Background: In the realm of nerve injury repair, acellularized nerve grafts (ANGs) are gaining recognition as a promising tissue engineering approach, offering better regeneration outcomes compared to traditional nerve conduits. This study, conducted on rat and chicken sciatic nerves (R-ANGs and C-ANGs, respectively), seeks to explore an optimized nerve decellularization technique that achieves effective decellularization while preserving the extracellular matrix (ECM) integrity. Methods: The Sondell decellularization (SD) protocol was evaluated against an optimized Sondell decellularization (OSD) method improved by DNase treatment and extra-washing with ultrapure water, and then MTT, DNA quantification, mechanical tensile, and histological evaluations were performed. Results and conclusion: Results showed that both R-ANGs and C-ANGs prepared by the OSD method had better biocompatibility, measured by MTT, than those prepared by the SD (p < 0.001). DNA quantification of samples obtained from the OSD method showed an improved elimination of residual DNA fragments (p < 0.0001). Mechanical tensile test also revealed a higher Young’s modulus of the OSD grafts (p < 0.05). Histological studies, including hematoxylin and eosin, Masson’s trichrome, Luxol fast blue, DAPI, and scanning electron microscopy (FESEM), confirmed that the OSD was superior to the SD in preserving ECM while equally removing cell nuclei and myelinated fibers. The OSD protocol yielded promising results for more reliable in vivo trials, as in vitro tests revealed better preservation of ECM components compared to the SD method. Notably, the OSD provided higher biocompatibility and improved viscoelastic integrity of the R-ANGs and C-ANGs.

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