Knee osteoarthritis (KOA) is a common degenerative cartilage disorder associated with structural and functional changes in the knee joint (1). Patients with KOA typically experience symptoms such as joint pain, stiffness, limited mobility, and functional impairment, which significantly reduce their quality of life and impose substantial burdens on both individuals and families (1). The knee, being one of the body's most weight-bearing joints, is particularly susceptible to structural damage from aging, wear, trauma, and developmental abnormalities, ultimately leading to KOA (2). It is reported that approximately 250 million people worldwide are affected by KOA, making it a major global public health concern (3).

The primary treatment goals for KOA are to alleviate pain and improve joint function (4). Management of KOA involves both pharmacological treatment and surgical interventions. Surgery, such as knee arthroplasty, is considered an effective therapy, primarily indicated for advanced-stage KOA patients (5). Therefore, conservative treatment remains the first choice for early-stage KOA patients (6). Nonsteroidal anti-inflammatory drugs (NSAIDs), while being primary treatment options for KOA, may cause gastrointestinal, cardiovascular, and renal adverse events due to systemic administration (6). Intra-articular injection therapy has gained increasing attention for its direct targeting of affected tissues and reduced adverse reactions (7).

Platelet-rich plasma (PRP) is a blood plasma derived from autologous blood, which contains high concentrations of platelets and growth factors, and acts as a reservoir for cytokines, including growth factors, chemokines, and various mediators (8). These growth factors can activate local stem cells and fibroblasts, mobilize them to damaged areas, stimulate angiogenesis in affected regions, control inflammation, block catabolism and cytokine production, and also prompt neighboring healthy cells to secrete additional growth factors (8). Studies have shown that the direct application of PRP to cartilage injury sites can effectively promote healing processes and accelerate the formation of repaired cartilage tissue (9). In recent years, intra-articular PRP injections for the treatment of KOA have gained increasing attention among orthopedic surgeons and researchers (10). It has been reported that intra-articular PRP injection therapy can help KOA patients resume movement and activities earlier by alleviating pain and improving endurance within 2–3 weeks (11). However, the optimal PRP injection dosage and frequency for KOA patients remain controversial (12).

Therefore, we conducted this retrospective single-center cohort study to compare the clinical efficacy of sequential and single intra-articular injections of PRP in the treatment of KOA.

All of the participants provided their written informed consent to participate in this study before their data were stored in the hospital database and used for research purposes. The work has been reported in line with the STROCSS criteria (13).

A total of 94 KOA patients were finally included, including 55 cases in single PRP group and 39 cases in sequential PRP group. The flow chart of the inclusion and exclusion of patients was shown in Figure 1. No significant differences were found in age (P = 0.394), gender (P = 0.832), body mass index (BMI) (P = 0.893), K–L grade (P = 0.355) and course of disease (P = 0.738) between the two groups (Table 1).

There were no significant differences in pre-treatment serum IL-1β, IL-6, and TNF-α between the two groups (P = 0.369, 0.365, and 0.571, respectively). At 6-month post-treatment, serum IL-1β, IL-6, and TNF-α were all significantly decreased compared with those pre-treatment in each group (P < 0.001 for all the three outcomes), and the sequential PRP group showed lower serum IL-1β, IL-6, and TNF-α than the single PRP group (P < 0.001 for all the three outcomes; Figure 2).

No significant differences were found in pre-treatment synovial fluid MMP-3 and TIMP-1 between the two groups (P = 0.250 and 0.417, respectively). At 6-month post-treatment, synovial fluid MMP-3 was significantly decreased (P < 0.001) and synovial fluid TIMP-1 was significantly increased (P < 0.001) compared with those pre-treatment in each group. Compared with the single PRP group, the sequential PRP group showed a lower synovial fluid MMP-3 (P < 0.001) and a higher TIMP-1 (P = 0.018) at 6-month post-treatment (Figure 3).

There were no significant differences in VAS score, WOMAC score and Lysholm score between the two groups at pre-treatment, 1 and 3 months post-treatment (VAS score: P = 0.149, 0.952, and 0.542, respectively; WOMAC score: P = 0.489, 0.789, and 0.893, respectively; Lysholm score: P = 0.499, 0.541, and 0.350, respectively). The VAS score, WOMAC score and Lysholm score were all significantly improved in each group at 1 month post-treatment compared with those pre-treatment (P < 0.001 for both groups), and all had a certain degree of efficacy loss at 3 and 6 months post-treatment (P < 0.001 for both groups). However, compared with single PRP group, sequential PRP had a lower VAS score and WOMAC score, and a higher Lysholm score at 6 months post-treatment (P = 0.005, P < 0.001, and P < 0.001, respectively; Figure 4).

A total of five patients had complications in sequential PRP group, including four cases of knee joint redness and one case of transient fever. While three patients of complications were found in single PRP group, including one cases of knee joint redness and two cases of knee joint pain. No significant difference was found in complications rate (12.82% vs 5.45%) between the two groups (P = 0.269) and all cases were cured after active treatment.

KOA is a degenerative disease characterized by pain and deformity, resulting from various etiological factors that lead to articular cartilage fibrosis, rupture, detachment, ulceration, and even cartilage loss (2). The destruction of chondrocytes and the cartilage matrix generates substantial metabolites, and the accumulation of type II collagen carboxyl-termini and chondrofibrillar proteins in the synovial cavity promotes the production and activation of inflammatory cytokines and related degrading enzymes, thereby exacerbating cartilage destruction and disrupting the equilibrium between progressive cartilage damage and extracellular matrix formation (2, 9). It has also been revealed that the development of KOA is closely associated with multiple cytokines and proteins (17). Notably, matrix metalloproteinases (MMPs) break down the extracellular matrix of articular cartilage, leading to thinning of the cartilage layer and reduced resistance to external stimuli, which further aggravates cartilage damage (18). Additionally, inflammatory factors also play a significant role in the progression of KOA (19).

MMP-3 directly damages the articular cartilage matrix by degrading type II collagen and proteoglycans. As a key enzyme in the cartilage degeneration associated with OA and rheumatoid arthritis (RA), MMP-3 activity correlates positively with the severity of articular cartilage damage, making it a biomarker for disease progression (20). TIMP-1 (tissue inhibitor of metalloproteinases-1) specifically inhibits MMP-3 and other matrix metalloproteinases to slow cartilage degradation. Imbalances in TIMP-1 levels (e.g., an elevated MMP-3/TIMP-1 ratio) exacerbate joint destruction (21). Additionally, TIMP-1 participates in cellular signaling and influences the inflammatory microenvironment (22). The dynamic balance between MMP-3 and TIMP-1 determines the direction of cartilage metabolism, and monitoring their levels helps assess arthritis severity and treatment efficacy.

Interleukins (ILs), cytokines secreted by leukocytes or immune cells, play core roles in immune regulation, inflammation, and hematopoietic functions. IL-1β, a pro-inflammatory factor secreted by macrophages and chondrocytes, activates NF-κB pathways to promote MMP expression, directly degrading collagen and proteoglycans, leading to cartilage destruction (23). In KOA patients, serum and synovial fluid levels of IL-1β are significantly elevated, showing a positive correlation with disease severity (24). IL-6, produced by synovial fibroblasts and chondrocytes, amplifies inflammatory responses through JAK-STAT pathways while inducing acute-phase protein synthesis (e.g., CRP), participating in subchondral bone remodeling (25). Studies demonstrate that IL-6 levels in synovial fluid of KOA patients are positively correlated with joint narrowing and osteophyte accumulation (26). TNF-α is a core pro-inflammatory factor in arthritis (especially rheumatoid arthritis). It drives the inflammatory cascade by activating the NF-κB pathway, promotes cartilage destruction and bone resorption, and is a key target for targeted therapy (27).

This study found that administering PRP therapy to early/mid stage KOA patients via single or sequential injections resulted in a significant reduction of serum inflammatory markers (including IL-1β, IL-6, and TNF-α levels). Synovial fluid analysis revealed markedly decreased MMP-3 levels and substantially increased TIMP-1 levels. These results demonstrated that both single and sequential PRP injections can effectively regulate inflammation and cytokine levels in KOA patients (28). The study findings also indicated that at 1, 3, and 6 months post-treatment, both groups showed significant improvements in VAS, WOMAC, and Lysholm scores compared to pre-treatment levels. Therefore, we confirmed that intra-articular injection of PRP can achieve satisfactory clinical efficacy in the treatment of KOA (29). However, at the 6-month follow-up, the single PRP group had lower VAS and WOMAC scores than the sequential PRP group, while the Lysholm score remained more favorable in the sequential PRP group. This suggested that single PRP treatments lack sustained long-term efficacy, with diminishing effects on knee pain and function improvement over time. Conversely, sequential PRP injections consistently yielded improved knee symptoms and functional outcomes, demonstrating excellent medium-to-long term therapeutic benefits. Therefore, for intra-articular PRP injection therapy, sequential treatments outperformed single-dose administration in effectively alleviating pain and enhancing joint function, offering superior efficacy and stability. No significant difference was found in complications between the two groups, and all complications cases were cured after active treatment, indicating the safety of the two PRP injection methods in KOA treatment. However, redness and pain in the knee joints were not uncommon in both groups. This indicated that during knee joint injections, close attention must be paid to aseptic operation and precise technique (such as puncture under ultrasound guidance). Additionally, measures such as applying ice packs within 48 h after injection to relieve swelling and using crutches or braces to assist walking to reduce joint load may also be beneficial.

This study had several limitations: first, the patient sample size was relatively small. Second, the short follow-up period made it impossible to evaluate long-term outcomes of sequential PRP treatments. Third, the assessment criteria were primarily subjective, lacking objective comparisons indicators such as knee MRI scans.

In conclusion, both sequential and single injection of PRP can achieve satisfactory clinical efficacy in the treatment of early/mid-stage unilateral KOA, but sequential intra-articular PRP injection has the advantages of sustained long-term efficacy in relieving pain and improving function of knee joint.