Spatial Distribution of Extracellular Vesicles, Autofluorescence and CD9 Positivity Around Chondrocytes in the Superficial Layer of Articular Cartilage.

This study investigates the distribution of extracellular vesicles (EVs) in superficial articular cartilage, hypothesizing that EVs (a) are unevenly distributed in this zonally organized tissue and (b) share a pattern similar to tissue autofluorescence. Fresh unfixed superficial cartilage from the femoropatellar groove of bovine knees was analysed using multiphoton microscopy (second harmonic generation, SHG, and multiphoton-autofluorescence, MPAF). Transmission electron microscopy (TEM) and immunostaining (CD9, membrane marker; collagen type VI, pericellular matrix marker) were performed on fixed tissue. Cartilage-bone cylinders were analysed in a simulated endomicroscopic setting. Additionally, EVs were isolated from synovial fluid and chondrocyte cell culture medium to demonstrate autofluorescence and staining properties. MPAF revealed a specific spatial distribution around superficial chondrocytes: lateral ring-like accumulations inside the cell lacunae and snow cap-like formations above cells outside the lacunae. CD9 staining was found outside the collagen type VI-positive matrix in MPAF-correlating locations. TEM confirmed a similar EV distribution. The endomicroscopic setting also visualized the lateral MPAF accumulations. In tissue with early osteoarthritic degeneration these patterns were not found. In conclusion, EVs/CD9 exhibit a specific spatial distribution, suggesting guided EV transport or binding within the extracellular matrix, which changes with tissue degeneration. These findings provide insight into the spatial relation between EVs and superficial cartilage architecture in health and disease and indicate a potential link between extracellular MPAF and EVs as a basis for the development of diagnostic methods and in vivo EV tracking.