Extracellular vesicles from primed Hoffa's fat pad mesenchymal stem/stromal cells in osteoarthritis therapy: effects on cells critical to osteoarthritis progression.

BACKGROUND: Since Hoffa's fat pad (HFP) is naturally involved in the joint environment, and given findings that HFP-derived mesenchymal stromal cells (MSCs) possess better chondrogenic differentiation capacity than other adipose tissue sources or bone marrow, we aimed to analyze HFP-MSCs derived extracellular vesicles (EVs) primed with blood products as potential cell-free osteoarthritis (OA) therapy. METHODS: HFP-MSCs were cultured under 3D conditions on microcarriers in vertical-wheel bioreactors. Prior to medium harvest, MSCs were primed with blood products, including fetal calf serum (FCS), platelet-rich plasma (PRP), or hyperacute serum (HypACT). EVs were isolated via ultrafiltration and characterized via cryo-electron microscopy, nanoparticle tracking analysis (NTA) in both scatter and fluorescence modes, as well as via Western blot (WB). CFSE-labeled EV internalization by chondrocytes and synovial fibroblasts was detected using fluorescent confocal microscopy in the presence or absence of polybrene. Polybrene was used to enhance EV internalization and potentially improve treatment efficacy in cells. To simulate an OA environment, chondrocytes and synovial fibroblasts were co-cultured with M1 macrophages. Gene expression changes were analyzed via RT-qPCR, while cytokine release was quantified using ELISA. RESULTS: HFP-MSC-EVs downregulated the expression of catabolic enzyme genes MMP3 and MMP13 in chondrocytes, as well as the inflammatory genes CCL5 and COX2 in synovial fibroblasts. COL2A gene expression was upregulated in chondrocytes treated with HFP-MSC-EVs from a young donor. Furthermore, HFP-MSC-EVs elevated the release of the anti-inflammatory cytokine IL-10 from chondrocyte and synovial fibroblast co-cultured with M1 macrophages. HFP-MSC-EVs also upregulated IL10 gene expression in M1 macrophages. Polybrene significantly enhanced the internalization of HFP-MSC-EVs by chondrocytes and synovial fibroblasts, but did not improve treatment efficacy. CONCLUSIONS: HFP-MSC-EVs may have therapeutic potential mediated by attenuating inflammation and downregulation of catabolic enzymes in cells critical to OA progression.