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Functional recovery following nerve injury declines when target re-innervation is delayed. Currently, no intervention exists to improve outcomes after prolonged denervation. We explored the neuroregenerative effects of glial cell line-derived neurotrophic factor (GDNF) and chondroitinase (CDN) in a chronic denervation animal model. A fibrin-based sustained delivery method for growth factors was optimized in vitro and in vivo, and then tested in our animal model. GDNF, CDN, and GDNF+CDN were injected into the denervated stump at the time of nerve repair. Histomorphometry and retrograde labeling were used to assess axonal regeneration. The mechanisms promoting such regeneration were explored with immunofluorescence. Five weeks after repair, the GDNF+CDN group had the highest number and maturity of axons. GDNF was noted to preferentially promote axonal maturity, whereas CDN predominantly increased the number of axons. GDNF favored motor neuron regeneration, and upregulated Ki67 in Schwann cells. CDN did not favor motor versus sensory regeneration and was noted to cleave inhibitory endoneurial proteoglycans. Early measures of nerve regeneration after delayed repair are improved by activating Schwann cells and breaking down the inhibitory proteoglycans in the distal nerve segment, suggesting a role for GDNF+CDN to be translated for human nerve repairs.

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Functional outcomes following nerve repair remain suboptimal. Scarring at the repair site is a major impediment to regeneration. A biomaterial scaffold applied around the coaptation site that decreases inflammation holds great potential in reducing scarring, enhancing axonal growth, and improving functional recovery. In this study, we evaluated the effect of a macroporous nanofiber wrap, comprised of nonwoven electrospun poly-ε-caprolactone (PCL), in improving axonal regeneration in a rat sciatic nerve cut and direct repair model. Controls consisted of conventional epineurial repair. We also evaluated our wrap against the commercially available AxoGuard wrap. At five weeks following repair, the nanofiber wrap group showed a significantly decreased intraneural macrophage invasion and collagen deposition at the repair site. This was associated with increased expression of the anti-inflammatory cytokine (IL-10), decreased expression of the pro-inflammatory cytokine (TNF-α), and a decrease in the M1:M2 macrophage phenotype ratio. These findings suggest that this nanofiber wrap, with its unique macroporosity, is modulating the inflammatory response at the repair site by polarizing macrophages towards a pro-regenerative M2 phenotype. Concomitantly, a higher number of regenerated axons was noted. At sixteen weeks, the nanofiber wrap resulted in enhanced functional recovery as demonstrated by electrophysiology, neuromuscular re-innervation, and muscle histology. When compared to the AxoGuard wrap, the nanofiber wrap showed similar inflammation at the repair site and similar nerve morphometric findings, but there was a trend towards a lower overall number of macrophages invading the wrap wall. These results demonstrate favorable outcomes of the macroporous nanofiber wrap in promoting neuroregeneration and functional recovery following nerve repair. STATEMENT OF SIGNIFICANCE: Electrospun nanofiber scaffolds, with specific fiber and pore sizes, were shown to modulate the immune response and create a regenerative environment. In this paper, we present a macroporous nanofiber wrap, made of poly-ε-caprolactone, to be applied at the coaptation site in primary nerve repair. We show that it regulates the inflammatory response at the repair site and decreases scarring/fibrosis. This results in enhanced axonal regeneration, allowing a higher number of axons to cross the suture line and reach the target muscle in a timely fashion. Functional outcomes are thus improved.

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Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.

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An imaging-coupled 3D printing methodology for the design, optimization, and fabrication of a customized nerve repair technology for complex injuries is presented. The custom scaffolds are deterministically fabricated via a microextrusion printing principle which enables the simultaneous incorporation of anatomical geometries, biomimetic physical cues, and spatially controlled biochemical gradients in a one-pot 3D manufacturing approach.

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Limitations in current nerve regeneration techniques have stimulated the development of various approaches to mimic the extrinsic cues available in the natural nerve regeneration environment. Biomaterials approaches modulate the microenvironment of a regenerating nerve through tailored presentation of signaling molecules, creating physical and biochemical guidance cues to direct axonal regrowth across nerve lesion sites. Cell-based approaches center on increasing the neurotrophic support, adhesion guidance and myelination capacity of Schwann cells and other alternative cell types to enhance nerve regrowth and functional recovery. Recent advances in presenting directional guidance cues in nerve guidance conduits and improving the regenerative outcomes of cell delivery provide inspirations to engineering the next generation of nerve repair solutions.

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