2.2. Chemiluminescence Analysis

SF levels of IL-1α, IL-1RA, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon gamma (IFN-y), TNF-α, and TNF-β were measured by an electrochemiluminescence immunoassay using a human customized U-Plex (Mesoscale, Rockville, MD). Matrix metalloproteinase (MMP)-1, MMP-3, and MMP-9 were measured using a human MMP-3 Plex Ultrasensitive kit (Mesoscale, Rockville, MD); transforming growth factor (TGF)-β1, TGF-β2, and TGF-β3 were measured using a human U-PLEX TGF-β Combo kit (Mesoscale, Rockville, MD); and basic fibroblast growth factor (bFGF) was measured using a Human V-PLEX bFGF kit (Mesoscale, Rockville, MD). Prior to measurement, the samples were diluted in Diluent 41, and MSD Discovery Workbench software was used for analysis (MESO QuickPlex SQ 120). Samples were run in duplex, and coefficient of variation (CV) values above 20% in individual analyses was considered high (Additional file 1). SF levels of C-terminal telopeptides of type 2 collagen (CTX-2) and Aggrecan (ACG) were analyzed by the enzyme-linked immunosorbent assay (ELISA) (MyBiosource, VersaMax™). Samples were run in duplex and performed according to the manufacturer's instructions.