Isolation and Cultivation of Murine SSCs

Murine SSCs were isolated from lone bones according to our previously reported procedure with minor revisions [12 (link), 25 (link), 26 (link)]. In the present study, the metaphysis was maintained because it has been proved as a pool of SSCs which showed stronger osteochondral potential than that of mesenchymal stem cells [7 (link), 8 (link)]. Briefly, femurs and tibiae from 1-week-old C57BL/6 N mice were dissected, and the bone marrow cells were flushed out before metaphyses and diaphyses were chopped and digested by collagenase II (Sigma-Aldrich, 0.1% vol/vol) at 37 °C for 1 h. The released cells were discarded, and the digested bone chips were cultured in minimum essential medium eagle, alpha modification (α-MEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT). The adherent cells were cultured in a 37 °C incubator with 5% CO₂. Cells from passages 3–6 were used for in vivo and in vitro experiments unless otherwise described. The PI3K/AKT inhibitor LY294002 (MCE, China) and NRF2 inhibitor ML385 (APExBIO, USA) were dissolved in DMSO to prepare 10 mM stock solutions and then diluted in cell culture medium to the appropriate experimental concentrations.