Peripheral Nerve Regeneration Using a Three Dimensionally Cultured Schwann Cell Conduit

Abstract

The use of artificial nerve conduit containing viable Schwann cells is one of the most promising strategies to repair peripheral nerve injury. To fabricate an effective nerve conduit whose microstructure and internal environment are more favorable in nerve regeneration than those currently existing, a new three-dimensional (3D) Schwann cell culture technique using Matrigel and dorsal root ganglion (DRG) was developed. Nerve conduit of 3D arranged Schwann cells was fabricated using direct seeding of freshly harvested DRG into Matrigel-filled silicone tubes (inner diameter 1.98 mm, 14 mm length) and in vitro rafting culture for 2 weeks. The nerve regeneration efficacy of 3D cultured Schwann cell conduit (3D conduit group, n = 6) was assessed using an Sprague-Dawley rat sciatic nerve defect of 10 mm and compared with that of a silicone conduit filled with Matrigel and Schwann cells prepared with the conventional plain culture method (two-dimensional [2D] conduit group, n = 6). After 12 weeks, sciatic function was evaluated with sciatic function index (SFI) and gait analysis, and histomorphology of nerve conduit and the innervated tissues of sciatic nerve were examined using image analyzer and electromicroscopic methods. The SFI and ankle stance angle in the functional evaluation were -60.1 +/- 13.9, 37.9 degrees +/- 5.4 degrees in the 3D conduit group (n = 5) and -87.0 +/- 12.9, 32.2 degrees +/- 4.8 degrees in the 2D conduit group (n = 4). The myelinated axon was 44.91% +/- 0.13% in the 3D conduit group and 13.05% +/- 1.95% in the 2D conduit group. In the transmission electron microscope study, the 3D conduit group showed more abundant myelinated nerve fibers with well-organized and thickened extracellular collagen than the 2D conduit group, and the gastrocnemius muscle and biceps femoris tendon in the 3D conduit group were less atrophied and showed decreased fibrosis with less fatty infiltration than the 2D conduit group. A new 3D Schwann cell culture technique was established, and nerve conduit fabricated using this technique showed much improved nerve regeneration capacity than the silicone tube filled with Matrigel and Schwann cells prepared from the conventional plain culture method.