Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep

Abstract

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Keywords: allograft; autograft; decellularization; nerve regeneration; sheep.

Figure 1

Decellularized nerve allograft (A) was trimmed into 7-cm nerve long and then it was placed into the gap created in the common peroneal nerve (B). The decellularized nerve allograft (C) or the same nerve segment resected used as autograft (D) were sutured bridging the gap.

Figure 2

Representative micrographs showing immunofluorescence of myelinated axons (A,F,K,G), Schwann cells (B,G,L,Q), nuclei (C,H,M,R), extracellular matrix proteins (D,I,N,S) and myelin (E,J,O,T), in a control rat sciatic nerve (A,B,C,D,E), in a decellularized rat nerve (F,G,H,I,J), in a control sheep peroneal nerve (K,L,M,N,O) and in a decellularized sheep nerve (P,Q,R,S,T). Scale bar 150 μm.

Figure 3

Results of the electrophysiological tests and histological evaluation after repair of a 15 mm long gap in the rat sciatic nerve. (A) Electrophysiological evaluation of nerve regeneration along 120 days follow-up after sciatic nerve section and repair with an autograft (AG, n = 5) or with a decellularized allograft (DRA, n = 5). Results are presented as mean ± SEM. Statistical analysis was performed using 2way ANOVA. Plots show the amplitude of CAMP of plantar (* p < 0.05 vs. AG), tibialis anterior (* p < 0.05 vs. AG) and gastrocnemius (* p < 0.05 vs. AG) muscles. (B) Representative micrographs showing immunofluorescence of myelinated axons labeled against Neurofilament 200, Schwann cells labeled against S100, nuclei labeled with DAPI, macrophages labeled with IBA1 and extracellular matrix labeled with laminin in cross sections of nerve graft in control, AG and DRA groups; scale bar 200 μm. (C) Representative transverse semithin sections of the mid graft and distal to the graft in AG and DRA groups, stained with toluidine blue. Scale bar 10 μm. Plots show the density and the number of myelinated axons in the sciatic nerve at mid graft and distal to the graft. Normal values in our laboratory for the sciatic nerve in rats average 8076 ± 215 myelinated axons, with a density of 11,336 ± 698 per mm².

Figure 4

Plots of the functional evaluation along the 9 months follow-up in the two groups of sheep. Similar evolution was observed for all the tests applied, without significant differences between the groups AG and DC. Regarding the time evolution, AG group (n = 5) showed a significant recovery in all the tests applied (# p < 0.05) vs. baseline at 30 days post-operation, while animals of DC group (n = 5) only showed a significant recovery in the withdrawal response-distal ($ p < 0.05) vs. baseline at 30 days post-operation.

Figure 5

Representative EMG recordings of the CMAP recorded in the TA muscle evoked by stimulation of the sciatic nerve in the intact left hindlimb (A), and in the operated right hindlimb of a sheep at 6.5 (B) and 9 (C) months after operation. Labels: “1” at the onset, “2” at the peak, “4” at the end of the CMAP. Scale in A: amplitude 10 mV/square and time 3 ms per square; in B and C: amplitude 500 μV/square and time 5 ms per square. Representative echographic images of the TA muscle recorded in the intact left hindlimb (D) and in the operated right hindlimb of a sheep repaired with an autograft (E) or with a decellularized nerve allograft (F).

Figure 6

Macroscopic representative pictures of a control sheep peroneal nerve (A), a peroneal nerve autograft (B), and a decellularized nerve allograft (C), both with a neuroma visible (marked with red arrows) at proximal and distal sutures, harvested at the end of the study. (D) Schema of the division in sections of nerves harvested. Section 1 includes the proximal suture and Section 3 includes the distal suture of the graft. Section 2 and Section 4 were divided into two different segments (dotted line). Micrographs of cross sections of the proximal segment of the graft of sheep stained with hematoxylin and eosin. (E,H) control nerve, (F,I) autograft and (G,J) decellularized nerve graft; E, F, G scale bar 200 μm, and H, I, J scale bar 100 μm. Representative semithin transverse sections of the graft of sheep in AG and DC groups, stained with toluidine blue. (K) control nerve, (L) autograft and (M) decellularized nerve graft, scale bar 10 μm. Red arrows in M point to the newly formed regenerative small fascicles.

Figure 7

Representative micrographs showing immunofluorescence of myelinated axons labeled against Neurofilament 200 in cross sections of the proximal graft in AG and DC groups. (A,D) control nerve, (B,E) autograft and (C,F) decellularized nerve graft. A, B, C scale bar 200 μm, and D, E, F scale bar 100 μm. Representative micrographs showing immunofluorescence labeling of Schwann cells with an antibody against the S100 protein in cross sections of the proximal graft in AG and DC groups. (G,J) control nerve, (H,K) autograft and (I,L) decellularized nerve graft. G, H, I scale bar 100 μm, and J, K, L scale bar 100 μm.

Figure 8

Representative micrographs of cross sections of the Tibialis Anterior muscle stained with hematoxylin and eosin from (A,D) the contralateral side (control), (B,E) an animal from AG group, (C,F) an animal from DC group. A, B, C scale bar 200 μm, and D, E, F scale bar 100 μm. Representative micrographs of perpendicular sections of the skin of the dorsum of the foot stained with hematoxylin and eosin from (G,J) the contralateral side (control), (H,K) an animal from AG group, (I,L) an animal from DC group. G, H, I scale bar 200 μm, and J, K, L scale bar 100 μm.