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

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.

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 µm sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Differential Effects of Varying Concentrations of Phosphorus, Iron, and Nitrogen in N2-Fixing Cyanobacteria.

Diazotrophs or N2-fixers are one of the most ecologically significant groups in marine ecosystems (pelagic and benthic). Inorganic phosphorus (PO4 3-) and iron (Fe) can limit the growth and N2-fixing capacities of cyanobacteria. However, studies investigating co-limitation of these factors are lacking. Here, we added different concentrations of PO4 3- and Fe in two cyanobacterial species whose relatives can be found in seagrass habitats: the unicellular Halothece sp. (PCC 7418) and the filamentous Fischerella muscicola (PCC 73103), grown under different nitrate (NO3 -) concentrations and under N2 as sole N source, respectively. Their growth, pigment content, N2-fixation rates, oxidative stress responses, and morphological and cellular changes were investigated. Our results show a serial limitation of NO3 - and PO4 3- (with NO3 - as the primary limiting nutrient) for Halothece sp. Simultaneous co-limitation of PO4 3- and Fe was found for both species tested, and high levels of Fe (especially when added with high PO4 3- levels) inhibited the growth of Halothece sp. Nutrient limitation (PO4 3-, Fe, and/or NO3 -) enhanced oxidative stress responses, morphological changes, and apoptosis. Furthermore, an extensive bio-informatic analysis describing the predicted Pho, Fur, and NtcA regulons (involved in the survival of cells to P, Fe, and N limitation) was made using the complete genome of Halothece sp. as a model, showing the potential of this strain to adapt to different nutrient regimes (P, Fe, or N).

"The Good, the Bad and the Double-Sword" Effects of Microplastics and Their Organic Additives in Marine Bacteria.

Little is known about the direct effects of microplastics (MPs) and their organic additives on marine bacteria, considering their role in the nutrient cycles, e.g., N-cycles through the N2-fixation, or in the microbial food web. To fill this gap of knowledge, we exposed marine bacteria, specifically diazotrophs, to pure MPs which differ in physical properties (e.g., density, hydrophobicity, and/or size), namely, polyethylene, polypropylene, polyvinyl chloride and polystyrene, and to their most abundant associated organic additives (e.g., fluoranthene, 1,2,5,6,9,10-hexabromocyclododecane and dioctyl-phthalate). Growth, protein overproduction, direct physical interactions between MPs and bacteria, phosphorus acquisition mechanisms and/or N2-fixation rates were evaluated. Cyanobacteria were positively affected by environmental and high concentrations of MPs, as opposed to heterotrophic strains, that were only positively affected with high concentrations of ~120 µm-size MPs (detecting the overproduction of proteins related to plastic degradation and C-transport), and negatively affected by 1 µm-size PS beads. Generally, the organic additives had a deleterious effect in both autotrophic and heterotrophic bacteria and the magnitude of the effect is suggested to be dependent on bacterial size. Our results show species-specific responses of the autotrophic and heterotrophic bacteria tested and the responses (beneficial: the "good," deleterious: the "bad" and/or both: the "double-sword") were dependent on the type and concentration of MPs and additives. This suggests the need to determine the threshold levels of MPs and additives concentrations starting from which significant effects can be observed for key microbial populations in marine systems, and these data are necessary for effective environmental quality control management.