Matrix Protective and Anti-Inflammatory In Vitro Potential of Adipose Mesenchymal Stromal Cell-Derived EVs Coupled...

The limited intrinsic repair capacity of articular cartilage impairs tissue regeneration in osteoarthritis (OA). Platelet-rich plasma (PRP) fibrin gels offer a viable autologous scaffold for cartilage repair but often promote fibrous tissue rather than native hyaline structures. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) can overcome this limitation by actively promoting hyaline cartilage formation and suppressing matrix degradation. This study describes and validates a protocol for embedding adipose-derived MSC (ASC)-EVs into PRP gels (PRP-EVs) and evaluates the protective efficacy in human pathologic chondrocytes and synoviocytes.

ASC-EVs were isolated via ultracentrifugation, characterized and their microRNA cargo profiled (quantitative reverse-transcription polymerase chain reaction, qRT-PCR). EVs (10 × 10⁹ particles/mL) were loaded into PRP activated with CaCl₂ to form a fibrin hydrogel. EV incorporation efficiency, 4-week release kinetics, cellular uptake (two-dimensional [2D] and three-dimensional [3D] microfluidic models) and penetration into OA cartilage explants using coherent anti-Stokes Raman scattering/two-photon excited fluorescence CARS/TPEF microscopy were assessed. To evaluate the biological effects of the PRP-EV gel secretome, we analyzed gene expression (qRT-PCR), collagen I/II/X deposition (immunofluorescence), metalloproteinase activity and nitric oxide release (ELISA) in interleukin-1β-stimulated 3D chondrocyte pellets, alongside gene expression in OA patient-derived synoviocytes (qRT-PCR).

The protocol demonstrated high EV encapsulation efficiency (60% ± 8 of the input) in PRP gels and steady kinetics yielding a 20% cumulative release over 4 weeks. Released EVs maintained functional integrity, showing matching 2D/3D uptake in target cells and penetrating to depths exceeding 200 μm within 48 hoursinto OA patients' cartilage explants. In 3D chondrocyte pellets, PRP-EVs significantly downregulated catabolic genes (MMP1, MMP3, MMP13), reducing cumulative matrix metalloproteinase (MMP) enzymatic activity, and blunted nitric oxide production and CCL5 expression. PRP-EVs counteracted PRP-induced fibrosis, significantly increasing the collagen II/I ratio and suppressing hypertrophic collagen X deposition. In synoviocytes, PRP-EVs significantly downregulated key inflammatory, chemotactic, and pain-signaling genes (CCL2, CXCL8, CXCL10, IL-6, COX2, NGF).

This study demonstrates that embedding ASC-EVs within a PRP fibrin hydrogel creates a bioactive delivery system. In vitro, the secretome released from this composite material successfully counteracts pathological processes in osteoarthritis patient-derived chondrocytes and synoviocytes, reducing matrix degradation and inflammation while promoting a hyaline-like cartilage phenotype.