关键词: 3D打印, 甲基丙烯酰化明胶, 水凝胶, 成纤维细胞, 人脐静脉内皮细胞, 促血管化

Abstract: BACKGROUND: Combining seed cells with 3D bioprinting technology enables the specific construction of various tissues and organs to meet the demands of tissue repair. However, further research is needed on the promotion of angiogenesis in damaged tissues. 
OBJECTIVE: By cultivating a 3D scaffold structure of methacrylated gelatin loaded with fibroblasts, obtaining the supernatant, and mixing it in different proportions with a complete culture medium to simulate the cellular microenvironment during tissue repair, this study aimed to explore the role of various cellular microenvironments in promoting angiogenesis in endothelial cells. 
METHODS: A methacrylated gelatin scaffold structure loaded with fibroblasts was prepared using an extrusion-based 3D bioprinting process. Hydrogel scaffold extract was prepared and mixed with a complete culture medium in ratios of 1:1, 1:2, and 1:4 to obtain conditioned medium. Mouse embryonic fibroblasts BALB3T3 and human umbilical vein endothelial cells were co-cultured with complete medium (control group) and hydrogel scaffold extract, respectively. Cell proliferation was assessed using the CCK-8 assay and cell viability was analyzed using live/dead staining. Three kinds of conditioned medium and complete medium (control group) were used to co-culture with human umbilical vein endothelial cells for tube formulation assay, vascular genetic testing, and immunofluorescence staining of CD31. 
RESULTS AND CONCLUSION: (1) Scanning electron microscopy revealed that the methacrylated gelatin scaffold exhibited a porous structure, and rheological results demonstrated excellent mechanical properties of the hydrogel. CCK-8 assay and live/dead cell staining showed that the hydrogel scaffold extract had no obvious cytotoxicity. (2) Tube formulation assay indicated that the hydrogel showed the total length of cell tubules in 1:1 conditioned medium group was smaller than that in the control group (P < 0.05). There were no statistical differences among the four groups in the number of vascular branches formed by endothelial cells (P > 0.05). (3) qRT-PCR results showed that for vascular endothelial growth factor mRNA expression, the 1:2 conditioned medium group was lower than the 1:1 conditioned medium group on day 1 (P < 0.01). On day 3, the expression level of vascular endothelial growth factor in the 1:2 conditioned medium group was higher than that in the control group (P < 0.01). On day 5, the cytokine expression level in the 1:2 conditioned medium group was significantly higher than that in the other three groups (P < 0.01 or P < 0.000 1). The expression in the 1:1 conditioned medium group was significantly lower than that in the other three groups (P < 0.05 or P < 0.01). On day 1, the expression level of basic fibroblast growth factor in the 1:1 conditioned medium group was significantly higher than that in the control group and 1:4 conditioned medium group (P < 0.01, P < 0.05). The expression was higher in the 1:2 conditioned medium group than that in the control group (P < 0.05). On day 3, the expression levels of cytokines in the 1:4 conditioned medium group was higher than that in the control group (P < 0.05). (4) On day 3, the expression of CD31 in the 1:2 conditioned medium group was higher than that in the control group and the 1:4 conditioned medium group (P < 0.05). (5) The results indicate that the resulting conditioned media can simulate the microenvironment of vascular regeneration after tissue damage, promoting the vascularization process of endothelial cells. The best promotion of vascularization in endothelial cells was observed when the ratio of supernatant to complete culture medium was 1:2.

Key words: 3D printing, methacrylated gelatin, hydrogel, fibroblast, human umbilical vein endothelial cell, promoting angiogenesis