Flow-Induced Vascular Network Formation and Maturation in Three-Dimensional Engineered Tissue

Engineered three-dimensional (3D) constructs have received much attention as in vitro tools for the study of cell-cell and cell-matrix interactions, and have been explored for potential use as experimental models or therapeutic human tissue substitutes. Yet, due to diffusion limitations, the lack of stable and perfusable blood vessel networks jeopardizes cell viability once the tissue dimensions extend beyond several hundred microns. Direct perfusion of 3D scaffold cultures has been shown to enhance oxygen and nutrient availability. Additionally, flow-induced shear stress at physiologically relevant levels, positively impacted endothelial cell migration and alignment in various two-dimensional (2D) culture models and promoted angiogenic sprouting in microfluidic systems. However, little is known about the effect of flow on vascularization in implantable 3D engineered tissue models. The present study investigated the effect of direct flow-induced shear stress on vascularization in implantable 3D tissue. The differential effect of various levels of shear stress, applied while maintaining constant culture conditions, on vascular parameters was measured. Samples grown under direct flow conditions showed significant increases (>100%) in vessel network morphogenesis parameters and increases in vessel and extracellular matrix (ECM) protein depth distribution, as compared to those grown under static conditions. Enhanced vascular network morphogenesis parameters and higher colocalization of alpha-smooth muscle actin (α-SMA) with endothelial vessel networks characterized the specific contribution of direct flow to vessel network complexity and maturation. These observations suggest that flow conditions promote 3D neovascularization and may be advantageous in attempts to create large-volume, clinically relevant tissue substitutes.