Tissue-engineered grafts exploit axon-facilitated axon regeneration and pathway protection to enable recovery after 5-cm nerve defects in pigs

Abstract

Functional restoration following major peripheral nerve injury (PNI) is challenging, given slow axon growth rates and eventual regenerative pathway degradation in the absence of axons. We are developing tissue-engineered nerve grafts (TENGs) to simultaneously "bridge" missing nerve segments and "babysit" regenerative capacity by providing living axons to guide host axons and maintain the distal pathway. TENGs were biofabricated using porcine neurons and "stretch-grown" axon tracts. TENG neurons survived and elicited axon-facilitated axon regeneration to accelerate regrowth across both short (1 cm) and long (5 cm) segmental nerve defects in pigs. TENG axons also closely interacted with host Schwann cells to maintain proregenerative capacity. TENGs drove regeneration across 5-cm defects in both motor and mixed motor-sensory nerves, resulting in dense axon regeneration and electrophysiological recovery at levels similar to autograft repairs. This approach of accelerating axon regeneration while maintaining the pathway for long-distance regeneration may achieve recovery after currently unrepairable PNIs.

Fig. 1.. Overview of clinical need and tissue-engineered solution.

(A) Clinical unmet need requires bridging + babysitting strategy. Functional recovery after PNI is often limited because of chronic denervation of the distal pathway resulting from slow axon regeneration across ultralong regenerative distances to muscle end targets. Although current repair strategies aim to accelerate regeneration, none of the commercially available approaches are designed to mitigate complications associated with chronic denervation. (B) TENG biofabrication. We have bioengineered stretch-grown axons from (1) embryonic rat or pig sensory DRG neurons that are cultured on two overlapping membranes in a custom-built mechanobioreactor, allowing for integration between the two populations for up to 5 DIV. (2) The two overlapping membranes are gradually separated via a microstepper motor to apply mechanical tension to the axons spanning the two neuronal populations, depending on the desired length. (3) Stretch growth occurs for days to weeks at 1 to 10 mm/day. Once the desired axon length is reached, the neurons and stretch-grown axons are encapsulated in collagen extracellular matrix (ECM) for stabilization. (4) Encapsulated constructs are “rolled” and (5) inserted into an NGT or nerve wrap to bridge segmental nerve defects. (C) TENG surgical implementation. Example of a 5-cm TENG-NGT and schematic of surgical implementation. (D) TENG proregenerative mechanism of action. Concept figure illustrating the dual mechanisms of TENG-mediated regeneration [as described in detail in the work of Katiyar et al. (25)] via AFAR whereby host axons rapidly grow directly along TENG axons, as well as host Schwann cells (SCs) migrating directly along TENG axons to efficiently fill the length of the graft zone.

Fig. 2.. Implanted TENGs integrate with denervated distal nerve sheath over 16 weeks in rodent chronic axotomy model.

(A) Proregenerative S100⁺ SCs were characterized in a rodent chronic axotomy model by securing to the otherwise denervated distal nerve with either a babysitting TENG or empty NGT (negative control). See Fig. 1B for additional information on encapsulation methodology. (B) Longitudinal tissue sections showing dense TENG neurons (predominantly showing the cell body region of the graft) transduced to express GFP survive transplantation and project axons into the otherwise denervated distal nerve stump. (b) Higher magnification revealed TENG axons growing in close proximity to host SCs. (C1 to C5) Following NGT implant alone, host SCs initially formed stereotypically aligned regenerative columns at 2 and 4 weeks but become disorganized over 6 to 9 weeks and disappear virtually completely by 16 weeks, suggesting a nonpermissive regenerative environment. (C6) In contrast, distal SCs were preserved in animals receiving TENGs at 16 weeks. (D) Semiquantitative scoring of SC presence and morphology revealed that TENGs preserve SCs in the otherwise axotomized distal nerve structure for prolonged time periods. (E1 to E3) Aligned SCs were found at 16 weeks after axotomy, distal to the babysitting TENG (E1) 0.2 cm, (E2) 1.0 cm, and (E3) 3.0 cm from the graft. (F) Oblique (45°) section of the distal nerve showing that GFP⁺ axons projecting from the babysitting TENG persist within the distal nerve stump at 16 weeks after transplantation, closely interacting with host SCs. Here, a relatively low “dose” of TENG axons may babysit an entire fascicle of host SCs. P values are represented as *P < 0.001. Scale bars, 250 μm and 50 μm (B and b), 50 μm (C), 20 μm (E), and 25 μm (F). n = 3 to 4 for NGT per time point; n = 4 for babysitting TENG at 16 weeks.

Fig. 3.. Primary porcine neurons subjected to axonal stretch growth to biofabricate TENGs.

E40 transgenic GFP⁺ porcine DRG neuron cultures (A) at 1 DIV in phase contrast and (B and C) at 7 DIV with fluorescent microscopy labeling nuclei (Hoechst), axons (Tuj1), and neuronal cell bodies and dendrites (MAP2). (D) Axons from porcine neurons extend beyond the towing membrane to integrate with the other neuron population on the base membrane. Aligned axon tracts are generated following mechanical stretch growth, as shown in phase-contrast microscopy. (E) Example of DRG populations that were stretch-grown over 1 cm shown with phase-contrast microscopy. (F) Phase-contrast image of the towing membrane side of a TENG showing healthy and robust axons. (G and H) Examples of a 5-cm-long TENG with dense fasciculation following stretch growth at 21 DIV (5 days before stretch, 16 days of stretch growth), shown in (G) phase contrast and with (H) fluorescent (GFP) microscopy. (I) Example of a 2.5-cm-long GFP⁺ TENG following collagen encapsulation before transplantation. Scale bars, 100 μm (A), 500 μm (B and C), 50 μm (D), 1000 μm (E), 100 μm (F), 1000 μm (G and H), and 2.0 mm (I).

Fig. 4.. TENGs maintain neuronal-axonal architecture and project axons into the host distal nerve that closely interact with host SCs and regenerating axons following a 1-cm porcine nerve repair.

(A) Acute regenerative mechanisms of action were evaluated at 2 weeks following a 1-cm repair of the mDPN and sDPN using an NGT, autograft (AG), or TENG. (B) Allogeneic TENG neurons (GFP⁺) survived transplantation in the absence of immunosuppression. (C) Robust axonal regeneration (SMI31/32⁺) was observed along TENG neurons/axons. (D) Host SC infiltration (S100⁺) and axon regeneration were visualized. (E) Within the graft, TENG neurons were located on both ends spanned by long-projecting axons, and (F) host axons were seen regenerating along TENG axons. (G) At higher magnification, AFAR was observed. (H) TENGs facilitated nerve regeneration using traditional SC-mediated axonal regeneration and also in the absence of SCs via AFAR mechanism (arrows). Host SCs aligned with TENG axons and likely support host axon regeneration (arrowheads). (I) Longitudinal section of the distal nerve showing columnar SCs or bands of Büngner and TENG axons. (J) Higher magnification revealed leading regenerator host axons within bands of Büngner in the distal nerve. (K) Accelerated regeneration was observed at 2 weeks following TENG repair compared to NGTs. Greater SC infiltration was also found in TENGs compared to NGTs for both nerves. No statistical differences were found for axon regeneration or SC presence between TENG and AGs. P values are presented as *P < 0.05, **P < 0.01, and ***P < 0.001 versus NGT. Scale bars, 50 μm (B and C), 1000 μm (D), 500 μm (E and F), 20 μm (G and H), 250 μm (I), and 100 μm (J). For the sDPN study, n = 4 for autograft, n = 4 for NGT, and n = 5 for TENG. For the mDPN study, n = 4 for all groups.

Fig. 5.. Nerve regeneration at 1 and 3 months following repair of long-gap (5-cm) nerve injury with TENGs in a porcine model.

(A) TENG efficacy was further assessed following a 5-cm mDPN repair. (B to H) At 4 weeks after repair, (B) the main bolus of regenerating axons (SMI31/32⁺, left) and SC infiltration (S100⁺) was visualized within the conduit (autofluorescing green). In this section, TENG neurons can be visualized in the proximal and distal conduit with some TENG axons in the center of the conduit, suggesting that the TENG axons span the entire length of the graft region. (C) Surviving TENG neurons/axons (GFP⁺) interacted with host SCs. (D) Dense host axon regenerative front and host SCs were found penetrating the graft. (E) Notably, host axons and aligned SCs grew along TENG axons near the conduit center. (F) TENG neurons/axons and (G) infiltrating host SCs were visualized within the graft. (H) Host axons were visualized in the distal nerve before complete SC infiltration, further suggesting that TENGs facilitated axon regeneration independent of SC infiltration. (I to O) At 12 weeks, (I) gross nerve structure shows the reformation of nerve and vascular tissue. (J) Surviving TENG neurons/axons interacted with host SCs. (K and L) Axial sections distal to the graft show (K) successful host axon regeneration and TENG axon penetration distally and (L) the presence of TENG axons, host SCs, and host axons at higher magnification. (M) Successful host axon regeneration and SC infiltration near both ends of the 5-cm graft. (N) Large-caliber host axons (arrowheads) and smaller-caliber TENG axons (arrows) were observed in the distal nerve. (O) CNAPs following TENG repair indicate significant axonal regeneration. Scale bars, 1000 μm (B), 100 μm (C), 50 μm (D to H, J, K, and M), 15 μm (L), and 5 μm (N).

Fig. 6.. Axon regeneration and muscle reinnervation following a 5-cm segmental repair of the mDPN in swine.

(A) At 7 months following TENG repair, the regenerated mDPN was isolated from the surrounding tissue for functional assessment. (B) Oblique nerve section from the extreme distal mDPN directly at the interface with the target muscle indicates that host axons (SMI31/32⁺) had reached the muscle by 7 months after repair. (C) Same nerve as (B) showing longitudinal nerve section from a more proximal zone. (D) Cross-sectional immunohistochemistry of the mDPN 5 mm distal to the TENG was performed to label host axons and SCs. (d) Higher magnification revealed a high density of host axons in the distal nerve surrounded by S100⁺ SCs. (E) At 9 months after repair with a TENG (arrow), the nerve gross structure and morphometry appeared indistinguishable from those of an adjacent, nonrepaired nerve. (F) Nerve morphometry showed fasciculation, dense axonal regeneration, and remyelination following TENG repair at 9 months after repair (5 mm distal to the repair zone). (f) Higher magnification of an individual fascicle revealed large- and small-diameter host axons surrounded by SCs, potentially indicating ongoing maturation. (G) Morphometric analysis revealed that axon density and percentage of myelinated axons were equivalent between animals repaired with a TENG or a sensory autograft (see fig. S4 for breakout of myelinated axon and g-ratio profiles). (H) An evoked hoof twitch was consistently achieved by 7 months after repair with robust CNAP and CMAPs, further suggesting successful axon regeneration, myelination, and muscle reinnervation. No significant differences were found (P > 0.05). Scale bars, 50 μm (B), 500 μm (C), 50 μm (D), 20 μm (d), and 50 μm (F and f). n = 3 for autografts and n = 5 for TENGs at this time point.

Fig. 7.. Deployment of distal TENGs to babysit the distal pathway following repair of 5-cm segmental defects in the CPN in swine.

(A) To evaluate the efficacy of simultaneously reconstructing proximal injuries (bridge) and preserving the distal nerve and motor targets (babysit), two babysitting TENGs were placed in continuity with the mDPN, distal to the primary CPN repair. (B and C) Immunohistochemistry distal to the primary CPN graft revealed axon regeneration and maturation in the (B) sDPN and (C) mDPN at 12 months following autograft or TENG repair with distal babysitting TENGs in the mDPN. (C2) GFP⁺ signal was found within the mDPN fascicles at 12 months following primary TENG repair and transplantation of distal babysitting TENGs. (D and E) Longitudinal sections showing host axons and aligned SCs present in the (D) sDPN and (E) mDPN at the babysitting graft zone, several centimeters distal to the primary CPN repair, further corroborating that host axons crossed the babysitting TENG. (F) High magnification of an mDPN fascicle revealed numerous TENG axons that closely interact with host SCs. (G) Detailed histological analysis revealed that g-ratio distribution profiles in the mDPN were similar across groups at this time point. Individual g-ratio distribution profiles were broken out and compared to the naïve contralateral nerve distribution (dotted line). No significant differences were found for the mDPN g-ratio distribution profiles between the groups or between the contralateral (naïve) mDPN nerve and the mDPN nerve distal to the autograft or TENG + BS repair at this time point. G-ratio distributions were compared between groups for each nerve using the two-sample Kolmogorov-Smirnov test. P values are represented as *P < 0.05. See figs. S5 and S6 for breakout of myelinated axon and g-ratio profiles. Scale bars, 500 μm (B and C) and 50 μm (b and c). At least n = 100 axons were measured per animal. ns, not significant.

Fig. 8.. Axon regeneration and functional reinnervation at 12 months following repair of 5-cm CPN segmental defects in swine.

(A) In this study, a 5-cm CPN segmental defect was repaired using a TENG (with or without distal babysitter grafts) or autograft. (B to D) Immunohistochemistry of a single representative fascicle of the (B) CPN segment distal to the repair zone, (C) mDPN, and (D) sDPN revealed successful axonal regeneration across the challenging defect and ongoing axon maturation. (E) Mean axon counts following TENG versus autograft repair for the repaired nerve (CPN) and two distal branches (mDPN and sDPN). (F) CMAPs were recorded from both muscles innervated by branches of the CPN: the tibialis anterior (TA; innervated ~8 cm distal to the primary repair zone) and the EDB (innervated ~27 cm distal to the primary repair zone). Nerve stimulation proximal and distal to the repair site elicited robust muscle movement, and positive hoof eversion was observed, corroborating the CMAP recordings. P values are represented as *P < 0.05. Scale bars, 100 μm. Divisions, 500 μV/25 ms. n = 3 for both groups at 12 months after repair.