Byline: Joo-Yul. Bae, Suk. Park, Young. Shin, Shin. Choi, Jae. Kim
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.
Damage to the peripheral nerve is a common injury pattern and is reported in up to 3% of all trauma patients (Rasulic et al., 2015; Zhu et al., 2017). It is a significant clinical challenge to overcome for microsurgeons (Sameem et al., 2011). If possible, primary tension-free end-to-end neurorrhaphy is the treatment of choice (Jesuraj et al., 2014). However, it is often difficult to repair the nerve primarily due to nerve defects or resection of the lesion. In this situation, autogenous nerve grafts are used alternatively (Beris et al., 2019). However, there is a limitation to the use of autogenous nerve grafts due to donor site morbidity, lack of sufficient tissue, or size mismatch between donor and recipient (Beris et al., 2019).
Recently, acellular nerve grafts have been studied to overcome these drawbacks (Beris et al., 2019; Rbia et al., 2019). To enable allogenic nerve grafting, decellularization methods should eliminate graft antigenicity by complete removal of cellular components and maintain the integrity of the basal lamina and extracellular matrix (ECM). Currently available decellularization techniques can be classified into physical methods (Zalewski and Gulati, 1982; Evans et al., 1998), chemical detergents (Sondell et al., 1998; Hudson et al., 2004), biological agents (Sridharan et al., 2015), and miscellaneous methods (Szynkaruk et al., 2013; Ishida et al., 2014)....