Impact of Vascular Endothelial Growth Factor Concentration on the Short-term Efficacy of Platelet-Rich Plasma (PRP) Therapy for Knee Osteoarthritis

Abstract

Introduction

This study investigates the association between vascular endothelial growth factor (VEGF) levels and platelet-rich plasma (PRP) treatment outcomes, as well as the role of other cytokines in symptom improvement.

Methods

Thirty-nine patients with knee osteoarthritis (KOA) who underwent PRP therapy were analyzed. Cytokine and growth factor levels in PRP were measured, and clinical outcomes were assessed using the visual analog scale (VAS) and the Knee Injury and Osteoarthritis Outcome Score (KOOS) before and 1 month after a single intra-articular PRP injection. Correlations between cytokine levels and clinical improvements were evaluated.

Results

Age correlated positively with C-X-C motif chemokine ligand 9 (CXCL9) (r = 0.50, p < 0.001). Body mass index (BMI) correlated negatively with interleukin-10 (IL-10) and positively with interleukin-18 (IL-18). Elevated IL-18 levels correlated with worse KOOS-Activities of Daily Living (ADL) improvements (r = -0.410, P = 0.01), linking obesity, inflammation, and reduced PRP efficacy. While VEGF showed no association with patient background, higher VEGF levels correlated with poorer VAS score improvements (r = −0.381, P = 0.017), suggesting reduced PRP efficacy. A VEGF cut-off of 120 pg/ml identified non-responders with 82.6% sensitivity, 56.2% specificity, and an area under the curve (AUC) of 0.71. Among patients with VEGF ≥120 pg/ml, the response rate was 26.9%, while those with VEGF <120 pg/ml had 75%.

Conclusions

Higher VEGF concentrations in PRP were associated with reduced short-term clinical efficacy in patients with knee osteoarthritis. VEGF may serve as a predictive biomarker for PRP treatment response.

Keywords

platelet-rich plasma vascular endothelial growth factor inflammatory cytokines knee osteoarthritis

Introduction

Knee osteoarthritis (KOA) is one of the most prevalent chronic diseases among older adults, leading to significant pain and functional impairment.¹ As the global aging population increases, KOA is becoming a major public health concern, with rising socioeconomic costs and a growing need for effective treatment options. While total knee arthroplasty remains the definitive treatment for end-stage KOA, many patients seek conservative therapies to delay surgery, necessitating alternative interventions with fewer side effects.

Platelet-rich plasma (PRP) therapy has gained attention in orthopedics as a promising option for managing KOA. Numerous randomized controlled trials (RCTs) and meta-analyses have demonstrated that PRP can be more effective than placebo, corticosteroids, and hyaluronic acid (HA) in improving clinical outcomes.^2-5 However, some studies have reported less favorable results, highlighting the inconsistency in PRP efficacy.⁶

The variability in PRP quality is a significant concern, as its composition and bioactive properties can differ based on preparation methods and individual patient factors. Upon activation, platelets in PRP release granular contents, including various growth factors essential for initiating and sustaining the healing response.⁷ In addition, PRP exerts anti-inflammatory effects by modulating the nuclear factor κB signaling pathway in various cell types, including synovial cells, macrophages, and chondrocytes.⁸ Moreover, the inflammatory state of the patient at the time of PRP preparation may influence the cytokine profile within PRP, potentially impacting its therapeutic efficacy. Despite its multifaceted role in promoting joint health, the precise mechanisms underlying PRP’s effectiveness remain unclear.

Several factors have been identified as determinants of PRP efficacy, including patient age, body mass index (BMI), severity of OA, concentrations of cytokines, and bioactive substances.^9-11 In particular, chronic inflammation in patients with KOA may contribute to the heterogeneity of PRP treatment outcomes, as inflammatory cytokines present in PRP could counteract its regenerative effects. Furthermore, aspects such as platelet concentration and the presence of growth factors may influence therapeutic outcomes by promoting angiogenesis and creating a favorable environment for articular cartilage repair.^12,13

While PRP therapy has emerged as a viable treatment option for KOA, it is becoming increasingly evident that there are “responders” who benefit from the treatment and “non-responders” who do not.¹⁴ Among the various growth factors present in PRP, vascular endothelial growth factor (VEGF) is a notable candidate due to its dual role in both promoting healing and potentially exacerbating KOA pathology.^15,16 Elevated VEGF levels may contribute to disease progression by enhancing synovitis and increasing bone metabolism in the subchondral bone.¹³ Some studies suggest that VEGF plays a key role in cartilage degeneration and osteophyte formation, further complicating its impact on KOA progression. Previous research has reported that the administration of anti-VEGF antibodies to the knee joints of mice has a chondroprotective effect. However, the effects of VEGF in humans are not fully understood.¹⁷

This study aimed to investigate the short-term clinical outcomes of a single dose of PRP while focusing on the concentrations of inflammatory cytokines and VEGF. By elucidating these relationships, we hope to gain insights into optimizing PRP therapy for patients with KOA. By identifying key cytokine profiles associated with treatment response, we also aim to clarify the mechanisms influencing PRP efficacy and provide insights into patient selection criteria for optimizing PRP therapy in KOA.

Materials and Methods

Patients

The study included 39 people who underwent PRP therapy for KOA at the hospital between October and December 2022. Informed consent was obtained from all participants prior to peripheral blood collection. This study was approved by the Ethical Committee of Juntendo University Hospital (approval number E21-0363). Patients with systematic inflammatory diseases such as rheumatoid arthritis, active infectious diseases, poorly controlled diabetes mellitus, or platelet disorders or diseases were excluded.

Knee osteoarthritis was evaluated radiographically, and only patients with Kellgren–Lawrence (KL) grade 3 were included in the study.

Platelet-Rich Plasma Preparation

The protocol and ethics of PRP therapy were certified by a special committee for regenerative medicine based on a law regulating the safety of regenerative medicine in Japan (approval number PB3150023). The PRP preparation was obtained by single centrifugation of whole blood using the MyCells autologous platelet preparation system (Kaylight Ltd., Israel). Each kit contains 1 ml of the anticoagulant acid citrate dextrose (ACD). Here, 22 ml of whole blood was aspirated from the median cubital vein, and 5 to 6 ml of PRP was obtained according to the manufacturer’s instructions. Briefly, centrifugation was performed at 2000×g for 7 min at 21 to 25°C, and the supernatant was discarded, leaving 2.5 to 3.0 ml. The buffy coat portion immediately above the gel was pipetted and mixed before collection, and 5 to 6 ml of leukocyte-poor PRP (LP-PRP) was adjusted.

Hematological Analysis

Venous peripheral blood (whole blood) samples were collected separately from the PRP preparation kit in ethylenediaminetetraacetic acid (EDTA) tubes normally used for peripheral blood counts. A portion of the PRP used for treatment was used for hematological analysis. According to the manufacturer’s recommendations, peripheral blood and PRP samples were analyzed using an XN-1000 automated hematology analyzer (Sysmex, Kobe, Japan). From 300 μl of peripheral blood and PRP, the concentrations of erythrocytes, neutrophils, lymphocytes, monocytes, platelets, and immature platelet fraction (IPF) were measured.

Measurement of Bioactive Substance Levels

The residual samples from the PRP administration were stored at −80 degrees and then thawed just before measurement. Various biomarkers were measured using a fully automated, highly sensitive immunoassay used in research (HI-1000 [Sysmex]). This device uses the chemiluminescent enzyme immunoassay measurement principle and can measure very small amounts of protein in blood and body fluids with high sensitivity. Bioactive substance measured 5 types of protein: interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-18 (IL-18), C-X-C motif chemokine ligand 9 (CXCL9), and VEGF. The bioactive factors measured in this study were selected because they could be simultaneously analyzed using the measurement system employed, even from a small-sample volume. The measurements were performed using only 300 μl of residual PRP remaining after treatment preparation.

Outcome Evaluation

Visual analog scale (VAS) and Knee Injury and Osteoarthritis Outcome Score (KOOS) were evaluated before and after treatment. The amount of improvement before and after treatment was calculated. The relationship between improvement in clinical scores and blood cell fractions in whole blood and PRP was analyzed, as well as biomarkers in PRP. In this study, a 100-point VAS was used to assess pain intensity. The 100-point VAS is a validated and widely accepted tool that enables more sensitive detection of subtle changes in patient-reported pain, particularly in small-sample studies. Its utility has been demonstrated in various musculoskeletal and osteoarthritis-related research settings.^18-20 Clinical outcomes were assessed at both 1 and 6 months following PRP injection; however, the primary analysis focused on 1-month outcomes to evaluate the short-term response to a single injection and its association with the composition of the administered PRP. This approach was adopted in consideration of the variability in bioactive factor levels among individual PRP preparations. In addition, early clinical responses may be predictive of longer-term therapeutic effects. To assess mid-term efficacy, outcome data at 6 months were also collected and analyzed in a subset of patients (n = 33).

Statistical Analysis

The VAS and KOOS improvements before and after treatment were analyzed by paired t-test. The correlation between each cell parameter and VAS improvement was analyzed by Spearman’s correlation. The significance level was set at less than 5%. Statistical analyses were performed with SPSS version 28.0 (IBM Corp., Armonk, NY, USA).

Results

Patient Characteristics

Table 1 summarizes the characteristics of the 39 patients analyzed. The mean age was 69.6 years (± 11.3), with a higher prevalence of females. The mean BMI was 23.8 (± 4.2), indicating that most patients were within a normal weight range. The femoro-tibial angle (FTA) of 179.8° suggests a tendency toward varus knee alignment, which is commonly associated with KOA progression.

Table 1.

Demographics of the Patients in This Study.

Number of patients	39
Age (mean ± SD)	69.6 ±11.3
Sex (female/male)	28/11
BMI (mean ± SD)	23.8 ± 4.2
FTA (mean ± SD)	178.1 ± 4.8

SD, standard deviation; BMI, body mass index; FTA, femoro-tibial angle.

Blood and PRP Cell Counts

The mean concentration of leukocytes in whole blood was 9,354 ± 1,177 /μl, while the mean platelet concentration was 25.5 ± 5.0 × 10⁴/μl. In the prepared PRP, the mean leukocyte concentration was 1,037 ± 575 /μl, and the mean platelet concentration was 35.5 ± 12.5 × 10⁴/μl. The leukocyte and platelet concentrations in PRP were 0.17 ± 0.09 and 1.44 ± 0.38 times that of peripheral blood, respectively ( Table 2 ). According to the PAW classification system described by DeLong et al.,²¹ this PRP preparation is classified as P2-Bβ PRP, which corresponds to LP-PRP.

Table 2.

Cell Parameters in Whole Blood and PRP.

Cell composition	Measurement parameters		Whole blood	PRP	Concentration rate
Leukocyte	Cell concentration	Leukocyte (/μl)	9354 ± 1177	1037 ± 575	0.17 ± 0.09
		Neutrophil (/μl)	3435 ± 1306	60 ± 94	0.01 ± 0.01
		Lymphocyte (/μl)	2055 ± 501	1351 ± 1771	0.61 ± 0.65
Platelet	Cell concentration	Platelet (×10⁴/μl)	25.5 ± 5.0	35.5 ± 12.5	1.44 ± 0.38
	Cell concentration	IPF (×10⁴/μl)	0.4 ± 0.2	0.5 ± 0.3	1.22 ± 0.72
	Fraction	IPF (%)	1.7 ± 0.9	1.3 ± 0.9

IPF, immature platelet fraction.

Bioactive Substances in Platelet-Rich Plasma

Table 3 presents the concentrations of various bioactive substances measured in PRP samples. The data indicate that the concentration of IL-6 is 8.0 ± 6.4 pg/ml, while IL-10 is found at a lower concentration of 0.4 ± 0.8 pg/ml. The IL-18 exhibits a higher concentration at 346 ± 162 pg/ml, and CXCL9 is measured at 67 ± 51 pg/ml. In addition, VEGF has a concentration of 195 ± 115 pg/ml. These results provide valuable insights into the composition of the PRP samples used in this study, particularly highlighting key inflammatory cytokines, chemokines, and growth factors, with notable variability observed in the concentrations of IL-18 and VEGF across the analyzed samples.

Table 3.

Concentration of Bioactive Substances in PRP.

	Concentration in PRP (pg/ml)
IL-6	8.0 ± 6.4
IL-10	0.4 ± 0.8
IL-18	346.9 ± 162.9
CXCL9	67.0 ± 51.9
VEGF	195.0 ± 115.2

Associations Between Patient Characteristics and Biomarkers

The relationships between patient characteristics and various biomarker concentrations were examined in this study. A significant positive correlation was observed between age and the concentration of CXCL9, an inflammatory chemokine (r = 0.50, P < 0.001). Furthermore, BMI demonstrated a significant negative correlation with the anti-inflammatory cytokine IL-10 (r = −0.49, P = 0.002) and a significant positive correlation with the pro-inflammatory cytokine IL-18 (r = 0.39, P = 0.015). These correlations are summarized in Table 4 . The VEGF showed no significant correlation with any patient background factors, indicating that its levels are not directly influenced by age, BMI, or other baseline characteristics.

Table 4.

Correlation Between Patient Background and Bioactive Substances.

		IL-6	IL-10	IL-18	CXCL9	VEGF
Age	R	-0.107	0.077	0.115	0.508**	0.075
	P	0.518	0.641	0.484	<0.001	0.649
BMI	R	0.305	-0.493**	0.398*	0.2	0.139
	P	0.067	0.002	0.015	0.235	0.411
FTA	r	0.069	-0.225	0.18	0.182	0.178
	P	0.719	0.232	0.342	0.335	0.348
%MA	r	-0.154	0.232	-0.022	-0.048	-0.131
	P	0.461	0.264	0.917	0.819	0.532

BMI, body mass index; FTA, femoro-tibial angle.

Bold-faced values indicate statistically significant differences.

p < 0.01; * p< 0.05.

Effects of Platelet-Rich Plasma on Clinical Outcomes

Table 5 presents the VAS scores and KOOS before and 1 month after a dose of PRP injection. The VAS score demonstrated a statistically significant improvement, decreasing from 52.9 ± 27.8 before treatment to 40.3 ± 27.8 1 month after the PRP injection (P = 0.007). In contrast, the KOOS showed mixed results across its subscales. The symptoms subscale improved slightly from 64.6 ± 18.1 to 67.7 ± 21.7; however, this change was not statistically significant (P = 0.24). The pain subscale improved from 55.2 ± 20.4 to 60.0 ± 21.7, but this also did not reach statistical significance (P = 0.10). Similarly, the activity of daily living subscale showed a minor increase from 76.2 ± 15.2 to 77.5 ± 15.1, with no significant difference (P = 0.40). Finally, the quality of life subscale improved from 37.5 ± 21.3 to 42.7 ± 20.6, yet this change was not statistically significant either (P = 0.10). In contrast, at the six-month follow-up, all clinical scores showed statistically significant improvements. The VAS score further declined to 31.5 ± 25.1 (P < 0.001), while KOOS subscales improved markedly: symptoms to 76.9 ± 18.5 (P = 0.005), pain to 74.1 ± 21.2 (P < 0.001), activity of daily living to 84.9 ± 16.0 (P = 0.004), and quality of life to 61.5 ± 30.1 (P < 0.001) ( Table 5 ). These findings suggest that while PRP provided early pain relief, its full therapeutic benefits—including functional and quality of life improvements—may become more evident with longer-term follow-up.

Table 5.

Visual Analogue Scale (VAS) scores and Knee Injury and Osteoarthritis Outcome Score (KOOS) Before and 1 Month After a Single PRP Injection One and Six Months After Treatment.

Clinical score	Before	After 1 month	P	After 6 months	P
VAS	52.9 ± 27.8	40.3 ± 27.8	0.007	31.5 ± 25.1	<0.001
KOOS (symptom)	64.6 ± 18.1	67.7 ± 21.7	0.24	76.9 ± 18.5	0.005
KOOS (pain)	55.2 ± 20.4	60.0 ± 21.7	0.10	74.1 ± 21.2	<0.001
KOOS (activity of daily living)	76.2 ± 15.2	77.5 ± 15.1	0.40	84.9 ± 16.0	0.004
KOOS (quality of life)	37.5 ± 21.3	42.7 ± 20.6	0.10	61.5 ± 30.1	<0.001

VAS, visual analogue scale; KOOS, Knee Injury and Osteoarthritis Outcome Score.

Bold-faced values indicate statistically significant differences.

Cytokine Correlations With Visual Analog Scale and Knee Injury and Osteoarthritis Outcome Score Improvements

Table 6 presents correlations between various bioactive substances (IL-6, IL-10, IL-18, and CXCL9) and clinical outcomes. No significant correlations were found between cytokines and VAS scores. However, IL-18 negatively correlated with KOOS-Activities of Daily Living (ADL) improvements (r = −0.410, P = 0.01), suggesting that higher IL-18 levels were associated with less improvement in daily activities. As shown in Table 6 , no significant correlations were observed between these cytokines and clinical outcomes at 6 months, indicating that the associations may be limited to short-term effects.

Table 6.

Correlation Between Bioactive Substances and Improvement in VAS and KOOS at 1 and 6 Months After Treatment.

Clinical score	Timepoint	IL-6(r / p)	IL-10(r / p)	IL-18(r / p)	CXCL9(r / p)
VAS	1 month	0.148 / 0.369	0.066 / 0.689	-0.148 / 0.367	0.063 / 0.701
VAS	6 months	0.134 / 0.457	0.019 / 0.916	0.265 / 0.137	0.170 / 0.343
KOOS (symptom)	1 month	-0.203 / 0.215	0.045 / 0.786	-0.063 / 0.705	-0.138 / 0.402
KOOS (symptom)	6 months	-0.025 / 0.893	-0.010 / 0.956	0.126 / 0.491	0.124 / 0.500
KOOS (pain)	1 month	-0.127 / 0.442	-0.010 / 0.954	-0.165 / 0.315	-0.057 / 0.728
KOOS (pain)	6 months	-0.040 / 0.829	-0.140 / 0.444	0.053 / 0.774	0.113 / 0.538
KOOS (activity of daily living)	1 month	-0.304 / 0.063	-0.043 / 0.798	-0.410 / 0.010*	-0.074 / 0.659
KOOS (activity of daily living)	6 months	-0.077 / 0.674	-0.198 / 0.278	0.155 / 0.398	0.121 / 0.510
KOOS (quality of life)	1 month	0.117 / 0.478	-0.173 / 0.293	0.275 / 0.090	0.107 / 0.518
KOOS (quality of life)	6 months	0.134 / 0.464	-0.115 / 0.530	0.171 / 0.348	0.032 / 0.860

VAS, visual analogue scale; KOOS, Knee Injury and Osteoarthritis Outcome Score.

Bold-faced values indicate statistically significant differences.

p < 0.05.

Vascular Endothelial Growth Factor and Clinical Outcomes

Table 7 illustrates the correlation between VEGF levels and improvements in VAS and KOOS. The VEGF negatively correlated with VAS score improvement (r = -0.381, P = 0.017), suggesting that higher VEGF levels may attenuate PRP’s pain-relieving effects. In contrast, no significant correlations were observed between VEGF and the KOOS subscales. As shown in Table 7 , no significant correlations were observed between these cytokines and clinical outcomes at six months, suggesting that the associations may be limited to short-term effects. However, it is also possible that patients who were non-responders after a single injection may have become responders following multiple injections. Since VEGF levels were evaluated only after the first injection in this study, it is conceivable that temporal changes in VEGF levels during subsequent injections influenced the clinical outcomes.

Table 7.

Correlation Between VEGF and Improvement in VAS and KOOS at 1 and 6 Months After Treatment.

Clinical score	Timepoint	r	P
VAS	1 month	-0.381	0.017*
VAS	6 months	0.005	0.976
KOOS (symptom)	1 month	-0.149	0.364
KOOS (symptom)	6 months	-0.022	0.905
KOOS (pain)	1 month	-0.134	0.415
KOOS (pain)	6 months	-0.095	0.604
KOOS (activity of daily living)	1 month	-0.037	0.825
KOOS (activity of daily living)	6 months	0.007	0.969
KOOS (quality of life)	1 month	-0.024	0.886
KOOS (quality of life)	6 months	-0.234	0.198

VAS, Visual Analogue Scale; KOOS, Knee Injury and Osteoarthritis Outcome Score.

Bold-faced values indicate statistically significant differences.

indicates p < 0.05.

Based on the criteria by Concoff et al.,²² a VAS improvement of ≥15 was defined as a responder. The mean VEGF level was 147.1 ± 99.1 pg/ml in responders and significantly higher at 228.3 ± 115.8 pg/ml in non-responders (P = 0.028) ( Fig. 1 ). A VEGF cut-off value of 120 pg/ml was identified for classifying non-responders, with 82.6% sensitivity, 56.2% specificity, and an AUC of 0.71 ( Fig. 2 ) (95% confidence interval (CI) = 0.54-0.88). The relative risk for non-response at VEGF ≥120 pg/ml was 4.75 (95% CI = 1.10-20.39).

Figure 1.

VEGF levels in the group of responders and the group of non-responders. We defined a VAS improvement of 15 or more as a responder and divided the subjects into a responder group and a non-responder group. VEGF concentration was significantly higher in the non-responder group (P = 0.028). * indicates P < 0.05.

Figure 2.

ROC curve for VEGF concentration. The cut-off value for VEGF concentration used to classify patients as non-responders was determined to be 120 pg/ml, with a sensitivity of 82.6%, specificity of 56.2%, and an area under the curve (AUC) of 0.71, indicating a relative risk of 4.75.

When stratified by VEGF levels, patients with VEGF ≥120 pg/ml (n = 26) had a response rate of 26.9% (7/26), whereas those with VEGF <120 pg/ml (n = 13) had a significantly higher response rate of 75% (9/13) ( Table 8 ) (χ² (1) = 6.41, P = 0.011). These results suggest that VEGF concentration may serve as a useful predictor of PRP treatment response in KOA patients, providing a potential basis for personalized treatment strategies. According to Table 8 , although the responder rate at 6 months remained higher in the group classified by an initial (first-injection) VEGF level of <120 pg/ml, the difference was not statistically significant (χ² (1) = 0.90, P = 0.45).

Table 8.

Response Rate in Patients Above and Below the VEGF Cut-Off at One and Six Months After Treatment.

	VEGFconcentration	Non-responder(patients)	Responder(patients)	Responder rate
One month after treatment	≥120 pg/ml	19	7	26.9%
One month after treatment	<120 pg/ml	4	9	75.0%
Six months after treatment	≥120 pg/ml	11	12	52.1%
Six months after treatment	<120 pg/ml	3	7	70.0%

Discussion

Vascular Endothelial Growth Factor and Platelet-Rich Plasma Treatment Response

The most significant finding of this study is the relationship between higher levels of VEGF and reduced effectiveness of PRP treatment. Specifically, this study’s analysis revealed a statistically significant negative correlation between VEGF levels and improvements in pain intensity, as measured by the VAS. This suggests that elevated VEGF concentrations may inhibit the therapeutic effects of PRP, possibly by exacerbating synovial inflammation and promoting angiogenesis in subchondral bone, both of which have been implicated in KOA progression. Prior studies have suggested that excessive VEGF activity contributes to joint degradation, osteophyte formation, and increased vascular permeability, leading to persistent pain and inflammation.¹⁶ These findings underscore the importance of considering VEGF levels when evaluating PRP's efficacy in clinical applications. Patients with VEGF <120 pg/ml showed a higher responder rate at both time points, though the difference was not statistically significant at 6 months. Since VEGF levels were measured only before the first injection, the influence of VEGF on long-term outcomes remains unclear. Given the variability in PRP composition and the heterogeneous response to treatment, VEGF concentration could serve as a potential biomarker for predicting treatment outcomes. Stratifying patients based on VEGF levels may help identify those who are more likely to benefit from PRP therapy, leading to more personalized treatment approaches. Additionally, modifying PRP preparation protocols to reduce VEGF concentrations or combining PRP with VEGF inhibitors could be explored as strategies to optimize therapeutic efficacy.

The Role of Vascular Endothelial Growth Factor in Osteoarthritis Progression

The VEGF plays a crucial role in the pathogenesis of KOA, contributing to cartilage degeneration, osteophyte formation, synovial inflammation, and pain.¹⁶ For instance, Nagao et al.¹⁷ demonstrated that administering anti-VEGF antibodies into the knee joints of rats effectively prevented cartilage degeneration. Other studies have indicated that local inhibition of VEGF signaling can reduce pain and mitigate cartilage degradation.²³ When PRP with reduced VEGF levels was administered to a rat model of OA, cartilage destruction was significantly less pronounced compared to PRP alone.²⁴ These findings suggest that adjusting VEGF concentrations in PRP could enhance its therapeutic effects, as excessive VEGF activity may counteract PRP’s regenerative properties by promoting angiogenesis and inflammation within the joint. To counteract the negative effects of VEGF in PRP, the use of anti-VEGF antibodies presents a promising avenue, potentially improving pain relief and cartilage protection in PRP therapy.

The Influence of Other Cytokines on Platelet-Rich Plasma Efficacy

Beyond VEGF, other cytokines also appear to influence PRP efficacy, particularly IL-18 and IL-10, which are closely linked to systemic inflammation and obesity. IL-18, a pro-inflammatory cytokine, was associated with poorer improvements in the KOOS-ADL subscale when present at higher levels in PRP ( Table 6 ). Furthermore, IL-18 showed a positive correlation with BMI ( Table 4 ), suggesting that obesity-related systemic inflammation may contribute to elevated IL-18 levels, which could impair the regenerative capacity of autologous PRP therapy. Conversely, the anti-inflammatory cytokine IL-10 exhibited a negative correlation with BMI ( Table 4 ), indicating that obese patients may have a reduced anti-inflammatory response, further exacerbating the inflammatory environment in KOA. Given that many KOA patients have elevated BMI, these findings highlight the importance of considering patient-specific inflammatory profiles when evaluating PRP efficacy. In addition, a significant positive correlation was observed between CXCL9 levels and patient age ( Table 4 ). CXCL9 is a chemokine involved in immune cell recruitment and is known to increase with age-related inflammation. Its elevation may reflect a pro-inflammatory state associated with immunosenescence, which could contribute to reduced PRP efficacy in older patients.^25,26 The presence of high levels of inflammatory cytokines in PRP prepared from KOA patients may diminish treatment efficacy, potentially explaining the heterogeneity in clinical responses. However, as shown in the 6-month follow-up data, none of the cytokines demonstrated significant correlations with clinical outcome scores, suggesting that their influence may be limited to short-term effects.

The Potential of Allogeneic Platelet-Rich Plasma and Platelet-Rich Plasma Modifications

This study raises the possibility that PRP derived from allogeneic sources, such as healthy donors or umbilical cord blood, could offer a more standardized and effective alternative for patients with chronic inflammation and obesity-related KOA.^27,28 However, challenges such as immunogenicity, ethical considerations, and regulatory approval must be addressed before allogeneic PRP can be widely adopted. Alternatively, autologous PRP modification strategies, such as selective cytokine removal or PRP conditioning, could provide a patient-specific approach to optimizing treatment outcomes.

Systemic Inflammation and Platelet-Rich Plasma Variability

In addition, this study’s analysis revealed that BMI was negatively correlated with IL-10 while IL-18 showed a positive correlation; CXCL9 was positively correlated with age ( Table 4 ). These findings suggest that, in obese and older patients, anti-inflammatory cytokines such as IL-10 may be reduced, while inflammatory cytokines like IL-18 may be elevated, potentially exacerbating the inflammatory environment in KOA. Inflammatory cytokines play a significant role in OA pathogenesis; when the balance between inflammatory and anti-inflammatory cytokines is disrupted—resulting in a predominance of inflammatory cytokines—catabolic enzymes, matrix metalloproteinases, and pro-osteogenic factors are secreted, accelerating disease progression.²⁸ This imbalance can lead to morphological changes within the joint, including cartilage degeneration, subchondral bone remodeling, and synovitis. Furthermore, blood levels of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha are known to be 2 to 4 times higher in elderly individuals compared to younger ones, even in the absence of chronic disease.²⁸ Obese patients are also reported to be more susceptible to inflammation due to adipose tissue-derived cytokines, known as adipokines, which further promote synovial inflammation and cartilage degradation.²⁹ However, accurately determining cytokine levels in PRP remains challenging due to fluctuations influenced by systemic inflammatory status, exercise, and circadian variations in platelet activity.^30,31

Limitations and Future Directions

This study has several limitations that should be considered when interpreting the findings. The relatively small-sample size may limit the generalizability of the results to the broader KOA population. In addition, the short follow-up period may not reflect long-term clinical outcomes, including the durability of PRP’s effects and delayed responses to VEGF levels. As the study focused on short-term efficacy following a single PRP injection, clinical outcomes such as VAS showed improvement, while KOOS scores demonstrated minimal change during this short observation period. This limited improvement in KOOS may reflect the need for longer-term follow-up to assess functional recovery more comprehensively.

Moreover, the concentration of bioactive factors in PRP can vary with each blood draw, even from the same patient. To minimize this variability and clarify the relationship between biomarker levels and treatment response, the outcomes of 1 month after a single injection were evaluated. Furthermore, only a limited number of bioactive factors were measured due to assay constraints and the small volume of residual PRP, which may not fully capture the complexity of PRP’s biological activity.

Cytokine levels may also be affected by systemic inflammation, recent physical activity, or circadian variation, contributing to variability. The inherent heterogeneity of PRP—due to centrifugation protocols, leukocyte content, and individual patient characteristics—may also influence treatment response. Furthermore, this study included only patients with advanced OA (KL grade 3), potentially limiting applicability to earlier disease stages. Finally, the absence of a control group makes it difficult to attribute the observed effects solely to PRP treatment or VEGF levels.

In the future, we aim to further investigate the efficacy of PRP and anti-VEGF strategies at the cellular and molecular levels, using in vitro models to elucidate underlying mechanisms. Integrating these findings with well-designed clinical trials incorporating larger cohorts, standardized PRP preparation protocols, and extended follow-up periods will be essential to validating PRP’s therapeutic potential and optimizing patient selection criteria.

This study demonstrates that higher levels of VEGF are associated with reduced PRP efficacy in patients with KOA, suggesting that VEGF may counteract PRP’s therapeutic effects. In addition, IL-18 and IL-10 were significantly correlated with BMI, indicating that systemic inflammation may influence treatment outcomes. These findings highlight the potential role of cytokine profiling in optimizing PRP therapy and suggest that VEGF and IL-18 could serve as biomarkers for predicting response. Personalized PRP formulations or strategies to modulate VEGF levels may enhance therapeutic efficacy. Future research should focus on refining PRP preparation techniques and exploring alternative sources of PRP to improve outcomes for patients with diverse inflammatory profiles.

Footnotes

Acknowledgements

The authors would like to thank the staff of the PRP therapy unit, including Hisako Homma, Maya Kobayashi, Akiko Mutaguchi, Naoko Tomoda, Kyoko Takamune, Saori Mizukawa, and Akiko Kawai, for their support with data collection.

ORCID iD

Yoshitomo Saita

Ethical Considerations

The Juntendo University Medical Ethics Committee approved this study (approval number E21-0363).

Consent to Participate

Informed consent was obtained from all participants prior to peripheral blood collection.

Consent for Publication

Written informed consent for publication of the clinical details and/or images was obtained from the patient.

Author Contributions

Study design: Y.S., N.Y., and M.I.; conducting the study: Y.S., Y.K., T.W., S.U., N.Y., H.K., and M.I.; data collection: Y.S. and N.Y.; data analysis: N.Y. and Y.S.; data interpretation: Y.S., Y.K., and N.Y.; drafting of the manuscript: Y.S. and N.Y.; revision of manuscript content: Y.S., Y.K., H.K., and M.I.; Y.S. took responsibility for the integrity of the data analysis. All the authors have read and agreed to the published version of the manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was partially funded by Sysmex Corporation (costs related to measuring the blood cells, cytokines, and growth factors using the Sysmex XN System and HI-1000 systems).

Declaration of Conflicting Interests

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

Data Availability Statement

Data supporting the findings of this study are available from the corresponding author, Y.S., upon reasonable request.

References

Yoshimura

Muraki

Oka

Mabuchi

En-Yo

Yoshida

, et al Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: the research on osteoarthritis/osteoporosis against disability study. J Bone Miner Metab. 2009;27(5):620-8. doi:10.1007/s00774-009-0080-8.

Yoshioka

Arai

Sugaya

Taniguchi

Kanamori

Gosho

, et al The effectiveness of leukocyte-poor platelet-rich plasma injections for symptomatic mild to moderate osteoarthritis of the knee with joint effusion or bone marrow lesions in a Japanese population: a randomized, double-blind, placebo-controlled clinical trial. Am J Sports Med. 2024;52(10):2493-502. doi:10.1177/03635465241263073.

Chu

Duan

Tao

, et al Intra-articular injections of platelet-rich plasma decrease pain and improve functional outcomes than sham saline in patients with knee osteoarthritis. Knee Surg Sports Traumatol Arthrosc. 2022;30(12):4063-71. doi:10.1007/s00167-022-06887-7.

Dubin

Leucht

Murray

Pezold

, Staff of the American Academy of Orthopaedic Surgeons on Behalf of the Platelet-Rich Plasma (PRP) for Knee Osteoarthritis Technology Overview Workgroup and Contributors. American Academy of Orthopaedic Surgeons technology overview summary: platelet-rich plasma (PRP) for knee osteoarthritis. J Am Acad Orthop Surg. 2024;32(7):296-301. doi:10.5435/JAAOS-D-23-00957.

Filardo

Previtali

Napoli

Candrian

Zaffagnini

Grassi

. PRP injections for the treatment of knee osteoarthritis: a meta-analysis of randomized controlled trials. Cartilage. 2021;13(suppl 1):364S-375S. doi:10.1177/1947603520931170.

Bennell

Paterson

Metcalf

Duong

Eyles

Kasza

, et al Effect of intra-articular platelet-rich plasma vs placebo injection on pain and medial tibial cartilage volume in patients with knee osteoarthritis: the RESTORE randomized clinical trial. JAMA. 2021;326(20):2021-30. doi:10.1001/jama.2021.19415.

Rendu

Brohard-Bohn

. The platelet release reaction: granules’ constituents, secretion and functions. Platelets. 2001;12(5):261-73. doi:10.1080/09537100120068170.

Marcu

Otero

Olivotto

Borzi

Goldring

. NF-kappaB signaling: multiple angles to target OA. Curr Drug Targets. 2010;11(5):599-613. doi:10.2174/138945010791011938.

Saita

Kobayashi

Nishio

Wakayama

Fukusato

Uchino

, et al Predictors of effectiveness of platelet-rich plasma therapy for knee osteoarthritis: a retrospective cohort study. J Clin Med. 2021;10(19):4514. doi:10.3390/jcm10194514.

10.

Park

Kim

Lee

. Clinical efficacy of platelet-rich plasma injection and its association with growth factors in the treatment of mild to moderate knee osteoarthritis: a randomized double-blind controlled clinical trial as compared with hyaluronic acid. Am J Sports Med. 2021;49(2):487-96. doi:10.1177/0363546520986867.

11.

Kobayashi

Saita

Nishio

Ikeda

Takazawa

Nagao

, et al Leukocyte concentration and composition in platelet-rich plasma (PRP) influences the growth factor and protease concentrations. J Orthop Sci. 2016;21(5):683-9. doi:10.1016/j.jos.2016.07.009.

12.

Uchino

Saita

Wada

Kobayashi

Wakayama

Nishio

, et al The immature platelet fraction affects the efficacy of platelet rich plasma therapy for knee osteoarthritis. Regen Ther. 2021;18:176-81. doi:10.1016/j.reth.2021.06.004.

13.

Andia

Maffulli

. Platelet-rich plasma for managing pain and inflammation in osteoarthritis. Nat Rev Rheumatol. 2013;9(12):721-30. doi:10.1038/nrrheum.2013.141.

14.

Zahir

Dehghani

Yuan

Chinenov

Kim

Burge

, et al In vitro responses to platelet-rich-plasma are associated with variable clinical outcomes in patients with knee osteoarthritis. Sci Rep. 2021;11(1):11493. doi:10.1038/s41598-021-90174-x.

15.

Vitale

Vandenbulcke

Chisari

Iacono

Lovato

Di Matteo

, et al Innovative regenerative medicine in the management of knee OA: the role of Autologous Protein Solution. J Clin Orthop Trauma. 2019;10(1):49-52. doi:10.1016/j.jcot.2018.08.019 [Epub 2018 August 23. Erratum. J Clin Orthop Trauma. 2020;11:1172-4. doi:10.1016/j.jcot.2020.10.044. doi:10.1016/j.jcot.2020.10.026. In: 1169-1171. doi:10.1016/j.jcot.2020.09.032.]

16.

Hamilton

Nagao

Levine

Chen

Olsen

. Targeting VEGF and its receptors for the treatment of osteoarthritis and associated pain. J Bone Miner Res. 2016;31(5):911-24. doi:10.1002/jbmr.2828.

17.

Nagao

Hamilton

Berendsen

Duan

Cheong

, et al Vascular endothelial growth factor in cartilage development and osteoarthritis. Sci Rep. 2017;7(1):13027. doi:10.1038/s41598-017-13417-w.

18.

Huskisson

. Measurement of pain. Lancet. 1974;2(7889):1127-31. doi: 10.1016/s0140-6736(74)90884-8.

19.

Salaffi

Stancati

Silvestri

Ciapetti

Grassi

. Minimal clinically important changes in chronic musculoskeletal pain intensity measured on a numerical rating scale. Eur J Pain. 2004;8(4):283-91. doi:10.1016/j.ejpain.2003.09.004.

20.

Bijur

Silver

Gallagher

. Reliability of the visual analog scale for measurement of acute pain. Acad Emerg Med. 2001;8(12):1153-7. doi:10.1111/j.1553-2712.2001.tb01132.x.

21.

DeLong

Russell

Mazzocca

. Platelet-rich plasma: the PAW classification system. Arthroscopy. 2012;28(7):998-1009. doi:10.1016/j.arthro.2012.04.148.

22.

Concoff

Rosen

Bhandari

Boyer

Karlsson

, et al A comparison of treatment effects for nonsurgical therapies and the minimum clinically important difference in knee osteoarthritis: a systematic review. JBJS Rev. 2019;7(8):e5. doi:10.2106/JBJS.RVW.18.00150.

23.

Sotozawa

Kumagai

Ishikawa

Yamada

Inoue

Inaba

. Bevacizumab suppressed degenerative changes in articular cartilage explants from patients with osteoarthritis of the knee. J Orthop Surg Res. 2023;18(1):25. doi:10.1186/s13018-023-03512-2.

24.

Lee

Guo

Klett

Hall

Sinha

Ravuri

, et al VEGF-attenuated platelet-rich plasma improves therapeutic effect on cartilage repair. Biomater Sci. 2022;10(9):2172-81. doi:10.1039/d1bm01873f.

25.

Campisi

d’Adda di Fagagna

. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007;8(9):729-40. doi:10.1038/nrm2233.

26.

Davalos

Kawahara

Malhotra

Schaum

Huang

Ved

, et al p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes. J Cell Biol. 2010;190(4):613-29. doi:10.1083/jcb.201001082.

27.

Gupta

Potty

Maffulli

. Allogenic platelet-rich plasma for treatment of knee and hip osteoarthritis. Front Pain Res (Lausanne). 2023;4:1216190. doi:10.3389/fpain.2023.1216190.

28.

Brüünsgaard

Pedersen

. Age-related inflammatory cytokines and disease. Immunol Allergy Clin North Am. 2003;23(1):15-39. doi:10.1016/s0889-8561(02)00056-5.

29.

Nedunchezhiyan

Varughese

Sun

Crawford

Prasadam

. Obesity, inflammation, and immune system in osteoarthritis. Front Immunol. 2022;13:907750. doi:10.3389/fimmu.2022.907750.

30.

Ahmadizad

El-Sayed

MacLaren

. Effects of time of day and acute resistance exercise on platelet activation and function. Clin Hemorheol Microcirc. 2010;45(2-4):391-9. doi:10.3233/CH-2010-1321.

31.

Feuring

Wehling

Ruf

Schultz

. Circadian variation of platelet function measured with the PFA-100. Platelets. 2009;20(7):466-70. doi:10.3109/09537100903226034.