Multimodal regulation of neurite extension by extracellular matrix composition and electrical stimulation on aligned...

Objective. Peripheral nerve regeneration is regulated by structural, biochemical, and electrical cues, yet their combined effects on neurite outgrowth remain unclear. In this study, an aligned fibrous scaffold was developed to evaluate how extracellular matrix (ECM) composition and electrical stimulation influence neurite extension and growth cone morphology.Approach. Type I collagen fibers incorporating fibronectin, collagen IV, and laminin I were fabricated by contact drawing. The contact drawn fibers were then consolidated into multifilaments, which were mounted on custom frames for cell culture. Primary dorsal root ganglia (DRG) explants were cultured on the multifilaments, and neurite outgrowth and alignment were quantified as a function of ECM composition. Functional activity was confirmed by patch clamp. High-resolution confocal imaging was performed to assess cytoskeletal organization and growth cone morphology. Electrical stimulation was applied to evaluate interactions between biochemical and bioelectrical cues.Main Results. Collagen multifilament scaffolds promoted a high degree of neurite alignment relative to DRGs cultured on isotropic surfaces. Fibronectin and collagen IV increased neurite length in a concentration-dependent manner, with maximal effects observed at intermediate ligand densities, while laminin I had no significant effect. Combinatorial ECM presentation produced non-additive responses. Electrical stimulation increased neurite length on collagen-only scaffolds but had a reduced effect or no effect with incorporation of additional ECM constituents. Growth cone morphology was altered by ECM composition, with fibronectin-, collagen IV-, and combinatorial scaffolds producing less circular growth cones, despite no significant differences in filopodia number or overall morphology-classification proportions.Significance. Neurite extension and growth cone structure are governed by non-linear interactions between multimodal cues. The scaffold system described here facilitates systematic comparison of multiple regenerative cues to inform the design of material for promoting nerve regeneration.