Low-Frequency Electrical Stimulation Optimizes Neurotrophic and Neuroimmune Signaling in Bisvinyl Sulfonemethyl-Based Nerve Guidance Conduits

Peripheral nerve injuries involving critical-sized gaps remain a major clinical challenge. Although autologous nerve grafting is considered the gold standard for peripheral nerve repair, its clinical application is limited by the availability of donor nerve tissue and the risk of donor-site morbidity, including sensory deficits and functional impairment. Therefore, nerve guidance conduits (NGCs) have emerged as a promising alternative when combined with bioactive modulation strategies. In this study, we evaluated bisvinyl sulfonemethyl (BVSM)-crosslinked gelatin conduits integrated with electrical stimulation (ES) at different frequencies (0, 2, 20, and 200 Hz) in a rat sciatic nerve defect model over a 4-week recovery period (n = 10 per group). Structural regeneration was assessed by morphometric analysis, electrophysiology, macrophage infiltration, CGRP immunoreactivity, retrograde Fluorogold tracing, quantitative PCR of growth factors and inflammatory cytokines, and behavioral testing. Among all stimulation paradigms, low-frequency ES at 2 Hz produced the most pronounced regenerative effects. The 2 Hz group demonstrated significantly greater axon number, axonal density, and regenerated nerve area compared with control and high-frequency groups (p < 0.05). Electrophysiological assessments revealed improved nerve conduction velocity, higher MAP amplitudes, and shorter latencies. Enhanced macrophage recruitment and elevated CGRP expression were observed, suggesting coordinated neuroimmune and neurochemical activation. Gene expression analysis indicated upregulation of neurotrophic factors and balanced inflammatory cytokine responses under low-frequency stimulation. In contrast, high-frequency stimulation (200 Hz) failed to enhance overall regeneration and showed reduced axonal metrics, suggesting possible overstimulation-associated suppression. Collectively, these findings demonstrate that BVSM-crosslinked conduits provide a stable and biocompatible regenerative scaffold, and that appropriately tuned low-frequency electrical stimulation (2 Hz) optimally enhances structural, molecular, and functional recovery. The integration of material engineering with bioelectrical modulation represents a promising strategy for next-generation bioelectronic interfaces in peripheral nerve repair.