The Efficacy of a Scaffold-free Bio 3D Conduit Developed from Autologous Dermal Fibroblasts on Peripheral Nerve Regeneration in a Canine Ulnar Nerve Injury Model: A Preclinical Proof-of-Concept Study

Abstract

Autologous nerve grafting is widely accepted as the gold standard treatment for segmental nerve defects. To overcome the inevitable disadvantages of the original method, alternative methods such as the tubulization technique have been developed. Several studies have investigated the characteristics of an ideal nerve conduit in terms of supportive cells, scaffolds, growth factors, and vascularity. Previously, we confirmed that biological scaffold-free conduits fabricated from human dermal fibroblasts promote nerve regeneration in a rat sciatic nerve injury model. The purpose of this study is to evaluate the feasibility of biological scaffold-free conduits composed of autologous dermal fibroblasts using a large-animal model. Six male beagle dogs were used in this study. Eight weeks before surgery, dermal fibroblasts were harvested from their groin skin and grown in culture. Bio 3D conduits were assembled from proliferating dermal fibroblasts using a Bio 3D printer. The ulnar nerve in each dog's forelimb was exposed under general anesthesia and sharply cut to create a 5 mm interstump gap, which was bridged by the prepared 8 mm Bio 3D conduit. Ten weeks after surgery, nerve regeneration was investigated. Electrophysiological studies detected compound muscle action potentials (CMAPs) of the hypothenar muscles and motor nerve conduction velocity (MNCV) in all animals. Macroscopic observation showed regenerated ulnar nerves. Low-level hypothenar muscle atrophy was confirmed. Immunohistochemical, histological, and morphometric studies confirmed the existence of many myelinated axons through the Bio 3D conduit. No severe adverse event was reported. Hypothenar muscles were re-innervated by regenerated nerve fibers through the Bio 3D conduit. The scaffold-free Bio 3D conduit fabricated from autologous dermal fibroblasts is effective for nerve regeneration in a canine ulnar nerve injury model. This technology was feasible as a treatment for peripheral nerve injury and segmental nerve defects in a preclinical setting.

Keywords: Bio 3D conduit; nerve regeneration; peripheral nerve injury; preclinical study; proof of concept; scaffold-free.

Figure 1.

Preparation of the Bio 3D conduit in vitro. Multicellular spheroids were skewered into the circular needle-array according to the pre-designed pattern. (A) viewed from the side, (B) viewed from above. Scale bar: 2 mm.

Figure 2.

Intraoperative pictures in animal experiments. (A) The obtained Bio 3D conduit after perfusion cultivation just prior to transplantation. (B) An 8-mm Bio 3D conduit was interposed into the nerve defect. The proximal and distal nerve stumps were pulled 1.5 mm into the conduit to create a 5-mm interstump gap. (C) Regenerated ulnar nerve 10 weeks after surgery. The diameter of the Bio 3D conduit remained larger than those of the proximal and distal stumps. (D) In the Bio 3D group, the mid portion of the conduit in the affected ulnar nerve and the distal portion, which is 5 mm distal to the distal sutures, were harvested. In the Intact group, the mid and distal portions of the unaffected ulnar nerve in the same positions were harvested.

Figure 3.

Transverse section of the mid portion. (A) White dotted line represents the cross-sectional area of the intact ulnar nerve. (B) White dotted line represents the boundary between the regenerated nerve and the inner wall of the Bio 3D conduit. Scale bars: 2 mm.

Figure 4.

Wet weight of the hypothenar muscle. Little muscle atrophy was observed in Bio 3D group. Scale bar: 10 mm.

Figure 5.

Immunohistochemistry of the mid portion of the Bio 3D conduit. NF-200 and S-100 expression indicate neurofilament and Schwann cells of regenerated nerve at transverse section. Scale bars: 50 μm.

Figure 6.

Histological and morphometric evaluation of the mid portions of the intact ulnar nerve and the Bio 3D conduit. (A–D) Semi-thin transverse section (toluidine blue staining) under light microscopy. Scale bars: 1000 μm (A, B) and 50 μm (C, D). (E, F) Ultra-thin transverse section under transmission electron microscopy. Scale bars: 2 μm. Although the myelinated axon diameter in the Bio 3D group is almost the same as that in the Intact group, the myelin thickness in the Bio 3D group is thinner.

Figure 7.

Histological and morphometric evaluation of the distal portions of the intact ulnar nerve and the Bio 3D conduit. (A–D) Semi-thin transverse section (toluidine blue staining) under light microscopy. Scale bars: 500 μm (A, B) and 50 μm (C, D). (E, F) Ultra-thin transverse section under transmission electron microscopy. Scale bars: 2 μm. Although elongated myelinated axons with proper myelin sheaths were confirmed in the Bio 3D group, their morphology was slightly less mature compared with that seen in the distal portion of the Intact group and in the mid portion of the Bio 3D group.