Motor Nerve Regeneration Using an Optimized Nerve Allograft in a Rat Model - An In vitro and In vivo Analysis: N/A - Not a clinical study

Abstract

In previous animal studies commercially available processed nerve allografts have been inferior to autograft nerve for motor recovery. The goal of this study is to create an optimized nerve allograft (in vitro) and subsequently challenge it to the nerve autograft regarding motor nerve regeneration (in vivo). For the in vitro experiment 50 rat nerves were processed. Based on previous research, standard decellularization protocols were used with different modifications and the addition of an highly potent enzymatic step Elastase. Subsequently, the nerve segments were stored at either 4 or -80°C for the duration of two weeks. Both processed and fresh control nerves were analyzed with confocal microscopy using immunohistochemical stainings on the basal lamina (laminin ?-1), Schwann cells (S100 protein) and immunogenicity (major histocompatibility complex class I). Morphology of the ultrastructure and amount of cellular debris was analyzed on cross sections of the nerves stained with toluidine blue and analyzed under electronmicroscopy. The superior method of this in vitro project was used for implementation in vivo to test the motor functional outcome of the nerve allograft in a rat. In the in vivo project, 60 rats sustained an 1 cm sciatic nerve reconstruction with either autograft (I), non-frozen allograft (II), or frozen allograft (III). Twelve and 16 weeks post-operatively motor functional outcome was determined with ankle angle, electrophysiology, isometric tetanic force, muscle mass and histomorphometry. Nerve ultrastructure was preserved with all decellularization protocols. Storage at -80°C severely altered nerve ultrastructure after any decellularization method. Elastase was found to significantly reduce the immunogenicity (MHC-I) and the amount of Schwann cells (S100), while maintaining good structural properties. When tested in vivo, significant differences were found between the three groups with regard to motor function at both 12 and 16 weeks. Cold storage of the processed allograft shows better results than the frozen stored allograft and reached the level of the nerve autograft. •Elastase, when added to nerve processing, reduced immunogenicity, diminished cellular debris and removed Schwann cells better while maintaining ultrastructure.•Storage at -80°C after the decellularization process heavily damaged nerve ultrastructure compared to cold storage.•The in vivo study, the optimized processed nerve allograft showed comparable to the golden standard, the nerve autograft, at both 12 and 16 weeks postoperatively.