All animal procedures were approved by the Italian Ministry of Health, by the IRCCS Ospedale Policlinico San Martino Ethical Committee (Authorization n. 55/2020‐PR) and performed in accordance with the national current regulations regarding the protection of animals used for scientific purpose.
E17.5 fetuses were removed from pregnant C57BL/6 wild‐type mice and metatarsals were dissected under stereomicroscope. The isolated metatarsals were cultured in 24‐well plates in 300 μL of α‐MEM‐GlutaMAX medium (Gibco, Waltham, Massachusetts), supplemented with 10% FBS (Gibco, Milan, Italy) and 50 mg/mL penicillin/streptomycin for 96 hours. After 96 hours, attached bones covered with fibroblasts were selected and medium was replaced with α‐MEM‐GlutaMAX supplemented with 2% FBS. For each replicate, experimental groups have been set as follows: (a) negative control: serum‐free α‐MEM GlutaMAX; (b) test groups: serum‐free α‐MEM GlutaMAX supplemented with 0.5 μg of either ADSC‐/BMSC‐mEVs or ADSC‐/BMSC‐sEVs; (c) positive control: α‐MEM‐GlutaMAX supplemented with 10% FBS. After 7 days, bones were analyzed for the expression of CD31 by immunohistochemistry (IHC) and immunofluorescence (IF), as previously described.21 For more information about IHC and IF, please see Supplemental Materials .
E17.5 fetuses were removed from pregnant C57BL/6 wild‐type mice and metatarsals were dissected under stereomicroscope. The isolated metatarsals were cultured in 24‐well plates in 300 μL of α‐MEM‐GlutaMAX medium (Gibco, Waltham, Massachusetts), supplemented with 10% FBS (Gibco, Milan, Italy) and 50 mg/mL penicillin/streptomycin for 96 hours. After 96 hours, attached bones covered with fibroblasts were selected and medium was replaced with α‐MEM‐GlutaMAX supplemented with 2% FBS. For each replicate, experimental groups have been set as follows: (a) negative control: serum‐free α‐MEM GlutaMAX; (b) test groups: serum‐free α‐MEM GlutaMAX supplemented with 0.5 μg of either ADSC‐/BMSC‐mEVs or ADSC‐/BMSC‐sEVs; (c) positive control: α‐MEM‐GlutaMAX supplemented with 10% FBS. After 7 days, bones were analyzed for the expression of CD31 by immunohistochemistry (IHC) and immunofluorescence (IF), as previously described.