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 axon‐facilitated axon regeneration, or “AFAR,” by simultaneously providing haptic and neurotrophic support. To extend this work, the current study investigated the efficacy of living versus nonliving regenerative scaffolds in preserving host sensory and motor neuronal health following nerve repair. 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 TENGs may 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.

Biopreservation of living tissue engineered nerve grafts

“…TENGs were found to survive biopreservation for up to 4 weeks, although overall construct health declined with increasing preservation time as evidenced by increased LDH enzyme release as well as a trend towards reduced expression of NeuN, a phenomenon often associated with neuronal stress or degeneration. 19,21,35 Similar findings were noted in vivo ; after transplantation for 2 weeks, biopreserved TENGs were again found to routinely survive, but with an apparent increase in health score standard deviation following 28 days of biopreservation as compared with 7-day and fresh TENGs.…”

“…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).…”

“…All antibodies used have been validated in our lab for use with TENGs and rodent tissue. 17,19,21,22…”

“…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 15,17–19 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 15,17–19 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.…”