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.

中文翻译：

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新型基于脱氧胆酸钠的周围神经化学脱细胞方法。

脱细胞的周围神经已被证明是对脊髓损伤后周围神经修复和临床前细胞载体的有效临床干预。但是，目前缺乏一种类似于先前建立的临床上已翻译的技术（Hudson方法）的方法，该方法可去除周围细胞并保持基质，该方法依赖于已停产的Triton X-200去污剂。因此，本研究的目的是基于目前可用的阴离子洗涤剂脱氧胆酸钠对一种优化的周围神经化学脱细胞方法。分离Sprague Dawley大鼠坐骨神经，在缓冲溶液中冷冻，然后依次在水，盐缓冲液，两性离子去污剂磺基甜菜碱-10和-16中洗涤，和不同浓度的脱氧胆酸钠（SDC）。为了优化SDC脱细胞后的DNA去除，对神经进行脱氧核糖核酸酶（DNase）孵育和盐缓冲液洗涤。免疫组织化学结果表明，在脱细胞过程中利用3％SDC可以保留细胞外基质（ECM）的成分和结构，同时与Hudson方法相比，可以显着更好地去除雪旺细胞，轴突和髓磷脂。与Hudson方法相比，在3％SDC脱细胞过程中添加3-h DNase孵育可显着去除细胞碎片。蛋白质组学分析表明，我们基于3％SDC + 3-h DNase与两性离子去污剂和盐缓冲液结合使用的新型脱细胞方法（使用3％SDC + 3-h DNase的新型脱细胞方法，与Hudson方法相比，两性离子去污剂和盐缓冲液[SDD方法]）产生了相似的蛋白质组学特征，并且细胞蛋白质的数量明显减少。最后，细胞毒性分析表明，SDD去细胞支架不含显着的细胞毒性残留，因为洗脱的介质在体外支持了代谢活跃的雪旺氏细胞。总体而言，这项研究表明，SDD脱细胞是利用目前可商购的化学试剂的一种新型替代方法。影响力陈述脱细胞神经是临床相关材料，可用于多种再生应用，例如周围神经和脊髓损伤修复。然而，停止使用已验证的化学脱细胞工艺中使用的关键洗涤剂需要优化等效或更好的方法。这项研究为该领域提供了一种新颖的化学脱细胞方法，以取代以前的验证标准。通过这种方法产生的支架提供了一种富含细胞外基质的材料，该材料可用于多种体外应用中，以了解细胞行为和体内应用，以促进神经损伤后的再生。