The Soluble Secretome and Extracellular Vesicles of Synovial Fluid Cells Closely Resemble Synovial Membrane Fibroblasts over Articular Chondrocytes in Donor-Matched Isolates from Osteoarthritis Patients

Background: Osteoarthritis (OA) is a whole-joint pathological condition driven by an intricate signaling network among immune cells, articular chondrocytes (ACs), fibroblast-like synoviocytes (FLSs) and non-immune synovial fluid cells (SFCs). While AC and FLS secretomes are well-characterized, the phenotypic identity of SFCs and their signaling cross-talk with structural joint cells remain poorly defined. This study aimed to comparatively characterize the secretome and extracellular vesicles (EVs) profile of donor-matched human ACs, FLSs and SFCs to identify shared and cell-specific signaling features within the osteoarthritic joint. Methods: Primary donor-matched ACs, FLSs and SFCs were isolated from five human osteoarthritic joints. EVs and soluble secretomes were isolated and analyzed by EV surface marker immunophenotyping, EV-microRNAs (miRNAs) profiling and soluble factor analysis. Data were evaluated using principal component analysis, hierarchical clustering, correlation matrices and bioinformatic analyses. Results: All three cell types shared a baseline OA secretome fingerprint dominated by inflammatory and catabolic factors. However, AC secretomes segregated distinctly. FLSs and SFCs displayed high phenotypic correlation across EV surface markers (r = 0.95), EV-miRNA signatures (r = 0.94) and soluble factor profiles (r = 0.94). Bioinformatic analyses showed that upregulated EV-miRNAs shared between FLSs and SFCs were specifically involved with pathways related to immune cell activation, cytokine signaling and apoptosis. In contrast, ACs demonstrated a distinct profile marked by the significant upregulation of soluble catabolic and pro-inflammatory mediators. Conclusions: Non-immune SFCs closely resemble FLSs in their secretome and EV profiles, supporting a similar phenotype and a potential role in EV-mediated immune and inflammatory signaling. In contrast, ACs display a distinct profile characterized by soluble catabolic and pro-inflammatory mediators. These findings highlight compartment-specific signaling patterns within the osteoarthritic joint and may inform future cell-free therapeutic strategies.