Engineering a long-lasting microvasculature in vitro model for traumatic injury research

Microvascular injuries can have systemic physiological effects that exacerbate other injuries and pose a danger to life. Reliable in vitro microvascular models are required to enhance understanding of traumatic injuries. This research aims to develop and optimise a three-dimensional (3D) hydrogel construct for the formation and long-term stability of an in vitro microvascular model for trauma research. First, we develop a 3D hydrogel scaffold using a physiologically relevant cell type to enable the formation of a durable microvascular endothelial network and validate it against the gold standard: HUVECs. Then, we explore the impact of modifying the hydrogel composition, specifically fibrinogen source and concentration, medium, and crosslinking ratio, on scaffold material properties and, consequently, the formation of endothelial networks, their architecture, and long-term integrity. Our results demonstrate that 3D hydrogel scaffolds are crucial for maintaining network stability beyond the initial 24 h. For trauma research applications, the material properties and mechanical behaviour of the hydrogels are critical. Microrheometry revealed that fibrinogen concentration significantly influences gelation times, absorbance rate, storage modulus (G'), loss modulus (G"), and complex viscosity, while also reducing creep compliance. Our multi-pronged approach to engineering microvasculature constructs revealed that variations in hydrogel composition, including fibrinogen concentration and source, crosslinking ratio and choice of medium, strongly affect the hydrogel material characteristics and, in turn, the resulting microvascular networks. Hydrogels made with high concentrations of human fibrinogen, a 200:10:1 crosslinking ratio, and endothelial basal medium (EBM) or EBM supplemented with VEGF performed best, demonstrating superior long-term network stability. The microvasculature construct developed here could be used as a potential platform for studying traumatic injuries, as well as testing interventions aimed at improving recovery and mitigating damage.