Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model

Abstract

Aims: Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants.

Methods: pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 10⁹ particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs.

Results: Significantly higher content of DNA and collagen was observed for the pEV-treated group compared to control and cEV groups. No differences were found in GAG quantification nor in EVs uptake within any treated group.

Conclusion: In conclusion, pEVs showed better performance than cEVs in our in vitro OA model. Although further studies are needed, pEVs are shown as a potential alternative to cEVs for cell-free regenerative medicine.

Fig. 1

Cartilage explants under osteoarthritis (OA)-like conditions after 14 days of treatment with platelet lysate-derived extracellular vesicles (pEVs) or human umbilical cord mesenchymal stromal cell-derived EVs (cEVs). a) DNA, c) glycosaminoglycan (GAG), and f) collagen quantification of cartilage explants after 14 days of in vitro culture under inflammatory stimulus to induce OA-like conditions and treated with 1 × 10⁹ particles of pEVs or cEVs. d) GAG quantification of spent media at days 1, 7, and 14 of explants cultured under OA-like conditions and treated with pEVs or cEVs. Representative images of 6 µm tissue sections stained with b) 4′,6-diamidino-2-phenylindole (DAPI) for nuclei identification (blue), e) toluidine blue for GAG staining (deep purple), or g) Sirius Red F3BA for collagen staining. Larger collagen fibres were detected as bright yellow or orange under polarized light microscopy, while the thinner ones including reticular fibres were detected as green. Scale bar represented on images. Nine different donors were used and the experiments were performed in triplicate. Results were compared for statistical significance using Kruskal-Wallis test with Mann-Whitney U test for DNA, collagen, and GAG in spent media quantification and using analysis of variance for GAG quantification in digested explants. ^ap < 0.05 vs control, ^bp < 0.05 vs OA, ^cp < 0.05 vs pEVs. *p < 0.05 vs day 1, ^#p < 0.05 vs day 7. C, control.

Fig. 2

PKH26 labelled-extracellular vesicle (EV) uptake by cartilage explants under osteoarthritis (OA)-like conditions at different time points. a) Corrected total cell fluorescence (CTCF) of cartilage explants under inflammatory stimulus to induce OA-like conditions, treated with PKH26 labelled-EVs (1 × 10⁹ particles of PKH-pEVs or PKH-cEVs) and monitored at one, two, three, four, and five hours. Results were normalized by the mean background signal of the control and OA groups, which were treated with PKH26 without EVs. b) Representative confocal images of 6 µm tissue sections of cartilage explants treated, as previously described, at the different timepoints. Cell nuclei marked with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and up-taken EVs labelled with PKH26 (red). Images were taken at 40× with their representative scale bar. Different areas from the same tissue section were photographed, and a total of 116 images were analyzed for PKH-pEV treatment and 174 images for PKH-cEV treatment. One donor was used for this experiment performing a triplicate for each treatment and time, but for the control one sample was used for each control and time. cEV, human umbilical cord mesenchymal stromal cell-derived extracellular vesicle; pEV, platelet lysate-derived extracellular vesicle. AU, arbitrary units.