Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA.

Cartilage breakdown, inflammation and pain are hallmark symptoms of osteoarthritis, and autologous blood products such as citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) have been developed as a regenerative approach to rebuild cartilage, inhibit inflammation and reduce pain. However, mechanisms of action of these blood derivatives are still not fully understood, in part due to the large number of components present in these medical products. In addition, the discovery of extracellular vesicles (EVs) and their involvement in intercellular communication mediated by cargo molecules like microRNAs (miRNAs) opened up a whole new level of complexity in understanding blood products. In this study we focused on the development of an isolation protocol for EVs from CPRP and hypACT that can also deplete lipoproteins, which are often co-isolated in EV research due to shared physical properties. Several isolation methods were compared in terms of particle yield from CPRP and hypACT. To gain insights into the functional repertoire conveyed via EV-associated miRNAs, we performed functional enrichment analysis and identified NFκB signaling strongly targeted by CPRP EV miRNAs, whereas hypACT EV miRNAs affect IL6- and TGFβ/SMAD signaling.

Fat tissue, due to its high concentration of stem cells, has a role in aesthetic medicine and reconstructive surgery. However, poor survival of the transplanted cells still limits the usefulness of this material in regenerative medicine. Several studies indicated that platelet-rich plasma (PRP) may improve adipose tissue viability due to its growth factor content. This study aimed at investigating the effects of PRP and hyperacute serum (HAS) on the adipogenic lineage in vitro . PRP was prepared by using two centrifugation steps in the presence of anticoagulants, and HAS was isolated from activated platelet-rich fibrin within 10 min of blood drawing to prevent the propagation of inflammatory cascades. Metabolic activity and proliferation rate of human bone marrow-derived mesenchymal stem cells (hMSCs) cultivated in media supplemented with three types of serum additives (fetal calf serum [FCS], human PRP, or HAS) was determined by using a tetrazolium assay. Adipogenesis was evaluated in standard and pro-adipogenic media and tested by oil red staining, triglyceride content, and expression of specific genes. Adipogenic regulators in the sera were measured by multiplex ELISA assays. We observed that proliferation of hMSCs was supported by both FCS and HAS in a time-dependent manner, but surprisingly, PRP had a much weaker effect (change in proliferation rate after 5 days relative to metabolic activity on day 0—FCS: 5.4-fold change, HAS: 5.8-fold change, serum free 1.9-fold change, PRP: 3.0-fold change, p < 0.05). Lipogenesis was only observed in groups with adipogenic differentiation medium, with HAS showing a significantly stronger effect than PRP. This was confirmed by intensive accumulation of lysochrome dye in lipid droplets, higher triglyceride concentration, and elevated expression of specific adipogenic genes. Measurement of lipogenic proteins in the sera revealed that both PRP and HAS are abundant in them; however, PRP also contains anti-adipogenic factors, which explains its weaker and less reliable effect. The results of this study suggest that HAS provides more robust support than PRP in hMSCs proliferation as well as lipogenic differentiation, indicating that it may be a better adjuvant in fat grafting procedures.

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. Besides risk factors including trauma, obesity or genetic predisposition, inflammation has a major impact on the development of this chronic disease. During the course of inflammation, cytokines such as tumor necrosis factor-alpha(TNF-α) and interleukin (IL)-1β are secreted by activated chondrocytes as well as synovial cells and stimulate the production of other inflammatory cytokines and matrix degrading enzymes. The mTORC1 inhibitor rapamycin is a clinical approved immunosuppressant and several studies also verified its chondroprotective effects in OA. However, the effect of blocking the mechanistic target of rapamycin complex (mTORC)1 on the inflammatory status within OA is not well studied. Therefore, we aimed to investigate if inhibition of mTORC1 by rapamycin can preserve and sustain chondrocytes in an inflammatory environment. Patient-derived chondrocytes were cultured in media supplemented with or without the mTORC1 inhibitor rapamycin. To establish an inflammatory environment, either TNF-α or IL-1β was added to the media (=OA-model). The chondroprotective and anti-inflammatory effects of rapamycin were evaluated using sulfated glycosaminoglycan (sGAG) release assay, Caspase 3/7 activity assay, lactate dehydrogenase (LDH) assay and quantitative real time polymerase chain reaction (PCR). Blocking mTORC1 by rapamycin reduced the release and therefore degradation of sGAGs, which are components of the extracellular matrix secreted by chondrocytes. Furthermore, blocking mTORC1 in OA chondrocytes resulted in an enhanced expression of the main chondrogenic markers. Rapamycin was able to protect chondrocytes from cell death in an OA-model shown by reduced Caspase 3/7 activity and diminished LDH release. Furthermore, inhibition of mTORC1 preserved the chondrogenic phenotype of OA chondrocytes, but also reduced inflammatory processes within the OA-model. This study highlights that blocking mTORC1 is a new and promising approach for treating OA. Low side effects make rapamycin an attractive implementation to existing therapeutic strategies. We showed that rapamycin’s chondroprotective property might be due to an interference with IL-1β triggered inflammatory processes.

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1β are secreted by activated chondrocytes, as well as synovial cells—including macrophages. Intra-articular injection of blood products—such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products—is gaining increasing importance in regenerative medicine for the treatment of OA. 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. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1β levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.