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Introduction: The use of nerve autografts remains as the gold standard method for critical nerve repair. However, due to their well-known disadvantage new nerve grafts are still needed. In this sense, decellularized peripheral nerve-derived extracellular matrices (ECMs) have emerged as a promising alternative, but these processes may affect the structure, composition and biomechanical properties of the ECMs generated. In this context, the present study aimed to investigate the impact of novel chemical-enzymatic decellularization method (CE) in the histology, ECM composition and biomechanical properties of rat-derived peripheral nerves. New decellularized nerves were compared to those obtained with the classical decellularization procedure described by Sondell (SD) and native nerve (NAT).
Methods: Rat sciatic nerves of 10-mm length were decellularized by the new CE method [distilled water, 3% Triton X-100, 1% SDS, 4% SDC and enzymatic mix (RNase and DNase)] and with SD method (1,2). Afterwards, the matrices obtained were characterized by histology, electron microscopy (scanning (SEM) and transmission (TEM)) and tensile test. Native nerves (NAT) were used as control group.
Results: Conventional histology conducted with HE, showed a better preservation of the different nerve stromal layers with the use of CE method respect to SD. Moreover, none of decellularized groups presented visible nuclear remnants with 4´,6-diamidino-2-phenylindole (DAPI) staining. MCOLL staining revealed an efficient myelin removal in both groups, being these results confirmed by SEM and TEM analyses. Furthermore, electron microscopy confirmed a complete decellularization process with both protocols but collagen organization was better preserved in CE as compared to SD. Finally, the tensile test showed no significant differences (p>0.05) in the strain at fracture and Young´s modulus between the matrices obtained by the new CE method and SD technique, being both closely comparable to NAT control group.
Discussion & conclusion: This ex vivo study demonstrated that the new CE method is a promising alternative to efficiently remove the cellular content from peripheral nerves. When compared with SD classical procedure, the new method showed better histological and ultrastructural features with a better preservation of the collagen network. From the biomechanical point of view, this study confirmed that this new method did not considerably affect the overall biomechanical properties of the matrices generated. Finally, further in vivo studies are needed to elucidate the regenerative potential of these new biomedical products.
Fundings: This study was supported by the Spanish “Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministerio de Economía y Competitividad (Instituto de Salud Carlos III)”, Grant FIS PI20-0318 co-financed by the “Fondo Europeo de Desarrollo Regional ERDF-FEDER European Union”; Grant No P18-RT-5059 by “Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020), Consejería de Transformación Económica, Industria, Conocimiento y Universidades, Junta de Andalucía, España”; and Grant A-CTS-498-UGR18 by “Programa Operativo FEDER Andalucía 2014–2020, Universidad de Granada, Junta de Andalucía, España”, co-funded by ERDF-FEDER, the European Union.
References:
- Sondell, M. et al., Brain Research. 795, 44–54, (1998).
- Philips, C. et al., Ann Biomed Eng. 46 (11):1921-1937, (2018).
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