Aligned Nanofibrillar Collagen Scaffolds Promote Repair Schwann Cell Phenotype in Peripheral Nerve Regeneration.

Introduction:: Peripheral nerve injury is a common clinical problem that can lead to debilitating neuropathy, loss of motor function and organ malfunction. Despite advances in tissue engineering and treatment strategies that mediate complication risk, nerve autograft remains the clinical standard for the treatment of nerve injuries exceeding 1cm. While the peripheral nervous system (PNS) has the ability to regenerate after injury, largely due to the plasticity of resident Schwann cells (SCs), the repair SCs phenotype and repair program is often insufficient to fully heal injured nerves.

Biomaterial scaffolds have been widely applied as nerve guidance conduits and have demonstrated the ability to promote regeneration. Nerve scaffold studies have also revealed that alignment of collagen fibrils encourages mirrored parallel alignment of repair SCs, and also, that nanoscale topographical features (smaller than the SC body) are ideal to promote motility. Our group previously fabricated aligned nanofibrillar collagen scaffolds that guide the cytoskeletal alignment of a diverse range of cell types including myogenic, osteogenic, and endothelial cells. In the current study, we extend these concepts of cell-material guidance to hypothesize that aligned nanofibrillar collagen scaffolds promote a repair phenotype in SCs. To investigate this, the SC migratory ability and morphology on scaffolds was evaluated. Our findings suggest that aligned nanofibrillar collagen promote a SC repair phenotype. Clinically, use of an aligned nanofibrillar collagen scaffold may reduce the need for autografts in PNS injury by utilizing the body’s natural repair program.


Materials and Methods:: Rat tail collagen I was dialyzed to ~30mg/mL and shear extruded into a pH7 Polyphosphate Buffered Saline at 37C to generate nano-alignment of collagen during fibrillogenesis. Randomly patterned scaffolds were fabricated by extrusion onto slides and submerged in neutral buffer solution to undergo delayed fibrillogenesis. Scaffolds were dried overnight and then sterilized and rehydrated prior to use. Sciatic nerves were harvested from C57BL/6 mice, and segmented and washed in Leibowitz medium before placement on either aligned or randomly-patterned scaffolds (Fig. 1a). Samples were subsequently cultured in Neurobasal medium supplemented with Neural Growth Factor for 14 days. Samples were fixed with 4% Paraformaldehyde on days 3, 9 and 14 post-explant for immunocytochemical analysis. Samples were permeabilized by incubation in 0.1% TritonX-100, and blocked in 5% goat serum, prior to incubation with an antibody to an SC-specific protein, s100b. Serial incubation with Alexa Fluor 555 goat anti-rabbit, followed by Phalloidin (actin) and DAPI was performed. Fluorescent images were acquired with a Zeiss LSM900 confocal scanning microscope and markers of a regenerative SC phenotype were analyzed in s-100b positive, migrating cells (Fig. 1b). Migratory cells were characterized as those that had at least 2/3 of their cell membrane detached from the nerve segment and their nucleus in the detached section. Measurements of migration distance were taken from the center of the cell nuclei to the closest point on the nerve segment, using the line tool in image J software. Protrusions exceeding 15um were counted in analysis.


Results, Conclusions, and Discussions:: To study the differences in migratory ability in SC outgrowth from explanted nerves, the migration distance of each cell from the terminal end of nerve segments was measured. Preliminary data suggests that SCs on aligned collagen scaffolds migrate 24% further compared to those on randomly oriented scaffolds (Fig. 1c). SCs adopt a regenerative phenotype after injury and are characterized by an elongated morphology with longer, more numerous protrusions. Morphological characteristics indicative of a repair phenotype were observed in SCs on aligned scaffolds. SCs had more protrusions on aligned scaffolds (p< 0.05), with 2.4 average protrusions, compared to 1.8 average protrusions on randomly oriented scaffolds (Fig 1d). Interestingly, actin fluorescence intensity was lower on aligned scaffolds as compared to random scaffolds, with a mean cell total corrected fluorescence of 5.8*10⁵ and 3.46*10⁶, respectfully (p < 0.05) (Fig. 1e). Initial data also suggests that circularity, an inverse measure of cell elongation, was lower on aligned scaffolds, indicating a more elongated phenotype (Fig. 1f). Together, these findings indicate a higher instance of a repair phenotype in SCs on the aligned scaffolds, suggesting that collagen scaffolds with nanofibrillar aligned patterning may be a potential engineered material to support peripheral nerve regeneration.

Supporting and enhancing SC’s regenerative capability is paramount to engineering tissues for PNS repair applications. Initial in vitro experiments show this capability in nano-aligned scaffolds. Future in vivo experiments will examine the potential regenerative effects of implanted nanofibrillar collagen scaffolds in an animal model of sciatic nerve gap injury. In addition, we will further investigate actin remodeling during the SC repair program on each scaffold pattern to further understand the effect of nano topography on migratory ability. This study furthers our understanding of the contribution that nano aligned fibrillar collagen scaffolds have in transitioning SCs towards a repair phenotype. By supporting the resident SCs or seeding with patient derived induced SCs, this material has the potential to improve existing peripheral nerve repair strategies and reduce the need for allografts and autografts in PNS injury.

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