Physiology of Nerve Regeneration: Key Factors Affecting Clinical Outcomes.

Functional recovery after peripheral nerve injuries is disappointing despite surgical advances in nerve repair. This review summarizes the relatively short window of opportunity for successful nerve regeneration due to the decline in the expression of growth-associated genes and in turn, the decline in regenerative capacity of the injured neurons and the support provided by the denervated Schwann cells, and the atrophy of denervated muscles. Brief, low-frequency electrical stimulation and post-injury exercise regimes ameliorate these deficits in animal models and patients, but the misdirection of regenerating nerve fibers compromises functional recovery and remains an important area of future research.

Decellularized porcine peripheral nerve based injectable hydrogels as a Schwann cell carrier for injured spinal cord regeneration.

Objective.To develop a clinically relevant injectable hydrogel derived from decellularized porcine peripheral nerves and with mechanical properties comparable to native central nervous system (CNS) tissue to be used as a delivery vehicle for Schwann cell transplantation to treat spinal cord injury (SCI).Approach.Porcine peripheral nerves (sciatic and peroneal) were decellularized by chemical decellularization using a sodium deoxycholate and DNase (SDD) method previously developed by our group. The decellularized nerves were delipidated using dichloromethane and ethanol solvent and then digested using pepsin enzyme to form injectable hydrogel formulations. Genipin was used as a crosslinker to enhance mechanical properties. The injectability, mechanical properties, and gelation kinetics of the hydrogels were further analyzed using rheology. Schwann cells encapsulated within the injectable hydrogel formulations were passed through a 25-gauge needle and cell viability was assessed using live/dead staining. The ability of the hydrogel to maintain Schwann cell viability against an inflammatory milieu was assessedin vitrousing inflamed astrocytes co-cultured with Schwann cells.Mainresults. The SDD method effectively removes cells and retains extracellular matrix in decellularized tissues. Using rheological studies, we found that delipidation of decellularized porcine peripheral nerves using dichloromethane and ethanol solvent improves gelation kinetics and mechanical strength of hydrogels. The delipidated and decellularized hydrogels crosslinked using genipin mimicked the mechanical strength of CNS tissue. The hydrogels were found to have shear thinning properties desirable for injectable formulations and they also maintained higher Schwann cell viability during injection compared to saline controls. Usingin vitroco-culture experiments, we found that the genipin-crosslinked hydrogels also protected Schwann cells from astrocyte-mediated inflammation.Significance. Injectable hydrogels developed using delipidated and decellularized porcine peripheral nerves are a potential clinically relevant solution to deliver Schwann cells, and possibly other therapeutic cells, at the SCI site by maintaining higher cellular viability and increasing therapeutic efficacy for SCI treatment.

The phenotypic changes of Schwann cells promote the functional repair of nerve injury.

Functional recovery after nerve injury is a significant challenge due to the complex nature of nerve injury repair and the non-regeneration of neurons. Schwann cells (SCs), play a crucial role in the nerve injury repair process because of their high plasticity, secretion, and migration abilities. Upon nerve injury, SCs undergo a phenotypic change and redifferentiate into a repair phenotype, which helps in healing by recruiting phagocytes, removing myelin fragments, promoting axon regeneration, and facilitating myelin formation. However, the repair phenotype can be unstable, limiting the effectiveness of the repair. Recent research has found that transplantation of SCs can be an effective treatment option, therefore, it is essential to comprehend the phenotypic changes of SCs and clarify the related mechanisms to develop the transplantation therapy further.

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro.

Schwann cells (SCs) are myelinating cells of the peripheral nervous system, playing a crucial role in peripheral nerve regeneration. Nanosecond Pulse Electric Field (nsPEF) is an emerging method applicable in nerve electrical stimulation that has been demonstrated to be effective in stimulating cell proliferation and other biological processes. Aiming to assess whether SCs undergo significant changes under nsPEF and help explore the potential for new peripheral nerve regeneration methods, cultured RSC96 cells were subjected to nsPEF stimulation at 5 kV and 10 kV, followed by continued cultivation for 3-4 days. Subsequently, some relevant factors expressed by SCs were assessed to demonstrate the successful stimulation, including the specific marker protein, neurotrophic factor, transcription factor, and myelination regulator. The representative results showed that nsPEF significantly enhanced the proliferation and migration of SCs and the ability to synthesize relevant factors that contribute positively to the regeneration of peripheral nerves. Simultaneously, lower expression of GFAP indicated the benign prognosis of peripheral nerve injuries. All these outcomes show that nsPEF has great potential as an efficient treatment method for peripheral nerve injuries by stimulating SCs.

Advances of Schwann cells in peripheral nerve regeneration: From mechanism to cell therapy.

Peripheral nerve injuries (PNIs) frequently occur due to various factors, including mechanical trauma such as accidents or tool-related incidents, as well as complications arising from diseases like tumor resection. These injuries frequently result in persistent numbness, impaired motor and sensory functions, neuropathic pain, or even paralysis, which can impose a significant financial burden on patients due to outcomes that often fall short of expectations. The most frequently employed clinical treatment for PNIs involves either direct sutures of the severed ends or bridging the proximal and distal stumps using autologous nerve grafts. However, autologous nerve transplantation may result in sensory and motor functional loss at the donor site, as well as neuroma formation and scarring. Transplantation of Schwann cells/Schwann cell-like cells has emerged as a promising cellular therapy to reconstruct the microenvironment and facilitate peripheral nerve regeneration. In this review, we summarize the role of Schwann cells and recent advances in Schwann cell therapy in peripheral nerve regeneration. We summarize current techniques used in cell therapy, including cell injection, 3D-printed scaffolds for cell delivery, cell encapsulation techniques, as well as the cell types employed in experiments, experimental models, and research findings. At the end of the paper, we summarize the challenges and advantages of various cells (including ESCs, iPSCs, and BMSCs) in clinical cell therapy. Our goal is to provide the theoretical and experimental basis for future treatments targeting peripheral nerves, highlighting the potential of cell therapy and tissue engineering as invaluable resources for promoting nerve regeneration.

Origin, identity, and function of terminal Schwann cells.

The highly specialized nonmyelinating glial cells present at somatic peripheral nerve endings, known collectively as terminal Schwann cells (TSCs), play critical roles in the development, function and repair of their motor and sensory axon terminals and innervating tissue. Over the past decades, research efforts across various vertebrate species have revealed that while TSCs are a diverse group of cells, they share a number of features among them. In this review, we summarize the state-of-knowledge about each TSC type and explore the opportunities that TSCs provide to treat conditions that afflict peripheral axon terminals.

N-cadherin directs the collective Schwann cell migration required for nerve regeneration through Slit2/3-mediated contact inhibition of locomotion.

Collective cell migration is fundamental for the development of organisms and in the adult for tissue regeneration and in pathological conditions such as cancer. Migration as a coherent group requires the maintenance of cell-cell interactions, while contact inhibition of locomotion (CIL), a local repulsive force, can propel the group forward. Here we show that the cell-cell interaction molecule, N-cadherin, regulates both adhesion and repulsion processes during Schwann cell (SC) collective migration, which is required for peripheral nerve regeneration. However, distinct from its role in cell-cell adhesion, the repulsion process is independent of N-cadherin trans-homodimerisation and the associated adherens junction complex. Rather, the extracellular domain of N-cadherin is required to present the repulsive Slit2/Slit3 signal at the cell surface. Inhibiting Slit2/Slit3 signalling inhibits CIL and subsequently collective SC migration, resulting in adherent, nonmigratory cell clusters. Moreover, analysis of ex vivo explants from mice following sciatic nerve injury showed that inhibition of Slit2 decreased SC collective migration and increased clustering of SCs within the nerve bridge. These findings provide insight into how opposing signals can mediate collective cell migration and how CIL pathways are promising targets for inhibiting pathological cell migration.

Cell Heterogeneity and Variability in Peripheral Nerve after Injury.

With the development of single-cell sequencing technology, the cellular composition of more and more tissues is being elucidated. As the whole nervous system has been extensively studied, the cellular composition of the peripheral nerve has gradually been revealed. By summarizing the current sequencing data, we compile the heterogeneities of cells that have been reported in the peripheral nerves, mainly the sciatic nerve. The cellular variability of Schwann cells, fibroblasts, immune cells, and endothelial cells during development and disease has been discussed in this review. The discovery of the architecture of peripheral nerves after injury benefits the understanding of cellular complexity in the nervous system, as well as the construction of tissue engineering nerves for nerve repair and axon regeneration.

Schwann Cells as Orchestrators of Nerve Repair: Implications for Tissue Regeneration and Pathologies.

Peripheral nerves exist in a stable state in adulthood providing a rapid bidirectional signaling system to control tissue structure and function. However, following injury, peripheral nerves can regenerate much more effectively than those of the central nervous system (CNS). This multicellular process is coordinated by peripheral glia, in particular Schwann cells, which have multiple roles in stimulating and nurturing the regrowth of damaged axons back to their targets. Aside from the repair of damaged nerves themselves, nerve regenerative processes have been linked to the repair of other tissues and de novo innervation appears important in establishing an environment conducive for the development and spread of tumors. In contrast, defects in these processes are linked to neuropathies, aging, and pain. In this review, we focus on the role of peripheral glia, especially Schwann cells, in multiple aspects of nerve regeneration and discuss how these findings may be relevant for pathologies associated with these processes.

A Novel Superparamagnetic Multifunctional Nerve Scaffold: A Remote Actuation Strategy to Boost In Situ Extracellular Vesicles Production for Enhanced Peripheral Nerve Repair.

Extracellular vesicles (EVs) have inherent advantages over cell-based therapies in regenerative medicine because of their cargos of abundant bioactive cues. Several strategies are proposed to tune EVs production in vitro. However, it remains a challenge for manipulation of EVs production in vivo, which poses significant difficulties for EVs-based therapies that aim to promote tissue regeneration, particularly for long-term treatment of diseases like peripheral neuropathy. Herein, a superparamagnetic nanocomposite scaffold capable of controlling EVs production on-demand is constructed by incorporating polyethyleneglycol/polyethyleneimine modified superparamagnetic nanoparticles into a polyacrylamide/hyaluronic acid double-network hydrogel (Mag-gel). The Mag-gel is highly sensitive to a rotating magnetic field (RMF), and can act as mechano-stimulative platform to exert micro/nanoscale forces on encapsulated Schwann cells (SCs), an essential glial cell in supporting nerve regeneration. By switching the ON/OFF state of the RMF, the Mag-gel can scale up local production of SCs-derived EVs (SCs-EVs) both in vitro and in vivo. Further transcriptome sequencing indicates an enrichment of transcripts favorable in axon growth, angiogenesis, and inflammatory regulation of SCs-EVs in the Mag-gel with RMF, which ultimately results in optimized nerve repair in vivo. Overall, this research provides a noninvasive and remotely time-scheduled method for fine-tuning EVs-based therapies to accelerate tissue regeneration, including that of peripheral nerves.

Enhancement of nerve regeneration through schwann cell-mediated healing in a 3D printed polyacrylonitrile conduit incorporating hydrogel and graphene quantum dots: a study on rat sciatic nerve injury model.

Despite recent technological advancements, effective healing from sciatic nerve damage remains inadequate. Cell-based therapies offer a promising alternative to autograft restoration for peripheral nerve injuries, and 3D printing techniques can be used to manufacture conduits with controlled diameter and size. In this study, we investigated the potential of Wharton's jelly-derived mesenchymal stem cells (WJMSCs) differentiated into schwann cells, using a polyacrylonitrile (PAN) conduit filled with fibrin hydrogel and graphene quantum dots (GQDs) to promote nerve regeneration in a rat sciatic nerve injury model. We investigated the potential of WJMSCs, extracted from the umbilical cord, to differentiate into schwann cells and promote nerve regeneration in a rat sciatic nerve injury model. WJMSCs were 3D cultured and differentiated into schwann cells within fibrin gel for two weeks. A 3 mm defect was created in the sciatic nerve of the rat model, which was then regenerated using a conduit/fibrin, conduit covered with schwann cells in fibrin/GQDs, GQDs in fibrin, and a control group without any treatment (n= 6/group). At 10 weeks after transplantation, motor and sensory functions and histological improvement were assessed. The WJMSCs were extracted, identified, and differentiated. The differentiated cells expressed typical schwann cell markers, S100 and P75.In vivoinvestigations established the durability and efficacy of the conduit to resist the pressures over two months of implantation. Histological measurements showed conduit efficiency, schwann cell infiltration, and association within the fibrin gel and lumen. Rats treated with the composite hydrogel-filled PAN conduit with GQDs showed significantly higher sensorial recovery than the other groups. Histological results showed that this group had significantly more axon numbers and remyelination than others. Our findings suggest that the conduit/schwann approach has the potential to improve nerve regeneration in peripheral nerve injuries, with future therapeutic implications.

Schwann Cell-Derived Exosomal Vesicles: A Promising Therapy for the Injured Spinal Cord.

Exosomes are nanoscale-sized membrane vesicles released by cells into their extracellular milieu. Within these nanovesicles reside a multitude of bioactive molecules, which orchestrate essential biological processes, including cell differentiation, proliferation, and survival, in the recipient cells. These bioactive properties of exosomes render them a promising choice for therapeutic use in the realm of tissue regeneration and repair. Exosomes possess notable positive attributes, including a high bioavailability, inherent safety, and stability, as well as the capacity to be functionalized so that drugs or biological agents can be encapsulated within them or to have their surface modified with ligands and receptors to imbue them with selective cell or tissue targeting. Remarkably, their small size and capacity for receptor-mediated transcytosis enable exosomes to cross the blood-brain barrier (BBB) and access the central nervous system (CNS). Unlike cell-based therapies, exosomes present fewer ethical constraints in their collection and direct use as a therapeutic approach in the human body. These advantageous qualities underscore the vast potential of exosomes as a treatment option for neurological injuries and diseases, setting them apart from other cell-based biological agents. Considering the therapeutic potential of exosomes, the current review seeks to specifically examine an area of investigation that encompasses the development of Schwann cell (SC)-derived exosomal vesicles (SCEVs) as an approach to spinal cord injury (SCI) protection and repair. SCs, the myelinating glia of the peripheral nervous system, have a long history of demonstrated benefit in repair of the injured spinal cord and peripheral nerves when transplanted, including their recent advancement to clinical investigations for feasibility and safety in humans. This review delves into the potential of utilizing SCEVs as a therapy for SCI, explores promising engineering strategies to customize SCEVs for specific actions, and examines how SCEVs may offer unique clinical advantages over SC transplantation for repair of the injured spinal cord.

Regenerative capacity of neural tissue scales with changes in tissue mechanics post injury.

Spinal cord injuries have devastating consequences for humans, as mammalian neurons of the central nervous system (CNS) cannot regenerate. In the peripheral nervous system (PNS), however, neurons may regenerate to restore lost function following injury. While mammalian CNS tissue softens after injury, how PNS tissue mechanics changes in response to mechanical trauma is currently poorly understood. Here we characterised mechanical rat nerve tissue properties before and after in vivo crush and transection injuries using atomic force microscopy-based indentation measurements. Unlike CNS tissue, PNS tissue significantly stiffened after both types of tissue damage. This nerve tissue stiffening strongly correlated with an increase in collagen I levels. Schwann cells, which crucially support PNS regeneration, became more motile and proliferative on stiffer substrates in vitro, suggesting that changes in tissue stiffness may play a key role in facilitating or impeding nervous system regeneration.

A scaffold containing zinc oxide for Schwann cell-mediated axon growth.

Objective.Schwann cells (SCs) transplanted in damaged nervous tissue promote axon growth, which may support the recovery of function lost after injury. However, SC transplant-mediated axon growth is often limited and lacks direction.Approach.We have developed a zinc oxide (ZnO) containing fibrous scaffold consisting of aligned fibers of polycaprolactone (PCL) with embedded ZnO nanoparticles as a biodegradable, bifunctional scaffold for promoting and guiding axon growth. This scaffold has bifunctional properties wherein zinc is released providing bioactivity and ZnO has well-known piezoelectric properties where piezoelectric materials generate electrical activity in response to minute deformations. In this study, SC growth, SC-mediated axon extension, and the presence of myelin basic protein (MBP), as an indicator of myelination, were evaluated on the scaffolds containing varying concentrations of ZnOin vitro. SCs and dorsal root ganglion (DRG) neurons were cultured, either alone or in co-culture, on the scaffolds.Main results.Findings demonstrated that scaffolds with 1 wt.% ZnO promoted the greatest SC growth and SC-mediated axon extension. The presence of brain-derived neurotrophic factor (BDNF) was also determined. BDNF increased in co-cultures for all scaffolds as compared to SCs or DRGs cultured alone on all scaffolds. For co-cultures, cells on scaffolds with low levels of ZnO (0.5 wt.% ZnO) had the highest amount of BDNF as compared to cells on higher ZnO-containing scaffolds (1 and 2 wt.%). MBP immunostaining was only detected in co-cultures on PCL control scaffolds (without ZnO).Significance.The results of this study demonstrate the potential of the ZnO-containing scaffolds for SC-mediated axon growth and its potential for use in nervous tissue repair.

Glial plasticity at nervous system transition zones.

The central and peripheral nervous systems (CNS and PNS, respectively) are two separate yet connected domains characterized by molecularly distinct cellular components that communicate via specialized structures called transition zones to allow information to travel from the CNS to the periphery, and vice versa. Until recently, nervous system transition zones were thought to be selectively permeable only to axons, and the establishment of the territories occupied by glial cells at these complex regions remained poorly described and not well understood. Recent work now demonstrates that transition zones are occupied by dynamic glial cells and are precisely regulated over the course of nervous system development. This review highlights recent work on glial cell migration in and out of the spinal cord, at motor exit point (MEP) and dorsal root entry zone (DREZ) transition zones, in the physiological and diseased nervous systems. These cells include myelinating glia (oligodendrocyte lineage cells, Schwann cells and motor exit point glia), exit glia, perineurial cells that form the perineurium along spinal nerves, as well as professional and non-professional phagocytes (microglia and neural crest cells).

[Deciphering the dynamic characteristics of non-neuronal cells in dorsal root ganglion of rat at different developmental stage based on single cell transcriptome data].

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4-L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype Ⅰ Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.

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.

Function Follows Form: Oriented Substrate Nanotopography Overrides Neurite-Repulsive Schwann Cell-Astrocyte Barrier Formation in an In Vitro Model of Glial Scarring.

Schwann cell (SC) transplantation represents a promising therapeutic approach for traumatic spinal cord injury but is frustrated by barrier formation, preventing cell migration, and axonal regeneration at the interface between grafted SCs and reactive resident astrocytes (ACs). Although regenerating axons successfully extend into SC grafts, only a few cross the SC-AC interface to re-enter lesioned neuropil. To date, research has focused on identifying and modifying the molecular mechanisms underlying such scarring cell-cell interactions, while the influence of substrate topography remains largely unexplored. Using a recently modified cell confrontation assay to model SC-AC barrier formation in vitro, highly oriented poly(ε-caprolactone) nanofibers were observed to reduce AC reactivity, induce extensive oriented intermingling between SCs and ACs, and ultimately enable substantial neurite outgrowth from the SC compartment into the AC territory. It is anticipated that these findings will have important implications for the future design of biomaterial-based scaffolds for nervous tissue repair.

Mesenchymal Stem Cells in Nerve Tissue Engineering: Bridging Nerve Gap Injuries in Large Animals.

Cell-therapy-based nerve repair strategies hold great promise. In the field, there is an extensive amount of evidence for better regenerative outcomes when using tissue-engineered nerve grafts for bridging severe gap injuries. Although a massive number of studies have been performed using rodents, only a limited number involving nerve injury models of large animals were reported. Nerve injury models mirroring the human nerve size and injury complexity are crucial to direct the further clinical development of advanced therapeutic interventions. Thus, there is a great need for the advancement of research using large animals, which will closely reflect human nerve repair outcomes. Within this context, this review highlights various stem cell-based nerve repair strategies involving large animal models such as pigs, rabbits, dogs, and monkeys, with an emphasis on the limitations and strengths of therapeutic strategy and outcome measurements. Finally, future directions in the field of nerve repair are discussed. Thus, the present review provides valuable knowledge, as well as the current state of information and insights into nerve repair strategies using cell therapies in large animals.

Schwann cells as a target cell for the treatment of cancer pain.

Tumor erosion and metastasis can invade surrounding tissues, damage nerves, and sensitize the peripheral primary receptors, inducing pain, which can potentially worsen the suffering of patients with cancer. Reception and transmission of sensory signal receptors, abnormal activation of primary sensory neurons, and activation of glial cells are involved in cancer pain. Therefore, exploring promising therapeutic methods to suppress cancer pain is of great significance. Various studies have found that the use of functionally active cells is a potentially effective way to relieve pain. Schwann cells (SCs) act as small, biologically active pumps that secrete pain-relieving neuroactive substances. Moreover, SCs can regulate the progression of tumor cells, including proliferation and metastasis, through neuro-tumor crosstalk, which emphasizes the critical role of SCs in cancer and cancer pain. The mechanisms by which SCs repair injured nerves and exert analgesia include neuroprotection, neurotrophy, nerve regeneration, neuromodulation, immunomodulation, and enhancement of the nerve-injury microenvironment. These factors may ultimately restore the damaged or stimulated nerves and contribute to pain relief. Strategies for pain treatment using cell transplantation mainly focus on analgesia and nerve repair. Although these cells are in the initial stages of nerve repair and pain, they open new avenues for the treatment of cancer pain. Therefore, this paper discusses, for the first time, the possible mechanism of SCs and cancer pain, and new strategies and potential problems in cancer pain treatment.