Individual immune cell and cytokine profiles determine platelet-rich plasma composition

Abstract

Objective: Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes.

Design: For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed.

Results: All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content.

Conclusions: Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual's immune profile and the concentration method appear to impact the final PRP product.

Trial registration: This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Keywords: Immune system; Inflammation; Orthobiologics; Osteoarthritis; Regenerative therapies.

Fig. 1

Cellular composition of different PRP products and corresponding donor blood samples. a Proportion of leukocytes (CD45⁺), b granulocytes (CD45⁺FSC/SSC NGr⁺), c lymphocytes and monocytes (CD45⁺FSC/SSC NGr⁻), d monocytes (CD45⁺CD14⁺), e B cells (CD45⁺CD14⁻CD19⁺CD3⁻), f T cells (CD45⁺CD14⁻LyCD19⁻CD3⁺), g NK cells (CD45⁺CD14⁻CD19^-CD3⁻CD56⁺), h CD4⁺ T helper cells (CD45⁺CD14⁻CD19⁻CD3⁺CD4⁺CD8⁻), and i CD8⁺ cytotoxic T cells (CD45⁺CD14⁻CD19⁻CD3⁺CD4⁻CD8⁺) in donor blood compared to PRP samples. Abbreviations: NK cells, natural killer cells; PRP, platelet-rich plasma

Fig. 2

Cytokine profile of different PRP products and corresponding donor blood samples. a Concentrations of IFN-γ, b TNF-α, c IL-1β, d IL-2, e IL-6, f IL-8, g IL-4, h IL-10, and i IL-13 in donor blood compared to PRP samples. Abbreviations: IFN-γ, interferon γ; TNF-α, tumor necrosis factor α; IL-1β, interleukin 1β; IL-2, interleukin 2; IL-4, interleukin 4; IL-6, interleukin 6; IL-8, interleukin 8; IL-10, interleukin 10; IL-13, interleukin 13; PRP, platelet-rich plasma

Fig. 3

2D and 3D cell cultures of primary human chondrocytes exposed to a pro-inflammatory environment. a Population doublings dependent on IFN-γ and b TNF-α at indicated concentrations, c metabolic activity dependent on IFN-γ and d TNF-α at indicated concentrations, e lactate dehydrogenase (LDH) release dependent on IFN-γ and f TNF-α at indicated concentrations, fold change in mRNA expression relative to control of gACAN, hCOL1A1, and iCOL2A1 and delta threshold cycles of jMMP3, kMMP9, and lMMP13 dependent on IFN-γ and TNF-α treatment, respectively. Abbreviations: PD, population doublings; CTRL, control; rel, relative; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α; LDH, lactate dehydrogenase; ACAN, aggrecan; COL1A1, collagen type 1A1; COL2A1, collagen type 2A1; MMP3, matrix metalloproteinase 3; MMP9, matrix metalloproteinase 9; MMP13, matrix metalloproteinase 13; ∆CT, delta cycle threshold