Abstract: BACKGROUND: Three-dimensional bioprinted hydrogels have become an important research direction for the repair of oral tissue defects. Pre-vascularization of hydrogels can be achieved by loading endothelial cells and stromal cells. However, the dense hydrogel fibers often limit cell viability and extension. Whether increasing the internal porosity of the hydrogel can improve pre-vascularization remains unclear.
OBJECTIVE: To construct porous three-dimensional bioprinted hydrogels loaded with human umbilical vein endothelial cells and human dental pulp stem cells, and to explore the relationship between hydrogel pore size and pre-vascularization.
METHODS: (1) Methacrylate gelatin solution and poly (ethylene oxide) solution were mixed at volume ratios of 2:1, 1:1, 1:1.5, 1:2, and 1:3, with pure methacrylate gelatin solution as a control. Three-dimensional bioprinting was performed, and after curing and crosslinking, poly (ethylene oxide) was leached out to form pores. Based on porosity measurements, the mixed solutions with methacrylate gelatin solution and poly (ethylene oxide) solution volume ratios of 1:1, 1:2, and 1:3, along with the pure methacrylate gelatin solution, were selected for subsequent experiments. (2) The four types of solutions mentioned above were used as bioinks, separately encapsulating human umbilical vein endothelial cells or human dental pulp stem cells for three-dimensional bioprinting. After curing and crosslinking, poly (ethylene oxide) was leached out to form pores. Live/dead staining was used to detect cell viability. Simultaneously, both cell types were encapsulated and three-dimensional bioprinting was performed. After curing and crosslinking, poly (ethylene oxide) was leached out to form pores, and a tube formation assay was used to detect vascular network formation. (3) The four groups of three-dimensional bioprinted hydrogels, with or without the two cell types, were implanted subcutaneously into CB17-SCID mice. After 14 days, samples were collected, and hematoxylin-eosin staining and CD31 immunohistochemical staining were performed to observe vascular formation within the hydrogels.
RESULTS AND CONCLUSION: (1) The hydrogel in the pure methacrylate gelatin group had the smallest pores. As the proportion of poly (ethylene oxide) solution in the bioink increased, the pore size of the hydrogel increased. The group with a 2:1 volume ratio of methacrylate gelatin solution to poly (ethylene oxide) solution had excessively small pores, while the 1:2 and 1:1.5 groups had similar pore sizes. Therefore, the 2:1 and 1:1.5 groups were excluded from subsequent experiments. (2) Live/dead staining showed that human umbilical vein endothelial cells in all four hydrogel groups did not show significant spreading. Human dental pulp stem cells showed significant spreading in the hydrogels with a 1:2 and 1:3 volume ratio of methacrylate gelatin solution to poly (ethylene oxide) solution. There was no significant difference in the survival rate of human umbilical vein endothelial cells or human dental pulp stem cells cultured in the hydrogels for 3 days. The pure methacrylate gelatin group hydrogel showed the least vascular formation, and as the proportion of poly (ethylene oxide) solution in the bioink increased, vascular formation in the three-dimensional bioprinted hydrogel increased, resulting in a denser network structure. (3) Hematoxylin-eosin staining and CD31 immunohistochemical staining showed no vascular formation in the cell-free hydrogels. No vascular formation was observed in the pure methacrylate gelatin group and the 1:1 volume ratio methacrylate gelatin solution to poly (ethylene oxide) solution group containing embedded cells. Significant vascular formation was observed in the other two hydrogel groups. (4) The results indicate that the internal pores of the three-dimensional bioprinted methacrylate gelatin hydrogel can promote the formation of in vitro vascular-like structures by human umbilical vein endothelial cells and human dental pulp stem cells, and promote in vivo vascularization of the hydrogel.

Key words: bioprinting, prevascularization, methacrylated gelatin, polyethylene oxide, human umbilical vein endothelial cells, dental pulp stem cells