Influence of Touch-Spun Nanofiber Diameter on Contact Guidance during Peripheral Nerve Repair

Abstract

Peripheral nerve regeneration across large gaps remains clinically challenging and scaffold design plays a key role in nerve tissue engineering. One strategy to encourage regeneration has utilized nanofibers or conduits to exploit contact guidance within the neural regenerative milieu. Herein, we report the effect of nanofiber topography on two key aspects of regeneration: Schwann cell migration and neurite extension. Substrates possessing distinct diameter distributions (300 ± 40 to 900 ± 70 nm) of highly aligned poly­(ε-caprolactone) nanofibers were fabricated by touch-spinning. Cell migratory behavior and contact guidance were then evaluated both at the tissue level using dorsal root ganglion tissue explants and the cellular level using dissociated Schwann cells. Explant studies showed that Schwann cells emigrated significantly farther on fibers than control. However, both Schwann cells and neurites emigrated from the tissue explants directionally along the fibers regardless of their diameter, and the data were characterized by high variation. At the cellular level, dissociated Schwann cells demonstrated biased migration in the direction of fiber alignment and exhibited a significantly higher biased velocity (0.2790 ± 0.0959 μm·min–1) on 900 ± 70 nm fibers compared to other nanofiber groups and similar to the velocity found during explant emigration on 900 nm fibers. Therefore, aligned, nanofibrous scaffolds of larger diameters (900 ± 70 nm) may be promising materials to enhance various aspects of nerve regeneration via contact guidance alone. While cells track along with the fibers, this contact guidance is bidirectional along the fiber, moving in the plane of alignment. Therefore, the next critical step to direct regeneration is to uncover haptotactic cues that enhance directed migration.

Quantification of cell migration: metrics selection to model application

“…Bias and errors in tracking exist whether cells are manually tracked or tracked using automation; additional specifics to avoid biased results are detailed in a review for immune cell migration ( Beltman et al, 2009 ; Svensson et al, 2018 ), which can be readily extended to other cell types. Images can be computationally expensive, particularly with long duration tracking, and tracking individual paths is time consuming; therefore, where fewer cells are tracked, care must be taken in generalizing the results ( Dolde et al, 2021 ; Cavanaugh et al, 2022 ). Faster moving cells may require shorter tracking durations than slow moving cells, but ultimately, similar amounts of data are collected ( Gorelik and Gautreau, 2014 ).…”

“…(C) Immature dendritic cells migrated less persistently with low persistent time and high turning angle distribution under mechanical loaded imposed by 18.1 kPa gels ( Choi et al, 2021 ). (D) MSD and superdiffusive migration was used to describe Schwann cell migration on aligned nanofibers of various diameters ( Cavanaugh et al, 2022 ). (E) Macrophage migration was subdiffusive in different concentrations of collagen hydrogels ( Pérez-Rodríguez et al, 2022 ).…”

“…Cell migration is critical to a number of fundamental biological processes, such as stem cell migration during embryogenesis ( de Lucas et al, 2018 ), angiogenesis ( Yang et al, 2020 ), and wound healing ( Rodrigues et al, 2019 ), but is also important to disease states, such as metastasis during tumor development ( Wu et al, 2021 ). Cell migration has long been studied ( Angevine and Sidman, 1961 ; Lauffenburger and Horwitz, 1996 ; Ridley et al, 2003 ; du Roure et al, 2005 ; Peyton and Putnam, 2005 ; Cattin et al, 2015 ; Motta et al, 2019 ; Cavanaugh et al, 2022 ; Brunetti et al, 2021 ), with a wide array of studies examining how and why cells move ( Isenberg et al, 2009 ; Roussos et al, 2011 ; Cortese et al, 2014 ; Wen et al, 2015 ), defining exogenous cues that quantitatively impact cell motility. It has long been argued that quantitative characterization of cell migration is critical to permit rigorous comparisons ( Dunn and Brown, 1987 ; Stokes et al, 1991 ).…”

Progress in methods for evaluating Schwann cell myelination and axonal growth in peripheral nerve regeneration via scaffolds

“…To encourage the growth of Schwann cells based on the aligned patterns, inducing newborn axonal sprouts from the proximal stump to the distal stump, poly(D,L-lactide-co-caprolactone) (PLCL) films with linear micropatterns of graphene oxide-or bioactive peptide-modified microscaled ridges and grooves have been developed to promote the aligned migration of Schwann cells to facilitate peripheral nerve regeneration (Zhang et al, 2020;Zhang et al, 2022b). Furthermore, the aligned electrospun fiber-based scaffolds containing anisotropic topological cues at the micro-nanoscale have also been fabricated to preciously mimic the microstructure of peripheral nerve tissue to improve the guidance and biological cues for supporting nerve regeneration (Yi et al, 2020;Cavanaugh et al, 2022;Chen et al, 2022) (Scheme 3).…”

Physical Cue‐Based Strategies on Peripheral Nerve Regeneration

“…Cavanaugh et al cultured SCs and DRG cells onto different nanofibers with various diameter distributions (300 ± 40 to 900 ± 70 nm), and found that larger diameters (900 ± 70 nm) fibers can increase SCs migration velocity and regulate growth orientation of DRG cells. [ 241 ] Although conduit filler has a positive effect on nerve regeneration‐related factors, the high‐density filler may in turn block the PNR process. One study explored the influence of fiber filling density in nerve conduits on nerve repair.…”