Nerve Growth Factor (NGF) And Glial Growth Factor (GGF2) Facilitate The Distribution Of Rat Schwann Cells Seeded Into Processed Nerve Allografts
Giulia Daneshgaran, BS, Michael Cooper, BS, Milan Stevanovic, MD, PhD, Alex K. Wong, MD, Erin L. Weber, MD, PhD.
Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA.
PURPOSE: When nerve injury necessitates graft repair and nerve autografts are not available, processed nerve allografts (PNAs) are a suitable reconstructive option. However, in the setting of larger nerve gaps, axonal regeneration across PNAs is suboptimal compared to autografts. To enhance axonal regeneration across PNAs, a variety of neurally supportive cell types have been seeded into the allografts through methods such as injection, microneedling, and immersion in pressurized solutions but have resulted in focal seeding of only a small proportion of the total allograft volume. We hypothesize that the use of chemokinetic growth factors will improve the uniform distribution of rat Schwann cells seeded into PNAs, with the goal of creating a more supportive environment for axonal regeneration.
METHODS: Rat sciatic processed nerve allografts (Axogen, Inc.) were centrally injected with 5x10^5 primary rat Schwann cells (RSCs) through a 27 gauge needle and incubated in media containing 10 ng/mL nerve growth factor (NGF) or glial growth factor 2 (GGF2) for up to 36 hours. Seeded allografts incubated in unenhanced media served as a negative control. Allografts were immunostained with collagen I and nuclear antibodies to visualize the location of RSCs within the allograft. ImageJ software was used to measure total cell number and the area of distribution.
RESULTS: RSCs injected and incubated in the absence of growth factors remained centrally located at the initial injection site. In allografts incubated with GGF2 or NGF, RSCs rapidly migrated towards the periphery, with many cells distributed just beyond the perineurium. There was a statistically significant difference in relative cell distribution between the plain media and GGF2 groups (9% vs 69% of total allograft area, p<0.0001) and the plain media and NGF groups (9% vs 77%, p<0.0001). In contrast, no significant difference was observed when comparing relative cell distribution between the GGF2 and NGF groups. There was no significant change in total cell number from 12h to 36h for the plain media or GGF2 groups while a decrease in cell number was observed in the NGF group (279 vs 72, p<0.01), suggesting that incubation in NGF may affect cell survival.
CONCLUSION: The addition of 10 ng/ml of GGF2 or NGF resulted in clear cell migration from the central site of injection towards the periphery of the allograft. Cell seeding by central injection, supplemented with immersion in chemokinetic growth factors, has the potential to induce Schwann cell migration and uniform distribution throughout the fascicle. We are currently investigating the role of altered growth factor concentration and time exposure to achieve optimal seeding of the complete volume of the processed nerve allograft. Ultimately, we theorize that more uniform seeding may help to support the regeneration of a greater number of axons across a PNA-bridged nerve gap.
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