Intra-articular injections of oxygen-ozone versus hyaluronic acid for the treatment of knee osteoarthritis: A randomized controlled trial

Abstract

Objective

The purpose of this paper was to evaluate the efficacy of intra-articular (AI) injections of Hyaluronic Acid (HA) versus Oxygen-Ozone (O₂O₃) in relieving pain and reducing disability in patients affected by knee osteoarthritis (KOA).

Methods

People with painful KOA for at least three months were randomly allocated to receive three IA injections, once a week, of HA (Group A) vs O₂O₃ (Group B). They were evaluated at baseline (T0), at 1 (T1), 3 (T2), 6 (T3), and 12 months (T4) after the treatment, using as outcomes: WOMAC pain score, WOMAC LK 3.1, Numeric Rating Scale (NRS), and KOOS.

Results

Of the 122 patients assessed for eligibility, 112 participants were enrolled. Both groups A and B showed significant improvements in all outcomes measures from 1 month after the treatment (HA Group: 6.77 ± 2.53 vs 3.66 ± 2.57; O2O3 Group: 6.50 ± 2.24 vs 3.45 ± 2.30). In contrast, at T2 and T3, the HA-treated group showed significant pain and functional improvement compared to O₂O₃ group.

Conclusion

Both HA and O₂O₃ might be considered safe and potential effective treatments for KOA, due to their anti-inflammatory effects. O₂O₃ seems to have a faster effect; rather, HA showed superior efficacy at 3 months onwards from the end of the treatment.

Keywords

Ozone oxygen-ozone therapy hyaluronic acid knee osteoarthritis pain

Introduction

Osteoarthritis (OA) is the most common degenerative musculoskeletal disease, characterized by progressive damage to articular cartilage with osteophyte formation, subchondral bone remodeling and joints inflammation.^1,2 Although almost all joints can be affected, knee osteoarthritis (KOA) represents the most common site, resulting in joint stiffness and impaired motor and gait function.^3,4 Therefore, early diagnosis is crucial to define a tailored therapeutic intervention to provide pain relief, improve joint function, and delay surgery.^5–7 Among the many conservative approaches to treat KOA,^8,9 the use of intra-articular (IA) injections of HA has been increased in recent years,^10,11 providing protective effect of cartilage in order to recover the viscoelastic properties of synovial fluid.^12,13 Different types of HA with various weights, concentrations, dosages, and cross-links are used in clinical practice.^14,15 This leads to difficulties in interpreting the results of efficacy studies and disagreement over guidelines for use in KOA management.^5,9,16–18

Recently, there has been increasing interest in the use of IA injections of oxygen-ozone (O₂O₃) therapy in KOA for its propriety of modulating inflammation in painful conditions.^19–24 In particular, ozone (O₃) can promote the generation of reactive oxygen species (ROS) and lipid oxidation products (LOP), which are oxidative stressors that can stimulate the endogenous antioxidant system. The resulting antioxidant response may be able to counteract the overexpression of pro-inflammatory and pro-oxidative factors present in KOA.

Despite recent systematic reviews and meta-analyses^25–27 showed the safety and efficacy of this approach in patients with KOA, there is still no precise consensus due to the poor methodologic quality of the studies and the variability of the treatment protocol applied. In this context, preliminary findings from a prior project²³ showed that O₂O₃ might be more effective than HA injections to obtain pain relief in the short-term.

Therefore, the aim of the present randomized controlled trial (RCT) was to compare clinical outcomes in terms of pain relief and disability in KOA patients treated with IA injections of HA versus IA O₂O₃ administrations.

Material and methods

Participants

This RCT was accepted by the Hospital Ethics Committee and Scientific Board (Authorization n° 2556, 27 May 2020), and the study protocol was registered on clinicaltrials.gov (NCT04426721) in June 2020.

People with KOA were enrolled in the Department of Rehabilitation and Functional Recovery of the Humanitas Research Hospital, Rozzano, Milan, Italy from January 2021 to September 2022.

A physician with experience in the Rehabilitation field screened the patients for eligibility, following these inclusion criteria: (1) painful unilateral knee, characterized by chronic pain (of at least 3 months) and not responsive to at least 1 conservative treatment (e.g., physical therapy or oral analgesics); (2) pain score evaluated by the WOMAC LK 3.1 subscale of ≤19; (3) X-ray imaging findings of osteoarthritis (Kellgren–Lawrence score of 2–3).

Exclusion criteria were based on: (1) age < 18 and > 80 years old; (2) BMI >40 kg/m2; (3) knee infection or inflammatory arthropathy; (4) knee ligament injury or knee replacement; (5) knee surgery in the latest years; (6) systemic disorders, including diabetes, rheumatologic disease, coagulopathy, severe cardiovascular issues; immunodepression; (7) cognitive disorders or psychiatric disease; (8) dependence on alcohol or drugs; (9) pregnancy or lactation status; (10) previous (3 months) IA knee-injections.

This study was conducted according to the Ethical Principles for Medical Research Involving Human Subjects outlined in the Declaration of Helsinki. The patients were adequately informed about the experimental procedures and signed a written informed consent form before participating.

Study design

The participants were randomly divided as follows: Group A received 3 weekly IA injections of high-molecular-weight HA, while Group B underwent 3 weekly administrations of O₂O_3.

An independent statistician generated the Randomization list using block sizes of eight participants and stored it in a dedicated office. Treatment allocation (HA or O₂O₃) was concealed in sealed envelopes. Immediately before each injection, the treating physician contacted the office to obtain the participant's assigned treatment group. The syringe was covered to keep the patients from knowing which substance they were receiving. The treatment is based on 3 injections at 1-week intervals. Participants were subject to evaluations at baseline and at one, three, six and 12 months after the injections. Ultimate the study, patients knew the type of the injected substance.

Intervention

A Medical Doctor of our institution, with experience in IA injections, administered the therapy as follows: a sterile 32 mm (22 G) needle was used in both groups. We also performed the IA injections through a masked syringe. The physician prepared the syringe, masking it for the injection, to ensure the blinding. We maintain sterile conditions by performing double skin disinfection with 7.5% iodopovidone on the knee flexed at 90°, positioning the patient in a supine position. The injection was administered anterolaterally to the patellar tendon, positioned between the lower border of the lateral femoral condyle and the upper edge of the tibial plateau. Furthermore, no premedication or anesthesia was applied.

Hyaluronic acid injections

Patients in Group A underwent HA (Sinovial^®, IBSA Farmaceutici, Lodi, Italy) injections into the knee according to the approach described above. Sinovial^® (sodium hyaluronate) is a linear polymer of HA, composed of N-acetyl-D-glucosamine and Na-D-glucuronate units linked by glycosidic bonds. It is categorized as a medical device and contains a 0.8% physiological solution of HA (32 mg/2 mL) in sodium chloride, supplied in a sterile, ready-to-use syringe for intra-articular administration, following the current Guidelines for the use of HA visco-supplementation in KOA.²⁸ The HA is obtained through biofermentation and undergoes a rigorous purification process, in absence of chemical modification, to yield a highly purified, non-pyrogenic polymer with a defined molecular weight (800–1200 kDa), utterly devoid of animal-derived proteins. Participants were instructed to avoid weight-bearing on the treated leg and refrain from physical activity for at least 24 h following the injection. Additionally, the application of ice or other cryotherapy modalities to the affected lower limb was recommended to manage potential post-injection inflammation or discomfort. During the treatment period, they were allowed to rest or engage in moderate activities, and then gradually returned to normal sports or recreational activities.

Oxygen-ozone injections

Patients of Group B underwent to IA injections of O₂O₃, with a total volume of 10 mL and an Ozone concentration of 10 μg/mL, as the therapeutic protocol established by the International Scientific Committee of Ozone Therapy (ISCO3).²⁸ It was generated by Multiossigen Medical 99 IR generator (Multiossigen s.r.l., Gorle, Bergamo, Italy). The device used an electrochemical process to obtain a medical oxygen with a percent of 0.05% of O₃ and 99.95% of O₂, through the presence of a photometer, which was calibrated following the conventional iodometric titration of O₃ combined with a voltage-controlled system that adjusted the concentration.

Outcome measures

Patients were evaluated at baseline (T0) and then at 1 (T1), 3 (T2), 6 (T3), and 12 months (T4) following the final injection. Because the first outcome was to compare the efficacy of the two treatments, pain relief in KOA was analyzed through the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain score using the Likert scale, Version 3.1 (WOMAC LK 3.1).²⁹ In addition, secondary outcomes were measured as follows: WOMAC LK 3.1 total score, Knee injury and Osteoarthritis Outcome Score (KOOS) and Numeric Rating Scale (NRS).³⁰

The WOMAC LK 3.1 is a standardized rating scale operated to evaluate knee pain, stiffness, and function. It consists of 24 items: five related to knee pain, two to knee stiffness, and 17 to physical function. Each item is scored on a 5-point Likert scale, ranging from 0 (none or never) to 4 (extreme or always). A higher score means worse pain, greater stiffness, and significant functional impairment.

The NRS is a validated and widely accepted measure of knee pain, consisting of an 11-point Likert-type scale from 0 (“absence of pain”) to 10 (“worst pain perceived”), where participants rate their average pain over the past 48 h.

The KOOS is a knee-specific assessment tool designed to capture the patient's perspective on knee-related issues. It includes 42 items divided into five separately scored subscales: pain, other symptoms, function in daily living (ADL), function in sport and recreation (Sport/Rec), and knee-related quality of life (QOL).

Finally, we evaluated safety by recording the number of adverse events. To ensure trial blinding, all clinical assessments were conducted by an independent medical doctor who was not involved in the injection procedure.

Statistical analysis

Regarding the sample size, a mean reduction of 4.2 points (with a standard deviation of 6.5) on the WOMAC Pain scale from baseline to 3-month follow-up (primary outcome) was assumed. Therefore, with an alpha error of 5% and a power of 90%, it was necessary to include 52 patients.

It was used frequency and percentage to summarize the categorical variables and mean, median standard deviation, minimum, and maximum, for the continuous variables, depending on distribution. Adherence to Gaussian distribution was check with Shapiro Wilks test. To be certain that the randomization was correct were used: (1) two- tailed independent sample t-tests for age, (2) Mann Whitney test for BMI, baseline WOMAC Pain and pain duration, (3) Chi-square test for gender distribution.

The missing data was handled with a single imputation based on the mean, median, and mode.

The first outcome of this study was the change in the WOMAC LK 3.1 pain score (from baseline to 3-month post-treatment). Difference between the two randomization groups for the change in WOMAC LK 3.1 pain score was explored with two-tailed independent sample T-test.

The same analysis was performed for difference between randomization group in change of secondary objectives between baseline and 3-month follow up.

The selected outcome measures, WOMAC LK 3.1, KOOS, and NRS pain, were summarized and comprehensively analyzed using appropriate descriptive statistics, including error measures.

Intra-group differences were assessed through the Wilcoxon-signed rank test and the between-group differences through the Mann Whitney U test.

The significance threshold was set to 0.05. The statistical analysis of the collected data was performed using the statistical package Stata version 18 (StataCorp LLC, College Station, TX).

Results

A total of 122 participants were recruited and evaluated for eligibility, ten of them were ruled out (eight did not comply the inclusion criteria, and two declined to participate). Thus, the remaining 112 participants were enrolled and randomly assigned into the two groups of treatment of 56 patients. During the study, ten patients dropped out: four decided voluntarily to discontinue the treatment (one assigned to O₂O₃ group, three to the HA group) and six decided to undergo other analgesic treatments or did not complete the scheduled follow-up (four assigned to O₂O₃ group and two to the HA group). Therefore, a total of 102 participants, randomized into two groups, reached the endpoints of the study at 12 months (PRISMA Flow Diagram reported in Figure 1).

Figure 1.

PRISMA flow diagram.

Group A was made of 56 patients (19 male, 33.93%) who received HA injections. Group B consisted of 56 patients (29 male, 51.79%) who underwent OT. The baseline characteristics of the patients are presented in Table 1. There are not significant differences groups were homogeneous in terms of gender, age, body mass index (BMI) and pain duration.

Table 1.

Baseline characteristics of study participants.

	HA Group (Group A)	O₂O₃ Group (Group B)	P value
Number of patients	56	56
Gender (male)	19 (33.93%)	29 (51.79%)	0.056
Age (years)	63.6 ± 13.0 (min:35; max:79)	67.5 ± 12.4 (min:40; max:79)	0.112
BMI (kg/m2)	26.8 ± 4.3	27.2 ± 4.2	0.840
Pain duration (months)	24 (120–240)	38 (12–144)	0.358

Continuous variables are expressed as means ± standard deviations; categorical data are expressed as counts and percentages. Abbreviations: O₂O₃: oxygen–ozone therapy; HA: hyaluronic acid; BMI: body mass index.

Intra-group differences in outcome measures are presented in Table 2.

Table 2.

Intra-group differences in outcome measures.

	Baseline		1 month		3 months		6 months		12 months
	HA	O₂O₃	HA	O₂O₃	HA	O₂O₃	HA	O₂O₃	HA	O₂O₃
WOMAC pain subscore	6.77 ± 2.52	6.50 ± 2.24	3.66 ± 2.57	3.45 ± 2.30	2.36 ± 1.67	3.89 ± 2.16	2.98 ± 1.89	4.50 ± 2.46	5.04 ± 1.97	5.63 ± 2.25
95% CI for difference	0.27 (−0.63; 1.16)		0.21 (−0.70; 4.00)		−1.54 (−2.26; −0.81)		−1.52 (−2.38; −0.66)		−0.59 (−1.42; 0.24)
WOMAC total score	43.7 ± 12.4	44.0 ± 12.3	25.8 ± 16.9	26.3 ± 13.7	20.4 ± 15.3	27.8 ± 15.4	22.8 ± 14.3	30.3 ± 16.5	29.5 ± 12.5	37.0 ± 11.4
95% CI for difference	0.34 (−4.95; 4.28)		−0.55 (−6.31; 5.20)		−7.45 (−13.19; −1.70)		−7.52 (−13.57;−1.47)		−7.55 (−12.24; −2.86)
KOOS	75.3 ± 19.2	78.5 ± 17.1	46.2 ± 25.7	45.5 ± 20.6	35.6 ± 24.8	45.2 ± 22.3	39.1 ± 23.9	50.0 ± 24.7	46.6 ± 22.0	37.0 ± 11.4
95% CI for difference	−3.21 (−10.01; 3.58)		0.66 (−8.06; 9.38)		−9.59 (−18.42;−0.76)		−10.88 (−20.39;−1.37)		−12.93 (−20.92;−4.95)
NRS	5.91 ± 1.55	5.82 ± 1.82	3.34 ± 2.09	2.79 ± 1.94	2.20 ± 1.76	3.39 ± 2.12	2.94 ± 1.80	4.00 ± 2.20	3.73 ± 1.72	5.08 ± 1.65
95% CI for difference	0.09 (−0.54; 0.72)		0.55 (−0.20; 1.31)		−1.20 (−1.93; −0.47)		−1.06 (−1.85; −0.27)		−1.35 (−2.01; −0.96)

Continuous variables are expressed as means ± standard deviations (95% confidence interval (CI). Abbreviations: O₂O₃: oxygen–ozone therapy; HA: hyaluronic acid; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index; KOOS: Knee injury and Osteoarthritis Outcome Score; NRS: Numeric Rating Scale.

WOMAC pain score

Data analysis showed a significant decrease in pain rating according to WOMAC subscore (WOMACp) in both groups. The baseline score was 6.77 ± 2.52 in Group A and 6.50 ± 2.24 in Group B. After reassessment at one-month follow-up, it was 3.66 ± 2.57 in Group A and 3.45 ± 2.30 in Group B. We found a similar trend in the subsequent evaluations: at 3 months’ follow-up, the score for Group A was 2.36 ± 1.67, while in Group B was 3.89 ± 2.16; at 6 months’ follow-up, it was 2.98 ± 1.89 in Group A and 4.50 ± 2.46 in Group B. At 12 months’ follow-up, the score for Group A was 5.04 ± 1.97, while in Group B was 5.63 ± 2.25 (Figure 2).

Figure 2.

Intra-group analysis of the primary outcome WOMAC LK 3.1 pain subscale score in the groups of HA (A) and O₂O₃ (B).

No significant differences were observed between the two groups at short-term (one month) and long-term (12 months) follow-up; although, at one-month follow-up, a more favorable trend was observed in the O₂O₃-treated group. At the three- and six-month follow-up assessments, statistically significant differences were observed between the treatment groups, favoring the HA-treated group (refer to Table 3 for detailed results).

Table 3.

Between-group differences in the primary outcome.

WOMAC LK 3.1 pain score	HA Group (Group A)	O₂O₃ Group (Group B)	P value
Baseline	6.77 ± 2.53	6.50 ± 2.24	0.735
1 month	3.66 ± 2.57	3.45 ± 2.30	0.501
Δ 1 month - baseline	3.10 (2.31; 3.91)	3.05 (2.38; 3.73)
3 months	2.36 ± 1.67	3.89 ± 2.16	<0.001
Δ 3 month - baseline	4.41 (3.73; 5.09)	2.61 (1.94; 3.27)
6 months	2.98 ± 1.89	4.50 ± 2.46	< 0.001
Δ 6 month - baseline	3.78 (3.09; 4.47)	2.00 (1.13; 2.77)
12 months	5.04 ± 1.97	5.63 ± 2.25	0.271
Δ 12 month - baseline	1.73 (1.10; 2.35)	0.88 (0.36; 1.40)

Continuous variables are expressed as means ± standard deviations; categorical data are expressed as counts (percentages). Abbreviations: O₂O₃: Oxygen-ozone therapy; HA: hyaluronic acid; Δ: delta; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index.

NRS

Pain assessment by NRS showed a significant decrease over time in both groups (Figure 3).

Figure 3.

Intra-group analysis of the NRS score in the groups of HA (A) and O₂O₃ (B).

The NRS score at baseline was 5.91 ± 1.55 in group A and 5.82 ± 1.82 in group B. In both groups, there was an improvement in pain as early as after 1 month of treatment: the NRS score was 3.34 ± 2.09 in group A and 2.79 ± 1.94 in group B, showing a more favorable trend in the O₂O₃-treated group although without statistical significance. An opposite trend was found in subsequent evaluations: at 3-month follow-up the score for Group A was 2.20 ± 1.76, while that for Group B was 3.39 ± 2.12; at 6-month follow-up it was 2.94 ± 1.80 in Group A and 4.00 ± 2.20 in Group B. Finally, at 12-month follow-up, the score of Group A was 3.73 ± 1.72, while that of Group B was 5.08 ± 1.65. In these latter intervals, the HA-treated group showed significant improvement over the O₂O₃ group.

Between baseline and 3-month follow up group A showed a bigger decrease (3.71 ± 2.10 vs 2.43 ± 2.22, p = 0.002) (see Table 4 for further details).

Table 4.

Between-group differences in the secondary outcomes.

	HA Group (Group A)	O₂O₃ Group (Group B)	P value
WOMAC total score (baseline)	43.7 ± 12.4	44.0 ± 12.3	0.727
WOMAC total score (1 month)	25.8 ± 16.9	26.3 ± 13.7	0.738
WOMAC total score (3 months)	20.4 ± 15.3	27.8 ± 15.4	0.013
WOMAC total score (6 months)	22.8 ± 14.3	30.3 ± 16.5	0.033
WOMAC total score (12 months)	29.5 ± 12.5	37.0 ± 11.4	0.002
KOOS (baseline)	75.3 ± 19.2	78.5 ± 17.1	0.507
KOOS (1 month)	46.2 ± 25.7	45.5 ± 20.6	0.854
KOOS (3 month)	35.6 ± 24.8	45.2 ± 22.3	0.022
KOOS (6 months)	39.1 ± 23.9	50.0 ± 24.7	0.036
KOOS (12 months)	46.6 ± 22.0	59.6 ± 18.7	0.001
NRS (baseline)	5.91 ± 1.55	5.82 ± 1.82	0.900
NRS (1 month)	3.34 ± 2.09	2.79 ± 1.94	0.231
NRS (3 month)	2.20 ± 1.76	3.39 ± 2.12	0.001
NRS (6 months)	2.94 ± 1.80	4.00 ± 2.20	0.012
NRS (12 months)	3.73 ± 1.72	5.08 ± 1.65	<0.001

Continuous variables are expressed as means ± standard deviations; categorical data are expressed as counts (percentages). Abbreviations: O₂O₃: Oxygen-ozone therapy; HA: hyaluronic acid; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index; KOOS: Knee injury and Osteoarthritis Outcome Score; NRS: Numeric Rating Scale.

WOMAC total score

At baseline, the mean WOMAC score was comparable in the two groups, with no statistical differences between Group A and Group B, 43.7 ± 12.4 and 44.0 ± 12.3 respectively (Figure 4).

Figure 4.

Intra-group analysis of the total WOMAC LK 3.1 score in the groups of HA (A) and O₂O₃ (B).

An improvement in the score at 13,6 and 12-month follow-up was recorded for both groups: at 1 month follow-up was 25.8 ± 16.9 in Group A and 26.3 ± 13.7 in Group B; at 3 months’ follow-up was 20.4 ± 15.3 and 27.8 ± 15.4 respectively; at 6 months 22.8 ± 14.3 and 30.3 ± 16.5; at 12 months 29.5 ± 12.5 and 37.0 ± 11.4. At intermediate to long-term follow-up (3–12 months), the HA-treated group showed significant improvement compared with the OT group. At the 3-month follow-up, group A showed a greater reduction (23.3 ± 15.7 vs. 16.2 ± 15.3, p = 0.017) (see Table 4 for further details).

KOOS score

The mean KOOS score at baseline was comparable in the two groups, with no statistical differences between Group A and Group B, 75.3 ± 19.2 and 78.5 ± 17.1 respectively (Figure 5). An improvement in the score at 1, 3,6 and 12-month follow-up was recorded for both groups: at 1 months’ follow-up was 46.2 ± 25.7 in Group A and 45.5 ± 20.6 in Group B; at 3 months’ follow-up was 35.6 ± 24.8 and 45.2 ± 22.3 respectively; at 6 months 39.1 ± 23.9 and 50.0 ± 24.7; at 12 months 46.6 ± 22.0 and 59.6 ± 18.7. At intermediate to long-term follow-up (3–12 months), the HA-treated group showed significant improvement compared with the OT group. Between baseline and 3-month follow up the two groups showed a similar decrease (39.6 ± 25.6 vs 33.3 ± 24.4, p = 0.180) (see Table 4 for further details).

Figure 5.

Intra-group analysis of the KOOS in the groups of HA (A) and O₂O₃ (B).

Safety profile

Both treatments demonstrated a good safety profile, with only a few mild and self-limiting adverse events occurring within 24 h post-injection. In the O₂O₃ group, 11% of patients reported temporary swelling immediately after the procedure, lasting for several hours. Meanwhile, in the HA group, 17% of patients reported transient pain and a localized bulky sensation following the injections.

Discussion

This RCT aimed to compare clinical outcomes of pain control and functioning changes in KOA patients treated with IA injections of HA versus O₂O_3. According to our findings, O₂O₃ and HA injections showed positive results in term of pain control and disability decrease in people affected by KOA. Both groups reached statistically significant improvements from baseline in all outcomes measured, starting from 1 month after treatment. Values remained stable, showing a worsening trend from month 6 to 1 year, as might be expected from the natural history of OA, however without returning to scores measured at baseline.

In the short-term observation period (1 month), O₂O₃-treated patients demonstrated better results in pain control, recovery of motor skills, and QOL compared to HA-treated patients, without a significant statistical difference. In contrast, at mid- and long-term follow-ups (3 months to 12 months after the end of the treatment), the HA-treated group showed statistically significant pain reduction and functional improvement compared with the O₂O₃ group. Regarding safety, both treatments were well-tolerated, with only a few mild and self-limiting adverse events reported.

Analysis of the results suggests that the long-term effects of O₂O₃ were significantly lower than those obtained with HA. This could be consistent with a slower analgesic response of HA, but with greater persistence. O₂O₃ instead demonstrated a faster therapeutic effect, probably related to the modulating properties on tissue inflammatory agents^31,32; in fact, O₂O₃ has an early anti-inflammatory action by inhibiting the synthesis of proinflammatory prostaglandins, the release of bradykinin, and the release of algogenic compounds.³³ At the same time, there is an increased release of antagonists or soluble receptors that neutralize proinflammatory cytokines, such as interleukin (IL)-1, IL-2, IL-8, and tumor necrosis factor- α (TNF-α).⁷ In this context, this effect may be generated by the transient and controlled hyperstimulation of the body's physiological antioxidant system mediated by O₂O₃³⁴ and, in particular, by decreasing the activation of the NF-Kβ pathway, with a consequent reduction in cartilage matrix degradation and chondrocyte apoptosis,³⁵ and by increasing serum levels of IGF-1, capable of reducing tissue inflammation while stimulating cellular repair.^36,37 Interestingly, when comparing them, it appears that the rate of action of O₂O₃ can vary depending on the musculoskeletal pathology being treated. For example, many studies on low back pain or radiculopathies have shown a slower onset of action but a much more persistent effect.³⁸ Probably the multiple properties of O₂O₃ operate with different and complex activities depending on the characteristics of the tissue and the pathogenesis of the disease. Conversely, we are aware that HA commonly provides a slow anti-inflammatory action, that is related to the molecular weight.^39,40 Indeed, low molecular weights determine a greater anti-inflammatory effect than medium-high molecular weights, which instead have a stronger viscoelastic propriety useful for joint shock absorbers.^41,42

The longer-lasting effects of HA on cartilage are attributed to the interaction between HA and the molecule CD44, which reduces chondrocyte apoptosis, promotes their proliferation, and inhibits the expression of interleukin (IL)-1β, decreasing the release of various matrix metalloproteinases and exerting an anti-inflammatory effect.²³ Moreover, due to its viscoelastic properties, HA contributes to joint lubrication, reduces friction-induced degeneration, and promotes an osteogenetic effect enhancement through the downregulation of BMP-2 antagonists.⁴³

The above concept confirms that OA, in addition to being a degenerative disease, involves chronic inflammatory processes and immune overactivation related to its pathophysiology.^44,45 As evidence of this, several studies of cartilage RNA sequence analysis have suggested the presence of two main pathogenetic phenotypes: one with a predominantly inflammatory component and another with a load-related pathway.^2,46,47 This could be crucial for the choice of appropriate treatment and the different mechanisms of action of O₂O₃ and HA could generate different results depending on the arthrosis profile.^18,48,49 In the future, it will probably be possible to identify tailored outline of patients with the highest probability of responding to treatment through dissection of key signaling pathways, thus providing indications of the best responders in KOA.

In the last few years, many studies compared the effectiveness of O₂O₃ and HA for treating KOA.^26,50–52 In 2020, de Sire et al. showed, in a group of 42 patients, a comparable pain relief efficacy in short-term between O₂O₃ and HA and, consequently, a significant difference in favor of HA in long-term follow-up of 31 weeks.⁷ Similar results were reported by Invernizzi et al.,⁵⁰ obtaining faster pain relief by O₂O₃ and long-term effectiveness by HA.

Giombini et al., in 2016, reported significant improvements in pain and disability at 2 months follow-up using combined injections of O₂O₃ and HA.⁵² In a cohort of 174 patients with KOA, Raeissadat et al. obtained pain relief through a higher O₂O₃ dosage rather than those used in our study (30 μg/mL, one injection weekly).²⁶ The various therapeutic protocols used for the intra-articular administration of O₂O₃ make it difficult to standardize the results.^24,25 Therefore, we decided to use the therapeutic protocol determined by the ISCO3.

A recent RCT by Aslan et al., instead, demonstrated the efficacy of O₂O₃ compared to steroid injections in functioning and pain relief among 96 patients with KOA diagnosis, obtaining improvements in knee function, pain, and QOL, without significant differences between the groups.⁵³

This study was conducted with scientific rigor, providing valuable evidence and insights useful for the conservative approach of KOA, particularly regarding pain and disability management. At the same time, it presents some limitations. First, although the patient sample size was statistically correct to obtain a significant analysis of the primary outcome data (WOMACp), it would be necessary to increase the study population to further confirm the results. No radiographic data are available for comparison between patients at the end of the study. Similarly, no biochemical data are available for comparison between patients. Looking forward, it would be very interesting to correlate the efficacy of O₂O₃ and HA on the basis of characterizing different OA patient populations (inflammatory or load-bearing) by analyzing molecular phenotypic differences.

Conclusion

Major findings of our trial showed that administration of O₂O₃ and HA injections might provide positive results in pain control and motor function recovery in patients affected by KOA. Both treatments were also comparably safe. O₂O₃ seems to have a faster effect than HA, but only in the first month of treatment. HA, on the other hand, is more effective in the medium to long term.

Therefore, O₂O₃ could be considered as a potential treatment for KOA, thanks to its anti-inflammatory and analgesic properties. especially in the short-term.

Footnotes

Ethical considerations

This paper was accepted by the Hospital Ethics Committee and Scientific Board (Authorization n° 2556, 27 May 2020). The study protocol was recorded in June 2020 on clinicaltrials.gov (NCT04426721).

Author contributions

Conceptualization, CS and AdS; methodology, CS and AdS; validation, CS, AP, and AdS; formal analysis, NM; investigation, CS, AP, and AdS; data curation, CS, AP, and AdS; writing—original draft preparation, CS, AP, and NM; writing—review and editing, AA, AB, and AdS; visualization, GF, DS, MV, and GLM; supervision, AdS. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Informed consent

Written informed consent was obtained from all participants.

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