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Previously, we developed a Bio3D conduit fabricated from human fibroblasts and reported a significantly better outcome compared with artificial nerve conduit in the treatment of rat sciatic nerve defect. The purpose of this study is to investigate the long-term safety and nerve regeneration of Bio3D conduit compared with treatments using artificial nerve conduit and autologous nerve transplantation.We used 15 immunodeficient rats and randomly divided them into three groups treated with Bio3D ( n = 5) conduit, silicon tube ( n = 5), and autologous nerve transplantation ( n = 5). We developed Bio3D conduits composed of human fibroblasts and bridged the 5 mm nerve gap created in the rat sciatic nerve. The same procedures were performed to bridge the 5 mm gap with a silicon tube. In the autologous nerve group, we removed the 5 mm sciatic nerve segment and transplanted it. We evaluated the nerve regeneration 24 weeks after surgery.Toe dragging was significantly better in the Bio3D group (0.20 ± 0.28) than in the silicon group (0.6 ± 0.24). The wet muscle weight ratios of the tibial anterior muscle of the Bio3D group (79.85% ± 5.47%) and the autologous nerve group (81.74% ± 2.83%) were significantly higher than that of the silicon group (66.99% ± 3.51%). The number of myelinated axons and mean myelinated axon diameter was significantly higher in the Bio3D group (14708 ± 302 and 5.52 ± 0.44 μm) and the autologous nerve group (14927 ± 5089 and 6.04 ± 0.85 μm) than the silicon group (7429 ± 1465 and 4.36 ± 0.21 μm). No tumors were observed in any of the rats in the Bio3D group at 24 weeks after surgery.The Bio3D group showed significantly better nerve regeneration and there was no significant difference between the Bio3D group and the nerve autograft group in all endpoints.

We previously reported that a nerve conduit created from fibroblasts promotes nerve regeneration in a rat sciatic nerve model. This study aims to determine whether a nerve conduit created from bone marrow stromal cells (BMSCs) can promote nerve regeneration. Primary BMSCs were isolated from femur bone marrow of two Lewis rats, and cells at passages 4-7 were used. We created seven Bio 3D nerve conduits from BMSCs using a Bio-3D Printer. The conduits were transplanted to other Lewis rats to bridge 5-mm right sciatic nerve gaps (Bio 3D group, n = 7). We created two control groups: a silicone group (S group, n = 5) in which the same nerve gap was bridged with a silicone tube, and a silicone cell group (SC group, n = 5) in which the gap was bridged with a BMSC injection. Twelve weeks after transplantation, nerve regeneration was evaluated functionally and morphologically. In addition, PKH26-labeled BMSCs were used to fabricate a Bio 3D conduit that was transplanted for cell trafficking analysis. Electrophysiological study, kinematic analysis, wet muscle weight, and morphological parameters showed significantly better nerve regeneration in the Bio 3D group than in the S group or SC group. In immunohistochemical studies, sections from the Bio 3D group contained abundant S-100-positive cells. In cell trafficking analysis, PKH26-positive cells stained positive for the Schwann cell markers S-100, p75NTR, and GFAP. Bio 3D nerve conduits created from BMSCs can promote peripheral nerve regeneration in a rat sciatic nerve model through BMSC differentiation into Schwann-like cells.

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.

Cells, scaffolds, growth factors, and vascularity are essential for nerve regeneration. Previously, we reported that the insertion of a vascular bundle and the implantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a nerve conduit promoted peripheral nerve regeneration. In this study, the efficacy of nerve conduits containing a vascular bundle, BM-MSCs, and thermally decellularized allogenic nerve matrix (DANM) was investigated using a rat sciatic nerve model with a 20-mm defect. Lewis rats were used as the sciatic nerve model and for the preparation of BM-MSCs, and Dark Agouti rats were used for the preparation of the DANM. The revascularization and the immunogenicity of the DANM were investigated histologically. The regeneration of nerves through nerve conduits containing vessels, BM-MSCs, and DANM (VBD group) was evaluated based on electrophysiological, morphometric, and reinnervated muscle weight measurements and compared with that of vessel-containing conduits that were implanted with BM-MSCs (VB group). The DANM that was implanted into vessel-containing tubes (VCTs) was revascularized by neovascular vessels that originated from the inserted vascular bundle 5-7 days after surgery. The number of CD8+ cells found in the DANM in the VCT was significantly smaller than that detected in the untreated allogenic nerve segment. The regenerated nerve in the VBD group was significantly superior to that in the VB group with regard to the amplitude of the compound muscle action potential detected in the pedal adductor muscle; the number, diameter, and myelin thickness of the myelinated axons; and the tibialis anterior muscle weight at 12 and 24 weeks. The additional implantation of the DANM into the BM-MSC-implanted VCT optimized the axonal regeneration through the conduit. Nerve conduits constructed with vascularity, cells, and scaffolds could be an effective strategy for the treatment of peripheral nerve injuries with significant segmental defects.

Bone marrow-derived cells (BMCs) are multipotent cells that have the potential to differentiate into bone, cartilage, fat, muscle, or neuronal lineages such as neurons and glial cells. A silicone tube model containing reverse-pedicled sural vessels was created in the sciatic nerves of Lewis rats. About 1 x 10(7) BMCs, removed from the bone marrow of synergetic rat femurs and cultured in vitro, were transplanted into the 15-mm-long chambers of the silicone tubes. Nerve regeneration in vessel-containing tubes that had received BMCs was significantly greater at 12 and 24 weeks after surgery than in tubes that did not receive cells. Transplantation of fibroblasts instead of BMCs into the vessel-containing tube resulted in reduced axonal regeneration, which was inferior to regeneration in the vessel-containing tube that did not receive cells. Polymerase chain reaction (PCR) studies revealed that in vessel-containing tubes containing transplanted BMCs, about 29% of cells in the regenerated nerve originated from BMCs. Cells identified by in situ hybridization and PKH26 prelabeling as being of BMC origin stained positively for S100 and GFAP. Transplanted BMCs differentiated into cells with phenotypes similar to those of Schwann cells under the influence of neurochemical factors and survived by obtaining nutrients from vessels that had been preinserted into the tube. They thus functioned similarly to Schwann cells, promoting nerve regeneration.

We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze-thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of beta-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.