Extracellular vesicles for cell-free regenerative medicine and its application for treatment of osteoarthritis

Abstract

Osteoarthritis (OA) is an articular cartilage degenerative joint disease with no current disease modifying treatments or successful regenerative therapies. Current regenerative therapies for OA are cell-based treatments, such as Autologous Chondrocyte Implantation (ACI) or Mesenchymal Stem Cells (MSCs) therapies, which present drawbacks that compromise its application to all OA patients. On the other hand, more common treatments include analgesic and non-steroid anti-inflammatory drugs, hyaluronic acid injections to help lubricate the joint area or platelet-rich plasma (PRP) injections. PRP are concentrates of plasma enriched in platelets, which are known to play a key role in wound healing processes. Moreover, platelets contain a high concentration of osteogenic growth factors with proven regenerative potential. However, its autologous use leads to a high variability from patient-to-patient. Furthermore, there is no consensus or standardised protocol to store and apply PRP in clinics. Since both, MSC-based therapies and non-MSC-based therapies, present serious limitations for their translation to clinics, other options need to be explored in the field of OA regenerative treatments. Extracellular Vesicles (EVs) have emerged as a promising alternative to cell-based treatments in regenerative medicine. EVs are membranous nanoparticles naturally secreted by all kind of cells and which function is cell-to-cell communication. EVs contain a diversity of active biomolecules protected from in vivo degradation thanks to their membrane, and since their main function is related to cell-to-cell communication they directly exert their effect onto their target cells, which is the feature of interest in the therapeutic applications. Thus, this Thesis aims to evaluate platelet lysate (PL)-derived EVs (pEVs) and human umbilical cord MSC-derived EVs (cEVs) as a cell-free regenerative treatment for OA. The isolation of EVs is a critical factor that influences their functionality. Ultracentrifugation, as gold standard isolation method, has limitations such as co-isolation of non-EV contaminants and potential damage to EV integrity. Therefore, size-exclusion chromatography (SEC) was explored in this Thesis as an alternative isolation method. EVs obtained from both ultracentrifugation and SEC were compared in terms of their regenerative effects in an OA in vitro model. SEC-derived EVs showed improved beneficial effects on the chondrocytes compared to ultracentrifuged derived EVs, which presented co-isolated deleterious proteins, highlighting the importance of working with well-purified EV preparations to observe their therapeutic effects accurately. To evaluate the regenerative potential of pEVs and cEVs, ex vivo and in vivo OA models were established using human cartilage explants from cadaveric donors and osteoarthritic rat knee joints, respectively. pEVs improved the osteoarthritic knee joint integrity and surpassed the regenerative effects of cEVs by promoting collagen deposition, reducing glycosaminoglycan degradation, and improving the integrity of articular cartilage explants, as well as recovering the knee joint integrity, especially in female rats, which exhibited greater susceptibility to OA damage. Overall, this Thesis demonstrates the regenerative potential of pEVs and highlights the use of PL as a feasible alternative EV source to MSCs for OA treatment. Thus, while pEVs are not yet ready for clinical implementation, their potential as cell-free regenerative treatment is undeniable. The findings derived from this Thesis shed light on the pEVs application as cell-free treatment for OA and other degenerative pathologies in the near future.