(178n) Bicontinuous Interconnected Porous Scaffolds As Extracellular Matrix Analogues for Driving Neurogenesis

The complexity and plasticity of the human brain make it susceptible to neurodegeneration, which is characterized by a gradual loss of neurons and synaptic connections. Tissue engineering has emerged as a promising strategy for direct neurogenesis stimulation. However, current approaches in brain tissue engineering are limited by the inability of materials to mimic key structural features of the native brain, namely its porosity, interconnectedness, and stiffness. To address this, we utilized a novel approach predicated on a bicontinuous interfacially jammed emulsion (BIJEL) of poly(ethylene glycol) diacrylate (PEGDA) and nanoparticles. By employing a custom-built fiber extrusion setup, we fabricated fibers with a nanoparticle-dependent hierarchical and interconnected microporous structure. The engineered scaffold exhibited a wide range of highly tunable physical properties, adaptable for neural tissue engineering. Cytocompatibility of human induced pluripotent stem cell-derived neural stem cells (i-HNSCs) was evaluated at the single-fiber level, with extracellular matrix (ECM)-coated 2D substrates serving as controls. Differentiation potential was determined via gene/protein markers and calcium imaging was performed to access neural activity. Our findings reveal remarkable growth, colonization, and infiltration of i-HNSCs seeded on the scaffolds, outperforming conventional ECM-coated 2D substrates. Live/dead assays indicated robust viability and strong affinity for the BIJEL fibers which were encapsulated by dense proliferating cell masses. The fiber’s interconnected domains and micro-interfacial surface area facilitated significant cell migration and proliferation compared to controls, as demonstrated by quantitative analysis of cell adhesion. Neuronal and glial cells were observed by Day 14, corroborated by the elevated expression of β-III-tubulin (B3T) and Glial fibrillary acidic protein (GFAP), respectively. BIJEL scaffolds promoted superior neurite elongation and density when compared to the coated substrates. Analysis of calcium ion flux confirmed spontaneous activity in differentiated neurons, indicating bioactivity comparable to the control. These findings underscore the potential of replicating key physical features of native brain tissue in creating a dynamic microenvironment that supports neurite outgrowth and 3D network formation. Such advancements have significant implications for neural tissue engineering and the design of physiologically relevant models.