Schwann Cell Morphology Research Articles

Gradient galectin-1 coating technology: bionic multichannel nerve guidance conduits promote neural cell migration

Engineered nerve guidance conduits have been widely used to repair peripheral nerve injuries. Galectin-1 is an important biological cue that promotes axon regeneration and Schwann cell migration. In this study, a series of polycaprolactone-based nerve guidance conduits were prepared. First, we determined the concentration of galectin-1 (a member of the galactose lectin family) via the proliferation and morphology of Schwann cells and the viability, morphology, and axon length of PC12 cells. On this basis, nanofiber yarns coated with a uniform or unidirectionally linear gradient coating layer of galectin-1 were prepared by electrospinning to investigate the viability and migration of Schwann cells and neural stem cells on the surfaces. The unidirectional linear gradient coating with increasing galectin-1 content was found to promote the migration of both Schwann cells and neural stem cells. To construct nerve guidance conduits with encapsulated nanofiber yarns, we fabricated nerve guidance conduit walls composed of conjugately electrospun nanofiber yarns and random polycaprolactone nanofibers as the inner and outer layers. With a biocompatible light-absorbing dye, the nanofibers can be sealed via light welding to obtain a hollow polycaprolactone conduit. Finally, we prepared nerve guidance conduits containing nanofiber yarns coated with graded galectin-1 as well as hyaluronic acid methacryloyl hydrogel in the lumen. We found that the topology (nanofiber yarns and hyaluronic acid methacryloyl) and biological cues (gradient galectin-1 coating) synergistically accelerated the migration of Schwann cells and neural stem cells along multiple channels of nerve guidance conduits.

Dual-layer conduit containing VEGF-A – Transfected Schwann cells promotes peripheral nerve regeneration via angiogenesis

Peripheral nerve injuries (PNIs) can cause neuropathies and significantly affect the patient's quality of life. Autograft transplantation is the gold standard for conventional treatment; however, its application is limited by nerve unavailability, size mismatch, and local tissue adhesion. Tissue engineering, such as nerve guidance conduits, is an alternative and promising strategy to guide nerve regeneration for peripheral nerve repair; however, only a few conduits could reach the high repair efficiency of autografts. The healing process of PNI is frequently accompanied by not only axonal and myelination regeneration but also angiogenesis, which initializes nerve regeneration through vascular endothelial growth factor A (VEGF-A). In this study, a composite nerve conduit with a poly (lactic-co-glycolic acid) (PLGA) hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with VEGF-A transfected Schwann cells (SCs) as the inner layer was established to evaluate its promising ability for peripheral nerve repair. A rat model of peripheral nerve defect was used to examine the efficiency of PLGA/GelMA-SC (VA) conduits, whereas autograft, PLGA, PLGA/GelMA, and PLGA/GelMA-SC (NC) were used as controls. VEGF-A-transfected SCs can provide a stable source for VEGF-A secretion. Furthermore, encapsulation in GelMA cannot only promote proliferation and tube formation of human umbilical vein endothelial cells but also enhance dorsal root ganglia and neuronal cell extension. Previous animal studies have demonstrated that the regenerative effects of PLGA/GelMA-SC (VA) nerve conduit were similar to those of autografts and much better than those of other conduits. These findings indicate that combination of VEGF-A-overexpressing SCs and PLGA/GelMA conduit-guided peripheral nerve repair provides a promising method that enhances angiogenesis and regeneration during nerve repair. Statement of significanceNerve guidance conduits shows promise for peripheral nerve repair, while achieving the repair efficiency of autografts remains a challenge. In this study, a composite nerve conduit with a PLGA hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with vascular endothelial growth factor A (VEGF-A)-transfected Schwann cells (SCs) as the inner layer was established to evaluate its potential ability for peripheral nerve repair.This approach preserves growth factor bioactivity and enhances material properties. GelMA insertion promotes Schwann cell proliferation and morphology extension. Moreover, transfected SCs serve as a stable VEGF-A source and fostering angiogenesis. This study offers a method preserving growth factor efficacy and safeguarding SCs, providing a comprehensive solution for enhanced angiogenesis and nerve regeneration.

Exploring Schwann Cell Behavior on Electrospun Polyhydroxybutyrate Scaffolds with Varied Pore Sizes and Fiber Thicknesses: Implications for Neural Tissue Engineering.

The placement of a polymeric electrospun scaffold is among the most promising strategies to improve nerve regeneration after critical neurotmesis. It is of great interest to investigate the effect of these structures on Schwann cells (SCs), as these cells lead nerve regeneration and functional recovery. The aim of this study was to assess SC viability and morphology when cultured on polyhydroxybutyrate (PHB) electrospun scaffolds with varied microfiber thicknesses and pore sizes. Six electrospun scaffolds were obtained using different PHB solutions and electrospinning parameters. All the scaffolds were morphologically characterized in terms of fiber thickness, pore size, and overall appearance by analyzing their SEM images. SCs seeded onto the scaffolds were analyzed in terms of viability and morphology throughout the culture period through MTT assay and SEM imaging. The SCs were cultured on three scaffolds with homogeneous smooth fibers (fiber thicknesses: 2.4 μm, 3.1 μm, and 4.3 μm; pore sizes: 16.7 μm, 22.4 μm, and 27.8 μm). SC infiltration and adhesion resulted in the formation of a three-dimensional network composed of intertwined fibers and cells. The SCs attached to the scaffolds maintained their characteristic shape and size throughout the culture period. Bigger pores and thicker fibers resulted in higher SC viability.

Electrospun aligned tacrolimus-loaded polycaprolactone biomaterials for peripheral nerve repair.

Background: Efficacious repair of peripheral nerve injury is an unmet clinical need. The implantation of biomaterials containing neurotrophic drugs at the injury site could promote nerve regeneration and improve outcomes for patients. Materials & methods: Random and aligned electrospun poly-ε-caprolactone scaffolds containing encapsulated tacrolimus were fabricated, and the gene expression profile of Schwann cells (SCs) cultured on the surface was elucidated. On aligned fibers, the morphology of SCs and primary rat neurons was investigated. Results: Both scaffold types exhibited sustained release of drug, and the gene expression of SCs was modulated by both nanofibrous topography and thepresence of tacrolimus. Aligned fibers promoted the alignment of SCs and orientated outgrowth from neurons. Conclusion: Electrospun PCL scaffolds with tacrolimus hold promise for the repair of peripheral nerve injury.

Neuromuscular junction denervation and terminal Schwann cell loss in the hTDP-43 overexpression mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with complex aetiology. Despite evidence of neuromuscular junction (NMJ) denervation and 'dying-back' pathology in models of SOD1-dependent ALS, evidence in other genetic forms of ALS is limited by a lack of suitable animal models. TDP-43, a key mediator protein in ALS, is overexpressed in neurons in Thy1-hTDP-43WT mice. We therefore aimed to comprehensively analyse NMJ pathology in this model of ALS. Expression of TDP-43 was assessed via western blotting. Immunohistochemistry techniques, alongside NMJ-morph quantification, were used to analyse motor neuron number, NMJ denervation status and terminal Schwann cell morphology. We present a time course of progressive, region-specific motor neuron pathology in Thy1-hTDP-43WT mice. Thy1-driven hTDP-43 expression increased steadily, correlating with developing hindlimb motor weakness and associated motor neuron loss in the spinal cord with a median survival of 21 days. Pronounced NMJ denervation was observed in hindlimb muscles, mild denervation in cranial muscles but no evidence of denervation in either forelimb or trunk muscles. NMJ pathology was restricted to motor nerve terminals, with denervation following the same time course as motor neuron loss. Terminal Schwann cells were lost from NMJs in hindlimb muscles, directly correlating with denervation status. Thy1-hTDP-43WT mice represent a severe model of ALS, with NMJ pathology/denervation of distal muscles and motor neuron loss, as observed in ALS patients. This model therefore provides an ideal platform to investigate mechanisms of dying-back pathology, as well as NMJ-targeting disease-modifying therapies in ALS.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Surface Modification of Titanium by Micro-Arc Oxidation in Promoting Schwann Cell Proliferation and Secretion of Neurotrophic Factors

Titanium and its alloys have been widely used in the field of oral implants over the past few decades. However, the effect of micro-arc oxidation modified titanium surface on Schwann cells has not been studied, which is of great significance for nerve regeneration around implants and improvement of osseoperception. In this study, the characterization of the titanium surface modified by micro-arc oxidation (MAO) was detected by scanning electron microscope (SEM), XPS and a contact angle measurement system. Schwann cells (SCs) were cultured on titanium surfaces of micro-arc oxidation (MAO) and pure titanium (Ti). At different time points, the morphology and adhesion of SCs on the titanium surfaces were observed by SEM. Cell proliferation activity was detected by the CCK-8 method. The expression levels of mRNA and proteins of nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) were detected by RT-PCR, immunofluorescence and western blot. The results of this in vitro study revealed that micro-arc-oxidation-modified titanium surfaces promoted Schwann cell proliferation and secretion of neurotrophic factors compared with pure titanium. CCK-8 results showed that the number of Schwann cells on MAO surfaces was significantly higher than that of the Ti group on day 7. The mRNA expressions of Ngf and Gdnf were up-regulated in both groups from day 1 to day 7. On day 3 and day 7, the gene expression of Ngf in the MAO group was significantly higher than that of the Ti group. On day 7, the MAO group appeared significantly up-regulated in gene expression level of Gdnf. The results of western blot were consistent. Micro-arc oxidation modification provides an accurate and effective method for promoting nerve regeneration of titanium microtopography coatings, which have potential significance for promoting patients’ osseoperception ability in clinical practice.

The impact of physical, biochemical, and electrical signaling on Schwann cell plasticity

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.

Analysis of Signal Transduction Pathways Downstream M2 Receptor Activation: Effects on Schwann Cell Migration and Morphology.

Background: Schwann cells (SCs) express cholinergic receptors, suggesting a role of cholinergic signaling in the control of SC proliferation, differentiation and/or myelination. Our previous studies largely demonstrated that the pharmacological activation of the M2 muscarinic receptor subtype caused an inhibition of cell proliferation and promoted the expression of pro-myelinating differentiation genes. In order to elucidate the molecular signaling activated downstream the M2 receptor activation, in the present study we investigated the signal transduction pathways activated by the M2 orthosteric agonist arecaidine propargyl ester (APE) in SCs. Methods: Using Western blot we analyzed some components of the noncanonical pathways involving β1-arrestin and PI3K/AKT/mTORC1 signaling. A wound healing assay was used to evaluate SC migration. Results: Our results demonstrated that M2 receptor activation negatively modulated the PI3K/Akt/mTORC1 axis, possibly through -arrestin downregulation. The involvement of the mTORC1 complex was also supported by the decreased expression of its specific target . Then, we also analyzed the expression of , a negative regulator of myelination that resulted in reduced levels after M2 agonist treatment. The analysis of cell migration and morphology allowed us to demonstrate that M2 receptor activation caused an arrest of SC migration and modified cell morphology probably by the modulation of β1-arrestin/cofilin-1 and PKCα expression, respectively. Conclusions: The data obtained demonstrated that M2 receptor activation in addition to the canonical Gi protein-coupled pathway modulates noncanonical pathways involving the mTORC1 complex and other kinases whose activation may contribute to the inhibition of SC proliferation and migration and address SC differentiation.

Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy.

Schwann cells (SCs) are known to produce extracellular vesicles (EV) that participate in cell-cell communication by transferring cargo to target cells, including mRNAs, microRNAs, and biologically active proteins. Herein, we report a novel mechanism whereby SC EVs may regulate PNS physiology, especially in injury, by controlling the activity of TNFα. SCs actively sequester tumor necrosis factor receptor-1 (TNFR1) into EVs at high density, accounting for about 2% of the total protein in SC EVs (~1000 copies TNFR1/EV). Although TNFR2 was robustly expressed by SCs in culture, TNFR2 was excluded from SC EVs. SC EV TNFR1 bound TNFα, decreasing the concentration of free TNFα available to bind to cells and thus served as a TNFα decoy. SC EV TNFR1 significantly inhibited TNFα-induced p38 MAPK phosphorylation in cultured SCs. When TNFR1 was proteolytically removed from SC EVs using tumor necrosis factor-α converting enzyme (TACE) or neutralized with antibody, the ability of TNFα to activate p38 MAPK in the presence of these EVs was restored. As further evidence of its decoy activity, SC EV TNFR1 modified TNFα activities in vitro including: (1) regulation of expression of other cytokines; (2) effects on SC morphology; and (3) effects on SC viability. SC EVs also modified the effects of TNFα on sciatic nerve morphology and neuropathic pain-related behavior in vivo. By sequestering TNFR1 in EVs, SCs may buffer against the potentially toxic effects of TNFα. SC EVs provide a novel mechanism for the spatial and temporal regulation of neuro-inflammation.

Effects of different implant surface properties on the biological behavior of Schwann cells.

This study investigated the effects of different implant surface properties on the biological behavior of Schwann cells. Schwann cells (SCs) were cultured on three types of implant surfaces including smooth polished (SMO), sand-blasted, large grit, acid-etched (SLA), and chemically-modified SLA (modSLA). At different time points, the morphology and adhesion of SCs on the implant surfaces were observed by scanning electron microscope. Cell proliferation activity was detected by MTT method. The expression levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were detected by enzyme-linked immunosorbent assay. Changes in the mRNA levels of NGF and BDNF were detected by real-time fluorescent quantitative polymerase chain reaction (PCR). SCs adhered, stretched, and proliferated well on the three types of implant surfaces. On the 3rd, 5th, and 7th days, the OD values of the SMO group were higher than those of the SLA group and the modSLA group, and the difference was statistically significant (P<0.05). On the 3rd day, the expression and mRNA levels of NGF and BDNF in the SLA group and the modSLA group were higher than those in the SMO group (P<0.05); in particular, the levels in the modSLA group were higher than those in the SLA group (P<0.05). Different implant surface properties have different effects on the biological behavior of SCs. Proliferation of SCs is significantly promoted by smooth surface, while secretion and gene expression of neurotrophic factors are significantly promoted by modSLA surface at early stage.

Fabrication and evaluation of porous and conductive nanofibrous scaffolds for nerve tissue engineering

Peripheral nerve repair is still one of the major clinical challenges which has received a great deal of attention. Nerve tissue engineering is a novel treatment approach that provides a permissive environment for neural cells to overcome the constraints of repair. Conductivity and interconnected porosity are two required characteristics for a scaffold to be effective in nerve regeneration. In this study, we aimed to fabricate a conductive scaffold with controlled porosity using polycaprolactone (PCL) and chitosan (Chit), FDA approved materials for the use in implantable medical devices. A novel method of using tetrakis (hydroxymethyl) phosphonium chloride (THPC) and formaldehyde was applied for in situ synthesis of gold nanoparticles (AuNPs) on the scaffolds. In order to achieve desirable porosity, different percentage of polyethylene oxide (PEO) was used as sacrificial fiber. Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FE-SEM) results demonstrated the complete removing of PEO from the scaffolds after washing and construction of interconnected porosities, respectively. Elemental and electrical analysis revealed the successful synthesis of AuNPs with uniform distribution and small average diameter on the PCL/Chit scaffold. Contact angle measurements showed the effect of porosity on hydrophilic properties of the scaffolds, where the porosity of 75–80% remarkably improved surface hydrophilicity. Finally, the effect of conductive nanofibrous scaffold on Schwann cells morphology and vaibility was investigated using FE-SEM and MTT assay, respectively. The results showed that these conductive scaffolds had no cytotoxic effect and support the spindle-shaped morphology of cells with elongated process which are typical of Schwann cell cultures.

P75 and S100 gene expression induced by cell‐imprinted substrate and beta‐carotene to nerve tissue engineering

AbstractHere we aimed to differentiate adipose derived stem cells (ADSCs) to Schwann cells (SCs), as one of the major and instrumental cell sources in nerve regeneration, by synergistic application of imprinting method and β‐carotene. Accordingly, the topography of Schwann cells was imprinted on poly dimethyl siloxane (PDMS) substrates via mold casting and human ADSCs seeded on substrates; moreover, β‐carotene was added to induce hADSCs differentiation. Physiochemical evaluations of PDMS by FTIR spectra presented its silicon‐methyl bond (SiCH3) at 1260 cm−1. Morphology analysis by crystal violet, picrosirus red staining, and SEM images illustrated that MSCs seeded on imprinted substrates have formed SC‐like morphology. Furthermore, according to q‐PCR and ICC evaluations, SCs specific markers; S100 and P75 in addition of 5 μl β‐carotene (BC) were upregulated (p‐value&lt;0.001). Also, the expression was detected on the imprinted surfaces without β‐carotene to a lesser degree. Our study revealed that Schwann cell imprinted substrates can mimic the morphology and topography of SCs and induce differentiation signals in mesenchymal stem cells (MSCs). In addition, the potency of β‐carotene as an organic substance in boosting and stimulating the neural differentiation was demonstrated. Relevantly, the reports have confirmed the synergistic pivotal roles of β‐carotene and patterned surfaces in directing MSCs into SC‐like cells differentiation without applying expensive and less safe chemical growth factors.

In Vitro Biocompatibility of Piezoelectric K0.5Na0.5NbO3 Thin Films on Platinized Silicon Substrates.

Lead-free piezoelectric ceramics like K0.5Na0.5NbO3 (KNN) represent an emerging class of biomaterials for medical technology, as they can be used as components in implantable microelectromechanical systems (MEMS) and bioactive scaffolds for tissue stimulation. Such functional materials can act as working components in future in vivo devices, and their addition to current implant designs can greatly improve the biological interaction between host and implant. Despite this, only a few reports have studied the biocompatibility of these materials with living cells. In this work, we investigate the biological response of two different cell lines grown on KNN thin films, and we demonstrate excellent biocompatibility of the KNN films with the cells. Undoped and 0.5 mol % CaTiO3-doped KNN thin films with nanometer-sized roughness were deposited on platinized silicon (SiPt) substrates, and cell proliferation, viability, and morphology of human 161BR fibroblast cells and rat Schwann cells grown on the KNN films and SiPt substrates were investigated and compared to glass control samples. The results show that proliferation rates for the cells grown on the KNN thin films were equally high or higher than those on the glass control samples, and no cytotoxic effect from either the films or the substrate was observed. The work demonstrates that KNN thin films on SiPt substrates are very promising candidates for components in implantable medical devices.

Rapidly formed stable and aligned dense collagen gels seeded with Schwann cells support peripheral nerve regeneration

Objective. Gel aspiration-ejection (GAE) has recently been developed for the rapid production of dense, anisotropic collagen gel scaffolds with adjustable collagen fibrillar densities. In this study, a GAE system was applied to produce aligned Schwann cells within a type-1 collagen matrix to generate GAE-engineered neural tissues (GAE-EngNT) for potential nerve tissue engineering applications. Approach. The stability and mechanical properties of the constructs were investigated along with the viability, morphology and distribution of Schwann cells. Having established the methodology to construct stable robust Schwann cell-loaded engineered neural tissues using GAE (GAE-EngNTs), the potential of these constructs in supporting and guiding neuronal regeneration, was assessed both in vitro and in vivo. Main results. Dynamic mechanical analysis strain and frequency sweeps revealed that the GAE-EngNT produced via cannula gauge number 16 G (∼1.2 mm diameter) exhibited similar linear viscoelastic behaviors to rat sciatic nerves. The viability and alignment of seeded Schwann cells in GAE-EngNT were maintained over time post GAE, supporting and guiding neuronal growth in vitro with an optimal cell density of 2.0 × 106 cells ml−1. An in vivo test of the GAE-EngNTs implanted within silicone conduits to bridge a 10 mm gap in rat sciatic nerves for 4 weeks revealed that the constructs significantly promoted axonal regeneration and vascularization across the gap, as compared with the empty conduits although less effective regeneration compared with the autograft groups. Significance. Therefore, this is a promising approach for generating anisotropic and robust engineered tissue which can be used with Schwann cells for peripheral nerve repair.

Fabrication and in vitro evaluation of 3D composite scaffold based on collagen/hyaluronic acid sponge and electrospun polycaprolactone nanofibers for peripheral nerve regeneration.

Replacement of peripheral nerve autografts with tissue engineered nerve grafts will potentially resolve the lack of nerve tissue especially in patients with severe concomitant soft tissue injuries. This study attempted to fabricate a tissue engineered nerve graft composed of electrospun PCL conduit filled with collagen-hyaluronic acid (COL-HA) sponge with different COL-HA weight ratios including 100:0, 98:2, 95:5 and 90:10. The effect of HA addition on the sponge porosity, mechanical properties, water absorption and degradation rate was assessed. A good cohesion between the electrospun PCL nanofibers and COL-HA sponges were seen in all sponges with different HA contents. Mechanical properties of PCL nanofibrous layer were similar to the rat sciatic nerve; the ultimate tensile strength was 2.23 ± 0.35 MPa at the elongation of 35%. Additionally, Schwann cell proliferation and morphology on three dimensional (3D) composite scaffold were evaluated by using MTT and SEM assays, respectively. Rising the HA content resulted in higher water absorption as well as greater pore size and porosity, while a decrease in Schwann cell proliferation compared to pure collagen sponge, although reduction in cell proliferation was not statistically significant. The lower Schwann cell proliferation on the COL-HA was attributed to the greater degradation rate and pore size of the COL-HA sponges. Also, dorsal root ganglion assay showed that the engineered 3D construct significantly increases axon growth. Taken together, these results suggest that the fabricated 3D composite scaffold provide a permissive environment for Schwann cells proliferation and maturation and can encourage axon growth.

Thrombin regulates the ability of Schwann cells to support neuritogenesis and to maintain the integrity of the nodes of Ranvier.

Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone- like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve ex vivo explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion.

Peptide nanostructures on nanofibers for peripheral nerve regeneration.

Self-assembled peptide nanofibrous scaffolds with designer sequences, similar to neurite growth promoting molecules enhance the differentiation of neural stem cells. However, self-assembled peptide nanofibrous scaffolds lack the required mechanical strength to suffice to bridge long critical-sized peripheral nerve defects. Hence, there is a demand for a potential neural substrate, which could be biomimetic coupled with bioactive nanostructures to regrow the denuded axons towards the distal end. In the present study, we developed designer self-assembling peptide-based aligned poly(lactic-co-glycolic acid) (PLGA) nanofibrous scaffolds by simple surface coating of peptides or coelectrospinning. Retention of secondary structures of peptides in peptide-coated and cospun fibers was confirmed by circular dichroism spectroscopy. The rod-like peptide nanostructures enhance the typical bipolar morphology of Schwann cells. Although the peptide-coated PLGA scaffolds exhibited significant increase in Schwann cell proliferation than pristine PLGA and PLGA-peptide cospun scaffolds (p<.05), peptide cospun scaffolds demonstrated better cellular infiltration and significantly higher gene expression of neural cell adhesion molecule, glial fibrillary acidic protein, and peripheral myelin protein22 compared to the pristine PLGA and PLGA-peptide-coated scaffolds. Our results demonstrate the positive effects of aligned peptide coelectrospun scaffolds with biomimetic cell recognition motifs towards functional proliferation of Schwann cells. These scaffolds could subsequently repair peripheral nerve defects by augmenting axonal regeneration and functional nerve recovery.

DC Electric Fields Induce Perpendicular Alignment and Enhanced Migration in Schwann Cell Cultures.

Schwann cells (SCs) are PNS glia that playnumerous support functions including myelination of axons. After PNS injury, SCs facilitate regeneration by phagocytosing cellular debris and providing physical and biochemical cues to guide axon growth. This reparative phenotype suggests SCs could be critical cellular targets for enhancing nerve regeneration. One method foraltering cell morphology and motility is the applicationof direct current (DC) electric fields (EFs). EndogenousEFs have physiologic relevance during embryogenesis and serve as guidance and polarization cues. While much literature exists on EFs and CNS and PNS neurons, the effects of EFs on SCs have not been extensively studied. In this work, cell alignment, migration, and morphology of rat SCs were measured in response to several EFstimulation regimes including constant DC, 50% duty cycle DC and oscillating DC. SCs were found to re-orient perpendicular to field lines and respond toDC EFs as low as 75mV/mm. EF exposure promoted directed migration, with travel towards the cathode at a mean rate of 7.5µm/h. The data highlight the utility of EFs in modulating SC morphology, alignment and migration. Results may have implications for using EFs to attract and realign SCs at the site ofPNS trauma.

Bio-fabrication of peptide-modified alginate scaffolds: Printability, mechanical stability and neurite outgrowth assessments

Peripheral nerve tissue requires appropriate biochemical and physical cues to guide the regeneration process after injury. Bioprinted peptide-conjugated sodium alginate (PCSA) scaffolds have the potential to provide physical and biochemical cues simultaneously. Such scaffolds need characterisation in terms of printability, mechanical stability, and biological performance to refine and improve application in nerve tissue regeneration. In this study, it was hypothesized that 3D scaffold printed with low concentrated multiple PCSA precursor would be supportive for axon outgrowth. Therefore, a 2% (w/v) alginate precursor was conjugated with either arginine-glycine-aspartate (RGD) or tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptides, or a mixture of RGD and YIGSR (1:2) and was bioprinted in this study. The printability of the composite PCSA scaffolds was tested in three different concentrations of crosslinker (i.e. 50, 100, and 150 mM of CaCl2), and was evaluated by measuring strand width, pore geometry, and angle-formation accuracy. Swelling, degradation, and compression experiments were conducted over a 3 week period to evaluate the mechanical stability of the composite PCSA scaffolds. Scanning electron microscopic (SEM) images were taken to study the surface morphology of the degraded scaffolds. Biological performance was assessed both for single and composite PCSA scaffolds by quantifying the viability and morphology of seeded or incorporated Schwann cells (SCs), amount of secreted brain derived neurotrophic factor (BDNF) by incorporated SCs, and directional neurite outgrowth of neuron cells in a 2D culture. Experimental results suggest that 30 kPa extrusion pressure and 18 mm/s needle speed are suitable to fabricate composite PCSA scaffolds with reasonable strand or pore printability (∼0.95–1.0), and 50 mM CaCl2 facilitated better strand and pore printability than the two other concentrations. Captured SEM images demonstrate that all the composite PCSA scaffolds preserved the initial biofabricated porous structure over 3 weeks, but they lost ∼70% of the initial elastic modulus. In terms of biological performance, composite PCSA scaffolds facilitated better viability and morphology of SCs, as well as supported superior directional neurite outgrowth compared to that of a single PCSA scaffold.