Biomaterial and Cellular Microenvironment Regulate Vascular Network Formation

Introduction:: Proper formation of vascular network in a thick engineered tissue is essential to its survival and functions in vivo. As engineered tissue are usually fabricated with biomaterials and multiple cell types, it is important to identify the material and cellular microenvironment that supports and promotes vascular network formation. In this study, we investigated the role of GelMA hydrogel weight percent on vascular self-assembly and host perfusion. The supportive role of several stromal cell types on vascular network formation was also identified and compared.


Materials and Methods:: Multiple materials and cellular conditions were studied utilizing the high-throughput highly parallel tissue grafting (HPTG) method (Figure 1). Primary human umbilical vein endothelial cells (HUVECs) were encapsulated in gelatin methacrylate (GelMA) hydrogel of different weight percent with or without the addition of stromal cells such as normal human dermal fibroblasts (NHDFs), mesenchymal stem cells (MSCs), dental pulp stem cells (DPSCs). 1µL of each designed combination of materials and cells was added to a 3D printed microarray and crosslinked using ultraviolet light to form 43 different microtissues. The array containing 43 microtissues made from different material and cell combinations were either sutured on perigonadal fat pad of athymic mice or cultured in vitro for 2 weeks. After explanting, arrays were stained for human CD31 and mouse Ter119 to identify self-assembled vascular network (huCD31) and sign of perfusion from the host vasculature (Ter119) using confocal microscope and Vesselucida 360 software.


Results, Conclusions, and Discussions:: In order to identify biomaterial and cellular environment that supports vascularization, we encapsulated HUVECs with or without NHDFs in GelMA hydrogel with various weight percent (Figure 1). Only at an extremely low weight percent of GelMA (2%), HUVECs can self-assemble into vascular networks. As the hydrogel weight percent increases, vascular self-assembly is reduced (3-10% GelMA) or even inhibited (12-15% GelMA). However, the presence of fibroblasts can rescue vascular network in hydrogel of 3-10% weight percent. When hydrogel weight percent is higher than 10%, the presence of NHDFs no longer promotes vascular self-assembly.

We further compared 3 stromal cell types. HUVECs were encapsulated in 5% or 10% GelMA hydrogel with NHDFs (EF), MSCs (EM) or DPSCs (ED). Although 10% GelMA hydrogel does not allow endothelial self-assembly by itself, the incorporation of stromal cells significantly enhanced vascular network formation. However, different stromal cells did not result in significantly different vascularization outcome.

In conclusion, only GelMA hydrogel with a low weight percent was able to support vascular self-assembly. The addition of NHDFs can promote vascular network formation and perfusion in vivo, even at a hydrogel with high weight percent of up to 10%. MSCs and DPSCs were even more potent to enhance vascular network formation and perfusion.


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