Tissue Engineered Axon Tracts Serve as Living Scaffolds to Accelerate Axonal Regeneration and Functional Recovery Following Peripheral Nerve Injury in Rats

Abstract

Strategies to accelerate the rate of axon regeneration would improve functional recovery following peripheral nerve injury, in particular for cases involving segmental nerve defects. We are advancing tissue engineered nerve grafts (TENGs) comprised of long, aligned, centimeter-scale axon tracts developed by the controlled process of axon "stretch-growth" in custom mechanobioreactors. The current study used a rat sciatic nerve model to investigate the mechanisms of axon regeneration across nerve gaps bridged by TENGs as well as the extent of functional recovery compared to nerve guidance tubes (NGT) or autografts. We established that host axon growth occurred directly along TENG axons, which mimicked the action of "pioneer" axons during development by providing directed cues for accelerated outgrowth. Indeed, axon regeneration rates across TENGs were 3-4 fold faster than NGTs and equivalent to autografts. The infiltration of host Schwann cells - traditional drivers of peripheral axon regeneration - was also accelerated and progressed directly along TENG axons. Moreover, TENG repairs resulted in functional recovery levels equivalent to autografts, with both several-fold superior to NGTs. These findings demonstrate that engineered axon tracts serve as "living scaffolds" to guide host axon outgrowth by a new mechanism - which we term "axon-facilitated axon regeneration" - that leads to enhanced functional recovery.

Keywords: axon guidance; axon regeneration; axon stretch growth; neural tissue engineering; tissue engineered nerve graft.

FIGURE 1

Tissue Engineered Nerve Graft (TENG) Inspiration, Biofabrication, and Surgical Implementation. LEFT: TENGs are inspired by axonal pathfinding during nervous system development, where (A) “pioneer axons” reach a target first, and then (B) serve as a physical guide for “follower axons” to reach that target. TENG axons are effectively (C) tissue engineered “pioneer axons,” thereby functioning as a (D) living scaffold to direct and target regenerating host axons across segmental nerve defects. RIGHT: TENGs are biofabricated in custom mechanobioreactors via the process of axon “stretch-growth.” Fully formed TENGs – comprised of longitudinally aligned axons encased in a collagenous matrix and rolled into a tubular form – are used to physically bridge segmental defects in peripheral nerve. Briefly, (1) Primary DRG neurons are plated in custom mechanobioreactors. (2) Traditional axon outgrowth integrates two neuron populations. (3) A computer-controlled micro-stepper motor is engaged to gradually separate the two neuron populations, applying mechanical tension to spanning axons. (4) Tension induces axon “stretch-growth,” resulting in increased length, diameter, and fasciculation. “Stretch-growth” occurs for days to weeks at 1–10 mm/day, depending on desired length. (5) Immediately prior to implant, neurons and stretch-grown axons are encased in ECM for stabilization. (6) The ECM containing neurons and stretch-grown axons is “rolled” and transferred into an NGT. (7) NGT containing the cylindrical TENG (neurons/axons embedded in ECM) is then sutured to sciatic nerve to bridge an excised segment.

FIGURE 2

Methods to Quantify Axon Regeneration and Schwann Cell (SC) Infiltration. (A) Cartoon depicting the staggered process of acute axonal regeneration across a segmental nerve lesion, where the main bolus of regenerating axons – termed the “Regenerative Front” – is preceded by an accelerated, but less dense, population of regenerating axons – termed the “Leading Regenerators.” These 2 populations of regenerating axons were measured for each animal at 2 weeks following repair of 1 cm segmental defects by quantifying the distance from the proximal end of the repair zone to the distal axonal projections, with multiple sections surveyed per animal to ensure that the maximal values were attained. (B) Examples of longitudinal tissue section of nerve regeneration across a 1.0 cm sciatic nerve gap (repaired by a TENG), with the repair zone beginning proximally on the left and proceeding distally to the right. (B, top) Example of axon regeneration measurements: neurofilament labeled with SMI31, and the measurements of the “Regenerative Front” and “Leading Regenerator” axons are denoted. Scale bar: 1000 μm. (B, bottom) Example of SC infiltration measurements: SCs are labeled with S100, and the measurements of both the proximal and distal infiltration are shown. Scale bar: 1000 μm. (b1) “Regenerative Front” showing colocalization of axons and SCs. (b2) “Leading Regenerator” axons found beyond the repair zone, within the distal stump. (b1,b2) Scale bar: 20 μm.

FIGURE 3

TENG Survival, Maintenance of Architecture, and Mechanism-of-Action following Allogeneic Transplants in Rats. Longitudinal sections across the graft zone at 2 weeks post-implant. (A–C) Implant of mCherry + TENGs into GFP + host rats to discriminate TENG neurons/axons (red) versus host cells/axons (green). (A) Full-width longitudinal section and (B) zoom-in of region of interest showing robust and directed host axon regeneration and support cell infiltration directly along TENG axon tracts. (C) Region-of-interest showing individual channels depicting TENG neurons/axons (red), axons (purple), all host cells (green), with overlay. Arrow depicts TENG axons co-localized with host axons, suggesting directed growth. Scale bars (A) 500 μm; (B) 250 μm; (C) 300 μm. (D–F) GFP + TENG neurons/axons into wild-type host rats. (D) Surviving TENG neurons/axons (green) exhibiting healthy morphology (scale bar: left, 100 μm; right 50 μm). (E,F) A nerve repaired using a TENG (GFP +) labeled with SMI31 (red) to show host axon regeneration. (E) Living TENG neurons and axons (green) were found across the entire nerve graft. TENG neurons/axons were placed off-center to demonstrate that host axons have a preference to follow the path created by the stretch grown axons: the white arrow points out the altered direction of host axon growth through the main cluster of TENG cell bodies and axons; the gray arrow points out the natural axon regeneration trajectory straight out from the proximal stump (scale bars: 1000 μm). This suggests that TENG neurons/axons actively direct and guide host axon regeneration. (F) Region-of-interest showing dense bundles of host axons that were intertwined with and appeared to grow directly along TENG axons. Collectively, these images provide evidence of a new mechanism of nerve regeneration: axon-facilitated axon regeneration (AFAR) denoted by host axon regeneration directly along tissue engineered axon tracts.

FIGURE 4

TENGs accelerate host axon regeneration. Longitudinal sections (A,B) across the graft zone and/or (C,D) into the distal stump at 2 weeks following repair using an NGT, a reverse autograft, or TENG. (A) Axon regeneration as labeled by SMI31 (red, host axons) and GFP (implanted TENG neurons/axons) showing the “regenerative front” – main bolus of regenerating axons – for each repair group. Regeneration through an NGT yielded retarded and disorganized axonal extension. Regeneration through a reverse autograft was highly organized owing to the presence of aligned autologous SCs throughout the graft (not shown). Regeneration through TENGs showed host axons following the length of the GFP-labeled TENG axons, with greater penetration and more organization than that found following NGT repair. Scale bar: 1000 μm. (B) Clear discrimination of TENG and host axons, as well as host SCs, across the graft zone. In addition to direct AFAR (hollow arrowhead), host SCs also migrated and organized along TENG axons (solid arrowheads), creating a “tripartite” regenerative complex of TENG axons: host axons: host SCs. Scale bar: 10 μm. (C) Following TENG repair, a group of host “Leading Regenerator” axons was present in the distal stump – far afield from the “Regenerative Front” – and were seen along with TENG axons that had also penetrated into the distal stump. Scale bar: 50 μm. (D) These accelerated “Leading Regenerator” axons were present following TENG or autograft repair, but were always absent following NGT repair. (E) Quantification of axon regeneration (mean ± standard deviation). Host “Regenerative Front” and “Leading Regenerators” were statistically equivalent following TENGs and autografts repair, and both were superior to NGT repair (**p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 5

TENGs Direct Host Schwann Cell Infiltration. Longitudinal sections across the graft zone at 2 weeks following repair using TENGs or NGTs. (Aa1) In cases where TENG axons (green) were present off-center (shown in upper half of the boxed section), SC infiltration was markedly directed upward toward TENG axons, indicating that SCs are attracted to TENG axons and alter their migration to infiltrate along TENG axons. (Bb) In NGTs, SCs generally infiltrated linearly from ends, typically observed only in center of NGT (tapered cone). (a2) TENG axons also projected into the distal nerve stump to grow along host SCs. (A,B) Scale bars: 500 μm. (a1,a2,b) Scale bars: 100 μm. (C) Host SCs also directly interacted with TENG neurons; scale bar: 100 μm. (D) Plot of quantified SC infiltration measurements (mean ± standard deviation). SC penetration from the proximal and distal ends was quantified. TENGs significantly increased infiltration of SCs compared to NGTs (**p < 0.01).

FIGURE 6

TENGs Facilitate Functional Recovery. (A–C) Functional recovery and structural regeneration at 16 weeks following repair of 1 cm nerve lesions using NGTs, reverse autografts, or TENGs. (A) Representative CMAP traces. (B) Representative nerve morphometry showing nerve cross sections (5 mm distal to repair zone) labeled for axons (red) and myelin (purple). Scale bar (top): 100 μm; scale bar (bottom): 50 μm. (C) Plots of mean recovery levels for CMAP, CNAP, muscle weight, and axon density (mean ± standard deviation; *p < 0.05 and **p < 0.01 versus NGT). (D–F) Proof-of-concept data showing structural and functional regeneration at 12 weeks following repair of 2 cm nerve lesions using TENGs. (D) Chronic nerve morphometry showing representative nerve cross sections (5 mm distal to repair zone) labeled for axons (purple) and myelin (red), also showing TENG neurons and axons (green). Scale bar: 50 μm. (E) Example CNAP across a 2 cm nerve segment repaired using a TENG. (F) Oblique nerve cross section (5 mm distal to repair zone) showing the architecture of numerous TENG axons (GFP+, green) interacting with host SCs (S100+, red) and growing along other axons (NF+, purple). Scale bar: 100 μm. (A,E) Electrophysiological traces were averaged over a train of 5 pulses with 1 s intervals between each pulse, with band pass filtering from 10 to 10,000 Hz for CMAP and 10–2000 Hz for CNAP.

FIGURE 7

TENGs Mechanisms-of-Action: The Value of Axons. Host axons favor paths created by transplanted TENG axons. TENGs serve as a living scaffold to facilitate regeneration via AFAR on the level of both (A) individual axons (scale bar: 100 μm) and (B) groups of axons (scale bar: 50 μm). (C,D) Tripartite regenerative mechanism: integration of TENG axons with both host axons and host SCs (scale bar: 100 μm). (d) Inset image from (D) showing colocalization of TENG axons with host axons and host SCs. TENGs enhance SC alignment as pointed out by the arrowheads, which then facilitate host axon growth. The arrows show instances of direct AFAR, illustrated by colocalization of TENG and host axons without the presence of host SCs (scale bar: 50 μm). (E) Conceptual schematic depicting the MoA of TENGs, leading to synergistic presentation of neurotrophic, chemotaxic and haptotaxic cues only possible with a “living scaffold.” Overall, TENGS possess novel mechanisms compared to NGTs & autografts: direct AFAR (not possible with autograft), increased SC infiltration and alignment (versus NGT), and robust presence of “leading regenerator” axons (versus NGT). This AFAR – unique to TENGs – compliments traditional SC-mediated axon regeneration.