Introduction

Osteoarthritis (OA) is a debilitating joint disease that significantly impacts both the physical and mental health of affected individuals1,2. Posttraumatic osteoarthritis (PTOA), a form of OA caused by joint injury, is increasingly prevalent, affecting over 5.5 million people in the United States, highlighting the substantial social and economic implications associated with this condition3. Joint injuries initiate a pro-inflammatory response, involving cytokines such as IL-1β, IL-6 and TNF-α, that can promote PTOA4,5,6. Treatment with platelet rich plasma (PRP) is a novel strategy to potentially mitigate PTOA progression7,8, as PRP contains cytokines and growth factors known to enhance healing responses9,10. Several studies have explored the efficacy of PRP in addressing cartilage degeneration and inflammation in OA8,11,12,13,14,15,16; however, its precise mechanism of action remains unclear.

Resolution of inflammation, once considered a passive process, is now known to be actively mediated by pro-resolving factors that modulate the chemokine and cytokine production, halt inflammatory leukocyte recruitment, promote macrophage polarization, and facilitate tissue repair and restoration of homeostasis17. Among these factors is a family of lipids derived from omega-3 and omega-6 fatty acids, collectively referred to as specialized pro-resolving mediators (SPMs). Two of these SPMs are Maresin 1 (MaR1) and Resolvin D1 (RvD1)18,19,20,21,22. Dysregulation of SPM levels has been associated with impaired resolution of inflammation in arthritis23,24,25. This dysregulation may also be relevant in PTOA, suggesting that the exogenous administration of SPMs could potentially mitigate cartilage breakdown and subsequent PTOA progression.

The aim of this study was to first show the efficacy of purified SPM administration on inflammation and cartilage degradation using an in vitro model of PTOA. Then we determined if human PRP contains detectable levels of SPMs and whether SPM concentrations correlated with anti-inflammatory and anti-degradative effect of PRP on human articular chondrocytes in vitro.

Materials and methods

Human subjects

This study was conducted in accordance with the principles outlined in the Declaration of Helsinki. Ethical approval was obtained from the Ethics Committee (Mass General Brigham #2022P003232), and informed consent was obtained from all participants prior to enrollment. Patients undergoing PRP injections for clinical indications in an outpatient physical medicine and rehabilitation clinic were included in this study; an aliquot of PRP was reserved for research investigation. For this pilot investigation, no restrictions were imposed regarding patients’ age, sex, or pathology being treated with PRP. Demographic data was collected.

PRP protocol

PRP was processed following the standardized protocol established by the harvest system manufacturer. Briefly, venous blood was aseptically drawn and processed using a double-spin protocol with an Executive Series Centrifuge (Plymouth Medical LLC). The first centrifugation at 3800 rpm for 1.5 min at room temperature separated the platelets and plasma suspension from other blood components. This suspension was then transferred to a secondary tube and subjected to a second centrifugation at 3800 rpm for 5 min, room temperature. This process allows the separation of PRP from platelet-poor plasma. Following the isolation, 750 uL of PRP was collected and stored for subsequent analyses. The remaining PRP was used to treat the patient.

Maresin 1 and resolvin D1 levels in PRP

Levels of MaR1 and RvD1 in PRP were measured using commercially available enzyme immunoassay kits (Cayman Chemical Company) according to the manufacturer’s protocol. Five samples with the lowest SPM concentration (PRP/SPM-low) and five with the highest SPM concentration (PRP/SPM-high) were determined, based on ranking by both MaR1 and RvD1 concentrations (Fig. 1). These ten samples were used in a subsequent in vitro experiment to investigate the effect of SPM concentration in PRP on human chondrocytes.

Proof-of-concept: effect of Maresin 1 and resolvin D1 in an in vitro model of PTOA

Osteochondral explant model

A bovine osteochondral (bOC) explant model was chosen for its potential to obtain multiple samples simultaneously, alongside the structural similarity between bovine and human articular cartilage26. Bovine forelimb joints (comprising the shoulder, elbow, and carpal joints) from 2 to 3-week-old calves were purchased from a local USDA certified slaughterhouse shortly after death. Young animals were used to minimize the likelihood of preexisting joint degeneration (e.g., articular cartilage damage). Osteochondral plugs (6 mm in diameter, 10 mm in depth) were aseptically harvested from humeral heads using an Autograft OATS 2.0 Set (Arthrex). The bOC explants were then washed in Dulbecco’s phosphate buffered saline (DPBS; Gibco), removing any bone marrow elements by flushing with a syringe and a 22-gauge needle.

Experimental design

To equilibrate, explants were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12; Gibco) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco) at 37 °C and 5% CO2 for 24 h. Following the equilibration period, explants were placed in 12-well untreated plates (one explant per well) containing 3 ml of fresh DMEM/F12 media with 10 ng/ml IL-1β (Bovine IL-1 beta Recombinant Protein; Invitrogen). This concentration of IL-1β was selected based on its ability to induce an inflammatory response in explants, as previously reported in preclinical studies27,28,29 and further confirmed by our group. After 24 h, IL-1β stimulated explants were randomly assigned to receive either a single dose of MaR1 (100 nM (36.05 pg/mL), Cayman Chemical Company), RvD1 (100 nM (37.65 pg/mL), Cayman Chemical Company) or vehicle (0.05% ethanol in DPBS) and were maintained in culture at 37 °C for an additional 48 h. The selected doses were based on concentrations shown to be effective in previous studies24,30,31. Supernatants were collected and frozen for further analyses.

Supernatant IL-6 and CTX-II level

Levels of IL-6 and CTX-II in the supernatant were quantified using commercially available ELISA kits (Invitrogen, Bovine IL-6 and Cartilaps, CTX-II) according to manufacturer’s protocols.

Impact of Maresin 1 and resolvin D1 concentration in PRP on human chondrocytes

Chondrocyte isolation and culture

To assess the influence of MaR1 and RvD1 concentration in PRP, an in vitro model of human chondrocytes was employed. Non-arthritic human articular cartilage was obtained from remnants of osteochondral allografts (OCA) (JRF Ortho) that had been used for operative cartilage restoration. Chondrocytes were obtained as previously described32. Briefly, articular cartilage was dissected and minced (< 1mm3) with a sterile scalpel, washed in PBS (Gibco), and enzymatically digested using collagenase II (0.2% in in Hank’s balanced salt solution (HBSS) with calcium and magnesium; Gibco) for 18–20 h at 37 °C and 5% CO2. After digestion, the cell suspension was filtered through a cell strainer (70 μm) and washed three times by centrifugation in Dulbecco’s phosphate buffered saline (DPBS; Gibco) without collagenase. The cell pellet was then resuspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin. Cells were seeded in tissue culture flasks (Thermo Scientific) at a density of 2.8 × 104 cells/cm2. Culture media was changed every three days. At 90% confluence, chondrocytes were trypsinized and transferred to a 48-well plate (Corning) at a density of 3 × 104 cells/cm2. 0.3 ml of culture media was added, and the plates were incubated at 37 °C, 5% CO2.

Treatment with PRP

Upon reaching 90–100% confluence, cells were exposed to 10 ng/ml of IL-1β (Human IL-1 beta Recombinant Protein; Thermo Scientific) to induce an inflammatory response33. After 24 h, chondrocytes were randomly treated with one dose of PRP/SPM-low (10%) or PRP/SPM-high (10%). Heparin (2 U/mL) was also added to prevent gelation34. Non-stimulated chondrocytes were cultured under identical conditions and served as non-inflamed controls. Cells were then incubated at 37 °C for an additional 48 h. Subsequently, culture media and chondrocytes were harvested for ELISA and qPCR analyses.

Fig. 1
figure 1

Study design. (A) PRP was collected from patients and its SPM (Maresin 1 – MaR1 and Resolvin D1 – RvD1) concentration were assessed by ELISA. (B) Proof-of-concept model using bovine osteochondral explants stimulated with IL-1β and treated with either Maresin 1 or Resolvin D1. (C) Primary human chondrocytes were obtained from osteochondral allografts, stimulated with IL-1β (10 ng/ml) and treated with PRP/SPM-low or PRP/SPM-high obtained from patients.

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RNA extraction and real time-qPCR

RNA was extracted from the chondrocytes using TRIzol and then subjected to DNase I treatment (Thermo Scientific). cDNA synthesis was conducted using a high-capacity cDNA reverse transcription kit (Applied Biosystems) following the manufacturer’s instructions. RT-qPCR was then performed using Fast SYBR green master mix (Applied Biosystems). The following human primers were used for analysis: MMP13 AGCTGGACTCATTGTCGGGC (forward), AGGTAGCGCTCTGCAAACTGG (reverse); IL6 GGTACATCCTCGACGGCATCT (forward), GTGCCTCTTTGCTGCTTTCAC (reverse); COL2A1 CCCAGAGGTGACAAAGGAGA (forward); CACCTTGGTCTCCAGAAGGA (reverse); 18 S GCAATTATTCCCCATGAACG (forward), AGGGCCTCACTAAACCATCC (reverse). Reactions were conducted using a StepOne Plus real-time PCR machine (Applied Biosystems). Average cycle threshold (Ct) values were calculated from duplicate technical replicates. Results were normalized to the housekeeping gene 18 S ribosomal RNA and calculated using the 2 − ΔΔCT method. 18 S ribosomal RNA was used as the reference gene due to its stable expression across experimental conditions, and primer efficiency was confirmed to be within the acceptable range through standard curve analysis35,36.

Measurement of cytokines and C-terminal cross-linked telopeptide-II (CTX-II)

Levels of pro-inflammatory cytokines (IL-1β, TNF, IL-2, IFN-γ) and anti-inflammatory/regulatory cytokines (IL-10, TGF-β, IL-4, IL-13), as well as CTX-II, were measured in the supernatant from chondrocyte cultures using commercially available enzyme immunoassay kits (Meso Scale Discovery for cytokines and Cartilaps for CTX-II). Measurements were performed according to the manufacturer’s protocols.

Statistical analyses

Continuous variables are expressed as means ± SD, while categorical variables are presented as absolute numbers and percentages. Data distribution was assessed using the Shapiro–Wilk test for normality. Spearman’s rank correlation analysis was used to examine correlations between SPM levels in PRP and patient demographics. To compare differences between the PRP/SPM-low and PRP/SPM-high groups, t-tests or Mann-Whitney U tests were applied as appropriate. For comparisons among multiple groups, one-way analysis of variance (ANOVA) with Tukey’s post hoc tests was used for continuous variables. A heat map was generated displaying the relative cytokine expression levels in chondrocyte supernatant, with color intensity indicating concentration variations. All statistical analyses were conducted using GraphPad Prism 9.3.1 (GraphPad Software, Inc.), with significance set at p < 0.05.

Results

Forty patients (57.5% male) were included in this study, with a mean age of 55.9 ± 18.0 years (range, 18 to 79) and a mean BMI of 25.7 ± 3.0 kg/m2 (range, 20.0 to 36.1; Table 1). The mean concentration of MaR1 was 667.5 ± 241.2 pg/ml (range, 349.3 to 1422.0), while the mean concentration of RvD1 was 139.5 ± 84.2 pg/ml (range, 41.9 to 351.2). Supplementary Table S1 demonstrates the concentrations of MaR1 and RvD1 in PRP from individual patients. PRP characteristics are presented in Table 2. No significant correlations were observed between the levels of MaR1 or RvD1 in PRP and age, sex, BMI, other comorbidities or previous corticosteroid injections. However, a significant correlation was found between MaR1 and RvD1 levels (r = 0.33; p = 0.04), and between MaR1 and platelet number (r = 0.38; p = 0.02; Fig. 2).

Table 1 Patient demographics. Medial collateral ligament (MCL). Data are presented as mean ± standard deviation or number (percentage).
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Table 2 Platelet-rich plasma (PRP) characteristics. Maresin 1 (MaR1); resolvin D1 (RvD1). Data are presented as mean ± sd.
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Fig. 2
figure 2

Correlation between Maresin 1 (MaR1) and Resolvin D1 (RvD1) levels in PRP (A), and between MaR1 and platelet number in PRP (B). Orange points represent PRP/SPM-low samples while green points represent PRP/SPM-high samples.

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Proof of concept: effect of Maresin 1 and Resolvin D1 in an in vitro model of PTOA

In an in vitro model of PTOA using bovine osteochondral explants, the application of 100 nM MaR1 to IL-1β-stimulated explants significantly reduced supernatant levels of both IL-6 (p = 0.035) and CTX-II (p = 0.043) compared to controls. In contrast, 100nM RvD1 was ineffective in reducing supernatant levels of IL-6 (p = 0.999), but significantly reduced CTX-II levels (p = 0.003; Fig. 3).

Fig. 3
figure 3

Effect of Maresin 1 and Resolvin D1 in an in vitro model of PTOA. Levels of IL-6 (A) and CTX-II (B) were measured in the supernatant of bovine osteochondral allografts cultures stimulated with IL-1β (10 ng/ml) followed 1 day later by treatment with vehicle (control), Maresin 1 (MaR1, 100 nM) or Resolvin D1 (RvD1, 100 nM). IL-6 and CTX-II levels were measured by ELISA. Data are presented as mean ± SD. *p < 0.05; **p < 0.01. N = 7–9 per group.

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Impact of Maresin 1 and resolvin D1 concentration in PRP on human chondrocytes

The five samples with the lowest SPM concentrations (PRP/SPM-low; mean MaR1: 457.3 ± 63.4 pg/ml, mean RvD1: 59.4 ± 13.9 pg/ml) and the five samples with the highest SPM concentrations (PRP/SPM-high; mean MaR1: 1166 ± 269 pg/ml, mean RvD1: 257.5 ± 81.9 pg/ml) were selected for in vitro assays. Supernatant analysis revealed that PRP with lower SPM content exhibited a more pronounced pro-inflammatory cytokine profile, whereas PRP with higher SPM content was associated with a more dominant anti-inflammatory and regulatory cytokine profile. However, these differences were not statistically significant (Fig. 4, Supplementary Fig. S1).

Fig. 4
figure 4

Cytokine levels in human chondrocyte supernatant. PRP with low SPM content (PRP/SPM-low) displayed a more pronounced pro-inflammatory cytokine profile (red panel) in comparison to high SPM content (PRP/SPM-high), whereas PRP/SPM-high exhibited a more pronounced anti-inflammatory and regulatory cytokine profile (blue panel). Cytokine levels were measured by a multiplex assay. Levels were normalized to those in chondrocytes without IL-1β treatment and are presented as fold change. N = 4–5 per group.

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Gene expression analysis in chondrocytes treated with PRP revealed a trend towards reduced IL6 expression in the PRP/SPM-high group compared to the PRP/SPM-low group (p = 0.056). PRP/SPM-high treatment also significantly reduced the gene expression of MMP13 (p = 0.034) and showed a trend toward lower COL2A1 expression (p = 0.054) compared to PRP/SPM-low. This reduction was also reflected in the supernatant levels of CTX-II, with the PRP/SPM-high group showing significantly lower concentrations than the PRP/SPM-low group (p = 0.014; Fig. 5).

Fig. 5
figure 5

Gene expression in human chondrocytes and supernatant levels of CTX-II. (A) PRP/SPM-high reduced the expression of IL6, MMP13, and COL2A1 compared to PRP/SPM-low. (B) PRP/SPM-high also led to a greater reduction in CTX-II levels in the supernatant compared to PRP/SPM-low. Gene expression in chondrocytes was quantified by qPCR and normalized to the housekeeping gene 18 S ribosomal RNA; CTX-II levels in the supernatant were measured by ELISA. Data are presented as mean ± SD. *p < 0.05. (A) N = 5 per group; (B) N = 8–9 per group.

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Discussion

The main findings of this study are that MaR1 and RvD1, two SPMs crucial for resolving inflammation, were found in high concentrations in human PRP. Notably, MaR1 was significantly correlated with both RvD1 and platelet number. In addition, PRP with higher concentrations of SPMs exhibited a more substantial effect in reducing inflammation and cartilage degradative enzymes in human chondrocyte cultures compared to PRP with lower SPM levels. These findings highlight the potential therapeutic roles of MaR1 and RvD1 in the efficacy of PRP.

Inflammation is a crucial component of the healing process37. However, when unresolved, it can lead to chronic diseases such as rheumatoid arthritis, pulmonary fibrosis, and potentially PTOA38,39,40. Following joint injuries, proinflammatory cytokines (e.g., IL-1β, IL-6, TNF) can persist, driving sustained inflammation, cartilage degradation, and ultimately, PTOA5. PRP, an autologous blood-derived product rich in platelets and bioactive molecules, has been explored as a therapy to counteract inflammation and PTOA progression due to its anti-inflammatory and regenerative properties14,15,16,41. Platelets are particularly important as they release growth factors and cytokines that are key to tissue healing and inflammation regulation42, and therefore, higher platelet counts in PRP, often achieved through double centrifugation methods, are associated with enhanced therapeutic outcomes43,44,45,46,47,48. In our study, PRP was prepared using a two-step centrifugation process, resulting in an average count of 8.5 billion platelets. This number is considered high and adequate for clinical applications according to previous studies49,50,51.

Recent systematic reviews and meta-analyses have demonstrated that PRP injections enhance cartilage and synovial integrity, reduce inflammatory markers, alleviate pain, and may improve patient-reported outcomes15,52,53,54. Despite these promising results, the exact mechanism of action of PRP remains unclear. We hypothesized that SPM concentrations influence the therapeutic effects of PRP. Specialized pro-resolving mediators, including maresins, resolvins, lipoxins and protectins, are omega-3 and omega-6-derived lipids that promote inflammation resolution and tissue repair. While their role in PTOA remains unclear, SPMs have been found in the blood and synovial fluid of patients with rheumatoid arthritis and OA55,56,57,58. Notably, lower levels of SPMs in rheumatoid arthritis patients correlate with higher pain scores59,60, and reduced serum levels of MaR1 and resolvins are associated with increased disease activity57,58. In this study, we found that MaR1 and RvD1 are present in PRP, but their concentrations vary dramatically between patients, as reflected in the wide range of values observed in our analyses. We did not find a correlation between MaR1 or RvD1 with patient demographics, although some studies have noted sex-based differences in SPM levels61,62,63. However, a correlation between MaR1 and the platelets count was found. This finding is consistent with the fact that lipoxygenases, enzymes that are crucial for the synthesis of SPMs, are also present in platelets64.

While the role of SPMs in PRP is still hypothetical, this study demonstrated that higher concentrations of MaR1 and RvD1 in PRP were associated with a greater effect in decreasing inflammation and chondrocyte breakdown, as well as increased anti-inflammatory and regulatory molecules. These findings support a potential role of SPM in PRP. Similarly, previous research has identified PRP as a significant source of bioactive lipids (not specifically SPMs) that enhance cell migration and proliferation, contributing to its healing properties65.

Our finding that higher SPM concentrations in PRP are associated with increased anti-inflammatory and regulatory responses aligns with previous research on SPMs and cartilage66,67. For instance, RvD1 has been shown to reduce COX-2, iNOS, and MMP-13 expression in IL-1β-stimulated human articular chondrocytes66, while lipoxin A4, another member of the SPM family, decreased MMP-13, NO, and PGE2 production in IL-1α-stimulated cartilage explants67. Not only were higher SPM concentrations associated with greater anti-inflammatory responses in our study, but lower SPM concentrations appeared to induce a less favorable immunomodulatory effect. Although more work is needed in this area, these results suggest that PRP with high SPM concentrations may be more beneficial or potentially lead to a greater treatment response, whereas lower SPM concentrations may result in a diminished treatment response. Optimizing PRP formulations by enhancing endogenous SPM biosynthesis through dietary supplementation with omega-3 fatty acids or purified SPMs, or exercise could help consistently yield PRPs with higher SPM content68,69.

To further validate the effects of MaR1 and RvD1 in an PTOA model, we used bovine osteochondral explants due to their structural similarity to human articular cartilage and the ability to obtain multiple samples from the same donor simultaneously. Interestingly, while MaR1 reduced both inflammation and cartilage breakdown, RvD1 dramatically reduced cartilage degradation markers, without significantly affecting inflammation. This suggests that each SPM may play distinct yet complementary roles in regulating inflammation and maintaining cartilage integrity. This may have practical relevance going forward as MaR1 and RvD1 seem to be acting synergistically and there may be benefit for both of them to be concentrated and optimized in PRP preparations in the future.

This study has several limitations. First, this study was designed as a pilot investigation, and future research could include longer-term experiments using 3D models to better evaluate structural effects on cartilage extracellular matrix components. Second, the investigation regarding the role of SPM concentrations in PRP on PTOA relied on in vitro models, which may not fully capture the complexity of in vivo conditions. While the effects of MaR1 and RvD1 on inflammation and cartilage degradation were clearly demonstrated in human chondrocytes and bovine osteochondral explants, it remains unclear whether these findings will translate to clinical outcomes. Additionally, variability in SPM concentrations among PRP samples could influence the consistency of therapeutic effects. The underlying reasons for these differences in SPM levels were not explored, nor were strategies to increase SPM concentrations in PRP. Further studies are warranted to explore ways to optimize their concentrations. SPMs are derived from omega-3 and omega-6 fatty acids; thus, investigating whether dietary supplementation or other interventions such as exercises can enhance SPM levels in PRP could be an interesting avenue for future research. Nonetheless, this study is the first to demonstrate the presence of SPMs in PRP and suggests they may play a role in attenuating inflammation and cartilage breakdown.

Conclusion

This study demonstrates that SPMs, specifically MaR1 and RvD1, are present in PRP and correlate with anti-inflammatory effects and reduced cartilage breakdown in vitro. MaR1 and RvD1 have different effects on osteochondral explant cultures and thus act more powerfully in tandem to address inflammatory and structural consequences of inflammatory cytokine action. In this study we show that purified MaR1 and RvD1 reduced inflammatory cytokine production and cartilage breakdown in a bOC explant model of PTOA. SPMs are more abundant in some patients’ PRP. These findings suggest that SPMs could contribute to the therapeutic potential of PRP. While further research is needed to clinically validate these results and understand SPM variability, this study opens new directions for understanding the mechanisms of action in PRP and optimizing its formulations to improve treatment outcomes for musculoskeletal disorders, including PTOA.