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Optimization of Decellularized Nerve Allografts: Comparison of Rat and Human Nerves
Caroline A. Hundepool, MSc1; Dimitra Kotsougiani, MD1; Patricia F. Friedrich, AAS1; Steven E.R. Hovius, MD, PhD2; Allen T. Bishop, MD1; Alexander Y. Shin, MD1
1Department of Orthopedic Surgery, Microvascular Research Laboratory, Mayo Clinic, Rochester, MN; 2Department of Plastic, Reconstructive and Handsurgery, Erasmus MC, University Medical Center, Rotterdam, Netherlands

Introduction: In previous animal studies, commercially available processed nerve allografts have been inferior to autograft nerve for motor recovery. We hypothesize that different processing and storage techniques may better maintain nerve ultrastructure, lower immunogenicity, and minimize cellular debris, resulting in an ‘optimized’ processed nerve. We tested several modifications to previously-described processing and storage protocols in both fresh rat sciatic and human sensory and motor nerves. The protocols evaluated used chemical detergents and irradiation as previously described, with the addition of variable exposure to the highly potent enzyme Elastase.

Materials & Methods: For this experiment 50 rat nerves and 70 human sensory and motor nerves were processed, followed by storage at either 4 or -80 °C for the duration of two weeks. Both processed and fresh control nerves were analyzed using immunohistochemical stainings and confocal microscopy. We evaluated basal lamina (laminin ?-1), Schwann cells (S100 protein) and immunogenicity (major histocompatibility complex class I, MHC-I). Ultrastructural integrity and amount of cellular debris were analyzed on cross-sections of the nerves stained with toluidine blue and by electron microscopy.

Results: Nerve gross morphology and internal structure and basal lamina were 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. Increased concentrations of the enzyme enhanced the decellularization process. It also significantly diminished cellular debris. Significant differences were found between rat and human nerve outcomes using the different decellularization protocols. However, both showed the same negative effect of the freeze storage of the nerve after the decellularization process.

Conclusions: Elastase, when added to nerve processing reduced its immunogenicity, diminished cellular debris and better removed Schwann cells while maintaining ultrastructure. Storage at -80° heavily damaged nerve ultrastructure. A following in vivo study will be needed to demonstrate superior functional outcome with the optimized processed nerve allograft.

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