In knee osteoarthritis patients, ozone injection showed prompt effects and better short-term results after two months. But, its effects did not sustain after six months. At the six-month follow-up, the platelet-rich plasma, plasma rich in growth factors, and hyaluronic acid displayed superiority over ozone. For long-term management, platelet-rich plasma and plasma rich in growth factors may be used since the improved symptoms were found to persist for 12 months.

Knee osteoarthritis, a degenerative and progressive joint disease, is one of the significant causes of disability. It affects the quality of life and poses a burden on society. Worldwide, the prevalence of symptomatic knee osteoarthritis is estimated to be 3.8% and impacts more than 20% of the over 45-year-old population. The available management options for knee osteoarthritis are not satisfactory for osteoarthritis individuals and pain has been reported by at least 40% of cases who underwent surgical arthroplasty.

There is a paucity of a single well-known or approved therapy that can cease the progression of knee osteoarthritis. Thus various studies have been carried out to develop non-operative or minimally invasive interventions to mitigate osteoarthritis symptoms or retard osteoarthritis advancement.

Concerning the standard management approach, no consensus has been yet reached. Among the minimally invasive approaches suggested for managing osteoarthritis, the intra-articular injections for which an enormous array of products have been utilized like hyaluronic acid, corticosteroids, dextrose, plasma derivatives including platelet-rich plasma, and plasma rich in growth factors, and ozone. Although injecting corticosteroids via intra-articular route have demonstrated efficacy, but in few circumstances, these products may be less favored due to their short-term activity and side effects.

Hyaluronic acid, a natural glycosaminoglycan present in the joints, offers the basis for viscoelastic characteristics of synovial fluid. Since synovial fluid hyaluronate degrades during knee osteoarthritis, thus intra-articular injection of the hyaluronic acid may lower knee joint pain and improve functional impairment. Hyaluronic acid has been therefore deemed as a pharmacologic choice and was granted approval by the Food and Drug Administration (FDA) to cure knee osteoarthritis in 1997. In 2000, the guideline of the American College of Rheumatology suggested it to be an effective therapeutic approach for knee osteoarthritis.

Due to the controversial outcomes, no agreement exists concerning the management of knee osteoarthritis with injections of hyaluronic acid. Few clinical guidelines do not suggest the usage of hyaluronic acid for knee osteoarthritis treatment, majorly because of less efficacy. However, it is still utilized as a safe agent with minimized side-effects alternative. Additionally, some other guidelines and numerous recent meta-analyses favor the usage of viscosupplements to alleviate osteoarthritis of the knee.

Recently, another biological product i.e  autologous platelet-rich plasma has gained more attention to manage patients suffering from osteoarthritis of the knee. Numerous studies have been performed worldwide favoring the usage of platelet-rich plasma injection as an effective methodology for  osteoarthritis of the knee. Various studies have utilized platelet-rich plasma in different settings and the procured outcomes demonstrate that platelet-rich plasma could serve as antinociceptive and stimulate cell proliferation.

The injection of platelet-rich plasma via intra-articular route has been found to promote cartilage formation, modulate joint environment, and hinder the deterioration of knee joint probably by lowering the generation of the pro-inflammatory mediators. The therapy effects of platelet-rich plasma may also be described by the supra-physiologic concentrations of growth factors and biological molecules. These exist in the platelets granules and could considerably reverse the catabolic environment in osteoarthritis, balancing the joint homeostasis, and consequently induce restoration of the destroyed cartilage.

In clinical practice, there is a discrepancy in the literature regarding the extensive usage of intraarticular platelet-rich plasma to manage knee osteoarthritis. The controversies have been linked to the release of growth factors from platelets after injection. Due to some reasons, a proportion of growth factors may not be released after injection, and therefore results in decreased therapeutic response. To avoid this obstacle, the biologic activators compatible with the body have been utilized to induce the platelets to discharge the granular content that results in plasma rich in growth factors creation. Plasma rich in growth factors (the final product of platelet-rich plasma without leukocytes and inflammatory cytokines) contains only a specific quantity of growth factors and cytokines.

In comparison to platelet-rich plasma, plasma rich in growth factors has more efficacy and reduced adverse effects like swelling and pain. Also, there has been a rising interest in the usage of ozone as a safe option to alleviate knee osteoarthritis. Advantages linked with ozone therapy include low cost of application and easy administration. The intra-articular injection of ozone is one of the effective therapies to mitigate knee osteoarthritis symptoms. The intra-articular injection of ozone, as a liquid form (a mixture of ozone and oxygen), has been proven to relieve mild to moderate knee osteoarthritis.

The mixture of oxygen and ozone can mitigate osteoarthritis symptoms by enhancing tissue oxygenation, accelerating reactive oxygen species production, and declining the discharge of proinflammatory cytokines, which subsequently counteracts the activation and recruitment of leukocytes and other types of immune cells at sites of inflammation. Although ozone treatment has been found to exert short-term effects, however, inconsistent results have been noted concerning its long-term effects.

Rationale behind research:

There is a paucity of general consensus on the choice and priority of the intra-articular hyaluronic acid, platelet-rich plasma, plasma rich in growth factors, and ozone injections to relieve knee osteoarthritis. Therefore, this randomized clinical trial was performed.

Objective:

A study was conducted to comparatively investigate the long and short-term effectiveness (two months and 12 months after interventions, respectively) of the intra-articular injections of hyaluronic acid, platelet-rich plasma, plasma rich in growth factors, and ozone to improve symptoms of mild to moderate knee osteoarthritis.

Study outcomes

• The outcomes were mean alterations from baseline (immediately from the initial injections) until two, six, and 12 months after intervention in the scores of Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Lequesne index, and visual analog scale (VAS)

Outcomes

Baseline: There were no significant differences reported at baseline.

Study outcomes

• In the two months follow-up, a substantial improvement in function, pain, and stiffness were witnessed in all the cohorts in comparison with the baseline. However, the ozone cohort displayed the best results
• In the six months follow up, the platelet-rich plasma, hyaluronic acid, and plasma rich in growth factors groups illustrated improved therapeutic effects in all scores when compared to ozone-treated subjects
• Only plasma rich in growth factors and platelet-rich plasma cohorts had improved results in all scores when compared to hyaluronic acid and ozone arms at the end of the 12th month
• Even though treatment with ozone depicted improved early results, its therapeutic effects start to wear off earlier when compared to other products and finally dissipate in 12 months

Participants in all four groups displayed considerably lower scores in VAS, WOMAC, and Lequesne in two months after injection in comparison with their primary evaluation prior to the injections (baseline levels). The trial outcomes indicated that the ozone cohort had remarkably reduced VAS, WOMAC, and Lequesne scores compared to the other cohorts at the second month of follow up. But, its effects were found to wipe out after 12 months.

It is apparent that ozone treatment has some early valuable but not long-lasting effects in patients with knee osteoarthritis. The findings of this randomized trial are in agreement with a previous meta-analysis conducted by Raeissadat et al. that illustrated that the effects of ozone wear off four-six months post-injection. According to the evaluation conducted by Dernek et al., ozone-treated patients reported earlier improvement in symptoms of osteoarthritis when compared to patients treated with platelet-rich plasma.

However, platelet-rich plasma was found to have long term effects compared to ozone therapy. Another analysis carried out by Gaballa et al. displayed that despite ozone being able to lower the WOMAC score in a comparable amount as platelet-rich plasma at one-month post-injection, however at the third month of follow-up, subjects who were administered ozone treatment had raised WOMAC scores. Although the outcomes procured by Gaballa et al. were somehow comparable to the outcomes of this randomized trial, in contrast, the platelet-rich plasma was found to have much long-term effects.

Based on the literature, ozone treatment can elevate the generation of reactive oxygen species in the sites of inflammation. This, in turn, may inactivate proteolytic enzymes and suppress the release of proinflammatory cytokines, and thus relieve the disease symptoms. But, in a short time, the dissolved ozone may be cleared up from the synovial fluid causing diminished therapy efficacy. Thus, it appears that the multiple ozone doses might be advantageous and can be added to the other therapy regimens.

In this trial, six months after injection, individuals treated with platelet-rich plasma, hyaluronic acid, and plasma rich in growth factors displayed better scores in comparison with ozone. The difference between plasma rich in growth factors, hyaluronic acid, and platelet-rich plasma was not noted to be significant. Similarly, Raeissadat et al. has demonstrated that hyaluronic acid and plasma rich in growth factors had comparable effects two and six months after with no vital difference between the groups.

According to the evaluation carried out by Duymus et al., the platelet-rich plasma, hyaluronic acid, and ozone displayed similar effects one-month post-injection. While six months after injection, the hyaluronic acid and platelet-rich plasma demonstrated superiority to ozone. The outcomes of another trial conducted in 2018 by Raeissadat et al. on 174 osteoarthritis patients showed no vital difference between ozone and hyaluronic acid six months after injection.

In this trial, 12 months after injection, only participants who were administered plasma rich in growth factors or platelet-rich plasma had considerably improved outcomes in comparison with those who had been managed with ozone or hyaluronic acid. In the analysis carried out by Duymus et al., the platelet-rich plasma had demonstrated significantly improved outcomes compared to ozone and hyaluronic acid after 12 months.

Superior effects have been witnessed for platelet-rich plasma in comparison with hyaluronic acid at 12 months after injection. A meta-analysis by Wen-Li-Dai in 2016 acknowledged the improved outcomes of platelet-rich plasma in comparison with hyaluronic acid in the 12-month follow-up. The discrepancies between these assessments may be due to the sample size or distinct methodologies utilized in these analyses. It has been proven that impairment of hyaluronate occurs in osteoarthritis. Although introducing the exogenous hyaluronic acid can relieve the symptoms and treat the functional impairment. However, it cannot suppress the inflammation in osteoarthritis of the knee.

With time, the exogenous hyaluronic acid is damaged at the site of inflammation, and therefore the symptoms again initiate after injection. Regarding the plasma rich in growth factors or platelet-rich plasma, it has been demonstrated that these products may initiate cartilage formation, regulate the intraarticular microenvironment and cellular composition and proliferation, and impact the expression of few prominent inflammatory mediators in the joint. Thus, their impact might sustain for a longer time in comparison with the hyaluronic acid or ozone.

Other investigations have shown comparable effects of platelet-rich plasma and plasma rich in growth factors in 12- month follow up. In 2012, a study by Filardo et al. showed that three plasma rich in growth factors or platelet-rich plasma injections three weeks apart had no vital differences concerning improvements in functioning and pain of osteoarthritis patients at two, six, and 12 months follow-ups, and both the products had proven effective in combating the symptoms.

Several factors should be considered while comparing the outcomes of different studies.

The reasons for the diversity of outcomes might be the differences in the platelet-rich plasma preparations utilized regarding purity (existence of red and white blood cells and their concentration), platelet dose (concentration and volume), and efficiency of the product preparation on the basis of the quality of the kit utilized and using/not using an activator.

The distinct concentrations and preparation methodology, despite having a similar product name, may lead to different products. This can have diverse effects in altering a destructive articular environment into a regenerative environment. In the case of hyaluronic acid, there could be

Differences may exist in the molecular weight, volume, concentration, being cross-linked or linear, and the source (fermentation or animal).

Taking ozone into consideration, the variances in concentration and volume may produce different therapeutic impact. The differences in the number of injections and the time intervals between them may also lead to variations in results. In several studies, a range of one to many injections has been carried out, that have been spaced between one week to three or four weeks apart. As can be noted in various studies, there is no agreement upon a number or standard frequency of injections. Considering the prior experience with plasma-based products and to attain a balance between the arms regarding the therapy cost, two injections with three weeks separation for platelet-rich plasma and plasma rich in growth factors were selected; while three weekly injections were taken into consideration for ozone and hyaluronic acid.

The other reasons for discrepancies in outcomes might stem from the variety in rehabilitative protocols used after injections and also the ways in which evaluation of response to therapy is carried out. The severity of osteoarthritis, demographic differences (age or sex), and activity amount also demonstrate a vital role in the study outcomes.