A. De Luna (Krems, AT)

Center for Regenerative Medicine Clinical Medicine and Biotechnology
Andrea De Luna earned a MSc. Degree from the University of Vienna in Biology with the specialization in Genetics and Microbiology followed by a PhD degree from the Medical University of Vienna where she investigated the role of the mTOR signaling pathway during chondrogenic differentiation of human amniotic fluid stem cells. De Luna´s postdoctoral training in the Center of Regenerative Medicine at the Danube University Krems focused on the use of blood derivatives such as platelet plasma and hyperacute serum for the regeneration of cartilage defects. In 2019; De Luna became an assistant professor for Regenerative Medicine and Tissue Engineering. Currently her work is centered around the characterization and application of extracellular vesicles from various sources; including blood-derived products or mesenchymal stem cells; in order to circumvent hurdles connected with the usage of cell-based therapies for cartilage repair.

Presenter Of 2 Presentations

Extended Abstract (for invited Faculty only) Growth factors, PRP and Cytokines

1.2.4 - Extracellular Vesicles in Blood-Derived Products

Abstract

Introduction

The degenerative disease osteoarthritis (OA) is one of the leading causes of disability especially in the Western world. One of the major drivers of OA progression is inflammation, mediated by the secretion of pro-inflammatory cytokines including IL-6, TNF-α and IL-1β by activated chondrocytes or macrophages. Depending on the disease stage, OA treatment involves pharmacological and non-pharmacological approaches, cell-based therapies, or total knee replacement as a last resort. In earlier stages of OA, application of blood derived products including platelet rich plasma (PRP) or the cell-free alternative hyperacute serum (hypACT) gain more and more popularity in regenerative medicine due to their beneficial outcome towards reduced inflammation and promotion of cartilage regeneration. Little is known about the mode of action of these blood derivatives and which of their components are the mediators of their regenerative capacity. Whereas most studies focus on the growth factor content of blood derived products, we aimed to investigate the role extracellular vesicles (EVs) isolated from PRP or hypACT in an inflammation model. 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 are the mediators of blood derived products for cartilage repair.

Content

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. OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from healthy donors as well as the monocytic cell line THP-1 were differentiated and activated into M1 macrophages according to published protocols. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, cryo-electron microscopy and Western blot. Cytokine secretion of the pro-inflammatory cytokines IL-6, TNF-α and IL-1β within the inflammation model was determined by ELISA.

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. EV-supplemented co-cultures showed lower levels of the pro-inflammatory cytokines IL-6, TNF-α and IL-1β compared to co-cultures which were supplemented with the respective blood derivatives from which EVs were isolated.

Conclusion

Compared to the whole blood products CPRP and hypACT, EVs isolated from these blood derivatives were able to decrease the secretion of pro-inflammatory cytokines in a model in which OA chondrocytes were co-cultured with inflammatory M1 macrophages mimicking an OA environment. This highlights the potential of EVs to be one of the main players in mediating the anti-inflammatory effects of blood derivatives which makes them potential candidates for new cell-free therapeutic approaches for OA.

Acknowledgments

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.

Collapse
Extended Abstract (for invited Faculty only) Growth factors, PRP and Cytokines

13.3.2 - EVs From Blood-Derived Products for Cartilage Repair

Presentation Topic
Growth factors, PRP and Cytokines
Date
14.04.2022
Lecture Time
07:50 - 08:10
Room
Potsdam 1
Session Type
Morning Workshop

Abstract

Introduction

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.

Content

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.

Acknowledgments

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.

Collapse