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Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol

机译：一种新型议定书的再生医学的人体神经基质的有效脱细胞化

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Injuries to the peripheral nerves represent a frequent cause of permanent disability in adults. The repair of large nerve lesions involves the use of autografts, but they have several inherent limitations. Overcoming these limitations, the use of decellularized nerve matrix has emerged as a promising treatment in tissue regenerative medicine. Here, we generate longer human decellularized nerve segments with a novel decellularization method, using nonionic, zwitterionic, and enzymatic incubations. Efficiency of decellularization was measured by DNA quantification and cell remnant analysis (myelin, S100, neurofilament). The evaluation of the extracellular matrix (collagen, laminin, and glycosaminoglycans) preservation was carried out by enzyme-linked immunosorbent assay (ELISA) or biochemical methods, along with histological and immunofluorescence analysis. Moreover, biomechanical properties and cytocompatibility were tested. Results showed that the decellularized nerves generated with this protocol have a concentration of DNA below the threshold of 50 ng/mg of dry tissue. Furthermore, myelin, S100, and MHCII proteins were absent, although some neurofilament remnants could be observed. Moreover, extracellular matrix proteins were well maintained, as well as the biomechanical properties, and the decellularized nerve matrix did not generate cytotoxicity. These results show that our method is effective for the generation of decellularized human nerve grafts. The generation of longer decellularized nerve segments would allow the understanding of the regenerative neurobiology after nerve injuries in both clinical assays and bigger animal models.Graphical AbstractEffective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Combination of zwitterionic, non-ionic detergents, hyperosmotic solution and nuclease enzyme treatment remove cell remnants, maintain collagen, laminin and biomechanics without generating cytotoxic leachables.
机译：周围神经损伤是成年人永久性残疾的常见原因。大型神经损伤的修复需要使用自体移植物，但它们有几个固有的局限性。为了克服这些局限性，脱细胞神经基质的使用已经成为组织再生医学中一种有前途的治疗方法。在这里，我们使用一种新的去细胞方法，使用非离子、两性离子和酶培养，生成更长的人类去细胞神经段。通过DNA定量和细胞残留分析（髓鞘、S100、神经丝）测定脱细胞效率。通过酶联免疫吸附试验（ELISA）或生化方法，以及组织学和免疫荧光分析，对细胞外基质（胶原蛋白、层粘连蛋白和糖胺聚糖）的保存进行评估。此外，还对其生物力学性能和细胞相容性进行了测试。结果表明，使用该方案生成的脱细胞神经的DNA浓度低于50 ng/mg干组织的阈值。此外，髓鞘、S100和MHCII蛋白缺失，尽管可以观察到一些神经丝残余。此外，细胞外基质蛋白以及生物力学特性得到了很好的维护，脱细胞后的神经基质没有产生细胞毒性。这些结果表明，我们的方法对脱细胞人神经移植物的生成是有效的。在临床试验和更大的动物模型中，更长的脱细胞神经段的产生将有助于了解神经损伤后的再生神经生物学。图形摘要：采用一种新方案对再生医学中的人类神经基质进行有效脱细胞。两性离子、非离子洗涤剂、高渗溶液和核酸酶处理的组合去除细胞残余，维持胶原蛋白、层粘连蛋白和生物力学，而不产生细胞毒性浸出物。

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