Fundamentals and Current Strategies for Peripheral Nerve Repair and Regeneration.

A body of evidence indicates that peripheral nerves have an extraordinary yet limited capacity to regenerate after an injury. Peripheral nerve injuries have confounded professionals in this field, from neuroscientists to neurologists, plastic surgeons, and the scientific community. Despite all the efforts, full functional recovery is still seldom. The inadequate results attained with the "gold standard" autograft procedure still encourage a dynamic and energetic research around the world for establishing good performing tissue-engineered alternative grafts. Resourcing to nerve guidance conduits, a variety of methods have been experimentally used to bridge peripheral nerve gaps of limited size, up to 30-40 mm in length, in humans. Herein, we aim to summarize the fundamentals related to peripheral nerve anatomy and overview the challenges and scientific evidences related to peripheral nerve injury and repair mechanisms. The most relevant reports dealing with the use of both synthetic and natural-based biomaterials used in tissue engineering strategies when treatment of nerve injuries is envisioned are also discussed in depth, along with the state-of-the-art approaches in this field.

Engineering Silk Fibroin-Based Nerve Conduit with Neurotrophic Factors for Proximal Protection after Peripheral Nerve Injury.

Artificial nerve conduits capable of adequately releasing neurotrophic factors are extensively studied to bridge nerve defects. However, the lack of neurotrophic factors in the proximal area and their visible effects in axonal retrograde transport following nerve injury is one of the factors causing an incomplete nerve regeneration. Herein, an advanced conduit made of silk fibroin is produced, which can incorporate growth factors and promote an effective regeneration after injury. For that, enzymatically crosslinked silk fibroin-based conduits are developed to be used as a platform for the controlled delivery of neurotrophic factors. Nerve growth factor and glial-cell line derived neurotrophic factor (GDNF) are incorporated using two different methodologies: i) crosslinking and ii) absorption method. The release profile is measured by ELISA technique. The bioactivity of the neurotrophic factors is evaluated in vitro by using primary dorsal root ganglia. When implanted in a 10 mm sciatic nerve defect in rats, GDNF-loaded silk fibroin conduits reveal retrograde neuroprotection as compared to autografts and plain silk fibroin conduit. Therefore, the novel design presents a substantial improvement of retrograde trafficking, neurons' protection, and motor nerve reinnervation.

Modern Trends for Peripheral Nerve Repair and Regeneration: Beyond the Hollow Nerve Guidance Conduit.

Peripheral nerve repair and regeneration remains among the greatest challenges in tissue engineering and regenerative medicine. Even though peripheral nerve injuries (PNIs) are capable of some degree of regeneration, frail recovery is seen even when the best microsurgical technique is applied. PNIs are known to be very incapacitating for the patient, due to the deprivation of motor and sensory abilities. Since there is no optimal solution for tackling this problem up to this day, the evolution in the field is constant, with innovative designs of advanced nerve guidance conduits (NGCs) being reported every day. As a basic concept, a NGC should act as a physical barrier from the external environment, concomitantly acting as physical guidance for the regenerative axons across the gap lesion. NGCs should also be able to retain the naturally released nerve growth factors secreted by the damaged nerve stumps, as well as reducing the invasion of scar tissue-forming fibroblasts to the injury site. Based on the neurobiological knowledge related to the events that succeed after a nerve injury, neuronal subsistence is subjected to the existence of an ideal environment of growth factors, hormones, cytokines, and extracellular matrix (ECM) factors. Therefore, it is known that multifunctional NGCs fabricated through combinatorial approaches are needed to improve the functional and clinical outcomes after PNIs. The present work overviews the current reports dealing with the several features that can be used to improve peripheral nerve regeneration (PNR), ranging from the simple use of hollow NGCs to tissue engineered intraluminal fillers, or to even more advanced strategies, comprising the molecular and gene therapies as well as cell-based therapies.

Enhanced performance of chitosan/keratin membranes with potential application in peripheral nerve repair.

Although surgical management of peripheral nerve injuries (PNIs) has improved over time, autografts are still the current "gold standard" treatment for PNIs, which presents numerous limitations. In an attempt to improve natural biomaterial-based nerve guidance conduits (NGCs), chitosan (CHT), a derivative of the naturally occurring biopolymer chitin, has been explored for peripheral nerve regeneration (PNR). In addition to CHT, keratin has gained enormous attention as a biomaterial and tissue engineering scaffolding. In this study, biomimetic CHT/keratin membranes were produced using a solvent casting technique. These membranes were broadly characterized in terms of their surface topography and physicochemical properties, with techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), contact angle, weight loss and water uptake measurements, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Biological in vitro assays were also performed, where a preliminary cytotoxicity screening with the L929 fibroblast cell line revealed that the membranes and respective materials are suitable for cell culture. In addition, Schwann cells, fibroblasts and endothelial cells were directly seeded in the membranes. Quantitative and qualitative assays revealed that the addition of keratin enhanced cell viablity and adhesion. Based on the encouraging in vitro results, the in vivo angiogenic/antiangiogenic potential of CHT and CHT/keratin membranes was assessed, using an optimized chick embryo chorioallantoic membrane assay, where higher angiogenic responses were seen in keratin-enriched materials. Overall, the obtained results indicate the higher potential of CHT/keratin membranes for guided tissue regeneration applications in the field of PNR.

Tunable Enzymatically Cross-Linked Silk Fibroin Tubular Conduits for Guided Tissue Regeneration.

Hollow tubular conduits (TCs) with tunable architecture and biological properties are in great need for modulating cell functions and drug delivery in guided tissue regeneration. Here, a new methodology to produce enzymatically cross-linked silk fibroin TCs is described, which takes advantage of the tyrosine groups present in silk structure that are known to allow the formation of a covalently cross-linked hydrogel. Three different processing methods are used as a final step to modulate the properties of the silk-based TCs. This approach allows to virtually adjust any characteristic of the final TCs. The final microstructure ranges from a nonporous to a highly porous network, allowing the TCs to be selectively porous to 4 kDa molecules, but not to human skin fibroblasts. Mechanical properties are dependent both on the processing method and thickness of the TCs. Bioactivity is observed after 30 days of immersion in simulated body fluid only for the TCs submitted to a drying processing method (50 °C). The in vivo study performed in mice demonstrates the good biocompatibility of the TCs. The enzymatically cross-linked silk fibroin TCs are versatile and have adjustable characteristics that can be exploited in a variety of biomedical applications, particularly in guidance of peripheral nerve regeneration.

Investigation of cell adhesion in chitosan membranes for peripheral nerve regeneration.

Peripheral nerve injuries have produced major concerns in regenerative medicine for several years, as the recovery of normal nerve function continues to be a significant clinical challenge. Chitosan (CHT), because of its good biocompatibility, biodegradability and physicochemical properties, has been widely used as a biomaterial in tissue engineering scaffolding. In this study, CHT membranes were produced with three different Degrees of Acetylation (DA), envisioning its application in peripheral nerve regeneration. The three CHT membranes (DA I: 1%, DA II: 2%, DA III: 5%) were extensively characterized and were found to have a smooth and flat surface, with DA III membrane having slightly higher roughness and surface energy. All the membranes presented suitable mechanical properties and did not show any signs of calcification after SBF test. Biodegradability was similar for all samples, and adequate to physically support neurite outgrowth. The in vitro cell culture results indicate selective cell adhesion. The CHT membranes favoured Schwann cells invasion and proliferation, with a display of appropriate cytoskeletal morphology. At the same time they presented low fibroblast infiltration. This fact may be greatly beneficial for the prevention of fibrotic tissue formation, a common phenomenon impairing peripheral nerve regeneration. The great deal of results obtained during this work permitted to select the formulation with the greatest potential for further biological tests.