0.22 μm polyethersulfone filter

The synovial fluid and plasma collected on days 0, 10, 35, 42, 49, 56 and 63 from OA and control joints were thawed, and synovial fluid was subsequently treated with 1 μg/mL hyaluronidase (from bovine testes; Sigma–Aldrich, Gillingham, WI, USA) for 1 h at 37 °C. Both the synovial fluid and the plasma were centrifuged at 2500× g for 10 min and 10,000× g for 10 min at 4 °C. EVs were subsequently isolated by size exclusion chromatography using qEV single columns (IZON, Lyon, France) following the manufacturer’s instructions. Briefly, 3.5 mL of phosphate-buffered saline (PBS; Sigma–Aldrich, Gillingham, United Kingdom), previously processed using a 0.22-μm polyethersulfone filter (Sartorius, Göttingen, Germany) was passed through the column, followed by 150 μL of synovial fluid or plasma. The first five 200 μL flowed through fractions were discarded, and the following five 200 μL fractions pooled (isolated EVs). Isolated EVs were subsequently concentrated to a volume of 100 μL using a 2 mL 100,000 kDa Vivaspin column (Sartorius).

The synovial fluid and plasma collected on days 0, 10, 35, 42, 49, 56 and 63 from OA and control joints were thawed, and synovial fluid was subsequently treated with 1 μg/mL hyaluronidase (from bovine testes; Sigma–Aldrich, Gillingham, WI, USA) for 1 h at 37 °C. Both the synovial fluid and the plasma were centrifuged at 2500× g for 10 min and 10,000× g for 10 min at 4 °C. EVs were subsequently isolated by size exclusion chromatography using qEV single columns (IZON, Lyon, France) following the manufacturer’s instructions. Briefly, 3.5 mL of phosphate-buffered saline (PBS; Sigma–Aldrich, Gillingham, United Kingdom), previously processed using a 0.22-μm polyethersulfone filter (Sartorius, Göttingen, Germany) was passed through the column, followed by 150 μL of synovial fluid or plasma. The first five 200 μL flowed through fractions were discarded, and the following five 200 μL fractions pooled (isolated EVs). Isolated EVs were subsequently concentrated to a volume of 100 μL using a 2 mL 100,000 kDa Vivaspin column (Sartorius).