Schwann Cell Migration and Elucidation of Sex-based Differences using Peptide-functionalized Aligned Fiber Scaffolds for Peripheral Nerve Reconstruction

Abstract

Peripheral nerve injuries remain a significant clinical challenge with limited tools availableto physicians and patients. Although autografts are the gold standard for nerve reconstruction, they are limited by donor site morbidity and availability. Commercially available nerve guidance conduits offer alternatives, yet their clinical application remains largely restricted to short nerve gaps, with limited success beyond 1 cm. To address these challenges, emerging innovations such as biofunctionalized materials and topographically engineered architectures are being readily explored to improve regenerative outcomes following neural injury. A key factor in the regenerative process is the behavior of Schwann cells; axonal regeneration can only proceed to the extent of which Schwann cells are able to migrate to and within the injury site. Thus, eliciting specified Schwann cell responses that are advantageous to nerve healing can be critical to the next-generation of nerve repair devices. Herein, we study the impact of aligned fiber scaffolds and peptide functionalization, both independently and synergistically, on Schwann cell response, targeting migratory parameters, such as speed, biased velocity, haptotaxis, and other cell cycle properties, such as adhesion and proliferation.Sexual dimorphism has been observed in many physiological and pathological responses,yet few studies incorporate both female and male experimental groups for preclinical work. For the development of biomaterial devices, understanding both female and male cell migratory behavior is a crucial design consideration. In this work, we thoroughly examined sex-based migration on flat controls, non-functionalized fiber scaffolds, and peptide-functionalized fiber scaffolds. Observed using single cell tracking, male and female cells exhibited significantly different migration on flat substrates, with female cells having greater speeds while male cells had higher persistence. Fiber scaffolds were fabricated using polycaprolactone-based copolymers and touch-spinning, allowing for the production of highly aligned and diameter-specific fibers. On non-functionalized fibers, persistence increased with the introduction of aligned fiber topography in both sexes, with smaller diameter fibers (0.9 and 1.2 μm) mitigating sex-based differences. Speed along the fiber axis was decreased in females compared to controls, while males were increased or unchanged. Again, cells on smaller fiber diameters showed no sex-based differences in parallel speed.To further harness and direct Schwann cell migration, we have developed aligned fiberscaffolds functionalized with variable concentration gradients of YIGSR, a laminin-derived peptide known to promote Schwann cell motility. Using thiol-ene click chemistries, we generated uniform and gradient patterns of YIGSR on the aligned fibers with spatiotemporal control over tethered peptide concentration during fabrication, yielding two uniform concentration scaffolds of 100 pmol/cm² and 420 pmol/cm² YIGSR, and three gradient profiles of slopes 7 pmol·(cm²·mm)⁻¹, 15 pmol·(cm²·mm)⁻¹, and 30 pmol·(cm²·mm)⁻¹. Schwann cell migration on functionalized scaffolds revealed that uniform YIGSR functionalization enhanced migration in a sex-specific and concentration-dependent manner. Female Schwann cells responded with greater motility on 100 pmol/cm² uniform YIGSR-functionalized fibers, while male Schwann cell motility was enhanced on both 100 and 420 pmol/cm² compared to non-functionalized fibers. Shallow YIGSR gradients (7 and 15 pmol·(cm²·mm)⁻¹) did not consistently bias Schwann cell directionality in the direction of the gradient. However, 30 pmol·(cm²·mm)⁻¹ gradients induced a haptotactic response, measured by directional velocity and haptotactic index, with both sexes migrating toward regions of higher peptide concentration. Furthermore, we developed mechanically tunable fiber scaffolds that can be orthogonally decorated with both RGD and YIGSR peptides. We show Schwann cell proliferation is enhanced on softer materials. Together, these findings highlight the potential of engineering fiber-based scaffolds with precisely controlled biochemical, topographical, and mechanical cues to elicit specified Schwann cell behaviors. Such strategies may ultimately enhance axonal regeneration by guiding neuronal components across segmental nerve defects.