Ozone: Science & Engineering, 34: 469–475
Copyright © 2012 International Ozone Association
ISSN: 0191-9512 print / 1547-6545 online
Application of Ozone Therapy in Patients
with Knee Osteoarthritis
José Luis Calunga,1Silvia Menéndez,1Rodolfo León,2Soulien Chang,3Dailen Guanche,1
Alberto Balbín,4José Zayas,4and Pedro García4
1Ozone International Clinic, Ozone Research Center, National Center for Scientiﬁc Research, Havana City, Cuba
2A.A. Aballí Pediatric Teaching Hospital, Havana City, Cuba
3Pharmaceutical and Food Institute, Havana University, Havana City, Cuba
4“Dr Fructuoso Rodríguez” Orthopedic Hospital, Havana City, Cuba
Osteoarthritis is a common degenerative joint disease.
Taking into account the ozone (O3) effects in cellular redox
balance and upon biomarkers of inﬂammation, the aim of this
study was to evaluate the action of ozone therapy in oxidative
stress parameters in synovial ﬂuid of patients suffering of knee
osteoarthritis and their clinical evolution. In 42 patients, O3
was administered rectally and by intra-articular applications.
Synovial ﬂuid was extracted for the measurement of param-
eters associated with oxidative stress. Also, evaluation of the
joint capacity, pain, and ultrasound imaging were performed.
Combined ozone therapy produced an intra-articular redox
balance and a signiﬁcant reduction of pain.
Keywords Ozone Therapy, Osteoarthritis, Synovial Fluids, Pain,
Osteoarthritis (OA) is the most common form of arthri-
tis and is deﬁned as a degenerative joint disease that causes
pain, stiffness, swelling and loss of motion in the joints which
is aggravated by prolonged activity (Martin and Buckwalter
2002; Poole 1999). It is a process of progressive deterioration
Received 5/24/2011; Accepted 8/4/2012
The opinions and conclusions expressed in this article are those
of the authors and contributors, and do not necessarily reﬂect those
of the International Ozone Association, the editors, Editorial Board,
or Taylor & Francis. Readers are to make their own decisions with
regard to the work presented. These medical articles are enclosed,
as in the past, as a service to the members of the IOA interested in
Address correspondence to Silvia Menéndez, Ozone
International Clinic, Ozone Research Center, National Center
for Scientiﬁc Research, P.O. Box 6414, Havana City, Cuba. E-mail:
of articular cartilage (it breaks down and becomes thin) and
formation of new bone (osteophyte) at the joint surface.
OA is becoming increasingly prevalent worldwide because
of the combination of an aging population and growing levels
of obesity. It mainly affects people over the age of 45, but it
can develop in younger people. It is estimated that 40 million
Americans and 70 to 90% of people older than 75 years are
affected by OA. Although symptoms of OA occur earlier in
women and appear to be more severe, the prevalence among
men and women is equal (Hinton et al. 2002).
Many studies have identiﬁed molecular characteristics of
ageing in OA cartilage or chondrocytes, which may contribute
to the onset of OA, including telomere genomic instabil-
ity, formation of advanced glycation end products, increased
apoptosis and senescence (Carrington 2005). Such changes
could be related to the increased levels of oxidative stress
that occur in OA, leading to cells unable to respond effec-
tively to normal loading regimens and potentially contributes
to disease onset (Henrotin et al. 2005; Ostalowska et al. 2006;
Plumb and Aspden 2005). Lipid peroxidation mediated by
free radicals is considered to be the major mechanism of cell
membrane destruction and cell damage. The body’s defense
mechanisms would play an important role in the formation of
anti-oxidants and try to minimize the damage, adapting itself
to the above stressful situation. Anti-oxidants are able to scav-
enge and suppress the formation of free radicals or oppose
their actions (Cotgreave et al. 1988; Sie 1991).
Oxidative phosphorylation is a major source of reactive
oxygen species (ROS); however, chondrocytes also express
NADPH oxidase and nitric oxide (NO) synthase family mem-
bers together with various oxygenases, which principally
generate the ROS, NO and the superoxide anion (O2•–)
(Henrotin et al. 2005). These ROS generate derivatives includ-
ing hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and
Ozone Therapy in Patients with Knee Osteoarthritis November–December 2012 469
hydroxyl radicals (OH•). Lipid peroxidation products and
nitrotyrosine residues have been observed in aged and OA
cartilage (Loeser et al. 2002; Tiku et al. 2000). ROS can
cause cartilage degradation directly by cleaving collagen
and activating matrix metalloproteinases (MMP), a family of
enzymes that have a key role in cartilage destruction in OA
(Klämfeldt and Marklund 1987; Rowan and Young 2007).
To prevent an accumulation of ROS-mediated damage chon-
drocytes produce a number of anti-oxidant enzymes, includ-
ing the superoxide dismutase (SOD), catalase, and glutathione
peroxidase (Henrotin et al. 2005).
OA is becoming increasingly prevalent worldwide because
of the combination of an aging population and growing lev-
els of obesity. Despite the increasing number of OA patients,
treatments to manage this disease are limited in controlling
pain and improving function and quality of life while limit-
ing adverse events (Hinton et al. 2002). Effective therapies
to regenerate damaged cartilage or to slow its degeneration
have not been developed. Current treatments such as: reha-
bilitation, exercise, modiﬁcation of activities of daily living,
pharmacotherapy, alternative medicine or surgery are focused
on symptomatic relief but they lack efﬁcacy to control the pro-
gression of this disease, which is a leading cause of disability
(Alcaraz et al. 2010). Therefore, the development of effective
disease-modifying drugs is urgently needed.
Taking into account different biological effects of ozone,
such as: the stimulation of the anti-oxidant defense system
counteracting the intracellular pro-oxidant status, improve-
ment in the oxygen deliver to tissues, the immunological
modulation, among others (Ajamieh et al. 2002, 2003, 2004,
2005; Al-Dalain et al. 2001; Bocci 2002, 2006; Borrego et al.
2004; Candelario-Jalil et al. 2001; Hernández et al. 2005;
León et al. 1988, 2008; Martínez et al. 2005; Menéndez
et al. 2008; Valacchi and Bocci 2000; Zamora et al. 2005),
the aim of this study was to evaluate the action of ozone
therapy in oxidative stress parameters in synovial ﬂuid of
patients suffering of knee osteoarthritis and their clinical
PATIENTS AND METHODS
This controlled clinical trial was approved by an insti-
tutional review board (Scientiﬁc and Ethics Committees
of the Institutions) in accordance with the principle of
the Declaration of Helsinki (1997). All patients provided
informed consent after receiving appropriate information
about the study (characteristics, beneﬁts, and possible side
effects). Before enrolling, all participants attended a training
program designed to familiarize them with the study objec-
tives and treatment plans. A complete clinical and personal
history of all the subjects involved in the study was recorded.
Inclusion criteria were: Adult patients (45–65 years
old), of both sexes and different ethnic origins that were
clinically and radiologically diagnosed as patients with
knee osteoarthritis. Exclusion criteria were: patients that
present severe hypertension, septic conditions, diabetic
complications, liver/hematological/cardiovascular diseases,
hypersensibility to the medication that will be used, inabil-
ity to cooperate with the requirements of the study or recent
history of alcohol or drug abuse. Subjects with supple-
menting anti-oxidant vitamins or receiving anti-inﬂammatory
drugs during the last 3 months were excluded. Forty-two
patients were involved in this study. There were two study
groups: (1) Control: 10 healthy age- and sex-matched sub-
jects, and (2) Ozone: 42 patients with clinically diagnosed
Ozone was administered by rectal way (20 sessions, daily,
Monday to Friday) at scaling doses, using ozone concentra-
tions between 25 and 40 mg/L and volumes of 100 to 200 mL
and by intra-articular (15 sessions, twice per week) applica-
tions with an ozone concentration of 20 mg/L and volumes
between 5 and 10 mL.
The evaluation criteria were based in the oxidant-anti-
oxidant status and in the clinical evolution of patients with
knee osteoarthritis. For the evaluation of the oxidant-anti-
oxidant status, 21 patients treated with ozone and a sam-
ple of 10 healthy volunteers (control group), 1–2 mL of
synovial ﬂuid was extracted, at the beginning and at the end
of the study, for the measurement of different parameters
related to oxidative stress, as: superoxide dismutase (SOD),
catalase (CAT), CAT/SOD ratio, reduced glutathione (GSH),
malondialdehyde (MDA), advanced oxidation protein prod-
ucts (AOPP), total hydroperoxides (ROOH) and peroxidation
For the clinical evolution, the pain, the evaluation of
the joint capacity and the imaging studies performed in all
patients were considered, at the beginning and at the end of
•For the pain measurement, the visual analog scale
(VAS) was used (10-maximum of pain and 0-no
•For the evaluation of joint capacity, four aspects
were taken into account: (1) Articular movements
(movements of ﬂexion and complete extension),
classifying the patients into 3 categories, accord-
ing to the movement limitation: null (between
0 and 10%), partial (between 11 and 65 %) and
total (between 65 and 100%); (2) Presence of
clover sign: In the knee appear 3 bulky zones in
form of a clover due to the presence of adipo-
sis; (3) Grade of tumefaction (swelling): severe,
moderate or slight; and, (4) Presence of palpable
•In respect to the imaging studies, they were
made by means of the ultrasound diagnose, at
470 J.L. Calunga et al. November–December 2012
the beginning and one month after the end of the
treatment. The radiologist classiﬁed the patient
synovitis into three levels: slight (scarce quan-
tity of synovial ﬂuid localized in the suprapatellar
region), moderate (when the synovial ﬂuid covers
the articular capsule in its external and internal
parts) and severe (when the synovial ﬂuid cov-
ers all the articular spaces with swelling of the
articular capsule that extends to the popliteal fossa
and prepatellar cavity).
All biochemical parameters were determined by
spectrophotometric methods using an Ultrospect Plus
Spectrophotometer from Pharmacia LKB, Sweden. CAT
activity was measured by following the decomposition of
hydrogen peroxide at 240 nm at 10 sec intervals for 1 min
(Boehringer 1987). SOD was measured using kits supplied
by Randox Laboratories Ltd., Ireland. Concentrations of
MDA were analyzed using the LPO-586 kit obtained from
Calbiochem (La Jolla, CA). In the assay, the production
of a stable chromophore, after 40 min of incubation at
45 oC, was measured at 586 nm. For standards, freshly
prepared solutions of malondialdehyde bis [dimethyl acetal]
(Sigma, St. Louis, MO, USA) were employed and assayed
under identical conditions (Esterbauer and Cheeseman,
Quantiﬁcation of ROOH was measured by Bioxytech
H2O2–560 kit (Oxis International Inc., Portland, OR, USA)
using xylenol orange to form a stable colored complex, which
can be measured at 560 nm. Total protein concentration was
determined by the method of Bradford with bovine serum
albumin as standard (Bradford 1976). PP was measured by
inducing lipid peroxidation by adding Cu+(2 mM) to serum
(incubated for 24 h at 37 oC). The difference between mal-
ondialdehyde levels, measured at 0 and 24 h after induction,
for each sample, was calculated (Özdermirler et al. 1995).
After precipitation of thiol proteins using trichloroacetic
acid 10%, GSH was measured according to the method
of Sedlak and Lindsay (1968), with Ellman’s reagent [55
dithiobis (2-nitrobenzoic acid) 10-2M (Sigma, St. Louis, MO,
USA). AOPP was measured as the oxidation of iodide anion
to diatomic iodine by advanced oxidation protein products
(Witko-Sarsat et al. 1998).
The OUTLIERS preliminary test for detection of error val-
ues was initially applied. Afterwards, data were analyzed by
one-way analysis of variance (ANOVA) followed by a homo-
geneity variance test (Bartlett-Box). In addition, the Wilcoxon
rank-sum test (Dalle-Donne 2006) and Student’s t-tests were
performed. The data were expressed as mean ±SD (stan-
dard deviation). The level of statistical signiﬁcance used was
RESULTS AND DISCUSSION
Reactive oxygen species (ROS) are involved in both bone
and cartilage physiology and play an important role in the
pathogenesis of osteoarthritis (Baur et al. 2011; Joki´
2010). ROS produced by abnormal chondrocyte metabolism
exceeds the physiological buffering capacity and results in
oxidative stress, causing cartilage degradation directly by
cleaving collagen and activating matrix metalloproteinases
(Klämfeldt and Marklund 1987). The excessive production of
ROS can damage proteins, lipids, nucleic acids, and matrix
components. They also serve as important intracellular sig-
naling molecules that amplify the inﬂammatory response
(Sutipornpalangkul et al. 2009a). Similar results of elevated
ROS levels have been reported in patients with rheumatic
disease (Mezes and Bartosiewicz 1983).
In this study, patients with knee osteoarthritis presented
a remarkable oxidative stress in synovial ﬂuid (high values
of AOPP and ROOH, as well as consumption of GSH) with
negative effects upon joint recovery. The mean ±SD of the
redox balance parameters, measured in synovial ﬂuid in both
groups, is shown in Table 1.
AOPP is an indicator of protein oxidative damage and pre-
cursor of advanced glycation end products (AGEs). Rise in
AOPP and ROOH could be due to the increased generation of
ROS and consequent excessive oxidative damage generated
in these patients, affecting the functions of bonding, trans-
portation and protein structure (García Villanova 1994). These
ROS, in turn, can oxidize many other important biomolecules,
including membrane lipids. It was demonstrated that at the
end of the ozone combined treatment, AOPP and ROOH levels
decreased signiﬁcantly in respect to the initial values, obtain-
ing for ROOH, similar ﬁgures to that of the control group,
which suggest the preservation of membrane integrity.
GSH presented signiﬁcant differences at the end of the
treatment, with values more similar to those of the con-
trol group. The level of erythrocyte GSH was signiﬁcantly
decreased in patients with osteoarthritis compared to controls,
at the beginning of the treatment. At the end of the study, even
maintaining a signiﬁcant low level in respect to the control
group, it had increased signiﬁcantly in respect to its initial one,
suggesting an increased defense against oxidant damage in
osteoarthritis. The decrease in the level of this nonenzymatic
anti-oxidant parameter may be due to the increased turnover
for preventing oxidative damage in these patients (Surapaneni
and Venkataramana 2007).
SOD activity had increased signiﬁcantly in synovial ﬂuid
of patients with osteoarthritis. Similar results of raised SOD
activity had been reported in patients with osteoarthritis
and rheumatic diseases (Mezes and Bartosiewicz 1983;
Surapaneni and Venkataramana 2007). SOD is an impor-
tant anti-oxidant enzyme having an antitoxic effect against
superoxide anion and to counter the effect of increased oxida-
tive stress. The over-expression of SOD might be an adaptive
response, and it results in increased dismutation of superoxide
to hydrogen peroxide. However, at the end of the treatment, its
Ozone Therapy in Patients with Knee Osteoarthritis November–December 2012 471
TABLE 1. Results of the Redox Balance Parameters Measured in Synovial Fluid after the Combined
Ozone Treatment (Rectal and Intra-articular Applications)
Ozone (n =21)
MDA (µM) 3.35 ±0.17 2.6 ±0.9 3.07 ±0.89
PP (µM) 7.72 ±1.17 5.4 ±1.5 6.02 ±1.70
CAT (U/L/min) 3810 ±156 3069 ±289 3668 ±307
SOD (U/L/min) 4.86 ±1.35a22. 9 ±7.5b12.5 ±3.8c
AOPP (µM) 2.50 ±0.70a21.8 ±6.3b11.3 ±2.4c
ROOH (µM) 33.9 ±11.5a64.5 ±15.5b40.7 ±10.7a
GSH (mg/L) 570.2 ±37.2a48.9 ±17.0b268.3 ±44.0c
Note: Superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), malondialdehyde (MDA),
advanced oxidation protein products (AOPP), total hydroperoxides (ROOH), and peroxidation potential (PP).
Different letters indicate signiﬁcant differences, p <0.05. Different letters indicate signiﬁcant differences.
level decreased signiﬁcantly, obtaining values more similar to
those of the control group, indicative of a better anti-oxidant
balance and therefore, a modulation of redox-controlled sig-
nalling pathways. Perhaps this can be an attempt to reduce
the expression of collagenases, enzymes that contribute to
cartilage degradation. Ostalowska et al. (2006) have also
reported increased activities of SOD, glutathione peroxidase
and glutathione reductase in synovial ﬂuid of patients with
primary and secondary osteoarthritis of the knee joint.
No signiﬁcant differences were observed in MDA, PP
and CAT in respect to the different groups, concentrations
were maintained to control levels. In synovial ﬂuid from
primary knee osteoarthritis patients with severe cartilage
damage, undergoing total knee replacement, in comparison
with those in the synovial ﬂuid from injured knee joint
patients with intact cartilage undergoing knee arthroscopy,
no signiﬁcant differences in MDA levels were also achieved
(Sutipornpalangkul et al. 2009a). Similar results, regarding
MDA and CAT levels, were reported by Ostałowska in his
knee posttraumatic arthritis patient study (Ostałowska et al.
The CAT/SOD ratio, which presented signiﬁcant
decreased value at the beginning of the treatment in compar-
ison with the control group, increased signiﬁcantly its level at
the end of the study, with values more similar to those of the
control, but with still signiﬁcant differences. This ratio can be
considered as a risk factor in the development of complica-
tions. All these suggest a regulation cellular redox balance.
With respect to pain evaluation, the results in the visual
analog scale (VAS) after 20 sessions of ozone by rectal
insufﬂation (administered daily) and the 15 sessions of intra-
articular ozone applications (twice per week) are shown in
Figure 1. At the beginning of the study, the mean value was in
9 according to the VAS; however, at the end of the treatment,
it diminished up to 1, demonstrating a signiﬁcant decrease of
pain with the application of the combined ozone treatment.
Initial 5 sessions 10 sessions 15 sessions
FIGURE 1. Pain evolution according to the Visual Analogous
Scale (VAS), after 15 sessions (twice per week) of ozone by
intra-articular knee application. Those patients have also received
20 sessions of rectal ozone administration (one daily).
This result was maintained in 80% of cases in 1-year
follow-up. It was also demonstrated that ozone administered
by rectal application favored the effect of intra-articular ozone
injection, improving the clinical symptoms, although this pro-
cedure needed more ozone sessions, but is the least invasive
and cheapest method.
The results of the evaluation of the joint capacity, accord-
ing to the limitation of articular movements and grade of
tumefaction, at the beginning and at the end of the study, are
shown in Table 2.
In respect to limitation of articular movements, all the
patients that presented a total limitation at the beginning of the
study improved signiﬁcantly their condition after the ozone
treatment, achieving 57% of patients without limitations in
their articular movements. Similar results were obtained in
respect to grade of tumefaction. At the end of the study, no
severe grade of tumefaction was observed, 50% of patients
472 J.L. Calunga et al. November–December 2012
TABLE 2. Evaluation of the Joint Capacity, According to the Limitation of Articular Movements and Grade of Tumefaction, at the
Beginning and at the End of the Study
Evaluation of the Joint Capacity Beginning of the Study End of the Study
Limitation of articular
Total Partial Null Total Partial Null
20 75 5 0 43 57
Grade of tumefaction (%) Severe Moderate Slight Severe Moderate Slight
35 55 10 0 5 45
TABLE 3. Results of the Radiological Study, at the Beginning and One Month after Finishing the Ozone Therapy Treatment
Beginning of the Study End of the Study
Radiological Evaluation Severe Moderate Slight Severe Moderate Slight
Presence of synovitis (%) 40 55 5 0 15 85
presented moderate or slight tumefaction, and in the other
50% of patients, no signs of tumefaction were observed in the
articular clinical exam.
The presence of clover sign and crepitations were also
improved at the end of ozone therapy. At the beginning of
the study, clover sign and crepitations were observed in 77%
and 85% of patients, respectively; however, at the end of the
treatment, they were seen only in 5%.
The results of the radiological studies performed one
month after ﬁnishing ozone therapy are shown in Table 3.
For all the patients that presented a severe synovitis, it dis-
appeared after the ozone therapy treatment, achieving 85% of
patients with only a slight synovitis remaining. A remarkable
improvement has been demonstrated in the clinical evolution
of patients with knee osteoarthritis. Patients presented less
disability, increasing their quality of life.
An understanding of oxidative stress involved in this dis-
ease might allow the use of anti-oxidant therapies in the
prevention and/or treatment of knee osteoarthritis (Afonso
et al. 2007). Ex vivo SOD3 gene transfer or SOD mimetics
(e.g., M40403) can reduce the severity of collagen-induced
arthritis in models (Cuzzocrea et al. 2005a, 2005b) and recom-
binant SOD1 can inhibit cartilage damage in hens. A trial
of intra-articular injections of bovine SOD1 (Orgotein) has
also been carried out in patients with OA with some success;
however, the drug was withdrawn, due to adverse side effects
(Afonso et al. 2007). As dietary supplements, vitamins have
been shown to decrease OA development and increase the
expression of anti-oxidant enzymes in an OA model (Afonso
et al. 2007; Sutipornpalangkul et al. 2009b).
However, epidemiological studies examining the beneﬁts
of anti-oxidants in human OA, especially vitamin E (α-
tocopherol), are contradictory (Henrotin et al. 2005). Also, the
failure of conventional treatments (analgesics or nonsteroidal
anti-inﬂammatory drugs) to satisfactorily control OA progres-
sion, combined with their frequent adverse side effects, justify
to recommend the use of ozone therapy in the treatment of
patients suffering of knee osteoarthritis. Ozone therapy was
able to control the oxidative stress achieving an intra-articular
redox balance and clinically, it reduced pain, decreased patient
disability and increased their quality of life, without the
presence of side effects.
The results of our study demonstrated higher oxygen-free
radical production in patients with knee osteoarthritis, as it
has been seen in the increased values of total hydroperox-
ides and advanced oxidation protein products, as well as a
decreased reduced glutathione level, obtained at the beginning
of the study, supporting the higher oxidative stress hypothesis
in osteoarthritis. This situation can contribute to the complica-
tions and progression of the disease. Combined ozone therapy
diminished the oxidative stress, achieving an intra-articular
redox balance, as well as a signiﬁcant reduction of pain, with a
maintained satisfactory response in 80% of patients in 1-year
follow-up. An increase in the quality of life of patients with
knee osteoarthritis was observed, without the presence of side
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