Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol,Cell and Tissue Research

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Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol
Cell and Tissue Research ( IF 3.2 ) Pub Date : 2021-01-20 , DOI: 10.1007/s00441-020-03317-3
N Nieto-Nicolau _{1,

2} , P López-Chicón _{1,

2} , O Fariñas _{1,

2} , S Bolívar ₃ , E Udina ₃ , X Navarro ₃ , R P Casaroli-Marano _{1,

2,

4} , A Vilarrodona _{1,

2}

Affiliation

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. Effective 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.

中文翻译：

用一种新的协议对用于再生医学的人神经基质进行有效的去细胞化

外周神经损伤是成人永久性残疾的常见原因。大神经病变的修复涉及使用自体移植物，但它们有几个固有的局限性。克服这些限制，脱细胞神经基质的使用已成为组织再生医学中一种有前途的治疗方法。在这里，我们使用非离子、两性离子和酶促孵育，通过一种新的脱细胞方法生成更长的人类脱细胞神经节段。脱细胞的效率通过 DNA 定量和细胞残留分析（髓鞘、S100、神经丝）来测量。细胞外基质（胶原蛋白、层粘连蛋白和糖胺聚糖）保存的评价通过酶联免疫吸附试验（ELISA）或生化方法进行，以及组织学和免疫荧光分析。此外，还测试了生物力学特性和细胞相容性。结果表明，使用该协议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，虽然可以观察到一些神经丝残余物，但髓鞘、S100 和 MHCII 蛋白均不存在。此外，细胞外基质蛋白以及生物力学特性得到很好的维持，脱细胞神经基质没有产生细胞毒性。这些结果表明，我们的方法对于脱细胞人神经移植物的产生是有效的。更长的脱细胞神经节段的产生将允许在临床试验和更大的动物模型中了解神经损伤后的再生神经生物学。使用新方案对用于再生医学的人类神经基质进行有效脱细胞。两性离子、非离子去污剂、高渗溶液和核酸酶处理的组合去除细胞残留物，保持胶原蛋白、层粘连蛋白和生物力学，而不会产生细胞毒性可浸出物。

更新日期：2021-01-20

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