Optimizing decellularization techniques to create a new nerve allograft: an in vitro study using rodent nerve segments

Abstract

OBJECTIVE Commercially available processed nerve allografts have been shown to be inferior to autografts in previous animal studies. The authors hypothesized that combining different processing and storage techniques will result in improved nerve ultrastructure preservation, lower immunogenicity, and minimized cellular debris. Different processing protocols were evaluated using chemical detergents, enzymes, and irradiation, with the addition the of enzyme elastase, were used. Additionally, the difference between cold and frozen storage was investigated. The goal of this study was to create an optimized nerve allograft. METHODS Fifty rat nerves were decellularized with modifications of previous protocols and the addition of elastase. Subsequently, the nerve segments were stored at either 4°C or -80°C. Both processed and fresh control nerves were analyzed with confocal microscopy using immunohistochemical staining on the basal lamina (laminin γ-1), Schwann cells (S100 protein), and immunogenicity using major histocompatibility complex-I (MHCI) staining. Morphology of the ultrastructure and amount of cellular debris were analyzed on cross-sections of the nerves stained with toluidine blue and H & E, and by using electron microscopy. RESULTS 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 and amount of Schwann cells, while maintaining good structural properties. CONCLUSIONS Reduced immunogenicity, diminished cellular debris, and the elimination of Schwann cells was observed when elastase was added to the nerve processing while maintaining ultrastructure. Storage at -80°C after the decellularization process heavily damaged the nerve ultrastructure as compared with cold storage. Further in vivo studies are needed to prove the nerve regenerative capacity of these optimized allografts.

Keywords: BL = basal lamina; MHCI = major histocompatibility complex–I; SB-16 = sulfobetaine-16; decellularization; elastase; nerve allograft; processing.