Ontogenetic stage and type of donor cells shape extracellular vesicles' therapeutic potential for osteoarthritis.

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Background: Osteoarthritis (OA) remains an intractable condition due to the limited regenerative capacity of adult cartilage. Extracellular vesicles (EVs) have emerged as promising therapeutics, yet the optimal donor cell source is still undetermined, as both donor cell type and age significantly influence EV therapeutic efficacy. This study evaluates the therapeutic potential of EVs derived from ovine fetal articular chondrocytes (fCCs) and ovine fetal umbilical cord blood mesenchymal stromal cells (fMSCs) compared to EVs from two immortalized human perinatal cell lines, Wharton's jelly (WJ-MSCs) and amnion MSCs (P-MSCs), on inflamed ovine adult chondrocytes and synoviocytes in vitro.Methods: EVs were isolated from conditioned media using tangential flow filtration and characterized by size, concentration, and EV markers. Inflamed adult articular chondrocytes and synoviocytes were treated with 1E + 09 particles/mL of each EV source. EV's cellular uptake was assessed via live-cell imaging, flow cytometry, and confocal microscopy. Therapeutic effects were evaluated through proliferation, wound healing assays, and multi-omics (RNASeq, proteomics) analyses at 24 and 48 h post-treatment.Results: All EVs were successfully internalized by inflamed ovine and human chondrocytes. Donor cell type significantly influenced incorporation with fCC-EVs achieving the highest uptake across conditions. All treatments reduced pro-inflammatory genes and upregulated growth and cell cycle-related genes. Fetal-derived EVs induced more robust transcriptional changes and enriched signaling pathways than perinatal-derived EVs. Notably, fCC-EVs exhibited the most pronounced effects on inflamed chondrocytes, while fMSC-EVs were most effective on synoviocytes. Donor cell age emerged as a more influential factor in therapeutic efficacy than cell type.Conclusions: The ontogenetic stage of donor cells plays a crucial role in EV's therapeutic efficacy, with fetal-derived EVs demonstrating superior outcomes compared to perinatal-derived EVs. The distinct effects of fCC-EVs and fMSC-EVs suggest that a combinatorial approach using both EV types could optimize therapeutic outcomes.