Biologic Augmentation in Peripheral Nerve Repair

Introduction

Peripheral nerve injuries remain a main source of morbidity and disability. Peripheral nerve injuries have life-altering impacts on the patients who suffer years of uncertainty while waiting for some level of recovery. Patients may be left with devastating sensory and motor deficits such as limb numbness, dysethesias, paralysis, and neuropathic pain which renders them disabled. The management of nerve injuries is multifactorial including the location of the injury, the type of injury, the size of segmental nerve defect, the timing of injury presentation, and accompanying soft tissue injury. Nerve injuries lead to both physiological and histopathological changes to the nerve and its surrounding soft tissue, including demyelination, degeneration, remyelination, and regeneration. Despite the permissive growth environment of the peripheral nervous system (PNS), major human nerve injuries have very limited potential for spontaneous recovery. Although there has been significant amount of research addressing the molecular biology of nerve injury and numerous surgical advances detailing peripheral nerve repair, the injured limb still cannot be restored to normal function. Unfortunately, optimization of outcomes has plateaued with surgical manipulation alone as issues with the rate of regeneration, specificity of regeneration, segmental nerve defects, and degeneration of target end organ remain unaddressed by current practices. Tension-free primary repair is the gold standard for peripheral nerve injuries that do not have gaps. Nerve injuries with gaps present a greater challenge for the reconstructive surgeons. Faced with this predicament, surgeons have been searching for alternative techniques to allow bridging of gaps, as well as tensionless nerve repair. The role of biologic augments in the repair of peripheral nerve injuries has long been studied in both animal models and humans to address this issue. It is, therefore, critical that we are made aware of the appropriate applications and the limitations of these tools to understand how we can promote an effective regenerative environment for damaged nerves. Adapted from our recent review, we include discussions regarding microanatomy, nerve response to injury, as well as limitations of peripheral nerve regeneration, as these topics provide invaluable information to further understand the role of biologic augmentation in peripheral nerve repair and reconstruction.

Microanatomy

Peripheral nerves are heterogeneous composite structures that are comprised of neurons, Schwann cells (SCs), macrophages, and fibroblasts. The neuron is a polarized cell that forms the foundation of the nerve and consists of dendrites, the cell body, and a single axon. Axons project toward their sites of innervation to form synapses with their target end organs. If the axonal diameter is greater than or equal to 1 μm, each SC will wrap its plasma membrane around one single region of an axon to form myelin. SCs produce myelin to encapsulate the axon and aid in action potential transmission as myelin allows for fast and efficient conduction and propagation of an action potential down an axon. The blood supply to the nerve is a complex vascular plexus formed from anastomoses of epineural, perineural, and endoneurial plexi, as well as segmental blood supply derived from a number of nutrient arteries. It is apparent that the blood supply to the nerve is fragile and may be disrupted due to trauma or tension during nerve repair. In addition, peripheral nerves have connective tissue layers to provide strength and protection to the nerve: (1) the epineurium, (2) perineurium, and (3) endoneurium. It is crucial to recognize that all surgical interventions are strictly directed at these connective tissue layers, whereas the axon and SCs must respond to injury and regenerate via their inherent biology.

Nerve Response to Injury

In contrast to chronic nerve injuries that are SC driven, acute nerve injuries are axonally mediated with Wallerian degeneration being initiated with granular disintegration of the axonal cytoskeleton. Within 48 hours after injury to the nerve, SCs break down myelin and phagocytose debris from the axon in the distal stump. Macrophages are then recruited to the area and start releasing growth factors that in turn encourage SC and fibroblast proliferation. SCs begin the reparative process by forming longitudinal bands of Bungner, which are essentially growth-promoting conduits for regenerating axons. Injection of predifferentiated SCs near injured nerves has been shown to aid remyelination in regenerating axons, reduce percent myelin debris, and improve functional recovery in rodents. At the tip of the regenerating axon is the growth cone, which is composed of cellular matrix from which fingerlike projections called filopodia extrude to explore the microenvironment. Proteases are released from the growth cone to clear a path toward a target organ, which is heavily influenced by different factors. After injury, SCs upregulate neurotrophic factors nerve growth factor (NGF) and brain-derived growth factor (BDNF), as well as their corresponding receptors in the distal stumps. This increase in expression of NGF and its low-density receptors is believed to promote extensive proliferation and migration of SCs and mainly affects properties of sensory neurons. BDNF levels are also increased and are postulated to act as an anterograde trophic messenger under the influence of NGF. Ciliary neuronotrophic factor (CNTF) is a neuronotrophic factor that is believed to affect survival and regeneration of motoneurons and is found to be reduced significantly in the SCs of the distal stump, with this reduction extending to the neuromuscular junction. Furthermore, there is increased retrograde axonal transport of CNTF after nerve injury. Neurite-promoting factors, such as laminin and fibronectin, and matrix-forming precursors, such as fibrinogen, are all synthesized in response to nerve injury.

In addition to the release of trophic factors, tubulin deacetylation is an important factor leading to decreased stability of microtubulin, which is required for axonal integrity and regeneration. Calcium-dependent activation of the histone deacetylases HDAC5 and HDAC6, in particular, leads to tubulin degeneration likely playing a role in inhibiting axonal regeneration after peripheral nerve injuries. The interaction between axons and SCs has also emerged as an important regulator of PNS development and regeneration. Fleming et al. identified that the receptor tyrosine kinase Ret genetically interacts with Er81 to control Nrg1-Ig in promoting the formation of Pacinian corpuscles. Taken together, these factors have the potential to promote regeneration; provide signaling for cell survival, neuronal differentiation, and proliferation; and influence synaptic function.

After a nerve injury, PNS neurons upregulate a number of regeneration-associated genes that may have direct role in neurite outgrowth. For example, the overexpression of transcription factor Activating transcription factor 3 (ATF-3) has been shown to promote neurite outgrowth after peripheral nerve injury. In their animal study, Bomze et al. concluded that growth-associated protein 43 and cytoskeleton-associated protein 23 were expressed after nerve injury and, together, were able to induce dramatic increase in the number of regenerated axons. Pathways associated with increased regeneration after a nerve injury have also been identified. The ERK pathway was shown to mediate axonal elongation, with the kinases Extracellular signal regulated kinase (ERK) and Akt promoting regeneration after axonal injury. After nerve injury, the cytokine interleukin-6 has been shown to work through the JAK-STAT3 pathway to overcome the inhibitory molecules and allow for axonal regeneration. In addition to pathways that are involved in axonal regeneration, there are also pathways that have been associated with inhibition of axonal regeneration. The small GTPase Rho signaling pathway, for instance, has a role in cytoskeletal reorganization and cell motility. Studies have shown that activation of Rho results to collapse of growth cones, and inhibiting Rho pathway allows for contractility and promotes neurite outgrowth.

Limitations of Peripheral Nerve Regeneration

Despite the promise of improved functional recovery, results are still limited as detailed by these studies with all currently available techniques. Outcomes of nerve repair after a traumatic injury are commonly influenced by factors outside of the control of the surgeon such as the age of the patient, location of injury, and timing of injury presentation and nerve repair. As initially reported by Sunderland and subsequently by many others, nerve reconstruction outcomes are better with younger patients, early repairs, repairs of single function nerves, distal injuries, and short nerve grafts. There are numerous challenges facing nerve repair, and optimization of outcomes has plateaued with surgical manipulations alone, offering limited capacity to effect true functional neural regeneration. The ultimate repair and regeneration is a complex biological process that we are just beginning to understand through clinical experiences as well as animal experimental studies. Rate of regeneration, specificity of regeneration, segmental nerve deficits, and degeneration of the target end organ are challenges that need to be overcome to achieve meaningful functional recovery ( Fig. 14.1 ).