Biological conduits based on spider silk for reconstruction of extended nerve defects.

Objectives: The availability of appropriate conduits remains an obstacle for successful reconstruction of long-distance nerve defects. In previous sheep trials, we were able to bridge 6 cm nerve gaps with nerve conduits based on spider silk fibers with full functional outcomes. Here, we describe the first application of spider silk for nerve repair in humans. Methods: Four patients with extended nerve defects (>20 cm) underwent nerve reconstruction by interposition of conduits that were composed of spider silk fibers contained in autologous veins. The longitudinal luminal fibers (approx. 2500 fibers per graft) consisted of drag line silk from Trichonephila spiders. All patients were evaluated between 2 and 10 years postreconstruction, clinically, and by neurography. Results: In all patients, primary wound healing and no adverse reactions to the implanted spider silk material were observed. Patients regained the following relevant functions: protective sensibility, full flexor function with near-normal grasp and powerful function after microvascular gracilis muscle transfer, and key grip function and gross finger flexion after additional tenodesis. One patient with sciatic nerve reconstruction developed protective sensibility of the lower leg, foot, and gait, enabling normal walking and jogging. No neuroma formation or neuropathic or chronic pain occurred in any of the patients. Conclusions: For patients with extended peripheral nerve defects in the extremities, use of conduits based on spider silk fibers offers the possibility of restoring sensory function and protection from neuroma. This kind of nerve bridges provides new perspectives for the reconstruction of complex and long-distance nerve defects.

[Research progress of silk-based biomaterials for peripheral nerve regeneration].

Objective: To describe the research progress of silk-based biomaterials in peripheral nerve repair and provide useful ideals to accelerate the regeneration of large-size peripheral nerve injury. Methods: The relative documents about silk-based biomaterials used in peripheral nerve regeneration were reviewed and the different strategies that could accelerate peripheral nerve regeneration through building bioactive microenvironment with silk fibroin were discussed. Results: Many silk fibroin tissue engineered nerve conduits have been developed to provide multiple biomimetic microstructures, and different microstructures have different mechanisms of promoting nerve repair. Biomimetic porous structures favor the nutrient exchange at wound sites and inhibit the invasion of scar tissue. The aligned structures can induce the directional growth of nerve tissue, while the multiple channels promote the axon elongation. When the fillers are introduced to the conduits, better growth, migration, and differentiation of nerve cells can be achieved. Besides biomimetic structures, different nerve growth factors and bioactive drugs can be loaded on silk carriers and released slowly at nerve wounds, providing suitable biochemical cues. Both the biomimetic structures and the loaded bioactive ingredients optimize the niches of peripheral nerves, resulting in quicker and better nerve repair. With silk biomaterials as a platform, fusing multiple ways to achieve the multidimensional regulation of nerve microenvironments is becoming a critical strategy in repairing large-size peripheral nerve injury. Conclusion: Silk-based biomaterials are useful platforms to achieve the design of biomimetic hierarchical microstructures and the co-loading of various bioactive ingredients. Silk fibroin nerve conduits provide suitable microenvironment to accelerate functional recovery of peripheral nerves. Different optimizing strategies are available for silk fibroin biomaterials to favor the nerve regeneration, which would satisfy the needs of various nerve tissue repair. Bioactive silk conduits have promising future in large-size peripheral nerve regeneration.

Design and Development of a Polymeric-Based Curcumin Nanoparticle for Drug Delivery Enhancement and Potential Incorporation into Nerve Conduits.

Peripheral nerve injuries (PNI) impact millions of individuals in the United States, prompting thousands of nerve repair procedures annually. Nerve conduits (NC) are commonly utilized to treat nerve injuries under 3 cm but larger gaps still pose a challenge for successful peripheral nerve regeneration (PNR) and functional recovery. This is partly attributed to the absence of bioactive agents such as stem cells or growth factors in FDA-approved conduits due to safety, harvesting, and reproducibility concerns. Therefore, curcumin, a bioactive phytochemical, has emerged as a promising alternative bioactive agent due to its ability to enhance PNR and overcome said challenges. However, its hydrophobicity and rapid degradation in aqueous solutions are considerable limitations. In this work, a nanoscale delivery platform with tannic acid (TA) and polyvinylpyrrolidone (PVP) was developed to encapsulate curcumin for increased colloidal and chemical stability. The curcumin nanoparticles (CurNPs) demonstrate significantly improved stability in water, reduced degradation rates, and controlled release kinetics when compared to free curcumin. Further, cell studies show that the CurNP is biocompatible when introduced to neuronal cells (SH-SY5Y), rat Schwann cells (RSC-S16), and murine macrophages (J774 A.1) at 5 µM, 5 µM, and 10 µM of curcumin, respectively. As a result of these improved physicochemical properties, confocal fluorescence microscopy revealed superior delivery of curcumin into these cells when in the form of CurNPs compared to its free form. A hydrogen peroxide-based oxidative stress study also demonstrated the CurNP's potential to protect J774 A.1 cells against excessive oxidative stress. Overall, this study provides evidence for the suitability of CurNPs to be used as a bioactive agent in NC applications.

Highly Bioadaptable Hybrid Conduits with Spatially Bidirectional Structure for Precision Nerve Fiber Regeneration via Gene Therapy.

Peripheral nerve deficits give rise to motor and sensory impairments within the limb. The clinical restoration of extensive segmental nerve defects through autologous nerve transplantation often encounters challenges such as axonal mismatch and suboptimal functional recovery. These issues may stem from the limited regenerative capacity of proximal axons and the subsequent Wallerian degeneration of distal axons. To achieve the integration of sensory and motor functions, a spatially differential plasmid DNA (pDNA) dual-delivery nanohydrogel conduit scaffold is devised. This innovative scaffold facilitates the localized administration of the transforming growth factor ß (TGF-ß) gene in the proximal region to accelerate nerve regeneration, while simultaneously delivering nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) to the distal region to mitigate Wallerian degeneration. By promoting autonomous and selective alignment of nerve fiber gap sutures via structure design, the approach aims to achieve a harmonious unification of nerve regeneration, neuromotor function, and sensory recovery. It is anticipated that this groundbreaking technology will establish a robust platform for gene delivery in tissue engineering.

Biodegradable Electrospun Conduit with Aligned Fibers Based on Poly(lactic-co-glycolic Acid) (PLGA)/Carbon Nanotubes and Choline Bitartrate Ionic Liquid.

Functionally active aligned fibers are a promising approach to enhance neuro adhesion and guide the extension of neurons for peripheral nerve regeneration. Therefore, the present study developed poly(lactic-co-glycolic acid) (PLGA)-aligned electrospun mats and investigated the synergic effect with carbon nanotubes (CNTs) and Choline Bitartrate ionic liquid (Bio-IL) on PLGA fibers. Morphology, thermal, and mechanical performances were determined as well as the hydrolytic degradation and the cytotoxicity. Results revealed that electrospun mats are composed of highly aligned fibers, and CNTs were aligned and homogeneously distributed into the fibers. Bio-IL changed thermal transition behavior, reduced glass transition temperature (Tg), and favored crystal phase formation. The mechanical properties increased in the presence of CNTs and slightly decreased in the presence of the Bio-IL. The results demonstrated a decrease in the degradation rate in the presence of CNTs, whereas the use of Bio-IL led to an increase in the degradation rate. Cytotoxicity results showed that all the electrospun mats display metabolic activity above 70%, which demonstrates that they are biocompatible. Moreover, superior biocompatibility was observed for the electrospun containing Bio-IL combined with higher amounts of CNTs, showing a high potential to be used in nerve tissue engineering.

Electrospun Composite PLLA-PPSB Nanofiber Nerve Conduits for Peripheral Nerve Defects Repair and Regeneration.

Peripheral nerve injury (PNI) is a common clinical problem and regenerating peripheral nerve defects remain a significant challenge. Poly(polyol sebacate) (PPS) polymers are developed as promising materials for biomedical applications due to their biodegradability, biocompatibility, elastomeric properties, and ease of production. However, the application of PPS-based biomaterials in nerve tissue engineering, especially in PNI repair, is limited. In this study, PPS-based composite nanofibers poly(l-lactic acid)-poly(polycaprolactone triol-co-sebacic acid-co-N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid sodium salt) (PLLA-PPSB) are aimed to construct through electrospinning and assess their in vitro biocompatibility with Schwann cells (SCs) and in vivo repair capabilities for peripheral nerve defects. For the first time, the biocompatibility and bioactivity of PPS-based nanomaterial are examined at the molecular, cellular, and animal levels for PNI repair. Electrospun PLLA-PPSB nanofibers display favorable physicochemical properties and biocompatibility, providing an effective interface for the proliferation, glial expression, and adhesion of SCs in vitro. In vivo experiments using a 10-mm rat sciatic nerve defect model show that PLLA-PPSB nanofiber nerve conduits enhance myelin formation, axonal regeneration, angiogenesis, and functional recovery. Transcriptome analysis and biological validation indicate that PLLA-PPSB nanofibers may promote SC proliferation by activating the PI3K/Akt signaling pathway. This suggests the promising potential of PLLA-PPSB nanomaterial for PNI repair.

Nerve Guide Conduits Integrated with Fisetin-Loaded Chitosan Hydrogels for Reducing Oxidative Stress, Inflammation, and Nerve Regeneration.

Peripheral nerve injuries (PNI) represent a prevalent and severe category of damage resulting from traumatic incidents. Predominantly, the deficiency in nerve regeneration can be ascribed to enduring inflammatory reactions, hence imposing substantial clinical implications for patients. Fisetin, a flavonoid derived from plants, is naturally present in an array of vegetables and fruits, including strawberries, apples, onions, and cucumbers. It exhibits immunomodulatory properties through the reduction of inflammation and oxidative stress. In the present research, a nerve defect is addressed for the first time utilizing a scaffold primed for controlled fisetin release. In this regard, fisetin-loaded chitosan hydrogels are incorporated into the lumen of polycaprolactone (PCL) nerve guide conduits (NGCs). The hydrogel maintained a steady release of an appropriate fisetin dosage. The study outcomes indicated that the fisetin/chitosan/polycaprolactone (FIS/CS/PCL) NGCs amplified Schwann cell proliferation and neural expression, curtailed oxidative stress, alleviated inflammation, and improved functions, electrophysiological properties, and morphology. This pioneering scaffold has the potential to contribute significantly to the field of neuroengineering.

Chitosan-Based Conduits with Different Inner Diameters at both Ends Combined with Modified Formula Radix Hedysari Promote Nerve Transposition Repair.

BACKGROUND: Severe peripheral nerve injuries, such as deficits over long distances or proximal nerve trunk injuries, pose complex reconstruction challenges that often result in unfavorable outcomes. An innovative approach to repairing severe peripheral nerve damage involves using conduit suturing for nerve transposition repair. Cylindrical nerve guides are typically unsuitable for nerve transposition repair. Moreover, postsurgical adjuvant treatment is essential to promote the development of axonal lateral sprouts, proximal growth, and the restoration of neurostructure and function. The purpose of this research is to assess the impact of chitosan-based conduits with varying inner diameters on nerve transposition repair when combined with modified formula Radix Hedysari (MFRH). METHODS: Using chitosan, we created conduits with varying inner diameters on both ends. These conduits were then utilized to repair the distal common peroneal and tibial nerves in SD rats using the proximal common peroneal nerve. Subsequently, MFRH was employed as a supplementary treatment. The assessment of the repair's effectiveness took place 16 weeks postsurgery, utilizing a range of techniques, including the neurological nerve function index, neuroelectrophysiological measurements, muscle wet weight, and examination of nerve and muscle histology. RESULTS: The outcomes of our study showed that following 16 weeks of postoperative treatment, MFRH had a significant positive impact on the recovery of neuromotor and nerve conduction abilities. Moreover, there was a significant increase in the ratio of wet weight of muscles, cross-sectional area of muscle fibers, quantity and structure of regenerated myelinated nerve fibers, and the count of neurons. CONCLUSIONS: A combination of chitosan-based chitin conduits possessing different inner diameters and MFRH can considerably promote the regeneration and functional recovery of damaged nerves, which in turn enhances nerve transposition repair efficacy.

History of peripheral nerve injuries.

This article reviews the history of peripheral nerve (PN) injuries and successive advances in their management by notable pioneers, an interesting topic that I chose for my Doctoral Thesis in 1990 in Madrid. Mentioning all their names and contributions is an obligatory tribute, and I offer my sincere apologies for inevitably leaving a few out. For half a century I have witnessed microsurgery advances, and also experienced frequent failures in my practice with the use of new techniques; a testimony that we are very far from achieving the 'Holy Grail' of complete PN recovery for these injuries. Our experience is often like a pendulum, from nihilism to optimism and vice versa. Many factors influence the results of PN repair. Fortunately, microsurgery has been a breakthrough but, too often, emergency surgery is carried out by surgeons without enough tools and experience, both very important factors in this field.

Chitin Conduits with Different Inner Diameters at Both Ends Combined with Dual Growth Factor Hydrogels Promote Nerve Transposition Repair in Rats.

Severe peripheral nerve injuries, such as deficits over long distances or proximal nerve trunk injuries, pose complex reconstruction challenges that often result in unfavorable outcomes. Innovative techniques, such as nerve transposition repair with conduit suturing, can be employed to successfully treat severe peripheral nerve damage. However, cylindrical nerve guides are typically unsuitable for nerve transposition repair. Furthermore, angiogenic and neurotrophic factors are necessary to stimulate the emergence of axonal lateral sprouts, proximal growth, and the rehabilitation of neuron structures and functions. In the current study, we used chitosan to make chitin conduits with different inner diameters at both ends, combined with gelatin methacrylate hydrogels that can continuously release dual growth factors, namely, the vascular endothelial growth factor (VEGF) and the nerve growth factor (NGF), and evaluated its impact on nerve transposition repair in rats. At 16 weeks after the operation, our findings showed that the conduit combined with the dual growth factor hydrogel significantly improved the restoration of both motor and conduction functions of the nerve. In addition, histological analysis showed significant recovery of nerve fibers, target muscles, and neurons. In conclusion, the combination of chitin conduits with different inner diameters and dual growth factor hydrogels can significantly improve the effect of nerve transposition repair, which has important potential clinical value.

Preparation and performance study of multichannel PLA artificial nerve conduits.

Compared with single-channel nerve conduits, multichannel artificial nerve conduits are more beneficial for repairing damaged peripheral nerves of long-distance nerve defects. Multichannel nerve conduits can be fabricated by the mold method and the electrospinning method but with disadvantages such as low strength and large differences in batches, while the braiding method can solve this problem. In this study, polylactic acid yarns were used as the braiding yarn, and the number of spindles during braiding was varied to achieve 4, 5, 6, 7 and 8 multichannel artificial nerve conduits. A mathematical model of the number of braiding yarn spindles required to meet certain size specification parameters of the multichannel conduit was established. The cross-sectional morphology and mechanical properties of the conduits were characterized by scanning electron microscopy observation and mechanical testing; the results showed that the multichannel structure was well constructed; the tensile strength of the multichannel conduit was more than 30 times that of the rabbit tibial nerve. The biocompatibility of the conduit was tested; thein vitrocell culture results proved that the braided multichannel nerve conduits were nontoxic to Schwann cells, and the cell adhesion and proliferation were optimal in the 4-channel conduit among the multichannel conduits, which was close to the single-channel conduit.

Development and preclinical evaluation of bioactive nerve conduits for peripheral nerve regeneration: A comparative study.

In severe peripheral nerve injuries, nerve conduits (NCs) are good alternatives to autografts/allografts; however, the results the available devices guarantee for are still not fully satisfactory. Herein, differently bioactivated NCs based on the new polymer oxidized polyvinyl alcohol (OxPVA) are compared in a rat model of sciatic nerve neurotmesis (gap: 5 mm; end point: 6 weeks). Thirty Sprague Dawley rats are randomized to 6 groups: Reverse Autograft (RA); Reaxon®; OxPVA; OxPVA + EAK (self-assembling peptide, mechanical incorporation); OxPVA + EAK-YIGSR (mechanical incorporation); OxPVA + Nerve Growth Factor (NGF) (adsorption). Preliminarily, all OxPVA-based devices are comparable with Reaxon® in Sciatic Functional Index score and gait analysis; moreover, all conduits sustain nerve regeneration (S100, ß-tubulin) without showing substantial inflammation (CD3, F4/80) evidences. Following morphometric analyses, OxPVA confirms its potential in PNI repair (comparable with Reaxon®) whereas OxPVA + EAK-YIGSR stands out for its myelinated axons total number and density, revealing promising in injury recovery and for future application in clinical practice.

Bridging Gaps in Peripheral Nerves: From Current Strategies to Future Perspectives in Conduit Design.

In peripheral nerve injuries (PNI) with substance loss, where tensionless end-to-end suture is not achievable, the positioning of a graft is required. Available options include autografts (e.g., sural nerve, medial and lateral antebrachial cutaneous nerves, superficial branch of the radial nerve), allografts (Avance®; human origin), and hollow nerve conduits. There are eleven commercial hollow conduits approved for clinical, and they consist of devices made of a non-biodegradable synthetic polymer (polyvinyl alcohol), biodegradable synthetic polymers (poly(DL-lactide-ε-caprolactone); polyglycolic acid), and biodegradable natural polymers (collagen type I with/without glycosaminoglycan; chitosan; porcine small intestinal submucosa); different resorption times are available for resorbable guides, ranging from three months to four years. Unfortunately, anatomical/functional nerve regeneration requirements are not satisfied by any of the possible alternatives; to date, focusing on wall and/or inner lumen organization/functionalization seems to be the most promising strategy for next-generation device fabrication. Porous or grooved walls as well as multichannel lumens and luminal fillers are the most intriguing options, eventually also including the addition of cells (Schwann cells, bone marrow-derived, and adipose tissue derived stem cells) to support nerve regeneration. This review aims to describe common alternatives for severe PNI recovery with a highlight of future conduits.

A comprehensive review on therapeutic application of mesenchymal stem cells in neuroregeneration.

Each year, thousands of people suffer from traumatic peripheral nerve lesions, which impair mobility and sensibility and frequently have fatal outcomes. The recovery of peripheral nerves on its own is frequently insufficient. In this regard, cell therapy is currently one of the most cutting-edge techniques for nerve healing. The purpose of this review is to highlight the properties of various types of mesenchymal stem cells (MSCs) that are critical for peripheral nerve regeneration after nerve injury. The Preferred Reporting term used to review the available literature are "nerve regeneration," "stem cells," "peripheral nerve damage," "rat," and "human" were combined. In addition, using the phrases "stem cells" and "nerve regeneration" in PubMed, a "MeSH" search was conducted. This study describes the features of the most often utilized MSCs, as well as its paracrine potential, targeted stimulation, and propensity for differentiation into Schwann-like and neuronal-like cells. For the repair of peripheral nerve lesions, ADSCs appear to be the most relevant and promising MSCs, because of their ability to sustain and increase axonal growth, as well as their outstanding paracrine activity, putative differentiation potential, low immunogenicity, and excellent post-transplant survival rate.

Peripheral Nerve Regeneration: Opportunities and Challenges.

Peripheral nerve injury has detrimental effects on the quality of life for patients and is a worldwide issue with high rates of morbidity. Research on the molecular mechanisms of nerve injury, microsurgical techniques, and advances in stem cell research have led to substantial progress in the field of translational neurophysiology. Current research on peripheral nerve regeneration aims to accelerate peripheral nerve development through pluripotent stem cells and potential use of smart exosomes, pharmacological agents, and bioengineering of nerve conduits. In this article critically reviewed and summarized various methods used for peripheral nerve regeneration and highlight the opportunities and challenges that come along with these strategies.

Polydopamine-Decorated PLCL Conduit to Induce Synergetic Effect of Electrical Stimulation and Topological Morphology for Peripheral Nerve Regeneration.

Due to the limited self-repairing capacity after peripheral nerve injuries (PNI), artificial nerve conduits are widely applied to facilitate neural regeneration. Exogenous electrical stimulation (ES) that is carried out by the conductive conduit regulates the biological behavior of Schwann cells (SCs). Meanwhile, a longitudinal surface structure counts to guide axonal growth to accelerate the end-to-end connection. Currently, there are no conduits equipped with both electrical conduction and axon-guiding surface structure. Herein, a biodegradable, conductive poly(l-lactide-co-caprolactone)/graphene (PLCL/GN) composite conduit is designed. The conduit with 20.96 ± 1.26 MPa tensile strength has a micropatterned surface of 20 µm groove fabricated by microimprint technology and self-assembled polydopamine (PDA). In vitro evaluation shows that the conduits with ES effectively stimulate the directional cell migration, adhesion, and elongation, and enhance neuronal expression of SCs. The rat sciatic nerve crush model demonstrates that the conductive micropatterned conduit with ES promotes the growth of myelin sheath, faster nerve regeneration, and 20-fold functional recovery in vivo. These discoveries prove that the PLCL(G)/PDA/GN composite conduit is a promising tool for PNI treatment by providing the functional integration of physical guidance, biomimetic biological regulation, and bioelectrical stimulation, which inspires a novel therapeutic approach for nerve regeneration in the future.

Stem Cells and Tissue Engineering-Based Therapeutic Interventions: Promising Strategies to Improve Peripheral Nerve Regeneration.

Unlike the central nervous system, the peripheral one has the ability to regenerate itself after injury; however, this natural regeneration process is not always successful. In fact, even with some treatments, the prognosis is poor, and patients consequently suffer with the functional loss caused by injured nerves, generating several impacts on their quality of life. In the present review we aimed to address two strategies that may considerably potentiate peripheral nerve regeneration: stem cells and tissue engineering. In vitro studies have shown that pluripotent cells associated with neural scaffolds elaborated by tissue engineering can increase functional recovery, revascularization, remyelination, neurotrophin expression and reduce muscle atrophy. Although these results are very promising, it is important to note that there are some barriers to be circumvented: the host's immune response, the oncogenic properties attributed to stem cells and the duration of the pro-regenerative effects. After all, more studies are still needed to overcome the limitations of these treatments; those that address techniques for manipulating the lesion microenvironment combining different therapies seem to be the most promising and proactive ones.

Graphene-based nanomaterials for peripheral nerve regeneration.

Emerging nanotechnologies offer numerous opportunities in the field of regenerative medicine and have been widely explored to design novel scaffolds for the regeneration and stimulation of nerve tissue. In this review, we focus on peripheral nerve regeneration. First, we introduce the biomedical problem and the present status of nerve conduits that can be used to guide, fasten and enhance regeneration. Then, we thoroughly discuss graphene as an emerging candidate in nerve tissue engineering, in light of its chemical, tribological and electrical properties. We introduce the graphene forms commonly used as neural interfaces, briefly review their applications, and discuss their potential toxicity. We then focus on the adoption of graphene in peripheral nervous system applications, a research field that has gained in the last years ever-increasing attention. We discuss the potential integration of graphene in guidance conduits, and critically review graphene interaction not only with peripheral neurons, but also with non-neural cells involved in nerve regeneration; indeed, the latter have recently emerged as central players in modulating the immune and inflammatory response and accelerating the growth of new tissue.

Development of Two-Layer Hybrid Scaffolds Based on Oxidized Polyvinyl Alcohol and Bioactivated Chitosan Sponges for Tissue Engineering Purposes.

Oxidized polyvinyl alcohol (OxPVA) is a new polymer for the fabrication of nerve conduits (NCs). Looking for OxPVA device optimization and coupling it with a natural sheath may boost bioactivity. Thus, OxPVA/chitosan sponges (ChS) as hybrid scaffolds were investigated to predict in the vivo behaviour of two-layered NCs. To encourage interaction with cells, ChS were functionalized with the self-assembling-peptide (SAP) EAK, without/with the laminin-derived sequences -IKVAV/-YIGSR. Thus, ChS and the hybrid scaffolds were characterized for mechanical properties, ultrastructure (Scanning Electron Microscopy, SEM), bioactivity, and biocompatibility. Regarding mechanical analysis, the peptide-free ChS showed the highest values of compressive modulus and maximum stress. However, among +EAK groups, ChS+EAK showed a significantly higher maximum stress than that found for ChS+EAK-IKVAV and ChS+EAK-YIGSR. Considering ultrastructure, microporous interconnections were tighter in both the OxPVA/ChS and +EAK groups than in the others; all the scaffolds induced SH-SY5Y cells' adhesion/proliferation, with significant differences from day 7 and a higher total cell number for OxPVA/ChS+EAK scaffolds, in accordance with SEM. The scaffolds elicited only a slight inflammation after 14 days of subcutaneous implantation in Balb/c mice, proving biocompatibility. ChS porosity, EAK 3D features and neuro-friendly attitude (shared with IKVAV/YIGSR motifs) may confer to OxPVA certain bioactivity, laying the basis for future appealing NCs.