Guosheng Cheng

Three-dimensional graphene foam as a biocompatible and conductive scaffold for neural stem cells

Neural stem cell (NSC) based therapy provides a promising approach for neural regeneration. For the success of NSC clinical application, a scaffold is required to provide three-dimensional (3D) cell growth microenvironments and appropriate synergistic cell guidance cues. Here, we report the first utilization of graphene foam, a 3D porous structure, as a novel scaffold for NSCs in vitro. It was found that three-dimensional graphene foams (3D-GFs) can not only support NSC growth, but also keep cell at an active proliferation state with upregulation of Ki67 expression than that of two-dimensional graphene films. Meanwhile, phenotypic analysis indicated that 3D-GFs can enhance the NSC differentiation towards astrocytes and especially neurons. Furthermore, a good electrical coupling of 3D-GFs with differentiated NSCs for efficient electrical stimulation was observed. Our findings implicate 3D-GFs could offer a powerful platform for NSC research, neural tissue engineering and neural prostheses.

The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates.

Graphene has been demonstrated in many biomedical applications and its potentials for neural interfacing. Emerging concerns on graphene, as a biomedical material, are its biocompatibility and how biologically targeted tissue/cells respond to it. Relatively few studies attempted to address the interactions of graphene or its derivatives with the tissues/cells, while very few reports on neural system. In this study, we tried to explore how neurites, one of the key structures for neural functions, are affected by graphene during the development until maturation in a mouse hippocampal culture model. The results reveal that graphene substrates exhibited excellent biocompatibility, as cell viability and morphology were not affected. Meanwhile, neurite numbers and average neurite length on graphene were significantly enhanced during 2-7 days after cell seeding compared with tissue culture polystyrene (TCPS) substrates. Especially on Day 2 of the neural development period, graphene substrates efficiently promoted neurite sprouting and outgrowth to the maximal extent. Additionally, expression of growth-associate protein-43 (GAP-43) was examined in both graphene and TCPS groups. Western blot analysis showed that GAP-43 expression was greatly enhanced in graphene group compared to TCPS group, which might result in the boost of neurite sprouting and outgrowth. This study suggests the potential of graphene as a material for neural interfacing and provides insight into the future biomedical applications of graphene.

Enhancement of electrical signaling in neural networks on graphene films

One of the key challenges for neural tissue engineering is to exploit supporting materials with robust functionalities not only to govern cell-specific behaviors, but also to form functional neural network. The unique electrical and mechanical properties of graphene imply it as a promising candidate for neural interfaces, but little is known about the details of neural network formation on graphene as a scaffold material for tissue engineering. Therapeutic regenerative strategies aim to guide and enhance the intrinsic capacity of the neurons to reorganize by promoting plasticity mechanisms in a controllable manner. Here, we investigated the impact of graphene on the formation and performance in the assembly of neural networks in neural stem cell (NSC) culture. Using calcium imaging and electrophysiological recordings, we demonstrate the capabilities of graphene to support the growth of functional neural circuits, and improve neural performance and electrical signaling in the network. These results offer a better understanding of interactions between graphene and NSCs, also they clearly present the great potentials of graphene as neural interface in tissue engineering.

Anti-inflammatory effects of three-dimensional graphene foams cultured with microglial cells

One of the key goals in nerve tissue engineering is to develop new materials which cause less or no neuroinflammation. Despite the rapid advances of using graphene as a neural interface material, it still remains unknown whether graphene could provoke neuroinflammation or not, and whether and how the topographical features of graphene influence the neuroinflammation induction. By immunofluorescence, Elisa technique, western blot, scanning electron microscope (SEM) methods, we investigated the pro- and/or anti-inflammatory responses of microglia in the graphene films (2D-graphene) or graphene foams (3D-graphene) culturing systems. Furthermore, the growth situations of the neural stem cells (NSCs) in the conditioned culture medium produced in the graphene substrates were evaluated. The results show that: 1) neither 2D nor 3D graphene induced distinct neuroinflammation when compared to the tissue culture polystyrene (TCPS) substrates; 2) the topographical structures of the graphene might affect the material/cell interactions, leading to disparate effects on lipopolysaccharide (LPS)-induced neuroinflammation; 3) 3D graphene exhibited a remarkable capability of rescuing LPS-induced neuroinflammation probably through the restriction of microglia morphological transformation by the unique topographical features on the surface, showing the ability of anti-inflammation against external insults, while 2D graphene failed to. These results provide insights into the diverse biological effects of the materials topographical structures and open new opportunity for the applications of graphene in neuroscience.

Label-Free MicroRNA Detection Based on Fluorescence Quenching of Gold Nanoparticles with a Competitive Hybridization.

MicroRNAs (miRNAs), critical biomarkers of acute and chronic diseases, play key regulatory roles in many biological processes. As a result, there is great demand for robust assay platforms to enable an accurate and efficient detection of low-level miRNAs in complex biological samples. In this work, a label-free and Au nanoparticles (NPs) quenching-based competition assay system was developed. In the designed system, Au NPs with diameter sizes of 10 and 20 nm displayed fluorescence quenching efficiencies of 84% and 82% for Cy3 dye on slide surface, whereas the quenching efficiency of commercial BHQ2 quencher was roughly 50%. Assay conditions were optimized for miRNA-205 detection. A limit of detection of 3.8 pM and a detection range covering from 3.8 pM to 10 nM were achieved. Furthermore, the proposed system was capable of specifically discriminating miRNAs with slight variations in their nucleotide sequence and was also qualified for assessing miRNA levels in human serum. Our strategy has the potential to provide new perspectives in profiling the pattern of miRNA expression and biomedical utilizations.

Enhanced Migration of Neural Stem Cells by Microglia Grown on a Three-Dimensional Graphene Scaffold

One of the key challenges in engineering neural tissues for cell-based therapies is to develop a biocompatible scaffold material to direct neural stem cell (NSC) behaviors. One great advantage for a scaffold would be to induce NSC migration toward pathological sites during regeneration and repair. In particular, the inflammatory responses in the pathological zone, which are mainly mediated by microglia in the central nervous system, affect the repair capacity of NSCs through NSC migration. Recently, graphene was used as a neural interface and scaffold material, but few studies have addressed the relationship between microglia and NSCs in a graphene culture system. In this study, we used a combination of immunofluorescence, Western blotting, enzyme-linked immunosorbent assays, and scanning electron microscopy to investigate how conditioned medium (CM) produced from microglia grown on two-dimensional graphene (2D-G) films or three-dimensional graphene (3D-G) foams govern NSC migration. The results revealed that the CM produced by microglia grown in 3D-G cultures could promote neurosphere formation, facilitate NSC migration from the neurospheres, and increase single cell polarization by activating the stromal cell-derived factor 1 α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) signaling pathway and enhancing cell adhesion on the substrate. By contrast, the 2D-G CM failed to achieve these results. Our study suggests the great potential of 3D-G as a neural scaffold for NSC-based therapy in tissue engineering and regenerative medicine.

The role of dimensionality in neuronal network dynamics

Recent results from network theory show that complexity affects several dynamical properties of networks that favor synchronization. Here we show that synchronization in 2D and 3D neuronal networks is significantly different. Using dissociated hippocampal neurons we compared properties of cultures grown on a flat 2D substrates with those formed on 3D graphene foam scaffolds. Both 2D and 3D cultures had comparable glia to neuron ratio and the percentage of GABAergic inhibitory neurons. 3D cultures because of their dimension have many connections among distant neurons leading to small-world networks and their characteristic dynamics. After one week, calcium imaging revealed moderately synchronous activity in 2D networks, but the degree of synchrony of 3D networks was higher and had two regimes: a highly synchronized (HS) and a moderately synchronized (MS) regime. The HS regime was never observed in 2D networks. During the MS regime, neuronal assemblies in synchrony changed with time as observed in mammalian brains. After two weeks, the degree of synchrony in 3D networks decreased, as observed in vivo. These results show that dimensionality determines properties of neuronal networks and that several features of brain dynamics are a consequence of its 3D topology.

Three-Dimensional Stiff Graphene Scaffold on Neural Stem Cells Behavior

Physical cues of the scaffolds, elasticity, and stiffness significantly guide adhesion, proliferation, and differentiation of stem cells. In addressable microenvironments constructed by three-dimensional graphene foams (3D-GFs), neural stem cells (NSCs) interact with and respond to the structural geometry and mechanical properties of porous scaffolds. Our studies aim to investigate NSC behavior on the various stiffness of 3D-GFs. Two kinds of 3D-GFs scaffolds present soft and stiff properties with elasticity moduli of 30 and 64 kPa, respectively. Stiff scaffold enhanced NSC attachment and proliferation with vinculin and integrin gene expression were up-regulated by 2.3 and 1.5 folds, respectively, compared with the soft one. Meanwhile, up-regulated Ki67 expression and almost no variation of nestin expression in a group of the stiff scaffold were observed, implying that the stiff substrate fosters NSC growth and keeps the cells in an active stem state. Furthermore, NSCs grown on stiff scaffold exhibited enhanced differentiation to astrocytes. Interestingly, differentiated neurons on stiff scaffold are suppressed since growth associated protein-43 expression was significantly improved by 5.5 folds.

Hydrophilic cell-derived extracellular matrix as a niche to promote adhesion and differentiation of neural progenitor cells

The natural extracellular matrix (ECM) offers a dynamic and intricate microenvironment, which serves as a structural support and regulates cell phenotype and its function. Recently, ECM has been revealed as a favorable and biocompatible architecture for stem cell adhesion and growth in cellular therapy to treat various diseases. However, cell-derived ECM is rarely used as a culture substrate for anchorage-dependent cells, such as neural progenitor cells (NPCs), which have the potential to differentiate into basal forebrain cholinergic neurons (BFCNs) for Alzheimers disease. Here, we report mouse embryonic fibroblast (MEF)-derived ECM, with an appropriately hydrophilic property (water contact angle of 66.8°) to mimic the neural niche for NPCs. In addition, MEF-derived ECM possesses a nanotopological surface, plentiful kinds of components and exhibits excellent adhesion properties for anchoring NPCs. Compared with a laminin-coated plate, MEF-derived ECM promotes NPC proliferation and differentiation into BFCNs by ∼1.6 fold and ∼3.1 fold, respectively, consequently enhancing the production of acetylcholine by ∼2.0 fold. This MEF-derived ECM could be a favorable cell culture carrier for NPC attachment, with great potential for applications in stem cell therapy for Alzheimers disease.