Mbmscs

The mouse bone marrow mesenchymal stem cells (mBMSCs; cells were kindly provided by Sciencell Biotechnology Co., Ltd, USA) were used to evaluate the osteogenic activity on different samples. The mBMSCs were cultured in DMEM medium (high glucose; Gibco, USA) with 15% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin serum (antibiotic/antimycotic; Gibco, USA) on the cell culture flask in a humidified atmosphere of 5% CO₂ at 37 °C. Cells were passaged at a ratio of 1: 3 every three days. The primary mBMSCs used in the experiments were only passaged 2–4 times.

Mouse bone marrow mesenchymal stem cells (mBMSCs; ATCC, USA), RAW264.7 (Chinese Academy of Sciences, China), and human umbilical vein endothelial cells (HUVECs; Sciencell, USA) were applied to assess the cell responses of the scaffolds. After confluence, the cells were digested by trypsin solution (Gibco) and collected. The culture medium for RAW264.7 and mBMSCs was DMEM (Gibco) with 10% fetal bovine serum (FBS; Gibco) and 1% P/S solution (Gibco). Endothelial cell medium (ECM; Sciencell) with supplements (FBS and growth factors) was used to culture HUVECs. The mBMSCs, RAW264.7, and HUVECs at 3–5 passages were used in this study. The culture medium was changed every 2 days.
SG, SrP/SG, and Rg1/SrP/SG scaffolds were prepared and sterilized under gamma-ray irradiation (cobalt source) of 5 kGy for about 4 h. Before cell seeding, the scaffolds were soaked in the basal medium for 24 h. Scaffold extracts were prepared by immersing a sample (Φ5 × 2 mm) in 1 mL basic medium and placed at 37°C for 24 h and/or 72 h. Then the supernatant was collected, supplemented with FBS and/or factors, and finally diluted by the corresponding complete culture medium with a ratio of 1:3. The concentrations of Sr, Ca, and P in DMEM extracts were measured by ICP, and the concentration of Rg1 was detected by high-performance liquid chromatography (Agilent, USA).