Edu kits

Fibroblasts, HUVECs, and HaCaT cells were treated with phosphate-buffered saline (PBS) or ADSC-EXOs (20 µg/mL) for 48 h; cell proliferation was evaluated by EdU kits (Beyotime Biotechnology). The migration abilities of fibroblasts, HUVECs, and HaCaT cells were assayed using 24-well Transwell Chambers (Corning Inc., Corning, NY, USA). In brief, cells suspended in Dulbecco’s modified Eagle medium without serum were added to the upper chamber and treated with PBS or ADSC-EXOs (5 µg/mL and 10 µg/mL). The lower chamber was filled with 600 µL of complete culture media consisting of 10% fetal bovine serum. After incubation at 37 °C for 24 h, cells that had migrated to the bottom surface were stained with crystal violet (Solarbio, Beijing, China). For the tube formation assay, HUVECs were seeded in 48-well plates that had been precoated with Matrigel Basement Membrane Matrix (BD Biosciences, NJ, USA). After incubation with PBS and ADSC-EXOs (10 µg/mL or 20 µg/mL) for 2 h, 4 h, and 8 h, tube formation was imaged by microscopy. The total number of loops, nodes, and branch points was calculated and used to quantify the tubular networks.

We chose a dose range of 1–50 µM because in extracellular fluid, the effective concentration of PPIs was relatively low (1–50 µM), which was almost equivalent to the clinical blood concentration of 20–40 mg/dl (Olbe et al. 2003 (link)). Pharmacokinetic analysis of clinical proton pump inhibitors showed that the maximum blood concentration of lansoprazole was in the range of 1–15 µM. CCK-8 (Beyotime, C0038) and EdU kits (Beyotime, C0078S) were used to study the effects of PPIs on the proliferation of MC3T3-E1 cells. MC3T3-E1 cells were seeded in 96-well plates (density: 8000 cells/well) until the cells reached ~ 70–80% confluence, after which the cells were treated with LPZ. MC3T3-E1 cells were incubated for 24 h in α-MEM/10% FBS containing different concentrations of LPZ (0, 5, 10, 20, 50 μM), and cells in the vehicle group were cultured in α-MEM/10% FBS containing 0.1% DMSO (v/v) (Sigma, D2650). Then, the cultures were washed with PBS, the medium in each well was replaced with 100 µl of FBS-free α-MEM, and 10% CCK-8 working solution was added. The mixture was incubated for 1 h at 37 °C in a humidified atmosphere of 5% CO₂ and measured at 450 nm using a microplate reader. The cells in the control group were cultured in α-MEM containing 0.1% DMSO.