Three-dimensional endothelial cell incorporation within bioactive nanofibrous scaffolds through concurrent emulsion...

Nanofibrous scaffolds hold great promise in tissue engineering owing to their extracellular matrix (ECM)-mimicking architectures. Electrospinning, with its ease for producing nanofibrous scaffolds, has therefore been widely employed for various tissue engineering applications. However, electrospun nanofibrous scaffolds have faced the inherent challenge of three-dimensional (3D) cell distribution due to the small sizes of interconnected pores in these scaffolds when conventional approach of scaffold fabrication with subsequent cell seeding is adopted, which severely limits their applications in repairing/regenerating human body tissues with thick and vascularized structures. In this study, we demonstrate a method to directly place living endothelial cells within bioactive nanofibrous scaffolds in 3D through concurrent emulsion electrospinning and coaxial cell electrospraying. Using this concurrent manufacturing method, endothelial cells are encapsulated in hydrogel microspheres and deposited along with vascular endothelial growth factor (VEGF)-containing nanofibers in the scaffold fabrication process, resulting in nanofibrous scaffolds with 3D embedded cell-encapsulated microspheres. After selective disruption of the hydrogel microspheres, the encapsulated endothelial cells are released, yielding bioactive nanofibrous scaffolds with tissue-like 3D cell-incorporated nanofibrous structures. It is shown that cell viability is well preserved (>98%) during the concurrent manufacturing process and that a deep cell distribution (~100 μm) through the scaffold thickness has been achieved. With combined structural and biochemical cues via the 3D cell-incorporated architectures, endothelial cells can freely stretch, display enhanced intercellular connections, and maintain the phenotype in the bioactive nanofibrous scaffolds. Our investigations offer a promising platform technology for creating bioactive nanofibrous scaffolds with 3D cell incorporation and for overcoming inherent problems of electrospun nanofibrous scaffolds, which should open new avenues for biomanufacturing tissue-mimicking constructs with vascularized structures and complex anatomy. STATEMENT OF SIGNIFICANCE: Electrospun nanofibrous scaffolds face challenges in three-dimensional (3D) cell incorporation and vascularization. Enhancing cell penetration via enlarged interconnected pores is a common strategy to address that. However, there are conflicts between cell penetration and structural integrity for scaffolds formed using such strategy, as deep cell penetration, if possible, can only achieve in highly loose architectures. In this investigation, we demonstrate a concurrent emulsion electrospinning and coaxial cell electrospraying technique, realizing 3D endothelial cell incorporation in electrospun nanofibrous scaffolds independent of cell penetration. Our technology appropriately addresses the conflict between deep 3D cell incorporation and structural integrity. In the scaffolds, the 3D incorporated endothelial cells show well-preserved viability, phenotype and functions, implying improved vascularization potential.