Microscopy, quantification, and statistical analyses

Neuronal constructs in vitro were imaged using phase-contrast or epifluorescence microscopy on a Nikon Eclipse Ti-S with digital image acquisition using a QIClick camera interfaced with Nikon Elements Basic Research software (4.10.01). For immunolabeled neuronal constructs, fluorescent imaging was completed using a Nikon Eclipse Ti-S or Nikon A1R confocal microscope.

All histological analyses were performed by trained scientists blinded to group identity. Longitudinal and axial tissue sections were imaged with a Nikon A1R confocal microscope (1024 × 1024 pixels) with a 10× air objective or 60× oil objective interfaced with Nikon NIS-Elements AR 3.1.0 (Nikon Instruments, Tokyo, Japan). Multiple confocal z-stacks were digitally captured and analyzed, with all reconstructions tiled across the full section and full z-stack thickness.

Schwann cell morphology in the distal nerve following chronic axotomy in rats was assessed from multiple high-resolution confocal reconstructions per animal. A semiquantitative scoring system was applied by two researchers blinded to the experimental group: 5, robust, aligned Schwann cells; 4, aligned Schwann cells; 3, reduced Schwann cells, semi-aligned; 2, scattered Schwann cells, little alignment; and 1, few/no Schwann cells, loss of alignment. This analysis was performed on the full cohort at the following time points: 2, 4, 6 to 8, 9, and 16 weeks after implant (NGT only, n = 3 to 4 rats per time point) or 16 weeks after implant (TENG, n = 4 rats).

In porcine experiments at the acute and subacute time points, axon regeneration and Schwann cell infiltration were measured from longitudinal sections including the 1-cm graft at 2 weeks after repair and the 5-cm graft at 1 month after repair using previously established methodology (26). Briefly, the length of the host axons (SMI31/32) was measured from the proximal end of the repair zone with TENG axons noted to be GFP⁺ (26). Host axons were grouped as the regenerative front, main bolus of regenerating host axons, or the leading regenerators, the longest-projecting host axons. Schwann cell infiltration was measured on the basis of the S100⁺ coverage from both the proximal and distal ends of the graft. This quantitative analysis was performed on the full cohort of 1-cm lesion repairs at 2 weeks after repair: sDPN segmental defect repaired with autograft (n = 4 pigs), NGT (n = 4), and TENG (n = 5); mDPN segmental defect repaired with autograft (n = 4), NGT (n = 4), and TENG (n = 4). At 1 month following repair of 5-cm lesions in the mDPN, this quantitative analysis was performed in animals repaired with autograft (n = 2 pigs) or TENG (n = 3).

In porcine experiments at intermediate time points following repair of 5-cm lesions in the mDPN or CPN, axon regeneration and Schwann cell presence/morphology were qualitatively assessed in longitudinal sections in the graft region and axial sections distal to the graft region. At chronic time points, axon regeneration and myelination were quantitatively assessed using axial sections distal to the graft region. Here, the total number of axons was determined by quantifying the number of neurofilament-positive axons. TENG axons were identified as GFP⁺ axons. The percentage of myelinated (MBP⁺) axons was also quantified. This quantitative analysis was performed in the chronic cohorts following 5-cm segmental defects in the mDPN repaired with autograft (n = 3 pigs) or TENG (n = 5) and in the CPN repaired with autograft (n = 3) or TENGs (n = 3; with or without distal grafts). Axon regeneration and Schwann cell presence/morphology were also qualitatively assessed at chronic time points, from longitudinal sections immediately distal to the babysitting grafts (in animals receiving these grafts).

All quantitative data (e.g., mean Schwann cell morphology, axon regeneration, Schwann cell infiltration, and axon myelination) were compared using either Student’s t test or one-way analysis of variance (ANOVA). When differences existed between groups following ANOVA, Tukey’s post hoc pairwise comparisons were performed. Axon fiber diameter and g-ratio for myelinated axons (ratio of the inner axonal diameter to the total outer diameter) were also calculated by sampling three 100-μm² areas. At least 100 measurements were obtained per animal. Data were binned in 0.5-μm increments from 0.5 to 7 μm (axon diameter) and 0.05 increments from 0.3 to 0.9 (g-ratio). The cumulative frequency distribution was plotted with the nonlinear Gaussian line of best fit. Myelinated axon diameter and g-ratio histograms were compared using the two-sample Kolmogorov-Smirnov test to evaluate the agreement between distribution profiles. For all statistical tests, P < 0.05 was required for significance (GraphPad Prism, La Jolla, CA, USA). Mean values are presented as means ± SEM unless otherwise noted.