Transparent dishes

A gap-closure assay was performed to evaluate the efficacy of SC migration among groups using two-well silicone culture inserts (Ibidi GmbH, Martinsried, Germany) set with transparent dishes (Ibidi GmbH). The two-well inserts were first filled with 10% FBS DMEM and then seeded with SCs (1 × 10⁴ cells/cm²) for 24 h to form a confluent monolayer, which was visible through the transparent dish. After the removal of the inserts, a 500 μm cell-free interval with clear demarcation was generated in the center of the dish. SCs were irrigated with PBS and then incubated in a serum-free medium at 37 °C. After replacing the serum-free medium with extracts of the control, PALD, and PALDE groups (in the same ratio as with the SC proliferation method), the migration of SCs from the medial edges of the interval across the 500 μm distance was observed for 48 h. Images were taken using light microscopy and digitalized (Leica QWin, Germany) after 0, 24, and 48 h of migration; data were quantified using ImageJ.

The efficiency of tenocyte migration was determined through gap-closing assay by the use of two-well silicone culture inserts (Ibidi GmbH, Martinsried, Germany) inset on the transparent dishes (Ibidi GmbH, Martinsried, Germany) without coatings. Tenocytes were seeded in the culture inserts (8 × 10³ cells/cm²) with 70 μL DMEM containing 20% FBS. After resting for 8 h, a single layer of confluent cells could be noted on the transparent dish. At this point, once the culture inserts were removed, a 500 μm gap was produced in the middle of the dish. Tenocytes were washed with PBS, and then incubated in a serum-free medium at 37 °C for 1 h. The medium was then replaced with EVs (100 μg/mL) diluted with culture media, and tenocytes were left to migrate for 24 h from the medial edges of the 500 μm gap. The migration assays were performed using the control group and EVs group. The migrated tenocytes were observed and captured under bright field microscopy with a camera (Leica QWin, Wetzlar, Germany) from 0 to 24 h. The percentage of remaining area was evaluated using Image J. All results were obtained in five independent experiments.