Abstract
Following peripheral nerve injury comprising a segmental defect, the extent of axon regeneration decreases precipitously with increasing gap length. Schwann cells play a key role in driving axon re-growth by forming aligned tubular guidance structures called bands of Büngner, which readily occurs in distal nerve segments as well as within autografts – currently the most reliable clinically-available bridging strategy. However, host Schwann cells generally fail to infiltrate large-gap acellular scaffolds, resulting in markedly inferior outcomes and motivating the development of next-generation bridging strategies capable of fully exploiting the inherent pro-regenerative capability of Schwann cells. We sought to create preformed, implantable Schwann cell-laden microtissue that emulates the anisotropic structure and function of naturally-occurring bands of Büngner. Accordingly, we developed a biofabrication scheme leveraging biomaterial-induced self-assembly of dissociated rat primary Schwann cells into dense, fiber-like three-dimensional bundles of Schwann cells and extracellular matrix within hydrogel micro-columns. This engineered microtissue was found to be biomimetic of morphological and phenotypic features of endogenous bands of Büngner, and also demonstrated 8 and 2× faster rates of axonal extension in vitro from primary rat spinal motor neurons and dorsal root ganglion sensory neurons, respectively, compared to 3D matrix-only controls or planar Schwann cells. To our knowledge, this is the first report of accelerated motor axon outgrowth using aligned Schwann cell constructs. For translational considerations, this microtissue was also fabricated using human gingiva-derived Schwann cells as an easily accessible autologous cell source. These results demonstrate the first tissue engineered bands of Büngner (TE-BoBs) comprised of dense three-dimensional bundles of longitudinally aligned Schwann cells that are readily scalable as implantable grafts to accelerate axon regeneration across long segmental nerve defects.
Highlights
Peripheral nerve injury (PNI) presents in 2–5% of all trauma cases, such as sports-related injuries, vehicle accidents, combat situations, or iatrogenic damage (Robinson, 2000; Pfister et al, 2011; Wang et al, 2017)
To biofabricate Tissue Engineered Bands of Büngner (TE-BoB), Schwann cells were seeded in an agarose hydrogel micro-column 5 mm long with outer diameter (OD) of 701 μm, inner diameter (ID) of 300 μm, and collagen-coated inner lumen
By 1 day in vitro, Schwann cells that were seeded in the agarose microcolumns had adhered to the collagen extracellular matrix (ECM) coating the inner lumen, began to exhibit a process-bearing morphology, and eventually self-assembled into a dense network along the inner lumen of the micro-column (Figure 2A)
Summary
Peripheral nerve injury (PNI) presents in 2–5% of all trauma cases, such as sports-related injuries, vehicle accidents, combat situations, or iatrogenic damage (Robinson, 2000; Pfister et al, 2011; Wang et al, 2017). The most severe nerve injuries are disconnections with a segmental defect that require implantation of grafting material, such as a biological or synthetic nerve conduit, to guide regeneration (Pfister et al, 2011). Poor regeneration is often associated with severe nerve injury, especially with long segmental defects and/or long total regenerative distances. Schwann cells distal to the injury dedifferentiate and align with the basal lamina forming highly longitudinallyoriented parallel tubular structures called the bands of Büngner (Salzer, 2015). These pro-regenerative micro-structures serve as a natural living scaffold that facilitates targeted reinnervation of the denervated end-target(s) (Gordon and Stein, 1982; Jessen and Mirsky, 2016)
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