Live dead assay kit

The viability of visualized cells was evaluated using a live/dead assay kit (Abcam, Cambridge, UK) following the standard protocol. Briefly, the RAW 264.7 cells and scaffold constructs were first washed twice with PBS and then incubated in standard working solution at room temperature for 10 min. The constructs were then washed twice with PBS and observed under a confocal laser scanning microscope (CLSM; NikonA1R; Nikon, Japan).
To investigate the morphology of RAW 264.7 cells on scaffolds, the cell/scaffolds were collected after being cultured for 7 days, rinsed in PBS and then fixed with 2.5% glutaraldehyde in PBS for 1 h. Then cell/scaffolds were washed with buffer containing 4% (w/v) sucrose in PBS and post-fixed in 1% osmium tetroxide in PBS, and then were dehydrated in a graded ethanol series (30, 50, 70, 90 and 100%) for 10 min each and twice in absolute ethanol, freeze dried, coated with gold, and examined by SEM.

Gelatin (Type B from bovine bone, average molecular weight 80,000 Da) was obtained from Dongbao Bio-tech (Baotou, China). Methacrylic anhydride (MA), poly (ethylene glycol) diacrylate (PEGDA), 2-Hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959), and deuterium oxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Collagenase Type I, FITC-BSA were purchased from Solarbio (Beijing, China). MC3T3-E1 (Mouse osteoblast cell line, 6 passages), fetal bovine serum, Alpha Modification Eagle Medium (α-MEM), and PBS buffer (pH 7.4) were purchased from Union Hospital (Beijing, China). The live/dead assay kit was purchased from ABcam (Britain, UK). All other reagents and solvents were of reagent grade.