As the incidence of the degenerative disease osteoarthritis (OA) is increasing, the establishment of new therapies which are not only palliative but also curative is getting in the focus of regenerative medicine approaches. Blood derivatives such as platelet rich plasma (PRP) are widely used in orthopedics with promising outcomes regarding their mediation of cell migration and proliferation, as well as anti-inflammatory signals. Drawbacks connected with the application of PRP include high donor variations and the availability of over 20 different devices on the market, which all produce different PRPs in the context of cellular content and growth factor profiles. To circumvent these problems, extracellular vesicles (EVs) are potential mediators of regenerative signals and are getting more and more in the focus of research. EVs are nanosized particles transporting bioactive molecules (mRNA, miRNA, proteins) from one cell to an other resulting in a specific biological response depending on their cargo. We seeked to determine whether these particles isolated from PRP and its cell-free alternative hyperacute serum (hypACT) can mediate cartilage repair. We focused on the miRNA content as well as on their anti-inflammatory properties in an OA-simulated inflammatory model.
Materials & Methods
Citrate anticoagulated PRP (CPRP) and hypACT were generated from whole blood according to published protocols involving two-step versus single step centrifugation protocols. EVs were enriched via ultracentrifugation (UC) from these blood products. OA chondrocytes were obtained from patients undergoing total knee replacement. Chondroprotective and anti-inflammatory effects were evaluated based on protein expression via Western Blot. EV-associated miRNA profiles were analysed by screening a 372 miRNA panel via RT-qPCR in EVs purified via UC and size exclusion chromatography (SEC).
Results
While mode sizes were similar between EVs from CPRP and hypACT, higher concentrations of EVs were obtained from CPRP compared to hypACT. Western blot analysis confirmed the enrichment of EV markers CD9, CD63 and Alix in P100 fractions after ultracentrifugation. EVs were devoid of buoyant lipoproteins indicated by the absence of ApoB100/48, which is found in (V)LDL particles. Within an inflammation model in which chondrocytes were treated with 10ng/ml IL-1β, EVs isolated from CPRP possessed anti-inflammatory properties indicated by similar morphological appearance of the cells as observed when compared to untreated cells. On the contrary, EVs isolated from hypACT did not mediate cell protective effects indicated by apoptosis of chondrocytes. Furthermore, CPRP blood product increased SOX9 protein expression whereas CPRP EVs decreased NFκB and COX2 expression in IL1β-stimulated chondrocytes compared to unstimulated cells. Analysis of the functional repertoire encoded in EV-associated miRNAs revealed that CPRP EV-associated miRNAs target NFκB signaling and hypACT EV-associated miRNAs affect IL6 and TGFβ/SMAD signaling.
Conclusion
EVs from blood derived products represent a new, powerful tool to create innovative approaches for cartilage repair. Major drawbacks of application whole autologous blood products to the site of injury such as high donor variability or induction of inflammation can be circumvented when using EVs due to their cell free nature. In addition, EVs from CPRP and hypACT carry diverse miRNA cargos with distinct functional repertoires, which might be sufficient to induce regenerative processes in cartilage and might supersede application of full blood products in clinics.
This work was jointly supported by the European Fund for Regional Development (EFRE) and the Fund for Economy and Tourism of Lower Austria, frant number WST3-F-5030664/003-2017.