Scaffoldless tissue-engineered nerve conduit promotes peripheral nerve regeneration and functional recovery after tibial nerve injury in rats

Abstract

Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fibroblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, respectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promoted nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.

Keywords: fibroblasts; nerve regeneration; neural cells; neural conduit; peripheral nerve repair; tissue engineering.

Figure 1

Surgical repair of a 10 mm sciatic nerve gap using EFC and ENC. (A) 10 mm gaps were surgically created in the tibial nerves of female Fischer 344 rats approximately 5 mm from the site where they innervate the gastrocnemius muscles. Autografts, ENCs, and EFCs were coapted to the nerve stumps as diagrammed to guide nerve regeneration. (B) The grafts were coapted to the nerve stumps of the tibial nerve on either side of the 1 cm acute damage site. An ENC is shown. EFC: Engineered fibroblast conduit; ENC: engineered nerve conduit.

Figure 2

Distal motor nerve conduction velocity after a 12-week recovery. Values are expressed as the mean ± SEM. *P < 0.05, vs. control; #P < 0.05, vs. autograft (one-way analysis of variance followed by Tukey's post hoc analysis. In the control group (n = 5), the contralateral legs of the autograft animals were used. In the autograft group (n = 5), a 10 mm long sciatic nerve segment was transected, reversed, and then coapted to the two newly formed nerve stumps. In the ENC (n = 8) and EFC groups (n = 8), 10 mm long nerve segments were removed and the engineered conduits were coapted to the distal and proximal nerve stumps. ENC: Engineered nerve conduit; EFC: engineered fibroblast conduit.

Figure 3

Effects of nerve repair on medial gastrocnemius muscle mass (A), maximal isometric force production (B), and specific force production (C) following a 12 week recovery. Values are expressed as the mean ± SEM. *P < 0.05, vs. control; #P < 0.05, vs. autograft (one-way analysis of variance followed by Tukey's post hoc analysis). In the control group (n = 5 in A–C), the contralateral legs of the autograft animals were used. In the autograft (n = 5 in A–C) group, a 10 mm sciatic nerve segment was transected, reversed and then coapted to the two newly formed nerve stumps. In the ENC (n = 8 in A, n = 6 in B, C) and EFC (n = 10 in A, n = 8 in B, C) groups, 10 mm nerve segments were removed and the engineered conduits were coapted to the distal and proximal nerve stumps. ENC: Engineered nerve conduit; EFC: engineered fibroblast conduit.

Figure 4

Immunohistochemical staining of transverse sections through native and surgically repaired tibial nerves after 12 weeks of recovery with antibodies against SM312 (red), WGA (connective tissue, green), and DAPI (nuclei, blue). All sections were taken from the center of the 10 mm repair site. (A) Native tibial nerve from contralateral side which did not undergo axotomy, (B) autograft repaired tibial nerve, (C) ENC repaired tibial nerve, and (D) EFC repaired tibial nerve. Scale bars: 200 μm. ENC: Engineered nerve conduit; EFC: engineered fibroblast conduit; WGA: wheat germ agglutinin; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 5

Characterization of structure and nuclei centralization in medial gastrocnemius muscle. Hematoxylin and eosin staining of the medial gastrocnemius muscle following 12-week recovery in (A) native contralateral leg, (B) autograft, (C) ENC, and (D) EFC repair. Immunohistochemical laminin (green) and nuclei (4′,6-diamidino-2-phenylindole: blue) staining of the medial gastrocnemius muscle in (E) native contralateral leg, (F) autograft, (G) ENC, and (H) EFC repair. Scale bars: 200 μm. Medial gastrocnemius muscle recovery from denervation and atrophy was assessed by (I) muscle fiber cross-sectional area (CSA), and (J) percentage of centrally nucleated fibers. Data regarding laminin and hematoxylin and eosin stained muscle sections were obtained using Image J software. Values are presented as the mean ± SEM from n = 3 animals in each group. *P < 0.05, vs. control (one-way analysis of variance followed by Tukey's post hoc analysis). The contralateral legs of the autograft animals (n = 3) were used for controls. In the autograft group, a 10 mm long sciatic nerve segment was transected, reversed and then coapted to the two newly formed nerve stumps. In the ENC (n = 3) and EFC (n = 3) groups, 10 mm long nerve segments were removed and the engineered conduits were coapted to the distal and proximal nerve stumps. ENC: Engineered nerve conduit; EFC: engineered fibroblast conduit.