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

1.2.4 - Extracellular Vesicles in Blood-Derived Products

Presentation Topic
Growth factors, PRP and Cytokines
Lecture Time
12:45 - 13:00
Potsdam 3
Session Type
Special Session
  • A. De Luna (Krems, AT)
  • A. De Luna (Krems, AT)
  • A. Otahal (Krems, AT)
  • K. Kramer (Krems an der Donau, AT)
  • L. Moser (Krems, AT)
  • Z. Lacza (Krems an der Donau, AT)
  • S. Nehrer (Krems, AT)



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.


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.


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.


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.


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.