Fig. 4.

In vitro assessment of the angiogenic ability of the piezoelectric hydrogels. a Schematic diagram of the pro-angiogenic cell experiment. First, the piezoelectric hydrogel was co-cultured with macrophages; then, vascular endothelial cells were cultured using the co-culture medium from macrophages. b Representative digital and microscopic images of the migrated HUVECs cells to the lower chamber after the macrophage medium was separately added to the lower chamber and cultured for 24 h. Scale bar: 200 μm. c Representative microscopic images of HUVECs after the cells were co-cultured with different macrophage media for 24 h. Scale bar: 200 μm. d Representative fluorescence images and quantitative analysis of HUVEC tube formation after the cells were co-cultured with different macrophage media for 8 h. Percentage of blood vessel area is the vessel density of vascular endothelial cells in a single image, total number of junctions is the cell intersections between vascular endothelial cells in a single image, tube formation was quantified using AngioTool (National Cancer Institute, NIH) for the percentage of blood vessel area and the total number of junctions. Scale bar: 250 μm. e Relative mRNA expression levels of angiogenesis-related genes in HUVECs after the cells were co-cultured with different macrophage media for 7 d, including VEGF, HIF-1α, bFGF, and Ang-1. f Representative immunofluorescence images of CD31 (red), VEGF (green), and nuclei (blue) in HUVECs after the cells were co-cultured with different macrophage media for 7 d. Scale bar: 25 μm. ^*P < 0.05, compared with the control group; ^#P < 0.05, compared with the CG group; ^$ P < 0.05, compared with the CG/PHA group. CG chitosan/gelatin, PHA polydopamine coated-hydroxyapatite, PBT polydopamine coated-barium titanate, VEGF vascular endothelial growth factor, HIF-1α hypoxia inducible factor-1α, bFGF basic fibroblast growth factor, Ang-1 angiopoietin-1, HUVECs human umbilical vein endothelial cells