Sustained bioactivity of nucleic acid-encoded EPO and NGF via functionalized collagen conduits for peripheral nerve regeneration

Peripheral nerve injuries (PNIs) remain among the most challenging conditions in reconstructive surgery, leading to chronic pain, muscle atrophy, and incomplete functional recovery. Autologous nerve grafts remain the gold standard for long-gap repair but are limited by donor morbidity and tissue availability, while hollow collagen conduits, though clinically approved, fail to provide the biological cues necessary for regeneration across critical defects. This thesis addresses these limitations through the development of collagen-based neural guidance conduits (NGCs) functionalized for sustained, localized gene delivery of erythropoietin (EPO) and nerve growth factor (NGF). In vitro dorsal root ganglion (DRG) cultures identified an optimal 1:5 EPO:NGF gene ratio, which enhanced neurite elongation and stimulus-related calcium-response dynamics and was associated with altered electrophysiological behaviour relative to single-factor treatments. Mechanistic analysis revealed convergence of NGF–TrkA and EPO–EPOR pathways on PI3K/AKT, MAPK/ERK, and JAK/STAT cascades, promoting complementary effects on axon extension, Schwann-cell behaviour, and neuroprotection. Aligned collagen fibre scaffolds demonstrated efficient plasmid uptake and sustained expression, confirming the feasibility of scaffold-mediated gene delivery. In vivo, gene-activated conduits bridged 15 mm rat sciatic nerve gaps, where clinically used conduits fail. Longitudinal imaging, transcriptomics, and cytokine profiling at early time points showed reduced inflammation, engagement of pro-regenerative signalling, and improved vascularisation. By 16 weeks, animals receiving EPO–NGF conduits exhibited superior axonal regeneration, myelination, and muscle xvi reinnervation, correlating with improved gait parameters and electrophysiological recovery comparable to autografts. Together, these studies validate dual gene delivery as a strategy to reconstitute the molecular and cellular programmes absent in conventional conduits. Gene-functionalized collagen scaffolds thus emerge as scalable, off-the-shelf, bio-instructive platforms capable of bridging critical nerve defects and advancing peripheral nerve repair beyond the current clinical standard.

Publisher

University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)