Extracellular vesicles from human adipose-derived stem cells relieve pain and inflammation in a rat model of knee osteoarthritis.

Inflammatory pain is a hallmark symptom of osteoarthritis (OA), characterized by spontaneous hypersensitivity resulting from tissue damage and chronic inflammation. This study investigates the pain-relieving and cartilage-protective potential of extracellular vesicles (EVs) derived from human adipose-derived stem cells (hASCs) as a cell-free therapeutic approach for OA. hASC-EVs were isolated via multi-filtrations based on tangential flow filtration (TFF) and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), zeta potential measurement, flow cytometry and Liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomic analysis. An in vitro inflammatory OA model was established by treating human osteoarthritic chondrocytes (HC-OA) with interleukin-1β (IL-1β). The expression of inflammation- and pain-related genes was assessed by quantitative PCR (qPCR), and modulation of the Phosphoinositide 3-kinase / Protein kinase B (PI3K/Akt) signaling pathway was analyzed using an antibody array. In vivo therapeutic effects were evaluated in seven-week-old male Wistar rats using a monosodium iodoacetate (MIA)-induced OA model following intra-articular injection of hASC-EVs. Pain behavior was assessed via paw withdrawal latency (PWL), paw withdrawal threshold (PWT), and weight-bearing tests. Cartilage protection was evaluated by histological and immunohistochemical stainings (IHC). hASC-EVs were efficiently internalized into chondrocytes and significantly suppressed IL-1β-induced expression of pain and inflammatory markers (TRPA1, COX-2, MMP-2, MMP-3, and MMP-9). Additionally, hASC-EVs down-regulated key PI3K/Akt signaling genes, such as PIK3CA and AKT1. In vivo, hASC-EV treatment markedly improved PWL, PWT, and weight-bearing performance compared with untreated OA rats. Histological and immunohistochemical analyses revealed reduction of inflammatory cytokine expression and preservation of collagen type II, indicating both anti-inflammatory and cartilage-protective effects. hASC-EVs exhibited robust pain-relieving and cartilage-preserving effects in an OA rat model, highlighting their potential as a promising cell-free therapeutic strategy for the management of OA-related pain and joint degeneration.