Enhancement of Schwann Cell Migration using Aligned Nanofiber Conduits for Peripheral Nerve Reconstruction
Yin Mei Chan, B.Sc.1, Yang Hu, B.Sc.2, Nicola G Judge, Ph.D.1, Neill Li, M.D.3, Rebecca K Willits, Ph.D.2,4 and Matthew L Becker, Ph.D.1,5, 1Department of Chemistry, Duke University, Durham, NC, 2Department of Chemical Engineering, Northeastern University, Boston, MA, 3Duke University Medical Center, Durham, NC, 4Department of Bioengineering, Boston, MA, 5Thomas Lord Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC
Introduction: Despite optimal peripheral nerve reconstruction for nerve defects, return of sensory and motor function is often slow and inconsistent. Key to the regenerative process are the behavior of Schwann cells (SCs) to impart biomolecular and topographical cues for regrowing axons. Axonal regeneration only proceeds to the extent of which SCs are able to migrate from the proximally intact nerve. To optimize SC migration, our lab has sought to develop synthetic nanofibrous scaffolds that provide affinity and guidance to enhance SC migration and subsequently, axonal outgrowth.
Materials and Methods: Touch-spinning was used to fabricate the nanofibers. Touch-spinning involves a rotating rod that comes in contact with and draws fibers from polymer droplets flowing out of a needle at a fixed rate via a syringe pump. As the droplet is pulled, the solvent evaporates, leaving behind a polymer nanofiber that is collected on a center, stationary collector (Figure 1A-D). Fiber diameter can be manipulated by varying the speed of the rotating platform. Using this technique, functional poly(caprolactone) (PCL) nanofiber conduits of well-defined diameters (1.65 ± 0.25, 1.18 ± 0.25, and 0.87 ± 0.27 µm) were produced.
To evaluate SC affinity and migration on the nanofibers, primary rat SCs were seeded at 10,000 cells/cm2 and incubated for 4 hrs in Dulbecco's Modified Eagle Medium (DMEM) + 1% v/v L-glutamine. Migration media (DMEM + 5% v/v fetal bovine serum + 1% v/v L-glutamine) was then added. In-situ free cell migration was recorded every 15 mins for 24 hrs on plates with and without nanofibers using Zeiss Axiom Observer 7. Individual cell positions were tracked using ImageJ.
Results: SCs seeded on fibers exhibited elongated morphology when compared to plates without fibers (Figure 2A-B). Migratory studies found that SCs specifically migrate in the x-axis (defined as the direction of aligned fibers) in the presence of nanofibers (Figure 2C-D). SCs on nanofibers moved at speeds of 0.24, 0.25, and 0.22 µm/min along the x-axis for diameters of 1.65, 1.18, and 0.87 µm, respectively, demonstrating higher rates than that of SCs on no fibers (0.19 µm/min) (Figure 3). Near-zero speeds were recorded for SC migration on nanofibers in the y-direction.
Conclusion: The use of synthetic, nanofibrous scaffolds improved the directionality and rate of migration of SCs in vitro. Efforts to implement such nanofibrous scaffolds during nerve reconstruction may enhance the efficiency and efficacy of nerve repair through directionally-guiding SCs in the immediate postoperative regenerative period.
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