Construction and performance evaluation of pre-vascularized three-dimensional porous bioprinted hydrogel

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, and pure methacrylate gelatin solution were selected for subsequent experiments. (2) The above four solutions were used as bioinks to encapsulate 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. Both cells were co-encapsulated for three-dimensional bioprinting, and after curing and crosslinking, poly (ethylene oxide) was leached out to form pores. Tube formation assay was used to detect vascular network formation. (3) The four groups of three-dimensional bioprinted hydrogels with or without encapsulated cells were implanted subcutaneously into CB17-SCID mice. After 14 days, samples were harvested, and hematoxylin-eosin and CD31 immunohistochemical staining were used to observe vascular formation within the hydrogels. RESULTS AND CONCLUSION: (1) 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 hydrogels increased. The 2:1 group had too small pores, and 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 the four groups did not spread significantly, while human dental pulp stem cells in the 1:2 and 1:3 groups spread significantly. There was no significant difference in cell viability of human umbilical vein endothelial cells or human dental pulp stem cells cultured for 3 days among the groups. The pure methacrylate gelatin group had the least vascular formation, and as the proportion of poly (ethylene oxide) solution increased, vascular formation in the three-dimensional bioprinted hydrogels increased, with denser network structures. (3) Hematoxylin-eosin and CD31 immunohistochemical staining showed no vascular formation in hydrogels without cells, and no vascular formation in the pure methacrylate gelatin group and the 1:1 group with cells, while obvious vascular formation was observed in the other two groups. (4) These results indicate that the internal pores of three-dimensional bioprinted methacrylate gelatin hydrogels can promote the formation of vascular-like structures in vitro by human umbilical vein endothelial cells and human dental pulp stem cells, and promote in vivo vascularization of the hydrogels.

1. Introduction

Tissue engineering technology has made great progress in recent years, and the use of three-dimensional bioprinting to construct tissues and organs is an important direction for clinical treatment of defective tissue repair. However, insufficient vascularization after implantation of printed tissue-engineered constructs has become one of the bottlenecks hindering their clinical application. On one hand, angiogenesis requires stringent conditions, involving the participation of multiple cell types and angiogenic factors; on the other hand, constructing an environment conducive to angiogenesis is difficult. After implantation, tissue-engineered constructs must rapidly establish sufficient blood circulation to ensure long-term survival of internal cells [1-3]; otherwise, the interior of the construct may undergo necrosis due to lack of blood supply under prolonged hypoxia [4-5]. Pre-vascularization aims to construct an internal vascular network within tissue-engineered constructs in vitro, which can rapidly anastomose with host vessels after transplantation, establishing blood supply in a short time, thereby facilitating the survival of the construct and tissue repair.

There are various methods for pre-vascularization, including introduction of growth factors, construction of channel scaffolds, use of perfusable bioreactors, co-culture of cells, and functional modification of cells. Among these, co-culture of endothelial cells and mesenchymal stem cells is one of the most commonly used methods. Studies have shown that co-culture of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells can form rich vascular network structures, with HUVECs lining the lumen and mesenchymal stem cells surrounding the lumen as pericytes [6-8]; after in vivo transplantation, the vascular network can remain stable and functional for a long time [9]. Dental pulp stem cells (DPSCs) are mesenchymal stem cells commonly used for oral and maxillofacial tissue repair [10], and can secrete various pro-angiogenic factors, such as vascular endothelial growth factor, promoting the migration and proliferation of HUVECs. DPSCs can also exert pericyte-like functions, further stabilizing the formed vascular network [11-13].

Three-dimensional bioprinting technology allows precise control of the internal structure and external shape of scaffolds, uniformly encapsulating cells within the scaffold material to mimic the natural three-dimensional cellular environment.