Decellularized Graft for Repairing Severe Peripheral Nerve Injuries in Sheep

Abstract

Background and objectives: Peripheral nerve injuries resulting in a nerve defect require surgical repair. The gold standard of autograft (AG) has several limitations, and therefore, new alternatives must be developed. The main objective of this study was to assess nerve regeneration through a long gap nerve injury (50 mm) in the peroneal nerve of sheep with a decellularized nerve allograft (DCA).

Methods: A 5-cm long nerve gap was made in the peroneal nerve of sheep and repaired using an AG or using a DCA. Functional tests were performed once a month and electrophysiology and echography evaluations at 6.5 and 9 months postsurgery. Nerve grafts were harvested at 9 months for immunohistochemical and morphological analyses.

Results: The decellularization protocol completely eliminated the cells while preserving the extracellular matrix of the nerve. No significant differences were observed in functional tests of locomotion and pain response. Reinnervation of the tibialis anterior muscles occurred in all animals, with some delay in the DCA group compared with the AG group. Histology showed a preserved fascicular structure in both AG and DCA; however, the number of axons distal to the nerve graft was higher in AG than in DCA.

Conclusion: The decellularized graft assayed supported effective axonal regeneration when used to repair a 5-cm long gap in the sheep. As expected, a delay in functional recovery was observed compared with the AG because of the lack of Schwann cells.

Graphical abstract

FIGURE 1.

The decellularized peroneal nerve of sheep A, was trimmed into 5-cm long segments. The same nerve segment resected was used as an autograft B, or the decellularized nerve allograft C, was used to bridge the gap.

FIGURE 2.

Representative micrographs showing immunofluorescence of Schwann cells A, F, K, and P, myelinated axons B, G, L, and Q, nuclei C, H, M, and R, extracellular matrix proteins D, I, N, and S, and myelin E, J, O, and T, A-J, in a control sheep peroneal nerve and K-T, in a decellularized sheep peroneal nerve. A-E and K-O, scale bar 150 μm, F-J, and P-T, scale bar 25 μm. Images F-J, correspond to higher magnification of a field in images A-E, respectively, and images P-T, correspond to higher magnification of a field in images K-O, respectively.

FIGURE 3.

Plots of functional tests scoring during the 9 months follow-up. Both AG and DCA groups showed a similar evolution in A, locomotion and in B, withdrawal reflex tests. Values are shown as the median. There were no significant differences between the 2 groups at any of the time points tested. AG, autograft; DCA, decellularized nerve allograft.

FIGURE 4.

Plots of the latency A, and the amplitude B, of the tibialis anterior CMAP recorded at 6.5 and 9 mps in AG (n = 4) and DCA (n = 6) groups, respectively, compared with the control contralateral hindlimb (n = 10). The CMAP amplitudes increased at 9 mps, without statistical differences between the AG and the DCA groups. In both groups, the amplitude was significantly lower than in control hindlimbs. *P < .05 vs DCA; #P < .05 vs control; $P < .05 vs control. AG, autograft; CMAP, compound muscle action potential; DCA, decellularized nerve allograft; mps, months postsurgery.

FIGURE 5.

Histograms of the perimeter A, and area B, of the TA muscle obtained by ultrasound imaging. The size of the TA muscle was decreased in AG and DCA groups compared with the contralateral hindlimb (****P < .0001 vs control). The TA muscle area was significantly higher in the AG group compared with the DCA group (*P < .05) at the end of the follow-up. AG, autograft; DCA, decellularized nerve allograft; mps, months postsurgery; TA, tibialis anterior.

FIGURE 6.

Representative micrographs of cross-sections at the middle of the nerve graft of the sheep stained with hematoxylin and eosin. A, D, Control nerve, B, E, AG, and C, F, DCA; A-C, scale bar 500 μm, and D-F, scale bar 150 μm. Representative semithin transverse sections of the nerve graft stained with toluidine blue. G, J, control nerve, H, K, AG, and I, L, DCA; G-I, scale bar 500 µm, and J-L, scale bar 25 µm. Red arrows in E, and K, point to the small regenerative clusters outside the defined fascicles in the AG group. AG, autograft; DCA, decellularized nerve allograft.

FIGURE 7.

Representative micrographs of cross-sections at the middle of the nerve graft showing immunofluorescence of myelinated axons labeled against Neurofilament 200 of A, D, and G, a control nerve, B, E, and H, an autograft, and C, F, and I, a decellularized allograft. Note that image H, is focused on some small regenerating clusters outside the main graft fascicles. A-C, Scale bar 500 μm; D-F, scale bar 100 μm; G-I, scale bar 25 µm. Representative micrographs of cross-sections of the nerve graft labeled against S100 protein of control J, M, A, G, K, N, and decellularized nerve allograft L, O, sheep. J-L, Scale bar 200 μm and M-O, scale bar 100 μm.