In Vivo Matrigel Plug Angiogenesis Assay

Angiogenesis was evaluated in vivo as the growth of blood vessels from subcutaneous tissue into a solid Matrigel plug containing the test sample according to a previously published protocol ²⁰. Growth factor‐reduced Matrigel (300 μl) was mixed in liquid form at 4°C with 100 μl of PBS (negative control), dBMSC‐exos, nBMSC‐exos, and basic fibroblast growth factor (bFGF, positive control). The final concentration of dBMSC‐exos and nBMSC‐exos was 1 × 10¹⁰ per milliliter, and the final concentration of bFGF was 150 ng/ml. The Matrigel mix was then injected subcutaneously into the ventral area of 6‐week‐old athymic nude mice. After 14 days, the mice were sacrificed, and the Matrigel plugs were excised. After fixation with 4% paraformaldehyde, the plugs were embedded in paraffin, sectioned, and stained with Masson's trichrome to visualize the endothelial cells/vessels as dark red and the Matrigel as blue. The number of migrated cells was counted in five randomly selected fields using a light microscope.