Enhanced Vascular-like Network Formation of Encapsulated HUVECs and ADSCs Coculture in Growth Factors Conjugated GelMA Hydrogels

Abstract

Tissue engineering primarily aimed to alleviate the insufficiency of organ donations worldwide. Nonetheless, the survival of the engineered tissue is often compromised due to the complexity of the natural organ architectures, especially the vascular system inside the organ, which allows food-waste transfer. Thus, vascularization within the engineered tissue is of paramount importance. A critical aspect of this endeavor is the ability to replicate the intricacies of the extracellular matrix and promote the formation of functional vascular networks within engineered constructs. In this study, human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured in different types of gelatin methacrylate (GelMA). In brief, pro-angiogenic signaling growth factors (GFs), vascular endothelial growth factor (VEGF₁₆₅) and basic fibroblast growth factor (bFGF), were conjugated onto GelMA via an EDC/NHS coupling reaction. The GelMA hydrogels conjugated with VEGF₁₆₅ (GelMA@VEGF₁₆₅) and bFGF (GelMA@bFGF) showed marginal changes in the chemical and physical characteristics of the GelMA hydrogels. Moreover, the conjugation of these growth factors demonstrated improved cell viability and cell proliferation within the hydrogel construct. Additionally, vascular-like network formation was observed predominantly on GelMA@GrowthFactor (GelMA@GF) hydrogels, particularly on GelMA@bFGF. This study suggests that growth factor-conjugated GelMA hydrogels would be a promising biomaterial for 3D vascular tissue engineering.

Keywords: bFGF; gelMA; hADSCs; hUVECs; vEGF; vascular formation.

Figure 1

(A) SEM micrographs of different type of GelMAs at different concentrations. (B) Pore-sized analysis result of different type of GelMAs at different content ratios based on SEM micrographs. (C) Oscillatory frequency sweep of 15% GelMA, GelMA@VEGF₁₆₅, and GelMA@bFGF hydrogels.

Figure 2

Swelling behaviors of (A) GelMA, (B) GelMA@VEGF₁₆₅, and (C) GelMA@bFGF hydrogels in ddH₂O.

Figure 3

Enzymatic degradation of (A) GelMA, (B) GelMA@VEGF₁₆₅, and (C) GelMA@bFGF hydrogels in collagenase-CaCl₂ contained in PBS.

Figure 4

Encapsulated HUVECs and hADSCs in 3D hydrogels. (A) Day 14 LIVE/DEAD imaging of encapsulated cells. The images are displayed in Z-projection and merged channels (Green: live cells, red: dead cells, blue: nuclei, scale bar: 100 μm). (B) CCK-8 assay cell growth profile of GelMA, GelMA@VEGF₁₆₅, and VEGF@bFGF in days 1, 3, 7, 14, and 28 of coculture in complete ADSC and complete M199:ADSC (50:50) medium.

Figure 5

Cell morphology and cell self-organization within the 3D hydrogel cultured in a 50:50 medium on day 7. Confocal live-cell imaging of prestained HUVECs (shown in red, CD31), a smooth muscle marker (shown in green, α-SMA), and hADSCs (shown in red, CD90). The images are displayed in Z-projection and merged channels images. Nuclei counter staining is shown in blue (scale bar: 100 μm).