Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion. Liver transplantation (LT) is the most efficient therapy for end-stage liver disease. The early outcome after LT has improved over the past years. In experienced centers, an 85–95% 1-year patient survival rate can be achieved [Restriction of blood supply (ischemia) followed by restoration of perfusion and reoxygenation (reperfusion) initiates a cascade of events in the transplanted organs, which ultimately leads to severely impaired organ function. While static cold storage (SCS) remains the standard for liver preservation, massive accumulation of metabolites derived from anaerobic respiration during the ischemic phase poses a hazard upon reperfusion. There is accumulating evidence, that gentle rather than abrupt rewarming of organs may ameliorate the events by significantly reducing succinate-driven reactive oxygen species (ROS) production and increasing adenosine triphosphate (ATP) levels. Ex vivo machine perfusion (MP) of livers may help preventing mitochondrial and tissue damage. Understanding the alterations leading to organ damage at the molecular levels and the possibility to optimize perfusion protocols to significantly minimize true ischemia times hold significant potential to limit organ damage in transplantation and beyond ,3.+ levels stimulate the Na+/H+ exchange and, in turn, intracellularly increased Na+ concentrations. Moreover, the ATP-dependent Na+/K+ and Ca2+ pumps fail during ischemia, resulting in an intracellular accumulation of Ca2+ [The interruption of blood and oxygen supply during organ transplantation remains a part of the routine. To minimize the negative impact on the outcome, ischemia times are kept at a minimum and organs are stored on ice. Because of restricted oxygen supply, the anaerobic metabolism leads to decreased intracellular ATP levels. Anaerobic glycolysis results in the production of lactic acid, thereby resulting in acidosis . Elevate of Ca2+ ,5,6. Blo of Ca2+ . The mit of Ca2+ ,8. DamagMitochondrial ROS (mROS) are generated during early reperfusion  and compEven if complex I may be the main source of mROS production during IRI, there is growing evidence that complexes II and III also contribute to mROS generation. Mitochondria need to be polarized for mROS production by complex I. Opening at the mitochondrial permeability transition pore (mPTP) leads to depolarization and limits complex I mROS production. In a situation where simultaneous inhibition of complex III by antimycin is induced, sustained mROS production by complex II is observed . InhibitROS production is not limited to the mitochondria. Xanthine oxidoreductase (XOR) is an enzyme which catalyzes the final two steps of purine catabolism and exists in two isoforms, which are interconvertible: Xanthine oxidase (XO) and xanthine dehydrogenase (XDH). The latter is dominating under physiological conditions, whereas XO contributes to the generation of ROS upon IRI. During the ischemic phase, hypoxanthine accumulates in the ischemic tissue through degradation of ATP. Following reperfusion, XO catalyzes the conversion of hypoxanthine in the presence of tissue oxygen to uric acid, thereby releasing superoxide, which may be dismutated to hydrogen peroxide .Moreover, also nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidases are sources of ROS. It has been shown that reduced oxidative phosphorylation results in elevated levels of NADPH . NADPH oc release is enabled, which triggers the activation of caspase-9 and thus induces apoptotic cell death via activation of caspase-3 [c, the second mitochondrial-derived activator of caspase/direct IAP inhibitor of apoptosis binding protein with low PI (Smac/DIABLO), high-temperature requirement protein A2 (HtrA2), endonuclease G or apoptosis-inducing factor (AIF) are released and may induce apoptosis [ROS are not detrimental per se, and physiological levels are actually required for cellular homeostasis. ROS induce reversible post-translational protein modifications in order to regulate signaling pathways . Diverseaspase-3 ,22. Furtpoptosis . Due to poptosis . The mospoptosis . Damagedpoptosis . Beclin-With regard to IRI, especially liver sinusoidal endothelial cells (LSECs), are highly vulnerable during the cold ischemic phase. These cells play a major role in vascular homeostasis and immune function. Upon reperfusion LSECs express a multitude of cytokines as well as DAMPs, while an imbalance of NO and endothelin (ET) results in luminal narrowing, thereby negatively impacting on the microcirculation ,28. The In recent decades, the gold standard for the preservation of solid organs for transplantation was SCS at 4 °C . Since oHMP is performed via the portal vein only . Due to SNMP displays a compromise of HMP and normothermic machine perfusion (NMP), and thus combines the respective advantages of HMP and NMP. The temperature is low enough to achieve sufficient oxygenation without the implicit addition of an oxygen carrier, which enables the restoration of ATP levels. At the same time, the temperature is high enough to allow for the partial viability assessment of the organ ,43,44.During NMP, organs are perfused at 37 °C to simulate near-to physiologic conditions . Thus, tThe most recently described perfusion strategy is COR. By gradually increasing the temperature after a cold perfusion period, it aims to minimize injury, which is normally triggered by a sudden temperature shift. After an initial period of HMP with a subsequent phase of COR to reach NMP conditions, the beneficial effects of HMP and NMP can be combined ,47.Implementing NMP in the clinical routine of liver transplantation has helped to reduce cold ischemic time in the course of organ preservation; however, cold ischemia cannot be totally averted in most cases. Most often, NMP of donor livers is started after a limited period of conventional SCS, e.g., after organ transportation. In NMP, livers are rewarmed and reperfused on the machine after a period of cold ischemia. While this is an equivalent of in situ reperfusion upon transplantation, the perfusion fluid used for NMP normally does not contain leukocytes and platelets, which are detrimental in the development of IRI . Togethec and caspase-3, which was observed in the study of Zhang in porcine livers [Several studies in liver grafts have already shown a benefit of NMP on IRI, even though complete prevention of IRI cannot be expected. Zhang et al.  conductee livers . This sue livers , and in e livers .One of the major advantages of NMP compared to SCS is the possibility to monitor an organ prior to transplantation and to assess its quality under close-to-physiological conditions. Therefore, the establishment of reliable markers and parameters suitable for organ viability testing during NMP, hence predicting organ function and transplant outcome, are urgently needed.For the liver, bile production may serve as a marker for the viability assessment as reported by Sutton et al. , since iIn search of biomarkers that are indicative of damage associated with IRI, De Vries et al.  recentlyAs protection of mitochondria is crucial to prevent IRI, the assessment of mitochondrial function may help to predict the extent of IRI damage and hence organ performance. Since ATP is depleted during ischemia, measurement of ATP tissue levels during MP is suggested to serve as a reliable mitochondrial function marker. During the preservation phase, the respective MP strategies contribute to the resynthesis of ATP, whereby the recovery of the energy status correlates with the functionality of the organ .Quantification of the DAMPs released also offers a potential parameter for the assessment of mitochondrial function during MP, since the release of DAMPs is induced by IRI. A key member of this group is the high mobility group box-1 protein (HMGB-1) . First ec, whereby an increase in oxygen consumption as a response is linked to membrane damage. The coupling status of the oxidation to the phosphorylation reports about inner membrane integrity [A well-established method for assessing mitochondrial function is high-resolution respirometry (HRR), which has already been used to analyse rat , pig 6060 and huntegrity ,65,66. LMitochondrial activity and, in general, metabolic activity are known to be temperature-dependent. Not surprisingly, different MP types operating at different temperatures, as displayed above, may exert various effects on mitochondrial activity and cellular responses. Karimian et al.  comparedWhile hypothermic preservation (HOPE) decreases mitochondrial respiration and enhances the ATP pool during preservation, NMP is the preferential preservation technique for viability testing as it restores metabolism under physiologic temperatures and hence allows functional monitoring under close-to-physiologic conditions. The direct effect of NMP and HOPE on liver cell injury have already been tested in rat liver grafts. NMP resulted in a reduced cell infiltration and IRI. This effect was further enhanced by HOPE . In the Thus, a combined perfusion technique consisting of HOPE followed by NMP not only attenuates IRI in livers in the course of transplantation, but also offers the possibility to assess declined livers for transplantation.MP offers the unique possibility to administer specific additives, compounds, substances or drugs with an advantageous effect on the perfused organ. Treatment can be added directly to the perfusion solution in an adequate concentration. While passing through the vasculature, substances may exert a direct and exclusive effect on the organ perfused ex situ.With regard to preventing IRI and restoring mitochondrial function during liver MP, the following candidates and concepts may be worth taking into consideration: Preventing succinate driven ROS accumulation may open the path to new therapies. A study in a heart IRI mouse model revealed a protective effect of inhibiting SDH against IRI . BecauseA determinant, which positively influences mitochondrial function during MP, is the supplement of an adequate oxygen carrier ,73. MattAs IRI significantly determines graft function after transplantation, a profound understanding of the molecular mechanisms leading to tissue and cell injury are inevitable. An excessive mitochondrial response during ischemia fuels mROS production and decreases ATP levels, which have been identified as the major cause of reperfusion injury, leading to oxidative stress and tissue inflammation. Due to the high metabolic activity of hepatocytes, a liver graft is especially vulnerable to IRI. The technique of MP prior to transplantation may help to mitigate IRI in livers and serve as a platform for ex vivo organ therapy. The combination of HOPE followed by NMP holds great potential to effectively restore mitochondrial function and avoid redox stress.


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.


Traumatic spinal cord injury (SCI) impedes signal transmission by disrupting both the local neurons and their surrounding synaptic connections. Although the majority of SCI patients retain spared neural tissue at the injury site, they predominantly suffer from complete autonomic and sensorimotor dysfunction. While there have been significant advances in the characterization of the spared neural tissue following SCI, the functional role of injury-induced interneuronal plasticity remains elusive. In healthy individuals, spinal interneurons are responsible for relaying signals to coordinate both sympathetic and parasympathetic functions. However, the spontaneous synaptic loss following injury alters these intricate interneuronal networks in the spinal cord. Here, we propose the synaptopathy hypothesis of SCI based on recent findings regarding the maladaptive role of synaptic changes amongst the interneurons. These maladaptive consequences include circuit inactivation, neuropathic pain, spasticity, and autonomic dysreflexia. Recent preclinical advances have uncovered the therapeutic potential of spinal interneurons in activating the dormant relay circuits to restore sensorimotor function. This review will survey the diverse role of spinal interneurons in SCI pathogenesis as well as treatment strategies to target spinal interneurons. Spinal cord injury (SCI) is a heterogeneous disorder that has devastating consequences on the patient’s well-being, independence, and quality of life  and glycine. Whereas, excitatory interneurons release excitatory neurotransmitters, such as glutamate  transcription factors, respectively , dorsal (DV0), cholinergic (CV0), and glutamatergic  transcription factor. These neurons project ipsilaterally and consist of four subpopulations, including V2a, V2b, V2c, and V2d  and macrophages into the extracellular matrix  via the activity of propriospinal interneurons that form new connections, referred to as crossed phrenic phenomenon (CPP).The glial reaction to trauma extends rostro-caudal from the astroglial scar into the perilesional zone of spared neural tissue. Within this region, astrocytes, microglia, and NG2-OPC induce spontaneous synapse and circuit reorganization. In contrast to the newly formed astrocytes in the astroglial scar border, hypertrophic reactive astrocytes in the perilesional zone maintain their connections with local neurons —tasked at reviewing SCI clinical trials in 2007—concluded that most SCI patients suffer from a complete functional loss distal to the injury level . These patients show minimal recovery after 1 year, despite having a rim of preserved neural tissue around the injury site. This suggests that either remyelination or synaptic readjustment can elicit improved recovery. Surprisingly, all other ASIA classifications  demonstrate behavioral improvements, but the majority of Grade A patients do not exhibit any improvements 1-year post-injury  ratio, leading to the functional inactivation of preserved tissue  is a neuron specific transmembrane protein responsible for maintaining chloride and potassium ion gradients in the synaptic cleft (Moore et al., A subtype of synaptopathy is channelopathy—the improper function of ion channels—which are critical for synaptic transmission and signal propagation in neural tissue Hanna, . Traumatvia pharmacological and gene therapy approaches.Therapeutic interneuronal modulation seeks to either reverse adverse maladaptive synaptic changes following injury or form new circuits to bypass the damaged injury site. The clinical translation of such therapeutics can restore vital autonomic functions, such as respiration, after high cervical injuries (Golder and Mitchell, via an electrode implanted over dura. Combined electrochemical neuroprosthesis and rehabilitation through treadmill-aid/robotic postural interface enabled the cortical control of locomotion through spared neural tissue (Lavrov et al., EDS refers to the introduction of electrical current A receptor antagonist bicuculline improves stepping in the absence of rehabilitative training (Robinson and Goldberger, The currently evolving gene therapeutic techniques, as well as pharmacological interventions, demonstrate new ways to influence neuroplastic change in spinal interneurons (Dimidschstein et al., The current clinical practice for traumatic SCI is limited to prevention and early neuroprotection (Chio et al., M-MZ and JH: literature review, manuscript writing, editing, and finalizing, approval of the final manuscript. MF: framing the concept and structure of the review, editing, and finalizing, approval of the final manuscript.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.


The field of tumor-specific fluorescence-guided surgery has seen a significant increase in the development of novel tumor-targeted imaging agents. Studying patient benefit using intraoperative fluorescence-guided imaging for cancer surgery is the final step needed for implementation in standard treatment protocols. Translation into phase III clinical trials can be challenging and time consuming. Recent studies have helped to identify certain waypoints in this transition phase between studying imaging agent efficacy (phase I–II) and proving patient benefit (phase III).Performing these trials outside centers of expertise, thus involving motivated clinicians, training them, and providing feedback on data quality, increases the translatability of imaging agents and the surgical technique. Furthermore, timely formation of a trial team which oversees the translational process is vital. They are responsible for establishing an imaging framework  and clinical framework  in which the trial is executed. Providing participating clinicians with well-defined protocols with the aim to answer clinically relevant research questions within the context of care is the pinnacle in gathering reliable trial data.If all these aspects are taken into consideration, tumor-specific fluorescence-guided surgery is expected be of significant value when integrated into the diagnostic work-up, surgical procedure, and follow-up of cancer patients. It is only by involving and collaborating with all stakeholders involved in this process that successful clinical translation can occur.Here, we discuss the challenges faced during this important translational phase and present potential solutions to enable final clinical translation and implementation of imaging agents for image-guided cancer surgery. Tumor-targeted imaging has changed our view on cancer diagnostics and therapy in the past two decades , 2. ThesTime Magazine [18F]-FDG-PET) gained FDA approval in 2002 [The potential for cancer-detection methods and vital-structure discrimination using exogenous contrast agents, or imaging agents, have been described extensively over the past years –6. It waMagazine  that spaagazine 1F-FDG-PET in 2002 –11. Furt in 2002 , 13. The in 2002 , 15. Thi in 2002 , 17. In Magazine F-FDG-PET in 2002 –21. Tech in 2002 , 22. HowWhen a multicenter phase III trial is initiated, meticulous preparations of the trial promote standardized data acquisition  to further facilitate general acceptance and implementation of FGS. Only a handful of randomized phase III trials with tumor-specific fluorescence imaging agents have been initiated in recent years and many more with non-tumor-specific fluorescent dyes like indocyanine green (ICG) , 23, 24.Assuming efficacy and safety of an imaging agent is determined earlier, a phase III study is initiated when a valid clinical research question is established Fig. . In ordeAlthough there is a need for a general applicable clinical trial endpoint for TS-FGS trials, the variation between surgical procedures, tumor types, and surgical site creates a difficulty in defining such an endpoint . We advoFor optimal imaging data in a phase III clinical trial, the imaging agent should be matched with a NIR fluorescent imaging system based on optical properties (choice of excitation source and filters to optimize fluorescent yield) and technical properties (procedure type). The trial team should take into account that all fluorescence imaging systems differ in their operational characteristics , signal max ~ 780 nm, Emmax ~ 810 nm), while other NIR imaging systems have their sensitivity for certain fluorophores [To date, a selection of NIR imaging systems are commercially available for intraoperative use and are designed for either open, laparoscopic, or robotic procedures and in some cases endoscopic examinations , 31. Therophores , 32 and Although most of the conjugatable fluorophores used to enable TS-FGS are excited around the same wavelength as ICG, the fluorescent yield is often suboptimal. Tumor-specific imaging agents are usually present in the tissue of interest at nanomolar concentrations, which requires sensitive cameras with low detection thresholds. Minimally invasive surgery eliminates some of the disadvantageous imaging conditions . Yet, current laparoscopic NIR fluorescence imaging systems have a significantly lower fluorescent yield as compared with open systems, mainly due to safety limitations concerning the power of the excitation laser. Bridging this technical gap is one that can only be achieved by collaborating intensively with optical engineers, camera developers, preclinical scientists, and surgeons. Only if the imaging agent, more specifically the fluorophore, is well matched with the clinical imaging system can standardized reporting really tell us something about the efficacy of the imaging agent itself (phase II study) and its potential for clinical use (phase III study). Standardized back-table pathology plays an important role in studying the biodistribution of an imaging agent in the tissue of interest , but lacPerforming TS-FGS requires background knowledge about the physiological behavior of NIR light, the pharmacodynamics, and pharmacokinetics of the imaging agent, the camera setup, and the intraoperative workflow. For TS-FGS to be applied in a standard clinical fashion, data acquisition and interpretation are both executed by a clinician. All these assets are therefore of influence on data quality and reproducibility. Although most clinically available camera systems are almost “plug-and-play” platforms, running a phase III trial, thus hands-on performing NIR fluorescence imaging, requires a surgical workflow designed specific for the trial. One should be aware that the combination of clinician and camera system together makes the imaging platform. Even before the first patient is included, participating surgeons need to be thoroughly trained. Especially in later phase trials where the influence of FGS on clinical decision-making or patient outcome is studied, data interpretation by the end-user is vital for the study’s overall quality, for example, dry-lab session to get familiar with the imaging system and acquire hands-on experience on the effect of imaging distance, camera angle, and overlaying tissue. Tissue perfusion studies with non-targeted dyes  or sentinel lymph node localization supervised by an expert can be used for this training purpose since the clinical application is relatively well studied in retrospective cohorts , 35. ThiFor TS-FGS to impact standard patient care, study data should be comparable and generalizable amongst clinicians, thus requiring minimal interobserver variability. Currently, most translational studies are performed in university medical centers or training hospitals, whereas 80–90% of cancer patients receive their care in a community hospital . InvolviBased on the CSE, imaging agent, and NIR fluorescence camera system, the trial team designs the imaging protocol. Technical feasibility and data quality prerequisites need to be discussed between the surgeon and data specialists. Both should be aware on how imaging data is acquired in the operating room, but also how it is eventually transferred into numerical data and further analyzed to answer the research question. This framework is then used by the surgeon to finalize the imaging protocol, taking in mind the technical specifications of the imaging system  and the procedure of interest  . In paraAfter the first inclusions, quality control and quality feedback by an expert of the field and study coordinator are important for correct interpretation of study results. Low compliance with the imaging protocol and data processing can encourage over and/or underestimation of the study results . PeriodiThough FGS provides new perspectives for surgeons, combining fluorescence imaging with other tumor-specific imaging modalities might further increase accurate patient selection and help to identify the population who might truly benefit from TS-FGS. FGS has many advantages over conventional preoperative imaging . Nonetheless, the majority of multidisciplinary team meetings still predominantly rely on 2D, anatomical, gray-scale images for disease staging and defining the treatment plan, in some cases combined with circulating biomarkers  , 40. DaiMultiplex imaging with an array of biomarkers is not a new idea, but is often done using the same imaging approach. Multimodal molecular imaging using different biomarkers will provide both molecular and spatial information. Such a multimodal imaging approach can provide the spatial information needed to plan or execute surgical treatment where in vitro diagnostics often stay behind. Also, blood drawn from the circulation is inherent to sample bias, created by a pooled sample of all biomarkers present in the circulation and is therefore lacking the spatial information.In this perspective, staging laparoscopies are routinely performed, and FGS is expected to be of added value there. However, multimodal molecular imaging should be able to provide this same information non-invasively, potentially preventing invasive procedures. This emphasizes the need for accurate preoperative staging with tumor-targeted imaging modalities with radionucleotides or for instance ultrasound to acquire this information beforehand. This enables the clinician to use TS-FGS to its best and, when needed, can be used for localization of the target lesion, check for distant  disease, or confirm a complete resection Fig. . PreferaOther opportunities and challenges of FGS lay in the increased use of minimally invasive surgery and neoadjuvant treatment. Surgeons lack the tactile information regarding benign or malignant tissue with robotic-assisted and laparoscopic surgery. Feedback from a fluorescent imaging signal could provide tactile information visually. Moreover, after neoadjuvant treatment, tumor-specific imaging enables a more accurate assessment of the treatment response and active regions of the tumor. With neoadjuvant treatment being applied in a growing number of cases, the need for tumor-specific molecular imaging becomes therefore increasingly evident. In this perspective, the better spatial resolution of FGS over PET may allow for more accurate assessment of vital tumor tissue. One should be cautious to study the survival impact of FGS on resection margins in patients whom are simultaneously being studied for new chemotherapy regimens as this can confound results. Also, radio-, chemo-, and/or immunotherapy might affect the receptor overexpression  and vascIn summary, challenges can be found in defining the incremental clinical value of using FGS and tumor-specific molecular imaging in particular. The gain of information provided by the spatial and biomolecular feedback of fluorescence guidance in the operating room will in some cases force clinicians to change treatment policy; the effect of which should be studied in order to establish new evidence-based decision models. New standards need to be set on trial design and reporting of results in order to successfully translate and implement FGS. Valuable examples can be found in the IDEAL framework describing the different stages of surgical innovations . Integra


Despite advances in chemotherapy and radiation, stage IV breast cancer presents a serious challenge to clinicians in light of the continued poor outcomes for patients. Stage IV breast cancer frequently metastasizes to the brain often necessitating neurosurgical intervention. The goals of the neurosurgeon are to adequately address metastatic disease to the central nervous system, limit morbidity for the patients, while preserving as much neurologic function as possible, and to help guide next steps regarding need for radiation and immunotherapy. In this review, we provide a background overview of the role of neurosurgery in managing stage IV metastatic breast cancer involving the brain, discuss what is known about brain metastasis, and highlight avenues for future study and investigation. Among patients with breast cancer about 10–15% will develop metastases ,3. AlthoImprovements in personalized medicine have provided additional options for some types of breast cancer. Targeted therapies with trastuzumab and lapatinib for Her2- positive patients have improved survival outcomes . Patientvia an Epithelial-to-Mesenchymal Transition (EMT), the actual mechanisms of which are poorly understood. What is known is that the process involves alteration of the adherens junction via down regulation of epithelial cell markers such as E-cadherin, and the upregulation mesenchymal markers such as N-cadherins, thus allowing cell separation and reduced cell-cell adhesion [Despite vast advances in our understanding of the biomechanics and the pathophysiology behind breast cancer metastasis, the exact mechanism of spread still remains a topic of ongoing debate. While cancer metastasis is a highly complex process brought about through spontaneous genetic adaptations developed by rapidly dividing tumor cells, there does appear to be established principles upon which this process functions as will be highlighted below Semanza pioneered a five-stage system for the mechanics of blood or lymphatic vessel metastasis. It consists of intravasation, circulation, margination, extravasation, and colonization . The proadhesion . Once a adhesion . The subadhesion .via studies examining the predisposition of breast cancer type and metastatic behavior. In fact, some studies have shown that breast cancer metastasis to the brain is increased in triple-negative and in HER-2+ subtypes in addition to cells exhibiting high expression of nestin, promonin-1, CK-5 and WNT/β-catenin signaling [2, Epidermal Growth Factor Receptor (EGFR) ligand HBEGF, and alpha-2,6-sialyltransferase ST6GALNAC5 have all been identified as mediators of breast cancer cell extravasation into the brain [The site specificity of cancer metastasis being dictated by the tumor cell compatibility with the end target tissue is a common notion, and was first suggested in Paget’s “seed and soil” hypothesis . We see ignaling . Others ignaling . One of he brain . FurtherOverall, the current body of evidence demonstrates an immensely complex and dynamic cascade involving countless transcriptional modifications and signaling proteins that allow breast cancer to metastasize to the brain. These molecular pathways permit both the predictability and spontaneity associated with cell metastasis and warrant further investigation.Systemic treatment is commonly implemented in conjunction with surgical resection or radiotherapy in the management of brain metastases of breast cancer and helps to reduce recurrence rates. Among ER positive patients’ hormonal therapy  can be offered . Many cyCurrent recommendations for the management of patients with brain metastasis of breast cancer vary based on the receptors expressed  and the degree of metastasis. Per ASCO guidelines, patients with a single central nervous system lesion that is surgically accessible, surgery should be offered with Stereotactic Radiosurgery (SRS) in post-op, whereas, SRS alone should be offered for patients who are unfavorable surgical candidates. If SRS is unavailable, Whole-Brain Radiotherapy (WBRT) or Fractionated Stereotactic Radiotherapy (FSRT) should be offered to reduce the recurrence risk . FurtherAs treatment modalities continue to evolve, one promising approach is Laser Interstitial Thermal Therapy (LITT). The benefit of LITT is that local control of tumor burden can be obtained in a quicker manner than radiation treatment . AnotherAnother up and coming therapy option is the use of Focus Ultrasound (FUS) on brain cancer metastases. FUS had demonstrated immense versatility with therapeutic utility in its ability for thermal ablation, penetration of the BBB, immunomodulation, sonoporation, sensitization to chemotherapy, and many others that currently remain under study . FurtherThe adopted standard for many centers for 2 or less brain metastases from breast cancer is surgical resection followed by stereotactic radiosurgery. Novel adjunctive chemotherapy is now being considered at multiple institutions. For 3 or more metastases to the brain, stereotactic radiation or whole brain radiation are the clinical options, with resection of a symptomatic lesion if easily accessible. Novel innovations such as immunotherapy with checkpoint inhibitors and LITT that target molecular pathways highlighted above may prove promising in the future. Further research regarding improved treatment modalities is warranted.


Reports on the modulatory role of the neuropeptide oxytocin on social cognition and behavior have steadily increased over the last two decades, stimulating considerable interest in its psychiatric application. Basic and clinical research in humans primarily employs intranasal application protocols. This approach assumes that intranasal administration increases oxytocin levels in the central nervous system via a direct nose-to-brain route, which in turn acts upon centrally-located oxytocin receptors to exert its behavioral effects. However, debates have emerged on whether intranasally administered oxytocin enters the brain via the nose-to-brain route and whether this route leads to functionally relevant increases in central oxytocin levels. In this review we outline recent advances from human and animal research that provide converging evidence for functionally relevant effects of the intranasal oxytocin administration route, suggesting that direct nose-to-brain delivery underlies the behavioral effects of oxytocin on social cognition and behavior. Moreover, advances in previously debated methodological issues, such as pre-registration, reproducibility, statistical power, interpretation of non-significant results, dosage, and sex differences are discussed and integrated with suggestions for the next steps in translating intranasal oxytocin into psychiatric applications. Regardlg., envy  that areg., envy , 21, sucg., envy –24, insug., envy –27, and g., envy .Owing to early reports of the beneficial effects of intranasal oxytocin on social cognition, it has been nominated as a promising pharmacological agent for the treatment of psychiatric disorders characterized by social and/or emotional dysregulations , such asCongruent evidence generated by diverse methodological strategies points to the role of oxytocin as a modulator of social behavior and social cognition in humans. Observational studies have reported associations between individual variations in social behavior and social cognition with endogenous oxytocin levels , candidaSeveral lines of research indicate that intranasally administered oxytocin reaches the brain. Given that only a small fraction of peripheral oxytocin passes the blood-brain barrier after intravenous administration , as compA comparison of the effects of intranasal and intravenous oxytocin administration demonstrates that intranasal oxytocin travels to the brain directly and that social-cognitive effects of intranasal oxytocin are not primarily due to effects on peripheral oxytocin receptors. Specifically, Quintana et al. demonstrated that despite comparable peripheral oxytocin levels after intravenous and intranasal administration (administered using a double-dummy design), social cognitive  and neurFunctionally relevant increases in oxytocin in CSF after intranasal administration demonstrates this approach can raise central levels for activation of oxytocin receptors in the brain, which can influence cognition and behavior. Peripheral concentrations of oxytocin are often measured after intranasal oxytocin administration to indicate an increase in central levels of oxytocin. Although a recent meta-analysis suggests that central and peripheral levels of oxytocin are strongly and significantly correlated after intranasal administration , peripheSome of the clearest evidence that intranasal oxytocin was effective to penetrate into the brain in rat and primate models was reported in recent studies , 84. Lee18F oxytocin receptor tracer with high potency and selectivity [The development of oxytocin receptor-specific radioligands would provide the means to directly trace the binding sites of exogenous oxytocin administration , 91. Howectivity  identifiectivity .AVPR1A receptor when high doses flood available oxytocin receptors [Dose-ranging research is a crucial and underrecognized step for translational oxytocin research . Withouteceptors . Of inteeceptors  examinedHuman intranasal oxytocin studies primarily employ dosages between 20 and 48 IU , with thbefore data is collected, based on strength of their rationale and methodological plan [Concerns over questionable research practices have led to skepticism regarding published effects in oxytocin research , 106, hocal plan . As wellcal plan  and toolcal plan  and fMRIcal plan . Second,cal plan , 113 moscal plan , 115. Ifcal plan , 116, mocal plan , 117. Focal plan , 119. Recal plan , which scal plan ). Fifth,cal plan , this atcal plan , 124 or cal plan  on approcal plan .Table 1Kp-value thresholds, which are recommended for hypothesis-driven studies [δ = 0.2 (with a Type I error rate of α = 0.05 and 80% power) using a between-participants design and a two-tailed test requires a sample size of 394 per group . However, using the same parameters with a between-participants design and a one-tailed test only requires the recruitment of 156 participants. In terms of meta-analyses, raw data studies can be combined for individual participant data meta-analysis, which facilitates more powerful moderation analysis  than conventional meta-analysis.Low statistical power is a well-known issue for intranasal oxytocin research . There a studies , can alsIn parallel with recent appeals to increase reproducibility by improving methodology, some have called for a greater focus on theory development to improve reproducibility –131. BroThe previous methodological recommendations largely pertain to issues in the biobehavioral science in general. We also provide a few additional suggestions specifically relevant to intranasal oxytocin research. First, future research should demonstrate the similarities and differences between the effects of oxytocin on cognition and physiology in healthy and clinical populations. In other words, are findings on the effect of intranasal oxytocin in neurotypical populations generalizable to clinical populations? Most claims regarding the therapeutic utility of oxytocin have been based on proof-of-principle studies that investigated the effects of oxytocin on social cognitive functions in healthy individuals . A serieSecond, it is unclear whether chronic administration of oxytocin will emerge as a long-term treatment for patients , or if sConverging translational evidence from recent studies suggests that oxytocin reaches the brain in biological and behavioral relevant amounts following intranasal administration. More recently, the field of oxytocin research has increasingly adopted more rigorous methodological practices, including replication designs in comparably large samples , 117, in


The development of an effective human immunodeficiency virus (HIV) cure is a critical global health priority. A major obstacle to this effort is the establishment of a latent reservoir of HIV infected cells, which necessitates lifelong therapy, causing both logistical and adherence burdens for infected individuals. However, in a subset of these individuals, cytotoxic T lymphocytes (CTLs) can durably suppress viral outgrowth in the absence of therapy, providing a path towards a viable HIV cure. In this review, we discuss the emerging role that CTLs have in HIV cure efforts, with particular emphasis on epitope specificity. Recent studies have demonstrated that successful in vivo containment of the virus is rooted in the specific targeting of fitness-constrained, mutation-resistant regions of the HIV proteome. We highlight these new insights, providing context with previous observations in HIV and other models of viral control, and delineate their translation into a therapeutic vaccine.  Despite over 3 decades of work, the human immunodeficiency virus (HIV)/AIDS epidemic continues to have enormous implications worldwide, with an estimated 37 million infected individuals . While cAmong the various strategies under investigation are the induction of cytotoxic T lymphocytes (CTLs), which constitute a major component of the host immune response to HIV. The emergence of HIV-specific CTLs during acute infection coincides with a decline in plasma viremia , 5 and, + T-cell decline, and progression towards AIDS. However, for HIV controllers, the absence of therapy does not prevent these individuals from durably suppressing plasma viremia to below the transmission/progression threshold (2000 RNA copies/mL) or maintaining normal CD4+ T-cell counts, making them compelling natural examples of a functional drug-free HIV cure. While these individuals do have smaller viral reservoirs with lower sequence diversity . Th. Th44]. The approach was validated on non-HIV proteins of known structure and function, which demonstrated highly significant inverse correlations between amino acid network score and experimentally determined mutational tolerance. This confirmed the value of the network score as a proxy for mutational resistance. Moreover, network score outperformed sequence conservation and relative solvent accessibility in its capacity to define mutation-resistant residues. Thus, the approach was applied to the HIV proteome and revealed that network score could distinguish residues with similar sequence conservation, but with disparate effects on viral fitness when mutated. Additionally, it demonstrated that the interconnectedness of residues in Gag p24 was significantly higher than other proteins, such as envelope, potentially providing a biophysical explanation as to why increased CTL breadth to Gag is associated with better control of viral replication.In the context of HLA associations with HIV control, the network approach revealed that protective HLA alleles preferentially presented epitopes comprised of residues with high network scores (highly networked epitopes), while risk HLA alleles rarely, if ever, presented such epitopes. These observations indicated that differences in protective/risk HLA allele odds ratios could potentially be explained by the stochastic probability of targeting a highly networked epitope . FurtherInterestingly, while median network score of presented epitopes differentiated protective and risk HLA alleles, several epitopes restricted by neutral HLA alleles were also found to be highly networked. This finding provided the opportunity to investigate whether HIV control could be decoupled from its association with HLA alleles, and instead linked to the targeting of highly networked epitopes by functional CTL responses. Indeed, evaluation of proliferative HIV-specific CTLs in a cohort of HIV-positive individuals with diverse clinical phenotypes revealed preferential targeting of highly networked epitopes by HIV controllers, but only rare and weak targeting of these epitopes by progressors, irrespective of HLA allele . In addiThe identification of highly networked epitopes that are presented by nonprotective HLA alleles, but which constitute major HLA supertypes  , provideWith renewed interest in T-cell–based cure strategies for HIV, it is vitally important that careful consideration is given to the specificity of CTL responses. Systematic study of HIV controllers has revealed that enrichment of specific HLA class I alleles within these individuals is likely the result of preferential presentation and functional CTL targeting of epitopes with higher-order sequence constraints, due to their presence at topologically important, mutation-resistant regions of the viral proteome. Moreover, the ability now to decouple HIV control from these specific HLA class I alleles and link to specific CTL epitopes provides a key step towards clinical translation. Developing a suitable therapeutic vaccine candidate that leverages these findings towards a functional HIV cure is an important next step.