Abstract
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 Abstract
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.
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Acknowledgments
We would like to thank the donors and their families which with their altruistic act of donation have allowed to perform this study. We would also like to thank transplant coordinators, donor center, and recovery teams for their efforts which make it possible to obtain the samples used in this study, and T. Van Eeckhout for his assistance in the final editing and preparation of this manuscript.
Funding
This work has been partially supported by grant from TERCEL (RD16/0011/0035) and CIBERNED (CB06/05/1105) from the Instituto de Salud Carlos III (Madrid, Spain), and internal competitive grant from BST (Ref I.2017.056).
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N.N.N. designed, performed experiments, analyzed data, wrote the manuscript, and final approval of the manuscript for publication. L.C.P., F.O., C.M.R., and V.A. critically revised the manuscript. B.S. performed experiments and analyzed data. U.E. and N.X. analyzed data and critically revised the manuscript.
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This study followed the ethical precepts of the Declaration of Helsinki (Fortaleza, Brazil, October 2013) and was approved by our local ethics committee (CEIC, Hospital Clinic de Barcelona; Ref 7365-12). Human samples were obtained, processed, and analyzed according to current guidance in relation to the collection and preservation of human tissues for clinical use (EEC regulations 2004/23/CE and 2006/17/CE) and in accordance with the protocol and legal requirements for the use of biological samples and biomedical research in Spain (Law 14/2007 and RD 1716/2011). In addition, the acquisition, processing, and preservation of the tissues of this study are in accordance with the Spanish Laws that regulates the quality and safety standards for donation, procurement, evaluation, processing, preservation, storage, and distribution of human cells and tissues (RD 9/2014).
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All the information provided before donation, together with informed consent, stressed that the samples obtained were to be used for clinical application and/or applied research. The use, protection, communication, and transfer of personal data complied with local regulations (Law 15/1999).
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Nieto-Nicolau, N., López-Chicón, P., Fariñas, O. et al. Effective decellularization of human nerve matrix for regenerative medicine with a novel protocol. Cell Tissue Res 384, 167–177 (2021). https://doi.org/10.1007/s00441-020-03317-3
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DOI: https://doi.org/10.1007/s00441-020-03317-3