Regenerative potential of targeting glycogen synthase kinase-3 signaling in neural tissues

Multiple roles of glycogen synthase kinase-3（GSK-3）in neural tissues：GSK-3 is a serine/threonine kinase that has two isoforms encoded by two different genes,GSK-3αand GSK-3β,in mammals.GSK-3 has several sites of serine and tyrosine phosphorylation.

Novel advancements in threedimensional neural tissue engineering and regenerative medicine

Neurological diseases and injuries present some of the great- est challenges in modern medicine, often causing irrevers- ible and lifelong burdens in the people whom they afflict. Conditions of stroke, traumatic brain injury, spinal cord injury, and neurodegenerative diseases have devastating con- sequences on millions of people each year, and yet there are currently no therapies or interventions that can repair the structure of neural circuits and restore neural tissue function in the brain and spinal cord. Despite the challenges of over- coming these limitations, there are many new approaches under development that hold much promise. Neural tissue engineering aims to restore and influence the function of damaged or diseased neural tissue generally through the use of stem cells and biomaterials. Many types of biomaterials may be implemented in various designs to influence the survival, differentiation, and function of developing stem cells, as well as to guide neurite extension and morphological architecture of cell cultures. Such designs may aim to reca- pitulate the cellular interactions, extracellular matrix char- acteristics, biochemical factors, and sequences of events that occur in neurodevelopment, in addition to supporting cell survival, differentiation, and integration into innate neural tissue.

Biotinylated dextran amine as a neural tracer in the rat corticospinal tract

BACKGROUND： The corticospinal tract is the core structure of cerebral control of extremity movement and plasticity, which are prerequisites for movement rehabilitation after brain injury. The measurement and assessment of plasticity changes within the corticospinal tract has become one of the key goals in this field. OBJECTIVE： To explore the effects of biotinylated dextran amine （BDA） as a neural tracer in the rat corticospinal tract and the possibilities of assessing plasticity within the corticospinal tract. DESIGN： An observational experiment. SETTING： Department of Acupuncture of Chinese Medical College, Chongqing Medical University, Department of Neurology, the Second Affiliated Hospital, Chongqing Medical University. MATERIALS： Eighteen male adult Sprague Dawley （SD） rats of clean grade, weighing 200-250 g, were provided by the experimental animal center of Chongqing Medical University. The animal procedures in this study were in accordance with the animal ethics standards. BDA was provided by Vector Laboratories Company （USA, catalogue Sp- 1140; serial number R0721 ）. METHODS. This experiment was performed in the Laboratory of Chongqing Medical University between September and December 2006. Adult SD rats were used in the experiment and 15% BDA was injected slowly with a mini-syringe through two round （3 mm diameter） holes into the left sensory and motor cortex. The center of one hole was located 3 mm anterior from the anterior fontanel and 1.5 mm left of the midline; the second hole was located 1.5 mm posterior from the anterior fontanel and 4 mm left of the midline. Three injections were made at each hole at three different levels： 1.4, 1.2, and 1 mm ventral from the surface of the flat skull. After 14 days, the brains and spinal cords were removed and frozen. Sections were cut on a cryostat and BDA transportation absorbed by axons was observed under a fluorescence microscope. MAIN OUTCOME MEASURES： Axonal absorption and transportation of BDA was observed under fluorescence microscope. RESULTS： Eighteen SD rats were enrolled in this experiment; 12 rats were included in the final analysis and six were eliminated, resulting in a dropout rate of 33% （6/18）. BDA injected into the left cortex was absorbed in the axons, and fluorescence was observed throughout the pyramidal neurons and axons of the left cerebral cortex. At 14 days after rejection, BDA was detected in the midbrain and cervical enlargement along the CST, and axonal structures and Ranvier nodes were clearly observed with 200x magnification. CONCLUSION： BDA injected into the cerebral cortex effectively traces the corticospinal tract and is biologically stable over long distance transportation. In addition, the method of BDA tracing is fairly simple to perform.

Engineering personalized neural tissue using functionalized transcription factors

Diseases and disorders of the central nervous system often require significant interventions to restore lost function due to their com- plexity. Examples of such disorders include Parkinson＇s disease, Alzheimer＇s disease, multiple sclerosis, traumatic brain injury, and spinal cord in）ury. These diseases and disorders result trom healthy cells being destroyed, which in turn causes dysfunction in the cen- tral nervous system, The death of these cells can trigger a cascade of events that affect the rest of the body, causing symptoms that become progressively worse over time. Developing strategies for repairing the damage to the central nervous system remains chal- lenging, in part due to its inability to regenerate.

Effect of SOD1 Overexpression on the 20S Proteasome during Aging

Metabolism of oxygen derivatives has been shown to be altered in Down syndrome (DS) due to the overexpression of the Cu/Zn superoxide dismutase gene ( SOD-1) on chromosome 21. Transgenic mice for the human SOD1 gene (h SOD1) exhibit some features of the syndrome. Oxidation of proteins and oxidative stress are involved in normal and pathological aging. The proteasome is an adaptative system to eliminate the modified proteins which can be deleterious. As SOD1 overexpression has been shown to be either deleterious or protective according to tissues and paradigms, we have measured in function of age the 20S proteasome activities in neural tissues (cerebral hemisphere, cerebellum and cortex) and in the thymus and the heart from control and transgenic mice. Indeed, although SOD1 overexpression is very deleterious in thymus and heart, it has little effect in cerebral hemisphere and cortex depending on the proteolytic activity measured. Conversely in the cerebellum the three proteolytic activities decrease dramatically in transgenic old mice while it was not modified in control mice during aging. The results of this study suggest that some phenotypes of DS present in thymus, heart and neural tissues of h SOD1 transgenic mice might be partially due to the modulation of the 20S proteasome expression during aging.

Restoring nervous system structure and function using tissue engineered living scaffolds

Neural tissue engineering is premised on the integration of engineered living tissue with the host nervous system to directly restore lost function or to augment regenerative capacity following ner- vous system injury or neurodegenerative disease. Disconnection of axon pathways - the long-distance fibers connecting specialized regions of the central nervous system or relaying peripheral signals - is a common feature of many neurological disorders and injury. However, functional axonal regenera- tion rarely occurs due to extreme distances to targets, absence of directed guidance, and the presence of inhibitory factors in the central nervous system, resulting in devastating effects on cognitive and sensorimotor function. To address this need, we are pursuing multiple strategies using tissue engi- neered ＂living scaffolds＂, which are preformed three-dimensional constructs consisting of living neural cells in a defined, often anisotropic architecture. Living scaffolds are designed to restore function by serving as a living labeled pathway for targeted axonal regeneration - mimicking key developmental mechanisms- or by restoring lost neural circuitry via direct replacement of neurons and axonal tracts. We are currently utilizing preformed living scaffolds consisting of neuronal dusters spanned by long axonal tracts as regenerative bridges to facilitate long-distance axonal regeneration and for targeted neurosurgical reconstruction of local circuits in the brain. Although there are formidable challenges in predinical and clinical advancement, these living tissue engineered constructs represent a promising strategy to facilitate nervous system repair and functional recovery.

Chitosan tube bridging autologous nerve segments for the repair of long-segmental sciatic nerve defects

BACKGROUND：Previous tissue-engineered nerve studies have focused on artificial nerve and nerve cell cultures.The effects of regeneration chambers with autologous nerve bridging for the repair of nerve defects remain unclear.OBJECTIVE：To explore the feasibility and advantages of chitosan tube bridging autologous nerve segments for repairing 12-mm sciatic nerve defects in rats.DESIGN,TIME AND SETTING：A randomized,controlled,animal study using nerve tissue engineering was performed at the Animal Laboratory and Laboratory of Histology and Embryology,Liaoning Medical University from June 2008 to March 2009.MATERIALS：Chitosan powder was purchased from Jinan Haidebei Marine Bioengineering,China.METHODS：A sciatic nerve segment of approximately 8 mm was excised from the posterior margin of the piriformis muscle of Sprague Dawley rats.The two nerve ends shrank to form a 12-mm defect,and the nerve defect was repaired using a chitosan tube bridging autologous nerve segment （bridge group）,a chitosan tube-encapsulated autologous nerve segment （encapsulation group）,and a chitosan tube alone （chitosan tube alone group）,respectively.MAIN OUTCOME MEASURES：Histological and ultrastructural changes of the injured sciatic nerve;number of regenerated myelinated nerve fibers; nerve conduction velocity; leg muscle atrophy; and sciatic nerve functional index.RESULTS：At 4 months after implantation,the chitosan tube was absorbed.The tube was thin,but maintained the original shape,and vascular proliferation was observed around the tube.In the bridge group,regenerative myelinated nerve fibers were thick and orderly,with a thick myelin sheath and intact axonal structure.The number of myelinated nerve fibers and nerve conduction velocity were significantly greater compared with the other groups （P〈 0.01）.Moreover,nerve and muscle function was significantly improved following chitosan tube bridging autologous nerve segment treatment compared with the other groups （P〈 0.05 or P 〈 0.01）.CONCLUSION：Chitosan tube bridging autologous nerve segments exhibited better repair effects on nerve defects compared with chitosan tubeencapsulated autologous nerve segments and a chitosan tube alone.This method provided a simple and effective treatment for long-segmental nerve defects.

Diffusion tensor imaging with multiple diffusion-weighted gradient directions

Diffusion tensor MRI （DT-MRI or DTI） is emerging as an important non-invasive technology for elucidating intemal brain structures. It has recently been utilized to diagnose a series of diseases that affect the integrity of neural systems to provide a basis for neuroregenerative studies. Results from the present study suggested that neural tissue is reconstructed with multiple diffusion-weighted gradient directions DTI, which varies from traditional imaging methods that utilize 6 gradient directions. Simultaneously, the diffusion tensor matrix is obtained by multiple linear regressions from an equation of echo signal intensity. The condition number value and standard deviation of fractional anisotropy for each scheme can be used to evaluate image quality. Results demonstrated that increasing gradient direction to some extent resulted in improved effects. Therefore, the traditional 6 and 15 directions should not be considered optimal scan protocols for clinical DTI application. In a scheme with 20 directions, the condition number and standard deviation of fractional anisotropy of the encoding gradients matrix were significantly reduced, and resulted in more clearly and accurately displayed neural tissue. Results demonstrated that the scheme with 20 diffusion gradient directions provided better accuracy of structural renderings and could be an optimal scan protocol for clinical DTI application.

The use of hydrogel-delivered extracellular vesicles in recovery of motor function in stroke:a testable experimental hypothesis for clinical translation including behavioral and neuroimaging assessment approaches

Neural tissue engineering,nanotechnology and neuroregeneration are diverse biomedical disciplines that have been working together in recent decades to solve the complex problems linked to central nervous system(CNS)repair.It is known that the CNS demonstrates a very limited regenerative capacity because of a microenvironment that impedes effective regenerative processes,making development of CNS therapeutics challenging.Given the high prevalence of CNS conditions such as stroke that damage the brain and place a severe burden on afflicted individuals and on society,it is of utmost significance to explore the optimum methodologies for finding treatments that could be applied to humans for restoration of function to pre-injury levels.Extracellular vesicles(EVs),also known as exosomes,when derived from mesenchymal stem cells,are one of the most promising approaches that have been attempted thus far,as EVs deliver factors that stimulate recovery by acting at the nanoscale level on intercellular communication while avoiding the risks linked to stem cell transplantation.At the same time,advances in tissue engineering and regenerative medicine have offered the potential of using hydrogels as bio-scaffolds in order to provide the stroma required for neural repair to occur,as well as the release of biomolecules facilitating or inducing the reparative processes.This review introduces a novel experimental hypothesis regarding the benefits that could be offered if EVs were to be combined with biocompatible injectable hydrogels.The rationale behind this hypothesis is presented,analyzing how a hydrogel might prolong the retention of EVs and maximize the localized benefit to the brain.This sustained delivery of EVs would be coupled with essential guidance cues and structural support from the hydrogel until neural tissue remodeling and regeneration occur.Finally,the importance of including nonhuman primate models in the clinical translation pipeline,as well as the added benefit of multi-modal neuroimaging analysis to establish non-invasive,in vivo,quantifiable imagingbased biomarkers for CNS repair are discussed,aiming for more effective and safe clinical translation of such regenerative therapies to humans.

Remodeling of skin nerve fibers during burn wound healing

Burn wound healing involves a complex sequence of processes. Recent studies have revealed that skin reinnervation may have an impact on physiological wound repair. Few studies have addressed the process of reinnervation and morphological changes in regenerated nerve fibers. The regeneration of neurites during full-thickness burn wound healing was determined by immunofluorescent staining using an anti-neurofilament protein monoclonal antibody, and three-dimensional morphology was observed under a laser scanning confocal microscope. Morphology and the volume fraction of collagen and nerve fibers were measured. Skin reinnervation increased during wound healing, peaked during the proliferative scar stage, and then decreased to lower levels during the maturation period. The results from the skin nerve fibers correlated with those from collagen using semi-quantitative analysis. Disintegration and fragmentation were observed frequently in samples from the proliferative stage, and seldom occurred during the maturation stage. There was a remodeling process of regenerated nerve fibers during wound healing, which comprised changed innervation density and topical morphology. The mechanism of remodeling for nerve fibers requires further investigation.

Interplay of SOX transcription factors and microRNAs in the brain under physiological and pathological conditions

Precise tuning of gene expression,accomplished by regulato ry networks of transcription factors,epigenetic modifiers,and microRNAs,is crucial for the proper neural development and function of the brain cells.The SOX transcription factors are involved in regulating diverse cellular processes during embryonic and adult neurogenesis,such as maintaining the cell stemness,cell prolife ration,cell fate decisions,and terminal diffe rentiation into neurons and glial cells.MicroRNAs represent a class of small non-coding RNAs that play important roles in the regulation of gene expression.Together with other gene regulatory factors,microRNAs regulate different processes during neurogenesis and orchestrate the spatial and temporal expression important for neurodevelopment.The emerging data point to a complex regulatory network between SOX transcription factors and microRNAs that govern distinct cellular activities in the developing and adult brain.Deregulated SOX/mic roRNA interplay in signaling pathways that influence the homeostasis and plasticity in the brain has been revealed in various brain pathologies,including neurodegenerative disorders,traumatic brain injury,and cancer.Therapeutic strategies that target SOX/microRNA interplay have emerged in recent years as a promising tool to target neural tissue regeneration and enhance neuro restoration.N umerous studies have confirmed complex intera ctions between microRNAs and SOX-specific mRNAs regulating key features of glioblastoma.Keeping in mind the crucial roles of SOX genes and microRNAs in neural development,we focus this review on SOX/microRNAs interplay in the brain during development and adulthood in physiological and pathological conditions.Special focus was made on their interplay in brain pathologies to summarize current knowledge and highlight potential future development of molecular therapies.

Self-assembled IKVAV Peptide Nanofibers Promote Adherence of PC12 Cells

Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile （IKVAV-PA） was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography （HPLC） and mass spectrometry （MS）. Then, by addition of hydrogen chloride （HC1）, self-assembly of IKVAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy （TEM）. The effect of IKVAV nanofibers on adherence of PCI2 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range （0.58 μg/cm^2 to 15.6 μg/cm^2）. However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general, we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PCI2 cells adherence is dosage-dependent within a certain range of densities.

A partition-type tubular scaffold loaded with PDGF-releasing microspheres for spinal cord repair facilitates the directional migration and growth of cells

The best tissue-engineered spinal cord grafts not only match the structural characteristics of the spinal cord but also allow the seed cells to grow and function in situ.Platelet-derived growth factor（PDGF） has been shown to promote the migration of bone marrow stromal cells;however,cytokines need to be released at a steady rate to maintain a stable concentration in vivo.Therefore,new methods are needed to maintain an optimal concentration of cytokines over an extended period of time to effectively promote seed cell localization,proliferation and differentiation.In the present study,a partition-type tubular scaffold matching the anatomical features of the thoracic 8–10 spinal cord of the rat was fabricated using chitosan and then subsequently loaded with chitosan-encapsulated PDGF-BB microspheres（PDGF-MSs）.The PDGF-MS-containing scaffold was then examined in vitro for sustained-release capacity,biocompatibility,and its effect on neural progenitor cells differentiated in vitro from multilineage-differentiating stress-enduring cells（MUSE-NPCs）.We found that pre-freezing for 2 hours at-20°C significantly increased the yield of partition-type tubular scaffolds,and 30 μL of 25% glutaraldehyde ensured optimal crosslinking of PDGF-MSs.The resulting PDGF-MSs cumulatively released 52% of the PDGF-BB at 4 weeks in vitro without burst release.The PDGF-MS-containing tubular scaffold showed suitable biocompatibility towards MUSE-NPCs and could promote the directional migration and growth of these cells.These findings indicate that the combination of a partition-type tubular scaffold,PDGF-MSs and MUSENPCs may be a promising model for the fabrication of tissue-engineered spinal cord grafts.

Neuron-fibrous scaffold interfaces in the peripheral nervous system: a perspective on the structural requirements

The nerves of the peripheral nervous system are not able to effectively regenerate in cases of severe neural injury.This can result in debilitating consequences,including morbidity and lifelong impairments affecting the quality of the patient’s life.Recent findings in neural tissue engineering have opened promising avenues to apply fibrous tissue-engineered scaffolds to promote tissue regeneration and functional recovery.These scaffolds,known as neural scaffolds,are able to improve neural regeneration by playing two major roles,namely,by being a carrier for transplanted peripheral nervous system cells or biological cues and by providing structural support to direct growing nerve fibers towards the target area.However,successful implementation of scaffold-based therapeutic approaches calls for an appropriate design of the neural scaffold structure that is capable of up-and down-regulation of neuron-scaffold interactions in the extracellular matrix environment.This review discusses the main challenges that need to be addressed to develop and apply fibrous tissue-engineered scaffolds in clinical practice.It describes some promising solutions that,so far,have shown to promote neural cell adhesion and growth and a potential to repair peripheral nervous system injuries.

Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

Vascularization of acellular nerves has been shown to contribute to nerve bridging.In this study,we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves.The rat nerve defects were treated with acellular nerve grafting（control group） alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein（experimental group）.As shown through two-dimensional imaging,the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation,and gradually covered the entire graft at day 21.The vascular density,vascular area,and the velocity of revascularization in the experimental group were all higher than those in the control group.These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

An NT-3-releasing bioscaffold supports the formation of TrkC-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury

The mechanism underlying neurogenesis during embryonic spinal cord development involves a specific ligand/receptor interaction,which may be help guide neuroengineering to boost stem cell-based neural regeneration for the structural and functional repair of spinal cord injury.Herein,we hypothesized that supplying spinal cord defects with an exogenous neural network in the NT-3/fibroin-coated gelatin sponge(NF-GS)scaffold might improve tissue repair efficacy.To test this,we engineered tropomyosin receptor kinase C(TrkC)-modified neural stem cell(NSC)-derived neural network tissue with robust viability within an NF-GS scaffold.When NSCs were genetically modified to overexpress TrkC,the NT-3 receptor,a functional neuronal population dominated the neural network tissue.The pro-regenerative niche allowed the long-term survival and phenotypic maintenance of the donor neural network tissue for up to 8 weeks in the injured spinal cord.Additionally,host nerve fibers regenerated into the graft,making synaptic connections with the donor neurons.Accordingly,motor function recovery was significantly improved in rats with spinal cord injury(SCI)that received TrkC-modified NSC-derived neural network tissue transplantation.Together,the results suggested that transplantation of the neural network tissue formed in the 3D bioactive scaffold may represent a valuable approach to study and develop therapies for SCI.

Increased connectivity of hiPSC-derived neural networks in multiphase granular hydrogel scaffolds

To reflect human development,it is critical to create a substrate that can support long-term cell survival,differentiation,and maturation.Hydrogels are promising materials for 3D cultures.However,a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction.Herein,granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell(hiPSC)-derived neural networks.A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules.Cells and hydrogel granules were combined using a weaker secondary gelation step,forming self-supporting cell laden scaffolds.At three and seven days,granular scaffolds supported higher cell viability compared to bulk hydrogels,whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions(65.52±11.59μm)after seven days compared to bulk hydrogels(22.90±4.70μm).Long-term(three-month)cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold.This approach is significant as it provides a simple,rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.

Scaffolds with anisotropic structure for neural tissue engineering

Nervous system injuries remain a great challenge due to limited natural tissue regeneration capabilities.Neural tissue engineering has been regarded as a promising approach for repairing nerve defects,which utilizes external biomaterial scaffolds to allow cells to migrate to the injury site and repair the tissue.Particularly,scaffolds with anisotropic structures biomimicking the native extracellular matrix(ECM)can effectively guide neural orientation and reconnection.Here,the advancements of scaffolds with anisotropic structures in the field of neural tissue engineering are presented.The fabrication strategies of scaffolds with anisotropic structures and their effects in vitro and in vivo are highlighted.We also discuss the challenges and provide a perspective of this field.

Three-dimensional direct laser writing of biomimetic neuron interfaces in the era of artificial intelligence:principles,materials,and applications

The creation of biomimetic neuron interfaces(BNIs)has become imperative for different research fields from neural science to artificial intelligence.BNIs are two-dimensional or three-dimensional(3D)artificial interfaces mimicking the geometrical and functional characteristics of biological neural networks to rebuild,understand,and improve neuronal functions.The study of BNI holds the key for curing neuron disorder diseases and creating innovative artificial neural networks(ANNs).To achieve these goals,3D direct laser writing(DLW)has proven to be a powerful method for BNI with complex geometries.However,the need for scaled-up,high speed fabrication of BNI demands the integration of DLW techniques with ANNs.ANNs,computing algorithms inspired by biological neurons,have shown their unprecedented ability to improve efficiency in data processing.The integration of ANNs and DLW techniques promises an innovative pathway for efficient fabrication of large-scale BNI and can also inspire the design and optimization of novel BNI for ANNs.This perspective reviews advances in DLW of BNI and discusses the role of ANNs in the design and fabrication of BNI.