Vascular Network Development in Response to Distinct Spatial Geometries

Background: The robust repair of large wounds and tissue defects critically relies on blood perfusion provided by a healthy vessel network. Vascularization is one of the major challenges in tissue engineering. A widely used approach to promote vascularization in engineered constructs consists of co-seeding endothelial cells, which form the vessel networks, and support cells, which provide chemical and mechanical support to the formed vessels, and later mature into smooth muscle actin-expressing cells (SMCs), stabilizing the vessels. Understanding the network formation and stabilization phenomena is of upmost importance to control the resulting vascularization within engineered tissue constructs. Methods: In this work, we study the vessel formation (vasculogenesis), subsequent angiogenesis, general branch orientation, and localized stabilization in response to varying pore geometries. For this, we seeded labeled endothelial cells and non-labeled support cells using a novel two-step seeding protocol on photoresist scaffolds with rectangular, squared, hexagonal, circular pores. The scaffolds were cultured for a week and imaged every two days. Later, we performed relevant fluorescent staining to observe proliferation, and maturation in response to the geometries. Using image analysis, we quantified key parameters of the network development. Results: The results showed distinct angiogenesis behaviors associated to each of the selected geometries. Furthermore, the localization of the newly differentiated SMCs among the different shapes varied significantly, heavily influenced by the pore geometry. Conclusion: This novel approach will help elucidate critical parameters of three-dimensional scaffold design and fabrication in order to improve vessel network functionality within engineered constructs.