PRIMING STRATEGIES TO ENHANCE THE EFFECT OF THE SECRETOME OF MESENCHYMAL STEM/STROMAL CELLS IN COUNTERACTING OSTEOARTHRITIS THROUGH THE ACTION OF THE EVS

Osteoarthritis (OA) is the most prevalent form of joint disease, affecting millions worldwide and leading to chronic pain, stiffness, and functional disability due to progressive degeneration of articular cartilage, synovial inflammation, and exposure of subchondral bone and nervous terminals. Further, OA is increasingly recognized as being influenced by “inflammaging”, an emerging oncept describing the chronic, low-grade inflammation associated with aging that contributes to tissue degeneration and impaired repair mechanisms. Despite its multifactorial pathophysiology, current OA treatments primarily address symptoms without targeting the underlying biological processes, ultimately necessitating surgical intervention. Additionally, OA can affect any individual since its early stages of aging, and considering the increasing average lifespan, disease-modifying or even disease-reverting approaches are urgently required, to overcome this social and economic burden. Among emerging therapeutic strategies, mesenchymal stem cell (MSC)-derived conditioned medium (CM) has gained significant attention for its ability to recapitulate many of the regenerative, immunomodulatory, and anti-inflammatory properties of stem cells through its complex cargo of cytokines, growth factors, and extracellular vesicles. However, translating CM into clinical practice is hindered by production variability and the absence of preclinical models and guidelines to test its efficacy, safety and dosage. With this project, we established a standardized protocol to produce CM from adipose-derived MSC (ASCs) and characterized it comprehensively. CM was then evaluated into increasingly complex OA models, both bidimensional (2D) and three-dimensional (3D), where the pathology was recapitulated by the exposure to inflammatory cytokines, namely TNFα and/or IL-1β. These platforms were used to test the effects of both naïve and primed CM on cellular and tissue responses, focusing on key molecular markers related to inflammation, matrix degradation, and tissue remodelling. CM treatment resulted in a significant downregulation of catabolic enzymes such as MMPs in cartilage representative models, moreover it demonstrated anti-inflammatory properties in both cartilage and synovial membrane representative ones. Importantly, in ex vivo settings, the response to CM varied among donors, with some explants exhibiting marked improvement while others showed minimal modulation, revealing inter-individual differences in therapeutic responsiveness. These findings not only confirm the potential of MSC-derived CM as a promising disease-modifying agent in OA but also underscore the value of patient-specific ex vivo models as predictive tools for therapeutic screening and for stratifying patients based on their molecular response profiles, paving the way toward personalized approaches in OA management.