Influence of cellular pre-conditioning on the therpuetic potential of adipose derived extracellular vesicles

As a result of the aging population, osteoarthritis (OA) became the fifteenth-highest leading cause of disability affecting over 7% of the global population. The number of diseased people aged 60 years old and above is predicted to double by 2050 and more than triple by 2100. OA not only is a disease affecting joints but also has a major impact on social life of patients as social isolation, depression, and involuntary unemployment due to disability make OA also a socioeconomic burden. Current treatment options only lead to alleviation of the symptoms including pain and inflammation but not to regeneration of the cartilage and therefore restoration of its mechanical function. Extracellular vesicles (EVs) represent promising cell-free tools for cartilage regeneration compared to conventional cell-based therapies. This study aims to evaluate whether cellular pre-conditioning of adipose-derived mesenchymal stem cells (AMSC) with pro-inflammatory or pro-chondrogenic factors as well as hypoxia impacts the therapeutic effect of EVs. AMSC will be expanded in a vertical wheel bioreactor and cultured in media supplemented with IL-1ß or TGFß-3 or will be grown under hypoxic conditions prior to EV isolation. Impact of this cellular priming on the regenerative effect of EVs will be tested by various biological assays as well as on expression of chondrogenic/osteoarthritic genes, miRNA composition within EVs, and activation status of signaling pathways involved in OA. This research question will also be evaluated within an inflammation model in which OA chondrocytes will be co-cultured with pro-inflammatory M1 macrophages. Impact of cellular pre-conditioning on EVs as anti-inflammatory mediators will be investigated in this setting. Furthermore, a defect model will be implemented via bovine osteochondral plugs which will be filled with gelatine methacryloyl hydrogels in which the respective EVs together with OA chondrocytes will be incorporated. Ability to promote healing of the defect will be evaluated by histochemical staining. This will be the first study comparing how cellular pre-conditioning of AMSC with various bioactive factors or hypoxia influences on the therapeutic efficacy of EVs in terms of OA. Elaborating this research question in an inflammation model in which OA chondrocytes are exposed not only to single inflammatory cytokine as in other studies but with pro-inflammatory macrophages secreting a multitude of bioactive factors gives this project another innovative aspect. Determining the repair ability of primed EVs in an ex vivo model for which no animals exclusively have to be harmed for the study rather as in in vivo models also adds the 3R aspect to the study.