Proof of Concept for Sustainable Manufacturing of Neural Electrode Array for In Vivo Recording.

Increasing requirements for neural implantation are helping to expand our understanding of nervous systems and generate new developmental approaches. It is thanks to advanced semiconductor technologies that we can achieve the high-density complementary metal-oxide-semiconductor electrode array for the improvement of the quantity and quality of neural recordings. Although the microfabricated neural implantable device holds much promise in the biosensing field, there are some significant technological challenges. The most advanced neural implantable device relies on complex semiconductor manufacturing processes, which are required for the use of expensive masks and specific clean room facilities. In addition, these processes based on a conventional photolithography technique are suitable for mass production, which is not applicable for custom-made manufacturing in response to individual experimental requirements. The microfabricated complexity of the implantable neural device is increasing, as is the associated energy consumption, and corresponding emissions of carbon dioxide and other greenhouse gases, resulting in environmental deterioration. Herein, we developed a fabless fabricated process for a neural electrode array that was simple, fast, sustainable, and customizable. An effective strategy to produce conductive patterns as the redistribution layers (RDLs) includes implementing microelectrodes, traces, and bonding pads onto the polyimide (PI) substrate by laser micromachining techniques combined with the drop coating of the silver glue to stack the laser grooving lines. The process of electroplating platinum on the RDLs was performed to increase corresponding conductivity. Sequentially, Parylene C was deposited onto the PI substrate to form the insulation layer for the protection of inner RDLs. Following the deposition of Parylene C, the via holes over microelectrodes and the corresponding probe shape of the neural electrode array was also etched by laser micromachining. To increase the neural recording capability, three-dimensional microelectrodes with a high surface area were formed by electroplating gold. Our eco-electrode array showed reliable electrical characteristics of impedance under harsh cyclic bending conditions of over 90 degrees. For in vivo application, our flexible neural electrode array demonstrated more stable and higher neural recording quality and better biocompatibility as well during the 2-week implantation compared with those of the silicon-based neural electrode array. In this study, our proposed eco-manufacturing process for fabricating the neural electrode array reduced 63 times of carbon emissions compared to the traditional semiconductor manufacturing process and provided freedom in the customized design of the implantable electronic devices as well.

Mineral Nanomedicine to Enhance the Efficacy of Adjuvant Radiotherapy for Treating Osteosarcoma.

It is vital to remove residual tumor cells after resection to avoid the recurrence and metastasis of osteosarcoma. In this study, a mineral nanomedicine, europium-doped calcium fluoride (CaF2:Eu) nanoparticles (NPs), is developed to enhance the efficacy of adjuvant radiotherapy (i.e., surgical resection followed by radiotherapy) for tumor cell growth and metastasis of osteosarcoma. In vitro studies show that CaF2:Eu NPs (200 µg/mL) exert osteosarcoma cell (143B)-selective toxicity and migration-inhibiting effects at a Eu dopant amount of 2.95 atomic weight percentage. These effects are further enhanced under X-ray irradiation (6 MeV, 4 Gy). Furthermore, in vivo tests show that intraosseous injection of CaF2:Eu NPs and X-ray irradiation have satisfactory therapeutic efficacy in controlling primary tumor size and inhibiting primary tumor metastasis. Overall, our results suggest that CaF2:Eu NPs with their osteosarcoma cell (143B)-selective toxicity and migration-inhibiting effects combined with radiotherapy might be nanomedicines for treating osteosarcoma after tumor resection.

Non-viral delivery of an optogenetic tool into cells with self-healing hydrogel.

Optogenetics offers unique, temporally precise control of neural activity in genetically targeted specific neurons that express light-sensitive opsin molecules. Three-dimensional (3D) delivery of optogenetics can be realized by co-injection of bacteriorhodopsin (HEBR) plasmid with a chitosan-based self-healing hydrogel with strong shear-thinning properties. The HEBR protein shows photoelectrical properties and can be used as an optical switch for cell activation. We optimize the shear force generated during the process of injection (∼100 Pa), which is transient because of the self-healing nature of the hydrogel. This transient force exerted by the self-healing hydrogel may allow the cytosolic delivery of HEBR plasmid with excellent cell viability and a high efficiency approaching 80%. When excited with green light, HEBR-delivered neural stem cells (NSCs) can proliferate and specifically differentiate into neurons in vitro and rescue the function of nerve impaired zebrafish in vivo. This novel optogenetic method combining 3D injectable self-healing hydrogel offers potential temporal-spatial approaches to treat neurodegenerative diseases in the future.

Neural differentiation on aligned fullerene C60 nanowhiskers.

Highly-aligned fullerene nanowhiskers (C60 NWs) are prepared by a modified liquid-liquid interfacial precipitation method. Neural stem cells on the aligned C60 NWs are oriented and have a high capacity to differentiate into mature neurons. The aligned C60 NWs can serve as a functional scaffold for neural tissue engineering.

Biomaterial Substrate-Mediated Multicellular Spheroid Formation and Their Applications in Tissue Engineering.

Three-dimentional (3D) multicellular aggregates (spheroids), compared to the traditional 2D monolayer cultured cells, are physiologically more similar to the cells in vivo. So far there are various techniques to generate 3D spheroids. Spheroids obtained from different methods have already been applied to regenerative medicine or cancer research. Among the cell spheroids created by different methods, the substrate-derived spheroids and their forming mechanism are unique. This review focuses on the formation of biomaterial substrate-mediated multicellular spheroids and their applications in tissue engineering and tumor models. First, the authors will describe the special chitosan substrate-derived mesenchymal stem cell (MSC) spheroids and their greater regenerative capacities in various tissues. Second, the authors will describe tumor spheroids derived on chitosan and hyaluronan substrates, which serve as a simple in vitro platform to study 3D tumor models or to perform cancer drug screening. Finally, the authors will mention the self-assembly process for substrate-derived multiple cell spheroids (co-spheroids), which may recapitulate the heterotypic cell-cell interaction for co-cultured cells or crosstalk between different types of cells. These unique multicellular mono-spheroids or co-spheroids represent a category of 3D cell culture with advantages of biomimetic cell-cell interaction, better functionalities, and imaging possibilities.

Glucose-sensitive self-healing hydrogel as sacrificial materials to fabricate vascularized constructs.

A major challenge in tissue engineering is the lack of proper vascularization. Although various approaches have been used to build vascular network in a tissue engineering construct, there remain some drawbacks. Herein, a glucose-sensitive self-healing hydrogel are employed as sacrificial materials to fabricate branched tubular channels within a construct. The hydrogel composes of mainly reversibly crosslinked poly(ethylene glycol) diacrylate and dithiothreitol with borax as the glucose-sensitive motif. The hydrogel is injectable and mechanically strong after injection. Moreover, it can be rapidly removed by immersion in the cell culture medium. To show the feasibility in building a vascularized tissue construct, the designed branching vascular patterns of the glucose-sensitive hydrogel are extruded and embedded in a non glucose-sensitive hydrogel containing neural stem cells. Vascular endothelial cells seeded in the lumen of the channels by perfusion can line the channel wall and migrate into the non-sacrificial hydrogel after 3 days. In long-term (∼14 days), the endothelial cells form capillary-like structure (vascular network) while neural stem cells form neurosphere-like structure (neural development) in the construct, revealing the morphology of "a vascularized neural tissue". The novel sacrificial materials can create complicated but easily removable structure for building a vascularized tissue construct particularly a neurovascular unit.

Water-Soluble Fullerene Derivatives as Brain Medicine: Surface Chemistry Determines If They Are Neuroprotective and Antitumor.

Delivering drugs to the central nervous system (CNS) is a major challenge in treating CNS-related diseases. Nanoparticles that can cross blood-brain barrier (BBB) are potential tools. In this study, water-soluble C60 fullerene derivatives with different types of linkages between the fullerene cage and the solubilizing addend were synthesized (compounds 1-3: C-C bonds, compounds 4-5: C-S bonds, compound 6: C-P bonds, and compounds 7-9: C-N bonds). Fullerene derivatives 1-6 were observed to induce neural stem cell (NSC) proliferation in vitro and rescue the function of injured CNS in zebrafish. Fullerene derivatives 7-9 were found to inhibit glioblastoma cell proliferation in vitro and reduce glioblastoma formation in zebrafish. These effects were correlated with the cell metabolic changes. Particularly, compound 3 bearing residues of phenylbutiryc acids significantly promoted NSC proliferation and neural repair without causing tumor growth. Meanwhile, compound 7 with phenylalanine appendages significantly inhibited glioblastoma growth without retarding the neural repair. We conclude that the surface functional group determines the properties as well as the interactions of C60 with NSCs and glioma cells, producing either a neuroprotective or antitumor effect for possible treatment of CNS-related diseases.

Substrate-mediated reprogramming of human fibroblasts into neural crest stem-like cells and their applications in neural repair.

Cell- and gene-based therapies have emerged as promising strategies for treating neurological diseases. The sources of neural stem cells are limited while the induced pluripotent stem (iPS) cells have risk of tumor formation. Here, we proposed the generation of self-renewable, multipotent, and neural lineage-related neural crest stem-like cells by chitosan substrate-mediated gene transfer of a single factor forkhead box D3 (FOXD3) for the use in neural repair. A simple, non-toxic, substrate-mediated method was applied to deliver the naked FOXD3 plasmid into human fibroblasts. The transfection of FOXD3 increased cell proliferation and up-regulated the neural crest marker genes (FOXD3, SOX2, and CD271), stemness marker genes (OCT4, NANOG, and SOX2), and neural lineage-related genes (Nestin, ß-tubulin and GFAP). The expression levels of stemness marker genes and neural crest maker genes in the FOXD3-transfected fibroblasts were maintained until the fifth passage. The FOXD3 reprogrammed fibroblasts based on the new method significantly rescued the neural function of the impaired zebrafish. The chitosan substrate-mediated delivery of naked plasmid showed feasibility in reprogramming somatic cells. Particularly, the FOXD3 reprogrammed fibroblasts hold promise as an easily accessible cellular source with neural crest stem-like behavior for treating neural diseases in the future.

Self-patterning of adipose-derived mesenchymal stem cells and chondrocytes cocultured on hyaluronan-grafted chitosan surface.

The articular cartilage, once injured, has a limited capacity for intrinsic repair. Preparation of functionally biocartilage substitutes in vitro for cartilage repair is an attractive concept with the recent advances in tissue engineering. In this study, adipose-derived adult stem cells (ADAS) and chondrocytes (Ch) were cocultured in different population ratios on the surface of hyaluronan-grafted chitosan (CS-HA) membranes. The two types of cells could self-assemble into cospheroids with different morphologies. In particular, when ADAS and Ch were cocultured at an initial ratio of 7:3 on CS-HA surface, the expression of chondrogenic markers was upregulated, leading to preferred chondrogenesis of the cospheroids. Therefore, using the ADAS/Ch 7:3 cospheroids derived on CS-HA surface instead of using only a single type of cells may be favorable for future therapeutic applications.

Increased cell survival of cells exposed to superparamagnetic iron oxide nanoparticles through biomaterial substrate-induced autophagy.

The cellular uptake of nanoparticles (NPs) can be promoted by NP surface modification but cell viability is often sacrificed. Our previous study has shown that intracellular uptake of iron oxide NPs was significantly increased for cells cultured on chitosan. However, the mechanism for having the higher cellular uptake as well as better cell survival on the chitosan surface remains unclear. In this study, we sought to clarify if the autophagic response may contribute to cell survival under excessive NP exposure conditions on chitosan. L929 fibroblasts and neural stem cells (NSCs) were challenged with different concentrations (0-300 µg ml(-1)) of superparamagnetic iron oxide NPs. The autophagic response as well as the metabolic activity of cells was evaluated. Results showed that culturing both types of cells on chitosan substrates significantly enhanced the cellular uptake of NPs. At higher NP concentrations, cells on chitosan showed a greater survival rate than those on TCPS. The expression levels of autophagy-related genes (Atg5 and Atg7 genes) and autophagy associated protein (LC3-II) on chitosan were higher than that on TCPS. The NP exposure further increased the expressions. We suggest that cells cultured on chitosan were more tolerant to NP cytotoxicity because of the increased autophagic response. Moreover, NP exposure increased the metabolic activity of cells grown on chitosan, while it decreased the metabolism of cells cultured on TCPS. In animal studies, iron oxide-labeled NSCs were injected in zebrafish embryos. Results also showed that cells grown on chitosan had better survival after transplantation than those grown on TCPS. Taken together, chitosan as a culture substrate can induce cell autophagy to increase cell survival in particular for NP-labeled cells. This will be valuable for the biomedical application of NPs in cell therapy.

Preparation and characterization of a biodegradable polyurethane hydrogel and the hybrid gel with soy protein for 3D cell-laden bioprinting.

3D printing shows great potential for fabricating customized scaffolds for tissue regeneration. Using hydrogel as a bioink for cell printing provides a biological platform for basic research and potential medical treatments. In this study, a waterborne poly(ε-caprolactone) (PCL)-based biodegradable polyurethane (PU) with a soft segment replaced with 20 mol% of poly(l-lactide) (PLLA) diol or poly (d,l-lactide) (PDLLA) diol was prepared. These two PUs formed compact packing structures at temperatures ≥37 °C. They responded differently to temperature changes and the presence of electrolytes because of the difference in the free volume. With their thermal-responsive properties, both PU dispersions could form a gel in 3 min with the gel modulus reaching about 6-8 kPa after 30 min. To enhance the structural integrity during layer-by-layer deposition, the hybrid hydrogel of PU and soy protein isolate (PU/SPI hybrid) was further developed. The PU/SPI hybrid dispersion could undergo rapid gelation at 37 °C with the modulus reaching 130 Pa in 1 min. Moreover, the PU/SPI hybrid gel was readily blended with cells and printed at 37 °C without preheating. Neural stem cells (NSCs) were embedded in the hydrogels and analyzed for cell viability, metabolism, proliferation, and gene expression of neural-related markers. Cells cultured in the PU/SPI hybrid construct had better survival and proliferation than those in the PU gel. The PU/SPI hybrid ink may provide unique rheological properties for direct cell/tissue printing at 37 °C and a biomimetic microenvironment for cell survival, growth, and differentiation.

3D bioprinting: A new insight into the therapeutic strategy of neural tissue regeneration.

Acute traumatic injuries and chronic degenerative diseases represent the world's largest unmet medical need. There are over 50 million people worldwide suffering from neurodegenerative diseases. However, there are only a few treatment options available for acute traumatic injuries and neurodegenerative diseases. Recently, 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. In this commentary, the newly developed 3D bioprinting technique involving neural stem cells (NSCs) embedded in the thermoresponsive biodegradable polyurethane (PU) bioink is reviewed. The thermoresponsive and biodegradable PU dispersion can form gel near 37 °C without any crosslinker. NSCs embedded within the water-based PU hydrogel with appropriate stiffness showed comparable viability and differentiation after printing. Moreover, in the zebrafish embryo neural deficit model, injection of the NSC-laden PU hydrogels promoted the repair of damaged CNS. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden constructs. Therefore, the newly developed 3D bioprinting technique may offer new possibilities for future therapeutic strategy of neural tissue regeneration.

Carboxyl-functionalized polyurethane nanoparticles with immunosuppressive properties as a new type of anti-inflammatory platform.

The interaction of nanoparticles (NPs) with the body immune system is critically important for their biomedical applications. Most NPs stimulate the immune response of macrophages. Here we show that synthetic polyurethane nanoparticles (PU NPs, diameter 34-64 nm) with rich surface COO(-) functional groups (zeta potential -70 to -50 mV) can suppress the immune response of macrophages. The specially-designed PU NPs reduce the gene expression levels of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) for endotoxin-treated macrophages. The PU NPs increase the intracellular calcium of macrophages (4.5-6.5 fold) and activate autophagy. This is in contrast to the autophagy dysfunction generally observed upon NP exposure. These PU NPs may further decrease the nuclear factor-κB-related inflammation via autophagy pathways. The immunosuppressive activities of PU NPs can prevent animal death by inhibiting the macrophage recruitment and proinflammatory responses, confirmed by an in vivo zebrafish model. Therefore, the novel biodegradable PU NPs demonstrate COO(-) dependent immunosuppressive properties without carrying any anti-inflammatory agents. This study suggests that NP surface chemistry may regulate the immune response, which provides a new paradigm for potential applications of NPs in anti-inflammation and immunomodulation.

3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair.

The 3D bioprinting technology serves as a powerful tool for building tissue in the field of tissue engineering. Traditional 3D printing methods involve the use of heat, toxic organic solvents, or toxic photoinitiators for fabrication of synthetic scaffolds. In this study, two thermoresponsive water-based biodegradable polyurethane dispersions (PU1 and PU2) were synthesized which may form gel near 37 °C without any crosslinker. The stiffness of the hydrogel could be easily fine-tuned by the solid content of the dispersion. Neural stem cells (NSCs) were embedded into the polyurethane dispersions before gelation. The dispersions containing NSCs were subsequently printed and maintained at 37 °C. The NSCs in 25-30% PU2 hydrogels (∼680-2400 Pa) had excellent proliferation and differentiation but not in 25-30% PU1 hydrogels. Moreover, NSC-laden 25-30% PU2 hydrogels injected into the zebrafish embryo neural injury model could rescue the function of impaired nervous system. However, NSC-laden 25-30% PU1 hydrogels only showed a minor repair effect in the zebrafish model. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden 25% PU2 constructs. Therefore, the newly developed 3D bioprinting technique involving NSCs embedded in the thermoresponsive biodegradable polyurethane ink offers new possibilities for future applications of 3D bioprinting in neural tissue engineering.

An Injectable, Self-Healing Hydrogel to Repair the Central Nervous System.

An injectable, self-healing hydrogel (≈1.5 kPa) is developed for healing nerve-system deficits. Neurosphere-like progenitors proliferate in the hydrogel and differentiate into neuron-like cells. In the zebrafish injury model, the central nervous system function is partially rescued by injection of the hydrogel and significantly rescued by injection of the neurosphere-laden hydrogel. The self-healing hydrogel may thus potentially repair the central nervous system.

Dner inhibits neural progenitor proliferation and induces neuronal and glial differentiation in zebrafish.

Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a single-pass transmembrane protein found to be a novel ligand in the Notch signaling pathway. Its function was previously characterized in the developing cerebellum and inner ear hair cells. In this study, we isolated a zebrafish homolog of DNER and showed that this gene is expressed in the developing nervous system. Overexpression of dner or the intracellular domain of dner was sufficient to inhibit the proliferation of neural progenitors and induce neuronal and glial differentiation. In contrast, the knockdown of endogenous Dner expression using antisense morpholino oligonucleotides increased the proliferation of neural progenitors and maintained neural cells in a progenitor status through inhibition of neuronal and glial differentiation. Through analysis of the antagonistic effect on the Delta ligand and the role of the potential downstream mediator Deltex1, we showed that Dner acts in Notch-dependent and Notch-independent manner. This is the first study to demonstrate a role for Dner in neural progenitors and neuronal differentiation and provides new insights into mediation of neuronal development and differentiation by the Notch signaling pathway.

Akt1 mediates neuronal differentiation in zebrafish via a reciprocal interaction with notch signaling.

Akt1 is well known for its role in regulating cell proliferation, differentiation, and apoptosis and is implicated in tumors and several neurological disorders. However, the role of Akt1 in neural development has not been well defined. We have isolated zebrafish akt1 and shown that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. Injection of akt1 morpholinos resulted in loss of neuronal precursors with a concomitant increase in post-mitotic neurons, indicating that knockdown of Akt1 is sufficient to cause premature differentiation of neurons. A similar phenotype was observed in embryos deficient for Notch signaling. Both the ligand (deltaA) and the downstream target of Notch (her8a) were downregulated in akt1 morphants, indicating that Akt1 is required for Delta-Notch signaling. Furthermore, akt1 expression was downregulated in Delta-Notch signaling-deficient embryos and could be induced by constitutive activation of Notch signaling. In addition, knockdown of Akt1 was able to nullify the inhibition of neuronal differentiation caused by constitutive activation of Notch signaling. Taken together, these results provide in vivo evidence that Akt1 interacts with Notch signaling reciprocally and provide an explanation of why Akt1 is essential for the inhibition of neuronal differentiation.

Zebrafish rgs4 is essential for motility and axonogenesis mediated by Akt signaling.

The schizophrenia susceptibility gene, Rgs4, is one of the most intensively studied regulators of G-protein signaling members, well known to be fundamental in regulating neurotransmission. However, little is known about its role in the developing nervous system. We have isolated zebrafish rgs4 and shown that it is transcribed in the developing nervous system. Rgs4 knockdown did not affect neuron number and patterning but resulted in locomotion defects and aberrant development of axons. This was confirmed using a selective Rgs4 inhibitor, CCG-4986. Rgs4 knockdown also attenuated the level of phosphorylated-Akt1, and injection of constitutively-activated AKT1 rescued the motility defects and axonal phenotypes in the spinal cord but not in the hindbrain and trigeminal neurons. Our in vivo analysis reveals a novel role for Rgs4 in regulating axonogenesis during embryogenesis, which is mediated by another schizophrenia-associated gene, Akt1, in a region-specific manner.

Zebrafish Her8a is activated by Su(H)-dependent Notch signaling and is essential for the inhibition of neurogenesis.

Understanding how diversity of neural cells is generated is one of the main tasks of developmental biology. The Hairy/E(spl) family members are potential targets of Notch signaling, which has been shown to be fundamental to neural cell maintenance, cell fate decisions, and compartment boundary formation. However, their response to Notch signaling and their roles in neurogenesis are still not fully understood. In the present study, we isolated a zebrafish homologue of hairy/E(spl), her8a, and showed this gene is specifically expressed in the developing nervous system. her8a is positively regulated by Su(H)-dependent Notch signaling as revealed by a Notch-defective mutant and injection of variants of the Notch intracellular regulator, Su(H). Morpholino knockdown of Her8a resulted in upregulation of proneural and post-mitotic neuronal markers, indicating that Her8a is essential for the inhibition of neurogenesis. In addition, markers for glial precursors and mature glial cells were down-regulated in Her8a morphants, suggesting Her8a is required for gliogenesis. The role of Her8a and its response to Notch signaling is thus similar to mammalian HES1, however this is the converse of what is seen for the more closely related mammalian family member, HES6. This study not only provides further understanding of how the fundamental signaling pathway, Notch signaling, and its downstream genes mediate neural development and differentiation, but also reveals evolutionary diversity in the role of H/E(spl) genes.