A novel technique for decellularization of allogenic nerves and in vivo study of their use for peripheral nerve reconstruction

Abstract

Autografts represent the gold standard for peripheral nerve reconstruction but their limited availability, the discrepancy of nerve caliber, and long surgical times are drawbacks. Allografts have therefore become a valid alternative option. In particular, acellular nerve allografts (ANAs) rather than fresh allografts do not need immunosuppression and appear to be safe and effective based on recent studies. An innovative method was conceived to obtain ANAs, so as to speed up nerve decellularization, without compromising nerve architecture, and without breaking the asepsis chain. Several detergent-based techniques, integrated with sonication and mechanical stirring, were tested in vitro on rabbit nerves, to identify, by microscopy and immunohistochemistry, the most effective protocol in terms of cell lysis and cellular debris clearance, while maintaining nerve architecture. Furthermore, a pilot in vivo study was performed: ANAs were implanted into tibial nerve defects of three rabbits, and autografts, representing the gold standard, in other three animals. Twelve weeks postoperatively, rabbits were clinically evaluated and euthanasized; grafts were harvested and microscopically and histomorphometrically analyzed. The method proved to be effective in vitro: the treatment removed axons, myelin and cells, without altering nerve architecture. The in vivo study did not reveal any adverse effect: animals maintained normal weight and function of posterior limb during the entire experimental time. A mild fibrotic reaction was observed, macrophages and leukocytes were rare or absent; ANAs regenerated fascicles and bundles were comparable versus autografts. Based on these results, this decellularization protocol is encouraging and deserves deeper investigations with further preclinical and clinical studies. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2228-2240, 2017.

Nerve Repair Using Decellularized Nerve Grafts in Rat Models. A Review of the Literature

“…Duration is important for the time needed to obtain the results, for the structural maintenance of the biological scaffolds, and for the cost of the procedure. In fact, the reduction of the entire process diminishes or eventually avoids the need for sterilization techniques, such as gamma irradiation, which has a controversial effect on decellularized matrix integrity ( Boriani et al, 2017 ). Another issue is the use of numerous chemical detergents with reduced incubation times during the decellularization protocol.…”

Exosomes and Their MicroRNA Cargo: New Players in Peripheral Nerve Regeneration

“…14,15 While tissue engineering techniques to produce synthetic conduits have been shown to be beneficial for PNI, outcomes are still far from optimized. [16][17][18][19][20] Recently, exosomes, nanosized extracellular vesicles released by nearly all cell types, have been shown to improve the nerve regeneration process. [21][22][23] To date, a sizeable body of literature have demonstrated that exosomes play a critical role in intracellular communication, transferring information via their cargo, which includes DNAs, messenger RNAs, microRNAs (miRNAs), and proteins.…”

“…14,15 While tissue engineering techniques to produce synthetic conduits have been shown to be beneficial for PNI, outcomes are still far from optimized. 16-20…”

Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure

“…Nevertheless, nerve autografting requires a separate surgery for tissue harvest and results in loss of function at the harvest site. To eliminate these drawbacks, several protocols have been developed to render allogeneic nerve tissue non-immunogenic through the process of decellularization [5–9].…”