Novel Sodium Deoxycholate-Based Chemical Decellularization Method for Peripheral Nerve

Abstract

Decellularized peripheral nerve has been proven to be an effective clinical intervention for peripheral nerve repair and a preclinical cell carrier after spinal cord injury. However, there are currently a lack of decellularization methods for peripheral nerve that remove cells and maintain matrix similar to the previously established, clinically translated technique (the Hudson method) that relies on the discontinued Triton X-200 detergent. Therefore, the aim of this study was to optimize a novel chemical decellularization method for peripheral nerves based on the currently available anionic detergent sodium deoxycholate. Sprague Dawley rat sciatic nerves were isolated, frozen in buffered solution, and then subject to sequential washes in water, salt buffer, zwitterionic detergents sulfobetaines -10 and -16, and varying concentrations of sodium deoxycholate (SDC). To optimize DNA removal after SDC decellularization, nerves were subjected to deoxyribonuclease (DNase) incubation and salt buffer washes. Immunohistochemical results demonstrated that utilization of 3% SDC in the decellularization process preserved extracellular matrix (ECM) components and structure while facilitating significantly better removal of Schwann cells, axons, and myelin compared with the Hudson method. The addition of a 3-h DNase incubation to the 3% SDC decellularization process significantly removed cellular debris compared with the Hudson method. Proteomic analysis demonstrated that our novel decellularization method based on 3% SDC +3-h DNase used in conjunction with zwitterionic detergents, and salt buffers (new decellularization method using 3% SDC + 3-h DNase, zwitterionic detergents, and salt buffers [SDD method]) produced a similar proteomic profile compared with the Hudson method and had significantly fewer counts of cellular proteins. Finally, cytotoxicity analysis demonstrated that the SDD decellularized scaffolds do not contain significant cytotoxic residuals as eluted media supported metabolically active Schwann cells in vitro. Overall, this study demonstrates that SDD decellularization represents a novel alternative utilizing currently commercially available chemical reagents.

Impact Statement

Decellularized nerves are clinically relevant materials that can be used for a variety of regenerative applications such as peripheral nerve and spinal cord injury repair. However, discontinuation of key detergents used in a proven chemical decellularization process necessitates the optimization of an equivalent or better method. This research presents the field with a novel chemical decellularization method to replace the previous validated standard. Scaffolds generated from this method provide an extracellular matrix-rich material that can be used in a variety of in vitro applications to understand cellular behavior and in vivo applications to facilitate regeneration after neural injury.