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Introduction

Osteoarthritis is a degenerative disease characterized by joint pain, tenderness, stiffness, joint swelling, restricted movement and joint deformities (1). In recent years, an increasing number of patients are being diagnosed with osteoarthritis, which has a notable impact on human health and quality of life (2,3). The causes of osteoarthritis are complex, and the pathogenesis related to this disease is not well understood (4). Osteoarthritis is divided into primary and secondary osteoarthritis according to the presence of local and systemic risk factors (5). Osteoarthritis is frequently diagnosed as rheumatoid arthritis or ankylosing spondylitis in clinical differential diagnosis (1,6). Previous studies have indicated that agents targeting rheumatoid arthritis are relatively ineffective at present (7,8). Therefore, there is an urgent requirement for more efficient treatments for osteoarthritis with minimal side effects.

Platelet-rich plasma (PRP) is an autologous blood sample that contains highly concentrated platelets and multiple cell growth factors. PRP promotes synovial cell proliferation and differentiation and may recover cartilage morphology (9). PRP also possesses multifunctional outcomes for the treatment of osteoarthritis, including osteonecrosis of the femoral head, cartilage injury and rheumatoid arthritis (9). Previous research has suggested that PRP exhibits benefits for injurious articular cartilage repair through the removal of harmful inflammation factors in patients with joint diseases (5). It has previously been reported that PRP was beneficial for rheumatoid arthritis without side effects through inhibition of inflammatory factor levels in synovial fluid (10). In addition, treatment-emergent adverse effects of PRP were not systematically reported in clinical investigation (10). The therapeutic outcomes of PRP isolated from autologous peripheral blood mononuclear cells, including blood products rich in cytokines, growth factors and other bio-active molecules, has been reported to be an efficient and innovative treatment protocol (11). Furthermore, a study by Sadabad et al (12) investigated the efficiency of PRP vs. hyaluronic acid for the treatment of knee osteoarthritis. A study Khoshbin et al (13) evaluated the available Level I and Level II literature on PRP as a therapeutic intervention in the management of symptomatic knee osteoarthritis in a systematic review. These reports demonstrated that intravenous injection of PRP was able to repair tendons and damaged articular bone, and primarily contribute to inflammatory elimination, which may have an important role in the morphology, collagen microarchitecture and subsequent mechanical properties of the injected vein.

Previous reports have indicated that inflammatory cytokines have an essential role in the initiation and development of osteoarthritis, targeting the synovium in joint diseases (14,15). A study by Battaglia et al (16) reported the efficacy of ultrasound-guided intra-articular injections of PRP vs. hyaluronic acid for hip osteoarthritis, which demonstrated that PRP was more effective at reducing inflammation and relieving pain. Furthermore, a study by Laudy et al (17) demonstrated that PRP injections in patients with knee osteoarthritis resulted in decreased pain, improved function and global assessment, and changes regarding joint imaging. A study by Meheux et al (18) suggested that PRP injection significantly improved validated patient-reported outcomes in patients with symptomatic knee osteoarthritis at 6 and 12 months post-injection and indicated similarities and differences in outcomes based on the PRP formulations used in the analyzed studies. These clinical reports suggest that PRP exhibits a potential efficacy in treatment of osteoarthritis.

In the present study, the efficacy and outcomes of PRP were evaluated in younger patients, aged between 18 and 30 years, with knee osteoarthritis. Inflammatory factors were analyzed following treatment with PRP or a placebo. Treatment-emergent adverse events in patients with knee osteoarthritis after PRP were also investigated in the present study. Therapeutic efficacy of PRP for knee osteoarthritis was evaluated by clinical arthritis scores. The present findings suggested that PRP has a therapeutic effect on knee osteoarthritis progression and highlighted its potential as an anti-inflammatory treatment agent for knee osteoarthritis.

Materials and methods

Ethics statement

The present phase-III study (XAJT00699978) was carried out in strict accordance with the recommendations in the Guide for Honghui Hospital of Xi'an Jiaotong University College of Medicine (Xi'an, China) between February 2009 and October 2014. Ethical approval was granted by the Defense Research Committee on the Ethics of Experiments (Honghui Hospital, Xi'an Jiaotong University College of Medicine, Xi'an, China). All patients were required to review trial protocols and amendments, and subsequently provided their informed consent.

Patients

A total of 366 patients with knee osteoarthritis, aged 18–30 years and with a Karnofsky performance status (19) ≥80% were enrolled between February 2009 and October 2014 in the present study. Patients were randomly divided into two groups and once-weekly, double-blind trials were conducted in Xi'an Jiangtong University College of Medicine. A detailed description of the inclusion/exclusion criteria, allocation method and other details can be found in previously published studies (20,21). A total of 8 ml blood was harvested from the cubital vein and centrifuged for 5 min at 1,500 × g. Patients with knee osteoarthritis received PRP (2, 4, 8, 10, 12 or 14 ml) treatment through intralesional injections and a placebo was used as a control. All patients were hospitalized throughout the duration of the study.

Study design

The present double-blind study was carried out in three phases: Baseline stage, double-blind treatment phase (4-week dose-titration treatment) and 4-week post-treatment stage for patients who volunteered to complete the ongoing extension study. Patients were randomized to once-weekly, double-blind treatment with PRP (2, 4, 8, 10, 12 or 14 ml) or placebo (10 ml normal saline). The optimal dosage of PRP was determined to be 10 ml.

Enzyme-linked immunosorbent assay (ELISA)

The plasma concentration levels of hepatocyte growth factor (HGF; ab100687), intercellular adhesion molecule 1 (ICAM-1; ab83760), osteopontin (OPN; ab91655), platelet-derived endothelial cell growth factor (PD-ECGF; ab193691), vascular endothelial growth factor (VEGF; ab119576), platelet-derived growth factor (PDGF; ab21234), insulin-like growth factor 1 (IGF-1; ab108873), transforming growth factor-β (TGF-β; ab92486), interferon-γ (IFN-γ; ab177743), interleukin (IL)-6 (ab46402), IL-17A (ab83688), tumor necrosis factor-α (TNF-α; ab181421), IL-1β and receptor activator of nuclear factor κB ligand (RANKL; ab100749) in patients with knee osteoarthritis were analyzed using ELISA kits (Abcam, Cambridge, UK). All procedures were carried out according to the manufacturer's instructions.

Magnetic resonance imaging (MRI) scanning

MRI was performed for all subjects to assess the therapeutic effects of PRP for knee osteoarthritis. A 3.0-T MRI scanner (Hitachi, Ltd., Tokyo, Japan) was used to evaluate the damaged area, joint inflammation and synovial proliferation as a marker for disease status. All data were transferred to the post-processing workstation. The data for the knee was recorded and used to calculate the degree of the lesion. Clinical osteonecrosis of the femoral head scores were evaluated using a scale of 0–2, as previously described (22). The degree of knee osteoarthritis in the joints was scored on a scale of 0–5, as previously described (23).

Efficacy and safety assessments

Efficacy assessments, including the median percent reduction scores and response rate, were analyzed in patients with knee osteoarthritis from baseline and during the 4-week treatment period. The median percent reduction scores were measured using the Karnofsky score and the analysis was conducted according to previous clinical studies (24,25). Furthermore, assessments of the most frequent treatment-emergent adverse events were evaluated in all randomized patients who received the study drug and had at least one post-dose safety assessment. Dose-response analysis was conducted after the last PRP injection (26).

Statistical analysis

All data were presented as the mean ± standard error of the mean. Differences between mean values were assessed using the Student's t-test for unpaired data. Comparisons of data between multiple groups were performed with analysis of variance followed by the Student-Newman-Keuls test. Continuous variables were reported as the mean with a 95% confidence interval (CI). Treatment effect was presented as the median reduction in knee osteoarthritis over the treatment period. Non-parametric Hodges-Lehmann estimates of median drug treatment effects and 95% CI were provided. Response rates and treatment-emergent adverse events were analyzed using the χ2 test by SPSS 20.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Patient characteristics

A total of 366 patients with knee osteoarthritis were recruited and included in the present analysis. The mean age of patients was 24 years. All patients received either the agent (PRP) or placebo. Patients were randomized into two groups and treated with PRP or placebo. At baseline, mean age, body mass index and time since knee osteoarthritis diagnosis were similar between the two groups. The characteristics of patients with knee osteoarthritis are summarized in Table I. Notably, there was a higher percentage of male patients than female patients with knee osteoarthritis. Overall, 350 patients with knee osteoarthritis completed the maintenance period of the phase III studies.

Table I. Characteristics of patients with knee osteoarthritis.

Table I.

Characteristics of patients with knee osteoarthritis.

Parameter	PRP group	Placebo group
Total patients (%)	310 (84.5%)	56 (15.5%)
Sex (F/M)	150/160	20/36
Performance status (Karnofsky)
100	121	13
90	112	25
80	77	18
Prior treatment
Surgery	85	20
Antibody therapy	103	18
Others	122	18

[i] PRP, platelet-rich plasma; F, Female; M, Male.

Duration of treatment, dose-limiting toxicity and maximum tolerated dose (MTD)

The median overall duration of PRP treatment was 8 weeks. Patients in the PRP group were treated with 2, 4, 8, 12 or 14 ml of PRP. As shown in Table II, 12 ml PRP once a week was identified as the MTD. Patients who received at least one dose of study therapy with post-baseline safety evaluation were included in the safety population. Following the last dose of PRP, it was observed that the common treatment-emergent adverse events were hypertension, diarrhea, vomiting, lethargy, rash, proteinuria, fatigue, constipation, weight decrease, appetite decrease, epistaxis, hypertriglyceridemia and peripheral edema (Table II). The most frequent treatment-emergent adverse events with a Common Toxicity Criteria grade ≥3 were hypertension and proteinuria (≥5% each; Table III). Accordingly, treatment of PRP also presented a dose-dependent effect, and the optimum therapeutic dose PRP was identified as 10 ml. The data for the 12 (n=28) and 14 ml (n=18) doses demonstrated a high number of adverse events, so these doses were considered inadvisable and few patients were treated at these dose levels.

Table II. Treatment-related adverse events.

Table II.

Treatment-related adverse events.

Adverse event	Total (n=54)	PRP 2–4 ml (n=12)	PRP 8–10 ml (n=24)	PRP 12–14 ml (n=18)
Hypertension	11	2	5	4
Diarrhea	4	1	2	1
Proteinuria	10	2	5	3
Vomiting	1	0	1	0
Lethargy	3	0	1	2
Rash	7	2	4	1
Fatigue	3	0	2	1
Constipation	3	1	0	2
Weight decreased	2	0	1	1
Decreased appetite	1	0	0	1
Epistaxis	4	2	1	1
Hypertriglyceridemia	2	1	1	0
Edema peripheral	3	1	1	1

[i] Treatment-emergent adverse events were analyzed by χ² test. PRP, platelet-rich plasma.

Table III. Treatment-related hypertension and proteinuria by Common Toxicity Criteria grade.

Table III.

Treatment-related hypertension and proteinuria by Common Toxicity Criteria grade.

Adverse event	Total (n=54)	PRP 2–4 ml (n=12)	PRP 8–10 ml (n=24)	PRP 12–14 ml (n=18)
Hypertension	11	2	5	4
Grade 1	4	0	2	2
Grade 2	4	1	2	1
Grade 3	3	1	1	1
Proteinuria	10	2	5	3
Grade 1	2	0	1	1
Grade 2	3	0	2	1
Grade 3	5	2	2	1

[i] Treatment-emergent adverse events were analyzed by χ² test. PRP, platelet-rich plasma.

Pharmacokinetic analysis

In the presence of PRP, it was observed that the majority of patients with knee osteoarthritis kept steady-state plasma concentrations after a last dose compared with placebo (Fig. 1A). Also, the dose response rates were observed via changes in Karnofsky performance score. An improvement in clinical score was observed regardless of the presence or absence of PRP (Fig. 1B). In addition, plasma concentrations of PRP increased in a dose-dependent manner in patients receiving PRP treatment (Fig. 2A). In the population pharmacokinetic analysis, PRP plasma concentration peaked at 12 h post-treatment (Fig. 2B).

Figure 1. Response rate and median reduction of score for patients with knee osteoarthritis after treatment with PRP or placebo. (A) Response rate of patients with knee osteoarthritis during the treatment period. (B) Median reduction of score for patients with knee osteoarthritis 8 weeks from the baseline over double-blind period. Data are presented as the mean + standard error of the mean. *P<0.05, **P<0.01 and ***P<0.001 vs. placebo. PRP, platelet-rich plasma.

Figure 2. Pharmacokinetic analysis of PRP in patients with knee osteoarthritis. (A) Plasma concentrations of PRP with increasing dose. (B) Plasma concentrations of PRP (10 ml) from 0–36 h post-treatment. Hodges-Lehmann estimates of median drug treatment effect and 95% confidence intervals are provided. Data are presented as the mean ± standard error of the mean. AUC, area under the curve; PRP, platelet-rich plasma.

Inflammatory factors

Plasma concentrations of inflammatory factors were analyzed in patients treated with PRP or placebo after the last dose of treatment. As demonstrated in Fig. 3A, plasma concentrations of IL-17A (P<0.001), IL-1β (P<0.01), TNF-α (P<0.01), RANKL (P<0.01), IL-6 (P<0.01) and IFN-γ (P<0.001) were significantly downregulated after PRP treatment compared with the placebo treatment in an 8-week observation. As demonstrated in Fig. 3B, plasma concentrations of HGF (P<0.001), ICAM-1 (P<0.01), OPN (P<0.01), PD-ECGF (P<0.001), VEGF (P<0.001), PDGF (P<0.01), IGF-1 (P<0.001) and TGF-β (P<0.001) were significantly upregulated after PRP treatment compared with the placebo treatment.

Figure 3. Inflammatory and pro-angiogenic factors in patients with knee osteoarthritis after treatment with PRP (10 ml) or placebo. (A) Plasma concentrations of IL-17A, IL-1β, TNF-α, RANKL, IL-6 and IFN-γ after an 8-week observation. (B) Plasma concentration of HGF, ICAM-1, OPN, PD-ECGF, VEGF, PDGF, IGF-1 and TGF-β after an 8-week observation. Data are presented as the mean + standard error of the mean. **P<0.01 and ***P<0.001 vs. placebo. PRP, platelet-rich plasma; IL, interleukin; TNF-α, tumor necrosis factor-α; RANKL, receptor activator of nuclear factor κB ligand; IFN-γ, interferon-γ; HGF, hepatocyte growth factor; ICAM-1, intercellular adhesion molecule 1; OPN, osteopontin; PD-EGCF, platelet-derived endothelial cell growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor, IGF-1, insulin-like growth factor 1; TGF-β, transforming growth factor β.

Clinical arthritis scores

The response rates to PRP for patients with knee osteoarthritis were evaluated by clinical arthritis scores in the presence and absence of PRP. As demonstrated in Fig. 4A, PRP alleviated osteoarthritis and repaired damaged tissue determined by MRI parameters as compared with the placebo. As shown in Fig. 4B, PRP presented beneficial effects in preventing joint inflammation and synovial proliferation compared with the placebo.

Figure 4. Analysis of joint inflammation and synovial proliferation in patients with knee osteoarthritis after treatment with PRP or placebo. (A) Effect of PRP treatment on joint inflammation in patients with knee osteoarthritis. (B) Effect of PRP treatment on synovial degeneration in patients with knee osteoarthritis. All patients underwent an 8-week maintenance period by injection of PRP (10 ml). Data are presented as the mean + standard error of the mean. ***P<0.001. PRP, platelet-rich plasma.

Neovascularization and size of damaged area

It was observed that neovascularization was significantly promoted (Fig. 5A) and the damaged area significantly decreased (Fig. 5B) by PRP treatment compared with the placebo treatment (both P<0.001). These clinical outcomes indicated that PRP at MTD 12 ml dose improved the clinical features of knee osteoarthritis.

Figure 5. Analysis of neovascularization and the damaged area in synovial grafts in patients with knee osteoarthritis after treatment with PRP or placebo. (A) Destruction of the cartilage and neovascularization analysis after treatment with PRP or placebo. (B) Histological analysis of synovial damaged area after treatment with PRP or placebo. Data are presented as the mean + standard error of the mean. ***P<0.001. PRP, platelet-rich plasma.

Discussion

The purpose of the present study was to demonstrate the efficacy and safety of PRP in patients with knee osteoarthritis, and in turn provide a rationale for PRP dosing recommendations. It was observed that PRP not only alleviated inflammation through humoral and cellular immune responses, but also had beneficial effects on MRI parameters., which was consistent with a previous study (20). As overall PRP therapeutic effects in the articular environment derive from an interaction with the pre-existing environment and other cells, and some surgical protocols involve the application of both platelets and cells, previous studies have investigated the effect of PRP on arthritis of different origins (25–27). Furthermore, the present investigation evaluated the MDT and treatment-emergent adverse events of PRP in the treatment of patients with knee osteoarthritis. The clinical data indicated that the most common treatment-emergent adverse events were hypertension and proteinuria (≥10%), which was consistent with adverse events in the overall phase II study (27). The present results also indicated that PRP-treated patients with knee osteonecrosis had markedly improved synovial hyperplasia, inflammatory cell influx, destruction of the cartilage and angiogenesis analyzed by histological staining, as compared with the placebo-treated patients. These preclinical data suggest that PRP may be an effective agent for treatment of osteonecrosis of the femoral head.

Osteoarthritis is a serious chronic degenerative disease that affects patient health and quality of life (24). Although osteoarthritis may affect all human joints, knee osteoarthritis is the most common type among adolescents and adults (25). In recent years, non-surgical treatments for knee osteoarthritis have become more widely used, such as PRP, corticosteroid injection and hyaluronic acid (25,28). A previous study described various methods for knee osteoarthritis treatment, including decompression with bone morphogenic proteins, growth factors, bone grafting and stem cells (29). In addition, a systematic review and meta-analysis compared the efficacy of PRP and hyaluronic acid for treatment of knee osteoarthritis and it was demonstrated that PRP injection was more effective than hyaluronic acid in a 2-year meta-analysis (12). These results were supported by the clinical outcomes of the present study in younger patients with knee osteoarthritis.

PRP is a prominent biomedical blood product that presents efficient outcomes for the treatment of patients with knee osteoarthritis, cartilage disorders and rheumatoid arthritis (30). As PRP has been approved as an agent for knee osteoarthritis therapy, it is important for clinicians to understand the potential pharmacokinetic interactions in order to maximize the therapeutic benefits of PRP and reduce the risk of treatment-emergent adverse events. The present study revealed that repeated administration of PRP (10 ml per week) relieved the pathogenesis of knee osteoarthritis. A previous report indicated that inflammatory cytokines form a complex regulatory signal network in osteonecrosis of the femoral head that is mediated by various intracellular kinase signaling pathways to regulate recruitment, stimulation and activation of autoimmune cells (31). Although the causes of knee osteoarthritis are not fully understood, laboratory and clinical evidence has suggested that inflammatory cytokines may contribute to its pathogenesis (32,33). Theoretically, blocking inflammatory factor pathways may interrupt the inflammatory process and limit joint damage (34,35). In the present study, clinical results indicated that inflammation factors were regulated following PRP treatment, which has not been previously reported. Furthermore, the stimulatory effects of PRP treatment have been demonstrated to promote proliferation and chondrogenic differentiation, which may produce beneficial molecules for the maintenance of articular cartilage perform (23,36). The results of the present study suggest that PRP treatment improves inflammatory cell influx and angiogenesis.

Although a previous study has reported the direct effects of various drugs and PRP on knee osteoarthritis, it is essential to investigate the overall role of PRP in affecting the entire joint cytokine homeostasis (37). PRP has a long half-life and therefore is beneficial for treatment of knee osteoarthritis as PRP may be expected to degrade slowly (32). The results of the present study suggested that pharmacokinetic interactions of PRP are important determinants in optimizing therapy for knee osteoarthritis. Therefore, it is necessary for clinicians to monitor clinical responses and tolerability when patients are treated with PRP. In conclusion, the present findings indicate that PRP treatment for patients with knee osteoarthritis had beneficial effects in regulating inflammatory factors, and alleviating joint inflammation, cartilage destruction and bone damage.

References