Comparison of systematically combined detergent and nuclease-based decellularization methods for acellular nerve graft: An ex vivo characterization and in vivo evaluation

Little consensus exists regarding which decellularization technique best removes the cellular components while maintaining structural integrity. We aimed to identify the most efficient and safest decellularization method by combining previously established chemical (detergent based) and biological (nuclease based) methods in a systematic manner. Sixty sciatic nerves were harvested from Sprague-Dawley rats and prepared in 120 nerve fragments with 1-cm length. Nerve fragments were randomly divided into six groups and decellularized with six different methods: A, nonionic detergent + amphoteric detergent; B, nonionic detergent + anionic detergent; C, anionic detergent + amphoteric detergent; D, nonionic detergent + nuclease; E, amphoteric detergent + nuclease; and F, anionic detergent + nuclease. The remaining cellular components were evaluated with H&E, DAPI, and S-100 immunohistochemical staining, and DNA content was measured in each sample. The remaining extracellular matrix (ECM) integrity was evaluated with H&E, Masson's trichrome, periodic acid-Schiff, Luxol fast blue, and laminin immunohistochemical staining, and collagen content was measured in each sample. The amphoteric detergent + nuclease method was the best protocol for both cell removal and ECM preservation. In the in vivo study, the nerve allograft that was decellularized with amphoteric detergent + nuclease showed an inferior recovery rate based on the tibialis anterior muscle weight to autograft, but considerable recovery was observed. In conclusion, among the possible systematic combinations of detergent- and nuclease-based methods, the combination of amphoteric detergent and nuclease is currently the most suitable for nerve decellularization in terms of adequate cell removal and sufficient preservation of the ECM.