Sodium barbiturate

All rabbits were anesthetized with an intravenous injection of 100 mg/kg sodium barbiturate (Sigma-Aldrich; Merck KGaA). The surgical region was shaved and aseptically prepared, with sterile barriers to limit the surgical field. A tongue-shaped incision was made over the head and the skin, and underlying tissues, including the temporal muscle, were subsequently retracted to expose the full extent of the cranium. The periosteum was resected to avoid any influence on bony regeneration. A 15-mm diameter full-thickness defect, which was demonstrated to be the critical-size defect (CSD) in previous studies (36 (link),37 (link)), was carefully prepared to avoid dural tears using a dental bar and continuous irrigation with sterile saline at room temperature. Rabbits were subsequently administered with their respective treatments. The scalp was repositioned and sutured to achieve primary closure. Each rabbit received a prophylactic intramuscular injection of penicillin (25,000 IU/kg).

Animals were anesthetized with an intravenous injection of 100 mg/kg sodium barbiturate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Bone marrow was subsequently aspirated from the hind limb bones under sterile conditions. MSCs were isolated from the aspirated bone marrow using the Percoll density gradient centrifugation method (Histopaque-1077; Sigma-Aldrich; Merck KGaA) and were cultured in α-Minimum Essential Medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere containing 5% CO₂ at 37°C. The culture medium was changed twice per week. When MSCs reached 80–90% confluence, they were trypsinized and re-seeded for further expansion.

After the successful establishment of the elderly female mouse model of T2DM + OP, mouse bone marrow mesenchymal stem cells (BMSCs) were extracted according to a previous study (18 (link)). First, the mice were anesthetized by an i.p. injection of sodium barbiturate (Sigma-Aldrich; Merck KGaA), and then the mice were sacrificed by cervical dislocation. The bilateral femur and tibia were isolated under sterile conditions; muscle tissue was removed, and the femur and tibia were exposed. Then BMSCs were cultured in high-glucose DMEM (Corning) containing 10% fetal bovine serum (FBS) (Cyagen), 100 U/ml penicillin (Beyotime) and 100 µg/ml streptomycin (Beyotime). The medullary cavity was rinsed repeatedly, the cells were extracted out of the marrow cavity, centrifuged with a high-speed centrifuge (Thermo Fisher Scientific, Inc.) for 5 min at 200 x g, and the cell pellet was placed in cell bottles. Then the cell pellet was placed in an incubator (Thermo Fisher Scientific, Inc.) with 5% CO₂ at 37˚C. BMSCs were purified by differential adherence method; culture medium was replaced every three days, and non-adherent cells were removed; the splitting ratio was 1:3. The above steps were repeated and passed to the third generation for subsequent experiments. The study was approved by the Animal Ethics Care Committee of Qiqihar Medical University (QMU-AECC-2019-51).