Growth Factor Enriched Multi-luminal Conduits for Axonal Regeneration and Maturation Across Critical Nerve Injuries

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In the United States, about 900,000 peripheral nerve repair surgeries are performed every year, to treat traumatic and iatrogenic injuries. Despite the robust regenerative capacity of peripheral axons, studies have found that in nerve injuries resulting in substantial tissue damage (≥ 3cm), as low as 25% patients gain full motor recovery and an even lower 3% gain complete sensory recovery. The current standard of care in clinic is surgical repair using an autologous nerve graft, extracted from a functionally less important nerve. While successful outcome depends on surgical precision and donor matching, in many cases, it can have deleterious co-morbid effects, formation of neuromas resulting in pain, in addition to loss of function in the donor nerve muscle. Although there are multiple biosynthetic nerve guides approved by the FDA, repair using autologous nerve grafts is still most preferred by surgeons, despite its risks. This is because currently available conduits have a hollow lumen, which fails to provide structural and biochemical support that is needed by peripheral axons to grow across long distances. Therefore, to enhance regeneration by mimicking the internal tubular architecture of the nerve, our lab previously designed a multiluminal Biosynthetic Nerve Implant (BNI). The BNI is a polyurethane tube with an agarose scaffold consisting of multiple microchannels filled with collagen matrix. In a 10mm transection sciatic nerve injury in a rat model, it was shown that the BNI provides structural support during regeneration, resulting in fascicular distribution of axons, and higher functional recovery, as compared to hollow lumen tubes. We next tested whether this effect can be further enhanced by the addition of growth factors, to repair critical nerve injuries with ≥3cm tissue loss, and how that affects functional outcome. In this dissertation, we provide specific neurotrophic and pleiotropic cues to promote axon growth, increase maturation and re-myelination after injury, uncover possible mechanisms that affect axon pathfinding and functional recovery. We also developed 3D printed S-shaped conduits to determine the effect of geometry and topography on growing axons and mimic a critical gap in the rat model. In Chapter 2, continuing from a previous study, we analyzed electron micrographs of distal end of nerve samples to determine the degree of myelination provided by pleiotrophin (PTN) and glialderived neurotrophic factor (GDNF) treatments. Although we observed a moderate effect of PTNGDNF in enticing axons and restoring function, there was a substantial lack of myelination, mainly contributed by abnormal radial sorting of the regenerating axons. We proposed that in addition to neurotrophic factors, incorporating biomolecules to stimulate Schwann cells into correctly sorting growing axons will improve re-myelination, and subsequent functional recovery. In Chapter 3, we reasoned that since axonal derived Neuregulin1 Type-III signals Schwann cells to sort out large axons and myelinate them, incorporating it into the BNI will enhance remyelination in a 4cm nerve gap. With an improvement in re-myelination and functional recovery, for the first time we show a neurotrophic effect of NRG1 Type-III alone. Here, we propose a possible signaling pathway wherein exogenous neuregulin binding to Schwann cells triggers release of endogenous growth factors from the Schwann cells, that bind to receptors on the nearest axon, triggering growth and myelination, and further release of the membrane bound isoform NRG1 Type-III, in a two-way axo-glial signaling. In Chapter 4, we developed 3D printed S-shaped nerve guide conduits (NGCs), to mimic a critical gap in the rat model and determine if axons can grow along curved paths. By comparing 10mm straight conduits with 13mm S-shaped conduits, we found that despite the 3mm difference in length, axons regenerated across the tubes within the same 7 week period, with no differences in the number, and with equal functional recovery. This model probes the importance of topological cues in axon guidance as well as provides a critical injury model that can be exploited for the standardized sensori-motor assessment techniques available for the rat. Together, the work described here highlights the complexity of growth factor mediated peripheral nerve regeneration and re-innervation and its effect on restoring function, provides possible mechanisms of axon maturation that can be further exploited to enhance the intrinsic regenerative capacity of peripheral axons.

Nerves, Peripheral -- Surgery, Myelination, Nervous system -- Regeneration