Mbmscs

Primary mouse bone mesenchymal stem cells (mBMSCs) were purchased from ATCC, while human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell. The cells used in the cell experiment were mBMSCs at passages 3–6 and HUVECs at passages 3–4. The culture medium for mBMSCs was Dulbecco's minimal Eagle medium (DMEM) high-glucose medium containing 1% double antibody (penicillin-streptomycin) and 10% foetal bovine serum (FBS). HUVECs were cultured in endothelial cell medium (ECM) with 1% endothelial cell growth supplement (ECGS), 1% double antibody (penicillin-streptomycin) and 5% FBS. The cells were digested and isolated by 0.25% ethylenediaminetetraacetic acid (EDTA)-trypsin. Before seeding, materials were sterilized in an autoclave (121°C, 30 min) and soaked in phosphate buffer overnight. When seeding, a high concentration cell suspension was added onto the surface of scaffolds. The cells were distributed over the whole surface. Then, culture medium was added after cells adhered preliminarily (approximately 1 h). In addition, the medium for osteogenic differentiation included osteogenesis-inducing reagents mixed with 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 mM ascorbic acid.

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).