Introduction

Osteoarthritis (OA) is a common degenerative joint disease characterized by pain, stiffness, swelling, and limited joint motion. It is the most prevalent form of arthritis and a major cause of chronic musculoskeletal pain and disability, particularly in older adults.1,2 Among weight-bearing joints, the knee is one of the most frequently affected sites.3,4 Knee OA is a multifactorial disorder influenced by mechanical forces and cellular and biochemical processes, and it commonly leads to pain, reduced range of motion, and functional limitation.5 Its prevalence continues to increase worldwide with age and obesity, and although it is more common after the age of 45 years, it may also occur in younger individuals.4

The pathophysiology of OA involves progressive degeneration of articular cartilage, subchondral bone remodeling, osteophyte formation, synovial inflammation, and periarticular soft tissue changes.6,7 These structural and inflammatory processes contribute to pain and progressive loss of function.8 In addition to radiographic changes, OA is associated with biochemical and inflammatory alterations, including increased levels of proinflammatory cytokines and oxidative stress, which may contribute to cartilage damage and disease progression.9,10

Current treatment of knee OA includes non-pharmacological approaches such as exercise, weight reduction, physical therapy, and rehabilitation, as well as pharmacological options including analgesics and non-steroidal anti-inflammatory drugs.11,12 More recently, technology-assisted rehabilitation approaches, such as virtual reality-based exercise programs, have also been investigated and shown to improve pain, WOMAC scores, physical function, balance, and quality of life in patients with symptomatic knee osteoarthritis.13 In advanced cases, total knee arthroplasty may be considered, although surgery may be postponed in some patients because of prosthesis lifespan and revision-related concerns.14,15 In addition, intra-articular treatments such as corticosteroids, hyaluronic acid, platelet-rich plasma, and ozone have been used to control symptoms with varying levels of success.16

Ozone is a triatomic form of oxygen that has been proposed to exert therapeutic effects through modulation of oxidative stress, stimulation of antioxidant enzyme systems, inhibition of inflammatory mediators, and improvement of tissue oxygenation.17–20 Recent studies and meta-analyses suggest that intra-articular ozone therapy may reduce pain and improve function in patients with knee OA.21–23 However, the optimal concentration for intra-articular ozone applications remains unclear, and considerable heterogeneity exists in dosing strategies, injection volumes, number of sessions, and treatment intervals across studies.23–25 Moreover, a recent dose-comparison randomized trial directly comparing 20 and 40 μg/mL intra-articular ozone concentrations reported no significant superiority of the higher concentration, suggesting that the dose–response relationship of intra-articular ozone in knee OA remains debated.25 Determining whether lower ozone concentrations provide comparable clinical benefit is important for treatment optimization and may have implications for both efficacy and safety. Therefore, this study aimed to evaluate the effects of different intra-articular ozone concentrations on pain, stiffness, physical function, and exercise capacity in patients with knee osteoarthritis.

The primary hypothesis of this study was that different intra-articular treatment concentrations would result in comparable clinical and functional outcomes in patients with knee osteoarthritis. Given the retrospective and non-randomized nature of the study, we did not prespecify a hypothesis that any particular concentration would be superior to the others. Therefore, the present study aimed to explore whether clinically meaningful differences in pain, function, and physical performance could be observed among patients treated with different concentrations in routine clinical practice.

Materials and MethodsStudy Design

This study was designed as a retrospective comparative study. The study protocol was approved by the Research Ethics Committee of Medipol University (Institutional Review Board approval no: 862; date of approval: 14.05.2026). All procedures were conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent for the intra-articular ozone injection procedure had been obtained from all patients as part of routine clinical practice before treatment. Because this study was based on anonymized retrospective medical record data and no additional intervention was performed for research purposes, the requirement for study-specific informed consent to participate was waived by the ethics committee.

Medical records of patients with knee osteoarthritis who underwent intra-articular ozone treatment between January 2026 and April 2026 were retrospectively reviewed at İstanbul Aydın University Medical Park Florya Hospital. The inclusion and exclusion criteria are presented in Box 1.

Box 1 Inclusion and Exclusion Criteria

Medical records of 52 patients were screened for eligibility. Of these, 7 patients were excluded due to not meeting the inclusion criteria (n=4) or incomplete clinical data (n=3). The remaining 45 patients were included in the final analysis (Figure 1).

Figure 1 Flow diagram of patient selection and grouping.

Sample Size

Because this was a retrospective, non-randomized observational study based on patients treated during the predefined study period, no a priori sample size or statistical power calculation was performed. The sample size was determined by the number of eligible patients who met the predefined inclusion and exclusion criteria during the study period.

Dose Selection and Group Allocation

This study was retrospective and non-randomized in design. The ozone concentration administered to each patient was determined by the treating physician as part of routine clinical practice, based on the patient’s clinical presentation, rather than through a predefined randomization or dose-finding protocol. Patients were subsequently and retrospectively categorized into the 1, 5, and 10 μg/mL groups according to the ozone concentration documented in their medical records. Because group allocation was not randomized, the possibility of confounding by indication — that is, systematic differences between groups related to the physician’s dosing decisions rather than to the treatment itself — cannot be excluded and is addressed further in the Limitations section.

Groups

Patients were classified into three groups according to the concentration of intra-articular ozone administered: 1, 5, and 10 μg/mL, as described above. Each group consisted of 15 patients. At our center, the treating physician has favored ozone concentrations at the lower end of the clinically used range, based on the rationale that low-dose ozone may induce a mild oxidative stimulus capable of activating endogenous antioxidant defense mechanisms, whereas higher concentrations may provoke excessive oxidative stress without additional therapeutic benefit — a hormetic dose–response relationship that has been proposed for ozone therapy.20 These concentrations are lower than those used in many previously published intra-articular ozone trials, which have typically ranged from approximately 15 to 40 μg/mL.17,22,24,25,28,29 Therefore, the present study aimed to compare whether low-dose ozone concentrations provide comparable clinical benefits in patients with knee osteoarthritis.

Treatment Protocol

All patients had received intra-articular ozone injections once weekly for 3 consecutive weeks. The treated knee was selected based on the presence of greater pain and functional limitation. In each session, a medical ozone–oxygen mixture had been injected intra-articularly according to the treatment protocol documented in the medical records: 5 mL at a concentration of 1 μg/mL in the 1 μg/mL group, 5 mL at a concentration of 5 μg/mL in the 5 μg/mL group, and 5 mL at a concentration of 10 μg/mL in the 10 μg/mL group. The injection volume was standardized at 5 mL in all three groups, and the treatment groups differed only in ozone concentration. All injections had been performed by the same physician (A.D.Y.) using a standardized technique.

The procedure was performed with the patient in a seated position and the knee flexed, under aseptic conditions. The entry point was identified at the femorotibial joint line, 1.5 cm medial to the patellar tendon and 1.5 cm below the apex of the patella. Ozone was injected intra-articularly using a standard anteromedial approach after confirming correct needle placement. Adverse events were retrospectively screened from medical records, including post-injection pain exacerbation, swelling, erythema, infection, vasovagal reaction, allergic reaction, and any other procedure-related complication.

Concomitant Treatments

At our center, all patients undergoing intra-articular ozone treatment for knee osteoarthritis routinely receive standardized home-based knee exercise recommendations and weight management counseling as part of usual clinical care, and none of the included patients received formal physical therapy during the study period. Analgesic use was permitted according to individual clinical need, but detailed information on the type, dose, and frequency of analgesic use was not systematically recorded in the medical records and therefore could not be reliably compared between the treatment groups. This lack of detailed analgesic-use data represents a potential source of confounding and should be considered as a limitation of this retrospective study.

Outcome Measurements

The primary outcome measure was pain intensity assessed using the Visual Analogue Scale (VAS). Secondary outcome measures included WOMAC pain, stiffness, physical function, and total scores, as well as walking capacity assessed using the 2-minute walking test (2MWT). All outcomes were recorded at baseline and at week 4.

Demographic and Clinical Characteristics

Age, sex, height, weight, body mass index (BMI), Kellgren–Lawrence grade, and the affected knee were recorded from the medical records at baseline.

Pain Assessment

Pain intensity was extracted from medical records and had been assessed using the Visual Analogue Scale (VAS) as part of routine clinical evaluation. Patients rated their knee pain on a 0–10 scale, where 0 indicated no pain and 10 indicated the worst imaginable pain. Higher scores indicated greater pain intensity.30

Functional Status

Functional status was obtained from medical records and had been assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) as part of routine clinical evaluation. The WOMAC consists of subscales evaluating pain, stiffness, and physical function in daily activities. The total score ranges from 0 to 96, with higher scores indicating worse symptoms.31

Exercise Capacity

Walking capacity was extracted from medical records and had been assessed using the 2-minute walking test (2MWT) as part of routine clinical evaluation. In this test, patients were instructed to walk as far as possible within 2 minutes in a designated walking area, and the total distance walked was recorded in meters. Higher distances indicated better walking capacity.32

Statistical Analysis

All statistical analyses were performed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expressed as mean ± standard deviation or median (minimum–maximum), as appropriate, and categorical variables as frequency (percentage).

The normality of data distribution was assessed using the Shapiro–Wilk test. Normally distributed variables (age, height, weight, body mass index, WOMAC total score, WOMAC physical function score, and 2-minute walking test results) were analyzed using two-way repeated-measures analysis of variance (ANOVA). For pairwise comparisons, the Least Significant Difference (LSD) test was used. Non-normally distributed variables (VAS score, WOMAC pain score, and WOMAC stiffness score) were analyzed using the Wilcoxon signed-rank test for within-group comparisons. Between-group comparisons of these variables were performed using the Kruskal–Wallis test based on change scores between measurements. Categorical variables were analyzed using the chi-square test. A p-value of <0.05 was considered statistically significant. To complement p-values with estimates of magnitude, between-group comparisons were further characterized using effect sizes (η2 for parametric outcomes; epsilon-squared for non-parametric outcomes). For the 2-minute walking test, where the overall between-group comparison was statistically significant, pairwise post hoc differences in change scores were expressed as mean differences with 95% confidence intervals derived from the Least Significant Difference (LSD) test. For outcomes with a non-significant overall between-group comparison, pairwise post hoc contrasts were not performed, in order to avoid inflating the type I error rate through additional unprotected comparisons.

Results

As described in the Methods (Figure 1), 45 patients met the eligibility criteria and were included in the final analysis, with 15 patients in each treatment group. No adverse events, including post-injection swelling, erythema, infection, vasovagal reaction, allergic reaction, or other procedure-related complications, were documented in any of the treatment groups during the study period.

Baseline demographic and clinical characteristics were similar across the treatment groups (Table 1). No statistically significant differences were observed among the groups in terms of age, height, weight, body mass index, sex distribution, or Kellgren–Lawrence grade (p>0.05 for all).

Table 1 Demographic and Clinical Characteristics of the Groups

Clinical outcomes at baseline and week 4 are presented in Table 2. Between-group comparisons of change scores showed no statistically significant differences for VAS (H=3.15, p=0.207, ε2=0.03), WOMAC pain (H=0.84, p=0.658, ε2<0.01), WOMAC stiffness (H=5.62, p=0.060, ε2=0.09), WOMAC physical function (F=1.11, p=0.338, η2=0.05), or WOMAC total scores (F=1.32, p=0.278, η2=0.06). For the 2-minute walking test, the between-group comparison was statistically significant (F=3.45, p=0.041, η2=0.14). Post hoc LSD comparisons indicated that the increase in walking distance was significantly greater in the 1 μg/mL group than in the 10 μg/mL group (mean difference 16.4 m, 95% CI: 3.4 to 29.3 m; p=0.015; Cohen’s d=0.87), whereas differences between the 1 μg/mL and 5 μg/mL groups (4.6 m, 95% CI: −8.4 to 17.6 m; p=0.482) and between the 5 μg/mL and 10 μg/mL groups (11.8 m, 95% CI: −1.2 to 24.8 m; p=0.074) were not statistically significant.

Table 2 Clinical Outcomes at Baseline and Week 4 in the Treatment Groups

Intragroup comparisons showed significant improvements in VAS, WOMAC pain, WOMAC physical function, and WOMAC total scores in all three treatment groups after treatment (p<0.05 for all). WOMAC stiffness scores improved numerically in all three groups, but this improvement reached statistical significance only in the 5 μg/mL group (p=0.006), while the improvements in the 1 μg/mL (p=0.054) and 10 μg/mL (p=0.078) groups did not reach statistical significance. In the 2-minute walking test, significant within-group improvement was observed in the 1 μg/mL and 5 μg/mL groups (p<0.05), whereas no significant change was found in the 10 μg/mL group (p=0.459).

The distribution of WOMAC total scores across treatment groups is presented in Figure 2.

Figure 2 Distribution of WOMAC total scores before and after treatment across the study groups.

The distribution of 2-minute walking test distances across treatment groups is presented in Figure 3.

Figure 3 Distribution of 2-minute walking test distances at baseline and week 4 across the treatment groups.

Discussion

The present study compared the short-term clinical outcomes of three different low-dose intra-articular ozone concentrations in patients with Kellgren–Lawrence grade II–III knee osteoarthritis. The principal finding was that all three treatment protocols resulted in significant improvements in pain intensity, physical function, and overall WOMAC scores after three weekly injections. However, no significant between-group differences were observed for most clinical outcomes, suggesting that increasing the ozone concentration within the evaluated range did not provide additional clinical benefit. Interestingly, patients treated with the lowest concentration protocol demonstrated greater improvement in walking capacity than those receiving the highest concentration protocol.

Pain relief remains the primary therapeutic goal in the management of knee osteoarthritis because pain is closely associated with disability, reduced physical activity, and impaired quality of life. Ozone therapy has attracted increasing attention as a minimally invasive treatment for chronic diseases such as OA, where limited benefit is often achieved with conventional pharmacological therapy, owing to its proposed analgesic, anti-inflammatory, and antioxidant properties.33,34 Ozone has been reported to provide clinically relevant benefits in reducing joint inflammation and relieving pain, stiffness, and physical limitation in patients with OA.33,34

The improvements in VAS and WOMAC scores observed in the present study are consistent with previous randomized controlled trials and recent systematic reviews demonstrating that intra-articular ozone therapy reduces pain and improves function in patients with knee osteoarthritis.14,17,18,28,29,35–40 However, unlike most previous investigations, our study directly compared three low-dose ozone concentrations used in routine clinical practice. The absence of clinically meaningful differences between protocols suggests that lower ozone concentrations may achieve analgesic effects comparable to those of higher concentrations, at least in the short term.

To assess the clinical relevance of these findings, we compared the observed mean changes with previously reported minimal important change (MIC) or minimal important difference (MID) thresholds for knee osteoarthritis outcome measures, converted to the scales used in the present study.41 For VAS pain (reported MIC ≈2.0 points on a 0–10 scale), the mean reduction exceeded this threshold in all three treatment groups. For the WOMAC pain subscale (reported MIC ≈3.4 points on a 0–20 scale), the mean improvement exceeded this threshold in the 5 and 10 μg/mL groups, while the 1 μg/mL group approached but did not reach it. For WOMAC physical function (reported MIC ≈11.6 points on a 0–68 scale), only the 5 μg/mL group exceeded this threshold. For the WOMAC total score (reported MID ≈6.5 points on a 0–96 scale), the mean improvement exceeded this threshold in all three groups. For the 2-minute walking test, the mean improvement exceeded the previously reported minimum detectable change (5.52 m)42 in the 1 μg/mL (19.8 m) and 5 μg/mL (15.2 m) groups, whereas the mean improvement in the 10 μg/mL group (3.4 m) did not exceed this threshold, suggesting that only the two lower-dose groups achieved a change likely to reflect a genuine improvement in walking capacity beyond measurement error. These comparisons should be interpreted cautiously, as published thresholds vary across studies and populations and were not derived from the specific cohort or ozone treatment context of the present study.

In the two-minute walking test after treatment, walking distance was significantly higher in the groups receiving doses of 1 and 5 μg/mL treatments compared to pretreatment values. Walking performance is an objective indicator of functional mobility and reflects the patient’s ability to perform daily activities beyond self-reported symptom scores. Although the mechanism underlying this finding cannot be established in a retrospective study, it may support the concept of ozone hormesis. According to this hypothesis, low ozone doses induce a mild oxidative stimulus that activates endogenous antioxidant pathways and adaptive cellular responses, whereas excessive oxidative stimulation may not provide additional therapeutic benefit. This mechanism remains speculative, as oxidative stress, antioxidant activity, and inflammatory biomarkers were not measured in the present study; therefore, the hormesis hypothesis should be regarded as a possible explanation for the observed findings rather than a confirmed mechanism. Similarly, other studies have reported a decrease in pain severity14,17,18,28,29,35–40 and functional limitation14,17,28,29,35–40,43 with ozone therapy in patients with knee OA. In the study of Hashemi et al, it was reported that VAS scores decreased after ozone treatment but WOMAC scores increased in contrast to other studies in the literature.35 According to the results of recent meta-analysis, intraarticular ozone treatment in knee OA patients has been reported to reduce pain and improve functionality.44–46

The significant finding observed in the 2-minute walking test should be interpreted cautiously. Given the relatively small sample size within each treatment group (n=15) and the number of outcomes and between-group comparisons performed, the possibility of a chance finding cannot be excluded. In addition, unlike VAS and WOMAC, which primarily reflect joint-specific pain and function, the 2-minute walking test was originally developed and validated as a measure of exercise capacity in respiratory disease and is influenced by cardiopulmonary fitness in addition to musculoskeletal factors.32 Because cardiopulmonary status was not formally assessed or compared between groups in this retrospective study, undetected between-group differences in cardiopulmonary function cannot be excluded as a contributing factor to this finding. Therefore, this result should be considered exploratory and hypothesis-generating rather than definitive evidence of a superior effect of the lower dose. Larger, adequately powered prospective randomized studies that also account for cardiopulmonary status are needed to determine whether the observed difference in walking performance is reproducible and whether dose-related differences in functional outcomes truly exist.

There are studies that report a decrease in the WOMAC Stiffness score in patients with knee OA after ozone treatment.14,17,28,29,36 In a study by Jesus et al, it was reported that WOMAC stiffness score did not show a significant change at the 8th week after ozone treatment in knee OA patients, but showed a significant decrease at the 16th week.17 In this study, after treatment, WOMAC Stiffness score was found to be significantly lower in the group receiving 5 μg/mL compared to pre-treatment results. If patients could have been followed for a longer duration, we may have been able to determine long-term alterations in this score.

Under various conditions, ozone treatment at different concentrations is recommended. For example, ozone treatment for lumbar disc herniation has been recommended with 27 μg/mL ozone, while knee osteoarthritis and degenerative diseases are suggested to benefit from a periarticular dose of 3–11 μg/mL and an intraarticular dose of 30–40 μg/mL. However, non-toxic concentrations have been reported to range from 1–40 μg/mL.40 In a study conducted by Yu et al in rats, it has been proved that excess ozone concentration is toxic. They reported that 35 μg/mL of ozone prevented cartilage damage in the joint and decreased free radicals, but 70 μg/mL ozone produced a peroxidatic reaction in tissue.47 Some previous studies investigating the effect of ozone on knee OA have used ozone at a concentration of 15–40 μg/mL.17,18,29,39,40 In this study, we used ozone at a concentration of 1, 5 and 10 μg/mL and found that knee OA symptoms were reduced in all three groups and there was no significant difference between the groups except for the 2-minute walking test. Similarly, Camelia et al used ozone at a concentration of 2–5 μg/mL and found it to be effective in knee OA.28 From a clinical perspective, our findings suggest that increasing ozone concentration should not automatically be considered synonymous with greater therapeutic efficacy. If lower concentration protocols provide comparable improvements in pain and physical function, clinicians may achieve satisfactory outcomes while minimizing unnecessary ozone exposure.

This finding may be related to the hormetic effect of ozone, in which low concentrations stimulate antioxidant defense mechanisms, whereas higher concentrations may induce excessive oxidative stress and reduce therapeutic benefit. Although the present study was not designed to evaluate cost-effectiveness or long-term safety, these findings may contribute to the optimization of intra-articular ozone treatment strategies in clinical practice. The findings suggest that lower ozone concentrations may achieve comparable pain relief and functional improvement while potentially minimizing unnecessary ozone exposure.

Several methodological limitations should be considered when interpreting these findings. Because dose selection was based on the treating physician’s clinical judgment rather than randomization, unmeasured confounding by indication cannot be excluded, and the treatment groups may have differed with respect to baseline clinical characteristics or physician-related treatment selection factors. Although standardized exercise recommendations and weight management counseling were provided to all patients as part of routine care, and no patient received formal physical therapy during the study period, detailed information on analgesic use could not be assessed and may have influenced the observed outcomes. Furthermore, the absence of a placebo or standard-treatment control group precludes any conclusion regarding the specific contribution of ozone therapy itself, as opposed to natural symptom fluctuation, regression to the mean, or nonspecific treatment effects.

Strengths This study is one of the few studies to compare the clinical effects of different concentrations of intra-articular ozone in patients with knee osteoarthritis. Both subjective and objective outcome measures, including VAS, WOMAC, and the 2-minute walking test, were used to evaluate treatment response. All injections were performed by the same physician using a standardized technique, which may have reduced procedural variability. Limitations The relatively small sample size may have limited the ability to detect subtle differences between treatment groups. The follow-up period was limited to 4 weeks, and therefore only short-term outcomes could be evaluated. Because of the retrospective study design, causal inferences regarding the comparative effects of different ozone concentrations should be made with caution. Longer follow-up may help clarify the efficacy of different ozone concentrations in knee osteoarthritis in both the short and long term. In addition, although exercise and weight management counseling were provided uniformly to all patients and no patient received formal physical therapy during the study period, detailed information on analgesic use was not systematically recorded, representing a further potential source of unmeasured confounding. The absence of an a priori sample size and power calculation, together with the relatively small number of patients in each treatment group, limits the statistical power of the study to detect potentially meaningful between-group differences. Accordingly, the findings should be considered exploratory and require confirmation in larger, prospectively designed studies. Conclusion

Low-dose intra-articular ozone therapy was associated with short-term improvements in pain, physical function, and overall clinical status in patients with Kellgren–Lawrence grade II–III knee osteoarthritis. Different ozone concentrations were associated with generally comparable clinical outcomes, indicating that increasing ozone concentration within the evaluated range did not confer additional clinical benefit. Although a significant between-group difference favoring the lowest concentration protocol was observed in the 2-minute walking test, this finding should be interpreted cautiously given the small sample size and the number of comparisons performed, and is considered exploratory rather than evidence that lower doses are preferable. Because of the retrospective, non-randomized design, causal inferences regarding the comparative effects of different ozone concentrations cannot be established from this study. Future well-designed, prospective randomized controlled trials with standardized treatment protocols, larger sample sizes, and longer follow-up are needed to determine the optimal ozone concentration and confirm these preliminary findings.

Data Sharing Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request, subject to ethical and privacy restrictions.

Ethics Approval and Consent to Participate

This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Research Ethics Committee of Medipol University (approval no: 862; date of approval: 14.05.2026). The approving ethics committee differs from the authors’ current institutional affiliations because the ethics application was submitted by the co-author Arzu Dinç Yavaş, who was listed as the responsible investigator in the ethics approval file. The application was processed through the Research Ethics Committee of Medipol University rather than a committee directly affiliated with the treatment site because this committee offered a more regular meeting schedule for non-interventional clinical research applications and was considered suitable for the retrospective, non-interventional design of the present study. The manuscript lists the authors’ current institutional affiliations; therefore, the difference reflects the ethics application and approval process rather than the institution at which the authors are currently affiliated.

Written informed consent for the intra-articular ozone injection procedure had been obtained from all patients as part of routine clinical practice before treatment. Because of the retrospective design of the study and the use of anonymized medical record data, the requirement for study-specific informed consent to participate was waived by the ethics committee.

Acknowledgments

The authors are grateful to the patients whose medical records contributed to this study.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

No funding was received for this study.

Disclosure

The authors report no conflicts of interest in this work.

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