2022 [52]
Reactive Nitrogen Species in Plasma-Activated Water Indirect 2020 [53]
Recent Advances in Plasma-Activated Water for Food Safety Indirect 2022 [54]
Influence of Plasma-Activated Water on Physical and Physical–Chemical
Indirect 2020 [55]
Soil Properties
PAW Triggers Plant Defense Responses Indirect 2020 [56]
PAW Generation, Origin of Reactive Species and Biological Applications Indirect 2020 [36]
Interactions of Plasma-Activated Water with Biofilms Indirect 2020 [57]
A Comprehensive Review of PAW for Enhanced Food Safety and Quality Indirect 2021 [58]
Applications of PAW in the Food Industry Indirect 2020 [59]
PAW for Cancer Treatment: Positives, Potentials and Problems of
Indirect 2020 [60]
Clinical Translation
Review on Discharge Plasma for Water Treatment Indirect 2020 [61]
PAW on Microbial Growth and Storage Quality of Fresh-cut Apple Indirect 2020 [62]
PAW Production and its Application in Agriculture Indirect 2021 [63]
Diagnostic Analysis of Reactive Species in PAW: Current Advances
Indirect 2021 [64]
and Outlooks
PAW from DBD as a source of Nitrogen for Agriculture Indirect 2021 [65]
PAW, Hydrogen Peroxide and Nitrates on Lettuce Growth Indirect 2021 [66]
Therefore, this review is divided as follows; Section 2 shows the main reactors used
for PAW/PAL and plasma-treated water (PTW). Another essential point demonstrated
in this section is the formation of RONS, the fundamental species for plasma medicine.
Section 3 explores the main text of this review, focusing on PAW applied to different areas
of dentistry. In this context, the following topics are explored: decontamination of dental
devices, treatment of oral infectious diseases, anti-inflammatory properties and wound
healing, anti-cancer therapy and tooth bleaching. Finally, Section 4 presents the conclusion
of the work.Page 5
Int. J. Mol. Sci. 2022, 23, 4131 5 of 26
2. Plasma-Activated and Plasma-Treated Water
In the literature, the terms treatment and activation are often misused. To avoid
doubt, treatment is defined as a practical means or refinements used to combat/mitigate
a problem. In the specific case of water treated by plasma, the term treated refers to
the process of elimination or complete mineralization in wastewater of synthetic dyes,
pharmaceuticals products and pathogenic bacteria, among other pollutants. Therefore,
plasma water treatment is commonly used to purify or decontaminate small or large
amounts of water. In contrast, activation is understood as increased activity, becoming
active, boosting, accelerating, or intensifying some specific property. In this case, the non-
pollutant water exposed to the plasma becomes activated, i.e., it obtains new properties.
Unlike treatment, activation is carried out in small amounts of water (between 1 mL to
1000 mL) [36,53,66], but the reactors used are the same, with a slight modification in both
cases. It is important to note that deionized water, distilled water, filtered water and potable
tap water, i.e., pure water without pollutants, are usually used for activation.
2.1. Main Reactors of Producing
Two main types of plasma reactors are used in PAW generation: dielectric barrier
discharges (DBDs) and plasma jets (PJs). Both are non-thermal plasmas, i.e., they are
NTAPP and have a wide range of types [65,67]. DBDs for PAW generation are considered
indirect sources of plasma, as the plasma is mainly produced between the reactor electrodes
without any contact with the water, taking advantage of the interactions of the gas with the
liquid. Some reactive plasma species can reach the water surface through ionizing wave
mechanisms using electric field propagation, convection through airflow and diffusion [65].
In contrast, the PJs used for PAW generation are considered direct sources of plasma,
i.e., there is direct contact between the plasma and the water and the water can act as a
counter electrode so that the discharge current can flow through the liquid. The PJs are
the most widely used reactors, as some of them generate a stable volume of plasma that is
controllable without the confinement between the electrodes, as in the case of the DBDs [67].
In both reactors, the formation of RONS occurs at the interface between the gaseous and
liquid phases and/or within the liquid, which drastically changes the concentration of the
RONS constituents [36]. In contrast, in the treatment of water by plasma, the reactors can
be immersed in the treated liquid [68,69]. Therefore, in this case, both DBD reactors and PJ
reactors can be used.
It is important to note that NTAPPs operate at a high voltage. As reported in the
literature, these voltages range from 1 to 50 kV, with operating frequencies that can start
in the tens to thousands of Hz (kHz) and powers that generally do not exceed values
greater than 10 W. Commonly used working gases are helium (He), argon (Ar), oxygen
(O2 ), nitrogen (N2 ), air or a mixture of these gases. Working gases are used with flow rates
ranging from 1 to 30 L/min [36,53–58]. Indeed, there is a range of reactors used for water
and liquid activation and, every day, a new article is published with new reactors that have
minor changes. Therefore, a dedicated review article would be needed to demonstrate the
NTAPP generation reactors and their characteristics, but the focus of the present work is
not that. Thus, below we show some reactors used to generate PAW.
As seen in Figure 2, discharges are used directly into the water and on the surface of
the water, which considerably affects the chemical composition of PAW. This difference in
chemical composition is basically due to the differences between the rupture forces in the
gas phase (discharge on the surface) and the water (discharge inside) [70,71]. However,
as reported in the literature, the most applied systems are those that operate with plasma
discharge in contact with the water, i.e., PJ and DBD as a plasma source [72,73]. As
demonstrated in the next section, these plasmas deliver the RONS from the plasma gas to
the liquid phase more efficiently.Page 6
Int. J. Mol. Sci. 2022, 23, 4131 6 of 26
Figure 2. Schematic drawing of different discharges used for the preparation of plasma-activated
water [47,74] (a–c) direct discharge into the water and (d) direct discharge into the water with multi-
electrodes; (e) discharge in the gaseous phase onto the water surface; (f) discharge in the gaseous
phase into the water; (g) discharge in the gas phase onto the water surface with plasma generated on
forward vortex flow reactor (FVFR); (h) discharges in bubbles into the water.
An important question is whether there is commercial equipment that can be applied
in clinical practice. Recently, Andrasch et al. [72], Pemen et al. [73] and Schnabel et al. [75]
developed pilot units with potential for practical applications. Andrasch et al. [72] and
Schnabel et al. [75] obtained a PAW production rate of 1 L/min. Pemen et al. [73] obtained
0.5 L/min. However, these devices cannot meet the application requirements in dentistry
and medicine, which are low pH and high concentrations of RONS. On the other hand, in
agriculture and in the food industry, the production of millions of liters of PAW at a low
cost is required. Therefore, it can be said that PAW-generating equipment is still in the pilot
phase and has great commercialization potential in the coming years.
2.2. Origin of Reactive Oxygen and Nitrogen Species
The RONS induced in PAW are dependent on many different parameters such as (i) the
composition of the solution, (ii) the distance between the plasma and the liquid surface,
(iii) the types of power supply used in the plasma generation, (iv) electrode configuration,
(v) applied voltage, (vi) voltage polarity, (vii) water volume, (viii) gas type and flow, among
other parameters [31,76–78]. It is also important to highlight that both the chemistry and
the reaction processes of PAW produced with the reactor in a few centimeters of water are
different from those made in the generation of PAW with the reactors submerged in water.Page 7
Int. J. Mol. Sci. 2022, 23, 4131 7 of 26
The gas–liquid system generated for electrical discharges placed in a few centimeters of
water is more interesting, as they can cause post-electrical discharge reactions capable of
forming other long-lived reactive species [59,70]. For example, when using compressed
air as the plasma-generating gas for water activation, the gas–liquid interface produces
numerous short-lived species, ranging from hydroxyl radicals, superoxide and nitrous
oxide to the generation of atomic oxygen and nitrogen [36]. Short-lived species later
generate long-lived species, such as nitrites, nitrates and hydrogen peroxide (see Figure 3).
Figure 3. Schematic diagram that shows the regions of reactive species generation induced by NTAPP
used to activate water. Note the three regions of RONS formation in an NTAPP device in contact with
water. The primary reactive species, short-lived species, electrons and electromagnetic radiation in
the ultraviolet range are found in region 1 and are in the gas phase. In region 2, we can observe the
secondary reactive species made up of short-lived and long-lived species, and this region is contained
in the plasma phase. Tertiary reactive species are short-lived and long-lived species, and their region
is limited to the plasma–water interface and liquid phase.
With the significant increase in PAW applications, it is necessary to understand which
RONS can be formed and how they are formed, in addition to the quantification of their
concentrations. In Appendix A, the generation and recombination mechanisms of the main
plasma-induced long-lived reactive species are described in water activation processes that
play a crucial role in healthcare applications.
3. PAW Applied in Dentistry
3.1. Decontamination of Dental Devices
The surfaces of medical and dental devices are constantly exposed to different mi-
croorganisms during procedures, working as a reservoir of pathogens and a potential
source of contamination for the patients [79]. To avoid the risk of contamination, different
methods and chemical products may be used to control pathogenic or nonpathogenic
microorganisms. However, limitations such as ineffective sterilization, low penetration,
incompatibility with materials that are sensitive to heat and corrosion, residue release,
toxicity to the environment and individuals [80,81] and an inability to penetrate biofilm
cell structures [82] support a constant search for new methods. In addition to being usefulPage 8
Int. J. Mol. Sci. 2022, 23, 4131 8 of 26
in the inactivation of microorganisms when applied over, inside, or touching the water
surface [83], NTAPPs are also efficient against prion proteins [84], which are resistant to
traditional cleaning methods [85].
The direct application of NTAPPs for the disinfection of heat-sensitive materials has
been explored in the literature [86,87]. The disinfection of endodontic devices [26], sili-
cone, diamond dental drills [88], metals [89], titanium alloy surfaces [90] and titanium
disc surfaces [91] were analyzed with satisfactory results. The effectiveness of biological
decontamination by NTAPPs depends on some parameters such as equipment configu-
ration (frequency, power), gas (type and flow), the geometry of the analyzed material,
distance, exposure time and also the position and design of the device [80,86,92]. Depend-
ing on the geometry of the material, multiple exposures may be required to eliminate
the microorganisms.
As an alternative method, solutions activated with plasma, such as PAW, have also
been evaluated for disinfection [93]. In fact, this in an interesting field of investigation
as the possibility of disinfecting a device with a simple washing seems easier, compared
to multiple exposures that may be needed to decontaminate a device/biomaterial with a
complex geometry. It was previously observed that microbicide action may be related to the
main reactive species and also the low pH acquired during aqueous plasma activation [94].
The use of PAW for the sterilization of medical devices was previously suggested, consid-
ering its antimicrobial properties [95]. One of the main advantages of this technology is
that plasma-activated liquids may keep the antimicrobial effect over one month after PAW
generation, when stored at a minimum of −80 ◦ C [96,97]. However, the antimicrobial activ-
ity may be reduced, depending on the storage temperature, by decreasing the number of
reactive species. Inactivation of S. aureus was observed after 20 min of treatment with PAW
stored at −80 ◦ C. However, this potential decreased significantly when plasma-activated
distilled water was stored at −20 ◦ C for 1, 7, 15 and 30 days [96]. Similarly, another study
observed that 60 min of exposure to PAW stored at –80 or –150 ◦ C may promote microbial
inactivation of S. aureus and E. coli, even after six months of storage. On the other hand,
temperatures equal to or higher than −16 ◦ C showed reduced antimicrobial properties in
short or long periods of storage [97]. In addition to the possibility of disinfecting dental
materials in the easiest way, the conservation properties of PAW indicate another advantage
of using this technology compared to direct NTAPP, since it would considerably reduce the
demand for using the plasma device.
The decontamination of dental unit waterline system tubes (DUWLs) is a challenge
for dentistry due to the risk of cross-contamination [98] and also the limitation of some
traditional disinfectants, which may be toxic [79,99]. The reduction in the viability of
mature Enterococcus faecalis biofilms formed in DUWLs for 5 days after PAW treatment was
detected. For this, distilled water was activated for 3 min by a continuous plasma jet of
compressed air gas. A significant reduction in viable cells was observed after 1–3 min of
treatment. Treatment for up to 5 min led to a 100% reduction. Moreover, 3 min of contact
showed similar effects to 1% H2 O2 and 10 mg/L NaOCl; antimicrobial agents that are
routinely used. The low pH (2.21) and the presence of NO, OH, NO3 and H+ species
probably contributed together to the bactericidal effect [99]. Further investigations on PAW
with this type of device and other microorganisms would be interesting.
The disinfection of stainless steel and polyethylene substrates by PAW has also been
explored [100]. It is important to highlight that these materials present a wide application in
the dental fields [101,102]. For these tests, 20 mL of sterile distilled water was activated for
5 min by non-thermal GlidArc plasma formed by moist air gas. After 30 min of exposure to
PAW, microbial reduction both in planktonic and biofilm forms, were observed on Gram-
positive and Gram-negative bacteria, as well as in yeasts. More than a 5 log reduction in
viable cells was observed for bacterial biofilms, while there was a decrease of approximately
3 log in yeasts. Advantageously, the disinfection of the solid materials with PAW did not
show damage to the materials, especially to stainless steel, which was free of corrosive
signs [100].Page 9
Int. J. Mol. Sci. 2022, 23, 4131 9 of 26
Resin based materials have an important applicability in dentistry [103]. Sterilization
by PAW of a duodenoscope coated with a polymer resin was also previously analyzed.
Distilled water (300 mL) was activated for 10 min, with a discharge of GlidArc plasma
operated with air gas. The treatment produced an acidified water of pH 2.78 that reduced
the viable cells of Escherichia coli and Acinetobacter baumannii after 20 min of exposure,
without any damage to the surface and composition of the equipment. The decontamination
effect was also observed for Klebsiella pneumoniae and Pseudomonas aeruginosa after 30 min
of PAW exposure [104].
Taken together, the studies with PAW have shown good decontamination properties of
materials that are widely used in dentistry, such as stainless steel, polyethylene and polymer
resin. These results bring insights for a new sterilization method in the dental field. Despite
that, new studies with other materials and mostly with polymicrobial biofilms, which are
closer to the clinical environment [105], would be interesting to explore this application.
3.2. Treatment of Oral Infectious Diseases
The use of PAW in the treatment of oral infectious diseases is a promising field of
investigation. Several groups of microorganisms are involved in the etiopathogenesis
and progression of the main infectious diseases that affect the oral cavity, such as caries,
periodontitis and candidiasis. Considering the antimicrobial properties of NTAPPs, the
control of these microorganisms by PAW or other plasma-activated solutions has also been
investigated as an alternative treatment to traditional therapies that have their limitations.
Biological, behavioral, psychosocial and environmental factors are related to the de-
velopment of caries [106]. The imbalance between demineralization and remineralization,
caused by fluctuations in pH, promotes tooth decay [107]. The metabolic activity of den-
tal biofilm, formed by microorganisms embedded in a matrix of extracellular polymeric
substances adhered to the teeth surface, is responsible for these pH fluctuations, especially
with the intake of a sugar-rich diet. The biofilm continues to grow progressively if undis-
turbed. In its composition, there are many groups of microorganisms and some of them are
especially involved in the carious process [108].
Streptococcus mutans and Lactobacillus spp. are considered the main cariogenic bacte-
ria responsible for producing acid and, consequently, the demineralization of the tooth
structure [109,110]. Interestingly, PAL has shown to be effective against cariogenic microor-
ganisms [22,111]. A previous study observed that PBS or a saline solution activated with a
non-thermal plasma of argon (Ar) and oxygen (O2 ) for 5 min are able to reduce the number
of S. mutans viable cells. This reduction was verified in both planktonic and biofilm forms
of S. mutans after 1 h of treatment. The authors further reported that the activated liquids
were not cytotoxic to fibroblasts [111].
Modifications in the composition of the oral biofilm may be observed due to dietary
habits, type of dentition (primary or secondary) and even with the disease progression.
Although S. mutans and Lactobacillus spp. are considered the most important microogas-
nisms related to dental caries, previous findings demonstrated that Actinomyces spp. may be
associated with the disease progression in root caries [108]. A previous study evaluated the
role of distilled water activated by a plasma jet of Ar and O2 (98% and 2%, v/v, respectively)
for 20 min, in the reduction in S. mutans, Porphyromonas gingivalis and Actinomyces viscosus.
The treatment that was performed from 0 to 120 s reduced the viability of all microbial
species. A significant reduction in A. viscosus was observed within 40 s while S. mutans
achieved a similar reduction after 60 s of treatment [22].
Candida albicans is another microorganism that may be found in the carious dentin of
active root carious lesions and some authors have suggested that this fungus might play
a role in the progression of the disease [112]. This species was detected on the biofilm in
cases of childhood caries [113] and it was associated with an increase in plaque glucosyl-
transferase (Gtf) enzyme activity, a virulence factor associated with caries, in children with
early carious lesions [114]. In addition to its possible role in dental caries, C. albicans is
the main species related to oral candidiasis, the most common oral fungal disease. LocalPage 10
Int. J. Mol. Sci. 2022, 23, 4131 10 of 26
and systemic factors such as impaired salivary gland function, inhaled steroids, dentures,
oral cancer/leukoplakia, broad-spectrum antibiotics, immunosuppressive drugs and condi-
tions, nutritional deficiencies, diabetes, smoking and Cushing’s syndrome may contribute
to oral candidiasis [115]. Previous studies demonstrated that the direct application of
NTAPPs is effective against C. albicans [116,117], both in planktonic and biofilm forms.
Investigations into the effects of PAW on C. albicans have also been conducted with different
methodologies and findings. A reduction in C. albicans viability after 5 min of treatment
with distilled water, activated by a dielectric barrier discharge (DBD) with atmospheric
air, for 5 and 10 min, was reported. The authors attributed the antimicrobial action to the
higher concentration of NO− 3 and lower concentrations of NO− 2 and H2 O2 in PAW [118].
Antimicrobial effects against C. albicans was also reported in hydrogels constituted by
plasma-activated deionized water, after 24, 48 and 72 h of contact time. An increase in
the inhibition zones of the microorganism was observed in longer exposure times, with
the best result after 30 min. Hydroxyl radical and NO− 3 were suggested to be the main
components with antifungal activity [119]. On the contrary, one study reported no effect
of plasma-activated tap water for 10 and 30 min on C. albicans planktonic cells. The water
was activated with an atmospheric air plasma, generated by a forward vortex flow reactor
(FVFR), for 30 min [16].
In the context of cariology, it is also important to emphasize the positive property of
NTAPPs in adhesive restorations. Plasma exposure generates the deposition of free radicals
and ions on the tooth substrate, changing the surface proteins of dentin, which has led to
an increased bond strength in adhesive restorations. The enhance in bone bond strength
avoids microleakage and consequently prevents secondary caries [47]. It is not known
whether PAW can also increase the bond strength, improving the restoration performance.
This process could also probably occur with plasma–liquid technology due to the action of
the reactive species on the surface of dentin.
In the evolution of the carious process, traumatic injuries and cracks allow that
pathogens and their products to pass through the dentin and reach the pulp. The pulpal
infection frequently progresses to necrosis of the tissue and the infection may spread to
the apex of the tooth, promoting periapical disease [120]. Enterococcus faecalis is commonly
found in primary and secondary endodontic infections [120–122]. This microorganism
expresses virulence factors and resistance mechanisms that favor its presence in the root
canal and consequently can lead to endodontic therapy failure [120,123]. Some studies
have investigated the action of PAW on E. faecalis [99,124], which is interesting for dentistry
as it could be used as an antimicrobial irrigator in endodontic treatments. Considering the
particularities of root canals, such as the presence of accessory canals, the antimicrobial
irrigation with PAW would be more useful than the direct application of NTAPPs. As
mentioned, a reduction in E. faecalis viable cells in 5-day DUWLs biofilms was observed
after 1–3 min of treatment with previously activated water. The treatment of the E. faecalis
suspensions, in deionized water, was also performed with satisfactory results. The bacterial
suspension was exposed to microjet plasma formed by atmospheric air for 10 to 90 s. The
antimicrobial effect occurred progressively after 45, 60 and 90 min of treatment, while
inhibitory effects on the biofilm formation was detected even in shorter exposure times (10,
20 and 30 s) [124]. Another microorganism that may be isolated from root canals, though
not often, is Escherichia coli [120]. It was demonstrated that the planktonic treatment with
PAW significantly reduced the number of E. coli colonies after 10 min of exposure [16]. In
this same study, 10 and 30 min of PAW contact was effective against Staphylococcus aureus, a
bacteria commonly found in chronic osteomyelitis of the jaw, in association with anaerobic
pathogens [125].
The development and progression of periodontal disease is related to specific groups
of Gram-negative bacteria, the so-called periodontopathogens. The transition from healthy
periodontium to periodontitis is related to three important factors: the polymicrobial syn-
ergy, the dysbiotic microbiota and a susceptible host [126]. The multifactorial etiology
of periodontal disease contributes to the difficulty in the treatment and alternative thera-Page 11
Int. J. Mol. Sci. 2022, 23, 4131 11 of 26
pies have been investigated [127]. Porphyromonas gingivalis, Treponema denticola, Tannerella
forsythia and Aggregatibacter actinomycetemcomitans have been considered important peri-
odontopathogens [128]. A progressive inhibition of P. gingivalis in planktonic and biofilm
forms was previously observed after 1, 3, 5 or 7 min of NTAPP exposure. Moreover, im-
proved periodontal tissue recovery was obtained after 5 min of exposure, proportionally to
the number of applications [129]. Interestling, a previous study demonstrated that PAW
may also be effective against P. gingivalis. A reduction of 5-log in planktonic bacteria was
observed after 20 s of exposure [22]. Considering the particularities of subgingival biofilm
and its relationship with the progression of the disease, the treatment with PAW would be
even more interesting since it would be able to reach areas of restricted access, such as the
subgingival sites.
The mechanisms of action suggested in all of these studies, evaluating the antimicrobial
properties of PAW, mainly involved the reactive oxygen and nitrogen species (RONS)
produced in the solutions by plasma activation. Different biological effects could be
observed in each study, which is probably related to differences in methodology and groups
of microorganisms. Further in vitro and in vivo investigations are needed to standardize
the best parameters for each solution and microorganism. Considering the potential
antimicrobial effects observed in these studies, they could probably contribute to the
treatment of oral infectious diseases.
3.3. Anti-Inflammatory Properties and Wound Healing
An important feature of non-thermal plasma is the possibility of tissue antisepsis
without causing damage, which makes NTAPPs a good alternative treatment for infectious
diseases. This selectivity probably occurs due to biochemical, metabolic and cell cycle
differences between eukaryotes and prokaryotes and also the surface/volume ratio of
mammalian cells, that is higher compared to bacterial and fungal ones [130]. It was
demonstrated that no important side effect occurs after oral application of NTAPP on the
mucosa of mice, in short-term experiments [131]. A previous study investigated the effect
of PAW intake in mice after 90 days of administration, as its use in dentistry may lead
to accidental ingestion. The mineral composition and surface micro-morphology of vital
mouse teeth after long-term exposure, as well as local and systemic toxicity, were evaluated.
The authors observed that there were not significant changes in the mineral composition
and surface micro-morphology of the teeth. Moreover, the long-term exposure was not
toxic to the tongue, oral mucosa, sublingual glands or other body organs, which presented
normal structure and physiology [132]. In addition to not being harmful to mammalian
cells, NTAPPs have been demonstrated to decrease inflammation and contribute to tissue
repair [133].
Studies on skin inflammatory diseases such as allergic contact dermatitis and atopic
dermatitis have shown anti-inflammatory effects of non-thermal plasma [134–136]. These
effects have also been observed in oral studies. The treatment of oral candidiasis in mice
showed a low occurrence of inflammatory alterations. After plasma exposure, the cell
inflammatory infiltrate was predominantly mononuclear and macrophage-rich, with scarce
polymorphonuclear cells [116]. Additionally, a study evaluating the role of NTAPP as
an adjuvant therapy for the treatment of periodontitis induced in rats, observed that
the expression of inflammatory-related cytokines such as TNF-α and IL-1β decreased
significantly in the group where NTAPP was used as an adjuvant approach, while the level
of the anti-inflammatory cytokine IL-10 showed a significant increase [137].
The behavior mast cells and keratinocyte cell line (HaCat) after the contact with non-
thermal plasma-activated medium has been analyzed. Interestingly, the plasma-activated
liquid prevented an enhancement of the pro-inflammatory genes and cytokines TNF-α, IL-6
and IL-13 in activated mast cells, by inhibiting the NF-κB signaling pathway. The activation
of NF-κB by TNF-α/IFN-γ was also inhibited in HaCat cells suggesting that this treatment
could be effective against, not only acute, but also chronic inflammation [136]. Similarly,
in another study the pro-inflammatory responses of HaCat, activated by TNF-α/IFN-γPage 12
Int. J. Mol. Sci. 2022, 23, 4131 12 of 26
or LPS, was also suppressed by PAL. Moreover, STAT3, which is an important pathway
for Th17 cell activation, was inhibited by PAL in IL-6-stimulated HaCaT [135]. In this way,
plasma-activated liquids have shown to act in different inflammatory signaling pathways
of keratinocytes. These findings are important for dentistry as NF-κB e STAT3 pathways
are involved in the etiopathogenesis of periodontitis [138]. Moreover, these pathways also
play a role in oral candidiasis as mucosal candidiasis promotes NF-κB activation [139] and
STAT3 signaling is related to IL-17-mediated immunity in oral mucosal candidiasis [140].
The possibility of treating autoimmune skin diseases by direct application of plasma
or plasma-activated liquids may also be interesting for dentists, who also have to deal
with some autoimmune conditions, such as oral lichen planus [141], pemphigus and
mucous membrane pemphigoid [142]. The effects of NTAPP on oral lichen planus (OLP)
were previously investigated [143]. For this, biopsies from healthy and OLP areas were
performed, followed by the application of NTAPP in the ex-vivo tissues for 3 min. From
these lesions, 24 were reticular, 3 erosive and 1 atrophic. The treatment decreased the
infiltration of T-cells in OLP, compared with healthy samples. Additionally, the levels of
IL1β, IL2, IL10 and GM-CSF decreased significantly after the treatment and a tendency to
decrease other inflammatory markers was observed, suggesting an immunomodulatory
role of NTAPPs in OLP. The authors also presented a clinical report from a 73-year-old
man suffering from erosive OLP. The treatment consisted of 5 min of application, two to
three times per week (12 sessions). It promoted relief of the burning sensation after four
sessions. During the treatment, the oral inflammation decreased and the ulcerated area of
the lesion healed almost totally [143]. Considering that the erosive presentation is usually
symptomatic, requiring treatment with topic steroids and sometimes systemic ones [141],
clinical studies evaluating a large number of these cases are welcome. The efficiency of
PAW should also be evaluated, since washing with a plasma-activated liquid could reach
the entire area of OLP without the need for direct application at several areas of the lesion.
The exposure of diabetic animals to NTAPPs has also shown anti-inflammatory prop-
erties [144,145] and improvement in wound healing [146], which brings perspectives for
dentistry, especially considering the proven relationship between diabetes and periodontal
disease. Severe periodontal destruction is usually observed in diabetic patients, while
poor glycemic control is more common in diabetic people who also have periodontal dis-
ease [147]. Interestingly, diabetic mice treated with NTAPP showed a decrease in oxidative
stress biomarkers, advanced glycation end products (AGEs) and inflammatory cytokines,
such as IL-1, IL-6 and TNF-α [145]. It has been suggested that increased accumulation of
AGEs and their interaction with specific receptors (RAGE) in diabetic gingival tissue could
promote the hyperproduction of proinflammatory cytokines, as well as vascular alterations
and a loss of tissue integrity, contributing to the worsening of periodontitis [148]. Thus, the
adjunct treatment for diabetes mellitus with plasma modalities could possibly act indirectly
and positively in periodontitis and in other inflammatory oral diseases. The direct action of
NTAPPs or PAW in periodontal disease should also be considered, since it is a multifactorial
condition in which the microbial biofilm activates the immune system with the production
of proinflammatory cytokines and, consequently, tissue loss [149]. The selective role of
plasma could be useful in both, in the elimination of periodontopathogenic microorganisms
and also in the gingival tissue, decreasing the inflammatory process. Considering the
generalized subtype of chronic periodontitis, treatment with PAW could be clinically easier,
reaching different affected areas in a single use.
The anti-inflammatory and microbicide functions of NTAPPs have been demonstrated
to favor tissue repair [133]. Clinical trials evaluating NTAPPs have already been per-
formed, in which this technique was considered safe, painless and effective against bacterial
load [150,151]. Solutions activated with plasma, such as medium, saline and water have
also shown good results in vitro and in vivo concerning the wound healing [19,133,152].
Cell proliferation and migration were observed in human keratinocytes exposed to 15 s of
medium activated with Helium-and-Argon (He/Ar)-generated NTAPP [133], which could
favor the re-epithelialization of wounds on the skin and also on the oral mucosa that hasPage 13
Int. J. Mol. Sci. 2022, 23, 4131 13 of 26
keratinocytes in the epithelial composition. There is no study evaluating the effect of PAW
or PAL on mouth wound healing, although two studies carried out in rats and mice have
observed a tendency to improve periodontal tissue loss after the experimental treatment of
periodontitis with NTAPPs [129,137]. Moreover, wound healing of some infected ulcerated
areas of advanced oral squamous cell carcinoma was observed after NTAPP exposure [153].
Considering that wound repair is important for different modalities of dentistry such as
oral surgery, periodontics, oral pathology and implantology, PAW could be an option to
accelerate the healing after oral diseases or oral surgeries.
NTAPPs were previously suggested as a good possibility for oral surgery because
NTAPP was tested with osteoblast-like cells (MG-63), leading to cell proliferation and
in vitro wound closure [154]. Oral implant modification with NTAPPs has also been
suggested since this treatment may enhance the roughness and wettability of the implant
surface, thus improving the cell adhesion and consequently the osseointegration [47]. These
results with the direct application of NTAPPs, open perspectives for the use of PAW in oral
surgery and implantology. PAW could be used even more easily in these procedures, such as
surgeries, for the removal of oral lesions and tooth extractions, especially in impacted third
molars. Thus, the use of PAW in dentistry should be considered, given the antimicrobial,
anti-inflammatory and wound healing properties of NTAPPs, in addition to their ability to
alter the surface of dental implants. The simplicity of the technique, considering the use of
PAW as a mouthwash or an irrigation agent and possibly the lower price of PAW compared
to the direct application of NTAPPs, which would necessarily demand a device in the
dental office, make PAW a potential adjuvant oral tool for conditions requiring tissue repair.
3.4. Anti-Cancer Therapy
Sensitivity of cancer cells to NTAPPs has been demonstrated in many studies. Reactive
oxygen and nitrogen species may penetrate cancer cells more easily, compared to healthy
ones, make them more vulnerable to their harmful effects. This fact may be explained
by the higher amount of water channels (aquaporins) in cancer cells, that facilitates the
transport of reactive species into cytosol. Additionally, the lipid peroxidation caused by free
radicals generates pores in the membrane, which also allow the entry of reactive species
into the cell. This process is attenuated by the condensation of membrane lipids in normal
cells that are rich in cholesterol. However, cancer cells usually present fewer amount of
lipids, which impairs this defense mechanism. The large influx of reactive species into
cancer cells triggers signaling cascade pathways that may culminate in different types of
cell death, such as apoptosis, necrosis or senescence, depending on the dose of exposure.
Another important anti-cancer molecular mechanism of NTAPPs is their capacity to reduce
the expression of some integrins. These molecules are essential for the adhesion, migration
and invasion of cancer, which indicates that NTAPPs can be useful against metastases [38].
Besides the direct action of NTAPPs in cancer cells, they may be useful in this approach
by their interaction with the tumor microenvironment. Reactive oxygen and nitrogen
species are able to damage important extracellular matrix components, such as collagen,
fibronectin and hyaluronic acid. The induction of an antitumor immunity has also been
proposed as an action mechanism [38]. Anti-cancer properties of NTAPPs have been
observed against many types of cancer cells [155–159]. Interestingly, clinical reports have
already been conducted showing the role of NTAPPs in advanced squamous cell carcinoma
(SCC) [153,160,161], most of them in intraoral sites [153,160]. SCC is the most prevalent
oral cancer. More than 90% of the cases occur in men over 45 years of age, exposed to
tobacco and/or alcohol. The lip is the most prevalent site, followed by the tongue [162].
An improvement in the quality of life of patients with advanced SCC located at intra-oral
sites or the jaw was described, after NTAPPs treatment, by the reduction in odor and pain.
Partial remission of the lesion occurred in some cases [153,160]. Additionally, a reduction
in microbial load, wound healing of some infected ulcerated areas [153] and enhancement
of apoptotic cells were described [161]. Partial or total remission of pre-malignant skin
lesions, resulting from chronic ultraviolet exposure, referred to as actinic keratoses, werePage 14
Int. J. Mol. Sci. 2022, 23, 4131 14 of 26
also observed after NTAPP treatment [163], which opens perspectives for actinic cheilitis,
the pre-malignant lip counterpart, that precedes the emergence of lip SCC [164]. The
adjunct treatment of initial SCC with NTAPPs has not been evaluated yet and it would be
interesting, considering some in vitro responses of oral SCC to NTAPPs. A synergistic effect
of cisplatin and NTAPP against oral SCC cells in vitro was described, associated with low
cytotoxicity to normal oral cells [165]. Moreover, a combination of NTAPP with cetuximab
inhibited invasion/migration of cetuximab-resistant oral SCC cells in vitro [166].
The possibility of using this technology of plasma-activated liquid is promising consid-
ering that PAL/PAW may be injected into large or deep tumors, facilitating the action in the
entire lesion. Moreover, this kind of treatment would probably be faster and easier for the
clinician compared to NTAPPs and more comfortable for the patient who is usually weak-
ened by radiotherapy and/or chemotherapy. Treatment using liquids activated directly on
the substrate or indirectly (activated first and then in contact with the substrate later) has
been performed satisfactorily in many types of cancer cells with the use of different liquids,
such as deionized water, cell culture media, Ringer’s solution and saline [17,18,167–171].
Apoptotic cells were observed in cancer cells exposed to activated deionized water [18,171].
Different studies have shown that PAL is also efficient against cancer, due to the toxic
effects of oxygen and nitrogen species that are accumulated in these solutions and also
by the immuno-stimulatory properties [172]. Different cancer cells may respond to PAL
with decreased proliferation and migration and increased cell death by apoptosis, necrosis,
autophagy and senescence [173]. A reduction in tumor burden and a metastasis-inhibitory
effect were also observed with the use of PAL [169]. It was suggested that RNS could play
a more relevant role in cancer cell death than ROS [171].
A previous study evaluating the effect of PAL on an oral squamous cell line (SCC15),
observed an anti-cancer capacity of the plasma-activated medium. A reduction in cell
viability was observed with an increasing incubation time. Moreover, they have demon-
strated that many signaling pathways, such as p-53 pathway, could play a critical role in
this process [174]. The effectiveness of PAW, as well as its possible mechanisms of action in
oral cancer, has not been evaluated yet. Considering that the treatment of SCC is the entire
removal of the lesion, this kind of treatment could be useful in two situations: (1) Prior
to surgery, by washing the lesion or through the injection of plasma-activated water in
deep neoplasms and (2) After the surgery, by reducing the microbial load and favoring
wound healing. The anti-cancer properties could also be positive to avoid recurrences.
The role of plasma-activated water in oral premalignant lesions, such as oral leukoplakia,
erythroplasia and actinic cheilitis, should also be evaluated. Different kinds of treatments
have been used in patients with actinic cheilitis, such as combinatory treatment with PDT
and laser ablation. However, carbon dioxide laser ablation and vermilionectomy, that are
invasive for the patients, have been considered the most effective treatments [164]. In this
way, plasma-activated water could represent a non-invasive approach to be used in oral
premalignant lesions with other therapies or even alone, depending on the results and risk
factors of each patient.
3.5. Tooth Bleaching
Some studies showed that NTAPPs may be efficient for tooth bleaching, with a syn-
ergistic effect with other whitening agents [175,176]. In addition to lower concentrations
of hydrogen peroxide solution (HP), its applicability might replace conventional light
sources that present some limitations, such as questionable efficacy and high tempera-
tures [176]. The association of HP and plasma exposure for 10 min generated a three-times
higher bleaching than only HP, which probably occurred due to the presence of •OH, that
was mostly present in the plasma-treated groups [175]. In addition to its efficiency for
tooth bleaching, it was demonstrated that NTAPPs do not promote thermal damage or
inflammatory responses in the pulp and oral soft tissues [177].
The role of NTAPPs applied to liquids for tooth bleaching has already been analyzed
with interesting results. A helium-based NTAPP applied to the tooth surface with salinePage 15
Int. J. Mol. Sci. 2022, 23, 4131 15 of 26
was evaluated. According to the authors, the wettability would enable the reactivity of
ROS in the solution, attenuating the tooth dye, and it also would reduce the amounts of
toxic gas produced using the air plasma method. The bleaching efficacy after 20 min of
treatment was improved. It was 2.4 times higher than the effect produced by the whitening
agent (35% of HP). The authors observed that H2 O2 and •OH were generated in the saline
solution, probably being a key factor for the observed effectiveness. Moreover, a scanning
electron microscope (SEM) indicated smoother surfaces in the group treated with NTAPP +
saline, which was probably less harmful to the enamel [178].
Satisfactory results of NTAPPs for tooth bleaching have also been observed in the pres-
ence of water. A previous study demonstrated that deionized water activated by NTAPP
for 5 or 10 min, on the surface of the teeth, showed similar whitening presented by HP, with
similar color stability [179]. Another piece of work that evaluated the whitening properties
of bleaching agents and deionized water activated by NTAPP, in the pulp chamber of
non-vital teeth, also obtained interesting findings. A total of 50 µL of water or bleaching
agents were put in the pulp chamber, followed by plasma discharge, for 5 min. Interest-
ingly, in addition to the improved bleaching of the whitening agents promoted by NTAPP,
the generation of PAW was also effective. These findings indicate that PAW generated
in the pulp chamber could be used as a substitute for conventional tooth bleaching in
cases of non-vital teeth [180]. Considering the available results, PAW or saline generated
on the surface of the teeth seems to be effective for tooth whitening, by the formation of
H2 O2 and •OH. Further studies are still required to investigate the effectiveness and to
rule out possible toxicity effects of the reactive species to vital teeth. Moreover, studies
evaluating the tooth bleaching potential of prior activated water (indirect method) or other
plasma-activated liquids are also needed, since its application would be easier and possibly
performed at home.
4. Conclusions
Plasma-activated water demonstrates antimicrobial activity, with promising applica-
bility in both the decontamination of dental devices and the treatment of oral infectious
diseases. The anti-inflammatory properties and wound healing benefits of PAW suggest
that, in addition to its antimicrobial effect, PAW could favor the repair of previously infected
lesions. In vivo studies are still needed to prove this effectiveness in oral diseases and rule
out damage to the host. While RONS generated in PAW seems to present a fundamental
role in decontamination and wound healing, the specific constituents H2 O2 and •OH
generated in plasma-activated liquids on the tooth surface may favor tooth bleaching. The
findings related to plasma-activated water and liquids indicate that they could play an
important role in the adjuvant treatment of some cancers by their antitumor response.
Studies of PAW involving oral cancer would be interesting to investigate its application in
oral neoplasms and the exact mechanisms of action inherent to this effect.
Author Contributions: N.V.M.M.—writing of abstract, introduction, Section 3 and conclusion, review
and editing; W.C.—writing of introduction, Section 2, Appendix A, review and editing; A.d.G.S.—
writing of Section 3.1 and review; M.R.d.C.V.—writing of Section 3.2 and review; R.S.P.—supervision
and review; and C.Y.K.-I.—supervision and review. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by The São Paulo Research Foundation (FAPESP), grant nº.
19/05856-7. C.Y.K.-I. and R.S.P. thank the research fellowship from National Council for Scientific
and Technological Development 308127/2018-8 and 405653/2016-6, respectively. N.V.M.M., W.C.
and A.d.G.S. thank the individual grants financed by FAPESP, nº. 21/00046-7; 20/10450-7 and
19/25652-7, respectively.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.Page 16
Int. J. Mol. Sci. 2022, 23, 4131 16 of 26
Appendix A
In this section we will show the main mechanisms for generating long-term RONS that
are found in PAW after the plasma–liquid phase interaction. Hydrogen peroxide (H2 O2 ),
nitrite (NO2 ), nitrate (NO3 − ), nitrous acid (HNO2 ) and ozone (O3 ) are the main reactive
species that will be presented below.
Appendix A.1. Nitrite: Nitrate and Nitrous Acid
Nitrite and nitrate ions, together with nitrous acid, are long-lived reactive species
formed as by-products from primary, secondary and tertiary species, generated in regions
1, 2 and 3 (as can be seen in Figure 3). These reactive species have a high microbial capacity
in acidic environments, mainly against bacteria [30,181].
In the gas phase, Equations (A1)–(A3) occur, accompanied by NO generation [33,182].
e− + N2 → • N + • N + e− (A1)
e− + O2 → •O + •O + e− (A2)
• N + •O → NO (A3)
In the plasma phase, Equations (A4)–(A7) occur with NOx dissociation [183].
NO + O → NO2 (A4)
NO + O3 → NO2 + O2 (A5)
NO2 + O3 ↔ NO3 + O2 (A6)
NO + NO3 ↔ NO2 + NO2 (A7)
In the plasma–water interface region (region 3), nitrites are generated in PAW through
the NOx conversion generated from Equation (A7) and shown in Equations (A8) and (A9).
In the Equations (A10) and (A11), we see the generation of NO3 − which is generated from
the coexistence of NO2 − with the hydrogen peroxide and ozone present in PAW [70,184].
NO2 + NO2 + H2 O → NO2− + NO3− + 2H + (A8)
NO + NO2 + H2 O → 2NO2− + 2H + (A9)
NO2− + O3 → NO3− + O2 (A10)
NO + O2− → NO3− (A11)
It is essential to highlight that together with the generation of nitrite and nitrate,
the pH of the water also decreases, which can be seen by Equations (A8) and (A9). As
demonstrated in the literature, the increase in NO2 − and NO3 − production is directly
related to two factors: the increase in the discharge voltage and the rise in the time of water
exposure to plasma [71,96,185].
In contrast, nitrous acid (HNO2 ) is generated in the destruction reaction of nitrous
oxide (NO) which is formed in the gas phase, as seen in Equations (A12)–(A15) [186].
The formation of NO at the plasma–water interface and at PAW itself is also observed, as
showed in the Equations (A16)–(A25).
N2
∗ e− + → • N + •O + e− (A12)
O2
• N + •O → • NO (A13)
• N + NO3 → NO2 + • NO (A14)
O2 + • N → • NO + •O (A15)Page 17
Int. J. Mol. Sci. 2022, 23, 4131 17 of 26
• N + •OH → • NO + • H (A16)
• H + NO2− → • NO + OH −
(A17)
N2 O3 ↔ • NO + • NO2 (A18)
HNO2 + • H → • NO + H2 O (A19)
HNO2 + HNO2 → • NO + • NO2 + H2 O (A20)
NO2− + H2 O2 → ONOO −
↔ • NO + •O2 (A21)
•OH + ONOOH → • NO + O2 + H2 O (A22)
• NO2 + •O → • NO + O2 (A23)
• NO2 + • N → 2• NO (A24)
NO2 + • H → • NO + OH − (A25)
Finally, the HNO2 formation reactions occur in the gas phase, at the plasma–water
interface and at PAW [36,186], as seen in Equations (A26) and (A27).
• NO + •OH → HNO2 (A26)
• NO + • NO2 + H2 O → 2HNO2 (A27)
Depending on the pH level of the PAW, an equilibrium dissociation reaction between
nitrous acid and nitrite can occur, as shown below [187].
HNO2 ↔ H + + NO2− (A28)
Appendix A.2. Hydrogen Peroxide
Due to the multiple functions of hydrogen peroxide in cellular redox pathways, this
reactive species is widely used in medicine and dentistry. The hydrogen peroxide generated
in PAW has been considered a potential method for inhibiting microorganisms, cancer
treatment, wound healing benefits, among other applications [188–190].
However, there is an excellent debate about the H2 O2 generation mechanism in PAW.
In the present work, we will approach the following mechanisms: (i) recombination of •OH
dissolved in solution (Equations (A29)–(A32)) and, (ii) dissociation of water molecules via
collisions with electrons.
In the first set of reactions, it is possible to see that OH− in the gas phase generates OH−
in the liquid phase with subsequent formation of H2 O2 in the liquid phase [77,191–193].
OHgas phase → OHliquid phase + OHliquid phase → H2 O2 liquid phase (A29)
UV
H2 O → Hliquid phase + OHliquid phase
z}|{
(A30)
OHgas phase + OHgas phase → H2 O2 gas phase → H2 O2 liquid phase (A31)
e− radiation
H2 O → Hliquid phase + OHliquid phase
z}|{
(A32)
In the second set of reactions, there is dissociation of water molecules via collisions
with electrons, as shown in Equations (A33) and (A34) [194,195]. Equations (A35) and (A36)
lead to the penning ionization reactions that occur at the plasma–water interface [194–196]. In
PAW, UV photolysis can be activated, as demonstrated by Equations (A37) and (A38) [194,195].
Finally, the OH radicals generated in Equations (A33), (A34), (A36) and (A38) form hydro-
gen peroxide through Equation (A39) [194,195,197].Page 18
Int. J. Mol. Sci. 2022, 23, 4131 18 of 26
e− + H2 O → H + OH + e− (A33)
e− + H2 O → H + OH (A34)
− 1 3
e + O2 → O( D ) + O( P) (A35)
O(1 D ) + H2 O → 2OH (A36)
UV + H2 O → H2 O ∗ (A37)
UV + H2 O ∗ → H + OH (A38)
OH + OH → H2 O2 (A39)
Appendix A.3. Ozone
Ozone is the reactive species with the highest redox potential among all oxidants [70].
According to Pavlovich et al., O3 has higher bactericidal effect compared to H2 O2 and
NO2 − [198]. It is worth highlighting that O3 is transported through the plasma–water inter-
face after its production. Another way is to generate ozone directly in water, for example,
using plasma in O2 bubbles [199]. The ozone generation reaction can be observed below:
O + •O2 → O3 → O3 liquid phase (A40)
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