Tissue Engineered Axon-Based “Living Scaffolds” Promote Survival of Spinal Cord Motor Neurons Following Peripheral Nerve Repair

Abstract

Peripheral nerve injury (PNI) impacts millions annually, often leaving debilitated patients with minimal repair options to improve functional recovery. Our group has previously developed tissue engineered nerve grafts (TENGs) featuring long, aligned axonal tracts from dorsal root ganglia (DRG) neurons that are fabricated in custom bioreactors using the process of axon "stretch-growth". We have shown that TENGs effectively serve as "living scaffolds" to promote regeneration across segmental nerve defects by exploiting the newfound mechanism of axonfacilitated axon regeneration, or "AFAR", by simultaneously providing haptic and neurotrophic support. To extend this work, the current study investigated the efficacy of living versus non-living regenerative scaffolds in preserving host sensory and motor neuron cell health, using retrograde dye transport as a proxy, following bridging of segmental nerve defects. Rats were assigned across five groups: naïve or repair using autograft, nerve guidance tube (NGT) with collagen, NGT + non-aligned DRG populations in collagen, or TENGs. We found that TENG repairs yielded equivalent regenerative capacity as autograft repairs based on preserved health of host spinal cord motor neurons and acute axonal regeneration, whereas NGT repairs or DRG neurons within an NGT exhibited reduced motor neuron preservation and diminished regenerative capacity.These acute regenerative benefits ultimately resulted in enhanced levels of functional recovery in animals receiving TENGs, at levels matching those attained by autografts. Our findings indicate that when used to bridge segmental nerve defects, TENGs preserve host spinal cord motor neuron health and regenerative capacity without sacrificing an otherwise uninjured nerve (as in the case of the autograft), and therefore represent a promising alternative strategy for neurosurgical repair following PNI.2 HIGHLIGHTS

Tissue Engineered Bands of Büngner for Accelerated Motor and Sensory Axonal Outgrowth

“…These findings suggest there may be preferential preservation of the MNs within the explant region in the presence of aligned Schwann cells. Interestingly, these findings corroborate other work suggesting SNs may be more resilient to extrinsic microenvironmental factors at acute time points following injury (Cheah et al, 2017;Maggiore et al, 2020).…”

“…In contrast, for nerve guidance conduits and acellular nerve grafts, infiltration of host Schwann cells from both nerve stumps is necessary to enable axon re-growth from the proximal stump and across the defect (Kaplan et al, 2015). This processinvolving Schwann cell proliferation, migration, and alignmentoccurs relatively slowly, likely contributing to reduced rates of axon regeneration across acellular bridging strategies as compared to autografts (Katiyar et al, 2020;Maggiore et al, 2020). In addition, acellular bridging strategies are generally inadequate in enabling axon regeneration across long segmental defects (e.g., >3 cm), which is believed to be due to an inability of host Schwann cells to fully infiltrate the grafts.…”

Biopreservation of living tissue engineered nerve grafts

“…Dorsal root ganglia (DRG) neurons were harvested as previously described. 17,19 Briefly, intact spinal cords were extracted from embryonic day 16 Sprague Dawley pups, and whole DRG were plucked from the spinal cord and placed in cold Leibovitz-15 (L-15) medium (ThermoFisher). Once the desired number of DRGs were harvested, they were rinsed twice in plating media consisting of Neurobasal © medium (ThermoFisher) supplemented with 2% B-27 (ThermoFisher, 500 μML-glutamine, 1% FBS (Atlanta Biologicals), 2.5 mg/mL glucose (Sigma), 20 ng/mL 2.5S nerve growth factor (BD Biosciences), 20 mM 5FdU (Sigma), and 20 mM uridine (Sigma).…”

“…18 Finally, TENGs have also been shown to exert protective effects proximally on host neuron populations. 19 By targeting multiple synergistic pathways, TENGs may provide a revolutionary approach to peripheral nerve reconstruction. Indeed, in both rodents 15,[17][18][19] and swine, 20 TENG transplantation has been shown to dramatically improve regeneration of host nerve as compared to nerve guidance conduits, performing at least as well as autografts.…”

“…19 By targeting multiple synergistic pathways, TENGs may provide a revolutionary approach to peripheral nerve reconstruction. Indeed, in both rodents 15,[17][18][19] and swine, 20 TENG transplantation has been shown to dramatically improve regeneration of host nerve as compared to nerve guidance conduits, performing at least as well as autografts. Clinical application of this strategy, however, necessitates shelf life, a major barrier to translation for all living TEMPs.…”