Leading-edge regenerative medicine can take advantage of improved knowledge of key roles played, both in stem cell fate determination and in cell growth/differentiation, by mechano-transduction and other physicochemical stimuli from the tissue environment. This prompted advanced nanomaterials research to provide tissue engineers with next-generation scaffolds consisting of smart nanocomposites and/or hydrogels with nanofillers, where balanced combinations of specific matrices and nanomaterials can mediate and finely tune such stimuli and cues. In this review, we focus on graphene-based nanomaterials as, in addition to modulating nanotopography, elastic modulus and viscoelastic features of the scaffold, they can also regulate its conductivity. This feature is crucial to the determination and differentiation of some cell lineages and is of special interest to neural regenerative medicine. Hereafter we depict relevant properties of such nanofillers, illustrate how problems related to their eventual cytotoxicity are solved via enhanced synthesis, purification and derivatization protocols, and finally provide examples of successful applications in regenerative medicine on a number of tissues. Graphene consists of an atomic honeycomb lattice composed of carbon atoms that can be considered as an indefinite large polycyclic aromatic hydrocarbon with an infinite number of condensed benzene rings. Graphene family is constituted by several derivatives such as graphene oxide (GO), reduced graphene oxide (RGO), graphene quantum dots (GQDs), graphene nanosheets, monolayer graphene, and few-layer graphene . A schemDue its high electrical conductivity, mechanical properties and aspect ratio, graphene has become attractive in many fields. In addition to being a rising star in scientific fields other than biology and medicine, graphene, GBMs and composites are widely used for important biotechnological and biomedical applications. Almost all graphene derivatives and composites are being used and tuned to develop special delivery carriers for theranostics , gene thConductivity and high transporter capability of graphene allow for tuning biosensor surface features and outperforming many other biosensor types in terms of speed, accuracy, specificity, selectivity and sensitivity. In general, proteins  are associated to the graphene-based biosensor surface via electrostatic interaction, covalent bond or by polymer mediated capture. To avoid electrostatic interaction may alter the protein conformation, GBMs are combined with polymers in nanocomposites where mild electrostatics combines efficient binding and maintenance of the original conformation . When inGBMs, and especially GO and RGO find plenty of applications in tissue engineering, where they are employed as scaffolds for tissue regeneration. Tissue engineering is an interdisciplinary technology that gains insights from material chemistry, engineering, cell biology, and immunology to develop biomaterials capable of restoring, maintaining, or improving tissue function or a whole organ . ScaffolGraphene-based scaffolds (GBSs) are a particular class of scaffold made from graphene, GO and/or RGO nanocomposites. Among the plethora of nanomaterials available, graphene and its derivatives are attractive candidates for developing tissue engineering scaffolds thanks to their tuneable electrical conductivity, excellent mechanical properties, biocompatibility, chemically modifiable surface, and nanoscale dimension matching cell surface receptors and extracellular matrix (ECM) nanoroughness/nanotopography. Morover, they display good capacity to adsorb proteins from the serum , favoring cell adhesion, proliferation and differentiation. 9 Pa), whereas nervous tissue requires much softer supports (E < 4·102 Pa) and muscles need substrate with intermediate stiffness (E > 104 Pa) [Graphene structural features and dimensions resemble many components of the extracellular environment such as proteins of the ECM (e.g. collagen), ion channels, signalling proteins and cytoskeletal elements . Therefo 104 Pa) . Being one of the toughest and strongest nanomaterials discovered so far, graphene incorporation into polymeric scaffolds enhances their mechanical properties, toughness and tensile strength . TherefoAt a glance, graphene ability to mimic the natural extracellular environment nanotopography, to retain signalling molecules, to be easily incorporated in both natural and synthetic polymers, and to modulate stiffness and conductivity of the scaffold make it the ideal nanomaterial to provide cues needed to guide cell behaviour and hence an invaluable tool for regenerative medicine applications. Nevertheless, the toxicology profile of graphene and its derivative has not been completely elucidated yet. Several drawbacks of GBMs employment for regenerative medicine approaches have been reported which might include membrane damage, hydrophobic interaction, oxidative stress, genotoxicity, mitochondrial disorders and autophagy. However, safety risks should be evaluated case by case based on the intrinsic properties of GBMs, such as purity, surface functional groups, lateral size, stiffness, hydrophobicity and structural defects. Moreover, several reports showed that graphene cytotoxicity is influenced by multiple parameters such as cell population tested as well as graphene dispersibility and functionalization ,25. As thoroughly reviewed by Wu and co-workers graphene synthesis can be performed through a plethora of bottom-up or top-down approaches . Among tCVD is often exploited to produce graphene for 2D composites and graphenic foams, described in the next sections. In CVD, gaseous precursors  are flowed at high temperatures over a metal surface, which acts as a catalyst for their decomposition and leads to the condensation of carbon atoms, forming a graphene sheet. In a typical process, graphene is grown onto a metal surface, supported with a polymer —PMMA), and the catalyst is etched by acidic treatment. Subsequently, the graphene foil is transferred on a substrate and the supporting polymer is appropriately dissolved. The choice of metal or alloy for deposition changes process thermodynamics and kinetics, and allows to finely tune the number of graphene layers of the resulting material. The most common metal catalysts for CVD are nickel ,28 and c-2) [However, bulk production of graphene is more conveniently achieved starting from graphite and weakening the van der Waals forces between its stacked monoatomic carbon layers. Examples for such top-down approaches are liquid-phase exfoliation, surfactant-assisted liquid-phase exfoliation and chemical functionalization. In the first two methods ,32,33,34-2) ; (ii) th-2) ,37.2 network, the enhanced hydrophilicity that results from the oxidation can be beneficial for its compatibility in different types of matrixes.Among the most common chemical top-down methods, there is the oxidation and subsequent exfoliation of graphite to GO, followed by either chemical reduction or thermal cleavage of oxidized groups to obtain RGO. In a typical procedure, graphite is mixed with sulfuric acid and oxidizing agents in an iterative and synergic action of intercalation and oxidation . SubsequOxidation is usually carried out at 40–50 °C. However, as demonstrated by Eigler and co-workers, working at lower temperatures could reduce damages to the basal plane. They demonstrated the possibility to synthesize a minimally damaged GO with an almost intact σ framework of C atoms  and supeRGO is obtained from GO with different synthetic methods, yielding materials with different properties. Indeed, thermal treatment  and chemical reduction of GO to RGO do not have the same effect on graphene structure, hence on the properties of the resulting materials. The disproportionation induced by thermal treatment of highly oxidized GO brings defective holes in the plane.N-doping in plane, while reduction with L-ascorbic acid leaves adsorbates on RGO that are not easily removed by washing procedures [2 network is partially restored, the performances are still lower than those of CVD graphene.Chemical reduction, on the other hand, can be achieved with different reactants  leading ocedures . Since cStability and reactivity of GO are also affected by other parameters such as pH of the dispersion, which is often neglected or underestimated. Indeed, Hirsch and co-workers  found evIn the next sections, graphene-based nanocomposites will be considered. In these materials, GBMs act as fillers while the matrix is typically an organic polymer , though bioglasses and ceramics are also used. In the first part, two-dimensional (2D) scaffolds are discussed, whereas the second part is devoted to three-dimensional (3D) scaffolds . In partTwo-dimensional scaffolds are relatively low cost and easy to fabricate, thus they are often used in preliminary studies to investigate the effect of a specific substrate on cell behavior.The simplest example of a graphenic 2D-scaffold is represented by CVD-grown graphene (one or more layers) on a PMMA-supported metal catalyst, and then transferred onto a substrate after etching of the metal catalyst. Jangho et al. used this technique to transfer the monolayer graphene onto glass to study its effects on the reciprocal interactions between cells and substrate and to test the possible promotion of human mesenchymal stem cell (hMSC) neurogenesis and neurite outgrowth . In a siAnother method to obtain a graphene-coated surface is based on the chemical modification of a substrate to enable specific interactions with graphene-based materials (GBMs). Ryoo et al. used (3-aminopropyl)triethoxylane (APTES) to decorate the surface of glass coverslips with aminic groups. As a result, they obtained a positively charged surface which could effectively interact with negatively charged GO. Similarly, they exploited (3-glycidyloxypropyl)trimethoxylane (GPTMS) to promote glass interaction with aminated carbon nanotubes (CNT). In vitro tests proved carbon nanomaterial-coated glass to be better at promoting the number and dimensionality of focal adhesions, suggesting good biocompatibility .Two-dimensional graphene-based scaffolds can also be obtained by vacuum filtration of material suspensions. For instance, Jasin and co-workers fabricated graphene-based paper as a substrate for cell growth, air drying vacuum filtrated dispersions of three different starting materials: (i) graphite oxide and graphene oxide with (ii) small and (iii) large average lateral dimensions. Although they did not observe any significant difference on cell adhesion, morphology or proliferation, the smaller release of lactate dehydrogenase (LDH) enzyme compared to control samples, suggested that their scaffold can enhance cell viability .Gluconacetobacter intermedius. GO was added to the growth media and G. intermedius bio-reduction capabilities were exploited to obtain BC-RGO composites. hMSCs seeded onto these materials showed higher proliferation compared to ones seeded onto films of RGO without the fibrous structure of cellulose [A higher degree of versatility is achieved with hybrid or composite scaffolds, where graphene is used as a filler or coating for polymeric matrices. As an example, Pandele et al. prepared chitosan/GO composites by solution blending, obtaining films with a rough surface useful for cell adhesion. The homogeneous dispersion of GO in a polymeric matrix led to an enhancement of the mechanical properties due to the large aspect ratio of the nanomaterial and its interaction with the polymer chains . Furtherellulose . Li et aThese scaffolds showed low resistivity (∼300 Ω/sq), increased mechanical strength and a specific surface micro-topography induced by RGO, which led to improved stem cell adhesion and osteogenic induction. Furthermore, their 2D-scaffolds could be employed with pseudo-3D stacked multilayered constructs that can be configured by rolling or folding, allowing designing a large number of different setups .To enhance their biological effects, two-dimensional scaffolds can be micro- or nanopatterned with specific topographical cues that can direct cell growth and differentiation. Different methods have been developed to this aim, and a pattern can be drawn with either the help of a positive photoresists spin-coated on graphene oxide surface , or by tAmong the plethora of chemical studies presenting new kinds of scaffolds, there is a modest number of works specifically focused on specific GBM functionalization strategies to improve biocompatibility or differentiation capabilities. As an example, Qi et al. functionalized GO with L-theanine, an amino acid that promotes neuronal differentiation. Its presence in a poly(lactic-co-glycolic acid (PLGA) film increased its hydrophilicity and enhanced neuronal differentiation of neuronal stem cells (NSCs) . In our Despite the aforementioned potentialities, 2D scaffolds have limitations. First of all, a two-dimensional environment is not suited to reproduce natural ECM. Then, nutrients are directly available to cells and wastes can diffuse to a limited extent. Lastly, altered cell–cell interactions may result in unpredictable cell responses. Therefore, in recent years the focus has shifted towards the study and design of 3D-scaffolds in order to overcome these limitations.As already mentioned, 3D scaffolds recapitulate tissue biophysical features thus are better candidates for in vivo applications. Scaffolds with a three-dimensional architecture should be endowed with a highly interconnected porous network. Recently, Lutzweiler and co-workers reviewed the effects of porosity, pore size and shape, interconnectivity and curvature in scaffolds used for tissue regeneration: not only these properties directly influence migration of nutrients and wastes inside the scaffold, but also the permeation and communication between cells . Recent The easiest method to fabricate porous scaffolds involve freeze-drying filtrates or suspensions. For example, Domínguez-Bajo et al. produced RGO foams by drying GO slurries, obtaining structures with 43% of porosity and 30 µm of pore size after thermal reduction. In addition, these scaffolds had a relatively low Young’s modulus (~1.3 kPa) and made a good candidate for nervous tissue engineering. When their applicability on neural repair after spinal cord injury was tested in vivo, not only scaffolds were populated by nerve cells, but the authors also observed full vascularization . In anotIn a similar way, Hermenean et al. fabricated a porous chitosan/GO scaffold with improved mechanical performance—i.e., increased compressive strength and tunable Young’s modulus while keeping scaffold flexibility—observing that the incorporation of 3% of GO significantly enhanced bone regeneration in vivo, compared to pure chitosan scaffolds, even in the absence of additional differentiating agents, confirming the active action of GO in facilitating cell infiltration and differentiation .Graphene foams are the first porous structures composed of single layer graphene, applied in tissue engineering. Besides porosity, these scaffolds are endowed with a wrinkled topography induced by the synthetic process, which is beneficial for cell adhesion and proliferation since it better mimics the ECM ,68. Li e−1 range) and density (around 3–4 mg cm−3). Such a scaffold was able to direct neuronal growth and align neurons along a defined path to form a network [In the techniques described so far, pore size and interconnectivity depended on the Ni foam features. However, Xiao et al. recently managed to finely tailor these properties, fabricating an ordered architecture of Ni: they used photolithography to define a mask in which Ni was deposited by electroplating and aligned. Graphene was then grown through CVD on the resulting Ni template . Thanks  network .Another method to fabricate porous structures has been employed by Rasch et al. . StartinAn alternative approach to induce porosity in scaffolds is supercritical foaming which allows to control scaffold morphology through a careful choice of experimental parameters, such as chamber pressure, temperature and decompression rate. Evlashin et al. exploited this process to manufacture RGO-reinforced polycaprolactone (PCL/RGO) and PCL/GO foams in a carbon dioxide atmosphere. Although the presence of RGO in the polymer matrix led to an increase of pore size, those foams showed poor cell adhesion properties. Conversely, they found PCL/GO scaffolds to enhance cell adhesion. However, both composites displayed lack of interconnected porosity, resulting in cells attaching only on scaffold surface . Polymerin vitro tests on the pre-osteoblast cell line MC3T3-E1 [A layer-by-layer (LBL) technique was followed by Song and co-workers who deposited a positively charged polymer, poly chloride (PDDA) on a negatively charged Ni template and subsequently placed negatively charged GO onto its surface, which was then thermally converted to RGO. Electrochemical deposition of polypyrrole (PPY) and hydroxyapatite (HA) on top, increased scaffold roughness and surface area, favoring cell adhesion and proliferation as confirmed by MC3T3-E1 .Besides metallic templates, polymeric organic foams are used for polymer replication technique, especially in the inorganic scaffold field. Deliormanlı et al. used polyurethane foam to fabricate HA scaffolds, eliminating the template and sintering HA by heat treatment. PCL/GO was added by dip coating, leading to a scaffold with improved mechanical performance and higher bioactivity . The same procedure can be applied to bioactive glass, another important class of useful scaffolds in tissue engineering. As an example, Turk et al. incorporated 10% graphene directly in the glass matrix before sintering borate-based porous scaffolds, doubling the compressive strength and obtaining an electrical conductivity (0.060 S/cm) which could be exploited to electrically stimulate cell growth . MoreoveAn alternative approach to porous structure design is 3D printing, which allows to accurately control scaffold geometrical features without the limitation of using a template. Jakus et al. exploited a PLGA-based ink where they incorporated graphene with the use of surfactants and plasticizers. The mechanical properties of composites are affected by graphenic particles, with an increase of elastic modulus to a value of 16 MPa at 20 vol% loading of graphene, but with detrimental effects at higher loadings (40–60 vol% of graphene). In addition, they observed an anisotropic alignment of graphene flakes, enhancing electrical conductivity due to shear forces produced during the 3D printing extrusion process, which increased with the decrease of the nozzle diameter . in situ by L-ascorbic acid treatment. When comparing mechanical properties of scaffolds incorporating GO or RGO they found the latter to better mimic bone Young modulus, thus their scaffold might be useful as a temporary support for bone regeneration [Cabral et al. used extrusion 3D printing to produce multicomponent scaffolds, based on tricalcium phosphate chitosan and gelatin, which mimicked the inorganic and organic components of bones, respectively. GO was added to this blend and reduced to RGO neration .Fiber-based scaffolds are largely employed in tissue engineering because they intrinsically resemble the microstructure of natural tissues. Fiber diameter, porosity, and orientation are the main features that influence cell growth and tissue regeneration . One of Generally, the smaller thickness induced by graphene-based nanofillers on electrospun fibers allows mimicking the structure of ECM even better. It is believed that the effect of GBMs on fiber diameter is due to the electrical conductivity of the feeder solution, which is a key factor in determining the diameter and size distribution of the electrospun fibers. Moreover, it is reported that fiber diameter is highly correlated to the viscosity of the feeder solution. Scaffaro and colleagues pointed out that the decrease of viscosity of a PCL solution by addition of GO induces electrospinning of thinner fibers. On the other hand, they observed an opposite effect on viscosity (and fiber diameter) with GO-grafted-PEG (GO-g-PEG). Functionalization of the filler not only increased fiber diameter, but also improved dispersion of the filler and maximized the filler/matrix interfaced area, making GO-g-PEG more effective than GO in reinforcing composite fibers, in particular at low concentration .-1 in PCL/GO-GRGDSP-Th (2% of GO), a 15-fold increase compared to neat PCL (0.95 μS cm−1). However, while scaffolds with higher content of GO (2%) showed higher electrical performances, the elastic modulus and tensile strength of 0.5% GO-scaffolds were found to be higher. Once again, this result was associated with the uniform dispersion of GO in the polymer matrix. Interestingly, this modification resulted in an increment of both electric conductivity and mechanical stability due to the ability of sulfur moieties to enable the crosslink between GO and PCL [In 2019, Basar and co-workers developed a PCL/GO composite scaffold  by funct and PCL . Scaffold properties can also be altered by combining different organic or inorganic fillers. Lui et al. developed electrospun PLA scaffolds reinforced with GO (1–3 wt %) and/or nano-HA (15 wt %). Interestingly, addition of 15 wt % nano-HA improved both elastic modulus and tensile strength, whereas concentrations of GO above 2% diminished them due to filler aggregation. Nanofiller addition slightly increased scaffold glass transition temperature and modified the hydrophobicity of PLA, enhancing the polymer water uptake, which in turn assisted cell adhesion and proliferation .Different strategies have been developed to obtain polymeric nanofiber scaffolds based on graphene and its derivatives. However, nanocomposites fail to provide a pure graphene interface. An alternative approach aims to immobilize nanostructures on the surface of polymeric nanofibers. The surface of aliphatic polyesters such as PCL and PLA can easily be functionalized with hydroxyl and amino groups by treating the polymeric scaffold with a diamine solution. In tissue engineering, aminolysis of polyesters improves their interactions with cells and allows them to form a stable graphenic coating . RecentlIndeed, coated fibers can be obtained without any surface treatment. Wang et al. developed a conductive graphene-based fibrous scaffold by coating RGO via an in situ redox reaction of GO on the surface of silk fibroin/poly(L-lactic acid-co-caprolactone) (ApF/PLCL) composite nanofibers . The aut−1) and a charge carrying capacity. This property is likely due to both RGO-CSNFM large surface areas and the extended π–π conjugated bond network generated over the surface of the RGO shell layer. Wu et al. developed an LBL method to coat electrospun nanofibers that mimic vascular ECM and enhance proliferation of endothelial cells. PLLA surface modification was achieved via electrostatic LBL self-assembly by alternately immersing PLLA fibers in a positively charged solution of 0.1 wt% chitosan and a negatively charged solution of 0.1 wt% heparin (PLLA-CS/Hep) or 0.1 wt% heparin/graphite oxide (PLLA-CS/Hep/GO). After the LBL coating, the hydrophilicity and mechanical properties of the modified PLLA nanofibers were greatly enhanced. Moreover, the CS/Hep/GO coating positively influenced cell attachment, viability, and proliferation of endothelial cells [Polymer core-CNS shell fibers can be obtained by electrospinning the polymer into a solution of graphene or one of its derivatives. Subsequently, it is possible to further functionalize or reduce the shell. Jin et al. exploited this principle to develop an RGO core-shell nanofiber (RGO-CSNFM) . The RGOal cells .The versatility of electrospinning allows to obtain complex and ordered structures. A compelling example has been reported by Shao and co-workers  who usedIn another work, Zhang and co-workers combined GO nanosheets and aligned aminolyzed PLLA nanofibers which favored nerve regeneration. The aminolysis of PLLA nanofibers allowed to form a stable GO coating. Schwann cells (SCs) cultured on these nanocomposite scaffolds displayed improved proliferation and elongation along the fiber direction compared to those grown on the aligned PLLA and aminolyzed-PLLA. The coated structure was also able to improve differentiation and neurite outgrowth of pheochromocytoma derived PC12 cell line. The authors suggest that these results may arise from the modification of surface chemistry and roughness induced by the GO coating .Hydrogels are three-dimensional entangled networks able to retain large amounts of water. Despite being mostly liquid, they display a solid-like rheological behavior and recently they have been employed as scaffolds for tissue engineering ,97. HydrGraphene and graphene derivatives in hydrogels may play the role of (i) self-assembling gelator molecule or (ii) filler in order to prepare multi-functional nanocomposite hydrogels. Self-assembly has been recognized as one of the most effective “bottom-up” strategies for building structured networks. Driven by non-covalent π-π interactions that arise from their 2D structure, graphene and graphene derivatives spontaneously re-organize into a 3D structure. Self-assembled hydrogels can be prepared through a one-step hydrothermal method starting from a graphene-based solution . For exaThe employment of pure graphene and/or graphene derivatives hydrogels is quite restricted, thus they are mainly used as high-quality nanofillers for composite hydrogels . Differe2+, Mg2+), alginate polymers form gels via non-covalent cross-linking of the carboxylate groups of the G blocks on the polymer backbone. Even if the concentration of crosslinker, percentage of G content and jellification time allows to tune the properties of alginate hydrogels, other limitations cannot be overcome without the use of specific fillers. Particularly, alginate-based hydrogels do not permit good control over their internal architecture, they lack cell receptors adhesion sites and suffer from low protein adsorption capability [Alginate is a natural polysaccharide composed of β-D-mannuroic acid (M) and α-L-guluronic acid (G) typically obtained from brown seaweed. In the presence of various divalent cation . Agarose (AG), on the other hand, is a polysaccharide obtained from red algae, displays a thermo-sensitive behavior and exhibits mechanical properties similar to that of soft tissues. However, its employment is limited by the lack of cell recognition sites. Sivashankari and Prabaharan used GO as a nanofiller for the fabrication of agarose/chitosan (AG/CS)-based scaffold . ThroughSelf-assembling peptide-based hydrogels (SAPHs) have been widely employed as vehicle for drug delivery, but they can also be employed in tissue engineering due to their biocompatibility and non-immunogenic nature ,111,112.Wang et al. prepared a silk fibroin scaffold incorporating exfoliated graphene . An aligin situ [w/v) composite hydrogels significantly increased swelling, protein adsorption and cell interaction compared to their GO-free counterparts. Moreover, GO introduction reduced gelation times and controlled degradation rates.In recent years, injectable hydrogels have drawn major attention since they need minimal invasive procedure to be administered and have reduced therapeutic costs. The hydrogel precursor should be injected as a controllable liquid  and must jellify into a robust hydrogel as quickly as possible in situ . Finallyin situ . Gelatioin situ ,118. GO-in situ . Due to Poor dispersion of GBMs within the polymer matrix causes aggregation , which mWu et al. took advantage of the abundant functional groups on the surface of nanosized GO to link starch chains via esterification . Starch Stem cells are non-specialized cells with self-renewal potential and the ability to differentiate into various cell types if directed with appropriate stimuli, making them a powerful tool for the regeneration of injured tissues . EmbryonBiochemical cues are provided by reciprocal interactions between the cell, soluble bioactive agents, and the ECM. Soluble molecules, such as growth factors, chemokines, and cytokines, diffuse to bind the cell surface receptors and have potent effects on cell growth, proliferation, and differentiation. Insoluble ECM macromolecules , glycoproteins , and polysaccharides  form a meshwork of fibers or fibrils with ECM glycoproteins incorporated into them. The resulting matrix is tissue-specific and functions as both a structural and signaling scaffold to cells .Many works showed that some of the aforementioned molecules—if administrated both in vitro and in vivo—are able to elicit specific cell responses ; moreoveCells are capable of sensing and responding to biophysical cues, over a wide range of length scales. Many of these cues are provided by the ECM, which acts as a cellular scaffold and is the primary extracellular component in tissues. In vivo, the ECM, through its structure and molecular composition, presents a variety of geometrically defined, three-dimensional (3D) physical cues in the submicron to micron scale, referred to as topographies. Cell response to topographies is mediated by a phenomenon called contact guidance, which is known to affect cell adhesion, morphology, migration, and differentiation . AnotherECM mimicry can be achieved using either natural or synthetic polymers interconnected by physical and ionic interactions and even covalent linkages . ElectroEndogenous electrical signals are present in many developing systems and influence crucial cellular behaviors—such as cell division, cell migration, and cell differentiation . Some ceElectrically conductive scaffolds not only enhance path finding of growing axons , but alsSince electroactive myocytes are responsible for heart and muscle contraction, electrically conductive materials found applications in cardiac and muscle tissue engineering as they support and maintain cell electrophysiology .Even though bone cells are non-excitable cells, stress-generated piezoelectricity has been shown to stimulate bone precursor cell proliferation and differentiation to restore the injured site, making electroactive materials and electrical stimulation a valid tool for bone regeneration strategies .Stem cells are also sensitive to electrical cues and their differentiation can be modulated by electrical stimulation and culture on electroactive materials. NSC ability to undergo neuronal and glial differentiation is boosted by electroactive material and exogenous electrical field ,158. TheCells have micro and nanoscale sensitivity because the extracellular environment presents a variety of spatially defined cues in the sub-micron to micron scale .At the nanometer level, the extracellular environment affects sub-cellular behaviors such as the organization of cell adhesion molecule receptors. At the micron level, the extracellular environment affects cellular and supracellular characteristics such as cell morphology and . The nanMolecular deposition and lithographic techniques allow the patterning of tissue-specific molecules with nanometer resolutions. For example, the deposition of molecules that promote and support neuronal adhesion, growth, and differentiation on regenerating scaffolds enables the selective adhesion and growth of neural cells and a controlled neurite extension along the geometric pattern . Apart fCombinations of stimulatory cues may be used to incorporate nanoscale topographical, biochemical, and electrical cues in the same scaffold to provide an environment for tissue regeneration that is superior to inert scaffolds. This approach—able to precisely regulate cell differentiation, morphology, and polarization—is fundamental for the development of next-generation scaffolds suitable for clinical applications.-1. Surface chemistry is key in tuning the strength and type of interactions. Epoxide, hydroxyl and carboxyl groups on the surfaces favor hydrogen and electrostatic bonding with proteins, facilitating adsorption on GO compared to pristine graphene or RGO. GBMs have been shown to strongly bind to different serum proteins such as albumin, fibronectin (Fn), collagen, and laminin [Interactions between the surface chemistry of nanomaterials and surfaces of biological components  are crucial to determine the effects on cells and tissues. As soon as a nanomaterial comes in contact with a biological fluid  it is coated with ions and proteins and develop a new interface which is often referred to as the protein corona or biomolecular corona (BC). This layer at the nanobio interface defines the biological identity of the nanomaterial, determining cell interactions, uptake and clearance . Protein laminin . Therefo laminin . In addi laminin .Not only graphene physical properties favor the adsorption of proteins, but they also offer tremendous opportunities for the covalent functionalization of protein active motifs and chemical groups . Such apThe use of graphene-based nanomaterials (GBMs) does not come without possible concerns about in vitro cytotoxicity and in vivo biocompatibility. As anticipated in Pristine GBMs have been shown to have a dose- and time-dependent in vitro toxicity in both procaryotic ,182,183 -1) compared to non-reduced GO. The authors also investigated the hemocompatibility profiles of the nanomaterials and revealed that both caused a hemolysis effects compared to negative controls. However, SRGO did not exhibit a direct proportionality between hemolytic activity and concentration, with hemolysis staying as low as ~4.9% in maximum concentration samples.To solve these problems, novel green approaches for nanoparticle synthesis and modification have been developed, involving the use of biocompatible surfactants and reducing agents. According to Askari et al. , graphenUnderstanding interactions between nanomaterials and blood is key to determining in vivo biocompatibility due to the unavoidable contact between the two. Thanks to the protein corona effect, nanoparticles that touch blood or enter the bloodstream are coated by a milieu of proteins that may undergo conformational changes, exposing new epitopes and promoting phagocytosis or elimination from the circulation . Nanomat-1. Pristine graphene showed the highest hemolysis (73%), followed by RGO (42%) and GO (27%), correlating with the degree of surface oxidation. Lower hemolytic concentrations and activity have been reported by other groups [−1) and under in vitro conditions. The authors also highlighted that pristine graphene had the potential to upregulate the production under sterile conditions of pro-inflammatory cytokines, such as IL-6 and IL-8. Cytokines are soluble glycoproteins released during an inflammatory response that recruit immune cells in order to tackle foreign bodies that have entered the organism.The hemolytic property of nanoparticles is influenced by their distribution size, shape, surface charge and chemical composition . Jaworskr groups . Howeverr groups  the hemor groups  provided−1 and therefore it is of little toxicity for M1 and M2 human macrophages, even though it triggers cell stress, ROS and inflammatory cytokines. Notably, neutrophils and macrophages are cleared from the circulation via the liver, spleen and kidneys and there is evidence that bone marrow may also play a major role in their clearance [Understanding interactions of GBMs with the immune system is of considerable relevance both from a toxicological and biomedical perspective. The BC of carbon-based nanomaterials is abundant in complement proteins. These proteins play a central role in modulating the immune and inflammatory responses towards intruders and may be a key factor in generating chronic ailments  by recruiting neutrophils and macrophages ,199. In learance . TherefoIn vivo biodistribution and pharmacokinetics of GBMs have been studied in small and large animal models ,208,209 Although the inherent toxicity of graphene and its derivatives is a major drawback for their biomedical applications, it is a well-known problem, and different strategies have been developed to overcome it. In an attempt to enhance their overall safety and minimize the risks for adverse reactions in humans from exposure, Bussy et al.  offered block-poly(propylene glycol-block-poly(ethylene glycol) (Pluronic P123), and sodium deoxycholate (DOC). The authors concluded that both materials had relatively good cytocompatibility in the 3.125–12.5 µg mL−1 range, with lowest toxicity detected in PEG-stabilized GO.Wojtoniszak et al.  showed tThe BC can be tuned and modified by exploiting the ability of GBMs to adsorb moieties from the culture medium. As previously discussed, biological and bioactive species  can be used as surfactants to stabilize graphene nanomaterials in aqueous solution, paving the way for different biomedical applications ,224,225.Covalent functionalization is another strategy for enhancing solubility in different matrices and is frequently used to obtain nanocomposites, as previously discussed in Finally, graphene and its derivatives have been used in combination with biocompatible polymeric matrices to obtain conductive nanocomposites with enhanced cell adhesion, differentiation and biocompatibility . These mBones possess a remarkable regenerative capacity, as they maintain the ability to remodel themselves throughout adult life and they can repair fractures spontaneously . After bFor instance, graphene was used to coat three-dimensional hydroxyapatite scaffolds to support the growth and osteogenic differentiation of hMSCs. Scaffolds were found to be self-standing, as hMSCs differentiation did not require common differentiative molecules  . MoreoveArnold and co-workers managed to enhance hMSCs osteogenic differentiation by directly functionalizing GO. Expressly, they set up an elegant universal synthetic procedure to covalently tether polyphosphates onto GO, generating a new phosphate-graphene material (CaPG) . Their aLi and co-workers employed graphene oxide and lysozyme films to favor bone regeneration while minimizing the possibility of infection. Precisely by depositing overlapping layers of GO and lysozyme onto a chitosan base they obtained a construct not only able to support dental pulp stem cell growth and differentiation but also with improved antimicrobial activity. While GO is responsible for scaffolds stiffness and roughness, lysozyme improves the antimicrobial activity of GO by degrading the bacterial cell wall .Among 3D scaffolds, Li and co-workers  providedGraphene oxide osteogenic potential was further investigated by Wu and co-workers, which grafted it with a peptide derived from the bone morphogenetic protein 2 . GO-BMP2Skeletal muscles made up most of the mass of the human body and are essential for motion and support. They are composed of multinucleated myofibers, which developed from mononucleated stem cell precursors during embryonic development. Satellite cells are unipotent stem cells that remain associated with adult myofibers and are responsible for muscle growth and regeneration ,249. BecAs muscle cells are aligned along the fiber axis, materials patterned with surface features resembling native extracellular environment can influence mechanotransduction and favor cell differentiation. Park and co-workers  employedBesides skeletal muscle, cardiac muscle regeneration has drawn much attention because of the severity of heart diseases. Cardiomyocytes are specialized muscle cells which have a crucial role in the propagation of electric signal throughout the heart. Unlike skeletal muscle cells, cardiomyocytes have a reduced regenerative potential, and, after damage, are often replaced by scar tissue, which may lead to pathological heart failure. In an elegant comparative study, Lee and co-workers  comparedIn the context of injectable gels, Choe et al. developed an RGO-modified alginate gel and studied its antioxidant activity for cardiac tissue repair post myocardial infarction (MI) . One of The nervous system represents the most intricate and vulnerable system in the human body, as, despite its pivotal importance, it is substantially unable to regenerate itself after injury. Because of its vital role, its organization is extraordinarily complex. Briefly, the nervous system comprises two main classes of cells: the glial cells and neurons. Neurons act as functional units, as they are characterized by peculiar electrophysiological features which allow them to rapidly transmit information between each other. Connections are established during neuritogenesis by the sprouting of dendrites and axons from the cell body. Specifically, each growing axon is tipped by the growth cone, a complex molecular machinery that senses environmental stimuli to guide growth toward the proper target . On the N-cadherin on PDA/RGD-G/PCL scaffolds rather than on control scaffolds, indicating that graphene can have a role in promoting cell adhesion. Western blotting and qRT-PCR analyses indicated that not only scaffolds were able to induce expression of neural markers  but also they increase the expression of neurotrophic factors , which are vital to establishing a permissive environment for nerve regeneration. Moreover, when Schwann cell-loaded PDA/RGD-G/PCL scaffolds were grafted onto Sprague Dawley rats, histological and immunohistochemical observations 18 weeks after surgery suggested that regenerated nerves were well organized, lacked scar tissue and, most importantly, functional recovery was comparable to autograft implants.Because of the inability of central neurons to regenerate, traumatic brain injury, and spinal cord injury have profound adverse effects on life quality and are a significant cause of mortality . Efforts2+ imaging in response to the addition of glutamate: encapsulated hippocampal neurons showed a coherent and coordinated response, and no delay with the response of external neurons was observed. Moreover, the formation of functional synapses between neurons was demonstrated both by immunocytochemistry against synapsin I, which is expressed by neurons at the synapse puncta, and by monitoring the synchronization of spontaneous Ca2+ waves. Besides serving as support for cell growth, the authors claim the graphene in their scaffold might serve as an electrode for electrophysiological recording and neuronal activity stimulation.As neurons require network formation to acquire proper function, tools to build 3D neuronal networks are required to enhance their function. An elegant method to encapsulate neurons onto a self-assembled micro-roll made of a bilayer of graphene and parylene-C , provideIn order to study the role of substrate conductivity in neuronal network formation and alignment, Wang and colleagues developed a 3D conductive GO-coated scaffold based on printed PLCL microfibers using a near-field electrostatic printing (NFEP) . NFEP isGlial cells are as necessary as neurons to ensure proper nervous system functionality, therefore their regeneration after injury is as crucial as neuronal restoration . SpecifiBesides rigid scaffolds, biocompatible conductive hydrogels have attracted much attention because of their ability to better reproduce the mechanical properties of host tissues. Javadi and co-workers  developeStarting from evidence that the cholinergic system is involved in several neuron protective processes, cortical plasticity, and functional recovery after brain injury, Pradhan and co-workers developed a choline-graphene oxide functionalized (CFGO) injectable hydrogel based on poly(acrylic acid). Not only their hydrogels were able to support neuronal cell growth and differentiation, but they also stabilized the actin cytoskeleton. As choline is involved in enhancing neural recovery in TBI treatment, they injected their hydrogels in mice with parietal cortex brain injuries. They found scaffolds were able to restore cortical loss in just 7 days of treatment .GBMs have also been employed as fillers for wound healing hydrogels. Rehman and co-workers  developeGraphene-based scaffolds have been proven to be versatile tools in mediating tissue regeneration, as highlighted by the examples of in vitro and in vivo applications that have been discussed in this review. However, much more effort is required from the scientific community to clarify and rationalize their mechanism of action. It is clear that different composites can be employed to obtain similar results and yet subtle changes in scaffold formulation may result in completely different results. Therefore, a systematic analysis of the effects of scaffold composition on differentiation is required in order to disentangle the role of each scaffold component on cell fate. This would allow for a direct comparison between different scaffolds and a finer rational design. Moreover, it needs to be stressed that most applications rely on qRT-PCR data to prove successful differentiation. However, this approach reveals only an average trend in cell differentiation, without detecting potentially significant and biologically important differences between cells seeded onto different spots of the scaffold. Even when immunocytochemical data are provided, most authors fail to mention whether or not those data are representative of the whole sample or are just isolate cases. Coupling scaffold engineering with single cell RNA sequencing would overcome those limitations, allowing both a better understanding of scaffold effects on cell physiology and comparisons between the in vitro culture and the in vivo reference.To date, scaffold engineering has focused on regeneration of a single tissue. However, clinical applications often require grafting of whole organs. Obtaining a scaffold that is able to efficiently reproduce a whole organ, or even multiple tissues , has proven challenging and further studies are required before any viable clinical usage. In our opinion, finding the rationale behind graphene regulation of cell fates will allow us to obtain scaffolds that can reliably support and differentiate cells in a number of pre-determined types at the same time.Although a molecular rationale for graphene-mediated effects is still lacking, it is remarkable that graphene-based scaffolds are able to determine cell fate more rapidly and efficiently than any other differentiation protocol, even without the addition of exogenous pro-differentiation factors. Indeed, in recent years research on mechanotransduction has unveiled several details on how nanotopography and stiffness stimuli are perceived and transduced by cells, whereas further efforts are needed to elucidate the contribution of other chemical and physical stimuli from the tissue environment. In particular, conductivity is of special interest to neuronal cell fate and differentiation, as it is specific to the nervous system. In addition to the aforementioned nanotopographic features, graphene and GBMs are endowed with tunable conductivity. Therefore, graphene-based nanomaterials represent a useful and cost-effective tool to enhance neuronal differentiation and tissue repair.


Background: Changes in calcium metabolism are quite common in sarcoidosis: hypercalciuria is linked to a persistent clinical phenotype and more active disease. No data is yet available on the specificity of parameters of calcium metabolism as biomarkers for distinguishing different chronic interstitial lung diseases (ILD). Here we assessed calcium metabolism in an Italian population of sarcoidosis patients, which included a group with stage IV fibrotic disease, and compared the results with those of idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis (cHP) patients.Population and Methods: We recruited sarcoidosis, IPF and cHP patients retrospectively. All patients were diagnosed through multidisciplinary discussion and were monitored at the Regional ILD Referral Centre in Siena. Clinical, radiological, functional, immunological and laboratory parameters were collected and entered in an electronic database for data analysis.Results: A total of 305 patients  were enrolled. Sarcoidosis patients included a predominance of females and were significantly younger than IPF and cHP patients (p < 0.0001 for both). In the sarcoidosis population, 17 patients (7.2%) showed radiological evidence of lung fibrosis, according the Scadding classification; fibrotic disease was also confirmed by CT scan. Concerning calcium metabolism, sarcoidosis patients showed significantly higher serum and urinary concentrations of calcium than IPF and cHP patients . These findings were also confirmed when comparing groups with fibrotic sarcoidosis, IPF and cHP (p = 0.0237 and p = 0.0138). According to receiver operating characteristics (ROC) curve analysis, urinary calcium showed better diagnostic accuracy than serum calcium in discriminating sarcoid and non-sarcoid lung fibrosis .Discussion: Our results confirmed that changes in calcium metabolism, particularly hypercalciuria, occur in a substantial percentage of patients with sarcoidosis. Higher serum and urinary concentrations of calcium were found than in IPF and cHP; the same results were observed when the comparison was limited to patients with fibrotic sarcoidosis, supporting the hypothesis that dysregulation of calcium metabolism may be a special feature of sarcoid granulomas. Hypercalciuria distinguished fibrotic sarcoidosis from IPF and cHP, suggesting that assessment of calcium metabolism may be useful in the diagnostic pathway of ILDs. Sarcoidosis is a systemic disease included in the wide group of interstitial lung diseases (ILDs) that commonly require a multidisciplinary approach for diagnosis and clinical management. Due to its systemic nature, sarcoidosis needs to be assessed by a holistic approach that should include all possible localizations and expressions of disease. Changes in calcium metabolism, including hypercalcemia and hypercalciuria, are quite common –5. TheirChanges in calcium metabolism and subsequent renal involvement  have been reported to have a prevalence of 5–60% in sarcoidosis patients , 11. It per se may influence bone status and fracture risk  . Moreoveure risk .Sarcoidosis is generally, but unwisely, viewed as a benign disease, although 9% of patients die from respiratory failure, particularly those with stage IV disease. Fibrotic lung sarcoidosis is observed in 5–15% of patients at presentation and is associated with poorer survival , 19. IsoIn the present study, we focused on changes in calcium metabolism in patients with granulomatous and non-granulomatous ILD, in order to evaluate their specificity as biomarkers in sarcoidosis and the clinical utility of differentiating stage IV sarcoidosis from other ILDs.We recruited sarcoidosis, IPF and cHP patients retrospectively from the population monitored at the Siena Regional ILD Referral Center. Diagnosis was made according to international guidelines , 21; allPatients were excluded if they were taking calcium or vitamin D supplements or drugs for osteoporosis. Demographic, radiological, immunological and functional data was collected from the medical records and entered in an electronic database.Serum samples were also obtained from the sarcoidosis cohort to measure the disease-specific biomarkers chitotriosidase, and ACE.All patients gave their informed consent to the study that was approved by the local Ethic committee. The study was conducted according to Declaration of Helsinki principles.The following lung function measurements were recorded according to American Thoracic Society/European Respiratory Society (ATS/ERS) standards , 24, usiHuman chitotriosidase activity was determined by a fluorimetric method using 22 μM 4-methylumbelliferyl β D-NNN-triacetylchitotriosidase  in citrate-phosphate buffer, pH 5.2; 100 μl substrate was incubated for 1 h at 37°C and the reaction was stopped with 1.4 ml 0.1 M glycine-NaOH buffer, pH 10.8. Fluorescence was read at 450 nm with a Perkin Elmer Victor X4 fluorimeter (excitation wavelength 365 nm). Serum chitotriosidase concentrations were expressed in mg/ml .ACE activity was measured previously described by a colorimetric method , widely used to determine ACE activity in serum, urine and tissues, as . The norSerum concentrations of calcium , phosphate, total alkaline phosphatase and creatinine were measured using standard automated laboratory techniques. Urinary calcium, phosphate and creatinine were determined by a colorimetric method  in 24-h urine samples. Serum PTH was assessed by immunoradiometric assay .t-test were used for group comparisons on the basis of normality of data. The Kruskall-Wallis test was used to compare more than two groups. The Spearman test was used to find correlations. The analysis was run in GraphPad version 5.0.Data was expressed as mean ± standard deviations. Study variables were tested for normal distribution. The Mann-Whitney test or p < 0.0001 for all comparisons). Concerning respiratory function, sarcoidosis patients showed normal lung volumes and diffusion capacity, while we observed mild restrictive impairment associated with moderate reduction in DLCO in IPF and cHP patients. Regarding radiological assessment, 17 sarcoidosis patients (7.2%) showed fibrotic lung disease, confirmed to be stage IV sarcoidosis by chest X-ray and HRCT. Despite their inclusion in stage 0 of disease according to Scadding classification, 91/99 sarcoidosis patients reported typical features of sarcoid lung involvement at HRCT; thus, only 8 patients showed (3.3%) an extrapulmonary disease phenotype, according to GenPhenResA assessment.A total of 304 patients  were enrolled retrospectively in the study. Demographic, clinical, radiological and immunological data and functional parameters are reported in p = 0.0007) (p = 0.0138). Similarly, sarcoidosis patients showed significantly higher serum concentrations of calcium . 0.0007) . This stp < 0.0001 vs. AUC 0.6195, 95% CI 0.5090–0.7300, p = 0.01756), with a sensitivity of 49.5% and a specificity of 89.7% for a cut-off value of 176.5 mg/24 h (likelihood ratio 4.67). Comparing stage IV sarcoidosis patients with IPF and cHP patients, urinary calcium was confirmed to have a moderate-to-good accuracy, significantly better than serum calcium   .No significant differences of serum and urinary parameters of calcium metabolism were found among different clinical phenotypes or radiological stages in the sarcoidosis group .r = 0.2564, p = 0.009), but not with ACE . Urinary calcium was also inversely correlated with DLCO  in patients with sarcoidosis, but not in IPF and cHP patients .We observed a significant direct correlation between urinary calcium and chitotriosidase activity in patients with sarcoidosis . The clinical features of calcium dysregulation in sarcoidosis have been repeatedly described , 15, 25,Our results showed that urinary and serum calcium are both significantly higher in sarcoidosis than in IPF and cHP. Twenty-four hour urinary calcium  showed promising accuracy in discriminating sarcoidosis from the other two ILDs. To our knowledge, the specificity of hypercalcemia or hypercalciuria in the setting of ILDs has never been researched, and no studies have ever investigated the potential of alterations in calcium metabolism as an indicator of non-sarcoid lung fibrosis. Sarcoid-related calcium alterations are possibly determined by overexpression of 1-alpha-hydroxylase and parathyroid hormone-related proteins by granulomatous macrophages –28, but The accuracy of urinary calcium for differential diagnosis was also confirmed when the comparison was limited to stage IV fibrotic sarcoidosis and IPF-cHP subgroups. Fibrotic sarcoidosis may have clinical onset and progression indistinguishable from IPF and cHP, with restrictive functional impairment and similar HRCT features in many patients. Stage IV sarcoidosis, like HP, is regarded as a “great mimicker” and may therefore be a diagnostic challenge in the differential diagnosis of ILDs , 31. SinOur study has some limitations: first, the sample size, though relevant for rare diseases such as sarcoidosis, IPF and cHP, is not sufficient to properly assess the reliability of our results, as well as the monocentric nature of the study. Second, the retrospective design is intrinsically prone to referral and reporting bias, that may significantly influence the analysis and the interpretation of data. Third, due to the lack of data contemporary to urinary sampling, we didn't include in the analysis serum 25-OH and 1, 25-OH vitamin D concentrations. Considering the prominent role of vitamin D in calcium metabolism, future and prospective studies will address the concentration of 25-OH and 1-25-OH forms to further clarify their potential value on this field.In conclusion, in this study we found a significant increase in serum and urinary concentrations of calcium in sarcoidosis patients with respect to IPF and cHP patients. The finding sustains the specificity of changes in calcium metabolism in this granulomatous lung disease. Urinary calcium revealed good specificity for fibrotic (stage IV) sarcoidosis, suggesting that it has potential as a biomarker for the differential diagnosis of ILDs and in the estimation of sarcoid disease activity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comitato Etico Area Vasta Sud Est (C.E.A.V.S.E.). The patients/participants provided their written informed consent to participate in this study.PC, CC, RR, SG, and EB: conception, study design, interpretation of results, and writing of the manuscript. LB, MT, MA, and Md'A: data acquisition and analysis, revision of the study, and interpretation of results. PC, CC, and PS: statistical analysis and revision of the study. All authors approved the final version of the study and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Idiopathic pulmonary fibrosis (IPF) is an independent risk factor for lung cancer (LC) development; however, its effect on recurrence after curative surgery remains unclear.This study aimed to determine the impact of IPF on recurrence-free survival following curative surgical resection of stage I–III non-small cell lung cancer (NSCLC) and investigate the effects of patient and surgical factors on the risk of recurrence.We reviewed retrospectively collected data of patients with surgically resected stage I–III NSCLC from two tertiary care hospitals in South Korea. By propensity score matching, patients with IPF (LC with IPF) were matched to those without IPF (LC without IPF).In total, 3416 patients underwent surgical resection, and 96 were diagnosed with underlying IPF. In the LC with IPF group, 89.6% patients were men, and the average age was 69.7 years. Sublobar resection was performed more frequently in the LC with IPF group than in the LC without IPF group, while the rate of mediastinal lymph node dissection and dissected node number were lower in the former group. The 5-year recurrence-free survival rate was significantly lower in the LC with IPF group (49.2%) than in the LC without IPF group . Multivariable Cox regression analysis revealed that IPF and postoperative stage III were independent risk factors for recurrence.IPF may increase the risk of recurrence after curative surgical treatment for NSCLC. Close surveillance for recurrence is mandatory for patients with underlying IPF. Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease characterized by progressive lung scarring and a histopathological pattern of usual interstitial pneumonia (UIP). The median survival period of IPF ranges from 2.5 to 3.5 years , 2. PatiThe reported prevalence of LC in patients with IPF is 2.7%–45.7% . MoreovePatients with stage I and stage II non-small cell lung cancer (NSCLC), as well as those with certain stage III NSCLCs, are considered eligible for curative resection. Complete resection, when possible, remains the mainstay of therapy and is associated with the highest probability of long-term survival. However, if patients with LC who were eligible for surgical resection have underlying IPF, several aspects must be considered. Previous studies have shown that the postoperative morbidity and mortality rates for patients with NSCLC are significantly higher if IPF is present, with the main cause of postoperative mortality being acute exacerbation of IPF (AE-IPF) –16. A feDeclaration of Helsinki. Local institutional review boards and independent ethics committees approved the study protocol .We retrospectively reviewed the medical records of patients with stage I–III NSCLC who underwent curative resection at Seoul National University Bundang Hospital and Severance Hospital between January 2003 and September 2016. Among these patients, we identified those who were diagnosed with underlying IPF. By propensity score matching of confounding variables, including age, sex, the histopathological subtype of NSCLC, the postoperative stage of NSCLC, and the year of NSCLC diagnosis, patients with IPF (LC with IPF group) were matched to those without IPF (LC without IPF group) in a 1:2 ratio. Flow chart of patient recruitment is shown in This study was approved by the Institutional Review Board and Ethics Committee of Seoul National University Bundang Hospital and Severance Hospital . Written informed consent was waived as the nature of retrospective study by IRB. The patient’s medical records between January 2003 and September 2016 were accessed. All data were fully anonymized before accessement.In accordance with diagnostic criteria defined by the International Consensus Statement of the American Thoracic Society and European Respiratory Society in 2018 [The LC stage was defined according to the 8th edition of the American Joint Committee on Cancer staging system. Recurrence-free survival was defined as the time from surgery to the first diagnosis of disease recurrence or the last follow-up visit. Death was considered a censoring event for recurrence. Overall survival was defined as the time from initial LC treatment to death or the last follow-up visit. Local recurrence was defined as evidence of tumor cells within the same lobe of the lung or ipsilateral pulmonary hilum, and regional recurrence was defined as evidence of tumor cells within another lobe of the ipsilateral lung or the ipsilateral or subcarinal lymph nodes (LNs), while distant metastasis was defined as evidence of tumor cells in the contralateral lung, contralateral mediastinal LNs, the pleural space, or areas outside the hemithorax.AE-IPF was defined as acute, clinically significant respiratory deterioration characterized by evidence of new widespread alveolar abnormality, in accordance with the diagnostic criteria proposed by Collard et al. in 2016 .CO; 0–3 points). Based on the score, IPF was categorized into three stages: I (0–3 points), II (4–5 points), and III (6–8 points). The predicted 1-year mortality rates for GAP stages I, II, and III were 6%, 16%, and 39%, respectively [The gender–age–physiology (GAP) index was applied for the assessment of the severity and prognosis of IPF. The index was calculated as follows: gender (0–1 point), age (0–2 points), forced vital capacity , and diffusing capacity of the lungs for carbon monoxide  were estimated using the stratified Kaplan–Meier method. Independent predictors for recurrence and overall survival were determined by stratified Cox proportional hazards models. A P-value <0.05 was considered statistically significant. All statistical analyses were performed using SAS software .In total, 3416 patients with NSCLC underwent surgical resection during the study period, and 96 were diagnosed with underlying IPF. Baseline characteristics of the overall cohort before propensity score matching are shown in CO) was significantly lower in the LC with IPF group than in the LC without IPF group . Other variables, including the location of the primary tumor, forced vital capacity (FVC), and forced expiratory volume in one second, were comparable between groups. In the LC with IPF group, stages I and II as per the GAP index were documented for 94 (97.9%) and two (2.1%) patients, respectively. There was no patient with GAP stage III.Baseline characteristics of the matched groups are shown in The treatment-related characteristics of the matched groups are shown in To assess the incidence of treatment-related complications, we defined the duration between the last treatment and the onset of complications as ≤4 weeks . The incDuring a median follow-up period of 49.2 months, 92 recurrences were documented for the entire matched study population. Recurrences were significantly more frequent in the LC with IPF group than in the LC without IPF group . Among the patients with recurrence, the type of recurrence  did not show significant difference between groups. The 5-year recurrence-free survival rate  and postoperative stage III  were independent predictors of recurrence .Univariable Cox proportional hazards regression analysis revealed that IPF, Eastern Cooperative Oncology Group score, FVC, postoperative stage, surgical extent, and surgery-related AE-IPF or ARDS were major covariates for overall survival. These variables and additional potential confounding variable were combined in multivariable analysis, which revealed that IPF , postoperative stage III NSCLC , and surgery-related AE-IPF or ARDS  were independent predictors of overall survival .In the present study, we demonstrated the impact of IPF on recurrence and survival following curative surgical resection of stage I–III NSCLC in a propensity score-matched study population. Both recurrence-free survival and overall survival after curative surgical resection were significantly compromised in patients with IPF. IPF and postoperative stage III were predictors of both recurrence and overall survival. Surgery-related AE-IPF or ARDS was also identified as an independent risk factor for mortality.Epidemiological studies have revealed that IPF is an independent risk factor for LC development , 11, 23,Patients with LC and underlying IPF are commonly encountered in clinical practice; however, for these patients, it is difficult to predict the efficacy of treatment for LC due to the increased incidence of treatment-related complications and poor prognosis of IPF itself –14, 30. Unlike previous studies on patients with NSCLC and IPF, we assessed detailed surgery-related variables, including whether or not mediastinal lymph node dissection was performed, the number of dissected LNs and resection margin status. However, none were found to be significant predictors of recurrence-free survival. Therefore, IPF itself appears to increase the risk of lung carcinogenesis and recurrence after curative resection. This may be explained by the aforementioned pathways for carcinogenesis in IPF, along with the possibility that tumors that developed within fibrotic lesions could be obscured by honeycombing or reticular changes in fibrotic lesions and consequently overlooked. Moreover, the probability of undiscovered positive LNs is higher for patients with IPF; this can be explained by the lower number of dissected LNs in our IPF group, which could lead to stage migration and remnant malignancy .CO, which showed a significant difference between the LC with IPF and LC without IPF groups in the propensity score-matched population, was excluded from the Cox regression model due to 39 patients with missing DLCO data in the LC without IPF group. The DLCO test was mainly missed in patients without underlying lung fibrosis and respiratory symptoms before surgery. Nevertheless, since DLCO has been reported as a risk factor for postoperative respiratory complication, patients with a low DLCO should be carefully evaluated, particularly for surgery-related AE-IPF or ARDS [Previous studies showed that morbidity and mortality rates after the surgical treatment of NSCLC were significantly higher in patients with IPF than in those without IPF, and inferred that AE-IPF is the main cause of morbidity and mortality –14, 30.  or ARDS .This study had several limitations. First, as the surgical extent was not included in propensity score matching due to lack of data, the LC with IPF group received sublobar resection more frequently compared to the LC without IPF group in the propensity matched population, which can be a risk factor for recurrence. To cope with these limitations, stratified Cox regression analysis was conducted including the surgical extent, and it revealed that IPF and cancer stage were independent risk factor for recurrence regardless of surgical extent. Second, more detailed surgical and tumor variables that can affect recurrence, including the distance from the tumor to the resection margin, visceral pleural invasion, angiolymphatic invasion, and the degree of tumor differentiation, were not available for analysis. Lastly, this study had a retrospective nonrandomized design, which is associated with various biases and confounding factors. However, we obtained the data from two tertiary care hospitals to minimize biases. Further studies with a larger patient sample are required to validate our findings.The present study revealed that the recurrence-free survival rate after curative surgical resection of stage I–III NSCLC was significantly lower for patients with IPF than for those without IPF, and that IPF and postoperative stage III were predictors of both recurrence and mortality. These findings suggest that close surveillance for recurrence after curative surgical resection of NSCLC is mandatory if the patient has underlying IPF. We suggest that the conventional regimen involving follow-up computed tomography every 6 months after surgery should be replaced with a regimen involving follow-up visits at shorter intervals in patients with underlying IPF, regardless of the surgical extent especially in higher cancer stage.S1 Fig(A) Cancer-specific survival was significantly inferior in the LC with IPF group compared to the LC without IPF group in the whole propensity score matched population (P = 0.008). (B) Cancer-specific survival was significantly inferior in the LC with IPF group compared to the LC without IPF group in patients who underwent lobectomy among the propensity score-matched population (P = 0.007).(PPTX)Click here for additional data file.S2 Fig(A) Recurrence-free survival was significantly inferior in the LC with IPF group than in the LC without IPF group (P = 0.003). (B) Overall survival was also significantly inferior in the LC with IPF group than in the LC without IPF group (P<0.001).(PPTX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


The specific capacitance is calculated on the weight of graphene. The device presents a large actuation peak-to-peak displacement of 24 mm at a frequency of 0.1 Hz under the stimulus potential of 2.5 V, and it can still reach a large value of 12 mm at a high frequency of 1 Hz. The free length of the device is 25 mm. Notably, the device exhibits excellent air-working stability at frequency of 1 Hz under 2.5 V with the actuation displacement retention of 98%, even after 10,000 cycles. This study presents insights into the design of smart actuators based on nanomaterials, and will accelerate the development of artificial intelligence.Electrochemical actuators have attracted tremendous attention worldwide because of their critical significance to artificial intelligence. The development of electrochemical actuators—with the merits of low driven-voltage, lightweight, flexibility and large deformation—is an urgent task in the development of smart technologies. Nanomaterials with special structures and superior properties provide the opportunity for the development and application of smart actuators. Here, we report an electrochemical actuator based on an ionogel graphene composite, which is assembled with simple casting methodology and can be driven with a low voltage of 2.5 V. The flexible sandwich-structured actuator operates under a capacitive mechanism based on asymmetrical volume expansion of active ions under electrical stimulus. It shows a high specific capacitance of 39 F g Eleg2+, Na+ . The devg2+, Na+ ,6,7. In g2+, Na+ ,9,10. ThWith the rapid development of nanomaterials in recent decades, there now exists a great opportunity for the preparation of high-performance flexible electrochemical actuators based on advanced nanomaterials, such as graphene ,12, grap4) ionogel electrolyte was applied for its high ionic conductivity and wide potential window. The actuator shows the high specific capacitance of 39 F g−1 at current density of 1 A g−1 under potential of 2.5 V, indicating its high accommodation for active ions. The driven voltage of the actuator is only as low as 2.5 V, which is easily achievable using the dry batteries used in everyday life and thus presents great potential for artificial intelligence applications. The device also displays a large actuation displacement of 24 mm at frequency of 0.1 Hz under 2.5 V, and the displacement retains as high as 12 mm at higher frequency of 1 Hz. It is found that the actuator shows excellent cycling stability with high displacement retention of 98%, even over 10000 cycles at 1 Hz under 2.5 V. This study provides a promising strategy for the fabrication of high-performance flexible electrochemical actuators, and will also shed light on the design of other flexible electrochemical devices.Here, we report a flexible and electroactive actuator based on graphene materials and ionogel electrolytes, which is fabricated through a simple solution casting method. Other previous works mostly utilized the hot-pressing method, which required sophisticated equipment and precise control of conditions ,24,25. G4 was purchased from Millipore Sigma company . Graphene powder (NO: XFGNP009) was obtained from XFNANO Materials Tech Co., Ltd., PVdF polymer and DMF (Dimethyl Formamide) solvent were obtained from Sinopharm Chemical Reagent Co., Ltd., . EMIBF4 were mixed in 30 mL DMF solvent at 30 °C. Then, the solution was poured into a mould and kept at 80 °C for 24 h. Finally, the PVdF/EMIBF4 electrolyte membrane was obtained by peeling off the mould after vacuum drying at 80 °C for 12 h.Firstly, 1.0 g PVdF and 1.5 g EMIBF4 electrolyte and then dried at 80 °C for 12 h. Following that, the actuator was obtained by casting the other side with graphene electrode with same method. The thickness of the actuator is 117 μm.Firstly, 50 mg graphene powder and 5 mg PVdF were dispersed in 10 mL DMF for 1 h through horn sonication in ice bath. Subsequently, the suspension was cast onto one side of PVdF/EMIBF−1. Electrochemical impedance spectroscopy was conducted from 10 mHz to 100 kHz with a voltage amplitude of 10 mV. Charge-discharge curve was conducted at 1 A g−1 under voltage of 2.5 V. The displacement of actuator was measured by Keyence LK-G800 laser positioning equipment .SEM and TEM images were made with Hitachi S-4800  and FEI Tecnai F200 , respectively. Electrochemical performances of ionic actuator were measured with Bio-logic Potentiostat VMP3 work station . The CV curve was measured in the potential range of 0–2.5 V with scan rate of 100 mV sThe mechanism and sandwich structure of ionogel graphene composite actuator are briefly illustrated in −1 are attributed to D (disordered and defective structure) and G (graphitic structure) bands of graphene. The XPS C 1s spectra can be fitted into three peaks, which are attributed to characterize the peaks of C–C, C=C and C–O. As illustrated by the scheme in 4 as an electrolyte. The electrochemical impedance spectroscopy (EIS) result is shown in −1 under a potential of 2.5 V is shown in The morphology of graphene materials is presented in The flexible bimorph cantilever actuator is evaluated through multi-step voltage stimulus in −1 at a current density of 1 A a−1, under a voltage of 2.5 V. Moreover, the actuator can operate under a low driven voltage of 2.5 V, which can be easily realized using the dry batteries used in everyday life. It shows large deformations both at high and low actuation frequencies, and the deformation amplitude and direction can be adjusted by controlling the driven voltages. In addition, the device displays excellent cycling stability, with actuation retention of 98% over 10,000 cycles in an air atmosphere, indicating promising practical applications. The research conducted in designing in this work will provide insights for the design of other flexible electrochemical devices with long cycling lives.In summary, a flexible and electroactive ionogel graphene composite actuator has been prepared based on graphene electrodes and ionogel electrolytes through a simple solution casting method. The porous structure of graphene facilitates the accommodation of active ions, and the chemically stable ionogel electrolyte promotes the cycling performances of the device. The as-prepared actuator displays a high specific capacity of 39 F g


The studies indicate the formation of a mixture of polymorphs, i.e., monoclinic and triclinic forms of PbPc with face-on (lying down) and edge-on (standing up) PbPc orientations, respectively. The formation of monoclinic fractions is attributed to the presence of the graphene layer directing the π interactions between the highly delocalized macrocycles. The competing interfacial van der Waals forces and molecule–molecule interactions lead to the formation of a small fraction of triclinic moieties. The nanoscale electrical characterization of the thin PbPc layer on graphene by means of conducting atomic force microscopy shows enhanced vertical conductance with interconnected conducting domains consisting of ordered monoclinic crystallites through which the charge transfer occurs via tunneling. These results show the importance of a templating layer to induce the formation of a required phase of PbPc suitable for specific device applications.The influence of single-layer graphene on top of a SiO H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS), MoO3 and CuI. A FDTS layer induces the edge-on arrangement regardless of the crystal phase, while a CuI interlayer induces the formation of triclinic PbPc moieties stacked face-on to the substrate yielding enhanced NIR absorption [60 layers on highly oriented pyrolytic graphite (HOPG) or SiO2, it has been reported that CuPc attains different orientations resulting in substantial differences in donor–acceptor energy level alignment at the interface. Thus, ordering and orientation of these molecules significantly affect charge carrier injection and transport in semiconductor devices [Organic semiconductors have been extensively used in, among others, organic light-emitting diodes and organic photovoltaics. In particular, metal phthalocyanines (MPcs) have gained considerable interest as they offer flexibility in the modification of their optoelectronic properties through their molecular packing, which in turn is governed by substrate–molecule interactions –4. Nonplsorption . Organicsorption –8. These devices .Pristine substrate surfaces of HOPG and Si themselves can induce orientation control over the growth of MPc structures without the aid of additional templating layers. In our earlier work, we have observed that orientation and molecular packing of nonplanar PbPc molecules are influenced by the nature of the substrate, which has been attributed to different substrate–molecule interactions . With th2/Si substrate, using synchrotron two-dimensional grazing incidence X-ray diffraction (2D-GIXRD). We show that although graphene induces the face-on stacking of monoclinic domains, the underlying SiO2 substrate can still cause edge-on triclinic orientations as well. We also present the electrical current mapping of PbPc on graphene revealing interconnected highly conducting domains.Most of the orientation studies of MPc on graphene deal with planar MPc molecules such as FePc, CoPc, CuPc and ZnPc, which form layers in a face-on configuration –19. A co2/Si substrate (referred to as SLG/SiO2/Si hereafter) is presented in −1 corresponds to the characteristic 2D band of SLG and can be fitted with a single Lorentzian function (FWHM = 35.47 cm−1). The peaks at 1587 and 1342 cm−1 correspond to the G- and the D-band, respectively [The Raman spectrum of CVD-grown single-layer graphene transferred onto a SiOectively .qz direction at 0.89 Å−1, d = 7.06 Å, corresponding to the (320) reflection of monoclinic PbPc crystallites. qz direction indicating the Braggs peaks as discussed above. The signature of monoclinic moieties along the qz direction indicates that the PbPc molecules exhibit ordered stacking, normal to the substrate plane. This type of vertical ordering known as cofacial arrangement can promote charge transport in the vertical direction [−1 along the qz direction, which may be assigned to the (200) reflection of the monoclinic phase or the (001) reflection of the triclinic phase, occurring at the same positions with d = 12.82 Å (−1 corresponding to the (100) reflection of the triclinic phase in the qxy plane with d = 11.87 Å. This signature in the qxy plane may be an indication of a tilted face-on arrangement of triclinic moieties with respect to the substrate  surface gives rise to disordered fractions due to the absence of long-range ordering [2 has a templating effect and induces a monoclinic packing of PbPc in the face-on configuration. Our previous theoretical studies have shown that the PbPc molecule exclusively adopts a face-on configuration on an unsupported graphene layer owing to π–π and van der Waals interactions. For multilayer depositions, ordered π stacking of the macrocycles with Pb-up configuration is expected due to the dominant π–π interactions between the Pc macrocycle and the graphene layer [The molecular orientation of both planar and nonplanar MPc molecules on oxide substrates such as SiOentation ,9,27–29.ordering . In the ne layer .2. The presence of triclinic moieties in an inclined configuration could be a result of competing interfacial van der Waals and forces and the π interactions between the graphene layer and the underlying SiO2. Thus more than one layer of graphene may be required to diminish the weak interfacial van der Waals forces from the SiO2 substrate [2/Si is inferred in In the present study, although there is a graphene layer, triclinic moieties with edge-on configuration are still observed. In the case of CVD graphene transferred on to substrates by the polymer method, it has been reported that the presence of polymer residues can cause edge-on orientation for pentacene on graphene . Howeverubstrate . The posubstrate . Based o2/Si substrate to study the effects of the molecular orientation on the interfacial electronic properties. The roughness of the film was reported to be 2.47 ± 0.28 nm [−1 using the Scherrer equation with a value of k = 0.9 assuming spherical particles. Crystallite sizes of 14.37 and 33.58 nm were obtained from the Bragg peaks of 0.49 and 0.89 Å−1, respectively.The topography of the PbPc layer was studied using atomic force microscopy AFM, . Figure  0.28 nm . The cryI–V curve acquired from one of the conducting domains in the vertical configuration.Further, we have carried out electrical studies using conducting-AFM (C-AFM). I–V plot can be fitted to a generalized Simmons equation indicating that the charge transport in a thin PbPc layer is governed by tunneling (I/V2) as a function of V−1 indicates a logarithmic dependence in the low-bias region showing direct tunneling, which transforms into a linear dependence in the high-bias region, suggesting Fowler–Nordheim (F-N) tunneling or injection tunneling. However, it is seen that the transition from direct to F-N tunneling is not a sharp transition. Instead, there is a seemingly linear slope between the two states. A sharp rise in linear current is noticed beyond this region, which corresponds to F-N tunneling [2-hybridized structure. The vertically stacked monoclinic domains on the graphene surface provide an uninterrupted path for electrical transport and thus give rise to higher current values.The curve is non-linear as expected for PbPc and higher current values are observed. The obtained unneling . A plot unneling . Graphen2/Si substrate show the presence of a mixture of monoclinic and triclinic polymorphs with, respectively, face-on and edge-on configurations. The presence of monoclinic fractions in a preferred face-on orientation is ascribed to the π–π interactions between the sp2-hybridized plane of graphene and the macrocycles of PbPc. However, the presence of SiO2 surface beneath graphene, which exerts interfacial van der Waals interactions and intermolecular interactions on the top layers, influences the orientation of PbPc molecules leading to the formation of a very small fraction of triclinic moieties in edge-on or tilted configuration. Further, electrical characterization of these films in a vertical configuration shows enhanced conduction. Monoclinic domains stacked cofacially on the substrate surface facilitate the charge transport by improved π-electron coupling. These studies demonstrate the possibility to fabricate device architectures with the desired orientation of the film by carefully choosing the substrate or introducing a templating layer.Structural studies of a thin layer of PbPc (10 nm) deposited on CVD-grown single-layer graphene supported on a SiO2/Si substrate (SLG/SiO2/Si) was deposited using physical vapor deposition. The PbPc film was deposited at a base pressure of 1 × 10−5 mbar while the substrate was held at 100 °C. The deposition rate was 1–1.5 Å·s−1. Single-layer graphene was synthesized by chemical vapor deposition (CVD) on a copper substrate and transferred by a standard technique using poly(methyl methacrylate) (PMMA) onto a SiO2 (300 nm)/Si substrate as reported elsewhere [A 10 nm thin PbPc film on single-layer graphene supported on a SiOo a SiO2 0 nm/Si sThe structural characteristics of the single-layer graphene were studied using a Horiba XploRA PLUS Raman spectrophotometer with a 532 nm laser and a 50× objective. The molecular orientation of the thermally deposited PbPc film on single-layer graphene was studied using 2D grazing incidence X-ray diffraction (2D-GIXRD) at the PETRA III P08 beamline  with a flat-panel Perkin Elmer detector with 2048 × 2048 pixels of the size of 200 µm at a beam energy of 25 keV, corresponding to λ = 0.496 Å. The obtained results were analyzed with the help of the GIXSGUI software with MATLAB interface [k = 0.18 N·m−1, MikroMasch, USA), serving as a top electrode.Morphological studies and electrical measurements of the PbPc layer were carried out using an Agilent 5500 AFM. The AFM was operated in C-AFM mode in which the PbPc layer is sandwiched between the conductive graphene layer, which served as a bottom electrode, and the Cr/Au tip (diameter < 35 nm and