Blood Vessels: The Pathway Used by Schwann Cells to Colonize Nerve Conduits.

The repair of severe nerve injuries requires an autograft or conduit to bridge the gap and avoid axon dispersion. Several conduits are used routinely, but their effectiveness is comparable to that of an autograft only for short gaps. Understanding nerve regeneration within short conduits could help improve their efficacy for longer gaps. Since Schwann cells are known to migrate on endothelial cells to colonize the "nerve bridge", the new tissue spontaneously forming to connect the injured nerve stumps, here we aimed to investigate whether this migratory mechanism drives Schwann cells to also proceed within the nerve conduits used to repair large nerve gaps. Injured median nerves of adult female rats were repaired with 10 mm chitosan conduits and the regenerated nerves within conduits were analyzed at different time points using confocal imaging of sequential thick sections. Our data showed that the endothelial cells formed a dense capillary network used by Schwann cells to migrate from the two nerve stumps into the conduit. We concluded that angiogenesis played a key role in the nerve conduits, not only by supporting cell survival but also by providing a pathway for the migration of newly formed Schwann cells.

Functionalizing nanofibrous platforms for neural tissue engineering applications.

Over the past two decades, electrospun nanofibers have shown great promise in developing functional nerve constructs resembling the structural organization of the fibrillar extracellular matrix (ECM). However, these niche nanofibrous structures are often hindered by inadequate cell infiltration and poor mechanical strength. Further challenge is presented by the intricate nature of neural regeneration and repair processes. The versatility of electrospun nanofibers allows extensive modifications with the overarching aim of optimizing the neurocompatibility and neuroinductivity, in addition to enhancing cellular adhesion, proliferation, migration, differentiation, and neurite outgrowth. In this review, we provide a comprehensive overview of the various optimization techniques for electrospun nanofibrous platforms in neural tissue engineering (NTE), including surface modifications to enhance cell-platform interactions, and techniques to facilitate drug and biomolecule delivery applications.

3D bioprinted silk fibroin hydrogels for tissue engineering.

The development of biocompatible and precisely printable bioink addresses the growing demand for three-dimensional (3D) bioprinting applications in the field of tissue engineering. We developed a methacrylated photocurable silk fibroin (SF) bioink for digital light processing 3D bioprinting to generate structures with high mechanical stability and biocompatibility for tissue engineering applications. Procedure 1 describes the synthesis of photocurable methacrylated SF bioink, which takes 2 weeks to complete. Digital light processing is used to fabricate 3D hydrogels using the bioink (1.5 h), which are characterized in terms of methacrylation, printability, mechanical and rheological properties, and biocompatibility. The physicochemical properties of the bioink can be modulated by varying photopolymerization conditions such as the degree of methacrylation, light intensity, and concentration of the photoinitiator and bioink. The versatile bioink can be used broadly in a range of applications, including nerve tissue engineering through co-polymerization of the bioink with graphene oxide, and for wound healing as a sealant. Procedure 2 outlines how to apply 3D-printed SF hydrogels embedded with chondrocytes and turbinate-derived mesenchymal stem cells in one specific in vivo application, trachea tissue engineering, which takes 2-9 weeks.

Reverse engineering of an anatomically equivalent nerve conduit.

Reconstruction of peripheral nervous tissue remains challenging in critical-sized defects due to the lack of Büngner bands from the proximal to the distal nerve ends. Conventional nerve guides fail to bridge the large-sized defect owing to the formation of a thin fibrin cable. Hence, in the present study, an attempt was made to reverse engineer the intricate epi-, peri- and endo-neurial tissues using Fused Deposition Modeling based 3D printing. Bovine serum albumin protein nanoflowers (NF) exhibiting Viburnum opulus 'Roseum' morphology were ingrained into 3D printed constructs without affecting its secondary structure to enhance the axonal guidance from proximal to distal ends of denuded nerve ends. Scanning electron micrographs confirmed the uniform distribution of protein NF in 3D printed constructs. The PC-12 cells cultured on protein ingrained 3D printed scaffolds demonstrated cytocompatibility, improved cell adhesion and extended neuronal projections with significantly higher intensities of NF-200 and tubulin expressions. Further suture-free fixation designed in the current 3D printed construct aids facile implantation of printed conduits to the transected nerve ends. Hence the protein ingrained 3D printed construct would be a promising substitute to treat longer peripheral nerve defects as its structural equivalence of endo- and perineurial organization along with the ingrained protein NF promote the neuronal extension towards the distal ends by minimizing axonal dispersion.

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing.

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the morphology of processes and substructures within these neurons. As such, much of what is known about brain function has arisen alongside developments in imaging technologies that allow further insight into how neurons are organized and connected in the brain. Improvements in tissue clearing have allowed for high-resolution imaging of thick brain slices, facilitating morphological reconstruction and analyses of neuronal substructures, such as dendritic arbors and spines. In tandem, advances in image processing software provide methods of quickly analyzing large imaging datasets. This work presents a relatively rapid method of processing, visualizing, and analyzing thick slices of labeled neural tissue at high-resolution using CLARITY tissue clearing, confocal microscopy, and image analysis. This protocol will facilitate efforts toward understanding the connectivity patterns and neuronal morphologies that characterize healthy brains, and the changes in these characteristics that arise in diseased brain states.

Bioabsorbable nerve conduits three-dimensionally coated with human induced pluripotent stem cell-derived neural stem/progenitor cells promote peripheral nerve regeneration in rats.

Peripheral nerve regeneration using nerve conduits has been less effective than autogenous nerve grafts. To overcome this hurdle, we developed a tissue-engineered nerve conduit coated with mouse induced pluripotent stem cell (iPSC)-derived neurospheres, for the first time, which accelerated nerve regeneration in mice. We previously demonstrated the long-term efficacy and safety outcomes of this hybrid nerve conduit for mouse peripheral nerve regeneration. In this study, we investigated the therapeutic potential of nerve conduits coated with human iPSC (hiPSC)-derived neurospheres in rat sciatic nerve defects, as a translational preclinical study. The hiPSC-derived quaternary neurospheres containing neural stem/progenitor cells were three-dimensionally cultured within the nerve conduit (poly L-lactide and polycaprolactone copolymer) for 14 days. Complete 5-mm defects were created as a small size peripheral nerve defect in sciatic nerves of athymic nude rats and reconstructed with nerve conduit alone (control group), nerve conduits coated with hiPSC-derived neurospheres (iPS group), and autogenous nerve grafts (autograft group) (n = 8 per group). The survival of the iPSC-derived neurospheres was continuously tracked using in vivo imaging. At 12 weeks postoperatively, motor and sensory function and histological nerve regeneration were evaluated. Before implantation, the hiPSC-derived quaternary neurospheres that three-dimensional coated the nerve conduit were differentiated into Schwann-like cells. The transplanted hiPSC-derived neurospheres survived for at least 56 days after implantation. The iPS group showed non-significance higher sensory regeneration than the autograft group. Although there was no actual motor functional nerve regeneration in the three groups: control, iPS, and autograft groups, the motor function in the iPS group recovered significantly better than that in the control group, but it did not recover to the same level as that in the autograft group. Histologically, the iPS group demonstrated significantly higher axon numbers and areas, and lower G-ratio values than the control group, whereas the autograft group demonstrated the highest axon numbers and areas and the lowest G-ratio values. Nerve conduit three-dimensionally coated with hiPSC-derived neurospheres promoted axonal regeneration and functional recovery in repairing rat sciatic nerve small size defects. Transplantation of hiPSC-derived neurospheres with nerve conduits is a promising clinical iPSC-based cell therapy for the treatment of peripheral nerve defects.

Unilateral section of the superior ovarian nerve induces first ovulation in the Zucker fatty (fa/fa) rat.

Hyperactivity in the sympathetic nervous system has been shown to be related to the development of ovarian pathologies. In addition, obesity has been found to be associated with multiple reproductive anomalies and is considered a chronic stress condition of low intensity with changes in the peripheral sympathetic activity. Therefore, in the present study, we aimed to evaluate if the information reaching the ovaries through the superior ovarian nerve (SON) modifies the ovarian function of Zucker fatty rats. We performed a unilateral section of the SON at 32 days of age and autopsies were carried out on the day of the first vaginal estrus. The results showed that fatty animals do not ovulate on the day of the first vaginal estrus and exhibit an increase in catecholaminergic fibers and the presence of precystic structures in the ovaries, without changes in the onset of puberty or in the secretion of ovarian and hypophyseal hormones. We also found that the section of the right SON resulted in ovulation on the day of the first vaginal estrus, which was accompanied by a decrease in ovarian noradrenaline content. The section of the left SON caused a delay in puberty without changes in the rest of the parameters. These results provide functional evidence that the peripheral sympathetic innervation participates in the regulation of ovarian functions in an animal model of genetic obesity.

High-resolution combinatorial 3D printing of gelatin-based biomimetic triple-layered conduits for nerve tissue engineering.

Peripheral nerve injury is a common clinical problem often requiring surgical nerve reconstruction. To this end, tissue-engineered conduit has been proved to be crucial for nerve reconstruction. Despite its progress in recent years, the design and fabrication of translational biomimetic nerve conduits is highly challenging. Therefore, this study aims to design and fabricate mechanically-tunable nerve conduits with biomimetic structural features of the human nerve suitable for nerve tissue engineering. Herein, we employed combinatorial approach comprising of electrohydrodynamic (EHD) jet printing, dip-coating, and electrospinning techniques for fabricating triple-layered conduits. The intricate structural details were achieved via high-resolution EHD jet printed PCL filaments with tunable directionality, as the innermost layer; followed by dip coating of gelatin hydrogels to form the middle layer, and lastly, wrapped with electrospun PCL nanofibers as an outer layer of the conduits. The mechanical properties, porosity, and biocompatibility of the fabricated conduits were studied and compared with control. The results of this study confirmed that the combinatorial approach has greater potential to fabricate mechanically-tunable triple-layered conduits with favorable neuronal precursor and vascular cell compatibility.

Fabrication of nanochitosan incorporated polypyrrole/alginate conducting scaffold for neural tissue engineering.

The utilization of conductive polymers for fabrication of neural scaffolds have attracted much interest because of providing a microenvironment which can imitate nerve tissues. In this study, polypyrrole (PPy)-alginate (Alg) composites were prepared using different percentages of alginate and pyrrole by oxidative polymerization method using FeCl3 as an oxidant and electrical conductivity of composites were measured by four probe method. In addition, chitosan-based nanoparticles were synthesized by ionic gelation method and after characterization merged into PPy-Alg composite in order to fabricate a conductive, hydrophilic, processable and stable scaffold. Physiochemical characterization of nanochitosan/PPy-Alg scaffold such as electrical conductivity, porosity, swelling and degradation was investigated. Moreover, cytotoxicity and proliferation were examined by culturing OLN-93 neural and human dermal fibroblasts cells on the Nanochitosan/PPy-Alg scaffold. Due to the high conductivity, the film with ratio 2:10 (PPy-Alg) was recognized more suitable for fabrication of the final scaffold. Results from FT-IR and SEM, evaluation of porosity, swelling and degradation, as well as viability and proliferation of OLN-93 neural and fibroblast cells confirmed cytocompatiblity of the Nanochitosan/PPy-Alg scaffold. Based on the features of the constructed scaffold, Nanochitosan/PPy-Alg scaffold can be a proper candidate for neural tissue engineering.

Zero-valent iron nanoparticles containing nanofiber scaffolds for nerve tissue engineering.

Regeneration of nerve tissue is a challenging issue in regenerative medicine. Especially, the peripheral nerve defects related to the accidents are one of the leading health problems. For large degeneration of peripheral nerve, nerve grafts are used in order to obtain a connection. These grafts should be biodegradable to prevent second surgical intervention. In order to make more effective nerve tissue engineering materials, nanotechnological improvements were used. Especially, the addition of electrically conductive and biocompatible metallic particles and carbon structures has essential roles in the stimulation of nerves. However, the metabolizing of these structures remains to wonder because of their nondegradable nature. In this study, biodegradable and conductive nerve tissue engineering materials containing zero-valent iron (Fe) nanoparticles were developed and investigated under in vitro conditions. By using electrospinning technique, fibrous mats composed of electrospun poly(ε-caprolactone) (PCL) nanofibers and Fe nanoparticles were obtained. Both electrical conductivity and mechanical properties increased compared with control group that does not contain nanoparticles. Conductivity of PCL/Fe5 and PCL/Fe10 increased to 0.0041 and 0.0152 from 0.0013 Scm-1 , respectively. Cytotoxicity results indicated toxicity for composite mat containing 20% Fe nanoparticles (PCL/Fe20). SH-SY5Y cells were grown on PCL/Fe10 best, which contains 10% Fe nanoparticles. Beta III tubulin staining of dorsal root ganglion neurons seeded on mats revealed higher cell number on PCL/Fe10. This study demonstrated the impact of zero-valent Fe nanoparticles on nerve regeneration. The results showed the efficacy of the conductive nanoparticles, and the amount in the composition has essential roles in the promotion of the neurites.

Bone marrow mesenchymal stem cells encapsulated thermal-responsive hydrogel network bridges combined photo-plasmonic nanoparticulate system for the treatment of urinary bladder dysfunction after spinal cord injury.

Spinal cord injury (SCI) is a distressing injury and an irretrievable dramatic event that can debilitate victims for lifespan. Recovery and treatment of SCI is critical challenges for medicine, to overcome the hurdles stem cells and hydrogel scaffolds implantation is a boon for SCI recovery. In this regard, we reported the synthesis of Gold nanoparticles (Au NPs) loaded Agarose/Poly (N-isopropylacrylamide) (PNIPAM) as promising materials for SCI treatment. Herein, Au NPs was synthesized by well-established citrate reduction method and the prepared materials were characterised by UV-visible spectroscopy (UV-vis), Transmission electron microscopy (TEM), Fourier- transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and EDAX analysis. The microscopic images showed an elliptical or ovoid porous structure nature of hydrogel, and successful and homogenous loading of photo plasmonic nanoparticles into the hydrogel structure. The in vitro cell viability and inflammation analyses data exhibited that prepared hydrogels have no toxic to the cells and displayed high anti-regenerative ability with bone marrow Mesenchymal stem cells (MSCs) and macrophages cells. The in vivo analysis study demonstrated that the treated materials with encapsulated MSCs have greater nerve tissue regeneration efficacy which was confirmed by the results of BBB scores. The hind limb locomotion of treated model animals was totally vanished after post-operational surgery. It's established that implanted nano-hydrogel materials combined with MSCs have quicker recovery of motor function after post-operative surgery, when compared to the other implanted animal groups.

Integrating Organs-on-Chips: Multiplexing, Scaling, Vascularization, and Innervation.

Organs-on-chips (OoCs) have attracted significant attention because they can be designed to mimic in vivo environments. Beyond constructing a single OoC, recent efforts have tried to integrate multiple OoCs to broaden potential applications such as disease modeling and drug discoveries. However, various challenges remain for integrating OoCs towards in vivo-like operation, such as incorporating various connections for integrating multiple OoCs. We review multiplexed OoCs and challenges they face: scaling, vascularization, and innervation. In our opinion, future OoCs will be constructed to have increased predictive power for in vivo phenomena and will ultimately become a mainstream tool for high quality biomedical and pharmaceutical research.

Determination of the electrical impedance of neural tissue from its microscopic cellular constituents.

The electrical properties of neural tissue are important in a range of different applications in biomedical engineering and basic science. These properties are characterized by the electrical admittivity of the tissue, which is the inverse of the specific tissue impedance. OBJECTIVE: Here we derived analytical expressions for the admittivity of various models of neural tissue from the underlying electrical and morphological properties of the constituent cells. APPROACH: Three models are considered: parallel bundles of fibers, fibers contained in stacked laminae and fibers crossing each other randomly in all three-dimensional directions. MAIN RESULTS: An important and novel aspect that emerges from considering the underlying cellular composition of the tissue is that the resulting admittivity has both spatial and temporal frequency dependence, a property not shared with conventional conductivity-based descriptions. The frequency dependence of the admittivity results in non-trivial spatiotemporal filtering of electrical signals in the tissue models. These effects are illustrated by considering the example of pulsatile stimulation with a point source electrode. It is shown how changing temporal parameters of a current pulse, such as pulse duration, alters the spatial profile of the extracellular potential. In a second example, it is shown how the degree of electrical anisotropy can change as a function of the distance from the electrode, despite the underlying structurally homogeneity of the tissue. These effects are discussed in terms of different current pathways through the intra- and extra-cellular spaces, and how these relate to near- and far-field limits for the admittivity (which reduce to descriptions in terms of a simple conductivity). SIGNIFICANCE: The results highlight the complexity of the electrical properties of neural tissue and provide mathematical methods to model this complexity.

Chitosan-g-oligo(L,L-lactide) copolymer hydrogel for nervous tissue regeneration in glutamate excitotoxicity: in vitro feasibility evaluation.

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 µM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

Electrospun natural polymer and its composite nanofibrous scaffolds for nerve tissue engineering.

Attributed to the excellent biocompatibility and desirable mechanical properties to natural tissue, natural polymer-based electrospun nanofibers have drawn extensive research interests in tissue engineering. Electrospun nanofibers have been explored as scaffolds in tissue engineering to modulate cellular behavior. Also, electrospun nanofiber matrices have morphological similarities to the natural extra-cellular matrix (ECM). Natural polymer and its composite nanofiber mats are the promising candidates in governing nerve cells growth and nerve regeneration due to their unique characteristics such as high permeability, stability, porosity, suitable mechanical performance and excellent biocompatibility. In this review, the progress in electrospun natural polymers and its composite nanofibers scaffold for neural tissue engineering are presented. The influences of fiber orientation and electrical stimulation on the nerve cell behavior and neurite growth are systematically summarized. Furthermore, the current application of natural polymer composite scaffold as in vivo implantable device for nerve regeneration is also discussed (see Figure 1).

Participation of cAMP/PKA-Mediated Signaling Pathways in Functional Activity of Regeneration-Competent Cells in the Nervous Tissue under Conditions of Ethanol-Induced Neurodegeneration.

We studied the involvement of cAMP/PKA signaling in the realization of the growth potential of neural progenitors and secretion of neurotrophic growth factors by glial elements under conditions of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP and PKA in cell cycle progression of the neural progenitor cells and in production of neurotrophins by the cells in nervous tissue under the optimal conditions to vital activity was demonstrated. Ethanol inverted the role of cAMP/PKA signaling pathways in determination of the proliferation-differentiation status of neural stem cells. Selective blockade of adenylate cyclase or PKA in neural stem cells increased the rate of their division against the background of relative decrease in differentiation rate. In addition, cAMP/PKA signaling does not longer participate in neurotrophin production by glial cells in neurodegeneration. These findings suggest that inhibitors of activity/expression of adenylate cyclase and PKA can be considered as possible drugs with regenerative activity for the treatment of nervous system pathologies provoked by alcohol.

Ultra-low percolation threshold POSS-PCL/graphene electrically conductive polymer: Neural tissue engineering nanocomposites for neurosurgery.

An ultra-low percolation threshold electrically conductive polymer nanocomposite incorporating graphene into a polyhedral oligomeric silsesquioxane polycaprolactone (POSS-PCL/graphene) is described in this paper. Multilayer graphene flakes were homogeneously dispersed into POSS-PCL at 0.08, 0.4, 0.8, 1.6, and 4.0 wt% concentrations. The impedance spectroscopy of 0.08 wt% and higher concentration of graphene in POSS-PCL represented major improvement in conductivity over pristine POSS-PCL. The percolation threshold occurred at 0.08 wt% graphene concentration, and at 4.0 wt% the electrical conductivity exceeded 10-4 Scm-1. Furthermore, the chemical, morphological, and mechanical of the POSS-PCL/graphene with various graphene concentrations were investigated. Finally, neural cells cultured on all POSS-PCL/graphene constructs indicated higher metabolic activity and cell proliferation in comparison with pristine POSS-PCL. Herein, we demonstrate a method of developing a neural-compatible and electrically conductive polymer nanocomposite that could potentially function as a neural tissue engineered platform technology for neurological and neurosurgical applications.

Human Neural Tissues from Neural Stem Cells Using Conductive Biogel and Printed Polymer Microelectrode Arrays for 3D Electrical Stimulation.

Electricity is important in the physiology and development of human tissues such as embryonic and fetal development, and tissue regeneration for wound healing. Accordingly, electrical stimulation (ES) is increasingly being applied to influence cell behavior and function for a biomimetic approach to in vitro cell culture and tissue engineering. Here, the application of conductive polymer (CP) poly(3,4-ethylenedioxythiophene)-polystyrenesulfonate (PEDOT:PSS) pillars is described, direct-write printed in an array format, for 3D ES of maturing neural tissues that are derived from human neural stem cells (NSCs). NSCs are initially encapsulated within a conductive polysaccharide-based biogel interfaced with the CP pillar microelectrode arrays (MEAs), followed by differentiation in situ to neurons and supporting neuroglia during stimulation. Electrochemical properties of the pillar electrodes and the biogel support their electrical performance. Remarkably, stimulated constructs are characterized by widespread tracts of high-density mature neurons and enhanced maturation of functional neural networks. Formation of tissues using the 3D MEAs substantiates the platform for advanced clinically relevant neural tissue induction, with the system likely amendable to diverse cell types to create other neural and non-neural tissues. The platform may be useful for both research and translation, including modeling tissue development, function and dysfunction, electroceuticals, drug screening, and regenerative medicine.

Preprocessing of Medical Image Data for Three-Dimensional Bioprinted Customized-Neural-Scaffolds.

IMPACT STATEMENT: Nerve damage, which can be devastating, triggers several biological cascades, which result in the insufficiencies of the human nervous system to provide complete nerve repair and regain of function. Since no therapeutic strategy exists to provide immediate attention and intervention to patients with newly acquired nerve damage, we propose a strategy in which accelerated medical image processing through graphical processing unit implementation and three-dimensional printing are combined to produce a time-efficient, patient-specific (custom-neural-scaffold) solution to nerve damage. This work aims to beneficially shorten the time required for medical decision-making so that improved patient outcomes are achieved.