Multimodal regulation of neurite extension by extracellular matrix composition and electrical stimulation on aligned collagen fiber scaffolds

Peripheral neurons respond to structural, biochemical, and electrical cues to regenerate following injury. However, how these cues contribute individually and in combination to promoting neurite outgrowth remains incompletely understood. Here, we developed an aligned collagen fiber-based scaffold to examine how combinations of extracellular matrix proteins contribute to neurite outgrowth with and without electrical stimulus. Type I collagen fibers were fabricated by contact-drawing, a dry spinning process capable of producing collagen fibers that can be twisted into multifilaments that support anisotropic cell growth. A custom frame enabled immobilization of the collagen multifilaments and quantification of longest neurite length. Aligned collagen scaffolds significantly enhanced neurite outgrowth and alignment compared to isotropic controls.Incorporation of fibronectin or collagen IV increased neurite elongation in a concentrationdependent manner, with the longest neurites observed at intermediate ligand densities. Laminin I did not significantly alter neurite length. Combinatorial presentation of fibronectin and collagen IV further demonstrated non-linear responses, with low-concentration combinations enhancing neurite extension and high-concentration combinations providing no additional benefit. Electrical stimulation increased neurite length on collagen-only scaffolds but exhibited diminished or negative effects when applied to ECM-enriched multifilaments, suggesting context-dependent integration of biochemical cues and electrical stimulus. Together, these findings demonstrate that scaffold anisotropy establishes directional guidance, whereas ECM composition and electrical stimulation modulate neurite extension in a non-additive manner. The collagen multifilament system functions as a tunable in vitro platform for systematically interrogating multimodal regenerative cues and defining design principles for next-generation nerve guidance biomaterials.