Multimodular Bio-Inspired Organized Structures Guiding Long-Distance Axonal Regeneration

Abstract

Axonal bundles or axonal tracts have an aligned and unidirectional architecture present in many neural structures with different lengths. When peripheral nerve injury (PNI), spinal cord injury (SCI), traumatic brain injury (TBI), or neurodegenerative disease occur, the intricate architecture undergoes alterations leading to growth inhibition and loss of guidance through large distance. In order to overcome the limitations of long-distance axonal regeneration, here we combine a poly-L-lactide acid (PLA) fiber bundle in the common lumen of a sequence of hyaluronic acid (HA) conduits or modules and pre-cultured Schwann cells (SC) as cells supportive of axon extension. This multimodular preseeded conduit is then used to induce axon growth from a dorsal root ganglion (DRG) explant placed at one of its ends and left for 21 days to follow axon outgrowth. The multimodular conduit proved effective in promoting directed axon growth, and the results may thus be of interest for the regeneration of long tissue defects in the nervous system. Furthermore, the hybrid structure grown within the HA modules consisting in the PLA fibers and the SC can be extracted from the conduit and cultured independently. This "neural cord" proved to be viable outside its scaffold and opens the door to the generation of ex vivo living nerve in vitro for transplantation.

Keywords: Schwann cell culture; dorsal root ganglion culture; hyaluronic acid conduit; long-distance axonal regeneration; neural cord; poly-lactic fibers.

Figure 1

Schematic view of the generation of the long-distance axonal regeneration strategy proposed. Longitudinal and transversal view of a unimodular conduit (left) and multimodular conduit (right) formed by three individual modules of HA and PLA fibers passing through its lumens. We seeded SC for 5 days and the SC formed a tapestry in the inner lumen and also grew on the PLA fibers. After 5 days of SC culture, we seeded a DRG explant at one end of the unimodular and multimodular conduits and culture for 21 days, expecting the axons of projecting neurons extent through the conduits. We observed the axon growth to prove the effectiveness of promoting directed axon growth in long distances.

Figure 2

Schematic view of the generation of the ‘neural cord’. Longitudinal and transversal schematic view of ‘neural cord’ after 5 days of SC culture and elimination of the HA conduits, obtaining a live hybrid pseudonerve in vitro for a possible transplantation.

Figure 3

Unimodular and multimodular conduits concept. Scanning electron microscope images from a transversal cut (A) of a hyaluronic acid conduit, and a longitudinal cut of (B) unimodular and (C) multimodular conduits (only the part with de PLA fibers is shown). Macroscopical view of hydrated (D) unimodular and (E) multimodular conduits, and a (F) multimodular conduit formed by 10 modules in order to cover a long and curved distance. The distances between the modules in multimodular conduits is exaggerated for better visualization. Scale bar: 5 mm (F), 2 mm (D,E), 500 μm (B,C), 100 μm (A).

Figure 4

Differences of Schwann cells density between unimodular and multimodular conduits after 5 days. (A) MTS assay on SC cultured for 5 days inside unimodular and multimodular conduits. In multimodular conduit cell proliferation was increased. An asterisk * indicates statistically significant differences, indicating a p-value below 0.05. (B) Macroscopical view of hydrated (top) unimodular and (bottom) multimodular conduits after MTS assay (reveled with color brown). Zone without cellular continuity in unimodular conduit is indicated with a black dash lines box. Scale bar: 6 mm.

Figure 5

Schwann cells distribution along the whole length and transversal sections of unimodular and multimodular conduits after 5 days. Representative fluorescence reconstruction of the conduit’s complete length of (A) unimodular and (G) multimodular conduits, after nuclear staining with DAPI (blue) and F-actin of SC staining with ActinRed™ 555 ReadyProbes™ Reagent (red). Confocal fluorescent image of a longitudinal section of unimodular conduit at (B) 0 mm, (C) 6 mm, (D) 12 mm, (E) 18 mm, and multimodular conduit at (H) 0 mm, (I) inter-module zone between modules 1 and 2, (J) inter-module zone between modules 2 and 3, (K) 18 mm, showing continuity in SC growth even in the absence of the HA conduit. Details in A and G are indicated with white dash lines boxes. Representative fluorescent images of a transversal section at 9 mm long of unimodular conduit (F), and multimodular conduit (L), after the same staining. Scale bar: 500 μm (A,G), 100 μm (B–F,H–L).

Figure 6

Axon growth in unimodular and multimodular conduits after 21-days dorsal root ganglion explant culture. Representative fluorescence reconstruction of the (A) unimodular and (B) multimodular conduit’s complete length after neuronal staining with Tuj1 (green), showing greater axonal extension of the multimodular conduit. Fluorescent image of a longitudinal section of (C) inter-module zone between modules 1 and 2 (m1, m2), (D) inter-module zone between modules 2 and 3 (m2, m3), (E) final zone in module 3. White dash lines delimit the modules of the multimodular conduit, and yellow dash lines box delimit the final zone of the module 3 (16–17 mm long from the DRG explant). Scale bar: 500 μm.

Figure 7

Schwann cells distribution in the ‘neural cord’ after 5 additional culture days. (A) Longitudinal and transversal schematic view of ‘neural cord’. (B) Representative phase-contrast reconstruction of the ‘neural cord’s’ complete length in culture before fixing. (C) Representative fluorescence reconstruction of the ‘neural cord’s’ complete length after F-actin of SC staining with ActinRed™ 555 ReadyProbes™ Reagent (red). (D) Representative SEM reconstruction of the ‘neural cord’s’ complete length, showing a compact structure after the manipulation of the sample. Details in reconstructions are indicated with dash lines boxes in (B’), (C’), and (D’). Scale bar: 500 μm.

Figure 8

Schwann cells distribution in multimodular conduits and ‘neural cord’. Scanning electron microscopic image from a longitudinal cut in multimodular conduits after (A) 5-days culture and (B) 10-days culture. (C,D) Scanning electron microscopic image of the ‘neural cord’ after 5 days of culture outside the HA conduit. Scale bar: 100 μm (A), 20 μm (B,C), 10 μm (D).

Figure 9

Schwann cells distribution in a transversal cut of the ‘neural cord’. Representative fluorescent images of a transversal section of the ‘neural cord’ after nuclear staining with DAPI (blue) and F-actin of SC staining with ActinRed™ 555 ReadyProbes™ Reagent (red) with (A) and without (B) the bright field view. Scale bar: 100 μm (A,B).

Figure 10

Axon growth in ‘neural cord’ after o-culture with dorsal root ganglion explant. (A) Longitudinal and transversal schematic view of obtention of the ‘neural cord’ and the co-culture with a dorsal root ganglion explant. (B) Representative confocal reconstruction of the first 4–5 mm of longitudinal sections of the ‘neural cord’ showing neuronal staining with Tuj 1 (green) after 10 days with HA conduits and 16 additional days without the HA conduits. (C) Analysis of relative mRNA expression of GAP43. Scale bar: 500 μm.