The side electrode structure is designed to reduce the mode loss induced by the graphene film and by heating the electrode. The graphene layer is designed to be located on the waveguide to assist in the conduction of heat produced by the electrode. The inverted ridge core is fabricated by etching and spin-coating processes, which can realize the flat surface waveguide. This core improves the transfer of the graphene layer and the compatibility of the fabrication processes. Because of the opposite thermal optical coefficient of polymer and silica and the high thermal conductivity of the graphene layer, the 3D hybrid TO switch with low power consumption and fast response time is obtained. Compared with the traditional TO switch without graphene film, the power consumption of the proposed TO switch is reduced by 41.43% at the wavelength of 1550 nm, width of the core layer (a) of 3 μm, and electrode distance (d) of 4 μm. The rise and fall times of the proposed TO switch are simulated to be 64.5 μs and 175 μs with a  Three-dimensional 3D) hybrid integrated chips have the advantages of a high integration degree of photonic chips, improvement and diversification in device function, and low signal crosstalk; consequently, these chips can be applied to optoelectronic integration fields and 3D integration fields ,2. A varD hybrid A key goal of 3D hybrid integrated optical devices is to realize the optical path switching between different layers. Therefore, the X-junction vertical structure optical switch has been proposed ,16,17. ITo further reduce the power consumption of the vertical optical devices, the polymer/silica hybrid MZI TO switches with a graphene film have attracted attention. Two-dimensional graphene material has many excellent optical properties such as large thermal conductivity, high carrier mobility, and wide optical response spectrum . TherefoIn this paper, a 2 × 2 MZI-inverted ridge TO switch of a graphene–polymer–silica hybrid waveguide with side electrode structure is proposed. The waveguide grooves are designed to be fabricated by the etching process. Then, the polymer core layer was obtained by polymer spin-coating in grooves. This process can realize the compatibility of silica and polymer materials and the flat surface waveguide, thus helping in the transfer of the graphene film. Because of the high thermal conductivity of the graphene layer and the opposite thermal optical coefficient of polymer and silica, the 3D-inverted ridge TO switch of the graphene–polymer–silica hybrid waveguide with low power consumption and fast response time can be obtained.The 3D structure of the MZI-inverted ridge 3D hybrid TO switch with a graphene film is shown in a = b = 3 μm. The parameters of m = 6 μm, eL = 1 cm, and 2d = 0.6 μm indicate the width of the electrode, length of the electrode, and coupling gap between two waveguide core layers, respectively. The thickness of the slab layer is h = 1.22 μm. The coupling length of the waveguide in the vertical direction is cL. The horizontal distance between the center of the core layer and the center of the electrode is d. cn1 = 1.48, cn2 = 1.478, and n1 = 1.444 are the refractive indices of the silica core layer, polymer core layer, and silica cladding layer, respectively. When the effective refractive index of the two core layers is equal, the optical field can be theoretically fully coupled in a 3-dB coupler. When heating the electrode, the effective refractive index of the waveguide changed, realizing the phase change of π.The dimensions of the core layers are −4/°C and 1.28 × 10−5/°C. The phase change ∆Φ can be expressed as:neff is the effective refractive index change, and λ is the wavelength of waveguide (set at 1550 nm). Because of the large thermal conductivity of a graphene layer , the graphene layer is coated on the waveguide to assist the conduction of heat produced by the electrode. This process helps realize the phase shift at a lower power consumption (P) of the 3D hybrid TO switch.The thermo-optical coefficients of the polymer and silica are −2.36 × 102n is the refractive index of the polymer cladding layer. To realize the complete coupling of light field, the structure of the waveguide has been optimized . a = b = 3 μm; c1n = 1.48; cn2 = 1.478; n1 =1.444; h =1.22 μm; d = 4 μm). The optical field distribution and the thermal field distribution of the devices are simulated by finite element analysis software. The simulation processes include geometry build, add material, physics analysis, meshing build, study, and results analysis. For the optical field analysis, the physics interfaces are “electromagnetic waves” and “frequency domain”. The meshing method and the study method are “physics-controlled mesh” and “mode analysis”, respectively. For the thermal field analysis, the physics interface is “heat transfer in solids”. The meshing method and the study method are “free triangular mesh” and “stationary”, respectively. First, the top electrode structure of the TO switch by lithography process has been simulated  induced by graphene film and electrode as a function of horizontal distances (d) between the center of the core layer and the electrode with the electrode width (a) from 1.5 to 3.5 μm. Five different polymer core layer dimensions are designed by COMSOL, realizing the complete coupling between two waveguide core layers. Because of the absorption of light by the side electrode, as shown in d > 4 μm).a = 3 μm and a = 2 μm, respectively. For the TO switch without a graphene film structure, the power consumption significantly increases as a function of electrode distance (d). The power consumption is 3.2 mW with a d of 0 μm, while the power consumption increases to 17.26 mW with a d of 7 μm. For the graphene TO switch structure, the power consumption is insensitive to the electrode distance because of the large thermal conductivity of graphene film, relaxing the tolerances during the fabrication work of the devices. The results show that the power consumption for the graphene TO switch with d of 4 μm is reduced by 23.13% compared with the top electrode TO switch structure (d = 0 μm) without graphene film.d = 4 μm) are shown in The calculated response time and the power consumption with different electrode widths (d of 4 μm, a of 2 μm, ∆T of 1.15 K, and P of 2.95 mW, respectively.d of 4 μm, a of 3 μm, electrode length of 1 cm, and wavelength of 1550 nm. Compared with the inverted ridge TO switch with a top electrode structure, the power consumption was reduced by 23.13% for the graphene TO switch with a d of 4 μm. Moreover, the rise and fall times were 64.5 μs and 175 with a d of 4 μm, a of 2 μm, ∆T of 1.15 K, and P of 2.95 mW, respectively.In summary, we have presented an inverted ridge 3D hybrid TO switch with graphene film. Because of the opposite thermal optical coefficients of polymer and silica and the high thermal conductivity of a graphene layer, a graphene-based side electrode TO switch with low power consumption and fast response time has been obtained. Compared with the traditional 3D polymer–silica hybrid TO switch with top electrode structure, the power consumption was reduced by 41.43% for the graphene TO switch with a


Idiopathic pulmonary fibrosis (IPF) is an incurable disease characterized by progressive lung fibrosis ultimately resulting in respiratory failure and death. Recurrent micro-injuries to the alveolar epithelium and aberrant alveolar wound healing with impaired re-epithelialization define the initial steps of the pathogenic trajectory. Failure of timely alveolar epithelial repair triggers hyper-proliferation of mesenchymal cells accompanied by increased deposition of extracellular matrix into the lung interstitium.We previously isolated fibrosis-specific mesenchymal stem cell (MSC)-like cells from lung tissue of patients with interstitial lung diseases. These cells produced factors bearing anti-fibrotic potential and changed their morphology from mesenchymal to epithelial upon culture in an epithelial cell (EC)-specific growth medium. Here, we set out to molecularly characterize these MSC-like cell-derived ECs using global gene expression profiling by RNA-sequencing. Moreover, we aimed at characterizing disease-specific differences by comparing the transcriptomes of ECs from IPF and non-IPF sources.Our results suggest that differentially expressed genes are enriched for factors related to fibrosis, hypoxia, bacterial colonization and metabolism, thus reflecting many of the hallmark characteristics of pulmonary fibrosis. IPF-ECs showed enrichment of both pro- and anti-fibrotic genes, consistent with the notion of adaptive, compensatory regulation.Our findings support the hypothesis of a functional impairment of IPF-ECs, which could possibly explain the poor clinical outcome of IPF that roughly compares to those of advanced-stage cancers. Our study provides a valuable resource for downstream mechanistic investigation and the quest for novel therapeutic IPF targets. Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease (ILD) characterized by progressive fibrosis and worsening dyspnea, ultimately leading to respiratory failure and death , 2. The bona fide (cobblestone-like) epithelial morphology when cultured in epithelial cell (EC)-specific growth medium. This distinct change in morphology was paralleled by induction of E-cadherin (CD324) expression, a canonical marker of ECs [Recently, we succeeded in isolating MSC-like cells from peripheral human fibrotic lung tissue. The MSC-like cells met the defining criteria of mesenchymal stem cells (MSCs) . Specifir of ECs .Here, we sought to determine the transcriptomic differences between these MSC-like cell-derived ECs from IPF and non-IPF sources. We further aimed at delineating the phenotypic properties of these ECs, following the goal to elucidate a possible functional involvement of these cells in IPF, which may help to explain the poor clinical outcome of IPF as compared to other types of ILDs.n = 5) and non-IPF ‘control’ patients (n = 5) between November 2015 and November 2016 as previously described [3) which were then cultured in tissue flasks under standard conditions  in DMEM supplemented with fetal bovine serum (10%), penicillin (20 U/l), streptomycin (20 μg/ml), and amphotericin B (2.5 μg/ml). MSC-like cells showed sprouting-like growth emanating from the tissue samples and finally formed a confluent cell monolayer around the tissue piece. The non-adherent cell fraction was washed away over the course of the culture through repeated exchange of medium. After 5 days, the tissue pieces were removed and the medium was exchanged for the EC-specific growth medium Cnt-17 . Total RNA was extracted after 7 days of culture in Cnt-17.Characteristics of the study population are specified in Table Total RNA was extracted from ECs (bulk populations) using the MicroElute Total RNA Kit . RNA was quantified using the QuantiFluor RNA System  and quality-controlled on the Bioanalyzer instrument using the RNA 6000 Pico Kit .Fifty ng total RNA were used for library generation using the TruSeq Stranded mRNA Library Prep Kit High Throughput , and 15 PCR cycles were performed. The Standard Sensitivity NGS Fragment Analysis Kit  was used to assess the quality of the libraries (123 ± 30 nmol/L average concentration and 321 ± 8 bp average library size). 1.8 pM of pooled sample was used for cluster generation on the NextSeq 500 instrument (Illumina). Single-read 76 bases (plus 8 + 8 bases for indexing) were sequenced using the NextSeq 500 High Output Kit 75-cycles (Illumina). Primary analysis of sequencing data was conducted using RTA version 2.4.11 and bcl2fastq v2.20.0.422 (both from Illumina).Total RNA was isolated from additional samples of an independent cohort and gene expression levels were determined using TaqMan® Gene Expression Assays . The following target genes were analyzed: CXCL8 (Hs00174103 m1), ICAM1 (Hs00164932 m1), PTGS2 (Hs00153133 m1), LIF (Hs01055668 m1), IL7R (Hs00902334 m1), and GAPDH/housekeeper (Hs03929097_g1). Reactions were performed at 50 °C for 2 min (1 cycle), 95 °C for 10 min (1 cycle), and 95 °C for 15 s followed by 60 °C for 1 min (40 cycles). Relative expression data are shown as -ΔCt of target gene-GAPDH.www.R-project.org). The Bioconductor package ‘edgeR’ (version 3.24.3) was used for the identification of differentially expressed genes. Exploratory analyses including Principal Component Analysis (PCA) were performed using the package ‘ade4’. Pathway enrichment analyses and visualization were done using the package ‘clusterProfiler’ (version 3.10.1) [http://geneontology.org), Kyoto Encyclopedia of Genes and Genomes  and DisGeNET (www.disgenet.org/rdf) was investigated using hypergeometric tests as implemented in clusterProfiler.All analyses were done using the R statistical software  . Differen = 5) and non-IPF lung disease     Table . While w Table . 5) Table a. Despit5) Table b, indica05) Fig. c. Of not05) Fig. d.Fig. 1n = 80) and downregulated genes in IPF (n = 119) (Supplementary Table p < 0.05)  could be validated as overexpressed in IPF, with the remaining gene (IL7R) showing enrichment in IPF samples as well without reaching statistical significance. Moreover, comparing these key genes to the publically available GSE134692 dataset [Supplementary Figure B). This suggested both similarities and characteristic differences between the datasets, thus corroborating our findings but also highlighting the distinct nature of our ex vivo model in comparison to primary explanted tissue. Moreover, the increasing appreciation of senescence as a major contributor to IPF development [We annotated the up- n = 0 and dow05) Fig. a+b. It s05) Fig. . Moreove05) Fig. a, consis05) Fig. . We next dataset , we founelopment  promptedWe moved on to interrogate the interconnection of the differentially regulated pathways and biological processes using the R package ‘clusterProfiler’. While pathways enriched for downregulated genes in IPF-ECs did not notably cluster Fig. a, activaversus non-IPF sources. Results from this study suggest that IPF-ECs (i) remain imprinted from the in vivo IPF milieu (hypoxia and dysbiosis), (ii) are more fibrotic than ECs from non-IPF sources, (iii) are enriched in various immune-related pathways, and (iv) carry signatures associated with organ development and cellular stemness. We therefore propose a distinct functional failure of these cells in IPF, which may help to explain the poor clinical outcome of IPF patients.In this article, we provide a comprehensive description of the transcriptomic landscape of MSC-like cell-derived ECs from IPF It is important to note that ECs were experimentally generated using a specific cell culture method and that their in vivo relevance is yet to be determined. Overlapping findings with the GSE134692 dataset based on primary tissue specimens from lung transplant recipients and donors  suggest,IPF is an age-related disease characterized by progressive and irreversible scarring , 24. TheAutophagy, i.e., the on-demand degradation and recycling of intracellular components and organelles, is yet another fundamental cell process that is deregulated in IPF. Diminished also during aging, the autophagy flux is typically reduced to insufficient levels in IPF, which in turn accelerates cellular senescence and provokes pro-fibrotic myofibroblast differentiation , 29. As Our study is based on fresh, native samples from a rare patient cohort and hence provides a valuable resource for IPF research. It is important, however, to mention also the limitations of our study: First, our study is based on a limited number of cases, which precludes that robust, general conclusions can be drawn. Second, the reference cohort was based on patients with various kinds of non-IPF lung diseases, rather than healthy individuals. Third, our study is descriptive, meaning that causal links or rational therapeutic targets cannot be established. Fourth, our study is ultimately based on a single technological platform, i.e., RNA-sequencing, and does not report protein-level data.Taken altogether, we here provide a comprehensive, molecular characterization of IPF-ECs generated ex vivo from IPF patients. Our results suggest that the transcriptome of IPF-ECs is clearly distinct from non-IPF-ECs and dominated by signatures whose underlying biological processes are plausibly involved in IPF pathogenesis, such as hypoxia, bacteria, metabolism and development. Further research is warranted to decipher the suitability of ECs as a cell-based medicinal product for anti-fibrotic, epithelium-regenerating treatment of IPF.Additional file 1: Supplementary Table 1. List of Differentially Expressed Genes. A total of 199 genes were differentially expressed in MSC-like cell-derived ECs from IPF versus non-IPF patients, with 80 genes upregulated and 119 genes downregulated in IPF-ECs. Abbreviations used: CPM, counts per million (reads mapped); EC, epithelial cell; FC, fold change; FDR, false discovery rate; IPF, idiopathic pulmonary fibrosis; MSC, mesenchymal stem cell.Additional file 2: Supplementary Table 2. Patient Characteristics of qPCR Validation Cohort (n = 16). Abbreviations used: IPF, idiopathic pulmonary fibrosis; ILD, interstitial lung disease.Additional file 3: Supplementary Figure 1. qPCR Validation of RNA-Sequencing Results and Comparison to a Published Dataset. (A) TaqMan-based qPCR analysis of 5 key genes in independent cohorts of IPF (n = 9) and non-IPF patients . (B) Expression levels of the 5 key genes in IPF and non-IPF patients from the GSE134692 dataset and two-tiered comparison to our data (same direction of regulation – yes/no). Abbreviations used: CPM, counts per million (reads mapped); IPF, idiopathic pulmonary fibrosis.


However, no differences were observed between the total doses of corticosteroids administered over days 31 to 60, 61 to 90, 91 to 120, and 151 to 180 after the AE. Furthermore, differences between the administration rates of immunosuppressive and antifibrotic treatments administered to the 2 patient groups were not significant. An increased total dose of corticosteroid administered over day 1 to 30 after an AE-IPF was associated with a decreased risk of subsequent recurrence of AE-IPF within 1 year after the first AE.Some patients with idiopathic pulmonary fibrosis (IPF) undergo recurrent acute exacerbations (AEs). This study aimed to elucidate the risk factors for recurrent AEs of IPF (AE-IPF). Consecutive patients with IPF admitted for their first AE-IPF between January 2008 and December 2018 were retrospectively recruited. Of 63 patients admitted for an AE-IPF and discharged alive, 9 (14.3%) developed a recurrence of AE within 1 year. The mean time to recurrence was 233 ± 103 days. Total doses (mg/month and mg/kg/month) of corticosteroids administered over day 1 to 30 after the AE were significantly higher in patients without recurrences of AE-IPF  than the doses in patients with recurrences  ( Over their clinical course, some IPF patients undergo rapid worsening of respiratory symptoms associated with new ground-glass opacities or consolidations on chest high-resolution computed tomography (HRCT). The event is termed “acute exacerbation (AE) of IPF” (AE-IPF), and its mortality rate is high2. The treatment for AE-IPF generally consists of high-dose corticosteroids and/or immunosuppressive agents, although there have been no supportive data from randomized controlled trials4. There is also no proven specific maintenance treatment after the initial treatment.Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown etiology5. However, specific risk factors for the subsequent recurrence of an AE in patients after their first AE remain unknown. The aim of this study was to test the hypothesis that as one of the treatments, the initial dosage of corticosteroid for the first AE is associated with early recurrence of an AE-IPF.AE-IPF sometimes recurs, even after it has resolved. A previous history of AE was reported to be associated with an increased risk of recurrenceDuring the study period, 86 patients with IPF were hospitalized for AE Fig. . Among tThe baseline clinical characteristics and treatments of the 63 patients admitted for the first time and discharged alive are shown in Table p = 0.02 and p = 0.03, respectively). However, no differences were observed between the total doses of corticosteroids administered over days 31 to 60, 61 to 90, 91 to 120, and 151 to 180 after the AE. Furthermore, differences between the rates of administration of immunosuppressive and antifibrotic treatments to the 2 patient groups were not significant. A multivariable analysis of the following independent variables: total doses (mg/month and mg/kg/month) of corticosteroids over days 1 to 30 after the diagnosis of AE, total doses (mg/month) of corticosteroids over days 61 to 90, sex, age, BMI, and use of antifibrotic agent at discharge; identified total doses (mg/month) of corticosteroids over days 1 to 30 as the only independent predictor of recurrence .Treatments for the first AE-IPF are shown in Table This study found that the total dose of corticosteroids over days 1 to 30 after the diagnosis of AE-IPF were associated with subsequent recurrence(s) of AE-IPF within 1 year after the date of admission for the first AE-IPF. A treatment that combined steroids with another immunosuppressive agent was not associated with recurrent AE. Furthermore, antifibrotic therapy administered within 1 year after AE-IPF was not associated with a subsequent recurrence. To our knowledge, this is the first study which has shown a significant association between the dose of corticosteroid used to treat a first AE-IPF and the subsequent recurrence of AE.6. However, the initial dose of corticosteroid for an AE-IPF is usually reduced gradually because of concern about a recurrence. Our study found that only the initial dose of a corticosteroid during the first month after the onset of an AE-IPF was important in regard to preventing a recurrence of an AE. It might be possible that the initial dose of a corticosteroid can be reduced rapidly after the first month of treatment for the AE-IPF. If this can be verified, the frequency and severity of side effects associated with corticosteroid therapy could be markedly reduced.Corticosteroids often cause serious side effects, especially at high doses administered over a long period of timeAn immunosuppressive agent tended to be more frequently used in addition to a corticosteroid for the patients who did not develop a recurrence of AE-IPF than for those who did develop a recurrence (11.1% use by patients with vs. 35.1% use by patients without), although statistical significance was not achieved. An immunosuppressive agent might be a possible treatment option not only for treating patients with an AE-IPF, but also for preventing recurrence of an AE, although further study is needed to test this hypothesis.7. Pirfenidon has also been reported to reduce the incidence of AE after surgical resection for patients with IPF and lung cancer8. In our study, however, antifibrotic therapies given within 1 year after the first AE-IPF were not associated with the recurrence of an AE-IPF. Antifibrotic agents have been usually started in our institution after the patient has stabilized. Given this practice for administration of antifibrotic agents, the duration of antifibrotic therapy given to some of the study patients over the year of observation after the diagnosis of an AE-IPF might have been too short. If the observation period had been longer than 1 year, a different result might have been observed. Another possible explanation for our finding is that because a small number of patients received the agents, the effects of antifibrotics were missed. The efficacy of antifibrotic agents for reducing the risk of a recurrent AE-IPF should be investigated further.The antifibrotic agents nintedanib and pirfenidone are approved drugs for the treatment of IPF. Nintedanib was reported to reduce the risk of AE-IPF in an efficacy study of patients with IPF9. Among the 7, two (28.6%) had a history of 3 episodes of AE-IPF. The recurrences reportedly occurred in most patients over a short period of time  after the previous AE. In our present study, the mean time to recurrence of an AE-IPF was 7.7 (range 2–12) months, which was similar to the previous report. There may be a phenotypic patient with IPF who develops frequent exacerbations.Another important finding of the study is that a recurrence of AE-IPF is also a risk factor for the subsequent recurrence of an AE. In this study, of the patients developing a recurrence of AE-IPF after their first AE, 42.8% developed a second recurrence. This result is similar to that of the previous study, which demonstrated that of 27 patients with an AE-IPF, 7 (25.9%) had more than 1 episode of AE4. A triggered AE includes an AE occurring after an infection, procedure, or surgery; or an AE associated with drug toxicity or aspiration. Distinguishing between infection-triggered AE and simple pulmonary infection is sometimes impossible. Fourth, data at the discharge from the chest HRCT, pulmonary function testing, the six-minute walk test, and echocardiography could not be used for the analysis, because they were not performed in most cases.This study has limitations. First, this was a single-center study with a relatively small number of patients. Second, the study was conducted in a retrospective fashion. A multicenter study employing prospective enrollment of a larger number of patients is needed to confirm the results of our small study. Third, our study might have included patients with pulmonary infections. Major efforts were made to exclude patients with obvious pulmonary infections. However, distinguishing clearly between an AE-IPF and pulmonary infection is sometimes quite difficult. The recent international working group report of the American Thoracic Society (ATS) proposed 2 types of AE-IPF, idiopathic versus triggered AE-IPFIn conclusion, when comparing IPF patients hospitalized for their first AE, which was treated by steroids, who were stratified according to whether or not they developed an early subsequent recurrence of an AE, a higher total dose of corticosteroids administered over days 1 to 30 after admission was associated with a lower risk of early recurrence of a subsequent AE-IPF.4. The diagnosis of IPF was made according to the criteria used in the INPULSIS trial7. If a surgical lung biopsy was not performed, a HRCT study was required, which showed the following findings: honeycombing and/or a combination of reticular abnormalities and traction bronchiectasis consistent with fibrosis with basal and peripheral predominance and without atypical features of UIP7. The study protocol was approved by the ethics committee of the Kindai University Faculty of Medicine (No. 31-228). Informed consent was waived, because this study was based on a retrospective analysis of case records from our university hospital. All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki).A retrospective review was performed of the files of consecutive patients with IPF who were admitted to the Kindai University Hospital for their first AE between January 2008 and December 2018 and were discharged alive. Whether or not the patient developed a recurrent AE within 1 year from the date of onset of the first AE was determined. The diagnosis of AE-IPF was made according to the recent international working group report of the ATS, as follows: 1) a previous or concurrent diagnosis of IPF; 2) acute worsening or development of dyspnea typically of less than 1-month’s duration; 3) computed tomography study of the lungs showing new bilateral ground-glass opacities and/or consolidations superimposed on a background pattern consistent with UIP; 4) deterioration not fully explained by cardiac failure or fluid overload11.Pulmonary function test results that were obtained during the stable phase of the disease within 1 year prior to the AE were adopted as baseline values. Pulmonary function testing was performed by the CHESTAC-8800 system , according to the standards proposed by the European Respiratory SocietyThe following baseline characteristics of the patients with AE-IPF were determined, as follows: gender, age, body mass index, smoking status, long-term oxygen therapy, and treatments for IPF. Admission and discharge results of routine blood tests were evaluated.The total dose and the total dose per body weight (kg) of corticosteroids administered over each month after the initiation of steroid therapy were evaluated. Whether or not immunosuppressive agents were used with corticosteroids was assessed. Use of an antifibrotic agent within 1 year after the day of admission for the first AE was also examined.The recurrence of an AE-IPF within 1 year after the date of admission for the first AE-IPF was reviewed in each patient. The time to recurrence was defined as the number of days from the date of admission for the first AE-IPF until the date of admission for the recurrent AE.t-test was used for continuous data and the χ2 test or Fisher exact test was used for categorical data between 2 groups. Multivariable analysis with logistic regression was used to identify the risk of AE-IPF recurrence. In this analysis, variables with p < 0.2 on univariate analysis and variables historically considered to be important in IPF were included as independent variables in the multivariable analysis. A p value of < 0.05 was considered statistically significant. Analyses were performed with Statflex, ver.6 software .Continuous variables were expressed as means ± standard deviation (SD), and categorical variables were expressed as actual numbers. The unpaired


Edge mode could disturb the ultra-subtle mass detection for graphene resonators. Herein, classical molecular dynamics simulations are performed to investigate the effect of edge mode on mass sensing for a doubly clamped strained graphene resonator. Compared with the fundamental mode, the localized vibration of edge mode shows a lower frequency with a constant frequency gap of 32.6 GHz, despite the mutable inner stress ranging from 10 to 50 GPa. Furthermore, the resonant frequency of edge mode is found to be insensitive to centrally located adsorbed mass, while the frequency of the fundamental mode decreases linearly with increasing adsorbates. Thus, a mass determination method using the difference of these two modes is proposed to reduce interferences for robust mass measurement. Moreover, molecular dynamics simulations demonstrate that a stronger prestress or a higher width–length ratio of about 0.8 could increase the low-quality factor induced by edge mode, thus improving the performance in mass sensing for graphene resonators. Since Geim and co-workers fabricated monolayer graphene by mechanical exfoliation in 2004 , graphen−24 g) with a mass responsivity of 0.34 GHz/yg. Then, Jiang et al. [To date, a number of authors have studied various methods to realize atomic-scale mass sensing by using graphene resonators. For example, Duan et al.  adopted g et al.  investigg et al. , utilizig et al. , and adjg et al. , have beg et al. , has rarThe molecular dynamics method, which directly computes the state of every atom and takes the scale effect into consideration, seems to be the most suitable method to investigate the edge mode of graphene sheets compared with finite element methods , nonlocaIn this paper, molecular dynamics simulation is performed to investigate the distinct responses between edge modes and fundamental modes to adsorbed mass on graphene sheets, and a novel mass determination method based on the frequencies of these two modes is proposed, which is expected to diminish the interferences caused by mutable stress in graphene. In previous studies, the mass was determined by the fundamental frequency shift, which is hypersensitive to the stress in graphene in addition to the absorbed mass. However, it is difficult to control the stress in graphene accurately and steadily. For example, temperature fluctuation can induce distinct thermal expansions between graphene and silicon oxide substrate, thereby changing the graphene inner stress significantly . MoreoveMolecular dynamics simulations have been used to study the impact of edge modes on mass sensing for strained graphene resonators ,45. The In the present MD simulation, the modeling schematic diagram of a graphene-based resonant mass sensor is shown in In L is the length of the graphene sheet and σ is the axial tension in graphene. The equivalent density ρ, in terms of the contributions from both graphene and adsorbates, is calculated considering the thickness of the monolayer graphene sheet to be 3.35 Å and the ideal density to be 2200 kg/m3 for pure grapheme [Referring to the previous literature ,6,53, doAs for the doubly clamped vibrating graphene sheet with a length of 100 Å and a width of 50 Å, as shown in To identify which vibration eigenmode the first peek represents, the mechanical vibrations of the graphene sheet were observed by visualization software OVITO , and notρ will increases, thus reducing the fundamental frequency. The frequency shift can be written byρ0 is the density of pure graphene and ∆ρ is the increment of equivalent density, assuming the absorbed mass is evenly distributed. The factor K is used to adjust the frequency shifts caused by distinct distributions of adsorbates and equals 1 when adsorbates are evenly distributed. Since the volume of graphene sheet can be regarded to be unchanged, Equation (2) is then rewritten asm0 is the mass of pure graphene sheets and ∆m represents the mass of adsorbates. In contrast, due to the unpredicted results when using standard elastic beam theory, the frequency of edge modes shows a considerably different response to central adsorbates.Resonant mass sensors are dependent on the fundamental frequency shifts in response to adsorbates. When the external mass is absorbed on the graphene sheet, the equivalent density 0.5 according to Equation (1), and it matches with the MD results considerably well, with an R-square of more than 0.99 and a maximum relative error of about 6%. Moreover, this relative error decreases to about 2% if a stronger tension (>15 GPa) is exerted, which can be explained by the fact that the stretched graphene sheet is closer to the ideal tensioned membrane structure. On the other hand, the two curves in biasC is a constant. Equation (4) is then used to fit the MD results, showing an excellent R-square value up to 0.99, as displayed in To compare the distinct response to the centrally distributed absorbates between edge modes and fundamental modes, 1–10 gold atoms as adsorbates were placed in the middle of the graphene sheet with a length of 100 Å and a width of 50 Å, along with the applied axial stress ranging from 10 to 50 GPa. The working principal of typical graphene-based resonant mass sensors complies with Equation 3), where the linear relationship between the fundamental frequency and adsorbate mass is critical for mass determination, assuming the stress σ in graphene is constant. However, the stress in graphene sheet is susceptible to numbers of factors, such as the temperature fluctuations , the var, where tm is absorbed mass; m0 is the mass of the graphene sheet (excluding fixed areas); biasC is a constant as illustrated before, which is irrelevant to the stress in graphene. Note that the above derivation is under the premise that the mass is centrally distributed; nevertheless these adsorbates cannot be located at the exact middle of graphene sheets. Thus, the acceptable range of the adsorbates where the mass determination method proposed above is applicable is worth discussing.The MD simulation results shown in To determine the appropriate range for centrally distributed adsorbates, two gold atoms as absorbed mass were moved from the center of graphene sheets to the edges along the width direction, and then the corresponding frequencies of the two modes were recorded and are depicted in edgef ≈ fundamentalf − 30. In this case, we cannot obtain two independent equations regarding absorbate mass and stress and solve them together like in the case of central distribution. That is, the frequency of edge modes cannot provide more independent and valid information for mass determination, so the mass determination method proposed above is not applicable for evenly distributed adsorbates.The discussions and conclusions above make sense only in the case of centrally distributed adsorbates. When adsorbed mass is evenly distributed, the frequencies of the fundamental mode and edge mode decrease synchronously, as can be seen in The quality factor (Q) is always an important issue for resonant sensors, and a higher Q factor means less energy dissipation per oscillation period and higher resolution for mass sensing ,56. The f is the fundamental frequency; t is duration of the traced vibration; A0 and tA are the amplitudes of fundamental oscillation at the beginning and end of simulation, respectively. In terms of Equation (6), the Q factors of these four samples are then calculated to be 1463, 2520, 2321 and 4405, respectively. It is apparent from these four charts that the graphene sheet with smallest width and stress has much more energy loss in oscillation, while that with the largest width and stress has the least energy loss, and its Q factor is three times bigger than that of the former. The intermediate two also clearly show less amplitude diminishing, with Q factors of 2520 and 2321, respectively. Incidentally, the ripples on the margins of these amplitude curves occur due to the incoherent mixing of vibrations of two modes with different frequencies, which is called the beating phenomenon. Comparing the four results, it can be seen that enlargement of the width and stress could effectively improve the Q factor of graphene resonators.To elucidate the edge effects on the Q factor for improving Q value, graphene sheets with different widths and inner stress were investigated by recording the diminishing amplitudes of fundamental vibrations that imply energy dissipations. For clarity, the impacts of the width and stress on the Q factor are therefore further studied individually. In conclusion, classical molecular dynamics simulations are adopted to investigate the impact of edge mode on mass sensing for a doubly clamped monolayer graphene resonator. The vibrations of edge mode were found to localize at the free edges and possess larger amplitude. Specifically, in the MD simulation for a 100 × 50 Å graphene sheet, the vibration amplitude of edge mode achieved 3.8 Å, nearly 2.5 times that of the fundamental mode. Moreover, the frequency gap of the two modes was calculated to be 32.6 GHz, which is irrelevant to the axial stress in graphene. Thus, considering that fundamental frequencies drop linearly with increasing centrally absorbed mass, while the frequencies of edge mode remain constant, a novel method of mass determination combining the two vibration modes was developed in order to diminish the interference of mutable stress in graphene sheets. More importantly, the MD results show that the vibration of edge mode broke the coherence of the mechanical fundamental oscillation, thus aggravating the energy dissipation and then resulting in a lower Q factor, which was as low as 1463 for the 100 Å × 50 Å graphene sheet with tensile stress of 10 GPa. However, increasing the stress in graphene can greatly increase the Q factor up to 7989 when an axial stress of 50 GPa is exerted. Moreover, appropriately widening the graphene sheets with a higher width–length ratio above 0.8 can double the Q factor, although excessive width does not contribute to an extremely high Q factor.


Observed contractile response indicated on effective SMCs stimulation mediated by graphene layer which constituted efficient cell to biomaterial interface.Tissue engineering allows to combine biomaterials and seeded cells to experimentally replace urinary bladder wall. The normal bladder wall however, includes branched neuronal network propagating signals which regulate urine storage and voiding. In this study we introduced a novel biocomposite built from amniotic membrane (Am) and graphene which created interface between cells and external stimuli replacing neuronal network. Graphene layers were transferred without modifying Am surface. Applied method allowed to preserve the unique bioactive characteristic of Am. Tissue engineered constructs composed from biocomposite seeded with smooth muscle cells (SMC) derived from porcine detrusor and porcine urothelial cells (UC) were used to evaluate properties of developed biomaterial. The presence of graphene layer significantly increased electrical conductivity of biocomposite. UCs and SMCs showed an organized growth pattern on graphene covered surfaces. Electrical filed stimulation (EFS) applied  Following this concept, a leading scenario aims to create a neobladder by combining different biomaterials with autologous urothelial and detrusor cells. These two components are expected to create a favourable environment for functional bladder wall regrowth. The biomaterial plays a pivotal role in this strategy as its biomechanical characteristic and bioactivity profile determine cellular events associated with regeneration2. Within the bladder wall, there is multilayer branched neuronal network delivering and propagating signaling from the central nervous system (CNS) to the detrusor smooth muscle layer. Synchronous initiation of action potentials mediates the wavelike contraction, gradually embracing the whole bladder increasing intravesical pressure and generating urine outflow3.The urinary bladder is a temporary storage reservoir for urine. Both continence and voiding phases are active processes involving detrusor contraction or relaxation controlled by peripheral and central nervous system. In addition, passive mechanical properties of the bladder wall, such as viscoelastic force determine static and dynamic behavior of urinary bladder4. Nevertheless, none of them was successful in clinical translation due to the high complication rate and lack of long-lasting function. The major problem hampering advances in the field of urinary bladder tissue engineering is the simplification of the targeted neobladder structure in conducted research. Accordingly, one of the critical challenges hardly ever addressed in experimental settings was the restoration of the neuronal network within the neobladder wall5.To date, urinary bladder reconstruction with tissue engineering methods testing different usually acellular scaffolds has been used in 131 patientsDe novo regeneration of convolutional neural network within the neobladder hasn’t been ever tried due to lack of applicable technology. There might be however difrent ways to approach this dilemma. The alternative solution is to completely replace a native neural network with biocompatible current conductive material that might be linked to an external unit generating electrical stimulation6. However, before such a biocybernetics organ replacing unit may be created, there is a need to develop a biomaterial scaffold providing the interface between external stimuli and host tissue. Physiologically, the contraction of the detrusor muscle is initiated by meditators like acetylcholine and ATP (Adenosine Triphosphate) released from efferent nerve fibres. Subsequently triggered, discharge of the dispersing action potential is the direct signal contracting the smooth muscle layer7. On account of this two-stage mechanism, the chemical mediated stimulation could be replaced by direct electrical stimulation in the tissue-engineered bladder.8. Research data derived from in vitro and in vivo models indicated that application of electrical stimuli directly to detrusor muscle may trigger effective contraction. Nevertheless the major challenge is to spread and sustain locally induced depolarization wave9. In comparison to heart urinary bladder doesn’t have recognized intramural conduction system delivering electrical stimuli to all bladder regions10. Creation of tissue engineered bladder offer possibility to develop de novo conduction system within bladder wall intended to distribute electrical stimulation aimed to trigger controlled contraction pattern of reconstructed smooth muscle layer.Inducing detrusor contraction by direct electrical stimulation is a concept which has been developed to initiate micturition reflex in patients with neurogenic bladderIn this study, we are proposing a new biocomposite biomaterial derived from the amniotic membrane (Am) covered with a graphene layer. The developed method of graphene transfer allowed to cover intact Am with solid graphene layers. The primary object was to obtain an electrical stimuli-responsive biomaterial tailored for provisional application in the tissue-engineered urinary bladder. The ability to deliver electrical stimuli opens a new perspective for the generation of dynamic tissue systems demanding a functional and responsive smooth muscle layer.11. Its excellent bioactivity profile is mediated by active molecules incorporated within the Am matrix. Am was applied for experimental reconstruction of the bladder wall in rat model and proved its ability to support regeneration of smooth muscle layer12. Furthermore, Am has universal capability to induce epithelization including urothelial layer regrowth13. Knowing this, the overriding assumption during interdisciplinary development of graphene transfer method was to avoid any modification of the Am structure impairing its biocompatibility profile. At any stage of biocomposite fabrication Am didn’t have any contact with cytotoxic, cross-linking or oxidizing substances.Am is used in urological tissue engineering due to its unique properties supporting the regeneration process14. Therefore, an innovative strategy involving non-damaging graphene layer transfer on a biomaterial surface may overcome this limitation and become a novel solution worth further development and testing.Conductive biomaterials are of particular interests for tissue engineering, and recently many new biomaterials were introduced. Despite intensified research efforts, there are several significant limitations. The most substantial is reported to have reduced biocompatibility, and challenging necessity to combine hard or brittle conductive layer with softer and compliant natural derived biomaterial15. In this context, the application of graphene in regenerative medicine enables to cross biological barriers which are inaccessible to more abundant commonly evaluated materials. The most crucial advantage of graphene is a neglectable influence on passive biomechanical characteristics of the tissue-engineered bladder wall. Therefore it doesn’t impair the ability to disdain during repetitive cycles of urine storage and micturition. Moreover, graphene, due to its nanostructure, might reach single effector cells analogously to healthy neurons and provide adequate interface replacing synapsis16. Graphene layers gradually overgrown with cells act as a nanoscale implant allowing to deliver electrical stimulation reestablishing cellular communication and regulating a cells’ behaviour or intrinsic features such as contraction17.Graphene has emerged in the last decade as one of the most exciting nanomaterials whose electrical, mechanical, chemical, and structural properties can’t be obtained from either individual molecules or bulk materialsThe sandwich-structured biocomposite material was constructed from frozen human Am and covered from the stromal side with two graphene layers. The stromal side was chosen due to its ultrastructure, which is irregular and offers a suitable scaffold for graphene attachment. Therefore the graphene layer could settle freely on pleated Am surface.Am was obtained according to the well-established protocol used in ophthalmology for Am transplantation Shortly, the donors was screened to exclude the risk of transmissible infectious diseases. The Am was obtained under sterile conditions after the elective caesarian section was washed out with an antibiotic solution, and the chorion was separated manually. With the epithelial surface up, Am was spread uniformly on nitrocellulose membranes. Am was then placed in a preservative medium and stored at −80 °C.Am harvesting and experiment protocols were approved by Ethics Committee of Collegium Medicum, University of Nicolaus Copernicus . All methods were carried out in accordance with national regulations on ethics and research in EU. Am was harvested from donors above 18 years old after obtaining written permission. All donors were informed consent for study participation.18.Transferring graphene onto scaffolds required an individual approach to the transfer methods. Traditional PMMA (Polymethyl-methacrylate) approaches can’t be used since graphene’s adhesion to polymers is stronger than to extended surfaces, which Am is. As a result, graphene is removed from the substrate scaffolds during the dissolving of PMMA. To overcome this issue, we applied a modified marker-frame method Fig. 18.Figure20. Then a PDMS (Polydimethylsiloxane) frame instead of marker frame was positioned on a surface of bilayer graphene. Next, a sample was placed on the surface of an aqueous solution of ammonium persulfate (1 M) . After approximately 2 hours when Cu foil was completely etched, and bilayer graphene inside the PDMS frame floats on the surface of the solution, the sample was cleaned with a continuous and controlled flow of DI water. In the end, the bilayer graphene with a PDMS frame was carefully fished by Am and left at room temperature to dry and to improve both adhesion and contact between graphene and Am. Finally, the PDMS frame was mechanically removed from the Am, leaving the graphene layer alone on its surface.In this work, we used commercially available graphene (Graphenea Inc. USA) on 18 µm thick copper (Cu) foil. Since the marker-frame method is very demanding in terms of handling fragile graphene, at the very first step, we aimed at creating a bilayer of graphene on the copper substrate. We transferred the monolayer of graphene from the copper foil onto graphene on the copper foil by using electrochemical delamination methods21. All biocomposite samples (n = 5) included the same Am used as control (n = 5). The tested samples  were mounted into flat grips with a gauge base of 10 mm. During the test, the specimen was longitudinally stretched at a rate of 0.2 mm/s until failure. The grip travel and specimen load were continuously measured over the test procedure with a precision force transducer  and a MTS system linear variable differential transformer . The Young’s elastic modulus (MPa) was estimated based on generated Stress/strain curves.Tensile tests were conducted on a load frame of a servohydraulic material testing machine  as it was described previouslyin vitro. It was designed also for Electrical Field Stimulation (EFS) of cells seeded on biomaterial fixed in CellCrown . To our knowledge, there isn’t any commercially available testing system for this purpose. Electrical conductivity was measured using a four-sensing probe. The custom made probe utilized four 4 mm diameter electrodes printed from graphene PLA filament   spaced 2 mm apart. This gap was adjusted to graphene layer dimensions (10mmx10mm). Graphene containing filament was recently shown to be a suitable material for the fabrication of customized 3D printed electrodes22. The low-frequency (100 Hz) electrical conductivity at room-temperature was calculated for samples (n = 5) of each material. As a control, Am without a graphene layer and porcine bladder wall were utilized. The measurement setup consisted of the following devices: AFG3251 Arbitrary Function Generator , DMM4050 multimeter  and DMM4040 Osciloscope . Raspberry Pi  computer gathered the data and calculated the conductivity of each material based on its electrical resistivity  filament  Fig.  All of tity Fig. .Figure 2et al.23. Accordingly, urinary bladders from male domestic pigs were obtained from the local abattoir. All isolation and culture procedures were carried out in a laminar flow hood, using sterile techniques. The dissected smooth muscle layer of the urinary bladder was cut into small pieces and digested in collagenase II  and dispase II  solution . After digestion, the resulting suspension was filtered through 100μm nylon cell strainer  and centrifuged at 1500 × g for 5 min. Isolated cells were counted and seeded at a density of 2 × 104 cells/cm2 in SmGM-2 culture medium . Bladder smooth muscle cells were cultured up to the third passage.Isolation and culture of bladder smooth muscle cells were carried out according to the protocol of Pokrywczyńska et al.24. Accordingly, urothelial cells were isolated from porcine urinary bladders harvested from male domestic pigs during the planned economic slaughter in a local slaughterhouse. All isolation and culture procedures were carried out in a laminar flow hood, using sterile techniques. Bladder tissue pieces (1 × 1 cm) were incubated for 15 h at 4 °C in the conical tubes containing 10 mL dispase II  dissolved in the Hank’s Balanced Salt Solution (HBSS) (2.4 U/mL). After the dispase inactivation with an equal volume of DMEM/Ham’s F12 medium supplemented with FBS, the urothelial cells were carefully scraped with the blunt side of a scalpel, and the remaining tissue was discarded. Separated white conglomerates were transferred into a falcon tube and centrifuged at 1000 × g for 5 min. Isolated cells were counted using a trypan blue and seeded at a density of 4 × 104 cells/cm2 in the commercially available CnT-57 growth medium . Urothelial cells were cultured up to the third passage in standard conditions at 37 °C with 5% CO2 atmosphere and 98% humidity until reaching 75–90% confluence.For isolation and culture of urothelial cells, we used the earlier optimized protocol of Pokrywczyńska 2) and cultured until confluency was reached. The first stage of analysis included evaluation of cells morphology and growth pattern . Then multi-staining  was used to determine the viability of cultivated cells on the graphene covered surface. The number of viable cells was estimated as follows. The number of ethidium positive cells was subtracted from the number of DAPI positive cells. Labeled cells were counted with a confocal microscope  within the circular ROI (Region of Interest) (n = 10). The analysis was performed at 40x magnification with particle analysis function in ImageJ (https://github.com/imagej/imagej).Glass coverslips covered with a graphene layer (n = 20) were applied to evaluate graphene cytotoxicity to SMCs. As a control group, coverslips without graphene were used (n = 20). SMCs were seeded (20 000 cells/cm2). Subsequently adherent cells were counted within ROI (n = 5) 3 h, 6 h and 12 h after seeding.Glass coverslips with a graphene layer (n = 5) were applied to evaluate SMCs adherence to surface covered with graphene. As a control group, coverslips without graphene were used (n = 5). SMCs were seeded  derived from SMCs seeded biocomposite grafts (n = 5) were fixed in 10% (v/v) neutral (pH = 7) buffered formalin and embedded in paraffin. Cross-sections of the entire area covered with graphene were prepared, and histological analysis using H&E staining was performed. Additional immunohistochemical staining using anti-SMA antibodies  and anti-smoothelin antibodies  was conducted to identify SMA and smoothelin cells and to distinguish them from the Am matrix. Stanning for smoothelin was conducted to verify whether in vitro cultured SMCs preserved their mature contractile phenotype. Digital images of anti-SMA and anti-smoothelin stained specimens were used for quantitative evaluation of SMC content. Immunohistochemistry Image Analysis Toolbox plugin (https://imagej.nih.gov/ij/plugins/) for ImageJ was applied to calculate SMA stained surface using generated histograms. The evaluation, according to this protocol, was done in square ROI (n = 10) for each specimen.After six days of in vitro culture, multi-staining  was used to determine viability and arrangement of UCs cultivated on biocomposite and Am surface. Labelled cells identified as alive were counted with a confocal microscope  by creating a square region-of-interest (ROI) (n = 10) at 40× magnification.After 7 days of in vitro cultured SMC was conducted using an electrode printed from conductive graphene PLA filament  . The measurement of the temperature was conducted 15 minutes after removing stimulator form cultivation incubator at room temperature. Following stimulation period cells were cultured an additional 48 hours. The multi-staining  was used to determine the impact of EFS on cell number and arrangement. Labelled cells identified as alive were counted with a confocal microscope  by creating a square (ROI) (n = 10) at 40× magnification. The orientation of cultivated SMC was established by a square ROI (80 × 80 pixels) (n = 10) analysis using ImageJ OrientationJ  plugin. SMC bodies were manually outlined on DAPI/Calceine threshold images by fitting an ellipse to the selected region. The average cell orientation was determined by averaging all the values of deflection angles obtained from the ROI (n = 10).Electrical stimulation of SA) Fig.  The gap We have designed an experimental setup to elicit false contractions from the cultivated muscle layer and record the increase of the pressure generated by the contracted graft Fig. . Each anCalculated results were reported as means with standard deviations. Statistical difference was evaluated using a one-way analysis of variance (ANOVA) with Tukey’s post hoc comparison at a significance level of 0.05.−1 and the 2D band at ~2688 cm−1, which confirms the presence of a graphene structure27. Evaluation of Young’s elastic modulus (E) indicated that either graphene layer itself or method of graphene transfer didn’t influence mechanical characteristic of Am which preserved its native elasticity  and reference scaffold substrate (red line) are present Fig. . For theity Fig. . The manDesign printed stimulator allowed to precisely lower the electrodes to provide comprehensive and stable contact points. The calculated electrical conductivity of the graphene-based biocomposite, Am alone, and porcine urinary bladder was demonstrated in Fig. . The graDue to the presence of the graphene layer, we didn’t notice an abnormal morphology of cultured SMCs. The cells were characterized by normal spindle shape, and the confluence of the cell populations was indistinguishable between groups. The graphene layer didn’t exert any cytotoxic effect or influence the SMCs adhesive properties. SMSs formed typical homogenous monolayer with single clusters. The number of cells cultivated on the graphene layer was comparable between groups, and the graphene layer didn’t decrease cell survival. The estimated cell number between groups is shown in Fig. in vitro cultivation. Graphene, as a hydrophobic carbon material didn’t impair the cells adhesive capability. Anti-SMA and anti-smoothelin staining were more intense on the biocomposite surface than Am alone . There is a need to analyze behavior of graphene layer in context of its elasticity and resistance for repeating load and unload cycles typical for urinary tract environment.Since the development of graphene, this carbon material has been evaluated for application in tissue engineering. Graphene is used to design multifunctional biomaterials that can provide physical, electrical, and structural signalling to cells and tissues11. An aqueous solution of ammonium persulfate is widely used for nanomaterial preparation. This agent, due to excellent solubility in water is easily washed away from the graphene layer. The concept of a stable graphene layer transfer is known in the electronics industry, but hitherto only metallic or polymeric materials acted as substrates33. This study introduced a new application for this technology in the field of biomaterials. The demonstrated method may be adopted to cover different biomaterials with graphene to fabricate conductive biohybrid materials that may find application in cardiologic or neurologic tissue engineering. The advantage of applied methodology for graphene transfer is the potential possibility to preserve intact Am bioactive content and mechanical properties. Nevertheless this assumption needs to be verified in further studies.Am was extensively evaluated for urological tissue engineering, also in clinical settings35. Bladder detrusor has a unique histological structure with a syncytial ultrastructure divided into autonomous functional units connected by distributed innervation and a network of intestinal cells36. In contrast to striated muscle tissue, bladder’s smooth muscle syncytium creates a different environment for automatic propagation of action potentials37. From the electrophysiological point of view, local electrical stimulation tends to “scatter” in syncytium evoking action potentials with different inefficient shapes and sizes without a contractile response. In this situation, the external electrical pulse aimed to trigger an action potential must be simultaneously delivered at multiple points. Due to the excellent electrical properties of graphene, it offers the technological possibility to address this challenge by establishing an efficient biomaterial-cellular interface mediated coordinated response. In this context, graphene may become a milestone technical solution allowing to replace neuronal networks in artificial organs.Graphene-based biomaterials were introduced as scaffolds supporting muscle cell proliferation. Most of them, however, were indented to promote myogenic differentiation and proliferation of striated muscle or cardiocytes38. We believe that the direct contact and exposure of SMCs to the electric charge was crucial to evoke an active contraction pattern. Moreover, graphene’s natural high flexibility prevented cells from detachment during construct manipulation and contraction itself. This graphene feature is particularly crucial for biomaterials indented to replace hollow organs that change volume during normal functioning such as the urinary bladder. The registered contractile activity of SMCs seeded on biocomposite was the result of graphene-supported stimulation propagation and a proper cell arrangement which enabled to generate stable force tensioning the Am. In comparison to cardiomyocytes electrophysiology of detrusor, SMCs is less known in vitro. Thus the applied stimulation parameters were chosen based on a few published studies concerning intact detrusor stimulation in vitro41. Parameters of electrical stimulation were chosen assuming that activation threshold of tissue engineered layer of SMCs should be similar to normal muscular layer of bladder wall.The graphene layer as a nanomaterial interacts with single cells promoting tight adhesion between cell membranes and the interfacing biomaterialin vitro, including the lack of a reference protocol. Kobayashi et al. determined that intestinal SMS seeded electrospun poly(3-caprolactone) scaffolds exhibited rhythmic contractions resulting in biomaterial contraction42. Krueger et al. reported the ability of muscle cells, forming myotubes seeded on graphene foam to mediate consolidated micromovements43. The designed, printed chamber, mimicking the bladder environment might become useful to evaluate the contraction ability of tissue-engineered grafts planned for reconstructive urology. We hypothesize that the fabricated chamber evaluating the tissue-engineered construct contractility based on pressure changes might be more sensitive in a tissue-engineered scenario than in stripe testing. It is challenging to obtain an SMCs seeded biomaterial with the ability to generate a singular vector contraction force recognized by a force transducer connected at both ends.There is a shortage of literature demonstrating contraction of tissue-engineered grafts 44. The exact mechanism responsible for this phenomenon is unknown. Mainly, it is unclear whether graphene acts as a passive molecular pathway for proliferating cells or instead actively interacting with adhesive membrane molecules. Graphene incorporated with a collagen scaffold likely created micro-topographic structures that were preferably recognized by migrating neurons or muscle cells45. Moreover, being over one hundred-fold smaller than cells, graphene shouldn’t negatively affect the cytoarchitecture of the regenerating tissue contributing to the functionality of the reconstructed bladder wall.The role of modern biomaterials must include the ability to influence the cell arrangement to restore proper tissue architecture. The graphene layer passively organized the distribution of seeded SMCs and UCs. In the case of SMCs, it also directed cell orientation during EFS. From the beginning of research on the biotechnological application, the graphene ability to order cell arrangement is underlined46. Hydrophobic character of graphene may also reduce contact of regenerating tissue without developed urothelial barrier with urine. Urine has recognized cytotoxic activity which may impair regeneration47.From the perspective application in urology, a graphene-based biomaterial would also have additional advantages particularly important for clinical practice. The known antibacterial activity of graphene should prevent infection and facilitate healing of urinary tract wall after future reconstructive proceduresin vitro. Further research are needed to evaluate behavior of this scaffold on in vivo models. Utilization of graphene for biomaterial design might become the first step to obtain biocybernetics platform aimed to control and regulate the function of tissue engineered bladder.The developed graphene based biocomposite successfully managed to deliver and to propagate external electrical stimulation to cultivated smooth muscle cells. The combination of conductive graphene and Am supporting cell growth resulted in creation of tissue engineered grafts capable of induced contraction


Molecular models of the proposed candidates are exposed to energy calculations at DFT level, in addition to geometry optimization processes at PM6 method. HOMO/LUMO orbitals, MESP maps and QSAR descriptors are calculated. Results ensure that graphene doped with NiO has the highest reactivity since it possesses the largest TDM and the smallest HOMO/LUMO band gap. MESP maps illustrate that the benzene rings of graphene are most probable to undergo nucleophilic interactions. Addition of Fe3O4 creates new negatively charged active sites that are ready for nucleophilic interactions. The calculated QSAR parameters demonstrate a hydrophobic nature for pure and modified graphene suggesting that they need further modification with further groups for usage in biological applications.Graphene has attracted great concern in recent years as one of the potential 2D materials in various applications. This work is devoted for assessing the feasibility of functionalizing 2D graphene sheets with ferromagnetic and antiferromagnetic metal oxides namely magnetite (Fe Materials chemistry, Graphene; Molecular modeling; DFT; PM6; QSAR and MESP. Like dynamic sealing [Graphene, material composed of two dimensional one-atom-size plane of spanoscale , 2. It wanoscale . This apanoscale  with theanoscale . While oanoscale  and dreaanoscale . It was anoscale  and effianoscale . Generalanoscale . Therefoanoscale , 12. Enhanoscale , 14. Recperature , 16. Bes sealing , biosens sealing , magneti sealing , therape sealing  targeted sealing . Nickel  sealing , 23. Ext sealing , 25, 26  sealing , the fab sealing  magnetis sealing , 31, 32. sealing , 34, 35. sealing , 37, 38. sealing . Such co sealing . Some mo sealing , 42. The sealing , 44, 45.3O4 and graphene/NiO with Density Functional Theory (DFT) and PM6 semiemripcial method as well as QSAR descriptors.Owing to the importance of functionalized grapheme, the present work is dedicate to model the graphene/Fe2All computations were carried out utilizing GAUSSIAN 09 software that is implemented at Spectroscopy Department, Physics Division, National Research Centre, NRC . These cThen, another optimization calculations were conducted using semiemprical quantum mechanical calculations at PM6 level  via SCIG33.13O4), nickel oxide (NiO), graphene functionalized with Fe3O4 (G- Fe3O4), and graphene functionalized with NiO (G-NiO). Graphene is built as a 2D sheet composed of 42C atoms linked together with single and double bonds alternatively. Then, Fe3O4 and NiO are added to graphene sheet at nearly its central benzene ring; exactly at C16 and C17 atoms.In order to describe the obtained results it is important to describe how the model molecules are built. 3.23O4) and antiferromagnetic one (NiO) on both its electronic and physical features. These calculations are carried out using DFT theoretical level at B3LYP method and 6-31G basis set. As stated earlier in the calculation details section, the zero-point vibration energy is not considered in the calculations, and also the basis set superposition error (BSSE) has not been included in the energy calculations. Calculations were conducted to investigate the effect of functionalizing graphene with ferromagnetic substance  is significant in determining structures' reactivity where those of large TDM values are considered more reactive relative to others. The resultant TDM of NiO is much larger than that of Fe3O4 with magnitudes 3.9303 and 0.2309 Debye, respectively reflecting higher reactivity for NiO with respect to Fe3O4. This can be attributed to that NiO has terminal reactive O atom while those of Fe3O4 are surrounded by Fe atoms. Therefore, TDM of graphene doped with NiO equals nearly one and half of that functionalized with Fe3O4. The calculated HOMO/LUMO band gap energies follow the same behavior of TDM ones ensuring the importance of band gap values in referring to the reactivity of chemical structures. The result of NiO is smaller than that of Fe3O4 and graphene functionalized with NiO possesses the smallest HOMO/LUMO band value of 0.0040 eV confirming its reactivity in comparison with the other proposed interaction with Fe3O4.Stability is one of the most important physical parameters that should be concerned when dealing with structures theoretically on the molecular scale. Total energy is the physical quantity which refers to stability level where the lower the structure's energy, the higher is its stability. The calculated energy of graphene sheet is about -43.8459 keV. NiO has a similar stability level to graphene while energy of Fe3.33O4), nickel oxide (NiO), graphene functionalized with Fe3O4 (G-Fe3O4), and NiO (G-NiO) which computed at B3LYP/6-31G theoretical level. Figures 3O4 show highly symmetric configurations indicating the homogeneity of electronic distribution through these orbitals either in the ground or excited states. However, they present asymmetric distribution for HOMO and LUMO of graphene functionalized with both Fe3O4 and NiO. For Fe3O4, the HOMO orbitals which are nearly concentrated around the added Fe3O4 group while the LUMO ones are distributed between both graphene sheet and Fe3O4. While the situation is reversed for graphene doped with NiO where the HOMO orbitals found in both entities and LUMO ones focused on NiO.3.43O4), nickel oxide (NiO), graphene functionalized with Fe3O4 (G-Fe3O4) and NiO (G-NiO). The constructed maps composed of several colors extending from red color to dark blue one representing the extreme negative for red and positive sites for blue. These colors appear in the order of red, orange, yellow, green, light blue and dark blue from the most negative to most positive regions where red is for the extreme negative potentials and dark blue for positive ones. Furthermore, yellow regions have less negative potentials with respect to the red one and green reflects regions of neutral potentials. The potential distributions and colors as well can be correlated in a certain way by the electronegativity of the attached atoms. Atoms of high electronegativity seem to be red when linked to another less electronegative ones. Presence of atoms of nearly the same electronegativity makes colors distribution much narrower. Hence, we can depend on the MESP maps as a physical feature in deciding whether the interested active sites can undergo either nucleophilic or electrophilic chemical interactions.Molecular electrostatic potential (MESP) maps are created for the studied chemical structures at DFT level of theory using B3LYP/6-31G. They are usually provide simple and quite positive way for clarifying the charge distribution over a substance and so the most probable active sites in it. 3O4, the graphene appears with light blue color indicating positive region while the added magnetite has a range of colors; yellow around the O atoms linked to the sheet, light blue in the middle surrounding the central Fe nuclei and red in the vicinity of the upper O atoms indicating highly electronegative region. This reflects the success of oxygen electronegativity on withdrawing the electrons from graphene sheet. However, graphene linked to NiO structure is characterized by general yellow color except for certain light red regions on the upper surface where O atoms are present. This suggest that NiO addition turn the graphene to less negative structure.Regarding MESP map of pure graphene sheet, there are three main colors composing the constructed map; red, blue and dark blue. It is noticeable that red is concentrated in the centers of benzene rings forming the sheet ensuring the effect of electron delocalization phenomenon within the rings suggesting that graphene core is most probable to undergo nucleophilic interactions. While the terminals are characterized by light and dark blue colors which may be attributed to presence of less electronegative H atoms. Hence, electrophilic reactions may be most likely to occur there. While magnetite structure is characterized by two main positive terminals due to the presence of Fe atoms colored by dark blue and nearly neutral central region colored by both yellow and light green. This may be due to the linkage of four O atoms to the central Fe atom canceling the effect of their high electronegativity. Such configuration propose that electrophilic chemical interaction is proper for magnetite ensuring the possibility of decorating graphene surface with it. NiO map contains two clear colors; red around O atom and blue surrounding Ni, reflecting the large difference in electronegativity between them. Thus, NiO may be involved in both electrophilic interactions utilizing Ni side and nucleophilic one from O terminal. Hence, NiO addition would be more convenient to interact with graphene sheet through Ni atom not oxygen. Turning to graphene functionalized with Fe3.5Quantitative structure-activity relationship (QSAR) descriptors provide a quite accessible and simple theoretical method for determining biological reactivity of chemical structures. QSAR always prove to be a point of research utilized when someone would like to assess biological activity , 53. Its3O4 and G-Fe3O4 equaling -281.949 and -84.614 kcal/mol, respectively. Ionization potential or IP is the quantity of energy required to ionize a certain structure. It reflects structures' reactivity as well. Magnetite has the lowest IP value of -10.687 eV while NiO owns the largest one of -6.006 eV. Addition of either Fe3O4 or NiO to graphene increases its IP values. Partition coefficient or Log P is one of the QSAR biological descriptors that reflects the solubility of a substance in either organic or aqueous solvents. It is the ratio between the dissolved amounts in organic solvent to that does in aqueous one. Log P results indicate that all the proposed structures are hydrophobic except for NiO suggesting that they need further modification with further groups for usage in biological applications. Graphene is the most hydrophobic while Fe3O4 is the least one with values 9.98 and 1.085, respectively. Graphene functionalization slightly lowers its hydrophobicity. The computed molar refractivity (MR) of graphene, Fe3O4 and NiO equal 171.344, 5.401 and 1.443, respectively. Their interactions does not have significant impact on the MR of the resultant structures whose values are 173.587 for G-Fe3O4 and 170.887 for G-NiO.For final heat of formation (FF) which is commonly known as the amount of heat change after the formation of a chemical substance of its individuals . FF valu43O4) and antiferromagnetic (NiO) materials in nanoscale. Based on DFT, PM6 and QSAR descriptors, one can drive some concluding remarks. Graphene doped with NiO has the highest reactivity since it possesses the largest TDM and the smallest HOMO/LUMO band gap. MESP maps illustrates that the benzene rings of graphene are most probable to undergo nucleophilic interactions. Addition of Fe3O4 to graphene create new negatively charged active sites that are ready for nucleophilic interactions as well. The calculated QSAR parameters demonstrate a hydrophobic nature for pure and modified graphene suggesting that they need further modification with further groups for usage in biological applications. Such results indicate that the reactivity of the given functionalized graphene can dedicate it for applications in the field of environmental pollution to control heavy metals for example from wastewater. This findings need some kind of experimental verifications which will be carried out in future based on the present computational study.Adding new functional groups for graphene is an important step towared directing its applications based on the functionalization process. Accordingly, it is aimed to assess the feasibility of functionalizing 2D graphene sheets with ferromagnetic (FeMedhat Ibrahim, Amal H. Al-Bagawi, Ahmed M. Bayoumy: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


The simulated results show that the absorptivity of the absorber reaches 90% in the range of 0.91–1.86 THz, and the normalized bandwidth of the absorptivity is 68.6% under normal incidence. In the simulation, the effects of the geometric parameters of the structure on the absorption band have been investigated. The results show that the absorber is insensitive to the incident polarization angle for both transverse electric (TE) and transverse magnetic (TM) under normal incidence. In addition, the absorber is not sensitive to oblique incidence of the light source under TE polarization conditions, and has an approximately stable absorption bandwidth at the incident angle from 0° to 50°. The absorption band can be adjusted by changing the bias voltage of the graphene Fermi level without varying the nanostructure. Furthermore, we propose that a two-layer graphene structure with the same geometric parameters is separated by a dielectric layer of appropriate thickness. The simulated results show that the absorptivity of the two-layer absorber reaches 90% in the range of 0.83-2.04 THz and the normalized bandwidth of the absorptivity is 84.3% under normal incidence. Because of its excellent characteristics based on graphene metamaterial absorbers, it has an important application value in the field of subwavelength photonic devices.A broadband terahertz (THz) absorber, based on a graphene metasurface, which consists of a layer of ring-porous patterned structure array and a metallic mirror separated by an ultrathin SiO The area between the far infrared and the ultra-microwave is terahertz (THz), whose frequency ranges from 0.1 to 10 THz. THz waves have important application significance. It can be used in imaging, drug inspection, biological detection, and intracellular protein composition analysis ,3,4. In In this paper, we propose a polarization-independent and wide-angle absorber based on a graphene metasurface at terahertz frequencies, which consists of a graphene array structure with ring-porous patterns, a dielectric layer and a metal layer to achieve a high-bandwidth absorption. The metal layer is used as the bottom-most layer and the dielectric layer is used as an intermediate layer. The patterned graphene array is periodically arranged on the dielectric layer silicon dioxide. The simulated results show that more than 90% of the broadband terahertz absorption in the range of 0.91 to 1.86 THz can be achieved. By optimizing the parameters of the absorber, a fractional bandwidth of 68.6% can be achieved under normal incidence and graphene Fermi level of 0.7 eV. Due to the axial symmetry structure, the absorber has insensitive polarization characteristics for the transverse electric (TE) and transverse magnetic (TM) polarized terahertz waves under normal incidence. For the TE polarization, the absorber has a relatively stable absorption bandwidth in the incident angle range from 0° to 50°. Finally, we study the tunability of the absorption band of the structure. By changing the graphene Fermi energy from 0.7 eV to 0.2 eV, the absorption rate of the absorber can be adjusted. Furthermore, a double sandwich graphene structure has also been studied, and the simulated result indicates that a wider absorption bandwidth (from 0.91 THz to 1.21 THz) and big fractional bandwidth (from 68.6% to 84.3%) can be achieved. This study provides new inspiration for the design of graphene-based tunable wideband absorbers that can be used in terahertz optoelectronic devices such as photodetectors and biosensors.R, r, and p denote the length of the outer and inner radius of the graphene ring and the radius of the small circle around the ring, respectively. The center of the small circle is at the center of the graphene ring. L refers to the width and length of a periodic unit along the x and y direction, which are both 15 μm. The middle dielectric layer comprises 28 μm-thick non-destructive SiO2 with a dielectric constant ε = 3.9 [7 S/m was used as the bottom metal layer, which is thick enough to meet the typical skin depth in the THz range. Therefore, wave transmission was completely suppressed [The structure of the absorber is shown in  ε = 3.9 . In the ppressed .T, Bk, and ħ are the absolute temperature of the environment, Boltzmann constant, and reduced Planck’s constant, respectively, and ω is the angular frequency, fE is the Fermi energy level, and τ is the electron-phonon relaxation time. The first term in Equation (1) is derived from the intra-band transitions, and the second term is derived from the inter-band transitions. For the THz frequency domain (hω << 2fE) at room temperature, according to the Pauli exclusion principle, the inter-band transitions in the graphene are negligibly small; therefore, Equation (1) can be safely simplified to the Drude model [Graphene is a two-dimensional honeycomb planar material with carbon atoms. Graphene supports surface plasmon resonance (SPR) in the infrared and terahertz bands . Becausede model :(2)σgra=graσ of graphene through the Fermi level. The relationship between the Fermi level and the carrier density can be described by FV is 1 × 106 m/s, and n represents the carrier density of graphene. We set the values of the Fermi level and relaxation time of graphene as fE = 0.7 eV and τ = 1 ps, respectively. The ambient temperature at room temperature was fixed at 300 K. In this work, we used the finite-difference time-domain (FDTD) numerical simulation method to analyze the three-dimensional absorber structure in the frequency domain. Plane wave occurred perpendicularly along the z-axis. We used periodic boundary conditions in the x and y directions and perfectly matched layer (PML) boundary conditions in the z direction. During the simulation, the simulation time and grid accuracy were set to 40,000 fs and 5, respectively. The size of the simulation area was 15 µm × 15 µm, and the grid size in graphene was ∆x = ∆y = 0.15 µm, Δz = 0.05 µm. In the calculation, a suitable non-uniform grid can be used to meet the conditions for good convergence results.It can be seen from Equation (2) that due to the existence of the carrier density, the carrier density can be changed by voltage or chemical doping, thereby adjusting the surface conductance Ef=ħVFπn . The FerA, the transmittance traT, and the reflectance R is A = 1 – traT − R. A high absorption can be achieved by minimizing R and traT simultaneously. The transmission is almost equal to zero (traT ≈ 0) in the total reflection geometry, so the absorption efficiency of the proposed absorber can be briefly expressed by A = 1 − R.Using the FDTD solutions, we studied a unit cell of a graphene-based THz absorber and obtained its electromagnetic response. The system model uses a plane wave as the light source. To investigate the absorption performance, the reflection and transmission spectrum of the absorber were captured. According to Kirchhoff’s current law, the relationship between the absorptivity Ef = 0.7 eV with normal plane-wave incidence. As expected, due to the symmetry of the structure, the absorber exhibited the same absorption characteristics under TE and TM polarization states. The simulated results show that nearly perfect absorption with a maximum absorption of 99.6% at 1.70 THz can be achieved. There are clearly two absorption peaks, with an absorption rate of 98.0% and 99.6% at 1.02 THz and 1.70 THz, respectively. The 90% absorbance bandwidth from 0.91 THz to 1.86 THz is 0.95 THz. Fractional bandwidth, which is the absolute bandwidth relative to the center frequency, is approximately 68.6%.To study the absorption characteristics of the periodically annular porous patterned graphene structure proposed, we first simulated the spectral distribution under TE polarization and TM polarization conditions. x-axis polarization direction, the amplitude of the incident electromagnetic wave is represented by incE and the amplitude of the reflected electromagnetic wave is represented by ref.E We can obtain the reflection coefficient of the metasurface absorber R as [r12 and r21 are the ratios of the reflected wave’s complex electric field amplitudes. Likewise, the transmission coefficients t12 and t21 are the ratios of the transmitted wave’s complex electric field amplitudes. The reflection coefficient of the bottom metal total reflection plate is r23 = −1, β = 2πn2/λ0 is the propagation constant, and n2 is the refractive index of the dielectric layer. According to the formula A = 1 − R, when R is equal to 0, the absorption rate A reaches the maximum value.The mechanism of the absorber is explained using Fabry–Perot interference theory. The structure of the absorber can be generally equivalent to a Fabry–Perot resonator, which is mainly composed of a partial reflector and a total reflector . The graber R as :(3)R=EreEf = 0.7 eV and plane-wave perpendicular incidence, we obtained the electric field amplitude (|E|) distribution of f = 0.21 THz, f = 1.02 THz, and f = 1.72 THz in TE and TM polarization modes, separately. Then, to further explore the absorption characteristics of the absorber, under the conditions of Fermi level r of the graphene ring on the absorption rate when other parameters of the absorber structure are kept at the optimal value under the normal incidence of the plane wave. When we only changed the value of the inner circle radius of the graphene ring (the inner circle radius r changed from 1.65 μm to 2.45 μm) and other parameters were unchanged , the first absorption peak showed a slight decrease, and the absorption wave had traces of blue shift. When r was gradually reduced, the bandwidth of the absorption wave slowly increases, but when r was less than 2.05 μm, the absorption rate of the middle part of the absorption band was reduced to below 90%.Next, we investigated the influence of the geometric parameters of the absorber and the graphene Fermi level on the absorption spectrum. According to the influence of the geometric parameters of the structure on the absorption spectrum, the geometric parameters with the best absorption performance are obtained after optimization. When the geometric parameters of the structure have been adjusted, the variation of the absorption band has the same trend, due to the symmetry of the absorber. p on the absorption spectrum is shown in p is similar to the parameter r. As R gradually decreases, the first absorption peak slightly decreases. The absorption bandwidth gradually increases, and the second absorption peak shifts slightly toward high frequencies. R of the outer circle of the graphene ring is changed. When the change value of the outer radius R gradually decreases, the first absorption peak shifts slightly toward blue, and the second absorption peak tends to shift slowly toward red. Obviously, as the value of R decreased, the band with an absorption rate of 90% gradually decreases. When R = 6.6 μm, the absorption rate of part of the absorption broadband is less than 90%. Ef changes from 0.2 eV to 0.7 eV, the absorbance of the absorber can be adjusted from 55% to 100%. In particular, at Fermi level Ef = 0.6 eV, the absorption spectrum has two absorption peaks at almost 100%, at 1.04 THz and 1.5 THz. At this time, the absorption characteristics of graphene begin to become saturated. At Fermi level Ef = 0.7 eV, the absorption rate of the absorber decreases, but there is a wider fractional absorption bandwidth. Graphene-based terahertz photoelectric devices such as absorbers, sensors, and detectors have the characteristics of small size and excellent performance. These are conducive to device integration and application.The influence of the value of the small circle radius L) and the relative permittivity (ε) of the dielectric layer on the absorption performance of the absorber. The simulated results are shown in L increases, the absorption bandwidth gradually decreases, and the left absorption peak begins to decrease. The absorption rate of the absorber reaches 99% in the range of 1.44 Thz to 1.73 THz when L = 13 μm, which is equal to the radius of the outer ring of the graphene ring. ε) of the dielectric layer on the absorber. As the relative permittivity gradually increases, the absorption bandwidth changes in small shifts to lower frequencies. The relative permittivity of the dielectric layer has little effect on the waveform of the absorption bandwidth of the absorber.Then, we studied the effect of the size of the periodic unit  are consistent with the single-layer graphene absorber. d1 increases, whereas the absorption rate of the middle of the absorption band will increase. Thus, there is an optimal d1 for the absorption rate and bandwidth. This is because the strength of the resonance coupling on the surface of the upper and lower layers of graphene depends, to a large extent, on the distance between them. Increasing or decreasing the coupling distance will reduce the near-field coupling to some extent, thus showing changes in absorption performance.An absorber with better absorption performance can be obtained by applying a multilayer stack structure. Here, the double-layer graphene metasurface is isolated by silica, and a model diagram of it is shown in In this paper, we have proposed an active tunable, wide-angle, and polarization-insensitive broadband terahertz absorber, based on a ring-porous patterned graphene metasurface. The simulated results show that the best characteristics can be achieved by optimizing the geometric parameters of the absorber. They also show that the bandwidth of the absorber is over 0.95 THz , for 90% absorbance under normal incidence. It is verified by simulation that the symmetrical structure of the absorber has the characteristics of wide-angle incidence and polarization independence. By changing the Fermi level of graphene, the absorption rate of the absorber can be flexibly adjusted. The absorber after coupling two layers of graphene structure obtains a fractional bandwidth of 84.3%. We think the absorber has potential applications in the fields of sensing, detection, and other optoelectronic devices, such as those used as an absorber in a Q-switched laser.