Interaction of graphene and WS2 with neutrophils and mesenchymal stem cells: implications for peripheral nerve regeneration.

Graphene and bidimensional (2D) materials have been widely used in nerve conduits to boost peripheral nerve regeneration. Nevertheless, the experimental and commercial variability in graphene-based materials generates graphene forms with different structures and properties that can trigger entirely diverse biological responses from all the players involved in nerve repair. Herein, we focus on the graphene and tungsten disulfide (WS2) interaction with non-neuronal cell types involved in nerve tissue regeneration. We synthesize highly crystalline graphene and WS2 with scalable techniques such as thermal decomposition and chemical vapor deposition. The materials were able to trigger the activation of a neutrophil human model promoting Neutrophil Extracellular Traps (NETs) production, particularly under basal conditions, although neutrophils were not able to degrade graphene. Of note is that pristine graphene acts as a repellent for the NET adhesion, a beneficial property for nerve conduit long-term applications. Mesenchymal stem cells (MSCs) have been proposed as a promising strategy for nerve regeneration in combination with a conduit. Thus, the interaction of graphene with MSCs was also investigated, and reduced viability was observed only on specific graphene substrates. Overall, the results confirm the possibility of regulating the cell response by varying graphene properties and selecting the most suitable graphene forms.

Scalable High-Mobility Graphene/hBN Heterostructures.

Graphene-hexagonal boron nitride (hBN) scalable heterostructures are pivotal for the development of graphene-based high-tech applications. In this work, we demonstrate the realization of high-quality graphene-hBN heterostructures entirely obtained with scalable approaches. hBN continuous films were grown via ion beam-assisted physical vapor deposition directly on commercially available SiO2/Si and used as receiving substrates for graphene single-crystal matrixes grown by chemical vapor deposition on copper. The structural, chemical, and electronic properties of the heterostructure were investigated by atomic force microscopy, Raman spectroscopy, and electrical transport measurements. We demonstrate graphene carrier mobilities exceeding 10,000 cm2/Vs in ambient conditions, 30% higher than those directly measured on SiO2/Si. We prove the scalability of our approach by measuring more than 100 transfer length method devices over a centimeter scale, which present an average carrier mobility of 7500 ± 850 cm2/Vs. The reported high-quality all-scalable heterostructures are of relevance for the development of graphene-based high-performing electronic and optoelectronic applications.

Structure-Property-Processing Correlations of Graphene Bioscaffolds for Proliferation and Differentiation of C2C12 Cells.

Graphene - an atomically thin layer of carbon atoms arranged in a hexagonal lattice - has gained interest as a bioscaffold for tissue engineering due to its exceptional mechanical, electrical, and thermal properties. Graphene's structure and properties are tightly coupled to synthesis and processing conditions, yet their influence on biomolecular interactions at the graphene-cell interface remains unclear. In this study, C2C12 cells were grown on graphene bioscaffolds with specific structure-property- processing-performance (SP3) correlations. Bioscaffolds were prepared using three different methods - chemical vapor deposition (CVD), sublimation of silicon carbide (SiC), and printing of liquid phase exfoliated graphene. To investigate the biocompatibility of each scaffold, cellular morphology and gene expression patterns were investigated using the bipotential mouse C2C12 cell line. Using a combination of fluorescence microscopy and qRT-PCR, we demonstrate that graphene production methods determine the structural and mechanical properties of the resulting bioscaffold, which in turn determine cell morphology, gene expression patterns, and cell differentiation fate. Therefore, production methods and resultant structure and properties of graphene bioscaffolds must be chosen carefully when considering graphene as a bioscaffold for musculoskeletal tissue engineering.

Local dielectric function of hBN-encapsulated WS2flakes grown by chemical vapor deposition.

Hexagonal boron nitride (hBN), sometimes referred to as white graphene, receives growing interest in the scientific community, especially when combined into van der Waals (vdW) homo- and heterostacks, in which novel and interesting phenomena may arise. hBN is also commonly used in combination with two-dimensional (2D) semiconducting transition metal dichalcogenides (TMDCs). The realization of hBN-encapsulated TMDC homo- and heterostacks can indeed offer opportunities to investigate and compare TMDC excitonic properties in various stacking configurations. In this work, we investigate the optical response at the micrometric scale of mono- and homo-bilayer WS2grown by chemical vapor deposition and encapsulated between two single layers of hBN. Imaging spectroscopic ellipsometry is exploited to extract the local dielectric functions across one single WS2flake and detect the evolution of excitonic spectral features from monolayer to bilayer regions. Exciton energies undergo a redshift by passing from hBN-encapsulated single layer to homo-bilayer WS2, as also confirmed by photoluminescence spectra. Our results can provide a reference for the study of the dielectric properties of more complex systems where hBN is combined with other 2D vdW materials into heterostructures and are stimulating towards the investigation of the optical response of other technologically-relevant heterostacks.

Axonal plasticity in response to active forces generated through magnetic nano-pulling.

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Human TrkAR649W mutation impairs nociception, sweating and cognitive abilities: a mouse model of HSAN IV.

A functional nerve growth factor NGF-Tropomyosin Receptor kinase A (TrkA) system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Indeed, mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, anhidrosis and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Here, we report the generation of a knock-in mouse line carrying the HSAN IV TrkAR649W mutation. First, by in vitro biochemical and biophysical analyses, we show that the pathological R649W mutation leads to kinase-inactive TrkA also affecting its membrane dynamics and trafficking. In agreement with the HSAN IV human phenotype, TrkAR649W/m mice display a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation, in addition to decreased sweating in comparison to TrkAh/m controls. Moreover, the R649W mutation decreases anxiety-like behavior and compromises cognitive abilities, by impairing spatial-working and social memory. Our results further uncover unexplored roles of TrkA in thermoregulation and sociability. In addition to accurately recapitulating the clinical manifestations of HSAN IV patients, our findings contribute to clarifying the involvement of the NGF-TrkA system in pain sensation.

Graphene-based nanomaterials for peripheral nerve regeneration.

Emerging nanotechnologies offer numerous opportunities in the field of regenerative medicine and have been widely explored to design novel scaffolds for the regeneration and stimulation of nerve tissue. In this review, we focus on peripheral nerve regeneration. First, we introduce the biomedical problem and the present status of nerve conduits that can be used to guide, fasten and enhance regeneration. Then, we thoroughly discuss graphene as an emerging candidate in nerve tissue engineering, in light of its chemical, tribological and electrical properties. We introduce the graphene forms commonly used as neural interfaces, briefly review their applications, and discuss their potential toxicity. We then focus on the adoption of graphene in peripheral nervous system applications, a research field that has gained in the last years ever-increasing attention. We discuss the potential integration of graphene in guidance conduits, and critically review graphene interaction not only with peripheral neurons, but also with non-neural cells involved in nerve regeneration; indeed, the latter have recently emerged as central players in modulating the immune and inflammatory response and accelerating the growth of new tissue.

Synthesis of Large-Scale Monolayer 1T'-MoTe2 and Its Stabilization via Scalable hBN Encapsulation.

Out of the different structural phases of molybdenum ditelluride (MoTe2), the distorted octahedral 1T' possesses great interest for fundamental physics and is a promising candidate for the implementation of innovative devices such as topological transistors. Indeed, 1T'-MoTe2 is a semimetal with superconductivity, which has been predicted to be a Weyl semimetal and a quantum spin Hall insulator in bulk and monolayer form, respectively. Large instability of monolayer 1T'-MoTe2 in environmental conditions, however, has made its investigation extremely challenging so far. In this work, we demonstrate homogeneous growth of large single-crystal (up to 500 µm) monolayer 1T'-MoTe2 via chemical vapor deposition (CVD) and its stabilization in air with a scalable encapsulation approach. The encapsulant is obtained by electrochemically delaminating CVD hexagonal boron nitride (hBN) from copper foil, and it is applied on the freshly grown 1T'-MoTe2 via a top-down dry lamination step. The structural and electrical properties of encapsulated 1T'-MoTe2 have been monitored over several months to assess the degree of degradation of the material. We find that when encapsulated with hBN, the lifetime of monolayer 1T'-MoTe2 successfully increases from a few minutes to more than a month. Furthermore, the encapsulated monolayer can be subjected to transfer, device processing, and heating and cooling cycles without degradation of its properties. The potential of this scalable heterostack is confirmed by the observation of signatures of low-temperature phase transition in monolayer 1T'-MoTe2 by both Raman spectroscopy and electrical measurements. The growth and encapsulation methods reported in this work can be employed for further fundamental studies of this enticing material as well as facilitate the technological development of monolayer 1T'-MoTe2.

Effect of Chemical Vapor Deposition WS2 on Viability and Differentiation of SH-SY5Y Cells.

In recent years, transition metal dichalcogenides have been attracting an increasing interest in the biomedical field, thus implying the need of a deeper understanding of their impact on cell behavior. In this study we investigate tungsten disulfide (WS2) grown via chemical vapor deposition (CVD) on a transparent substrate (sapphire) as a platform for neural-like cell culture. We culture SH-SY5Y human neuroblastoma cells on WS2, using graphene, sapphire and standard culture well as controls. The quality, thickness and homogeneity of the materials is analyzed using atomic force microscopy and Raman spectroscopy. The cytocompatibility of CVD WS2 is investigated for the first time by cell viability and differentiation assessment on SH-SY5Y cells. We find that cells differentiated on WS2, displaying a viability and neurite length comparable with the controls. These findings shine light on the possibility of using WS2 as a cytocompatible material for interfacing neural cells.

Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions.

The set-up of an advanced imaging experiment requires a careful selection of suitable labeling strategies and fluorophores for the tagging of the molecules of interest. Here we provide an experimental workflow to allow evaluation of fluorolabeling performance of the chemical tags target of phosphopantetheinyl transferase enzymes (PPTases), once inserted in the sequence of different proteins of interest. First, S6 peptide tag was fused to three different single-pass transmembrane proteins (the tyrosine receptor kinases TrkA and VEGFR2 and the tumor necrosis factor receptor p75NTR), providing evidence that all of them can be conveniently albeit differently labeled. Moreover, we chose the S6-tagged TrkA construct to test eight different organic fluorophores for the PPTase labeling of membrane receptors in living cells. We systematically compared their non-specific internalization when added to a S6-tag negative cell culture, the percentage of S6-TrkA expressing cells effectively labeled and the relative mean fluorescence intensity, their photostability upon conjugation, and ratio of specific (cellular) versus background (glass-adhered) signal. This allowed to identify which fluorophores are actually recommended for these labeling reactions. Finally, we compared the PPTase labeling of a purified, YBBR-tagged Nerve Growth Factor with two differently charged organic dyes. We detected some batch-to-batch variability in the labeling yield, regardless of the fluorophore used. However, upon purification of the fluorescent species and incubation with living primary DRG neurons, no significant difference could be appreciated in both internalization and axonal transport of the labeled neurotrophins.

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes.

Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first 2 days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications.

Peripheral Neuron Survival and Outgrowth on Graphene.

Graphene displays properties that make it appealing for neuroregenerative medicine, yet its interaction with peripheral neurons has been scarcely investigated. Here, we culture on graphene two established models for peripheral neurons: PC12 cells and DRG primary neurons. We perform a nano-resolved analysis of polymeric coatings on graphene and combine optical microscopy and viability assays to assess the material cytocompatibility and influence on differentiation. We find that differentiated PC12 cells display a remarkably increased neurite length on graphene (up to 27%) with respect to controls. Notably, DRG primary neurons survive both on bare and coated graphene. They present dense axonal networks on coated graphene, while they form cell islets characterized by dense axonal bundles on uncoated graphene. These findings indicate that graphene holds potential for nerve tissue regeneration and might pave the road to novel concepts of active nerve conduits.

Saturable absorption of femtosecond optical pulses in multilayer turbostratic graphene.

We investigate the nonlinear transmission of a ~280-layer turbostratic graphene sheet for near-infrared amplifier laser pulses (775 nm, Ti:sapphire laser) with a duration of 150-fs and 20-fs. Saturable absorption is observed in both cases, however it is not very strong, amounting to ~13% transmittance change for the 20-fs (150-fs) pulses at a peak intensity of 30 GW/cm2 (4 GW/cm2). The dependence on incident peak intensity is reproduced well using a theoretical model for the time-dependent saturable absorption, where the excited carriers vacate the photo-excited energy range within 3-5 fs, which we attribute to energy redistribution due to carrier-carrier scattering. This is also supported by spectrally resolved measurements for the 20-fs pulses, which show a marked dependence of the degree of saturation on the photon energy. A key result is that the shorter pulses do not yield a lower saturation fluence, due to the combined effects of the broader excitation bandwidth, and the rapid and broad energy redistribution. We also predict the potential performance of multilayer graphene samples for removing pedestal and pre-pulse structure from ultrafast high-energy pulses.

THz saturable absorption in turbostratic multilayer graphene on silicon carbide.

We investigated the room-temperature Terahertz (THz) response as saturable absorber of turbostratic multilayer graphene grown on the carbon-face of silicon carbide. By employing an open-aperture z-scan method and a 2.9 THz quantum cascade laser as source, a 10% enhancement of transparency is observed. The saturation intensity is several W/cm2, mostly attributed to the Pauli blocking effect in the intrinsic graphene layers. A visible increase of the modulation depth as a function of the number of graphene sheets was recorded as consequence of the low nonsaturable losses. The latter in turn revealed that crystalline disorder is the main limitation to larger modulations, demonstrating that the THz nonlinear absorption properties of turbostratic graphene can be engineered via a proper control of the crystalline disorder and the layers number.

Electroburning of few-layer graphene flakes, epitaxial graphene, and turbostratic graphene discs in air and under vacuum.

Graphene-based electrodes are very promising for molecular electronics and spintronics. Here we report a systematic characterization of the electroburning (EB) process, leading to the formation of nanometer-spaced gaps, on different types of few-layer graphene (namely mechanically exfoliated graphene on SiO2, graphene epitaxially grown on the C-face of SiC and turbostratic graphene discs deposited on SiO2) under air and vacuum conditions. The EB process is found to depend on both the graphene type and on the ambient conditions. For the mechanically exfoliated graphene, performing EB under vacuum leads to a higher yield of nanometer-gap formation than working in air. Conversely, for graphene on SiC the EB process is not successful under vacuum. Finally, the EB is possible with turbostratic graphene discs only after the creation of a constriction in the sample using lithographic patterning.