Effects of Different Concentrations of Platelet-Rich Plasma-Derived Extracellular Vesicles on Cellular Activity and Lineage-Associated Marker Expression in Tendon Stem Cells

Background: Tendon stem cells (TSCs) contribute to tendon maintenance and repair through their self-renewal capacity and multilineage differentiation potential. Platelet-rich plasma-derived extracellular vesicles (PRP-EVs) may regulate tissue repair; however, their effects on TSC cellular activity and lineage-associated marker expression remain unclear. Objective: To investigate the effects of different concentrations of PRP-EVs on CCK-8 signal and the expression of tendon-associated and alternative lineage-associated markers in TSCs. Methods: Rat TSCs were isolated from Achilles tendon tissue by enzymatic digestion and characterized using flow cytometry, immunofluorescence staining, colony formation assays, and multilineage differentiation assays. PRP-EVs were isolated from pooled rat PRP and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and Western blotting. TSCs were treated with PRP-EVs at concentrations of 0, 5, 20, and 40 μg/mL. CCK-8 absorbance was measured as an indicator of cellular metabolic activity. The mRNA and protein expression levels of tendon-associated markers (ScxA, Col I, and Tnmd) and alternative lineage-associated markers (Osx, RUNX2, SOX9, and PPARγ) were assessed using quantitative real-time PCR and Western blotting, respectively. Results: TEM revealed vesicle-like structures in the PRP-EV preparation, with a mean particle diameter of 198.6 ± 94.5 nm measured by DLS. Western blotting detected the EV-associated proteins TSG101, CD9, and CD81. PRP-EV treatment increased the CCK-8 signal, with 40 μg/mL generally producing the strongest overall response among the concentrations tested, although differences between concentrations were not consistently significant. Treatment was also associated with increased expression of tendon-associated markers and generally decreased expression of alternative lineage-associated markers. Conclusion: PRP-EV treatment increased the CCK-8 signal and altered the expression of tendon-associated and alternative lineage-associated markers in cultured rat TSCs. These findings indicate that PRP-EVs modulate the molecular phenotype of TSCs in vitro.