Pooled Platelet-Rich Plasma Lysate Therapy Increases Synoviocyte Proliferation and Hyaluronic Acid Production While Protecting Chondrocytes From Synoviocyte-Derived Inflammatory Mediators

Abstract

Platelet-rich plasma (PRP) preparations are being used with moderate success to treat osteoarthritis (OA) in humans and in veterinary species. Such preparations are hindered, however, by being autologous in nature and subject to tremendous patient and processing variability. For this reason, there has been increasing interest in the use of platelet lysate preparations instead of traditional PRP. Platelet lysate preparations are acellular, thereby reducing concerns over immunogenicity, and contain high concentrations of growth factors and cytokines. In addition, platelet lysate preparations can be stored frozen for readily available use. The purpose of this study was to evaluate the effects of a pooled allogeneic platelet-rich plasma lysate (PRP-L) preparation on equine synoviocytes and chondrocytes challenged with inflammatory mediators in-vitro to mimic the OA joint environment. Our hypothesis was that PRP-L treatment of inflamed synoviocytes would protect chondrocytes challenged with synoviocyte conditioned media by reducing synoviocyte pro-inflammatory cytokine production while increasing synoviocyte anti-inflammatory cytokine production. Synoviocytes were stimulated with either interleukin-1β (IL-1β) or lipopolysaccharide (LPS) for 24 h followed by no treatment or treatment with platelet-poor plasma lysate (PPP-L) or PRP-L for 48 h. Synoviocyte growth was evaluated at the end of the treatment period and synoviocyte conditioned media was assessed for concentrations of hyaluronic acid (HA), IL-1β, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Chondrocytes were then challenged for 48 h with synoviocyte conditioned media from each stimulation and treatment group and examined for gene expression of collagen types I (COL1A1), II (COL2A1), and III (COL3A1), aggrecan (ACAN), lubricin (PRG4), and matrix metallopeptidase 3 (MMP-3) and 13 (MMP-13). Treatment of inflamed synoviocytes with PRP-L resulted in increased synoviocyte growth and increased synoviocyte HA and IL-6 production. Challenge of chondrocytes with conditioned media from PRP-L treated synoviocytes resulted in increased collagen type II and aggrecan gene expression as well as decreased MMP-13 gene expression. The results of this study support continued investigation into the use of pooled PRP-L for the treatment of osteoarthritis and warrant further in-vitro studies to discern the mechanisms of action of PRP-L.

Keywords: HA; IL-1β; LPS; MMP-13; aggrecan; collagen type II; osteoarthritis; platelet-rich plasma lysate.

Figure 1

Schematic of the study design.

Figure 2

PRP-L but not PPP-L treatment increases synoviocyte growth and production of hyaluronic acid regardless of synoviocyte stimulation. Equine synoviocytes were left unstimulated or stimulated with 10 ng/mL of IL-1β or 100 ng/mL of LPS for 24 h before being non-treated (NT) or treated with platelet-poor plasma lysate (PPP-L) or platelet-rich plasma lysate (PRP-L) for 48 h. (A) Synoviocyte growth was measured via optical density and displayed as a fold change from the unstimulated, non-treated group. (B) Production of hyaluronic acid (hyaluronan; HA) was measured in the media using a commercial ELISA kit. Data is shown as the mean ± standard deviation of n = 5. Differing letters indicate significant differences between groups (p < 0.05); statistical analysis was performed within each stimulation and not between stimulation groups. Asterisks (^*) denote significant differences, when present, in stimulated NT groups from the unstimulated NT control. Blue bars = unstimulated synoviocytes, red bars = 10 ng/mL IL-1β stimulated synoviocytes, and green bars = 100 ng/mL LPS stimulated synoviocytes.

Figure 3

PRP-L treated synoviocytes produce more anti-inflammatory IL-6 under all stimulations conditions and less pro-inflammatory TNF-α in response to LPS stimulation compared to PPP-L treated synoviocytes. Conditioned media from cultured equine synoviocytes was collected after stimulation with 10 ng/mL of IL-1β or 100 ng/mL of LPS for 24 h and subsequent non-treatment (NT) or treatment with platelet-poor plasma lysate (PPP-L) or platelet-rich plasma lysate (PRP-L) for 48 h. Media concentrations (pg/mL) of (A) IL-1β, (B) TNF-α, and (C) IL-6 were measured using a commercial equine multiplex assay. Data is shown as the mean ± standard deviation of n = 5. Differing letters indicate significant differences between groups (p < 0.05); statistical analysis was performed within each stimulation and not between stimulation groups. Asterisks (^*) denote significant differences, when present, in stimulated NT groups from the unstimulated NT control. Blue bars = unstimulated synoviocytes, red bars = 10 ng/mL IL-1β stimulated synoviocytes, and green bars = 100 ng/mL LPS stimulated synoviocytes.

Figure 4

Conditioned media from synoviocytes treated with PRP-L under all stimulation conditions increases collagen type II but not collagen type I or type I in cultured chondrocytes. Equine chondrocytes were challenged for 48 h with conditioned media from synoviocytes stimulated with IL-1β or LPS and either non-treated (NT) or treated with platelet-poor plasma lysate (PPP-L) or platelet-rich plasma lysate (PRP-L). Relative gene expression is represented as the 2^−ΔΔCt of (A) collagen type I (COL1A1), (B) collagen type II (COL2A1), and (C) collagen type III (COL3A1). Data is shown as the mean ± standard deviation of n = 5. Differing letters indicate significant differences between groups (p < 0.05); statistical analysis was performed within each stimulation and not between stimulation groups. Asterisks (^*) denote significant differences, when present, in stimulated NT groups from the unstimulated NT control. Blue bars = unstimulated synoviocytes, red bars = 10 ng/mL IL-1β stimulated synoviocytes, and green bars = 100 ng/mL LPS stimulated synoviocytes.

Figure 5

Conditioned media from synoviocytes treated with PRP-L under all stimulation conditions increases aggrecan expression in cultured chondrocytes. Equine chondrocytes were challenged for 48 h with conditioned media from synoviocytes stimulated with IL-1β or LPS and either non-treated (NT) or treated with platelet-poor plasma lysate (PPP-L) or platelet-rich plasma lysate (PRP-L). Relative gene expression represented at the 2^−ΔΔCt of (A) aggrecan (ACAN), and (B) lubricin (PRG4) was measured in cDNA made from extracted chondrocyte RNA. Fold changes were generated from the chondrocytes cultured with unstimulated, non-treated synoviocyte conditioned media. Data is shown as the mean ± standard deviation of n = 5. Differing letters indicate significant differences between groups (p < 0.05); statistical analysis was performed within each stimulation and not between stimulation groups. Asterisks (^*) denote significant differences, when present, in stimulated NT groups from the unstimulated NT control. Blue bars = unstimulated synoviocytes, red bars = 10 ng/mL IL-1β stimulated synoviocytes, and green bars = 100 ng/mL LPS stimulated synoviocytes.

Figure 6

Conditioned media from synoviocytes treated with PRP-L or PPP-L under all stimulation conditions decreases MMP-13 but not MMP-3 gene expression in cultured chondrocytes. Equine chondrocytes were challenged for 48 h with conditioned media from synoviocytes stimulated with IL-1β or LPS and either non-treated (NT) or treated with platelet-poor plasma lysate (PPP-L) or platelet-rich plasma lysate (PRP-L). Relative gene expression, 2^−ΔΔCt, was generated using the unstimulated, non-treated conditioned media cultured chondrocytes as the control for (A) MMP-3 and (B) MMP-13. Data is shown as the mean ± standard deviation of n = 5. Differing letters indicate significant differences between groups (p < 0.05); statistical analysis was performed within each stimulation and not between stimulation groups. Asterisks (^*) denote significant differences, when present, in stimulated NT groups from the unstimulated NT control. Blue bars = unstimulated synoviocytes, red bars = 10 ng/mL IL-1β stimulated synoviocytes, and green bars = 100 ng/mL LPS stimulated synoviocytes.