Insights into mesenchymal stem/stromal cell conditioned media as a long-lasting treatment for pain and psychiatric comorbidities in a murine model of osteoarthritis

• MSC secretome alleviates osteoarthritis pain and mood alterations in mice. • Priming of MSC does not improve the efficacy of secretome. • Single i.v. secretome dose exerts multi-target, long-lasting therapeutic effects. • It rebalances peripheral and central neuroinflammation associated to OA pain. • MSC secretome emerges as a safe, cell-free biological therapy for OA comorbidities. Osteoarthritis (OA) is a prevalent musculoskeletal condition characterized by chronic pain and often accompanied by psychiatric comorbidities, such as anxiety and depression. The mesenchymal/stromal cell (MSC) secretome has emerged as promising therapy due to its immunomodulatory and regenerative properties. This study aimed to compare the therapeutic efficacy of standard (CM) and cytokine-primed conditioned media (CM + ) derived from human adipose-derived MSCs in mitigating pain, mood alterations and neuroinflammation in a murine OA model. OA was induced by intra-articular injection of monoiodoacetate in male C57BL/6J mice. On day 7, mice received a single intravenous dose of CM or CM + at two concentrations. Pain-related behavior was assessed by mechanical and thermal nociceptive tests. Anxiety- and depression-like behaviors were evaluated through open field, light/dark box, and forced swim tests. Pro- and anti-inflammatory cytokines and (neuro)inflammatory markers were analyzed in OA joint, and in the peripheral and central nervous system. Peripheral innervation and substance P were assessed in OA paw skin. Both CM and CM + reduced painful and affective symptoms in a dose-dependent manner. However, CM (from non-primed MSCs) exhibited faster onset and longer-lasting effects, up to 17 days post-injection, compared to CM + . CM also led to greater improvements in neuroinflammatory profiles, rebalancing cytokine pattern and macrophage polarization across peripheral and central tissues. MSC secretome, especially when generated without inflammatory priming, represents a safe and long-lasting therapeutic option for OA pain and associated mood disorders. These findings highlight its clinical potential as an innovative multi-targeted biological therapy.