These novel histologic findings are based on the pathology of cardiac sarcoidosis. We also discussed the principal histologic differential diagnoses of cardiac sarcoidosis, such as tuberculosis myocarditis, fungal myocarditis, giant cell myocarditis, and dilated cardiomyopathy.Sarcoidosis is a rare disease of isolated or diffuse granulomatous inflammation. Although any organs can be affected by sarcoidosis, cardiac sarcoidosis is a fatal disorder, and it is crucial to accurately diagnose it to prevent sudden death due to dysrhythmia. Although endomyocardial biopsy is invasive and has limited sensitivity for identifying granulomas, it is the only modality that yields a definitive diagnosis of cardiac sarcoidosis. It is imperative to develop novel pathological approaches for the precise diagnosis of cardiac sarcoidosis. Here, we aimed to discuss commonly used diagnostic criteria for cardiac sarcoidosis and to summarize useful and novel histopathologic criteria of cardiac sarcoidosis. While classical histologic observations including noncaseating granulomas and multinucleated giant cells  are the most important findings, others such as microgranulomas, CD68 Sarcoidosis is a systemic inflammatory disease of unknown etiology and is characterized by noncaseating granulomas that may involve any organs of the body, particularly the lungs, lymph nodes, skin, liver, spleen, and eyes . CardiacCardiac sarcoidosis typically occurs in the 20s to 60s age group and is more common in women than in men ,6. The iCardiac sarcoidosis is discovered at autopsy in 15% to 90% of cases of systemic sarcoidosis, and it can affect anywhere in the heart ,7,8,9,10It is often difficult to recognize cardiac sarcoidosis via antemortem endomyocardial biopsies. It is typically diagnosed when endomyocardial biopsy demonstrates noncaseating granulomas; however, endomyocardial biopsy has low sensitivity (approximately 20% to 30%) for the diagnosis owing to the small size and high sampling error rate associated with biopsy specimens ,13. In aHere, we introduce commonly used diagnostic criteria for cardiac sarcoidosis and discuss the significance of endomyocardial biopsy. Thereafter, we summarize useful and novel histopathologic criteria of cardiac sarcoidosis. Finally, we discuss the principal differential diagnoses of cardiac sarcoidosis.The most commonly used diagnostic criteria for cardiac sarcoidosis are the 2014 Heart Rhythm Society Expert Consensus Statement on the Diagnosis and Management of Arrhythmias Associated with Cardiac Sarcoidosis see  16] and and16] aMost pathologists diagnose cardiac sarcoidosis depending on the presence of sarcoidal granulomas. However, the diagnostic sensitivity of endomyocardial biopsy is low because sarcoidal granulomas are rarely detected in many cases. Here, we emphasize the histologic findings of cardiac sarcoidosis based on the temporal evolution and significance of the morphologic alterations.+ helper T cells are predominantly recruited, expanded oligoclonally, activated, and polarized into the T helper 1 (Th1) phenotype, which results in a CD4/CD8 T-cell ratio of approximately 10:1 ..59].Giant cell myocarditis and cardiac sarcoidosis are sometimes notably difficult to distinguish because they have many similar clinical manifestations, histopathological findings, and pathoetiology. Although there is some controversy regarding whether giant cell myocarditis and cardiac sarcoidosis are different phenotypes of a single disease or two distinct entities, some histological differences have been identified . Granulo+ helper T cells are dominantly recruited in cardiac sarcoidosis as described above, whereas CD8+ cytotoxic T cells more often occur in giant cell myocarditis [Moreover, some reports indicate that immunohistochemistry may be helpful for diagnosis; although both are caused by T cell-mediated autoimmune inflammation, CD4carditis .The histologic alterations in dilated cardiomyopathy are nonspecific because they do not point to a specific etiology. Dilated cardiomyopathy occasionally exhibits similar clinical symptoms and histopathology to those of cardiac sarcoidosis. In particular, when typical granulomas are not formed in endomyocardial biopsy, it is occasionally difficult to distinguish between the interstitial phase of cardiac sarcoidosis and dilated cardiomyopathy. A fibrotic pattern is one of the histologic findings that can distinguish between both conditions. Fibrosis is pronounced in the myocardium with a mosaic pattern in cardiac sarcoidosis, whereas diffuse fibrosis in extensively injured cardiomyocytes is often seen in dilated cardiomyopathy ,36.Additionally, fatty infiltration can occur within loose connective tissue around granulomas in cardiac sarcoidosis, whereas there is no fatty infiltration within fibrous granulation tissues in dilated cardiomyopathy . It is uC. acnes components (detected by PAB antibody), confluent fibrosis, and fatty infiltration may be helpful to improve the sensitivity of endomyocardial biopsy. These novel histologic findings are based on the pathology of cardiac sarcoidosis.Sarcoidosis is a rare disease of isolated or diffuse granulomatous inflammation. Cardiac sarcoidosis is sometimes a fatal disorder, and it is important to accurately diagnose it to prevent sudden death due to fatal dysrhythmia. Although endomyocardial biopsy is invasive and has limited sensitivity for detecting granulomas, it is the only procedure that provides a definitive diagnosis of cardiac sarcoidosis. While classical histologic observations including noncaseating granulomas and multinuclear giant cells  are the most crucial, other findings such as microgranulomas, M1 macrophage accumulation, lymphangiogenesis, Cardiac sarcoidosis is an involvement of a major organ and should be thoroughly studied and treated to avoid the risk of arrhythmia and of sudden death. Therefore, pathologists should carefully observe for granulomas and other histologic findings that indicate cardiac sarcoidosis, in order to achieve a timely and accurate diagnosis of the condition.


The G peak thermal lineshift rates dω/dT showed that the H2 intercalation significantly weakened the pinning effect in epitaxial graphene. Furthermore, the G peak dω/dT value showed a perspicuous pinning effect disparity of QFSBEG samples. Additionally, the anharmonic phonon effect was investigated from the Raman lineshift of peaks. The physical mechanism responsible for dominating the G-mode temperature-dependent behavior among samples with different substrate coupling effects was elucidated. The phonon decay process of different samples was compared as the temperature increased. The evolution from in situ grown graphene to QFSBEG was determined. This study will expand the understanding of QFSBEG and pave a new way for its fabrication.Epitaxial graphene on SiC without substrate interaction is viewed as one of the most promising two-dimensional (2D) materials in the microelectronics field. In this study, quasi-free-standing bilayer epitaxial graphene (QFSBEG) on SiC was fabricated by H ComPb, etc. ,7,8,9, Hrication ,11,12.2 intercalation condition, and how the evolution proceeds from the in situ graphene and QFSBEG have not been determined. The extent of H intercalation at the interface of the SiC substrate and the graphene has a direct influence on transport properties. Therefore, it is of vital importance to conduct a study on the progress from in situ graphene to QFSBEG.The difference between the structure and properties of in situ grown monolayer graphene and QFSBEG has been researched elsewhere ,12,13,14 407 GHz . However2 intercalation condition using a Hall instrument. However, the sample can be damaged by the probe contact even under the simple four-probe Van der Pauw configuration. It is well known that Raman spectroscopy is one of the most effective and nondestructive methods for graphene characterization. In addition to the layer number and structure characteristics of graphene, the Raman spectra can also provide the anharmonic phonon effect by analyzing the temperature-dependent spectra. The anharmonic phonon effect is the collective effect of thermal expansion and electron-phonon and phonon-phonon interactions, which play an important role in electronic transport properties [For the characterization of QFSBEG, a mobility test is the most intuitive way to reveal the Hoperties ,18,19,202 intercalation conditions in the temperature range from 213 K to 663 K. The substrate effect was compared through the line shift rates of G mode. Furthermore, the anharmonic phonon process was studied by conducting theoretical model calculations.In this study, the Raman spectra were measured as a function of temperature on in situ graphene and QFSBEG obtained under different H2 etching procedure to obtain a regular and micron-scale step structure in a 5 sccm flow of H2 at a temperature of 1500–1600 °C and pressure of 800–900 mbar for 20 min. Graphene growth was conducted at 1600–1700 °C, 800–900 mbar in 20 sccm argon atmosphere for 60 min. The sample was then cooled to room temperature, corresponding to in situ graphene. For H2 intercalation, two samples were selected for H2 intercalation processing in a hydrogen flux of 50 sccm at a temperature of 1600 °C and pressure of 800–900 mbar. The operation time was selected as 30 min and 60 min to study the evolution process from in situ grown graphene to QFSBEG, corresponding to samples QFSBEG-1 and QFSBEG-2, respectively.Epitaxial graphene was grown on home-made 4 inch SiC wafers in a self-developed furnace. The (0001) face was processed by chemical mechanical polishing (CMP) and followed by cleaning with acetone , ethanol , and deionized water cleaning to remove surface contamination. The SiC substrate was subjected to a HFor the Raman measurement, HR 800 by Horiba Jobin Yvon  was used in the backscattering geometry with a 532 nm laser for excitation. For surface enhanced Raman spectra testing, the 100× objective was used with a 600 diffraction grating with the assistance of Ag particles at room temperature. For the temperature variation measurement, a Linkam  hot/cold stage was used and cooled with liquid nitrogen. The 50× long-focus objective was used with a 600 diffraction grating restricted by the distance to the sample stage. The samples were measured from 213 K to 663 K in steps of 15 K. In the case of temperature interference on SiC substrate for substrate subtraction, a SiC substrate was placed in the hot/cold stage and its spectra were synchronously collected under different temperatures. Before each measurement, the samples were stabilized for about 5 min to ensure that the temperature had reached the set value and the optical system was refocused to avoid the thermal effect. For the accuracy of data, the positions of four samples  were recorded, and after each measurement, the stage was set at the same coordinate to ensure that the spectrum was taken at the same spot.2 at different time periods were studied systematically. First, in order to get intuitive understanding of graphene after H2 intercalation, the surface enhanced Raman spectra of these three samples were measured with the assistance of Ag particles. as shown in −1 appeared in the Raman spectra of samples after H2 intercalation. It was considered that the perpendicular stretch mode of the Si-H bond was excited by the near-field plasmonic effect, thus, the H2 intercalation could be proved straightforward in comparison with the conventional method [2 was intercalated successfully or failed, but they did not provide information about the inner condition of QFSBEG.The in situ grown graphene and the samples intercalated by Hl method ,21. HoweRaman spectra of three samples were measured in the temperature range of 213K–663K, and the spectra obtained in three typical temperatures  are shown in dω/dT are summarized in dω/dT rate of −0.048 cm−1/K, which was consistent with the reported in situ grown epitaxial graphene on SiC rate of −0.043 cm−1/K [2 intercalation, the value declined dramatically and varied by the time duration of H2 intercalation. The dω/dT rates of samples QFSBEG-1 and QFSBEG-2 were −0.035 cm−1/K and −0.022 cm−1/K, respectively. It is clear that the dω/dT rate is an effective parameter for characterizing the intrinsic state of the substrate pinning effect under different H2 conditions. As the time extended, the substrate effect would be further released but beyond the reach of free-standing graphene at the rate of ~−0.010 cm−1/K [For the analysis of the G peak, the Raman spectra of graphene were subtracted by the SiC Raman spectra simultaneously taken at a specific temperature. The subtraction process was shown in 3 cm−1/K . After H0 cm−1/K .2 intercalation deepening from It is worth noting that, with the extent of Hsp2 carbon hybridization and resulted from the degenerate phonon mode 2gE at the center of Brillouin zone. The D peak is contributed by a one-phonon process assisted by the defect near the K point of the Brillouin zone, thus it is sensitive to the crystalline defects. The 2D peak is an overtone of the D peak, which originated from a second-order phonon process and is sensitive to the number of graphene layers. For a Raman spectrum taken at room temperature, the domain size or the layer of graphene could be extracted through these three peaks using an empirical equation. Temperature-dependent Raman spectra are an excellent tool to investigate anharmonic effects. According to the ab initio calculation, the Raman peak position is the real part of the phonon self-energy, and the Raman linewidth is the imaginary part of the self-energy. The Raman shift of G peak at a certain temperature can be expressed using the following formula [γ is the Gruneisen parameter of graphene for the Raman G mode; here, we take Gγ = 1.99 [For a typical Raman spectrum of graphene, it is of vital importance to study the G, D, and 2D peaks. The G peak is representative of  formula ,29.(1)ΩG = 1.99 ; βT is twas used .(3)βT=αM1 and M2 are constants representing the three-phonon and four-phonon processes to the Raman shift, respectively. The term wHere, the simple Klemens model was used to clarify the Raman shift variation caused by the pure temperature effect. In this model, the effect can be considered as three-phonon and four-phonon interactions, and can be expressed as follows:Ω0, M1, and M2 are shown in Ω0 of the QFSBEG samples exhibited a redshift, in contrast with the in situ graphene. In consideration of the phonon process, the reported studies showed that the four-phonon effect was the dominating factor over the thermal expansion and three-phonon process G mode with the temperature [1/M2 value of QFSBEG-2 decreased in contrast with that of QFSBEG-1, which indicated that the three-phonon effect decreased with the extent of H intercalation deepening.perature ,32. In cperature . Indeed,tructure . It can Usually, the phonon damping rate is proportional to the linewidth Γ(T) of the Raman peak. Hence, the phonon lifetime τ can be calculated from the peak linewidth Γ through the energy-time uncertainty relation, and the equation can be expressed as follows :(6)Γћ=1τ−1∙ps. Based on the G mode, the peak width was extracted by Lorentz fitting and the phonon lifetime was calculated by the above equation. The linewidth of the Raman G peak and phonon lifetime of these three samples are shown in the Here, τ, it was affected by two main factors, and the equation can be expressed as [For the measured phonon lifetime essed as ,36:(7)12 processing, the Si-C bonds were broken and the Si dangling bonds were partially saturated by H atoms, as shown in Based on the experimental data, the transformation evolution process from in situ graphene to QFSBEG is summarized in 2 intercalation time periods were fabricated. Temperature-dependent Raman spectra were collected in the temperature range of 213–663 K. In contrast to the in situ grown graphene that the G peak dω/dT value at changed at the rate −0.048 cm−1/K, the G peak thermal lineshift rates dω/dT declined to −0.035 cm−1/K after 30 min H2 management and −0.022cm−1/K after 60 min H2 management, respectively. The anharmonic effect analysis showed that the three-phonon process was the dominant decay pathway, and the ratio of the three-phonon process to the four-phonon process dropped from 4.74 to 3.69 as the H2 treatment time increased. At the conclusion, the evolution process of transformation was demonstrated.In conclusion, in situ grown graphene on SiC and QFSBEG under different H


Accurate and imperceptible monitoring of electrophysiological signals is of primary importance for wearable healthcare. Stiff and bulky pregelled electrodes are now commonly used in clinical diagnosis, causing severe discomfort to users for long-time using as well as artifact signals in motion. Here, we report a ~100 nm ultra-thin dry epidermal electrode that is able to conformably adhere to skin and accurately measure electrophysiological signals. It showed low sheet resistance , high transparency, and mechano-electrical stability. The enhanced optoelectronic performance was due to the synergistic effect between graphene and poly  polystyrene sulfonate (PEDOT:PSS), which induced a high degree of molecular ordering on PEDOT and charge transfer on graphene by strong π-π interaction. Together with ultra-thin nature, this dry epidermal electrode is able to accurately monitor electrophysiological signals such as facial skin and brain activity with low-motion artifact, enabling human-machine interfacing and long-time mental/physical health monitoring. Novel dry electrodes with enhanced mechano-electrical stability and conformability are attractive for long-time electrophysiological signal monitoring. Here, the authors report polymer-covered CVD-grown graphene with enhanced optoelectronic performance for biopotential monitoring. Skin-contact electrophysiology, such as electromyography (EMG), electrocardiography (ECG), electrooculogram (EOG), and electroencephalogram (EEG), relying on the recording of biopotential, are important clinical measurements to assess health status. The ability to accurately and imperceptibly monitor electrophysiological signals is the most basic requirement for wearable electronics8. Skin-contact electrodes in clinical use are mainly pregelled Ag/AgCl11. Although they provide good signal quality, the electrolytic gel may irritate the skin and will dry in a certain period, making it impossible for long-term monitoring and diagnosis. Moreover, with the gel interfacing on skin, electrode displacement with respect to skin will cause severe motion artifact13. Specifically, when detecting facial electrophysiological signals, gel electrodes are easily detached from skin, resulting in poor electrophysiological signal resolution. This is because superficial portions of the face muscles are uneven, full of wrinkles, and inevitably large-shape changed. To achieve high signal quality with low-motion artifact for long-time monitoring, dry electrodes being ultra-conformably adhesive to skin and sufficiently mechano-electrical stable are highly desirable for biopotential monitoring.Epidermal electronics refer to electronic devices that are mounted on the epidermis and able to acquire information about the body precisely or to provide therapy to the body. It can open new pathways for wearable healthcare18 and gold film17 are two frequently studied dry electrodes for electrophysiological recordings. As a representative conducting polymer, PEDOT:PSS blended with waterborne polyurethane and D-sorbitol (~20 µm) was reported to be adhesive on skin and electrically stable upon strain, being able to measure electrophysiological signals19. But the detected signal quality  is not good, because thick material is difficult to be ultra-conformal on skin and blending with polymer results in poor conductivity (545 S/cm)  was proved to be self-adhesive to the arbitrary 3D biological surface and able to monitor surface biopotentials with less motion artifact2. However, Au-film dry electrodes are expensive and visible on the skin, which may bring light-induced photoelectric artifact when applied in certain scenarios. To make dry electrodes more suitable for epidermal electronics, low-cost, optical transparency, ultra-conformability to skin and mechano-electrical stability are essential characteristics to be introduced into dry electrodes for biopotential monitoring.Conducting polymer32 (Supplementary Table 8. Poly(methyl methacrylate) (PMMA)—assisted monolayer CVD graphene electronic tattoo, with a total thickness of 463 ± 30 nm, the optical transparency of ~85%, the sheet resistance of 1994.33 ± 264 Ω/sq and stretchability of ~40%, was applied on skin to detect electrophysiological signals. To fabricate dry electrodes that are easy to handle, polymer thin film (herein PMMA) was utilized. However, the PMMA polymer carrier layer would introduce contamination, cracks, or defects into the CVD-grown graphene during transfer, leading to a significant decrease in the conductivity of final electrodes34. Therefore, to improve the quality of electrophysiological recordings for graphene-based ultra-conformal dry electrodes, a modified PEDOT:PSS layer, not only capable of handling graphene as well as enhancing its superior electronic performances, was proposed in this work.As the thinnest electrode material, graphene demonstrates superior optical transparency, high electrical conductivity, excellent mechanical property, and low electrochemical reactivityHere, we elaborated a strategy for the fabrication of transparent, highly conductive, and ultra-thin dry electrodes by using PEDOT:PSS thin film as a polymer carrier layer to handle and enhance the electrical performance of CVD-grown graphene for biopotential monitoring. To enable a thin, uniform, continuous and highly conductive PEDOT:PSS layer covered on top of graphene, surfactants and ionic compounds were added since pristine PEDOT:PSS solution did not wet graphene and exhibited limited conductivity. Optimized dry electrodes of PEDOT:PSS transferred CVD graphene film (PTG) demonstrated a total thickness of ~100 nm, a sheet resistance of ~24 Ω/sq (4142 S/cm), high optical transparency, and a sufficient mechano-electrical stability for skin electronics. The dramatic increase in electrical conductivity is not merely a consequence of a parallel connection of two conductive units, but a synergistic effect of improved molecular packing of PEDOT and charge transfer on underlying graphene. This observation has been recognized by Grazing-incidence wide-angle X-ray scattering (GIWAXS), Raman, electron spin resonance (ESR) spectroscopy, and so on. PTG exhibited outstanding capability for electrophysiological signal recording such as facial skin and brain activity in motion, enabling human-machine interfacing and long-time mental/physical health monitoring. Our synergistic strategy towards ultra-conformal, transparent, and dry conductive electrodes will pave a new way for epidermal electronics.35 method on copper foil and used conducting polymer PEDOT:PSS 38, as it is a safe additive that has been widely used in detergents for laundry  sulfonimide lithium salt as an example, BSL in short), was also introduced into PEDOT:PSS solution to further improve the electrical conductivity and mechanical stretchability of PTG as skin electrodes. As-obtained PEDOT:PSS (with SDS and BSL)/graphene/Cu foil was annealed at 120 °C for several minutes to enhance the interaction between PEDOT:PSS and graphene, and subsequently immersed in (NH4)2S2O8 solution to etch the underlying Cu foil, followed by rinsing in deionized water to remove possible residues . The resultant PTG thin film was then floated on water and scooped onto arbitrary substrates, such as a thin elastomer, glass, decal transfer tattoo paper and even skin, working as a dry electrode immediately.To achieve dry electrodes with superior electronic performance for biopotential monitoring, we synthesized graphene via CVDPSS Fig. a, b as tPSS Fig. . Adding dry Fig. . In addiDue to the amphiphilic nature of these additives, the addition of SDS and BSL to PEDOT:PSS solution improves its wettability on the hydrophobic surface and helps to form a continuous and uniform PEDOT:PSS conductive layer on graphene. To verify the improvement in electrical conductivity of PTG with the addition of SDS and BSL, we measured 4-probe sheet resistance and transmittance on a variety of PTGs as a function of SDS and BSL. The sheet resistance of PTG thin film was clearly reduced with increased SDS concentrations in the PEDOT:PSS solution Fig. . This is41. Thus, to compromise the transparency, conductivity and mechano-electrical stability of PTG, our optimal condition for following epidermal electrophysiology is bi-layer graphene transferred by PEDOT:PSS at both SDS and BSL concentrations of 1.0 wt%.Next, the mechano-electrical stability of PTG was evaluated (see “Methods”). We placed PTG onto poly (PET) and bent it at various angles. Almost no resistance change was observed when PTG was at a bending radius from 12 to 2 mm Fig. . To eval2/Si substrates are similar  in PEDOT:PSS solution, PEDOT:PSS only (~185 nm) film is ~2.3 times thickness of PTG  film  was shifted to 1599.5 cm−1 in PTG. Such blue-shifts indicated that there is strong π–π interaction between PEDOT:PSS layer and graphene, which will engender the delocalization of π electrons, thus increasing the charge carrier mobility of PTG thin film.To Figure out the synergistic effect between PEDOT:PSS and graphene, we performed a comprehensive study on the structure of PTG. Raman spectroscopy is a powerful tool to characterize carbon-based materials. For the PEDOT:PSS film only, there was a vibration peak at 1434.5 cmThe charge delocalization of π electrons was also confirmed by UV–vis-NIR spectroscopy. Compared with pristine PEDOT:PSS film, a strong absorbance occurred in PTG over a broad range, both at the ~800-nm peak and the free-carrier tail of the near-infrared region. This observation proved the charge delocalization between graphene and PEDOT:PSS, leading to the formation of polaron pairs, that is, bipolarons. The π-π interaction effect was then studied by electron spin resonance (ESR), which probed the presence of unpaired electrons Fig. . The strz and qxy directions were obtained from the 2D GIWAXS patterns , 3.46 Å (PEDOT:PSS with SDS), 3.47 Å (PEDOT:PSS with SDS and BSL) 3.40 Å (PTG with SDS) and 3.37 Å (PTG with SDS and BSL). With the addition of SDS, BSL, and mainly the underlying graphene, the π-π stacking distance of PEDOT decreases from 3.54 to 3.37 Å  and graphene on the molecular packing of PEDOT was further investigated by Grazing-incidence wide-angle X-ray scattering (GIWAXS), an advanced technique to analyze thin-film morphology. Fig. 7 Å Fig. , which r44. The more ordered PEDOT packing in PTGs can be explained by the strong π–π interaction between graphene and PEDOT:PSS. Overall, the synergistic enhancement between PEDOT:PSS and graphene will induce a higher crystallinity in PEDOT, so as to achieve a high conductivity for PTG to accurately detect electrophysiological signals.The highly ordered molecular packing in PTGs can also be reflected in the surface morphology. We used atomic force microscopy (AFM) to observe the morphological evolution of pure PEDOT:PSS and PTGs Fig. . With thh represents thickness, 45. Transferred onto a human ring finger full of wrinkles, PTG indeed exhibited intimate contact on it  pulses to drive the servo motor  recording is to measure the electrical activity of the brain, providing helpful information for the diagnosis or treatment of the brain disorders. Due to the very weak signal strength , long-time monitoring of EEG is extremely challenging, in particular for the status of exercising. Normally, for EEG monitoring, the volunteer sits in a comfortable position peacefully and experiences brain stimulus, such as doing math quiz, watching videos or listening to music. As PTG is ultra-thin and the attachment is comfortable, we, therefore, applied PTG on a volunteer for long-time monitoring of EEG without taking off the electrode pairs. During the monitoring of 12 h, the volunteer slept, exercised, and recovered to calm with two PTG electrodes mounted in the frontal region (Fp1 and Fp2) of the brain and one reference electrode behind the earely Fig. . The intely Fig. . The capIn conclusion, by leveraging two well-known skin electrodes together, we have demonstrated a transparent, highly conductive, and ultra-conformal dry electrode, which is PEDOT:PSS thin film-transferred CVD-grown graphene (PTG). Graphene transferred by PEDOT:PSS without the step of removing the carrier layer was confirmed to be electron doped with higher conductivity; on the other hand, the PEDOT backbones showed improved molecular packing due to the underlying graphene with strong π–π interaction. Thus, the synergistic enhancement effect between graphene and PEDOT:PSS resulted PTG in a very high conductivity (4142 S/cm). Together with the ultra-thin feature, PTG dry electrode exhibited excellent capability of long-time and low-motion artifact electrophysiological signal recording. For facial muscle evaluation, it showed superior advances in lower-motion artifact electrophysiology and less disturbance to laser speckle contrast imaging than commercial Ag/AgCl gel electrodes, and being able to stably and accurately control the robotic hand. For the very weak EEG signals, the user can continuously wear the PTG for 12 h monitoring regardless the extension/contraction and sweat secretion of skin. Our proposed ultra-thin, transparent, and conductive dry electrodes will pave a new way for epidermal electronics.PEDOT:PSS solution (Clevios PH 1000) was purchased from Han Feng, the sodium dodecyl sulfate (99%) was obtained from Innochem, the SEBS and BSL were obtained from Aladdin Industrial Corporation. The decal transfer paper was purchased from The Magic Touch Ltd., UK.2 and 35 sccm CH4. After 40 min growth at 1000 °C, both sides of Cu foil were covered with monolayer graphene. The SDS of 0.25 wt%–1.5 wt% was added to PEDOT:PSS solution and stirred vigorously for 2 h. The BSL was then added to PEDOT:PSS/SDS solution and stirred for another 2 h. Before spin-coating, the prepared PEDOT:PSS solution was filtered by 0.45 µm PES syringe filter. And the PEDOT: PSS solution was spin-coated on the Cu foil with graphene at 2000 rpm for 60 s, forming a PEDOT:PSS/graphene/Cu structure. The PEDOT:PSS coated graphene on Cu foil was annealed at 120 °C. Subsequently, Cu foil was etched by ammonium persulfate [(NH4)2S2O8] solution about 2 h, then the PEDOT:PSS/graphene film was washed in deionized water for 3 times, The film can be transferred to arbitrary substrates, such as silicon wafer, glass, decal transfer tattoo paper, and flexible substrates.Cu foil was put in the quartz tube under a constant pressure of 20 sccm (standard cubic centimeter per minute) HThe sheet resistances were measured using a four-probe resistance measuring meter, with a Keithley 2400 Source Measure Unit. The transparency of the thin film was tested by an ultraviolet-visible spectrometer (UV-2450). The AFM images were obtained in tapping mode . The Grazing-incidence wide-angle X-ray scattering (GIWAXS) measurement was performed on a laboratory beamline system (Xenocs Inc. Xeuss 2.0) with an X-ray wavelength of 1.54 Å and sample to detector distance of 17.1 cm. An incidence angle of 0.2° was used. Diffraction images were recorded on a Pilatus 1 M detector (Dectris Inc.) and processed using the Nika software package, in combination with WAXS Tools. The Raman spectra were obtained at 532 nm excitation (LabRAM HR Evolution). The electrochemical impedance spectroscopy (EIS) measurements were conducted using a CHI600e electrochemical workstation (CH Instruments).Two working electrodes were placed on forearm, and the reference electrode was placed on the back of wrist for sEMG. The electrodes were connected with commercial signal recording equipment (DueLite) and sEMG signals were digitally filtered in MATLAB by a six-order Butterworth filter. And the low cut-off frequency is 10 Hz while the high cut-off frequency is 450 Hz. For ECG, two working electrodes and reference electrodes were placed on arm by using the Einthoven triangle principle. During EOG measurement, the working electrodes were placing on the forehead and lower eyelids, respectively. For easy handling and stable connection to electrophysiological recording instrument, SEBS or decal transfer tattoo paper were sometimes assisted as an additional substrate, and evaporated Ag films were aided in the connection between soft-hard interfaces.The electromechanical vibrator was placed near the working electrodes (about 2 cm) to induce skin vibration, which is similar to vibration in arm movement.Two working electrodes of PTG were placed on levator labii superioris, and one reference electrode was placed behind the ear. Ag/AgCl and PEDOT:PSS electrodes were placed on the same position on the other side of face as control. Laser speckle contrast imaging was recorded in SIM BFI-WF System . The monitor detector was set 25–30 cm above the volunteer’ face, with a maximum field of view of 14.8 × 14.8 cm so that the entire facial area could be illuminated. The frequency used for the blood perfusion image was 1 s, and the size of the image was 512 × 512 pixels. To control the robotic hand, three pairs of PTG were attached on zygomaticus, risorius and corrugator to extract sEMG signals of the index, middle finger, and wrist of the robotic hand, respectively.Brain activity was evaluated by EEG signals using a commercial signal recording equipment (DueLite). The EEG signal was measured by mounting two working electrodes (PTG on tattoo paper with Ag as connections) in the frontal region of the brain and one reference electrode behind the ear. During the monitoring of 12 hours, the volunteer slept for 1 h, and exercised for 0.5 h, finally recovered to calm without taking off PTG electrodes. The EEG signal was analyzed by Matlab and Fast Fourier Transformation.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Biocompatible neural guidance conduits are alternatives to less abundant autologous tissue grafts for small nerve gap injuries. To address larger peripheral nerve injuries, it is necessary to design cell selective biomaterials that attract neuronal and/or glial cells to an injury site while preventing the intrusion of fibroblasts that cause inhibitory scarring. Here, we investigate a potential method for obtaining this selective cellular response by analysing the responses of rat Schwann cells and human dermal fibroblasts to isoleucine-lysine-valine-alanine-valine (IKVAV)-capped dendrimer-activated collagen films. A high quantity of nanoscale IKVAV-capped dendrimers incorporated onto pre-crosslinked collagen films promoted rat Schwann cell attachment and proliferation, and inhibited human dermal fibroblast proliferation. In addition, while pre-crosslinked dendrimer-activated films inhibited fibroblast proliferation, non-crosslinked dendrimer-activated films and films that were crosslinked after dendrimer-activation (post-crosslinked films) did not. The different cellular responses to pre-crosslinked and post-crosslinked films highlight the importance of having fully exposed, non-covalently bound biochemical motifs (pre-crosslinked films) directing certain cellular responses. These results also suggest that high concentrations of nanoscale IKVAV motifs can inhibit fibroblast attachment to biological substrates, such as collagen, which inherently attract fibroblasts. Therefore, this work points toward the potential of IKVAV-capped dendrimer-activated collagen biomaterials in limiting neuropathy caused by fibrotic scarring at peripheral nerve injury sites. Nerve autografts, which are nerve tissues transplanted from one part of the body to another in the same individual, are the gold standard for severed peripheral nerve reconstruction ,2. Howev2 and gold surfaces [Attaching small biochemical motifs to a larger scaffold is an attractive method to engage a specific subset of cells on biocompatible materials for neural applications . Small psurfaces . It is usurfaces , while ssurfaces . UnderstAn important consideration when activating collagen scaffolds with small molecules is to ensure that appended biochemical motifs are physically accessible to cells. Recent research has looked into the use of poly ε-lysine dendrimers, which are branched peptides, to activate different scaffold materials such as collagen ,17,18,19Given the potential of dendrimer-activated biomaterials for improved neural repair, the objective of this work is to integrate nanoscale IKVAV-capped poly ε-lysine dendrimers onto collagen surfaces and study the response of rat Schwann cells (RSCs) and human dermal fibroblasts (HDFs). By adding IKVAV-capped dendrimers to non-crosslinked collagen films, pre-crosslinked collagen films or by crosslinking collagen films after dendrimer addition (post-crosslinking), the response of Schwann cells and fibroblasts to different surface presentations of dendrimers can be studied. Previous studies have shown that non-crosslinked collagen films possess a lower tensile modulus compared to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/ N-hydroxy-succinimide (NHS) crosslinked collagen films, thus providing different environments for cellular attachment and proliferation . What is® CRL-2765™, Manassas, VA, USA). Unless stated otherwise, all other reagents were analytical grade as obtained from Sigma-Aldrich, Gillingham, UK.Insoluble bovine dermis type I collagen powder (product code: 01AWB003) was obtained from Devro PLC, . 16-arm IKVAV-capped poly ε-lysine dendrimers  were obtained from Tissue Click Ltd., Brighton, UK. Human dermal fibroblasts (HDFs) were sourced from Sigma Aldrich . RSC-96 rat Schwann cells (RSCs) were obtained from the ATCC cell biology collection (product code: ATCCw/v) suspension of collagen powder in 50 mM acetic acid at 4 °C for 3 days. Using a commercially available blender, the hydrated powder was blended for 2 min, rested for 2 min, and blended again for another 2 min at 22,000 rpm. Air bubbles were removed from the suspension by centrifuging at 2500 rpm for 30 s. After cooling the suspension at 4 °C overnight, films of micron-scale thickness were cast by reverse pipetting 200 µL of slurry per well into a CytoOne TC-treated 48-well plate  and drying for 48 h in a laminar flow cabinet [Collagen slurries were prepared by swelling a 0.5%  = 708.11 Da). Atto 565 nm NHS ester stock solution was prepared by dissolving 1 mg of powdered ester dye in 0.5 mL of dry dimethyl sulfoxide (DMSO). One mg of dendrimer powder was suspended in 1 mL of phosphate-buffered saline (PBS). For dye conjugation, it was assumed that a 1:1 molar ratio of dye to free primary amine would tag each terminal amine with Atto dye. To adjust to pH 9, 1M NaHCO562) immediately after incubation using a Fluostar Optima plate reader . The absorbance values of the control samples were subtracted from the absorbance values of the experimental samples to correct for background dye absorption.To remove non-conjugated dye, the agitated solutions were dialysed for 24 h in Slide-A-Lyzer dialysis cassettes . The concentrations of purified dye-conjugated dendrimers were measured colourimetrically with a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit,  using a Bovine serum albumin (BSA) protein standard. Prior to measurement, the mixtures were agitated on a rocker for 30 s covered in foil and then incubated at 37 °C for 40 min (78% dye-conjugated dendrimers) or agitated on a rocker for 20–30 min at room temperature (8% dye-conjugated dendrimers). Absorbance values were read at 562 nm (ADye-conjugated IKVAV-capped dendrimers were added to non-crosslinked films (A), pre-EDC/NHS crosslinked films (B), or non-crosslinked films that were crosslinked with EDC/NHS agents after dendrimer addition (C). The three varieties of dendrimer-activated collagen films were simultaneously fabricated over three days to ensure that they were exposed to the same experimental conditions. To coat the collagen films, which were used as dried, with dye-conjugated dendrimers, 120 µL of dendrimer solution (µg of dendrimer/mL of PBS) was added to non-crosslinked  or crosslinked (B) collagen films. The films were incubated for 1.5 h on a rocker at room temperature covered in foil. After incubation, excess dendrimer solution was removed, and the films were washed with 3 × 200 µL PBS. The films were dried in a dark fume hood overnight.v/v) ethanol was added and incubated for 1.5 h. These conditions were deemed optimal by Olde Damink and co-workers [To chemically and covalently crosslink the collagen films, a 5:2:1 molar ratio of EDC/NHS/COO-(Col) in 75% (-workers  for stanv/v) in deionised water, 0.1% Tween (v/v) in PBS, saturated saline, or 0.1 M citrate buffer (pH 3). Collagen films were analysed at 0 h (pre-wash), and after 2 h, 1 day, 2 days, 3 weeks, and 7 weeks. Fluorescent images of the collagen films were taken with a Zeiss Axio Observer Z1 microscope fitted with an Axiocam 530 camera  after each time interval. Fluorescent images were taken at 10× magnification and at 565 nm and 488 nm to detect the dye-conjugated dendrimers and collagen autofluorescence, respectively. Prior to imaging, the samples were washed with 3 × 200 µL deionised water, and all images were taken of collagen films that were hydrated in 200 µL of either fresh PBS or deionised water. In between imaging time points, the samples were incubated in their respective washing solutions on a rocker at room temperature covered in foil. ImageJ software was used to quantify the dendrimers bound to the collagen films after each time interval.To probe dendrimer-collagen interactions, all samples were washed with various detergents for increasing lengths of time. The samples were washed with PBS, 75% ethanol (EtOH) (v/v) foetal bovine serum and 1% (v/v) streptomycin/penicillin antibiotics in a humidified incubator with 5% CO2 at 37 °C. RSCs were passaged 1:20 and HDFs were passaged 1:4 every 3–4 days. Cell layers at ~75% confluence were harvested for analysis by detaching the cells from T75 flasks with 0.05% (w/v) trypsin/0.02% (w/v) EDTA, centrifuging the cells at 180 g for 3 min, and resuspending the cells in serum-free DMEM.RSCs and HDFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) BSA solution in PBS and washed with 3 × 300 µL PBS to prevent non-specific cell binding. The cells were seeded at a density of 5 × 105 cells/mL for RSCs and 3.06 × 105 cells/mL for HDFs. These cell densities were determined using a haemocytometer. Three hundred µL of appropriately diluted cells were added to the films and tissue culture plastic (TCP) controls and incubated for 1.5 h in a humidified incubator with 5% CO2 at 37 °C. Loosely bound cells were gently removed with 3 × 300 µL PBS washes.Before seeding the cells, the dendrimer-activated collagen films were blocked for 1.5 h with 300 µL of 2% (v/v) in deionised water was added to all the samples and incubated at 4 °C overnight. According to manufacturer recommendations, 100 µL of LDH substrate  was made and was added to the samples and left to incubate until a significant colour change was apparent (20–30 min). Absorbance values were read at 490 nm (A490) using a Fluostar Optima plate reader .The number of cells bound to the collagen films were quantified colourimetrically by measuring the amount of lactate dehydrogenase (LDH) released from lysed cells into the media ,24. To lv/v) foetal bovine serum at a density of 1.46 × 104 cells/mL and 2.2 × 104 cells/mL for RSCs and HDFs, respectively. Eight hundred µL of these cell suspensions were incubated on the collagen samples for 72 h in a humidified incubator with 5% CO2 at 37 °C to allow for ~75% confluent proliferation.Dendrimer-activated collagen films were blocked with BSA solution and seeded with cells as for cell attachment analysis, except the cells were resuspended in media containing 10% (w/v) glutaraldehyde directly to the cell media ) for 30 min at room temperature. After the glutaraldehyde solution was removed, the films were washed 3× with sterile deionised water. The cells were stained with 200 µL of 5 µM DAPI  in water. The DAPI was incubated for 10–20 min at room temperature and then removed. The collagen films were washed 3× with sterile deionised water before they were imaged with a Zeiss Axio Observer Z1 microscope fitted with an Axiocam 530 camera. Fluorescent images were taken at 10× magnification and at 358 nm excitation wavelength to detect the DAPI stain. The number of cells per field of view were quantified with CellProfiler .To image the cell number on dendrimer-activated collagen films, the cells were fixed by adding 200 µL of 25% , a 2-way analysis of variance (ANOVA) was still conducted when sample sizes were roughly equivalent. Mean values were compared using 2-way ANOVA with Tukey’s post-hoc tests to determine statistical significance (p ≤ 0.05). Bonferroni comparisons were used for multiple comparisons. The values reported are means of triplicate measures ± standard deviations, unless stated otherwise. Error bars indicate standard deviations from the mean. N/S denotes p ≥ 0.05.The data were analysed for normality and equal variances using Shapiro-Wilk (or Kolmogorov-Smirnov) and Levene’s tests, respectively, with To visualise and quantify the IKAV-capped dendrimers integrated onto collagen substrates, the dendrimers were first conjugated with Atto 565 nm fluorescent probes. The efficiency of Atto dye conjugation was investigated and the dendrimer:dye ratio was optimised. The percent conjugation was defined as the ratio of the molecular weight of the free 16-arm dendrimer  ,15 to thDye-tagged IKVAV-capped dendrimers were incorporated onto collagen substrates using three different methods to yield three different types of collagen surface environments. Dye-tagged IKVAV-capped dendrimers were coated onto (1) non-crosslinked collagen films, (2) pre-crosslinked films that were initially crosslinked with EDC/NHS, and (3) non-crosslinked films that were subsequently crosslinked with EDC/NHS after the addition of dendrimers to yield “post-crosslinked” films. It was thought that crosslinked surfaces would be more hydrophobic given the ablation of exposed free amines (lysine-based) and carboxylates (glutamate and aspartate-based) upon EDC/NHS treatment ,25. Conv3+ groups) than 78% dye-tagged dendrimers, and it was hypothesised that differences between their interactions with the collagen films could be attributed to differences in steric bulk and overall charge.Dendrimers with low extent of Atto 565 nm dye-conjugation (8%) and high extent of dye-conjugation (78%) were investigated in this study. Of the dye-tagged dendrimers, 8% have a lower molecular weight and higher overall charge (fewer dye-consumed NHv/v) in deionised water, 0.1% Tween (v/v) in PBS, saturated saline, and 0.1 M citrate buffer (pH 3). These solvents were chosen to probe different types of chemical interactions that could exist between the dendrimers and the surface of the collagen films. To probe non-ionic interactions, 75% EtOH was used, and saline and PBS solutions, which are salt-based buffers, were used to probe ionic interactions. To probe ionic and non-specific, non-covalent interactions, 0.1% Tween in PBS—a salt-based buffer containing a non-ionic detergent—was used. Citrate buffer (pH 3) was used to probe interactions at an acidic pH. It was thought that by incubating the films in wash buffer for an extended period of time and taking fluorescence measurements of the films at various time points, the relative rates of dendrimer dissociation from the collagen substrates could be compared. These trends would provide insight into how bulky, charged nanomaterials, such as dendrimers, interact with different collagen surface chemistries long-term, and how this could ultimately impact cell binding and proliferation.Long-term interactions between charged, dye-tagged IKVAV-capped dendrimers and non-crosslinked, pre-crosslinked, and post-crosslinked collagen films were probed by studying how dendrimers dissociate from the films when exposed to various solvents. As mentioned in the methods above, the solvents selected were PBS, 75% ethanol (EtOH)  to dye-conjugated dendrimers (565 nm), was initially measured at each time point to ensure that collagen substrate breakdown was not a confounding factor for dendrimer dissociation . After cp ≥ 0.05) unlike the other wash buffers. For the non-crosslinked films activated with 8% dye-tagged dendrimers, none of the wash buffers significantly dissociated dendrimers from the films (p ≥ 0.05). These results suggest that the overall charge on the dendrimers affects how they interact with non-crosslinked collagen substrates over time.p ≤ 0.001), with limited dissociation for the remaining incubation time period. On the other hand, there was no significant dissociation (p ≥ 0.05) of either 8% or 78% dye-tagged IKVAV-capped dendrimers incorporated into post-crosslinked films when washed with any of the buffers. These results provide evidence that dendrimer-collagen interactions in the pre-crosslinked films are easily disturbed with a variety of aqueous solvents, and that dendrimer-collagen interactions in the post-crosslinked films are too strong to be disturbed by aqueous, ionic wash buffers.Unlike the non-crosslinked films, for which dendrimer dissociation was dependent on both the extent of dye-conjugation and also the type of solvent used to dissociate the dendrimers, dendrimer dissociations from pre-crosslinked and post-crosslinked films were unaffected by these parameters. When dendrimer-activated pre-crosslinked films were washed with any of the wash buffers, there was drastic dissociation of both 8% and 78% dye-tagged dendrimers in <2 h   via elecAfter the mode of chemical interaction between the dendrimers and the various collagen films were probed, cellular responses to the various dendrimer-activated collagen films were studied. Rat Schwann cells (RSCs) were used to model how glial cells, which nourish neurites during regrowth , respondv/v) Triton-X 100 in water. As all cells were lysed, the quantity of bound cells could be determined given a linear relationship between LDH detection and cell density [To probe cellular adhesion, a modified LDH assay, as previously reported, was employed ,24. WhilWhile fibroblasts had similar binding affinity to collagen films, irrespective of both the degree of crosslinking and extent of dendrimer activation, RSCs only had low binding affinity to non-crosslinked and crosslinked collagen films with 0.12 µg/mg or no dendrimer activation . Increasp ≥ 0.05) by dendrimer activation. This data supports that dendrimer-activated collagen surface properties affect HDF proliferation, and that a relatively large quantity of untethered dendrimers integrated into pre-crosslinked films provides the desired ablation of fibroblast proliferation.In addition to the cellular binding trends analysed after one day of incubation on the collagen substrates, cellular proliferation was probed after three-day incubation on the collagen surfaces. Cellular proliferation was calculated as a fold-change at day 3 from the initial loading density on day 0. A fold-change of 1 indicated no change in cell density. For non-crosslinked and post-crosslinked films, lower dendrimer quantities (0.12 and 0.36 µg/mg) did not reduce HDF proliferation relative to films without dendrimer activation . DespiteDAPI-stained fluorescent images of HDFs after 3 days in culture were obtained to show the cell distribution . This shRSC proliferation was dependent on the collagen substrate and peptide coating. Intermediate dendrimer coating concentrations  increased RSC attachment to both non-crosslinked and pre-crosslinked films. However, only the highest dendrimer coating concentration (0.72 µg/mg) increased RSC proliferation, relative to non-dendrimer-activated films, for all three collagen substrate varieties . This inAs for HDF proliferation, DAPI-stained fluorescent images of RSCs on the different collagen substrates with various extents of dendrimer activation were obtained . Unlike In this study, IKVAV-capped dendrimers were integrated into three substrate varieties: non-crosslinked collagen films, pre-crosslinked collagen films or initially non-crosslinked collagen films that were crosslinked after dendrimer addition (“post-crosslinked collagen films”). Prior to studying the cellular responses to these three substrate varieties, the chemical interactions between the dendrimers and the collagen films were probed.It was predicted that dendrimers with higher MW and lower charge (78% dye-tagged dendrimers), due to their hydrophobicity and steric bulk, would preferentially associate with similarly hydrophobic, crosslinked collagen substrates. On the other hand, it was anticipated that dendrimers with lower MW and higher charge (8% dye-tagged dendrimers), which are more hydrophilic, and less sterically encumbered, would favour ionic or electrostatic associations with non-crosslinked collagen. However, both 78% and 8% dye-tagged IKVAV-capped dendrimers had higher initial association to post-crosslinked films than non-crosslinked films, even though both substrates were non-crosslinked at the point of dendrimer addition . Unlike While the association studies suggested that dendrimer interactions with non-crosslinked films are dependent on local environments, the dissociation assays elucidated the dendrimer binding mechanisms for pre-crosslinked and post-crosslinked films, in addition to further supporting that dendrimers make ionic associations with non-crosslinked films.p ≤ 0.001) to a significant extent. Ethanol may wash away non-covalently associated dendrimers like 0.1% Tween solutions. In addition, while water molecules stabilise collagen triple helices via hydrogen bonded hydration shells [For dendrimer-coated non-crosslinked films, the salt containing solutions, saline, PBS and 0.1% Tween (in PBS) dissociated a large quantity of 78% dye-conjugated dendrimers . It is ln shells , larger Interestingly, citrate buffer (pH 3) did not significantly dissociate dendrimers from the non-crosslinked films. As an acidic buffer, citrate buffer was expected to increase dissociation of dendrimers from their collagen substrates as negatively charged glutamate (pKa 4.25) and aspartate (pKa 3.65) residues  become pp ≤ 0.05, not denoted in Furthermore, the dissociation assays also suggested that dendrimers are physisorbed onto pre-crosslinked films. Dendrimers, which are sterically bulky, likely entangle into pre-crosslinked substrates via weak non-covalent interactions, as these bioactive motifs quickly and completely dissociate when washed with any of the solvents. The lack of strong chemical interactions also explains why the dissociation of dendrimers from pre-crosslinked films is not dependent on the initial quantity of associated dendrimers . While 0Finally, dendrimers may be more permanently bound to post-crosslinked films than either non-crosslinked or pre-crosslinked films as there was little dissociation for all wash buffers. The detergents used in these dissociation studies targeted ionic, non-ionic, and non-covalent interactions, showing that these are not the only bond types present between the peptide and collagen after EDC/NHS crosslinking. Although covalent interactions cannot be probed directly, as detergents such as SDS that disrupt all non-covalent interactions in proteins can also breakdown collagen films, these results suggest that covalent linkages may form between the peptide and collagen post-crosslinking. Like for non-crosslinked films, these stronger chemical interactions also explain why dissociation was proportionally increased when higher dendrimer quantities were initially integrated into the collagen films. These studies show that while EDC/NHS crosslinked collagen has been shown to have superior mechanical stability relative to non-crosslinked collagen, albeit at the expense of reduced bioactivity ,22,33,34In designing biomaterials for neural applications, it is important to not only consider the response of neurite forming cells, but to also consider the responses of helper glial cells, such as Schwann cells, and inhibitory cells, like scar-forming fibroblasts. In this study, human dermal fibroblast (HDF) and rat Schwann cell (RSC) adhesion and proliferation on various IKVAV-capped dendrimer-activated collagen substrates were studied to better understand how various cell types interact with laminin-derived peptides, such as IKVAV, which have been thought to be promising for neural applications .IKVAVSADR coated onto tissue culture plastic was found to increase fibroblast adhesion at or above the mass of IKVAV used per sample in this study [For biomaterials targeted at neural repair, the response of fibroblasts is extraordinarily important given that scar-forming fibrotic tissue is one of the greatest hinderances to attaining full-functional recovery after injury . The datis study . As showin vivo [Although there was an increase in HDF adhesion to non-crosslinked films with greater dendrimer-activation, this increase was minimal relative to the already high affinity of HDFs to both non-crosslinked and crosslinked collagen films without dendrimer activation. When non-crosslinked collagen films, with or without IKVAV-capped dendrimers, are used, HDFs may bind exposed biochemical motifs on native collagen such as GFOGER, or laminin-derived peptides such as IKVAV, via β1-type cell-surface integrins . This isin vivo  and humain vivo . While tin vivo . HoweverUnlike HDF adhesion, HDF proliferation, as well as both RSC attachment and RSC proliferation (proliferation defined here as fold-change in cell number over 3 days in culture), were affected not only by the surface properties of collagen but also the presence and quantity of IKVAV motifs present. As discussed above, dendrimers can bind ionically/non-specifically, non-covalently, or covalently to non-crosslinked, pre-crosslinked and post-crosslinked films, respectively. Ionic or non-specific interactions, and non-covalent physisorption do not alter the functional groups on IKVAV motifs. However, covalent binding reduces primary amines to amides on lysine or terminal valine residues on IKVAV. Higher RSC adhesion  and prolp ≤ 0.05) and HDF proliferation on non-crosslinked films, relative to pre-crosslinked films, even though both substrate varieties had high quantities of unaltered IKVAV motifs. These lower-than-expected cellular responses may be affected by the decreased stiffness of non-crosslinked surfaces [When lower dendrimer quantities were used, there was lower than expected RSC attachment  was incorporated into pre-EDC/NHS crosslinked collagen films, rat Schwann cell attachment and proliferation were promoted while human dermal fibroblast proliferation was inhibited, as desired. These results show the potential of IKVAV-capped dendrimer-activated collagen biomaterials for neural applications since glial Schwann cells help neurite organisation and reduced fibroblast activity can limit detrimental fibrotic scarring at peripheral nerve injury sites. The different cellular responses to pre-crosslinked and post-crosslinked films also highlighted the importance of having fully exposed, non-covalently hindered biochemical motifs in attracting the desired cell type.


Polyamide 46 (PA46) is used in various automotive parts because of its excellent heat resistance and mechanical properties. This study aims to improve the frictional properties of PA46 using the lubricating ability of graphene. Nanocomposites are prepared via two mixing methods: Graphene powder is compounded directly with PA46 pellets through a twin-screw extruder, or PA46 powder is added to graphene dispersion for self-adsorption, and subsequently, it is dried and compounded with PA46 through the twin-screw extruder. Application of the nanocomposite in the friction field is evaluated via the pin-on-disk method. The coefficient of friction of the nanocomposite prepared by self-adsorption is lower than that of the nanocomposite prepared by direct compounding. The mechanical properties of the nanocomposite fabricated by self-adsorption are superior to those of other materials. This can be attributed to the uniform dispersion of graphene and the strong attractive force between the PA46 matrix and graphene. Polyamide (PA) is used in the automotive industry as a representative engineering plastic. Polyamide 46 (PA46) exhibits higher crystallinity, melting temperature, and thermal stability than the commonly used polyamide 6 (PA6) and polyamide 66 (PA66) because of its high amide content per unit length chain length and its symmetrical chain structure. Further, PA46 exhibits excellent wear properties because of its high crystallinity, which incentivizes its application in highly heat-resistant frictional parts, such as valves and gears ,2,3. RecGraphene is a two-dimensional single layer of graphite, and it is an emerging two-dimensional carbon-based nanomaterial. Graphene exhibits excellent thermal properties, electrical conductivity, mechanical strength, moisture barrier properties, and wear resistance. Consequently, graphene-polymer nanocomposites are expected to have numerous potential applications ,6,7,8,9.In this study, graphene was composited in various ways to improve the wear resistance of PA46. A chemically modified graphene (CMG) dispersion was used to increase the dispersibility of graphene, and composite particles were prepared and processed using PA46 nanoparticles and by exploiting self-adsorption behavior. For comparison, a composite was prepared by directly compounding graphene powder with PA46 pellets. The frictional properties were compared and analyzed based on the manufacturing method.® TW341 grade from DSM  was used to produce PA46.Graphene powders of BGF-H grade  and BGF-R grade  were provided by BESTGRAPHENE . For the production of functionalized graphene, 150 μm grade graphite flakes purchased from Graphene Supermarket were used. Stanyl2SO4/H3PO4 (3600 mL:400 mL) and KMNO4  at a 9:1 ratio and stirred for 12 h. The reaction mixture was cooled to room temperature (23 °C) and diluted by immersing it in ice water (4000 mL) containing 30% H2O2 (3 mL) after the reaction was completed. The non-oxidized graphite was removed by filtration, whereafter the oxidized graphite was precipitated by centrifugation at 4000 rpm for 2 h. The precipitate was washed a minimum of two times in a mixed solution of DI water (2000 mL), 30% HCl (2000 mL), and ethanol (2000 mL), and subsequently, it was freeze-dried to obtain fine non-agglomerated particles. Distilled water (2000 mL) and oxidized graphite (2 g) were mixed and sonicated for a minimum of 4 h using an ultrasonic grinder to obtain an aqueous solution of GO.Chemically modified graphene (CMG) was designed to substitute -OH, -COOH, and epoxide groups of graphene oxide with -CN, amide, and amine through functionalization to enable molecular structural interaction with PA46. An improved, recently developed, and widely used method was used to produce graphene oxide (GO) . At 50 °+) of the substituted 4,4-oxidianiline generated an electrostatic repulsive force between the graphene monolayers to yield a chemically modified graphene colloid with high dispersibility.A graphene surface-modification reaction was performed using 4,4-oxidianiline to obtain a chemically modified graphene colloid that can undergo physicochemical interactions with PA46. A 500 mL aqueous solution of 0.1% GO was prepared by sonicating for a minimum of 2 h using an ultrasonic homogenizer. The aforementioned solution was mixed with a solution of 20 g of 4,4-oxidianiline in 500 mL of N,N-dimethylformamide (DMF). This mixture was stirred at 90 °C for 40 h. Thereafter, the mixture was cooled to room temperature (23 °C), a precipitate was separated by centrifugation, washed three to five times with acetone, and filtered to obtain graphene. The as-obtained graphene was immediately immersed in ethanol, and the positive charge (-N50 = 385 μm) PA46 powder was mixed with a graphene dispersion, precipitated through chemical self-adsorption, and dried  to prepare a master batch . The MB was injected into the main feeder of the twin-screw extruder with PA46 pellets. The graphene content was adjusted to 0.05, 0.10, and 0.15 wt% by adjusting the ratio of 0.5 wt% MB and PA46 pellets (sample name S05). The mixing ratios are listed in Nanocomposites were fabricated using two methods. In the first method, PA46 pellets were injected into the main feeder of a twin-screw extruder , graphene powder was injected into the side feeder, and the two feeds were compounded at 300–330 °C. Samples with graphene powder  contents of 0.05, 0.1, and 0.2 wt%  were prepared. In the second method, finely ground , transmission electron microscopy , zeta potentials measurement , and FT-IR . A pin-on-disc tester  was used to determine the frictional properties of the material. The testing was conducted at 25 °C, a motor speed of 400 RPM, a test load of 10 N, and by using a stainless-steel ball of 6 mm diameter as the pin. Following the frictional evaluation, the frictional surface was analyzed using an optical microscope . The tensile strength, flexural strength, and flexural modulus were measured using a universal testing machine .−1 are assigned to the amide vibrations. Peaks between 3330 and 3300 cm−1 were attributed to the secondary amide group. The C-N stretching vibration gave rise to bands in the region 1200–1100 cm−1.−1) and G band (near 1580 cm−1) peaks of graphene can be identified [−1) was identified in the spectrum of CMG that underwent reduction and surface-functionalization. The D/G ratio decreased to 0.927 for CMG compared to 1.049 for GO. The smaller D/G ratio of CMG than that of GO indicates that reduction and functionalization decrease defects in the CMG structure, which increases crystallinity. This suggests the formation of a crystalline structure that possesses the functionality of graphene. Since the 2D peak near 2700 cm−1 is caused by the double resonance of a phonon with the energy of 1350 cm−1, the particular peak appears around 2700 cm−1, which is double the energy of 1350 cm−1. In the case of multilayered graphene, various scattering processes take place because of the higher number of energy bands, and this increases the number of 2D peaks and thereby leads to a broad 2D band [The structural characteristics of the as-synthesized graphene oxide and CMG were identified through their Raman spectra. In the Raman spectrum of graphene, a D peak is caused by defects within the crystal and the edge of the sample, whereas a G peak is attributed to the vibration of the hexagonal crystal structure composed of sp2 carbon atoms commonly seen in graphite-based materials. entified . The 2D  2D band . As showThe number of layers of graphene is the main parameter in basic research conducted to improve frictional properties using graphene. Interactions between graphene layers occur through van der Waals forces. The mechanical properties of graphene change significantly as the number of graphene layers increases because van der Waals forces have low bonding strength compared to the covalent bonds connecting carbon atoms in individual planes . PreviouThe effect of the friction coefficient can be examined from The friction coefficient of the S05 sample compounded through the self-adsorbing powder was smaller than that of the GH and GR conditions directly compounded with the polymer matrix, as shown in The uniform dispersion of the nanoadditives and the attractive force between graphene and the matrix are critical to the mechanical properties of nanocomposites. The frictional and mechanical properties of graphene-reinforced polyamide 46 were investigated. The uniform dispersion of the nanoadditives and attractive force between the graphene and matrix are critical to the frictional and mechanical properties of nanocomposites. The dispersibility and compatibility with the matrix were higher when the graphene was self-adsorbed onto the PA46 surface. Thereafter, it was subjected to secondary melt processing when the graphene powder was directly compounded. Thus, the friction coefficient decreased by more than 50%. The findings of this study may guide the application of PA46 composite materials in friction-driving parts to reduce the weight of automotives.


For expanding applications of spongy graphene aerogels (GAs) cost-effectively, we report a marriage of the two-step hydrothermal reduction and atmospheric drying method to fabricate a spongy CNC-graphene aerogel (CNG) with oil/water selectivity and tunable mechanical strength by a low-cost and straightforward approach. The reduced graphene oxide (rGO) with CNC by the ice-templated method can give rise to forming the hierarchical structure of hybrid GAs within the PUS network. Meanwhile, the fractured structure of PUS with a pre-compressive step arouses more versatility and durability, involving its selective and high-volume absorbability (up to 143%). The enhanced elastic modulus and more significant swelling effect than pure sponge materials give it a high potential for durable wastewater treatment. Assembling graphene to a three-dimensional 3D) porous monolith while keeping the intrinsic advantage of the building blocks is of great promise to achieve the practical application ,2. CelluD porous Additionally, 3D graphene/cellulose monoliths utilizing APD showed excellent hydrophobicity and elasticity, emerging as promising large-scale and low-cost absorbent materials in water treatment ,12,13. XHerein, porous cellulose graphene aerogel (CNG) was prepared using a two-step reduction process without lyophilization, where the cellular walls of aerogels enclosed by macroscopic-scale sponge porous scaffold led to a primarily enhanced mechanical performance. Even when the impact load with maximum (99%) strain is applied, the CNG exhibits resilience capacity without fracture and degradation. In addition, the cooperation of GAs and sponge porous cell walls endows a water droplet “sticky” hydrophobicity, which can absorb n-heptane over 1.3 times its volume and 120 times its weight and reuse manually about 1000 times. More importantly, these graphene sponges display a noticeable swelling effect, which means that adsorbed oil extended storage in more volume space of the nanocomposite. The CNG enabled a combination of visible polymer framework and microscale graphene scaffold, which can be successfully used for efficient and cost-effective water purification. These properties also demonstrate that, if graphene sheets are assembled into polymer skeletons where graphene nanosheets are kept, novel and combined properties will be observed.http://sciencek.com/, accessed on January 2018) and its length nanoscale is approximately 100 to 200 nm size range, with the length–diameter ratio being 10–20. Graphite powder (CP), L-Ascorbic acid (AR), sulfuric acid (98% ), sodium hydroxide (AR), potassium permanganate (AR), and hydrogen peroxide (AR) were supplied by Shanghai Aladdin Bio-Chem (China) and used without further purification. Graphene oxide as a raw material was synthesized according to a modified Hummer’s method [The recycled PUS samples were obtained from Shenzhen Tenghuida Electronic auxiliary material Co., Ltd. The Cellulose Nanocrystals (CNCs) were obtained from ScienceK (s method . The radCNG was characterized with a Nicolet 6700 Fourier transform infrared spectroscopy (FT-IR). Scanning electron microscopy (SEM) images were obtained by a Hitachi SU8010 at 20 kV. The X-ray diffraction (XRD) analysis is done with an X-ray source of CuKA high efficient and energy-saving wet shaping method under atmospheric pressure was demonstrated in this work. The typical fabrication procedure was depicted in Moreover, these steps towards a more facile and green route, CNC/GA as an essential building block, are assembled into the 3D architecture of PUS. When the intermediate is frozen, GO nanosheets are expelled by the grown ice crystals, also known as ice templating, and for the formation of the micro-pore network in each sponge nest. The freezing temperature and the amount of oxygen-containing groups of GO are adjusted via reduction time . These eame time . The uniThe XRD diffraction patterns of CNC, the prepared GO and the rGO/PUS and after the CNC adding (CNG) are presented in s nature . After aarticles . These cTypical architectures of CNG sponge were prepared by the hydrothermal reduction and frozen by dry ice, as shown in Comparable results were obtained when different concentrations of the dispersed GO solution were tested. Previous research has shown the mechanical properties of the pre-treated sponge relied mainly on the skeleton contact area and being verified that the fractured sponge network shows an increase of the contact area with the compressive stress in contrast to the network without pre-compression treatment . Aside fIn contrast to what is observed in the CNG specimens, the micropores structure strongly affects the stress–strain response without a plateau stress region such as deformation over the above strain region. This phenomenon can be attributed to the high modulus of whisker-like CNCs nanoparticles and changes induced by the dual network, which combined polymer networks and graphene cellular porosity. The previous research also revealed that the compressive yield stress of porous graphene materials increases linearly with an increase in porosity . In ordeOwing to the hydrophobic porous structure and ultra-lightweight, the oil adsorption capacity  of cellulose/graphene aerogels up to 198 times their weight . PristinIn particular, the volume ratio of absorbed liquid is much higher than lightweight aerogel and other graphene-based foams as shown in In summary, spongy CNC/graphene aerogels have been successfully prepared via an ambient-drying method with a pre-compaction treatment. The corresponding hybrid structure allows CNGs to have unique compressive strength to afford compression cycling numbers and maintain a linearly mechanical property. Moreover, optimizing the freezing and drying process and controlling the internal microstructure with hierarchical pores simultaneously led to a high volumic oil-absorption capacity (up to 142%). The composite developed here also demonstrated a swelling effect with easy recyclability. Significantly, this energy saving synthesis process would be a key progress for realizing large-scale commercial production, and this graphene-based hybrid material provided access to assembling a monolith by embedding porous structures of graphene aerogel inside another porosint. The networks’ interactions changed the material’s bulk properties to find broader promising applications for graphene aerogels, including pressure sensors and petroleum contamination remediation.


Aberrant activation of Wnt/β-catenin signaling and downstream β-catenin-TCF target genes is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin-like cell adhesion receptor L1CAM (L1) as a target of β-catenin-TCF transactivation in CRC cells. Overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis, and liver metastasis, and L1 is exclusively localized at invasive areas of human CRC tissue. Several genes are induced after L1 transfection into CRC cells by a mechanism involving the L1-ezrin-NF-κB pathway. We conducted a secretomic analysis of the proteins in the culture medium of L1-overexpressing CRC cells. We detected a highly increased level of biglycan, a small leucine-rich ECM component, and a signaling molecule. We found that induction of biglycan is required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis, and liver metastasis. The suppression of endogenous biglycan levels or a point mutation in the L1 ectodomain that regulates cell–cell adhesion mediated by L1 blocked the enhanced tumorigenic properties conferred by L1. The mechanism of biglycan induction by L1 involves the L1-NF-κB pathway. Blocking NF-κB signaling in L1 expressing cells suppressed the induction of biglycan and the tumorigenic properties conferred by L1. Biglycan expression was undetectable in the normal colonic mucosa, but expressed at highly increased levels in the tumor tissue, especially in the stroma. The therapeutic strategies to target biglycan expression might provide a useful approach for CRC treatment in L1-overexpressing tumors. Aberrant activation of the Wnt/β-catenin pathway and its downstream β-catenin-T cell factor (TCF) target genes is a characteristic feature of colorectal cancer (CRC) development . Among sTo identify proteins secreted by L1-expressing CRC cells whose levels are increased after L1 transfection, we performed mass spectrometry analysis on proteins in the secretome of LS 174T L1-expressing and control LS 174T cells. As shown in To examine the role(s) of changes in biglycan expression on the growth and motile properties of L1-expressing CRC cells, we isolated biglycan-overexpressing CRC cell clones A and L1-We wished to determine whether the changes in CRC cell proliferation and motility observed in cultured CRC cells in which biglycan levels were modulated can also be detected in mouse models in vivo. CRC cell clones expressing L1 and CRC cells where the endogenous levels of biglycan are suppressed see B were inPrevious studies have shown that the tumorigenic properties conferred by L1 expression in CRC cells require an intact L1 ectodomain that mediates cell-cell adhesion  and invoWe wished to determine the localization of biglycan in human CRC tissue. We used 38 paraffin-embedded human CRC tissue samples and immune-stained them with anti-biglycan antibodies. The results summarized in The studies described above demonstrated that we have identified biglycan among the proteins whose levels are highest in the secretome of L1-expressing CRC cells and that the induction of biglycan by L1 is required for CRC progression. We found that suppressing the increase in biglycan in L1-transfected CRC cells blocks the multiple pro-tumorigenic properties conferred by L1 overexpression in CRC cells, including increased proliferation under stress, cell motility, tumorigenesis and liver metastasis in mouse models. Biglycan is a small leucine-rich proteoglycan (SLRP) and is mainly known for its role in the ECM and infiltrating immune cells in the stroma ,19, whicA unique property of L1 expression in CRC cells is that L1 is induced only in invasive CRC cells at the tumor edge but not in the homeostatic colonic mucosa, or even in adenocarcinoma tissue of the colon . FurtherBy what mechanism/s is increased biglycan expression in L1-expressing CRC cells contributing to CRC progression? A wide variety of mechanisms have been implicated in biglycan-mediated tumor progression, including upregulation of VEGF ,28, the Taken together, the targeting of biglycan expression in L1-expressing CRC cells promises to be a useful strategy to detect novel therapies of CRC in invasive CRC cells that are positive for L1.The LS 174T cell line was cultured in RPMI-1640  supplemented with 10% FBS  and 1% penicillin/streptomycin solution . LS 174T-L1, LS 174T+biglycan, LS 174T-L1/D598N, and LS 174T-L1/H210Q cells were cultured in RPMI-1640 medium with neomycin (800 µg/mL) .Transfection of LS174T cells was performed using the Xfect™ transfection reagent , according to the manufacturer’s instructions. For cell proliferation assays, 2500 cells were seeded in 12-well plates containing RPMI-1640 with 0.1% FBS, and the proliferation rate was assessed by cell counting over a period of 5 days. Cell motility was determined by the artificial “scratch wound” closure assay as described [The biglycan cDNA expression vector was obtained from Dr. Liliana Schaefer  . pSUPER.The following antibodies were used for immunoblotting: rabbit anti-L1  at 1:2000 dilution, mouse anti-biglycan, SC-100857  at 1:1000 dilution, rabbit anti-phospho-IκBα #2859  at 1:1000 dilution, rabbit anti-NF-κB p65, sc-109  at 1:1000 dilution, mouse anti-β-tubulin  at 1:5000 dilution. RIPA lysis buffer supplemented with 1% complete protease inhibitor cocktail was used to prepare cell lysates as described [Immunofluorescence was performed on cells grown on glass coverslips. Cells were permeabilized with 0.5% Triton X-100, fixed with 4% paraformaldehyde , incubatTotal RNA was isolated from cells using the Bio-Tri reagent  according to the manufacturer’s protocol. Synthesis of first-strand cDNA was carried out using SuperScript™ II Reverse Transcriptase  according to the manufacturer’s protocols. The primers used for amplification are described in 6 cells in 100 μL PBS were injected at different sites in the flanks of male nude mice [6 cells in 20 μL PBS into the distal tip of the spleen of 4–5-week-old male nude mice as described [Subcutaneous tumor growth was induced as previously described ,30. Brieude mice . Mice weescribed . Mice weThe Weizmann Institutional Animal Care and Use (IACUC) ethics committee reviewed, approved, and supervised the animal studies.Immunohistochemistry was performed on 38 paraffin-embedded human colorectal adenocarcinomas using a rabbit polyclonal anti-biglycan antibody , at a dilution of 1:650, as previously described .t-test. A p-value of <0.05 was considered significant and marked by an asterisk.Statistical analysis was performed on SigmaPlot v11 software and the significance of results was calculated using the student’s non-paired


Hydrogel-based bio-inks have been extensively used for developing three-dimensional (3D) printed biomaterials for biomedical applications. However, poor mechanical performance and the inability to conduct electricity limit their application as wearable sensors. In this work, we formulate a novel, 3D printable electro-conductive hydrogel consisting of silicate nanosheets (Laponite), graphene oxide, and alginate. The result generated a stretchable, soft, but durable electro-conductive material suitable for utilization as a novel electro-conductive bio-ink for the extrusion printing of different biomedical platforms, including flexible electronics, tissue engineering, and drug delivery. A series of tensile tests were performed on the material, indicating excellent stability under significant stretching and bending without any conductive or mechanical failures. Rheological characterization revealed that the addition of Laponite enhanced the hydrogel’s mechanical properties, including stiffness, shear-thinning, and stretchability. We also illustrate the reproducibility and flexibility of our fabrication process by extrusion printing various patterns with different fiber diameters. Developing an electro-conductive bio-ink with favorable mechanical and electrical properties offers a new platform for advanced tissue engineering. Engineered materials that can imitate native tissue properties are promising tools for treating a wide variety of clinical problems in patients and for use in research labs. In particular, hydrogels engineered to be conductive can be used to produce flexible, biocompatible sensors for the in vivo recording of bio-signals and biomolecules, or in tissue engineering applications . HoweverHydrogel-based bio-inks should be flexible, physically robust, and capable of withstanding the host tissue’s physical activity and movement. Additionally, the hydrogel must have suitable electro-conductive properties for conducting currents generated by in vivo sensors or by the living tissue itself . Hydrogels with shear-thinning properties are good candidates for use as inks with applications in 3D printing of tissue and soft electronics . This isSome electro-conductive materials that have been used for engineering bio-inks include metals such as gold , copper operties . Graphenoperties .The use of hydrogel-based electroconductive bio-inks has the advantage of offering a soft substrate at the interface between the electronic circuit and the tissue, a feature that overcomes the issues associated with a mechanical mismatch between current flexible electronic systems and soft tissue. An ideal bio-ink needs to be printed on a substrate that is flexible, durable, and is not cytotoxic. However, providing a stretchable, durable, and cost-effective method remains a constant challenge . VariousIn the present work, we report a novel silicate-based conductive bio-ink composed of alginate, Laponite, and rGo. This material composition provides a printable and flexible hydrogel with low electrical resistance that can be embedded into a multilayered hydrogel bed using a multistep process. The properties of the novel bio-ink, including its structural morphology, rheological characteristics, and conductivity under different physical conditions, are analyzed. Employing rGo in the structure of the bio-ink resulted in the enhanced electro-conductivity of the final hydrogel. The shear-thinning properties, electro-conductivity, and biocompatibility of this hydrogel can be used for engineering a three-dimensional network that can mimic the extracellular matrix (ECM) structures by 3D printing a desired form . The othPartially exfoliated graphite crystals were used in this research. The exfoliation process involves expanding the graphite platelets up to hundreds of times in order to eliminate aggregation and improve the lubricity and flexibility of this material . Partialin water ,52. Thisin water .w/v) alginate and vortexing the solution to dissolve all of the particles, 6% (w/v) Laponite was added to the 4 °C solutions of alginate/graphene oxide and vortexed vigorously for 5 min to form a final gel, as shown in In this study, the aqueous suspension of graphene oxide was prepared by, first, dispersing 5 and 3 mg/mL graphene oxide in deionized water and then sonicating the dispersion for 5 min to form a uniform colloid, as shown in Zeta potential analysis of the prepared hydrogels provides insight when discussing the electrostatic interactions of the nanocomposite hydrogels. According to the literature, the zeta potential of the Laponite is consistently negative in both the acidic and the basic pH range . The zetw/v)% of gel) is a minor component of the current nanocomposite hydrogel, some of the alginate’s characteristic peaks overlap with the intense peaks of the Laponite. The characteristic peak of the Laponite is observed at 1005  spectra . As showectively . Additio spectra . Furthera [cm−1,  while th1, [cm−1 . Since atructure . MoreoveLaponite . SimilarOH bands . This peThe XRD analysis was carried out to prove the presence of graphene oxide nanosheets in the structure of the dried Laponite/alginate hydrogel. As depicted in The unique isomorphic substitution of Laponite can enhance the shear-thinning properties, plastic behaviors, and the high elastic response of the hydrogel composites . SpecifiThe electrostatic interactions between Laponite and graphene oxide lead to a stable colloid . LaponitLaponite has been reported to improve the shear-thinning properties of alginate . In ordeAs observed in The shear-thinning properties of the different formulations of the bio-ink can also be determined by measuring the viscosity under constant shear. The viscosity increased with the addition of 3 mg/mL graphene oxide to the alginate/Laponite bio-inks. This increase in viscosity was attributed to the electrostatic interactions between the negatively charged graphene oxide and the positively charged Laponite, as previously mentioned. As shown in In order to characterize the mechanical integrity of the bio-ink upon printing, the recovery of the nanocomposite hydrogel was measured from high (100% oscillatory strain) to low strain (1% oscillatory strain) conditions. Previously, Laponite and gelatin showed a rapid elastic recovery in less than 10 s . The stoAs previously mentioned, printing is gaining popularity for fabricating biomimetic scaffolds for tissue engineering and drug screening; however, developing a biocompatible bio-ink remains a challenge . ParticuOnly a few existing bio-inks are electro-conductive, and even those are only slightly electro-conductive . Metalliw/v), 3% (w/v) Laponite, and 0, 3, and 5 mg/mL of graphene oxide, respectively. These bio-inks were unsuitable for extrusion printing due to the low mechanical integrity of the stacked layer, or their inability to form consistent and cylindrical filaments following extrusion.w/v) Laponite generated cylindrical filaments upon extrusion and provided the desired mechanical integrity for the stacked layer, which hinders the deformation of the pattern. w/v) Laponite disrupted extrusion, while employing 3% (w/v) Laponite resulted in droplet formation and the deformation of the stacked-layer after printing.In w/v) alginate, 6% (w/v) Laponite, and 0, 3, and 5 mg/mL graphene oxide, respectively, were chosen as the optimum formulations of the electro-conductive bio-ink. The feeding pressure and moving nozzle speed are determinative parameters for controlling the printability, filament formation consistency, and fiber diameter. w/v) alginate, 3% (w/v) Laponite, and 0, 3, and 5 mg/mL of graphene oxide, respectively. These formulations were unsuitable for extrusion printing due to their low mechanical integrity, the deformation of the stacked-layer, or the formation of a droplet instead of the desired cylindrical filaments following extrusion, as shown in w/v) w/v) alginate, 6 (w/v) Laponite and 0 (i), 3 (ii), and 5 (iii) mg/mL graphene oxide, respectively, show the printability of different formulations of the bio-ink  agarose solution was added to 4% (w/v) of w/v) alginate solution was added to the top of the agarose sheet, as shown in w/v) with 4% (w/v) of In order to fabricate the alginate substrate, 1.5% , graphene oxide, gelatin from porcine skin , agarose, and calcium chloride were all obtained from Sigma-Aldrich, St. Louis, Missouri. Laponite XLG, a synthetic nanosilicate, was acquired from BYK Additives, Germany.w/v) alginate, 6% (w/v) Laponite, and 5 mg/mL graphene oxide were prepared by, first, dispersing the graphene oxide in deionized water. The suspension was then sonicated for 5 min, alginate was dissolved in the graphene oxide suspension, and the solution was cooled to 5 ℃. Laponite was added to the solution and immediately vortexed for 5 min to ensure the homogenous distribution of Laponite nanoplatelet in the solution. In order to prepare different formulations of the bio-ink, different concentrations of the graphene oxide and Laponite were added to the solution, and the rest of the procedure was followed as explained above. A list of the different formulations of the bio-ink is given in Preparation of Bio-inks: A total of 0.5% (w/v) alginate, 6% (w/v) Laponite, and 0, 3, 6% (w/v) graphene oxide, respectively, were diluted to 0.2% (w/v) and sonicated for 5 min to ensure a uniform distribution of graphene oxide and Laponite. This test was conducted to study the effect of the introduction of different concentrations of graphene oxide on the zeta potential of the bio-ink.Zeta Potential: Zeta potential tests were conducted using a Brookhaven BI-ZR3 Zeta Potential Analyzer with a 600 nm wavelength laser at room temperature. Samples with 0.5% (Fourier Transform Infrared Spectroscopy (FTIR): The chemical structure of Laponite/alginate with various concentrations of graphene oxide was analyzed by a PerkinElmer Spectrum Two Fourier transform infrared spectrometer (USA) to obtain FTIR spectra by a potassium bromide (KBr) technique. Samples were firstly freeze-dried and then ground into powder and mixed with KBr to collect the spectra and determine the interactions between the components within the range of 400–4000 X-ray Diffraction (XRD): Firstly, samples were freeze-dried and ground into a powder. The crystal structure of the bio-ink with different concentrations of graphene oxide was analyzed using a PANalytical Empyrean X-ray Diffractometer at room temperature with a current of 40 mA and a voltage of 45 kV over the diffraction angle range of 10°–90° (2θ).w/v) alginate and 6% (w/v) Laponite.Scanning Electron Microscopy (SEM): SEM images were acquired using the Hitachi S-4800 FESEM microscope at an accelerating voltage of 1 kV. Samples were freeze-dried by storing them in a −80 °C freezer for 24 hr, lyophilized for 2 days, tapped to the SEM stubs, and then coated with gold–palladium. SEM images were employed to investigate the microstructural differences between bio-ink formulations with various concentrations of graphene oxide , with 0.5%  were measured using an Anton Paar MCR 502 rheometer  over shear rate sweeps of 0.001 to 10 Hz at room temperature. Viscosity was measured under a constant shear rate over 300 s, and stress recovery measurements were conducted at 1 Hz by alternating the application of 100% strain and 1% strain for 5-min intervals on the bulk bio-inks.w/v) Laponite, 5 mg/mL graphene oxide, and 0.5% (w/v) alginate. The electro-conductive bio-inks with alginate 0.5% (w/v), Laponite 6% (w/v), graphene oxide 5 mg/mL, and 2% (w/v) 3D printing Process: All 3D printing experiments for the present work were conducted employing a Cellink Inkredible+  microextrusion 3D printer with pneumatic pressure for extruding materials. Repetier and Slic3r software were used to manually generate codes for the 3D printing of rectangular patterns. In order to print serpentine and spiral patterns, AutoCAD was employed to develop the drawings and Cura was used to develop the final codes for 3D printing. Different formulations were used to study the printability of the bio-inks B. In ord5 mm see G. Besidew/v) alginate was chosen to develop a flexible substrate for printing an electrically conductive pattern. The first step to develop a flexible printed pattern was spreading the 4% (w/v) alginate solution in a 100 mm Petri dish and crosslinking it with 4% (w/v) w/v) agarose solution was prepared by suspending 1.5 g agarose powder in 100 mL deionized water and dissolving at 70 °C to form a clear solution. A 4% (w/v) w/v) alginate was spread to the same Petri dish at the top of the agarose sheet. The agarose sheet containing 4% (w/v) 3D Bioprinting on Alginate Substrate: A 4% (w/v) agarose sheet containing 4% (w/v) w/v) alginate solution to the agarose sheet for 20 min, 2 mm of the alginate solution was crosslinked to provide sufficient support and to maintain the printed pattern on the alginate sheet. Partially crosslinking the alginate solution is necessary for forming layers of alginate sheets from the bottom to the top of the solution. The layers of alginate sheets tolerate the weight of the bio-ink and prevent any deformations of the bio-ink after printing on alginate and eliminate sinking the pattern to unwanted levels of alginate substrate. After printing the pattern, the sample was crosslinked for 20 more minutes so to be fully crosslinked. During the next 20 min, the printed patterns and the alginate sheet were crosslinked in parallel.After preparing the alginate sheet, developing the printing codes, and choosing an optimum formulation of the bio-ink, different patterns were printed on the substrate. The most significant challenge in this process was trapping the printed pattern inside a half-crosslinked solution to provide a strong attachment between the printed patterns to the alginate sheet. Therefore, the prolonged crosslinking method played a significant role in providing control over the crosslinking procedure. The various volumes of alginate solution were prepared and crosslinked with a 1.5%  length and glued to sandpaper from two ends, and then the sandpapers were fixed inside two grips. This method successfully prevents any undesired ruptures in the prepared samples as a result of direct contact with the alginate sheets and the grips during the measurement. For the cyclic tensile test, the samples were stretched with a linear velocity of 1 mm/min and the test was repeated for 7 cycles with a maximum strain of 10%. The electrical conductivity of the printed pattern on the alginate sheets was measured after each cycle and during the first cycle using a multimeter.2 incubator at 37 °C. Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS (Invitrogen 16000), 100 IU/mL of penicillin, and 100 mg/mL of streptomycin was used as the cell media and was changed every other day. Cells were cultured up to 90% confluency. Afterwards, the U251 cells were detached into a single cell suspension followed by centrifuging and diluting, and ready for counting using a hemocytometer to obtain a certain number of cells.Cell culture: U251 Glioblastoma cell lines were cultured in T-25 flasks in a 5% COv/v of fetal bovine serum at 37 °C, in 5% CO2 and fully humidified air for 1, 3, 7 days, respectively. The liquid extraction medium (500 µL each) was collected for the PB assay at each time interval and was substituted with the fresh DMEM + FBS solution. Then, 200 µL of each periodically collected liquid extract was added to each well and incubated for 24 h. Finally, the cell metabolic activity was determined using a PB cell viability reagent . PB reagent was added to each well at 10% concentration of the total well volume, and the plate was incubated for 15 min. Excitation and emission wavelengths of 560 and 590 nm were used to measure the fluorescent intensity of the incubated cells. Subsequently, the viability of the samples was calculated using the following equation:Cell viability assay: To measure the cell viability of the fabricated hydrogels with different GO concentrations, 96-well plates  were seeded with 10,000 cells per well and cultured in 0.2 mL of media for 24 h. Presto blue (PB) assay was used as a standard method for rapidly evaluating the viability and proliferation of a wide range of cell types by measuring the metabolic activity of the live cells. An indirect extraction method was used to assess the cell viability of the experimental samples. Equal weights (0.3 g) of GO-incorporated hydrogels in triplicate were incubated in 2 mL DMEM with 10% Cell Viability: (Average fluorescent density of the samples/Average fluorescent density of the control) ∗ 100.