Chitosan/PLGA-based tissue engineered nerve grafts with SKP-SC-EVs enhance sciatic nerve regeneration in dogs through miR-30b-5p-mediated regulation of axon growth

Extracellular vesicles from skin-derived precursor Schwann cells(SKP-SC-EVs)promote neurite outgrowth in culture and enhance peripheral nerve regeneration in rats.This study aimed at expanding the application of SKPSC-EVs in nerve grafting by creating a chitosan/PLGA-based,SKP-SC-EVs-containing tissue engineered nerve graft(TENG)to bridge a 40-mm long sciatic nerve defect in dogs.SKP-SC-EVs contained in TENGs significantly accelerated the recovery of hind limb motor and electrophysiological functions,supported the outgrowth and myelination of regenerated axons,and alleviated the denervation-induced atrophy of target muscles in dogs.To clarify the underlying molecular mechanism,we observed that SKP-SC-EVs were rich in a variety of miRNAs linked to the axon growth of neurons,and miR-30b-5p was the most important among others.We further noted that miR-30b-5p contained within SKP-SC-EVs exerted nerve regeneration-promoting effects by targeting the Sin3a/HDAC complex and activating the phosphorylation of ERK,STAT3 or CREB.Our findings suggested that SKP-SC-EVs-incorporating TENGs represent a novel type of bioactive material with potential application for peripheral nerve repair in the clinic.

RNA sequencing of exosomes secreted by fibroblast and Schwann cells elucidates mechanisms underlying peripheral nerve regeneration

Exosomes exhibit complex biological functions and mediate a variety of biological processes,such as promoting axonal regeneration and functional recove ry after injury.Long non-coding RNAs(IncRNAs)have been reported to play a crucial role in axonal regeneration.Howeve r,the role of the IncRNA-microRNAmessenger RNA(mRNA)-competitive endogenous RNA(ceRNA)network in exosome-mediated axonal regeneration remains unclear.In this study,we performed RNA transcriptome sequencing analysis to assess mRNA expression patterns in exosomes produced by cultured fibroblasts(FC-EXOs)and Schwann cells(SCEXOs).Diffe rential gene expression analysis,Gene Ontology analysis,Kyoto Encyclopedia of Genes and Genomes analysis,and protein-protein intera ction network analysis were used to explo re the functions and related pathways of RNAs isolated from FC-EXOs and SC-EXOs.We found that the ribosome-related central gene Rps5 was enriched in FC-EXOs and SC-EXOs,which suggests that it may promote axonal regeneration.In addition,using the miRWalk and Starbase prediction databases,we constructed a regulatory network of ceRNAs targeting Rps5,including 27 microRNAs and five IncRNAs.The ceRNA regulatory network,which included Ftx and Miat,revealed that exsosome-derived Rps5 inhibits scar formation and promotes axonal regeneration and functional recovery after nerve injury.Our findings suggest that exosomes derived from fibro blast and Schwann cells could be used to treat injuries of peripheral nervous system.

Pathogenesis, diagnosis, and treatment of epilepsy: electromagnetic stimulation-mediated neuromodulation therapy and new technologies

Epilepsy is a severe,relapsing,and multifactorial neurological disorder.Studies regarding the accurate diagnosis,prognosis,and in-depth pathogenesis are crucial for the precise and effective treatment of epilepsy.The pathogenesis of epilepsy is complex and involves alterations in variables such as gene expression,protein expression,ion channel activity,energy metabolites,and gut microbiota composition.Satisfactory results are lacking for conventional treatments for epilepsy.Surgical resection of lesions,drug therapy,and non-drug interventions are mainly used in clinical practice to treat pain associated with epilepsy.Non-pharmacological treatments,such as a ketogenic diet,gene therapy for nerve regeneration,and neural regulation,are currently areas of research focus.This review provides a comprehensive overview of the pathogenesis,diagnostic methods,and treatments of epilepsy.It also elaborates on the theoretical basis,treatment modes,and effects of invasive nerve stimulation in neurotherapy,including percutaneous vagus nerve stimulation,deep brain electrical stimulation,repetitive nerve electrical stimulation,in addition to non-invasive transcranial magnetic stimulation and transcranial direct current stimulation.Numerous studies have shown that electromagnetic stimulation-mediated neuromodulation therapy can markedly improve neurological function and reduce the frequency of epileptic seizures.Additionally,many new technologies for the diagnosis and treatment of epilepsy are being explored.However,current research is mainly focused on analyzing patients’clinical manifestations and exploring relevant diagnostic and treatment methods to study the pathogenesis at a molecular level,which has led to a lack of consensus regarding the mechanisms related to the disease.

Multilevel analysis of the central-peripheral-target organ pathway:contributing to recovery after peripheral nerve injury

Peripheral nerve injury is a common neurological condition that often leads to severe functional limitations and disabilities.Research on the pathogenesis of peripheral nerve injury has focused on pathological changes at individual injury sites,neglecting multilevel pathological analysis of the overall nervous system and target organs.This has led to restrictions on current therapeutic approaches.In this paper,we first summarize the potential mechanisms of peripheral nerve injury from a holistic perspective,covering the central nervous system,peripheral nervous system,and target organs.After peripheral nerve injury,the cortical plasticity of the brain is altered due to damage to and regeneration of peripheral nerves;changes such as neuronal apoptosis and axonal demyelination occur in the spinal cord.The nerve will undergo axonal regeneration,activation of Schwann cells,inflammatory response,and vascular system regeneration at the injury site.Corresponding damage to target organs can occur,including skeletal muscle atrophy and sensory receptor disruption.We then provide a brief review of the research advances in therapeutic approaches to peripheral nerve injury.The main current treatments are conducted passively and include physical factor rehabilitation,pharmacological treatments,cell-based therapies,and physical exercise.However,most treatments only partially address the problem and cannot complete the systematic recovery of the entire central nervous system-peripheral nervous system-target organ pathway.Therefore,we should further explore multilevel treatment options that produce effective,long-lasting results,perhaps requiring a combination of passive(traditional)and active(novel)treatment methods to stimulate rehabilitation at the central-peripheral-target organ levels to achieve better functional recovery.

Sustainable biomedical microfibers from natural products

Microfibers from natural products are endowed with remarkable biocompatibility,biodegradability,sustainable utilization as well as environmental protection char-acteristics etc.Benefitting from these advantages,microfibers have demonstrated their prominent values in biomedical applications.This review comprehensively summarizes the relevant research progress of sustainable microfibers from natural products and their biomedical applications.To begin,common natural elements are introduced for the microfiber fabrication.After that,the focus is on the specific fabri-cation technology and process.Subsequently,biomedical applications of sustainable microfibers are discussed in detail.Last but not least,the main challenges during the development process are summarized,followed by a vision for future development opportunities.

Biophysical Regulation of Cell Behavior-Cross Talk between Substrate Stiffness and Nanotopography

The stiffness and nanotopographical characteristics of the extracellular matrix （ECM） influence numerous developmental, physiological, and pathological processes in vivo. These biophysical cues have therefore been applied to modulate almost all aspects of cell behavior, from cell adhesion and spreading to proliferation and differentiation. Delineation of the biophysical modulation of cell behavior is critical to the rational design of new biomaterials, implants, and medical devices. The effects of stiffness and topographical cues on cell behavior have previously been reviewed, respectively; however, the interwoven effects of stiffness and nanotopographical cues on cell behavior have not been well described, despite similarities in phenotypic manifestations. Herein, we first review the effects of substrate stiffness and nanotopography on cell behavior, and then focus on intracellular transmission of the biophysical signals from integrins to nucleus. Attempts are made to connect extracellular regulation of cell behavior with the biophysical cues. We then discuss the challenges in dissecting the biophysical regulation of cell behavior and in translating the mechanistic understanding of these cues to tissue engineering and regenerative medicine.

Tissue Engineering and Regulatory Science

Tissue engineering has successfully evolved from its original concept[1]into medical products with a rapid pace of develop-ment and a multi-billion dollar market[2].Compared with tradi-tional medical products,tissue-engineered medical products(TEMPs)have distinct characteristics that provide unique benefits for the repair and regeneration of damaged or diseased tissues or organs[1,2].

Current Challenges for the Practical Application of Electroencephalography-Based Brain-Computer Interfaces

It has been almost 50 years since the term“brain–computer interface”(BCI)was first proposed by Jacques J.Vidal in 1973[1].Unlike traditional electronic interfaces that transmit nonliving information between devices,BCIs set up a communication bridge between a living brain and nonliving devices.Technically speaking,a BCI is a system that measures brain activity and converts it into the artificial outputs that replace,restore,enhance,supplement,or improve the natural central nervous system outputs[2].At present,electroencephalography(EEG)is the most commonly used brain signal for BCIs.

The Achyranthes bidentata polypeptide k fraction enhances neuronal growth in vitro and promotes peripheral nerve regeneration after crush injury in vivo

We have previously shown that Achyranthes bidentata polypeptides （ABPP）, isolated from Achyranthes bidentata Blume （a medicinal herb）, exhibit neurotrophic and neuroprotective effects on the nervous system. To identify the major active component of ABPP, and thus optimize the use of ABPP, we used reverse-phase high performance liquid chromatography to separate ABPP. We obtained 12 fractions, among which the fraction of ABPPk demonstrated the strongest neuroactivity. Immunocytochemistry and western blot analysis showed that ABPPk promoted neurite growth in cultured dorsal root ganglion explant and dorsal root ganglion neurons, which might be associated with activation of Erk1/2. A combination of behavioral tests, electrophysiological assessment, and histomorphometric analysis indicated that ABPPk enhanced nerve regeneration and function restoration in a mouse model of crushed sciatic nerve. All the results suggest that ABPPk, as the key component of ABPP, can be used for peripheral nerve repair to yield better outcomes than ABPP.

Gene expression profiling of the rat sciatic nerve in early Wallerian degeneration after injury

Wallerian degeneration is an important area of research in modern neuroscience. A large number of genes are differentially regulated in the various stages of Wallerian degeneration, especially during the early response. In this study, we analyzed gene expression in early Wallerian degeneration of the distal nerve stump at 0, 0.5, 1,6, 12 and 24 hours after rat sciatic nerve injury using gene chip microarrays. We screened for differentially-expressed genes and gene expression patterns. We examined the data for Gene Ontology, and explored the Kyoto EncycLopedia of Genes and Genomes Pathway. This allowed us to identify key regulatory factors and recurrent network motifs. We identified 1 546 differentially-expressed genes and 21 distinct patterns ofgene expression in early Wallerian degeneration, and an enrichment of genes associated with the immune response, acute inflammation, apoptosis, cell adhesion, ion transport and the extracellular matrix. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed components involved in the Jak-STAT, ErbB, transforming growth factor-13, T cell receptor and calcium signaling pathways. Key factors included interleukin-6, interleukin-1, integrin, c-sarcoma, carcinoembryonic antigen-related cell adhesion molecules, chemokine （C-C motif） ligand, matrix metalloproteinase, BH3 interacting domain death agonist, baculoviral lAP repeat-containing 3 and Rac. The data were validated with real-time quantitative PCR. This study provides a global view of gene expression profiles in eady Wallerian degeneration of the rat sciatic nerve. Our findings provide insight into the molecular mechanisms underlying early Wallerian degeneration, and the regulation of nerve degeneration and regeneration.

Neurological function following intra-neural injection of fluorescent neuronal tracers in rats

Fluorescent neuronal tracers should not be toxic to the nervous system when used in long-term labeling. Previous studies have addressed tracer toxicity, but whether tracers injected into an intact nerve result in functional impairment remains to be elucidated. In the present study, we examined the functions of motor, sensory and autonomic nerves following the application of 5% Fluoro-Gold, 4% True Blue and 10% Fluoro-Ruby （5 pL） to rat tibial nerves via pressure injection. A set of evaluation methods including walking track analysis, plantar test and laser Doppler perfusion imaging was used to determine the action of the fluorescent neuronal tracers. Additionally, nerve pathology and ratio of muscle wet weight were also observed. Results showed that injection of Fluoro-Gold significantly resulted in loss of motor nerve function, lower plantar sensibility, increasing blood flow volume and higher neurogenic vasodilatation. Myelinated nerve fiber degeneration, unclear boundaries in nerve fibers and high retrograde labeling efficacy were observed in the Fluoro-Gold group. The True Blue group also showed obvious neurogenic vasodilatation, but less severe loss of motor function and degeneration, and fewer labeled motor neurons were found compared with the Fluoro-Gold group. No anomalies of motor and sensory nerve function and no myelinated nerve fiber degeneration were observed in the Fluoro-Ruby group. Experimental findings indicate that Fluoro-Gold tracing could lead to significant functional impairment of motor, sensory and autonomic nerves, while functional impairment was less severe following True Blue tracing. Fluoro-Ruby injection appears to have no effect on neurological function.

Developmental Temporal Patterns and Molecular Network Features in the Transcriptome of Rat Spinal Cord

The molecular network features of spinal cord development that are integral to tissue engineering remain poorly understood in placental mammals,especially in terms of their relationships with vital biological processes such as regeneration.Here,using a large-scale temporal transcriptomic analysis of rat spinal cord from the embryonic stage to adulthood,we show that fluctuating RNA expression levels reflect highly active transcriptional regulation,which may initiate spinal cord patterning.We also demonstrate that microRNAs(miRNAs)and transcriptional factors exhibit a mosaic profile based on their expression patterns,while differential alternative splicing events reveal that alternative splicing may be a driving force for the development of the node of Ranvier.Our study also supports the existence of a negative correlation between innate immunity and intrinsic growth capacity.Epigenetic modifications appear to perform their respective regulatory functions at different stages of development,while guanine nucleotidebinding protein(G protein)-coupled receptors(including olfactory receptors(ORs))may perform pleiotropic roles in axonal growth.This study provides a valuable resource for investigating spinal cord development and complements the increasing number of single-cell datasets.These findings also provide a genetic basis for the development of novel tissue engineering strategies.

S100 protein expression during induced Schwann cell-like cell differentiation of rat bone marrow mesenchymal cells in vitro

BACKGROUND： S100 protein can promote axonal growth. Therefore, transplantation of induced bone marrow-derived mesenchymal stem cells （MSCs） that can secrete S100 may provide a beneficial microenvironment for neural regeneration. OBJECTIVE： To explore the changes in S100 expression during rat MSCs differentiation into Schwann ceils in vitro. DESIGN, TIME AND SETTING： This cytology experiment was performed at the Jiangsu Key Laboratory of Neuroregeneration, Nantong University in China, from January 2006 to May 2007. MATERIALS： The rabbit anti-S100 polyclonal antibody was purchased from Dako, Denmark; the mouse anti-rat S100 monoclonal antibody was purchased from Sigma, USA. METHODS： MSCs were cultured from adult Sprague-Dawley rat femur and tibia. Cell proliferation was determined by the MTT method and CD markers, and cell cycle was measured by flow cytometry. MSCs were induced to differentiate into SC cells. SC cells were stained for S100 protein, glial fibrillary acidic protein, and low-affinity nerve growth factor receptor. S100 protein and mRNA levels were evaluated by flow cytometry, Western blot, and reverse transcription-polymerase chain reaction. MAIN OUTCOME MEASURES： S100 protein and mRNA expression. RESULTS： MSCs exhibited high amplification potential over eight passages. Prior to induction, the majority of MSCs were at the G0/G1 phase of the cell cycle. After induction, MSCs displayed morphology changes similar to Schwann cells. Moreover, induction increased S100 mRNA levels. Immunofluorescence showed that MSCs expressed S100 protein, glial fibrillary acidic protein, and low-affinity nerve growth factor receptor at 7 days of induction. Induction also increased S100 protein levels compared with untreated MSCs. CONCLUSION： MSCs are capable of differentiating into Schwann cells-like cells under conditional induction in vitro, with increasing S100 mRNA and protein expression.

Protective effects of nerve regeneration factor and brain-derived neurotrophic factor on retinal ganglion cells in a rabbit model of acute hyper-intraocular pressure

BACKGROUND： Previous studies have shown that nerve regeneration factor （NRF） provides neuroprotective effects. However, the neuroprotective effects on retinal ganglion cells in an animal model of glaucoma remain uncertain. OBJECTIVE： To determine the neuroprotective effects of NRF on retinal ganglion cells in a rabbit model of acute hyper-intraocular pressure and to compare the effects on brain-derived neurotrophic factor （BDNF）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at Jiangsu Provincial Key Laboratory of Neural Regeneration from September 2006 to August 2007. MATERIALS： Sterone, a major component of NRF, was provided by the Key Laboratory of Neural Regeneration, Nantong University in China; BDNF was provided by BioDesign Inc., USA. METHODS： A total of 24 healthy rabbits were randomly assigned to NRF, BDNF, and phosphate buffered saline groups, with 8 rabbits per group. The left eyes were considered normal controls, and acute hyper-intraocular pressure was induced in the right eyes via anterior chamber perfusion. The right camera vitrea bulbi was injected with 4.5 μg NRF, 3.75 μg BDNF, or 5 μL 0.1 mol/L phosphate buffered saline, respectively. MAIN OUTCOME MEASURES： Retinal ganglion cells were reverse-labeled using horseradish peroxidase to quantify cell density at 2, 4, and 6 mm from the optic disc edge. RESULTS： NRF increased the number of surviving retinal ganglion cells at the optic disc edge （P 〈 0.01 or P 〈 0.05）. The density of surviving retinal ganglion cells decreased with increasing distance from the optic disc. The number of retinal ganglion cells in the BDNF group was similar to the NRF group （P 〉 0.05）. At 2, 4, and 6 mm away from the optic disc edge, there was no significant difference in retinal ganglion cell density between NRF and BDNF groups （P〉 0.05）. CONCLUSION： NRF provided protection to retinal ganglion cells in a rabbit model of acute hyper-intraocular pressure, Le., NRF enhanced the survival rate of retinal ganglion cells. The neuroprotective effect was similar to BDNF.

Three-dimensional reconstruction of human spinal cord based on histological serial sections

BACKGROUND： It is not possible to reconstruct the inner structure of the spinal cord, such as gray matter and spinal tracts, from the Visual Human Project database or CT and MRI databases, due to low image resolution and contrast in macrosection images. OBJECTIVE： To explore a semi-automatic computerized three-dimensional （3D） reconstruction of human spinal cord based on histological serial sections, in order to solve issues such as low contrast. DESIGN, TIME AND SETTING： An experimental study combining serial section techniques and 3D reconstruction, performed in the laboratory of Human Anatomy and Histoembryology at the Medical School of Nantong University during January to April 2008. SETTING： Department of Anatomy, Institute of Neurobiology, Jiangsu Province Key Laboratory of Neural Regeneration, Laboratory of Image Engineering. MATERIALS： A human lumbar spinal cord segment from fresh autopsy material of an adult male. METHODS： After 4% paraformaldehyde fixation for three days, serial sections of the lumbar spinal cord were cut on a Leica cryostat and mounted on slides in sequence, with eight sections aligned separately on each slide. All sections were stained with Luxol Fast Blue to reveal myelin sheaths. After gradient dehydration and clearing, the stained slides were coverslipped. Sections were observed and images recorded under a light microscope using a digital camera. Six images were acquired at x25 magnification and automatically stitched into a complete section image. After all serial images were obtained, 96 complete serial images of the human lumbar cord segment were automatically processed with ＂Curves＂, ＂Autocontrast＂, ＂Gray scale 8 bit＂, ＂Invert＂, ＂Image resize to 50%＂ steps using Photoshop 7.0 software. All images were added in order into 3D-DOCTOR 4.0 software as a stack, where serial images were automatically realigned with neighboring images and semi-automatically segmented for white matter and gray matter. Finally, simple surface and volume reconstruction were completed on a personal computer. The reconstructed human lumbar spinal cord segment was interactively observed, cut, and measured. MAIN OUTCOME MEASURES： The reconstructed human lumbar spinal cord segment. RESULTS： Compared with serial images obtained from other image modalities, such as CT, MRI, and macrosections from The Visual Human Project database, the Luxol Fast Blue stained histological serial section images exhibited higher resolution and contrast between gray and white matter. Image processing and 3D reconstruction steps were semi-automatically performed with related software. The 3D reconstructed human lumbar cord segment were observed, cut, and measured on a PC. CONCLUSION： A semi-automatically computerized method, based on histological serial sections, is an effective way to 3D-reconstruct the human spinal cord.

Noncoding RNAs and Their Potential Therapeutic Applications in Tissue Engineering

Tissue engineering is a relatively new but rapidly developing field in the medical sciences. Noncoding RNAs(ncRNAs) are functional RNA molecules without a protein-coding function; they can regulate cellular behavior and change the biological milieu of the tissue. The application of ncRNAs in tissue engineering is starting to attract increasing attention as a means of resolving a large number of unmet healthcare needs, although ncRNA-based approaches have not yet entered clinical practice. In-depth research on the regulation and delivery of ncRNAs may improve their application in tissue engineering.The aim of this review is: to outline essential ncRNAs that are related to tissue engineering for the repair and regeneration of nerve, skin, liver, vascular system, and muscle tissue; to discuss their regulation and delivery; and to anticipate their potential therapeutic applications.

Biodegradable Materials and the Tissue Engineering of Nerves

1.Introduction Over the past 100 years,the development of biodegradable materials has helped to advance innovation in tissue-engineering technology by making such materials more feasible,resulting in technological breakthroughs and new clinical applications[1,2].Classical tissue-engineering theory involves three elements:biological materials,seed cells,and factors.Research progress suggests that biodegradable materials with low immunogenicity,high biodegradability,good biocompatibility,and favorable regeneration microenvironments are of great significance[3].

Tissue Engineering Is Under Way

Tissue engineering involves bioma- terials, stem cells, associated factors and construction technologies, and clinical translation. Researchers from around the world have put great ef- fort into making breakthroughs in the translation and application of tissue engineering. In recent years within China, scholars in this field have conducted highly successful work, such as： artificial skin substitutes for burn therapy （Xiaobing Fu et al.：

Biocompatibility and biosafety of butterfly wings for the clinical use of tissue-engineered nerve grafts

In a previous study, we used natural butterfly wings as a cell growth matrix for tissue engineering materials and found that the surface of different butterfly wings had different ultramicrostructures, which can affect the qualitative growth of cells and regulate cell growth, metabolism, and gene expression. However, the biocompatibility and biosafety of butterfly wings must be studied. In this study, we found that Sprague-Dawley rat dorsal root ganglion neurons could grow along the structural stripes of butterfly wings, and Schwann cells could normally attach to and proliferate on different species of butterfly wings. The biocompatibility and biosafety of butterfly wings were further examined through subcutaneous implantation in Sprague-Dawley rats, intraperitoneal injection in Institute of Cancer Research mice, intradermal injection in rabbits, and external application to guinea pigs. Our results showed that butterfly wings did not induce toxicity, and all examined animals exhibited normal behaviors and no symptoms, such as erythema or edema. These findings suggested that butterfly wings possess excellent biocompatibility and biosafety and can be used as a type of tissue engineering material. This study was approved by the Experimental Animal Ethics Committee of Jiangsu Province of China(approval No. 20190303-18) on March 3, 2019.

Protective effects of Achyranthes bidentata polypeptides on retinal ganglion cells post-optic nerve crush in rats

Achyranthes bidentata polypeptides（ABPP） have been reported to inhibit apoptosis of retinal ganglion cells（RGCs）.The present study investigated the protective effects of ABPP on RGCs in a rat model of optic nerve injury.With prolonged injury time,RGC densities were gradually decreased.ABPP（5 μg） significantly increased RGC densities and upregulated growth associated protein 43 expression in rats with optic nerve injury.Results demonstrate that ABPP can protect RGCs and promote axonal growth after optic nerve crush.