Clinical Uses and Molecular Aspects of Ozone Therapy: A Review

Abstract

Ozone was discovered over a hundred years ago and since then, it has been widely used in many areas, with the primary use as a disinfectant in various ways, but with applications in diverse conditions. More recently, the use of ozone has extended to other fronts, using it to treat different pathologies. Extensive studies have shown its effects, as well as the safety of its application in various modalities of use. Other studies showed its toxicity, which depends on the doses of use. Emerging evidence revealed that ozone also plays an important role in the wound healing and modulation of immune cells, describing the molecular pathways responsible for these actions and describing therapeutic actions in the treatment of wounds, pain, postoperative and infectious diseases. Ozone use has already been documented in different doses, forms of use and routes of application, depending on the clinical situation and an adaptation is necessary for a better result. Thus, this review summarizes the main clinical uses of ozone, presenting the molecular pathways responsible for its actions, as well as discussing the main routes of use, doses and, vehicles used in the clinic.

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© 2020 Ana Paula Pivot to, Fernanda Weyand Ba nhuk, Izabela Virgi nia Staffen, Maiara Aline Daga, Thaís Soprani Ayala and
Rafael Andrade Menoll i. This open access article is dist ributed under a Creative Co mmons Attr ibution (CC-BY) 3.0 l icense.
OnLine Journal of Biological Sciences
Literature Reviews
Clinical Uses and Molecular Aspects of Ozone Therapy: A
Review
Ana Paula Pivotto, Fernanda Weyand Banhuk, Izabela Virginia Staffen,
Maiara Aline Daga, Thaís Soprani Ayala and Rafael Andrade Menolli
Laboratory of Applied Immunology, Center of Medical and Pharmaceutical Sciences,
UNIOESTE-Western Parana State University, Cascavel/PR, Brazil
Article history
Received: 07-01-2020
Revised: 12-02-2020
Accepted: 24-02-2020
Corresponding Author:
Rafael Andrade Menolli
Laboratory of Applied
Immunology, Center of
Medical and Pharmaceutical
Sciences, UNIOESTE-Western
Parana State University,
Cascavel/PR, Brazil
Email: rafael.menolli@unioeste.br
Abstract: Ozone was discovered over a hundred years ago and since then,
it has been widely used in many areas, with the primary use as a
disinfectant in various ways, but with applications in diverse conditions.
More recently, the use of ozone has extended to other fronts, using it to
treat different pathologies. Extensive studies have shown its effects, as well
as the safety of its application in various modalities of use. Other studies
showed its toxicity, which depends on the doses of use. Emerging evidence
revealed that ozone also plays an important role in the wound healing and
modulation of immune cells, describing the molecular pathways responsible
for these actions and describing therapeutic actions in the treatment of
wounds, pain, postoperative and infectious diseases. Ozone use has already
been documented in different doses, forms of use and routes of application,
depending on the clinical situation and an adaptation is necessary for a
better result. Thus, this review summarizes the main clinical uses of ozone,
presenting the molecular pathways responsible for its actions, as well as
discussing the main routes of use, doses and, vehicles used in the clinic.
Keywords: Ozone, Mechanism of Action, Immunomodulation, Antioxidant,
Wound Healing
Introduction
Ozone (O3) is an allotrope of oxygen, which at room
temperature, is an explosive gas that absorbs UV
radiation in the range 220-290 nm (Eriksson et al.,
2007). Ozone is a gas with a pungent and characteristic
smell. It is formed through electrical discharges on the
oxygen molecule, which breaks down, releasing atoms
that bind to the other two molecules, generating O3. At
high concentrations in air, it turns blue, while at low
concentrations, it is a colorless gas, lighter than the air
(Wysok et al., 2006). Ozone loses only to fluorine in its
oxidizing power and oxidizes most inorganic compounds to
their final oxidative state, converting ferrous, manganous
and chrome ions to their respective higher oxidation states
(Loegager et al., 1992). Because it is exceptionally
oxidizing and unstable, ozone quickly returns to its
molecular oxygen form, but for medical use, it needs to be
synthesized through specific generators. Most medical
generators on the market use the corona effect to produce
the oxygen-ozone gas mixture. Some studies demonstrate
the generation of ozone in the antigen-antibody complex
in the human body, which proves that the production of
this molecule happens physiologically via the immune
system too (Nathan, 2002).
Ozone is an unstable molecule, so it is usually mixed
with oxygen in ozone therapy (Biçer et al., 2016). This
product has powerful oxidizing properties, forming
oxygen-free radicals that lead to the destruction of
microorganisms and improves stimulation of the process
of healing. As a result, the immune system is activated to
fight pathogens such as bacteria, fungi and virus
infections (Stübinger et al., 2006). Ozone therapy is
effective in managing many skin conditions, mainly
wound healing, primarily because of its ability to
promote inflammation in the body such as the secretion
of immunomodulatory products and, reduction of the
oxidative damage. Ozone therapy has been widely
studied in infected wounds, gangrene, burns and,
circulatory disorders, showing to be highly effective in
the outcome of these conditions (Gulmen et al., 2013).
Its characteristics as an activator of the antioxidant
system make this molecule capable of stimulating tissue
healing and an anti-inflammatory environment at low

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doses, but when used in high concentrations, it can lead
to an exacerbation of inflammation (Bocci et al., 2011).
Thus, this article aims to review the main aspects of
ozone therapy regarding its clinical uses, routes of
administration, toxicity, as well as the molecular
mechanisms, highlighting the benefits and also the main
challenges and difficulties of its use.
Uses of Ozone
Ozone was first suggested as a potable water
disinfectant in the nineteenth century because of its
powerful ability to inactivate microorganisms (Ohmuller,
1892) and it has been later confirmed by other
researchers that this substance, when used for limited
periods, can effectively disinfect the water supply
(Broadwater et al., 1973). It has been considered an
effective alternative antimicrobial agent to chlorine, even
with adverse toxicological problems arising from its use
in high concentrations (Morris, 1971).
Due to its ability to oxidize substances and destroy
microorganisms, ozone has been widely used to sterilize
air, clean water and remove odors. In addition, its
application is intended for medical use. Ozone therapy
has been used and studied extensively for many decades.
It has been used to treat infections, wounds and various
diseases (Elvis and Ekta, 2011).
It was considered as an autohemotherapeutic agent in
the treatment of HIV-infected patients in the 1980s
(Garber et al., 1991), however, subsequent studies have
proven that ozone therapy neither improves nor worsens the
dynamics of HIV-1 replication in vivo (Bocci et al., 1998).
Although ozone has been applied to treat over 100
diverse diseases with varying results, the supporting
evidence for most medical ozone applications is limited. In
1976, inhalation of ozone gas was able to induce lung
inflammation and pulmonary edema, and this led the US
Food and Drug Administration (FDA) to declare concern
about its toxicity, which was reiterated in 2006 (USFDA,
2019). The FDA also emphasized that effective therapeutic
ozone concentration should be as far as possible from
tolerable doses by humans and other animals (Wang, 2018).
Ozone has wide medical applications due to its
effective antimicrobial function (Ozturk et al., 2017),
antioxidant defense and the anticipation in wound
healing (Delgado-Roche et al., 2017; Elvis and Ekta,
2011; Mirowsky et al., 2016) and in the control of
bleeding (Gupta and Mansi, 2012). The effects on
immunoregulation and antioxidant defenses, as well as
the effectiveness of ozone therapies, have been gradually
recognized, although the mechanisms involved in
various conditions are still unclear. Due to its role as an
activator of antioxidant defenses, ozone is widely used
for cosmetic-related services (Borrelli et al., 2012) and
general health services (Di Paolo et al., 2005).
In another direction, depending on the exposure time
and dose, ozone may promote harmful effects to the
individual, knowing that inhalation of ozone gas, as well
as its intake, is harmful to health (Bocci et al., 2012), as
well as attaining motor and cognitive activity, leading to
brain dysfunction and memory loss, seen in experiments
with rats exposed to ozone (Rivas-Arancibia et al., 1998).
Route of Administration
Many clinical and laboratory in vitro and in vivo
animal studies have shown the effectiveness of ozone
therapy used in several pathways (Gautam et al., 2011;
Izadi et al., 2019; Martínez-Sánchez et al., 2005).
Generally, ozone therapy was conducted by introducing
the mixing of ozone with gases or liquids into the body
through various ways, including intravaginal, intrarectal,
intramuscular, subcutaneous or intravenous routes
(Wang, 2018). Ozone has hemodynamics and can also be
introduced into the body through autohemotherapy
(Garber et al., 1991).
The topical route is the main route used for wounds
and ulcers, such as those occurring in the diabetic foot
(Izadi et al., 2019; Martínez-Sánchez et al., 2005) and
also in cases of burns (Campanati et al., 2013). Also, the
use of topical ozone therapy was widely used in dental
procedures (Sivalingam et al., 2017). The primary
vehicles for topical uses are water, saline, or oil and the
ozonation of these vehicles can occur before or during
the procedure, the area to be treated can be submerged in
the ozonated vehicle or spread/applied directly over the
area (Campanati et al., 2013; Izadi et al., 2019).
Parenteral ozone therapy encompasses systemic and
local injection and is widely used mainly in the
treatment of low back pain, arthritis, coronary artery
disease, herniated disc, osteoarthritis, hepatitis C, muscle
pain, among other conditions (Babaei-Ghazani et al.,
2018; Celakil et al., 2017; Gautam et al., 2011;
Martínez-Sánchez et al., 2005; Zaky et al., 2011). Beyond
the practices of systemic ozone therapy are rectal
insufflation, as in the case of coronary artery disease and
also in the treatment of hepatitis C (Martínez-Sánchez et al.,
2012; Zaky et al., 2011) and intravenously, performing
autohemotherapy to treat the symptoms of hepatitis C
too (Zaky et al., 2011) and also in cases of diabetic foot
(Izadi et al., 2019). Intramuscular infiltration for acute
spinal pain (Paoloni et al., 2009), intradiscal for lumbar
disc herniation (Gautam et al., 2011) and intrapatellar
injection for osteoarthritis treatment in the knee (Babaei-
Ghazani et al., 2018) could be classified as local
injections but not systemic or topical applications.
Vehicles for the Use of Ozone Therapy
Ozone therapy is available through different types of
vehicles. It is commonly used in the form of gas, under

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topical treatment, where the skin is covered with a
plastic bag filled with ozone gas-oxygen mixture (Wang,
2018). It also can be used in the form of ozonated saline,
which consists of prior saturation of physiological saline
with an oxygen-ozone mixture (Clavo et al., 2013).
An ozone generator is used when the aqueous ozone
is needed, which mixes ozone with water. This format is
highly valid for many skin-related disorders and is
widely used to treat infectious diseases. However, the
disadvantage of ozone hydrotherapy is the short shelf
life and its instability, which makes this treatment an
option available only in equipped clinics (Wang, 2018).
Its short half-life is approximately 20 minutes in water
at 20°C, it is partially soluble in water and, like most
gases, increases its solubility as the temperature
decreases (Kim et al., 1999; Wysok et al., 2006). This
solubility of ozone in aqueous media will depend on the
content of organic matter in the medium, as the lower the
concentration of organic matter, the longer the half-life
of ozone in water. The decomposition of ozone in the
liquid phase is characterized by a rapid decrease in initial
concentration, with a later phase in which ozone
concentration decreases according to first-order kinetics,
with hydroxyl radicals (OH) being the main products of
this decomposition (Almeida et al., 2004; Bocci, 2004).
Ozone can react with organic compounds in aqueous
solution by a direct ozone reaction or by an indirect
reaction of OH radicals, formed from the decomposition
of ozone. This indirect reaction can promote an attack on
organic compounds approximately 100 times faster than
some oxidizing agents such as H2O2 and ozone itself.
Predominantly, the disinfection processes occur via
molecular ozone, whereas the oxidation processes can
occur through both direct and indirect processes
(Almeida et al., 2004; da Silva et al., 2011).
Ozonated water is applied to wounds and ulcers, at
different concentrations depending on the outcome
expected (disinfect or regenerate) and the type of tissue
to which it will be applied (Clavo et al., 2013). Ozonated
water is indicated for pain relief, disinfection and anti-
inflammatory effects in acute and chronic lesions, with
or without infection (Wang, 2018).
Another option is the ozonated oil, obtained by
bubbling ozone into cold-pressed oils such as olive oil,
sunflower oil and many other unsaturated fatty acids
(Menéndez et al., 2011). It has the advantage of the shelf
life of 2 years if stored in refrigeration and the topical
application of this formulation can be performed at home,
which decreases the visits of the patients to the medical
institutions. Ozone oil is used in many conditions,
including wounds, anaerobic, viral and fungal infections,
as well as ulcers, anal fissures, and vulvovaginitis,
without any visible side effects (Bocci, 2004).
The vehicle by which ozone is applied can also play a
role in the mechanism of ozone therapy. Ozonated oils and
water act as stimulators, with both forms improving blood
circulation, oxygen delivery, and regularized antioxidant
enzymes to activate the immune system and promote the
release of growth factors. These phenomenons happen
because ozone reacts with the polyunsaturated fatty acids
present in the stratum corneum to generate reactive
oxygen species and lipo-oligopeptides, substances that
can be partially absorbed by skin, capillary and
lymphatic antioxidants (Sega et al., 2010).
Direct ozonation of vegetable oils with unsaturated
fatty acids leads to the formation of the 1,2,4-trioxolane
portion (Sega et al., 2010), which represents an active
form of ozone in these materials. The trioxolane ring in
the ozonated vegetable oil rapidly produces compounds
that accelerate the healing process in wet wounds or ulcers
and are responsible for antimicrobial and antimycotic
treatments (Menéndez et al., 2011). In addition, the oil
itself can act as a moisturizer and protector, especially
for patients with the compromised skin barrier.
Studies using vegetable oils in their pure form have
found that oils obtained from plants popularly used as
healers have demonstrated their healing effect. In a study
by Oliveira et al. (2013), it was found that pumpkin oil
increased tensile strength and increased collagen
synthesis, possibly as a result of the action of fatty acids
(Oliveira et al., 2013). The sunflower seed oil has
positively contributed to wound healing (Coelho et al.,
2012; Rodrigues et al., 2004). Oleic and linoleic acids,
the main components of sunflower oil, also present in
extra virgin coconut oil, demonstrated that when
topically applied, promoted rapid and satisfactory
healing in mice (Cardoso et al. 2004).
Ozone Clinical Uses
Ozone therapy has been used to treat different
conditions as summarized in Table 1, seen in different
Randomized Clinical Trial (RCT) and reviewed by many
authors. The conditions already studied includes:
Tissue healing from inflammatory processes showed by
a systematic review that included only RCT for chronic
wounds (Fitzpatrick et al., 2018) and revised by
Travagli et al. (2010) and Valacchi et al. (2005); for
back pain revised by Braidy et al., 2018 and also showed
by Andrade et al. (2019) in a RCT; osteoarthritis showed
in a systematic reviews (Anzolin and Bertol, 2018;
Noori-Zadeh et al., 2019) and seeing in an RCT
(Babaei-Ghazani et al., 2018; Lopes de Jesus et al.,
2017) and in a clinical trial phase 2 (Calunga et al.,
2012); diabetic foot ulcers as showed by a RCT
(Zhang et al., 2014) and revised by others (Braidy et al.,
2018); dental problems such as caries and periodontitis
summarized in a review (Almaz and Sönmez, 2015); in
the treatment of systemic sclerosis, as seeing in a RCT
(Hassanien et al., 2018); multiple sclerosis, seeing in a
clinical trial phase 1 (Delgado-Roche et al., 2017;
Nowicka, 2017); among other conditions showed in
clinical trials (Vanni et al., 2016).

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Table 1: Ozone therapy results in different clinical conditions
Condition Outcome
Wound healing Increase in vessel numbers, collagen deposition and myofibroblasts numbers; Stimulation of
the growth factors and peroxide releases; Improvement in oxygenation of the affected area.
Gingival graft surgery Improvement in the healing process; Diminishment of the pain intensity.
Cardiovascular diseases Increase in oxygenation in ischemic sites
Chronic inflammatory diseases Improvement of the standard treatment; Reduction in pain sensation.
Balanitis xerotica obliterans Anti-inflammatory effect; Reduction in the levels of the gene transcription of cytokines;
Reduction in the TG2 enzyme.
Atopic dermatitis Reduction in the exudation, improvement of the erosion process.
Osteoarthritis Positive effect on the intra-articular redox balance; Pain reduction.
Multiple and systemic sclerosis Decrease inflammatory process and cellular oxidative stress; Improved joint mobility;
Reduction in the thickness of the skin.
Asthma Reduction in immunoglobulin (Ig)E and HLA-DR levels; Improvement of lung function
reduction of the symptoms related.
Fig. 1: The main mechanism behind ozone treatment
Ozone therapy is considered an alternative therapy
and has several positive effects, but depending on the
dose, it may result in harmful effects for the individual
(Travagli et al., 2010). The primary reported effects in
humans are based on three main functions: Antimicrobial
(Ozturk et al., 2017), in the antioxidant/oxidant balance
(Calunga et al., 2012) and immunomodulatory effects
(Zeng and Lu, 2018) (Fig. 1). It is as a topical treatment
that ozone therapy has been most widely used
(Solovăstru et al., 2015; Travagli et al., 2010; Zeng and
Lu, 2018), as ozone gas is highly toxic to the airways,
whereas and in skin, a transient exposure at low and
controlled doses (range from 10 to 50 µg/mL), may be
beneficial (Valacchi et al., 2005). In the different
scenarios of different RCT, the use of ozone acts
mainly to anticipate the healing process, such as
reducing the size of the diabetic foot injury in
individuals with different wound sizes (Zhang et al.,
2014), as well as improving the treatment and complete
closure of these wounds in association with usual
treatment (Wainstein et al., 2011). It also showed this
characteristic in the effective healing of the treatment of
digital ulcers of patients with systemic sclerosis,
improving from 44% to 96% the effectiveness of the
treatment when combining calcium channel blocker with
topical aqueous ozone (Hassanien et al., 2018).
Another RCT showed that the application of ozone
on the gingival graft surgery site improved the patient's
Oxidation of ph ospholipids
and lipoproteins
Treatment of m any skin
diseases: diabetic foot atopic
dermatitis psoriasis vulgaris
Damage to
the capside
Bacterias Virus
Antimic robian activit y
Improved lu ng
function
↑
Myofibroblasts
↑ Collagen
Neovascularization
↓ IgE
Wound healing
↑
IFN-
γ
,
↑
TNF-
α
,
↑
IL-2
improved in flamma tory
response Imunno modulation
Release of growth
factors, VEFG, TGF-β,
FGF-2, PDGF
O3
M1 macrophages, IL-12
Pro inflam atory action
M2 macrophages, IL-10
anti infla matory action
Oxidant-antio xidant
balance
H2O2 regulation
↑ O2 affect area
↑
Antioxidant e nzymes
↓
doses O2
↑ NrF2
↓ NFkB
↑
doses O2
↓ NrF2
↑ NFkB
COX2
PGE2
Cytokines
Beneficial ef fect Toxicity
Prevention of metabolic,
inflam matory and
infectious diseases
Or

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quality of life, as well as improved healing along with
lower pain intensity (Taşdemir et al., 2016). Ozone, due
to its antioxidant effect (Vanni et al., 2016), has had
positive effects in the treatment of cardiovascular diseases,
improving the patient's prognosis, such as increasing
oxygenation in ischemic sites, as has been demonstrated
by a RCT to be beneficial in coronary artery disease and
heart failure (Martínez-Sánchez et al., 2012) and showed
in a clinical trial to treat peripheral arterial disease
(Coppola et al., 2002), besides, reviewed by Mauro et al.
(2019), ozone also showed to be beneficial in the
prevention of ischemic damage in an in vitro experiment
of ischemia (Frosini et al., 2012) and in a myocardial
infarction animal model (Di Filippo et al., 2010).
In chronic inflammatory diseases, the use of ozone
therapy as an adjuvant has been shown to improve the
reference treatment of back pain in patients studied under
an RCT (Andrade et al., 2019; reviewed by Bocci et al.,
2015). Another clinical practice is in the treatment of
disc herniation, which consists in injecting a gaseous
oxygen-ozone mixture into the intrasomatic space, site of
the disc herniation, or paravertebral muscle, showed in
an RCT to be beneficial to the patients, decreasing the
pain mainly. This practice was successful in treating this
condition (Paoloni et al., 2009). In 2011, a case report of
oral osteonecrosis was treated with an ozonated oil
suspension, achieving therapeutic success effectively and
safely (Ripamonti et al., 2011).
Ozone therapy has also been used in diseases such as
Balanitis Xerotica Obliterans (BXO). An RCT with male
children showed that ozonized olive oil produced a
powerful anti-inflammatory effect demonstrated by the
reduction in the levels of gene transcription of cytokines
examined in the treated tissues. In addition, it was
discovered that ozone treatment was able to reduce the
expression of the levels of the Transglutaminase 2 (TG2)
enzyme, supporting its anti-inflammatory role in the
foreskin affected by BXO (Currò et al., 2018).
Recent data shows the use of ozone also in
autoimmune diseases, such as atopic dermatitis and
psoriasis vulgaris. An RCT divided sixty children into
two groups, the first received ozonated water 3 to 5
times a week and ozonized oil twice a day, the second
group received only base oil and a moisturizer for 2
weeks. After 3-5 days of topical ozone therapy, the
exudation of the skin was reduced, and the erosion
healed. The effective rates were 80.0% and 20.0% in the
treatment and control group for 1 week and 89.6% and
30.7% for 2 weeks, respectively, with a significant
difference between the 2 groups (P<0.001), these data
show the effectiveness of topical ozone therapy in this
condition (Qin et al., 2018). A clinical trial phase 1
shows the efficiency of ozone in the treatment of
psoriasis vulgaris, in which 39 patients whit this
condition received medical ozone therapy during the
nursing intervention. The cure rate was 38.5% and the
total efficiency was 84.7%, showing that this substance
can be also a good alternative in the treatment of this
disease (Le, 2012).
An RCT to study the effect of ozone as a therapy
to treat osteoarthritis also showed to be a successful
approach to treat and relieve the pain of the patients
under this condition (Babaei-Ghazani et al., 2018;
Lopes de Jesus et al., 2017). Also, a clinical trial
showed that a combination of the usual treatment to
knee osteoarthritis with ozone therapy had a positive
effect on the intra-articular redox balance, reducing
the pain of these patients, increasing their quality of
life (Calunga et al., 2012).
Ozone was also beneficial to treat multiple sclerosis and
systemic sclerosis (Delgado-Roche et al., 2017; Nowicka,
2017). In a clinical trial phase 1, Delgado-Roche et al.,
2017, showed that ozone therapy decreases inflammatory
process and cellular oxidative stress, promoting a
possible neuroprotective effect, where ozone therapy
could be used together with the usual treatment,
diminishing its toxicity (Delgado-Roche et al., 2017).
Also, in a systemic sclerosis clinical trial, Nowicka
(2017) presented results where ozone, alone or together
with the usual treatment, improved the clinical
parameters and skin temperature of these patients,
increasing the movability of the joints and decreasing the
thickness of the skin (Nowicka, 2017).
Safety Dose and Clinical Protocol
The safe dose of ozone is variable depending on the
location of therapy and the goal of the treatment
(Smith et al., 2017). Currently, it has been proposed that
ranging from 10 up to 80 µg/mL of ozone, is beneficial
leading to the positive effects and no toxicity, depending
on the tissue, shown in in vitro and in vivo in animal
models (El-Sawalhi et al., 2013; Frosini et al., 2012;
Orakdogen et al., 2016; Thiele et al., 1997; Sweet et al.,
1980; Smith et al., 2017). In humans studies, the safe
dose range varies between 15 and 50 µg/mL and it will
depend on the type and goal of treatment as well as the
local of application.
Several clinical protocols have been used to treat the
different pathologies already mentioned here. Depending
on the extent of the disease and the goal of the treatment,
the protocol may vary from one week up to months. In the
case of diabetic foot ulcers, many studies use ozone
treatment in conjunction with conventional treatment
(Liu et al., 2015). Clinical studies perform submersion
treatment of the lower limb in a bag of water and constant
ozonation for 30 min, at an approximate concentration of
50 µg/mL (Izadi et al., 2019; Martínez-Sánchez et al.,
2005; Zhang et al., 2014). Different studies have different
endpoints, some of them, in the case of the diabetic foot or

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open wounds, treat until complete wound closure or until
the reepithelialization process is identified (Izadi et al.,
2019), others establish a limit of days, such as 20
consecutive days or so (Zhang et al., 2014). Besides, the
use of ozone oil is widely used in topical therapy and the
preparation is spread over the wound and left to act for
30 minutes, at an average concentration of 40 µg/ml
(Fitzpatrick et al., 2018).
To treat disc herniated low back pain and acute back
pain, patients received for five consecutive weeks, three
intramuscular injections of an oxygen and ozone mixture
at an average concentration of 20 µg/mL (Paoloni et al.,
2009). Protocols that perform rectal insufflation,
generally indicated for the treatment of low back pain,
osteoarthritis, arthritis, multiple sclerosis, among others,
use an ozone concentration of approximately 20 µg/mL
during approximately 2-3 times per week for 3 months
(Anzolin and Bertol, 2018; Delgado-Roche et al., 2017)
and also 20 applications for 4 weeks in the case of
rheumatoid arthritis (León Fernández et al., 2016). In the
case of coronary artery disease, a concentration of 40
µg/mL once a day for 20 days was effective in
decreasing oxidative biomarkers generally present in this
condition (Martínez-Sánchez et al., 2012). The
autohemotherapy protocols use the patient's collected
blood, which is then ozonated at a dose of 20 µg/mL
and reinserted intramuscularly or intravenously, as in
the treatment of complications of hepatitis C (Zaky et al.,
2011) and individuals with peripheral arterial disease
(Di Paolo et al., 2005).
In local injection, such as intradiscal injection, used
mainly for back pain such as disc herniation, an average
of 50 µg/mL of ozone is usually injected into an
ozone/oxygen mixture (Niu et al., 2018). Also, there are
intrapatellar ozone injection protocols at an average
concentration of 15 µg/mL that is effective in treating
osteoarthritis (Babaei-Ghazani et al., 2018).
Molecular Mechanisms of Action
The mechanism of action of ozone therapy lays
mainly in three aspects: Immunomodulation (Bocci,
2006; 2007), antimicrobial activity (Polydorou et al., 2012;
Thanomsub et al., 2002) and the capacity to interplay with
the oxidant/antioxidant balance (Soares et al., 2019).
Altogether, another main aspect of the mechanism of the
ozone treatment is the wound healing property and the
main mechanism behind this is the capacity to interact
with the skin epithelization system (Borges et al., 2017;
Eroglu et al., 2018), as summarized in Fig. 1.
Ozone therapy, especially in its topical forms, has
been used clinically as an adjuvant in wound healing
(Borges et al., 2017). Ozone promotes an increase in the
epithelialization process increasing matrix deposition and
cell proliferation. Ozone-treated wounds also demonstrate
a granulation tissue with fewer inflammatory cells and a
higher number of myofibroblast-compatible spindle cells.
As well as increased vessel numbers and collagen
deposition (Soares et al., 2019). Several endogenous
growth factors, such as vascular Endothelial Growth
Factor (VEGF), Transforming Growth Factor-beta
(TGF-β), Fibroblast Growth Factor (FGF) and Platelet-
Derived Growth Factor (PDGF), play an essential role in
early wound healing (Eroglu et al., 2018), so it becomes
crucial for the healing process the stimulation of the
expression of these growth factors.
Another mechanism by which ozone leads to wound
healing involves its interaction with wound exudates that
lead to the breakdown of ozone into peroxides and
stimulates tissue repair, improving oxygenation in the
affected area. Oxygen-responsive species stimulate
platelet aggregation and lead to the release of growth
factors, that as mentioned above, play a key role in wound
healing (Wang, 2018). According to recent researches,
ozone increases endogenous growth factors (Eroglu et al.,
2018) such as the expression of VEGF, TGF-β and PDGF,
all involved in the cicatrization process (Zhang et al.,
2014). Fibroblast Growth Factor 2 (FGF2), also known as
basic FGF, is an essential protein that activates
myofibroblastic angiogenesis-associated pathways and
proliferation (Svystonyuk et al., 2015). FGF2 expression
was associated with fibroblast activation, resulting in
higher tissue collagen production. Soares et al. (2019), who
used ozone to treat skin wounds in rats, observed that FGF2
was overexpressed in macrophages and myofibroblasts,
which may indicate an improvement in wound healing
events. This finding corroborates with the hypothesis that
these cells, under topical ozone treatment, secrete growth
factors and cytokines that are essential to the healing
process. Besides, ozone has been reported to induce an
increase in the number of macrophages associated with a
higher number of myofibroblasts, as well as proliferative
neovascularization and vascular regression in a wound
remodeling phase. So, the mechanism of action of ozone
may be associated with FGF2 overexpression and
myofibroblastic differentiation (Soares et al., 2019).
Additionally, ozone activates the immune system by
inducing secretion of interferon-γ and tumor necrosis
factor-α, Interleukin 2 (IL-2) and other factors involved
in the inflammatory response (Bocci 2006; 2007). The
underlying mechanism involves the interaction of ozone
with wound exudates, which leads to the breakdown of
ozone into peroxides and stimulates tissue repair,
improving oxygenation in the area (Xiao et al., 2017).
Species of oxygen, released by the macrophages,
stimulate platelet aggregation and lead to the release of
growth factors, which aid in wound healing
(Valacchi and Bocci, 1999). However, studies should be
performed to highlight the phenotype of macrophages
that produce and secrete FGF2 during wound healing
(M1 or M2 macrophages). Characteristically, M1

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DOI: 10.3844/ojbsci.2020.37.49
43
macrophages induce inflammation, negatively regulating
the early healing process, producing IL-12 and M2
macrophages positively regulate the healing process in
an IL-10 mediated process (Mills, 2012).
Ozone therapy has been shown to increase tissue
oxygenation and metabolism, interfering with
oxidant/antioxidant balance. Based on this mechanism,
ozone is capable of reducing the levels of hydrogen
peroxide (H2O2) by regulating the levels of antioxidant
molecules. Also, peroxides produced through ozone
improve oxygen availability by regulating antioxidant
molecules in red blood cells, favoring the metabolism
and the release of cytokines, autacoids and growth
factors that are fundamental in the treatment of
metabolic, inflammatory and infectious diseases
(Soares et al., 2019). However, this release of peroxides
in a large amount can cause harm to humans, as is
evidenced in some studies that show structural changes in
lung tissue (Buell et al., 1965), low resistance to
respiratory bacterial infection and reduced lung function
(Holzman et al., 1968). In addition, a pre-clinical study on
the reaction of ozone with amino acids indicates that
cysteine and methionine are easily oxidized, as well as
tryptophan, histidine, tyrosine, cystine, and phenylalanine
(Mudd et al., 1969). This oxidation of amino acids can be
responsible for the toxic reactions of ozone to plants and
animals. The studies above were performed mainly at
ozone levels between 1.0 and 5.0 ppm (Leh, 1973).
Despite the possible deleterious dose-dependent
effect, ozone therapy can increase oxygen metabolism in
erythrocytes, increasing the rate of glycolysis,
consequently leading to increased oxygen release into
the tissues (Di Filippo et al., 2015). Ozone increases
oxidative pyruvate carboxylation and ATP production
during the Krebs’ cycle, reducing NADH and oxidizes
cytochrome C. Indeed, it increases the production of
enzymes such as glutathione peroxidase, catalase, and
superoxide dismutase that serve as sequestrators of free
radicals and protectors for healthy cells (Nathan, 2002).
Ozonization in asthmatic patients effectively reduces
Immunoglobulin (Ig)E and HLA-DR levels, thus
improving lung function and reducing related symptoms.
A study suggested that the therapeutic efficacy of ozone in
reducing IgE and inflammatory mediators are associated
with its immunomodulating and regulating properties of
the oxidative stress (Wang, 2018). Another study showed
a decrease in IgE and HLA-DR levels in asthmatic
patients treated with systemic ozone therapy with the
consequent increase in lung function and improvement in
the symptoms (Hernández Rosales et al., 2005).
Evidence shows that moderate oxidative stress
induced by low doses of ozone activates nuclear factor
erythroid 2-related factor 2 (Nrf2), suppresses
transcriptional factor-kappa B (NF-κB) and reduces
inflammatory responses. Over the past decade, evidence
has suggested that Nrf2 activation induces transcription of
Antioxidant Response Elements (ERA). ERA
transcription leads to the production of antioxidant
enzymes such as Superoxide Dismutase (SOD),
Glutathione Peroxidase (GPx), Glutathione S-Transferase
(GST), Catalase (CAT), Heme-Oxygenase-1 (HO-1),
NADPH-Quinone Oxide reductase (NQO-1), phase 2
enzymes of drug metabolism and heat shock proteins.
Thus, the Nrf2 system contributes to the protection against
carcinogenesis, liver toxicity, chronic respiratory and
inflammatory diseases, neuronal ischemia and renal
problems. Systemic administration of ozone through
ozonated saline could activate this mechanism (Re et al.,
2014). In a study by Pecorelli et al. (2013), it was shown
that ozonated plasma can positively regulate HO-1
expression in endothelial cells and activate Nrf2 in dose-
dependent serum ozone concentration.
On the other hand, severe oxidative stress triggered
by high concentrations of ozone activates NF-κB,
leading to elevated inflammatory responses and tissue
damage by the production of Cyclooxygenase 2 (COX2),
Prostaglandin E2 (PGE2) and cytokines. So, the strength
of oxidative stress determines the effectiveness and
toxicity of ozone (Wang, 2018). This effect was shown
on the skin, demonstrating that ozone is capable of
interacting with skin antioxidant molecules (Thiele et al.,
1997), besides causing an increase in the expression of
the COX-2, with an increase in heat shock protein (HSP)
(Valacchi et al., 2005). The ozone dose that can cause
toxicity is not yet defined, varying according to the
clinical condition, route of administration and the
individual's own organism (Bocci, 2006).
Ozone inactivates bacteria, viruses, fungi, yeast, and
parasites through different mechanisms. In bacteria,
ozone disrupts the integrity of the bacterial cell wall
through oxidized phospholipids and lipoproteins
(Polydorou et al., 2012; Thanomsub et al., 2002). In
fungi, ozone inhibits its growth, while in viruses, ozone
damages the viral capsid, preventing contact between the
virus and the cell. Cells vulnerable to virus invasion are
coated with oxidation-susceptible enzymes and can be
eliminated from the body by interacting with ozone
(Gupta and Brintnell, 2013; Travagli et al., 2010).
Main Challenge and Difficulties
The biggest challenge is the lack of standardization of
ozone doses used in the studies, many not even
mentioning this concentration. This may be due to the fact
that ozone is a very unstable gas, which makes it difficult
to remain in vehicles and, consequently, to measure the
dose (Bocci et al., 2011). Another problem is the fact that
in many countries medicine does not consider the use of
ozone as a therapy, which makes it difficult to develop
clinical treatment protocols (Re et al., 2012).

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44
Conclusion
The use of ozone in clinical practice has numerous
benefits in the treatment of various diseases, mainly
through the modulation of the immune system and of the
oxidant/antioxidant balance and is applied to treat a wide
of clinical conditions, both on local skin and systemic.
The significant routes of use include the topical,
intrarectal, subcutaneous and intravenous routes, carried
out by different vehicles, as the main ones being saline,
water, and ozone oil. The main characteristic of
ozonetherapy is the anticipation of the healing process,
which is related to the increase of endogenous growth
factors and in the increase of oxygenation of the affected
area. As well as its physiologic effect, ozone also
inactivates microorganisms by its oxidizing capacity.
This review evidenced that the toxicity of this compound
is inherent to the concentration of ozone, being a higher
dose responsible for causing exacerbation of the immune
system with detrimental effects and a lower dose
contributing to the reduction of inflammatory responses
and increasing the production of antioxidant enzymes,
thus improving the treatment of a range of pathologies.
Altogether, ozone as a therapy has been used
successfully in a wide range of medical conditions,
however, it should be used in the correct concentration,
paying attention to the dose range to avoid possible
adverse effects due to toxicity of high concentration.
Acknowledgment
The authors thank Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES) - the Brazilian
government funding agency, for the fellowships
received.
Funding Information
FWB. and IVS received a master's degree and TSA
received a postdoctoral fellowship from the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) - the Brazilian government funding agency.
Author’s Contributions
Conceptualization: APP and RAM; Literature review:
APP, FWB, IVS, MAD and TSA; Validation: RAM;
Writing – original draft: APP, FWB, IVS, MAD, and
TSA; Review and editing: RAM and TSA.
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