Preparation of human decellularized peripheral nerve allograft using amphoteric detergent and nuclease.
Preparation of human decellularized peripheral nerve allograft using amphoteric detergent and nuclease.
复制标题
两性去污剂和核酸酶联合应用制备人脱细胞周围神经同种异体移植物。
DOI：
10.4103/1673-5374.306091
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发表时间：
2021-09
影响因子：
6.1
通讯作者：
Kim JK
中科院分区：
文献类型：
--
作者：
Bae JY;Park SY;Shin YH;Choi SW;Kim JK
动物研究表明，两性去污剂和核酸酶（DNA酶I和核糖核酸酶A）是最可靠的外周神经脱细胞方法。然而，最佳的化学试剂组合用于人同种异体神经脱细胞还需要进一步的研究。为了找到去除神经组织中免疫原性细胞成分并保留基底层和细胞外基质的最佳方案，以及最佳方案是否可以应用于较大直径的人类周围神经，在本研究中，我们用两种不同的方法对尸体的正中神经和腓肠神经进行脱细胞：非离子和阴离子去污剂（Triton X-100和脱氧胆酸钠）和两性去污剂和核酸酶（3-[（3-胆酰胺丙基）二甲基铵基]-1-丙磺酸盐（CHAPS）、脱氧核糖核酸酶I和核糖核酸酶A）。所有的细胞成分被成功地从正中神经和腓肠神经的两性去污剂和核酸酶。非离子型和阴离子型去污剂不能完全去除正中神经的细胞成分。用两性去污剂和核酸酶处理的正中神经和腓肠神经都保持了完全完整的细胞外基质。用非离子和阴离子去污剂处理降低正中神经和腓肠神经中的胶原含量，而两性去污剂和核酸酶处理不降低胶原含量。此外，接触细胞毒性试验表明，两性去污剂和核酸酶脱细胞的神经是生物相容的。强度失效测试表明，用两性去污剂和核酸酶脱细胞的神经的生物力学性质与新鲜对照的那些相当。与使用非离子和阴离子去污剂的去细胞化相比，使用两性去污剂和核酸酶的去细胞化更好地去除细胞组分并更好地保护细胞外基质，即使在大直径的人外周神经中也是如此。在韩国，尸体研究在法律上尚未接受机构审查委员会的审查。
Animal studies have shown that amphoteric detergent and nuclease (DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents (Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review.