Optimized Acellular Nerve Graft Is Immunologically Tolerated and Supports Regeneration

Abstract

The long-term goal of our research is to engineer an acellular nerve graft for clinical nerve repair and for use as a model system with which to study nerve-extracellular matrix interactions during nerve regeneration. To develop this model acellular nerve graft we (1) examined the effects of detergents on peripheral nerve tissue, and (2) used that knowledge to create a nerve graft devoid of cells with a well-preserved extracellular matrix. Using histochemistry and Western analysis, the impact of each detergent on cellular and extracellular tissue components was determined. An optimized protocol was created with the detergents Triton X-200, sulfobetaine-16, and sulfobetaine-10. This study represents the most comprehensive examination to date of the effects of detergents on peripheral nerve tissue morphology and protein composition. Also presented is an improved chemical decellularization protocol that preserves the internal structure of native nerve more than the predominant current protocol.

Decellularisation and histological characterisation of porcine peripheral nerves

“…The most commonly utilised methods for decellularisation of nerve tissue include cold preservation; (Evans et al, 1998), thermal (Ide et al, 1998; Gulati, 1988) radiation (Hiles, 1972) and detergent based chemical treatments (Hudson et al, 2004; Sondell et al, 1998). …”

“…There have however been reports that the freeze‐thawing process fails to remove the cell debris from the tissue, in addition to fracturing the basal laminae that surround the axons (Hudson et al, 2004). In vivo studies have demonstrated cellular remnants left behind from this process have led to cellular invasion in the form of Schwann cells and macrophages invading the basal lamina to clear away the debris.…”

“…Studies have shown that this process does not damage the tissue morphology (Marmor, 1964). Nevertheless similar to thermal decellularisation, cellular debris still remains inside the basal laminae causing cellular invasion post implantation (Hudson et al, 2004). …”

“…It has been hypothesised that the inadequate preservation of the ECM structure in these detergent‐treated decellularised graft is a contributing factor to this failure (Hudson et al, 2004). Studies using milder detergents have demonstrated superior preservation of native ECM and equivalent levels of decellularisation compared with previous chemical processing techniques (Hudson et al, 2004). The protocol used in this present study uses a ten‐fold lower concentration of SDS (0.1% w/v).…”

“…A comparative study between an acellular nerve grafts produced by the Hudson detergent based method (sulfobetaine‐10; SB‐10) and Triton X‐200/sulfobetaine‐16 (SB‐16) and NeuraGen ® (an FDA‐approved and commercially available type I collagen NGC) saw the acellular graft outperforming the synthetic NGC (Hudson et al, 2004). The study proposed that the presence of intact basal lamina within the acellular nerve graft contributes to the enhanced regenerating capacity to support axonal growth (Whitlock et al, 2009).…”