Osteoarthritis (OA) is a degenerative joint disease with limited therapeutic options, where inflammation plays a critical role in disease progression. Extracellular vesicles (EV) derived from mesenchymal stromal cells (MSC) have shown potential as a therapeutic approach for OA by modulating inflammation and alleviating degenerative processes in the joint. This study evaluated the therapeutic effects for the treatment of OA of two types of EV—exosomes and matrix-bound nanovesicles (MBV)—both derived from the human umbilical cord MSC (UC-MSC) via differential ultracentrifugation. Different phenotypes of human monocyte-derived macrophages (MDM) were used to study the anti-inflammatory properties of EV in vitro, and the medial meniscectomy-induced rat model of knee osteoarthritis (MMx) was used in vivo. The study found that both EV reduced pro-inflammatory cytokines IL-6 and TNF-α in MDM. However, exosomes showed superior results, preserving the extracellular matrix (ECM) of hyaline cartilage, and reducing synovitis more effectively than MBVs. Additionally, exosomes downregulated inflammatory markers (TNF-α, iNOS) and increased Arg-1 expression in macrophages and synovial fibroblasts, indicating a stronger anti-inflammatory effect. These results suggest UC-MSC exosomes as a promising therapeutic option for OA, with the potential for modulating inflammation and promoting joint tissue regeneration.

Macrophages play a crucial role in regulating immune responses, inflammation, and tissue repair. Depending on environmental cues, they polarize into pro-inflammatory M1 or anti-inflammatory, pro-regenerative M2 phenotypes. Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have emerged as key mediators of intercellular communication and immune modulation. This study investigates the effects of matrix-bound vesicles (MBVs) and small extracellular vesicles (sEVs) derived from human umbilical cord MSCs (UC-MSCs) on human monocyte-derived macrophages (MDMs) in vitro. Both MBVs and sEVs reduced pro-inflammatory activation of M1 macrophages, downregulating the expression of CXCL10 and CD86 while increasing the M2 marker CD206. MBVs exerted a stronger suppressive effect on M1 MDM phenotype markers as well as on STAT1, STAT2, and IRF9 mRNA levels in M1 macrophages, indicating the inhibition of the JAK/STAT1 signaling pathway involved in the pro-inflammatory activation of macrophages. Functionally, both vesicle types enhanced phagocytosis of FITC-labeled E. coli by M1 and M0_GM macrophages, promoting a shift toward an M2-like phenotype. Moreover, MBVs and sEVs attenuated reactive oxygen species (ROS) production, with sEVs showing a more pronounced effect both on ROS generation and on the expression of NOX2 complex subunits (p47^phox, p67^phox) in M1 macrophages. These findings demonstrate that MBVs and sEVs from UC-MSCs possess distinct yet complementary immunomodulatory and antioxidant properties on MDMs, suggesting their potential as promising cell-free therapeutic agents for inflammatory and degenerative diseases.

Implantation of scaffolds causes a local inflammatory response whereby the early recruitment of neutrophils is of great importance not only for fighting the infection, but also for facilitating effective regeneration. We used luminol-dependent chemiluminescence, flow cytometry, ELISA, and confocal microscopy to assess the responses of neutrophils after the exposure to the scaffold-decellularized bovine pericardium (collagen type I) crosslinked with genipin (DBPG). We demonstrated that DBPG activated neutrophils in whole blood causing respiratory burst, myeloperoxidase (MPO) secretion, and formation of neutrophil extracellular trap-like structures (NETs). In addition, we studied platelets, another important player of the immediate immune host response. We found that platelets triggered redox-activation of isolated neutrophils by the pericardium scaffold, and likely participate in the NETs formation. Free radicals generated by neutrophils and hypochlorous acid produced by MPO are potent oxidizing agents which can oxidatively degrade biological structures. Understanding the mechanisms and consequences of redox activation of neutrophils by pericardium scaffolds is important for the development of new approaches to increase the efficiency of tissue regeneration.

RSL3 and ML-162, inhibitors of glutathione peroxidase 4 (GPX4), and erastin, an inhibitor of cystine/ glutamate transport, were used to induce ferroptosis in THP-1 macrophages. The progression of ferroptosis was monitored using three independent methods: reduction of Alamar blue by live cells, measurement of lactate dehydrogenase in the medium, and the LIVE/DEAD assay. Ferroptotic cell death was proven by using the specific inhibitor ferrostatin-1 and by detecting lipid oxidation in cells using the BODIPY 581/591 C11 fluorescent probe. RSL3 and ML-162 dose-dependently induced ferroptosis in cells. THP-1 macrophage ferroptosis is a slow process and begins ~5 h after inducer addition. Erastin was a weak ferroptosis inducer; however, it enhanced ferroptosis induced by GPX4 inhibitors. We compared the ability of two NO donors with different half-lives to affect THP-1 macrophage ferroptosis: DEA NONOate (2 min) and DTPA NONOate (3 h). Donors were added either once after the inducer at a concentration of 100–120 μM or repeatedly until reaching the final concentration. DEA had no effect on THP-1 macrophage ferroptosis, whereas DPTA completely inhibited ferroptosis. DTPA, being an NO donor with a half-life of 3 h at 37°С, can be used to inhibit ferroptosis in THP-1 macrophages, which develops within 17–19 h. Therefore, there are mechanisms of prolongation of NO action in cells that should be studied to use NO donors for regulation of cellular ferroptosis.