While numerous clinical studies examined the use of PRP for treating KOA at various injection frequencies, considerable variations exists in PRP preparation methods, such as inclusion of white blood cells in PRP, injection techniques, dosage, use of activators and local anesthetics, and other factors. Moreover, different studies employ varying evaluation methods for assessing efficacy and have diverse follow-up periods. Consequently, no current definitive consensus exists on the ideal treatment frequency for intra-articular PRP injections to effectively manage KOA.

Currently, 9 clinical studies investigated the effects of different injection regimens on the efficacy of PRP treatment forKOA (Table 4). Of these studies, eight compared the efficacy of multiple versus single PRP injection, while one study compared the efficacy of two against four injections. All of these studies concluded that PRP injections are effective in treating KOA, resulting in significant pain reduction and improvement in knee joint function [19,20,21,22,23,24,25,26,27]. One study found no statistically significant difference in efficacy between single and double injections [19], as well as between single and triple injections [22]. Six studies reported that multiple injections were more effective than single injections [19,20,21,22,23,24,25,26,27]. In one study comparing the efficacy of single and double injections, double injections were found to be superior [24]. Five studies compared the efficacy of single and triple injections and concluded that triple injections were more effective. Two studies suggested that triple injections were more effective than both single and double injections [22], with one study recommending a minimum of two injections. One study compared the clinical efficacy and levels of inflammatory factors in synovial fluid between two and four injections for the treatment of KOA. Both doses significantly improved the clinical symptoms of patients with KOA, with no statistical difference between the two groups. However, neither regimen had an impact on the levels of inflammatory factors, such as interleukin-6, interleukin-1β, and TNF-α n the synovial fluid.However, the use of PRP activation and application of activators vary among the studies.In five studies, calcium chloride was used to activate PRP, while one study utilized 10% calcium gluconate for activation. Three studies did not use any activator to activate PRP. Most of the studies did not administer local anesthesia at the injection site before PRP injection, except for two studies that used lidocaine for local anesthesia prior to injection. Six studies did not classify the PRP preparations used. One study classified them as P2Bb according to the Platelets, Activation, White cells (PAW) classification, and two studies classified them as 2 A and 4B according to the Mishra classification. Additionally, the volume of PRP per dose used in each study also varies, with most studies using a volume of 4-5mL and the maximum volume being 8mL. The time intervals between the two PRP injections also varied significantly. Two studies had a one-week interval, three studies had a two-week interval, three studies had a six-week interval, and one study had a one-month interval. The follow-up duration also differed among the studies with a minimum follow-up of three months and a maximum follow-up of two years. Four studies had a six-month follow-up, while three studies had a one-year follow-up. The aforementioned 9 clinical studies exhibit substantial variations in research methods, inclusion criteria, PRP preparation methods, types of PRP, presence of white blood cells, injection methods, PRP activation and activators, injection doses, use of local anesthetics, follow-up period, efficacy evaluation methods, and other aspects. Consequently, their findings diverge significantly and even result in contradictory conclusions (Table 4). In addition, the effectiveness of PRP treatment for KOA is not only influenced by the frequency of treatment, but also by the quality of the PRP, which is determined by factors such as patient age, immune status, presence of concurrent metabolic diseases, and medications used [28].

Based on these studies, three intra-articular PRP injections can yield favorable clinical outcomes for knee joints. However, we hypothesize that additional injections may be necessary to achieve even better clinical results. For instance, undergoing a treatment consisting of five injections might lead to improved outcomes. Therefore, we compared the effectiveness between groups receiving three and five injections. Considering the insights derived from these studies, we adopted a standardized approach in preparing PRP, ensuring consistency in the composition of its various components. Depending on the concentration of white blood cells, it can be divided into leukocyte-poor and leukocyte-rich PRP. The leukocyte-rich PRP used in this study has a better anti-inflammatory effect [29]. Research has shown that local anesthetics may have a toxic effect on knee joint cartilage cells and can affect platelet activation by altering the intra-articular pH. Therefore, local anesthetics were not used prior to PRP injection [30]. Based on the substantial clinical evidence supporting the efficacy of PRP in treating KOA, we excluded a placebo control group or other injection interventions, such as steroids or hyaluronic acid,, as comparators. We selected patients with bilateral KOA at KL 1–3 stages as our study population, with simultaneous injection and functional assessment conducted on both knees of each patient. Building upon previous research that primarily compared the effects of three PRP injections with single or two PRP injections, we investigated the differences in efficacy between single, three and five PRP injections. Each injection consist of 4mL of PRP per joint, with a one-week interval between injections.

Ultrasound-guided injection was demonstrated to be more precise and accurate compared to blind injections in existing literature [31]. Therefore, we employed ultrasound guidance for intra-articular PRP injections, which was one of the contributing factors for achieving improved therapeutic outcomes in this study.

The results of this study demonstrate that, compared to baseline, the VAS scores and WOMAC index of all three groups improved at 6 weeks, 12 weeks, 24 weeks and 52 weeks after intervention. This indicates that intra-articular PRP injections significantly enhance the function and alleviate joint pain in patients with KOA, which is consistent with the findings of previous studies. However, at the same time points after intervention, both the VAS and WOMAC scores of the PRP3 and PRP5 groups were statistically superior to those of the PRP1 group (P < 0.05). This suggests that multiple injections have better clinical efficacy than a single injection, which aligns with the conclusions of previous studies. Görmeli G et al. [20] discovered that three injections of PRP showed superior efficacy compared to single injections for patients with early KOA. Similarly, Kavadar G et al. [21] suggested that two PRP injections had a better prognosis than a single injection. Mehdi et al. [23] found that two PRP injections were more effective than a single injection and both were more effective than hyaluronic acid injections, which aligns with our findings. A most recent meta-analysis incorporating seven clinical studies involving 575 patients suggests that triple-dose PRP therapy may be more effective than single-dose PRP in the treatment of KOA [32]. Additionally, the VAS and WOMAC scores showed a decreasing trend after 6 months of PRP intervention, which became more pronounced after one year of intervention. Although this result suggests that multiple are more effective than single injections in the treatment of KOA, the principle of “more is better” does not necessarily apply. The results of this study revealed no significant difference (P > 0.05) in VAS and WOMAC scores between the PRP3 and PRP5 groups at the same time points following PRP intervention. Our research findings primarily suggest that three injections provide the maximum benefit to patients and may serve as a potential option for delaying KOA surgery or as substitute surgical treatment.

The possible mechanism for the better effect of multiple injections compared to a single injection in this study could be attributed to the growth factors in PRP that continue to promote cartilage and tissue metabolism with a cumulative effect. In vitro experiments on PRP therapy for KOA have shown significant dose and time dependence in terms of the quantity and metabolic activity of chondrocytes [33]. Moreover, a clinical study indicates that PRP therapy has a dose-dependent cumulative effect. In this study, multiple injections of PRP combined with PRF (Platelet Rich Fibrin) were administered, with a maximum of four injections. This treatment approach could alleviate pain, improve knee joint function, function, and delay the need for knee replacement surgery, with the clinical effects becoming more evident with a higher number of treatment sessions [34].

However, there was no significant difference in clinical efficacy between the 3 and 5 injections, and the reasons for this result are remain unclear. Nevertheless, this finding is consistent with many clinical studies that recommend a three-injection treatment protocol. These findings align with that of Ngarmukos et al. (25) where they observed that despite dosage variations between the group receiving two and four injections, both groups demonstrated improvement in clinical scores without any statistically significant difference in clinical efficacy.

PRP contains a variety of growth factors and cytokines, such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), and vascular endothelial growth factor (VEGF), which promote cell proliferation, differentiation, and tissue repair. PRP is extensively used in clinical practice for conditions such as tendinopathies and cartilage injuries. The mechanism of action behind PRP injections for KOA lies in its high concentrations of platelets and growth factors, including PDGF, TGF-β1, basic fibroblast growth factor (bFGF), and VEGF. These substances can stimulate the production of fibroblasts and collagen, regulate the joint microenvironment, enhance cell viability [35, 36] promote angiogenesis, and facilitate myogenic cell proliferation [37]. KOA is characterized by the degeneration of articular cartilage, local synovial inflammation, joint capsule contracture, and ligamentous laxity or contracture [38]. The synergistic effects of growth factors present in PRP can promote cartilage regeneration and metabolism, potentially exerting a chondrogenic effect.

In KOA, which is characterized by synovial inflammation, cartilage erosion, and infiltration of inflammatory cells, PRP treatment can work through several mechanisms: (1) First, it promotes tissue repair and regeneration. The growth factors and cytokines in PRP stimulate the proliferation and synthesis of chondrocytes, promoting the repair and regeneration of damaged tissues [39, 40]. (2) Second, PRP exhibits anti-inflammatory properties. The growth factors in PRP also possess anti-inflammatory effects, reducing the infiltration of inflammatory cells and alleviating associated inflammatory reactions. This helps to relieve the symptoms of arthritis [41]. (3) Third, PRP improved joint lubrication and function. The introduction of PRP into affected joints can enhance the viscoelasticity and lubricating properties of synovial fluid, leading to improved joint mobility and function [42]. (4) Finally, PRP therapy offers analgesic effects. PRP therapy can potentially relieve pain symptoms by reducing inflammation and promoting tissue repair. These mechanisms collectively contribute to the effectiveness of PRP in the treatment of KOA [41]. Additionally, neovascularization facilitated by PRP can nourish contracted muscles and tissues, thereby slowing down the progression of KOA. These effects can function independently or even interact additively and synergistically [37].

This study has certain limitations. While implementing patient blinding would have bolstered the validity of our findings, it would have necessitated the administration of placebo injections, a course of action deemed ethically inappropriate. Furthermore, this study is constrained by its exclusive reliance on a single clinical research center and a relatively small sample size. To ascertain the optimal frequency of PRP treatment for KOA, more robust clinical evidence is still warranted. Conducting large-scale, multicenter clinical trials utilizing randomized double-blind controlled methodologies, extending the follow-up duration, assessing treatment efficacy comprehensively, and observing the long-term effects of PRP are imperative. Additional fundamental research is indispensable in elucidating the precise mechanisms underlying the therapeutic use of PRP in the treatment of KOA.