Adjuvant neurotrophic factors in peripheral nerve repair with chondroitin sulfate proteoglycan-reduced acellular nerve allografts

Abstract

Background Acellular nerve allografts are now standard tools in peripheral nerve repair due to decreased donor site morbidity and operative time savings. Preparation of nerve allografts involves several steps of decellularization and modification of extracellular matrix to remove chondroitin sulfate proteoglycans (CSPGs), which have been shown to inhibit neurite outgrowth through a poorly understood mechanism involving RhoA and ECM-integrin interactions. Chondroitinase ABC (ChABC) is an enzyme that degrades CSPG molecules and has been shown to promote neurite outgrowth following injury of the central and peripheral nervous systems. Variable results following chondroitinase ABC treatment make it difficult to predict the effects of this drug in human nerve allografts, especially in the presence of native extracellular signaling molecules. Several studies have shown cross-talk between neurotrophic factor and CSPG signaling pathways, but their interaction remains poorly understood. In this study, we examined the adjuvant effects of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth post-injury in CSPG-reduced substrates and acellular nerve allografts. Materials and Methods E12 chicken DRG explants were cultured in medium containing ChABC, ChABC + NGF, ChABC + GDNF or control media. Explants were imaged at 3 d and neurite outgrowths measured. The rat sciatic nerve injury model involved a 1-cm sciatic nerve gap that was microsurgically repaired with ChABC pre-treated acellular nerve allografts. Prior to implantation, nerve allografts were incubated in NGF, GDNF or sterile water. Nerve histology was evaluated at 5d and 8wk post-injury. Results The addition of GDNF in vitro produced significant increase in sensory neurite length at 3 d compared to ChABC alone (P < 0.01), while NGF was not significantly different from control. In vivo adjuvant NGF produced increases in total myelinated axon count (P < 0.005) and motor axon count (P < 0.01), while significantly reducing IB4+ nociceptor axon count (P < 0.01). There were no significant differences produced by in vivo adjuvant GDNF. Conclusions This study provides initial evidence that CSPG-reduced nerve grafts may disinhibit the pro-survival effects of NGF in vivo, promoting motor axon outgrowth and reducing regeneration of specific nociceptive neurons. Our results support further investigation of adjuvant NGF therapy in CSPG-reduced acellular nerve grafts.

Side‐to‐side supercharging nerve allograft enhances neurotrophic potential

“…In most clinical situations in which inadequate graft supply is a legitimate challenge—long defects, multiple extremities involved, failed previous nerve reconstruction, and others—the size of the defects are generally too long for either conduits or commercially available PNA. Efforts to enhance PNA are ongoing and include seeding with cultured Schwann cells, the addition of growth factors, and supplementation with stem cells,…”

Comparing Processed Nerve Allografts and Assessing Their Capacity to Retain and Release Nerve Growth Factor

“…The cleavage of GAG side chains of chondroitin sulfate proteoglycans (CSPGs) is known to attenuate inhibitory activity and promote axon regeneration. Chondroitinase ABC (ChABC) is an enzyme used to cleave GAG side chains from CSPGs and has shown promise as a treatment for central and peripheral nerve lesions (14). Sondell allografts also had the best preservation of extracellular matrix architecture and clearance of cellular debris.…”

“…Advances in tissue processing have overcome the problem of immunological rejection with the development of detergents and protocols capable of decellularizing and removing immunogenic components from nerve allograft, removing the need for immunosuppression (13). Additionally, these techniques can remove inhibitors of axonal outgrowth such as chondroitin sulfate proteoglycans (CSPGs) (14). Unfortunately, in the process of decellularizing the nerve allograft there is also a loss of Schwann cells and their products, such as neurotrophic factors, one being nerve growth factor (NGF) (8).…”

Diffusion tensor tractography to visualize axonal outgrowth and regeneration in a 4-cm reverse autograft sciatic nerve rabbit injury model

“…The peripheral nervous system, unlike the central nervous system, has a remarkable resilience to injury because of its ability to regenerate axons. 3 Axons in peripheral nerves can grow distally at a rate of about 1 mm per day. 4 This recovery, however, requires a complex interaction between cells, growth factors, and the extracellular matrix.…”

“…5 After being transected, proximal segments form growth cones that respond to neurotrophic and chemotropic signals to direct neurite outgrowth. 3 Fibroblasts and Schwann cells produce growth factors that promote neuron survival or cell death. 6 Extracellular matrix can stimulate or inhibit growth of the nerve and serve as a guiding substrate for nerve regeneration.…”