Surprisingly, this nanosheet exhibited sonodynamic ability, in which CaO2 caused the in situ oxidation of Ti3C2 MXene to produce acoustic sensitiser TiO2 on its surface. In addition, this nanosheet displayed chemodynamic features, which promoted a Fenton reaction triggered by self-supplied H2O2. We detected that C-T@Ti3C2 nanosheets increased reactive oxygen species (ROS) production in response to sonodynamic therapy, which displayed an ideal antibacterial effect. Furthermore, these nanoreactors facilitated the deposition of Ca2+, which promoted osteogenic transformation and enhanced bone quality in osteomyelitis models. Herein, a wound healing model and prosthetic joint infection (PJI) model were established, and the C-T@Ti3C2 nanosheets played a protective role in these models. Taken together, the results indicated that the C-T@Ti3C2 nanosheets function as a multifunctional instrument with sonodynamic features, which might reveal information regarding the treatment of bacterial infections during wound healing.Prolonged and incurable bacterial infections in soft tissue and bone are currently causing large challenges in the clinic. Two-dimensional (2D) materials have been designed to address these issues, but materials with satisfying therapeutic effects are still needed. Herein, CaOThe online version contains supplementary material available at 10.1186/s12951-023-01933-z. Staphylococcus aureus (S. aureus) slow the healing process and lead to bone loss [Although prosthetic joint replacements provide excellent functional improvements, improve the quality of life of patients and offer pain relief , approxione loss –5. The eone loss –9. There2O2, which is less efficient in treating infection, into ROS with higher cytotoxicity by catalysing metal ion-mediated Fenton or Fenton-like reactions in acidic microenvironments [2 secreted through this synergistic therapeutic strategy inhibits the growth of anaerobic bacteria [Recent research has indicated that MXene, a new two-dimensional (2D) nanomaterial with ultrathin layer structure topology, has been widely used in the biomedical field. As transition metal carbides, MXenes feature excellent metallic conductivity, photothermal-conversion capability, hydrophilicity and mechanical properties , 11 as wronments , 29–33. ronments . In addibacteria –37. It ibacteria –40.2O2-triggered and SDT-strengthened Fenton reaction based on the constructed 2D therapeutic CaO2-TiOx@Ti3C2 (designated C-T@Ti3C2) nanomedicine with biosafety and multifunctions, including antibacterial effects and enhancement of bone repair. The C-T@Ti3C2 nanoworks employ calcium peroxide (CaO2) as an efficient source of H2O2 to sustain the Ti3C2 MXene nanosheet-mediated Fenton catalytic reaction, releasing more toxic ·OH to induce bacterial death. Intriguingly, by utilizing CaO2 oxidation, the in situ oxidation of Ti3C2 MXene generates sonosensitizer TiO2 on the MXene surface, accompanied by the production of a small amount of trivalent titanium ions (Ti3+) [2 from H2O2 through a Fenton-like reaction [2 can increase ROS  production and subsequently mediate bacterial apoptosis. Additionally, MXenes can effectively trap photogenerated holes to compensate for the defects in TiO2 with a fast electron (e−)-hole (H+) complexation rate (50 ± 30 ns) [x (TiO2 and Ti3+). The regeneration process of bone defects includes antibacterial reactions, which might otherwise cause delayed healing or even osteomyelitis, and the deposition of calcium ions in the infected bone defect, providing the raw material for bone repair [3C2 in terms of morphology, particle size and lattice structure and the combined effects of C-T@Ti3C2 in the direction of bacterial inhibition and osteogenesis in vivo and in vitro.Herein, we report a self-supplied Hs (Ti3+) , 42, whireaction , 42. Thr± 30 ns) ; thus, Me repair , 45. Ove3C2 MXene was synthesized by a facile exfoliation and intercalation process [3AlC2 by chemical etching using a hydrofluoric acid (HF) solution. The layers were then further stripped by intercalation of tetrapropylammonium hydroxide (TPAOH) to reduce interlayer interactions, followed by centrifugation to obtain few-layer (FL) Ti3C2 MXene  [3C2 MXene, and the corresponding original selected area electron diffraction (SAED) in the upper right corner confirms its hexagonal symmetrical structure. The lattice spacing was calculated to be 0.231 nm [3C2 MXene nanosheets 2 with H2O2 under alkaline conditions. TEM  by vigorous stirring to obtain C-T@Ti3C2 nanosheets. Figure 3C2, respectively. The typical morphology of the nanosheets and the uniform distribution of CaO2 NPs on the surface of the nanosheets were observed  . Figure 0.231 nm . In addi3C2 MXene and C-T@Ti3C2 nanosheets to illustrate the surface oxidation in their nanostructures  is an efficient source of H2O2, which sustains the Fenton-catalysed reaction mediated by Ti3C2 MXene nanosheets. The intensities of the D and G peaks in the C-T@Ti3C2 Raman spectra are significantly increased, indicating the carbon content in the C-T@Ti3C2 nanosheets is enhanced. Moreover, a sharp characteristic peak appears at 727 cm−1 in the Raman spectra of the C-T@Ti3C2 nanosheets. The result fully demonstrates the combination of TiOx and CaO2 generated on Ti3C2 MXene.We tested the Raman spectra of Tires Fig. o. There 610 cm−1 , 49. How2 MAX, Ti3C2 MXene nanosheets and C-T@Ti3C2 nanosheets  at 2θ ≈ 6°. The most prominent peak at 2θ ≈ 39° disappears for the etched pristine Ti3AlC2 MAX by HF etching and TPAOH intercalation, which further indicates that the Al atoms in the pristine Ti3AlC2 MAX have been etched away effectively. Additionally, 2θ values of 30.1°, 35.6°, 47.3° and 52.8° correspond to the (002), (110), (112) and (103) lattice planes of CaO2 NPs in the XRD pattern of C-T@Ti3C2 nanosheets, which illustrates that CaO2 NPs are present on the surface of Ti3C2 nanosheets. This characterization also further demonstrates that the Ti3C2 MXene nanosheets were successfully constructed and CaO2 NPs immobilized onto their surface.We further used X-ray diffraction (XRD) to analyse the structure of TiAlCets Fig. p. Compar3C2 nanosheets, X-ray photoelectron spectroscopy (XPS) characterization of Ti3C2 MXene and C-T@Ti3C2 nanosheets was performed to obtain more detailed information. In the full spectra of Ti3C2 MXene and C-T@Ti3C2 nanonetworks  due to the hydroxyl groups on the surface, so amine groups were introduced using (3-aminopropyl)triethoxysilane (APTES). Hydrolysis reaction between silanol and hydroxyl groups on MXene covalently anchored amino groups on the surface of nanosheets. The groups produced the negative Zeta potential positive (+ 33.8 mV) (pH = 6.0), which facilitated subsequent loading with the negatively charged CaO2 NPs. To improve the stability of the nanosheets in the physiological environment, the surface of the C-T@Ti3C2 nanosheet was modified with polyvinylpyrrolidone (PVP)  as a molecular probe for ROS (1O2) detection. The intensity of the characteristic absorption peak of DPBF at 416 nm decreased markedly in the presence of C-T@Ti3C2 nanosheets , while no significant difference was observed in the absorption spectra without the addition of C-T@Ti3C2 nanosheets  was used as a probe for ·OH without US activation. The reaction mechanism of H2O2-mediated oxidation of TMB could be divided into two steps. The O–O bond in the H2O2 molecule was broken to form ·OH, and TMB was subsequently oxidized by ·OH to form ox TMB. After different reaction times, C-T@Ti3C2 nanosheets showed the ability to induce a Fenton-like reaction to produce ·OH.To further validate the ability of the C-T@Ti3C2 nanosheets to generate ROS in vitro was measured using nonfluorescent 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA), which can be oxidized by ROS to generate fluorescent 2',7'-dichlorofluorescein (DCF). As shown in Fig. 3C2 + US  was stronger than that of Pd@Pt-T790, indicating that CDT combined with US-triggered SDT produced more ROS than that of CDT alone. Ti3C2 MXene produced less ROS with ultrasound or without ultrasound treatment, while DCFH-DA alone or DCFH-DA treated with US showed almost no fluorescence, indicating negligible ROS production.The ability of C-T@Ti1O2 and ·OH by using the electron trapping agents 2,2,6,6-tetramethylpiperidine (TEMP) and dimethyl pyridine N-oxide (DMPO). The 1O2 peak intensity of the C-T@Ti3C2 + US group was significantly more robust than that of the US and Ti3C2 MXene groups  spectroscopy was used to evaluate the production of  and CDT . Without surface .3C2 nanosheets, Escherichia coli , Staphylococcus aureus  and methicillin-resistant Staphylococcus aureus  were treated with C-T@Ti3C2 nanosheets , and we compared the antibacterial property of Ti3C2 and C-T@Ti3C2 nanosheets .To evaluate the antibacterial properties of C-T@Ti3C2 nanosheets on the morphology of bacteria was observed by SEM  and propidium iodide  was performed and analysed regarding the signal intensity and ratio Fig. e, f. As 3C2-induced SDT, DCFH-DA was used to detect ROS in MRSA  is plotted in Fig. 3C2 + US group, respectively, compared to the control group. A heatmap was used to analyse the difference in gene expression between the control and C-T@Ti3C2 + US groups , were downregulated. In contrast, the expression levels of rpsS and the gene (SAOUHSC_01185), etc., regarding ribonucleoprotein complex biogenesis, ribosome biogenesis, and S-adenosylmethionine-dependent methyltransferase activity, respectively, were markedly upregulated .As shown in Fig. ted Fig. g. Furthe3C2 nanosheets, rat bone marrow mesenchymal stem cells (rBMSCs) were chosen to evaluate the biocompatibility and osteogenic differentiation of C-T@Ti3C2 in vitro. The rBMSCs (1 × 106/mL) were seeded with different concentrations of C-T@Ti3C2 in a 96-well plate and cultured for one day. The cell viabilities in this plate were then detected by using the Cell Counting Kit-8 (CCK-8) assay  and PI . The fluorescent images of rBMSCs treated with C-T@Ti3C2 nanosheets showed intense green fluorescence, further validating the negligible cytotoxicity of C-T@Ti3C2  or an equivalent amount of saline into the tail vein of mice (n = 3). The routine blood parameters, including red blood cells (RBCs), mean corpuscular haemoglobin (MCH), haemoglobin (HGB), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean platelet volume (MPV), number of neutrophils (GRAN), red cell distribution width (RDW), number of lymphocytes (LYMPH), haematocrit (HCT) and platelets (PLT), were within normal limits and were not significantly different in either group. In addition, biochemistry parameters, including alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), and creatinine (CREA), did not show any significant abnormalities , osteocalcin (OCN), runt-related transcription Factor 2 (RUNX2), and osteopontin (OPN) were detected by the fluorescence intensity. Obviously, the C-T@Ti3C2 group showed stronger fluorescence signals than that of the other groups. Encouraged by this, RT‒PCR was selected to evaluate osteogenic differentiation at the mRNA level by measuring osteogenic genes, including COL-I, BMP2, OPN, and RUNX2. Compared to those in the other groups, the mRNA expression of cells was much higher at Day 14 , which resulted in the rapid release of large amounts of ROS; as a result, the ROS antagonized infection and promoted wound healing. Due to the burden of MRSA, the wound area of the control group with a final size of approximately 10 mm2 exhibited an ulcerated appearance and a reluctance to form scabs.Based on the great biocompatibility and strong bactericidal effect, the therapeutic efficiency of the C-T@Tiice Fig. a. The sutigation , 20, 54.ics Fig. b–d. Surg3C2 + US treatment resulted in the highest levels of epithelial and dermal regeneration, with a great reduction in the inflammatory response as well as follicle regeneration. The control and US only groups showed ruptured epithelial and dermal tissue, along with an infiltration of granulation tissue consisting of neutrophils and endothelial cells; these observations all indicated a severe inflammatory response to MRSA infection in the wound. Masson staining was used to detect collagen deposition and directional alignment, which are important in tissue regeneration. Masson staining showed massive collagen deposition and better skin recovery in the NIR group, while in the other groups, little or no collagen was observed. Double immunofluorescent labelling of CD31 was used to detect angiogenesis, which is a crucial indicator of the angiogenic process [3C2 nanosheets did not cause side effects. These macroscopic and microscopic observations suggest that C-T@Ti3C2 nanosheets activated SDT, which in combination with CDT is highly effective in the treatment of wound infections caused by US-induced MRSA.Wound samples from mice at Day 11 were subjected to different staining tests for further histological analysis Fig. e, which  process –57. Duri3C2 nanosheets prompted us to establish a bone defect model with MRSA injection in male Sprague Dawley (SD) rats and to assess the antibacterial efficacy and bone regeneration ability of C-T@Ti3C2 in vivo . Thereafter, all the rats were randomly divided into five groups, and different treatments were administered to the indicated group. Then, the muscle and skin were sutured. Femurs were collected from rats at 14 and 28 days postoperatively and examined for bone defect repair and tissue regeneration via micro-CT scanning  and the highest expression of transforming growth factor-β (TGF-β) in the C-T@Ti3C2 + US group. This was because macrophages tended to polarize towards M2 due to the elimination of infection, which facilitated bone regeneration  was purchased from Adamas-beta Inc. . Calcium hydroxide (Ca(OH)2), tetramethylammonium (TMAOH) hydroxide solution (25%), 3,3ʹ,5,5ʹ-tetramethylbenzidine dihydrochloride hydrate (TMB), and (3-aminopropyl)triethoxysilane (APTES) were obtained from Macklin Inc. . 1,3-Diphenylisobenzofuran (DPBF) was kindly provided by Adamas-beta Inc. . Polyvinyl pyrrolidone (PVP), penicillin‒streptomycin-amphotericin B solution, and 2,7-dichlorofluorescein diacetate (DCFH-DA) were purchased from Solarbio Co., Ltd. . The Live & Dead Bacterial Staining Kit was purchased from YEASEN Biotech Co., Ltd. . Cell Counting Kit-8 (CCK-8) was obtained from Biosharp . Calcein/PI Cell Viability/Cytotoxicity Assay Kit, radioimmunoprecipitation assay (RIPA) buffer and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were from Beyotime Institute of Biotechnology . 2,2,6,6-Tetramethylpiperidine (TEMP) was obtained from Aladdin Ltd. . Hydrogen peroxide (H2O2) was purchased from Sigma‒Aldrich Trading Co., Ltd. . Ammonia water , hydrogen fluoride aqueous solution (40%), dimethyl sulfoxide (DMSO) and alcohol were provided by Sinopharm Chemical Reagent Co., Ltd. . Anti-BMP2 antibody (#ab284387), anti-collagen I (#ab284387), anti-osteocalcin antibody (#ab133612), anti-RUNX2 antibody (#ab236639) and anti-beta actin antibody (#ab8226) were obtained from Abcam Inc. . Osteopontin antibody (#AF0227) was purchased from Affinity Biosciences . Foetal bovine serum (FBS), phosphate buffered saline (PBS) and Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F-12) were provided by Gibco. Paraformaldehyde was purchased from Wuhan Servicebio Technology Co., Ltd. . Deionized (DI) water with a resistivity of 18.2 Ω was used throughout this project.Layered titanium aluminium carbide  solution was carefully mixed with 4G Ti3AlC2 MAX in an autoclave with a Teflon liner for 72 h at room temperature (RT) to remove the Al layer. Then, the precipitate was collected by high-speed centrifugation  and washed several times with ethanol and DI water until the pH of the supernatant reached 6.0. The bulky precipitate was obtained and stirred with 60 mL of a TMAOH aqueous solution for three days at RT. After centrifugation , single- and few-layered Ti3C2 MXenes were collected from the colloidal supernatant. PVP (6 g) was dissolved in 12 mL of DI water. Then, Ca(OH)2 (0.6 g) was added and stirred for 20 min. Then, 4 mL H2O2 (4.0 mL) was added dropwise with vigorous stirring afterwards. After 15 min, stable CaO2 NPs formed on the Ti3C2 MXene. Finally, the product was obtained through centrifugation and washed with DI water and ethanol. Since the surface potential of Ti3C2 MXene was negative, surface modification was performed. In short, APTES (100 μL) was added to Ti3C2 MXene  and stirred at 50 °C for 24 h to form Ti3C2-NH2. To obtain CaO2-TiOx@Ti3C2 nanosheets, Ti3C2-NH2 composite nanosheets were stirred with CaO2 NPs in ethanol for 24 h and then centrifuged to obtain CaO2-TiOx@Ti3C2 (C-T@Ti3C2) composite nanosheets. The resulting C-T@Ti3C2 nanosheets were stored at 4 °C for further experiments.To synthesize single- and few-layered Ti1O2 and ·OH production were detected using a JEOL-FA200 ESR spectrometer . Confocal laser scanning microscopy (CLSM) images were captured by a high-speed confocal platform . Tissue sections were observed by Olympus VS120 .Transmission electron microscopy (TEM) images were obtained on a JEM-2100F transmission electron microscope at an acceleration voltage of 200 kV. Scanning electron microscopy (SEM) images and bio-SEM images were acquired on an SU8220 microscope  and Regulus 8100 , respectively. High-resolution STEM, HAADF/ABF-STEM images, elemental mapping and relative EDS analysis were carried out on a JEM-ARM 300F Grand ARM  at 80 kV with two spherical aberration correctors. XPS measurements were performed on ESCAlab250 electron spectrometers . X-ray diffraction analysis (XRD) was performed by a Rigaku automated multipurpose X-ray diffractometer . Atomic force microscopy (AFM) was performed by a Bruker Dimension Icon . Raman spectroscopy was performed using an inVia confocal Raman microscope . Raman spectra were recorded by a Horiba Jobin Y’ von (LabRAM HR) micro-Raman spectrometer . Dynamic light scattering (DLS) and zeta potential examinations were obtained from the Malvern Nano-ZS90 Zetesizer . UV‒vis–NIR absorption spectra were obtained on a UV-1800 spectrometer . The 2-TiOx@Ti3C2 was modified to help stabilize the nanosheet in a physiological environment. Ten milligrams of CaO2-TiOx@Ti3C2 nanosheets were dispersed in an aqueous PVP solution  at 50 °C and stirred for 8 h. After centrifugation and washing with water and ethanol, the CaO2-TiOx@Ti3C2 composite nanosheets were obtained and stored at 4 °C for the following in vivo investigation.The surface of CaO3C2 was mixed with 20 μL of DPBF (1 mg mL−1 in ethanol) along with DI water to form a 3 mL reaction system. The absorbance changes in DPBF were observed at the characteristic peak (416 nm) after ultrasonic stimulation  for different times ranging from 0 to 8 min. Similarly, TMB (20 mg mL−1 in DMSO) was utilized to detect the generation of hydroxyl radicals (·OH) after different reaction times (from 0 to 30 min) by monitoring the increase in absorbance at 662 nm with a UV‒vis spectrometer. Furthermore, ROS generation by ultrasonically activated C-T@Ti3C2 was recorded by DCFH-DA, fixed to 3 ml with DI water and subjected to different treatments. The fluorescence intensity of DCF was detected by a FLS1000 spectrofluorometer  with an excitation wavelength of 488 nm.To detect ROS generated by sonodynamic therapy (SDT), 1 mL of C-T@Ti3C2 nanosheets and Ti3C2 MXene were detected for 1O2 and •OH production by ESR spectroscopy at room temperature. In brief, TEMP and DMPO, as spin trapping agents for singlet oxygen and hydroxyl radicals, respectively, were incubated with DI water, Ti3C2 MXene and C-T@Ti3C2 nanosheets at room temperature and ultrasonicated  for 5 min. Afterwards, the ESR spectra were recorded for three samples.The synthesized C-T@Ti3C2 nanosheets and Ti3C2 MXene was evaluated through a spread plate assay. Escherichia coli  was used as the gram-negative bacterial strain, while Staphylococcus aureus  and methicillin-resistant Staphylococcus aureus  represented the gram-positive bacterial model. All of them were purchased from BeNa Culture Collection Co., Ltd. . They were cultured in sterile Luria–Bertani (LB) medium at 37 °C in an aerophilic environment for 12 to 16 h. The diluted bacterial suspension (109 CFU mL−1) was utilized for the following experiments after testing the absorbance at 600 nm by a microplate reader. The bacterial suspension was diluted with sterile DI water to 106 CFU mL−1 and incubated with C-T@Ti3C2 nanosheets at different concentrations  at 37 °C for 4 h. Batch assays were performed with continuous orbital shaking at 150 rpm. The resulting bacterial suspension (20 µL) was further diluted and spread on prepared LB agar plates by bacterial spread rods with three replicates per sample. The plates were incubated for an additional 12–16 h at 37 °C, and bacterial viability was measured by counting the numbers of bacterial colonies. To further validate the sonodynamic effect of the nanosheets, four experimental groups were set up, and two of the groups were added to coculture the bacterial suspension with the C-T@Ti3C2 nanosheets (the remaining two groups were not treated) for 1 h. The bacterial mixture from one of the blank groups and one of the C-T@Ti3C2 groups was also sonicated  for 5 min (US only group and C-T@Ti3C2 + US group). The resulting bacterial suspension was diluted and spread evenly on agar plates and then incubated for 16 h at 37 °C.The antimicrobial efficiency of C-T@TiS. aureus, MRSA and E. coli were incubated at 37 °C for 12–16 h. They were then immersed in a Live/Dead Baclight Viability Kit of SYTO9  and propidium iodide  for 15 min in the dark and then rinsed with PBS. Finally, photographs were obtained with a confocal fluorescence microscope .For live/dead staining, samples with To further illustrate the antibacterial activity, the bacteria were examined morphologically using SEM. The samples were fixed in 2.5% glutaraldehyde for 6 h. Subsequently, the cells were washed three times with PBS, followed by dehydration with an ethanol gradient  for 15 min. Finally, the samples were observed under SEM.MRSA suspension (109 CFU mL−1 in LB medium) was placed in 96-well plates with different treatments . Next, the samples were coincubated with 10 μL of DCFH-DA (10 μM) in DMSO solution for 30 min at room temperature and washed with sterilized DI water to remove excess dye. The samples were then observed under a confocal fluorescence microscope as mentioned above.Fluorescence imaging was performed to investigate the level of intercellular ROS. A 200 μL MRSA, ATCC 43300) at a concentration of 109 CFU mL−1 was treated with C-T@ Ti3C2 nanosheets for 5 min under ultrasonication . RNAseq analysis was performed by technical staff at Hangzhou Kaitai Biolab. Sequencing was carried out on a MiSeq instrument . Quality examination was analysed by an Agilent Bioanalyzer  and real-time PCR . Fastp was used to process raw data (https://github.com/OpenGene/fastp). Gene expression was calculated by Stringtie (https://ccb.jhu.edu/software/stringtie), and genes with FDR < 0.05 and |log2FC|≥ 1 were considered differentially expressed genes (DEGs). Additionally, KEGG pathway enrichment analysis was performed using the R language to detect paths associated with DEGs.Methicillin-resistant Staphylococcus aureus  were obtained from SD rats and further cultured at 37 °C in Dulbecco's Modified Eagle Medium (DMEM), 5% foetal bovine serum (FBS) and 1% penicillin streptomycin, 95% humidity and 5% CO2. The rBMMSCs were incubated with C-T@Ti3C2 nanoworks at gradient concentrations  for 24 h. Cell viability was tested by a microplate reader  using the Cell Counting Kit-8 (CCK8) assay. For the live/dead staining assay, rBMMSCs were cultured in confocal dishes and incubated with C-T@Ti3C2 nanoworks at different concentrations  separately after washing with PBS twice. Next, the dishes were placed in the cell incubator for another 8 h. Then, the cells were stained with Calcein-AM/PI for 20 min before washing with PBS twice. The ultimate images were observed through CLSM.The surfaces of TiThe rBMMSCs were fixed on 24-well plates with 4% paraformaldehyde for 15 min and then washed 3 times with PBS and 0.2% Triton X-100 (W/V) in PBS for 15 min. The cells were then blocked with 5% bovine serum albumin (BSA) for 30 min. The cells were incubated for 12 h at 4 °C with primary antibodies , followed by a one-hour incubation with secondary antibodies. In addition, the nuclei of the cells were stained with DAPI. Images were captured with CLSM.3C2 MXene or C-T@Ti3C2 was assessed by detecting the mRNA expression of osteogenic genes, including collagen type I (COL-I), morphogenetic protein-2 (BMP2), osteomucin (OPN) and runt-related transcription Factor 2 (RUNX2). After osteogenic induction for 14 days, total RNA was extracted from stimulated rBMMSCs using TRIzol reagent, and the purified RNA was reverse transcribed into complementary DNA (cDNA) using PrimeScript RT Master Mix.The mRNA transcript levels of osteogenic-specific genes were detected via real-time quantitative reverse transcription PCR (RT‒qPCR). The osteogenic differentiation of rBMMSCs treated with Ti3C2 MXene and C-T@Ti3C2), the cells were collected on ice and washed with ice-cold PBS solution twice. To prepare the cell lysates, the samples were resuspended in RIPA buffer containing protease inhibitors. After incubation on ice for 30 min, the cell lysates were clarified by centrifugation  at 4 ℃ to remove debris, and the protein content was detected via the BCA protein assay kit. Equal amounts of extracts were diluted in sample buffer, subjected to 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Next, nonspecific binding was blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Immunoblotting was carried out with antibodies. The samples were immunoblotted with the following primary antibodies: β-actin (1:1000 dilution), BMP2 (1:1000 dilution), RUNX2 (1:1000 dilution), COL-I (1:1000 dilution) and OPN (1:1000 dilution) at 4 °C overnight. After washing three times with TBST, the PVDF membrane was incubated with the secondary antibody for 1 h at room temperature. The target proteins were detected using a Luminescent Immunoanalyzer . Anti-β-actin was used as a reference to monitor equal protein loading in the amounts of protein among samples.After 14 days of different treatments  for 14 days of observation. After 14 days, the mice were euthanized, and major tissues  along with blood samples were obtained for subsequent H&E staining for pathological evaluation. Routine blood and biochemical tests  were carried out using automatic blood cell analysers .Six C57BL/6JNifdc mice  were purchased from Vital River Laboratory Animal Technology in Beijing. The ethics of animal experiments were approved by the Ethics Committee on animal experiments of Shandong University Qilu Hospital . Six mice were randomly divided into the following groups: the control group was administered 100 μL of physiological saline through tail vein injection, and the other group was treated with 100 μL of C-T@TiMRSA (ATCC 43300) used for the infection model was in the mid-exponential growth phase at 107 CFU mL−1. Mice were anaesthetized using isoflurane by airway inhalation at a rate of 600 mL/min for 5 min, then their backs were depilated and disinfected with iodophor and 75 v/v% alcohol. A circular wound 8–10 mm in diameter was made on the back, and twenty mice were randomly divided into four groups . In the control and US only groups, we added approximately 60 µL to the wounds, while in the other two groups, we added equal amounts of C-T@Ti3C2 nanosheets. In particular, the wounds of the US only group and C-T@Ti3C2 + US group were implemented with ultrasound treatment  for 5 min each time every two days. The body weight and wound size were recorded at 1, 3, 5, 7, 9, and 11 days. All mice were sacrificed after 12 days, and the granulation tissue from the wounds and the major organs  were excised for further histological analysis. Mouse tissue sections were observed using an Olympus VS120 panoramic digital section scanning microscope .All animal surgical procedures were approved by the Ethics Committee on Animal Experiments of Shandong University Qilu Hospital in China . The mouse wound defects model was operated on male C57BL/6JNifdc mice , which were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The 3C2 and C-T@Ti3C2 + US). The skin was incised with sterilized surgical instruments, the muscle layer was opened, and the femur was exposed. Then, a 1.5–2 mm diameter hole was formed in the lower end of the femur with a bone drill. The suspension of MRSA (ATCC 43300) was injected into the bone marrow of four groups , and C-T@Ti3C2 nanoworks were added to the two groups (C-T@Ti3C2 and C-T@Ti3C2 + US), while an equal amount of saline was added to the other two groups (control and US only), and the wound was finally closed layer by layer. SDT treatments were performed every 2 days, with each rat treated at 1.5 Wcm−2, 50% duty cycle, 1 MHz, for 15 min. To assess the effectiveness of the treatment, five groups of rats were sacrificed at the second and fourth postoperative weeks, and the femur was removed to observe the condition of the bone tissue under micro-CT . All rats were sacrificed after four weeks, and the bone tissue was excised for further histological analysis with H&E, iNOS, TGF-β dye, Masson, COL-I and OCN. In addition, the major organs  were collected for H&E staining evaluation. Images of tissue slices were captured by an Olympus VS120 panoramic digital section scanning microscope .Sprague Dawley rats  were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The surgeries on animals were performed under the approval of the Ethics Committee on Animal Experiments of Shandong University Qilu Hospital . Fifteen rats were anaesthetized, shaved around the knee joint and disinfected with iodophor and alcohol, and they were randomly divided into five groups  and were repeated at least three times. The significance of the data in this work was analysed by two-tailed t test and two-way analysis of variance test using GraphPad Prism 9.3 (Graph Pad Software Inc.). The tests were regarded as statistically significant with signs of *Additional file 1: Fig. S1. Digital photographs of Ti3AlC2, Ti3C2 MXene, CaO2-PVP and CaO2-TiOx@Ti3C2 (C-T@Ti3C2). Fig. S2. SEM image of multilayered Ti3C2 MXene. Fig. S3. TEM, dark-field STEM images and corresponding element mappings  of C-T@Ti3C2 nanosheets. Fig. S4. DLS analysis of CaO2-PVP in water. Fig. S5. DLS analysis for Ti3C2 MXene in water and saline. Fig. S6. UV‒vis absorption spectra of time-dependent DPBF degradation under US treatment. Fig. S7. Digital photos of spread plates containing E. coli, S. aureus, and MRSA with various treatments. Fig. S8. Fluorescence microscopy images of MRSA stained with DCFH-DA after various treatments for ROS detection . Fig. S9. Haematological index of C57BL/6JNifdc mice intravenously administered C-T@Ti3C2 nanosheets dispersion for 14 d (n = 3). Data are presented as the mean ± SD. Routine blood parameters included red blood cells (RBCs), mean corpuscular haemoglobin (MCH), haemoglobin (HGB), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean platelet volume (MPV), number of neutrophils (GRAN), red cell distribution width (RDW), number of lymphocytes (LYMPH), haematocrit (HCT) and platelets (PLT). Biochemistry parameters included alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), and creatinine (CREA). Fig. S10. Histological assessments of the major organs  of C57BL/6JNifdc mice after intravenous injections with C-T@Ti3C2 nanonetworks for 14 d. Fig. S11. Histological assessments of the major organs  of C57BL/6JNifdc mice after different treatments in the MRSA-infected wound model. Fig. S12. Micro-CT images of femurs in different groups after 2 weeks of treatment. Fig. S13. Corresponding quantitative analysis showing BV/TV, Tb. Th and Tb.N. (n = 3) in different groups after surgery for 2 weeks. Data are presented as the mean ± SD. Statistical significance was calculated with a two-tailed t test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Fig. S14. Histological assessments  of bone tissue in different groups after 2 weeks of treatment. Fig. S15. Histological assessments  of the bone tissue in different groups after 2 weeks of treatment. Fig. S16. Histological assessments of the major organs  of SD rats from the bone defect model with MRSA infection at 2 weeks. Fig. S17. Histological assessments of the major organs  of SD rats from the bone defect model with MRSA infection at 4 weeks.


Here, we demonstrate an additional iron-sensing mechanism of NCOA4. Our results indicate that the insertion of an iron–sulfur (Fe–S) cluster enables preferential recognition of NCOA4 by the HERC2 (HECT and RLD domain containing E3 ubiquitin protein ligase 2) ubiquitin ligase in iron-replete conditions, resulting in degradation by the proteasome and subsequent inhibition of ferritinophagy. We also found that both condensation and ubiquitin-mediated degradation of NCOA4 can occur in the same cell, and the cellular oxygen tension determines the selection of these pathways. Fe–S cluster–mediated degradation of NCOA4 is enhanced under hypoxia, whereas NCOA4 forms condensates and degrades ferritin at higher oxygen levels. Considering the involvement of iron in oxygen handling, our findings demonstrate that the NCOA4–ferritin axis is another layer of cellular iron regulation in response to oxygen levels.To ensure proper utilization of iron and avoid its toxicity, cells are equipped with iron-sensing proteins to maintain cellular iron homeostasis. We showed previously that nuclear receptor coactivator 4 (NCOA4), a ferritin-specific autophagy adapter, intricately regulates the fate of ferritin; upon binding to Fe Iron is an essential trace element that functions as a cofactor in a wide variety of biological processes, such as oxygen delivery, ATP production, and DNA synthesis and repair, by binding to proteins , 2. Howe3+) ions  ions . In iron3+) ions , 11, 12.via canonical macroautophagy to release the stored iron  of NCOA4 induces condensation of the protein. The condensates are sequestered away from ferritin in the early phase of iron repletion, allowing ferritin to be accumulated for storage of excess iron and the maintenance of iron homeostasis. In prolonged iron-replete conditions, to prevent iron deficiency caused by excessive iron storage and reduced iron uptake by TfR1, NCOA4 condensates promote the delivery of ferritin to lysosomes. Therefore, NCOA4 functions as a crucial regulator of iron metabolism by coordinating with the IRP system to fine-tune the fate of ferritin  cells. Our results demonstrate that HERC2 preferentially recognizes NCOA4 harboring an Fe–S cluster, leading to degradation of NCOA4 and subsequent accumulation of ferritin. A NCOA4 mutant lacking the Fe–S cluster insertion formed condensates in HEK293T cells, suggesting that both FeA). In six of the seven iron-treated cell lines, the majority of NCOA4 was found in the insoluble fraction, suggesting iron-dependent condensation (A) (B). As mentioned previously, a previous report suggested that NCOA4 is recognized by the HERC2 ubiquitin ligase and subsequently degraded by the proteasome (B). Overall, these results indicate that iron treatment promotes the formation of insoluble NCOA4 condensates in most cell lines tested, with the exception of HEK293T cells.To determine whether iron-induced condensation of NCOA4 is a common mechanism determining the fate of ferritin, we first examined the effect of the cellular iron status on NCOA4 expression in seven human cell lines. The addition of ferric ammonium citrate (FAC) to the culture medium increased the amount of ferritin (FTH1) in all cell lines tested A. In sixensation A 16). B. BA). Intion (A) B. As menoteasome . Here, soteasome B. Overalvia a noncanonical autophagy pathway under prolonged iron repletion (A). Therefore, the fate of NCOA4 is differentially regulated by iron in a cell type–specific manner.We showed previously that insoluble NCOA4 condensates deliver ferritin to lysosomes epletion . Consistepletion , 14, 16,epletion A. TherefC). We showed previously that binding of Fe3+ to the IDR (amino acids 167–334) of NCOA4 promotes condensation of the protein. Thus, to investigate what form of iron binds to amino acids 383 to 522 of NCOA4 to enable its recognition by HERC2, we expressed and purified a glutathione S-transferase (GST)-fused NCOA4 (amino acids 383–522) protein using a bacterial expression system. UV–visible spectrometry of GST-NCOA4 (amino acids 383–522) showed peaks at 330 and 420 nm as well as a broad shoulder at a longer wavelength , a spectrum that is similar to that of 2Fe–2S cluster proteins (via four cysteine (Cys) or histidine residues  are conserved among NCOA4 orthologs (A). UV–visible spectrometry analyses of GST-NCOA4 (amino acids 383–522) mutants in which one of the five Cys residues was substituted with serine revealed that the C404S, C410S, C416S, and C422S mutants exhibited attenuated absorption at 330 and 420 nm, whereas the C418S mutant did not (B and C and S2A). Furthermore, simultaneous mutation of C404, C410, C416, and C422 to serine abrogated the characteristic spectrum almost completely (B and C). Collectively, these results suggest that iron binds to the HERC2 binding region (amino acids 383–522) of NCOA4 as an Fe–S cluster.To probe the mechanisms underlying the two modes of regulation of NCOA4 under iron-replete conditions, we examined the regulation of NCOA4 by HERC2 in more detail. A previous study showed that upon binding to iron, amino acids 383 to 522 of NCOA4 are preferentially recognized by HERC2 . Here, aFig. S1proteins . Regardlresidues . An in srthologs A. UV–vismpletely , B and CD and E). In addition, an in vitro binding assay using purified GST-NCOA4 (amino acids 383–522) mutants and HERC2 (amino acids 2540–2700), as used in a previous study (F).To examine their cellular fate, the NCOA4 mutants were expressed exogenously in HEK293T cells. The levels of the four NCOA4 mutants that were unable to bind an Fe–S cluster  were not decreased upon FAC treatment, and these mutants did not interact with the HERC2 (amino acids 2292–2923) fragment , D and Eus study , revealeus study F.G). In addition, knockdown of ISCU affected Fe–S synthesis, as indicated by an increase in the amount of IRP2, which is recognized by 2Fe–2S ligated FBXL5 in the SCFFBXL5 ubiquitin ligase complex and is destined for degradation in iron-replete conditions . Taken together, these results suggest that HERC2 preferentially recognizes and degrades NCOA4 harboring an Fe–S cluster.Next, we investigated the fate of NCOA4 following the disruption of Fe–S cluster biogenesis. The generation of Fe–S clusters is initiated in mitochondria, and the first step of biogenesis involves the scaffold protein ISCU , 20, 21.Fig. 2nditions . Further3+ to its IDR, NCOA4 forms insoluble condensates in mouse embryonic fibroblasts (MEFs) and other cell lines (A) (A). Lysis of the cells with SDS lysis buffer confirmed that iron treatment reduced the total amount of WT NCOA4 but did not affect that of NCOA4 C410A (B). These results indicate that disrupting the insertion of an Fe–S cluster into NCOA4 does not alter the total amount of endogenous NCOA4 in HEK293T cells but does alter its biochemical characteristics to match those of WT NCOA4 in MEFs and other cell lines (A) . T. T3+ to ines (A) . In viewines (A)  23, 24), 243+ toines (A) A. Lysis A4 C410A B. These ll lines A (16).FiC and D). A faster migrating FTH1 band was observed in immunoblot analyses of iron-replete HEK293T cells expressing endogenous NCOA4 C410A (A and B). Immunoprecipitation and immunoblotting analyses using two different anti-FTH1 antibodies confirmed that the faster migrating band was partially degraded ferritin in the lysosomes, as reported in previous studies (E and F). These findings indicate that insoluble NCOA4 condensates deliver ferritin to lysosomes upon inhibition of Fe–S cluster insertion in HEK293T cells. Overall, these results demonstrate that both Fe–S cluster–mediated NCOA4 degradation and iron-induced NCOA4 condensation can operate in iron-replete HEK293T cells, but the former seems to predominate.Consistent with our previous observation that NCOA4 in the insoluble fraction forms dot-like structures , endogenA4 C410A , A and B studies , 14, 16. studies , E and FA and B), indicating that a defect in noncanonical ferritinophagy in WT cells might induce a response typical of cellular iron deficiency in normal culture medium conditions. By contrast, the observed reduction in the level of TfR1 and increase in the level of ferritin in cells expressing NCOA4 C410A likely counteracted the ferritinophagy-mediated increase in iron delivery by reducing iron uptake and augmenting iron storage (A and B). We then added iron to the culture medium of the WT and NCOA4 C410A HEK293T cells and examined time-dependent changes in the expression levels of FBXL5 and iron-related proteins. The expression level of FBXL5 increased rapidly in WT cells and C410A cells at an early time point (3 h) after iron administration (C and D). However, the amount of FBXL5 in WT cells was decreased continuously over the observation period (24 h), whereas FBXL5 was maintained at higher levels in C410A cells at later time points. In addition, a faster migrating band representing a partial degradation product of ferritin was observed in C410A cells but not WT cells (C). In accordance with the belief that FBXL5 expression reflects the amount of available cellular iron, mitochondrial iron levels were increased in HEK293T C410A cells 24 h after iron administration (E). Overall, these results indicate that NCOA4-dependent noncanonical ferritinophagy modulates cellular iron metabolism by providing ferritin-stored iron to cells in steady-state conditions or at late time points after iron administration.Next, we evaluated the effect of NCOA4-mediated noncanonical ferritinophagy on cellular iron metabolism. The expression level of the iron sensor FBXL5 is increased in iron-replete conditions, and FBXL5 mediates the degradation of IRP2 , 26. The storage , A and Bstration , C and DWT cells C. In accstration E. Overal3+ (2+) ion to the ferric (Fe3+) ion requires oxygen (2+). In addition, several studies have shown that hypoxia rescues Fe–S availability in cells by protecting Fe–S clusters against molecular oxygen (2) and found that the amount of NCOA4 in the insoluble fraction was lower in iron-replete MEFs at 1% versus 21% O2 (A). As mentioned previously, most NCOA4 was detected in the insoluble fraction of the majority of human-derived cell lines examined here (A). Consistent with this finding, immunofluorescence analysis confirmed that NCOA4 formed condensates in iron-treated HepG2 cells (B). As expected, NCOA4 was downregulated (A), and NCOA4 condensates were not overtly observed (B and C) in iron-replete HepG2 cells under hypoxia (1% O2). Correspondingly, the faster migrating partial degradation products of ferritin disappeared in hypoxic conditions (A).Next, we examined whether HERC2-mediated degradation of NCOA4 occurs in cell lines other than HEK293T. In our previous study, we found that NCOA4 preferentially forms insoluble condensates in iron-treated MEFs upon binding to Fe3+ . Becauses oxygen , most cyr oxygen , 29. Thus 21% O2 A. As menned here A. ConsisG2 cells B. As expegulated A, and NCobserved , B and Cnditions A.Figure via recognition of Fe–S cluster–containing NCOA4 by HERC2, we depleted HERC2 expression using specific siRNAs. Under hypoxic conditions, HERC2 depletion substantially increased the amount of insoluble NCOA4 (B). Moreover, knockdown of ISCU effectively inhibited NCOA4 degradation under hypoxic and iron-replete conditions, whereas it did not affect iron-dependent NCOA4 condensation under normoxic (21% O2) conditions (D). These results confirm that Fe–S cluster biogenesis is dispensable for iron-dependent NCOA4 condensation, and that reduced oxygen tension prioritizes Fe–S cluster–dependent NCOA4 degradation in cells. Notably, as observed in HEK293T cells, our findings demonstrate that NCOA4 can be regulated by both Fe–S cluster–mediated degradation and Fe3+-mediated condensation in iron-treated HepG2 cells, and the latter was predominant in normoxic conditions.To examine whether the decrease in NCOA4 expression under hypoxic and iron-replete conditions is mediated le NCOA4 B. Moreovnditions D. These C). We supposed that the sensitivity to oxygen levels may be altered in HEK293T cells and thus speculated that NCOA4 might form insoluble condensates at high oxygen tension. To test this possibility, we cultivated HEK293T cells under hyperoxic conditions (70% O2). A substantial amount of NCOA4 was located in the insoluble fraction of iron-treated HEK293T cells cultured under hyperoxia, and immunofluorescence analysis confirmed that NCOA4 formed condensates in the hyperoxic and iron-replete condition (E–G). Moreover, a partial degradation product of ferritin was observed in iron-treated HEK293T cells cultured under hyperoxia (E).As described previously, Fe–S cluster–dependent degradation of NCOA4 was predominant in iron-treated HEK293T cells cultured under normoxia . Since wFig. S4ondition , E–G. Moyperoxia E.2) (D) but not those cultured under hypoxia (1% O2) (I). Consistently, a faster migrating FTH1 band disappeared under hypoxia. Cells expressing NCOA4 ΔIDR were generated by reconstituting NCOA4 ΔIDR into NCOA4 KO HEK293T cells (J and K). Upon iron administration under hyperoxia, partially degraded ferritin and insoluble NCOA4 were detected in cells expressing WT NCOA4 but not in those expressing NCOA4 ΔIDR (L), whereas loss of IDR did not overtly affect NCOA4 degradation under normoxia (M). Taken together, these findings demonstrate that while NCOA4 is degraded in an Fe–S cluster–dependent manner in HEK293T cells under low oxygen tension (including normoxia), it forms insoluble condensates, and ferritin is degraded under high oxygen tension. Although the threshold of oxygen tension that determines the fate of NCOA4 differs among cells, and it is unclear why HERC2-mediated degradation operates predominantly in normoxic HEK293T cells  as well as that of the NCOA4 ΔIDR mutant, which cannot bind to ferric iron. NCOA4 C410A was detected in the insoluble fraction of iron-treated HEK293T cells cultured under hyperoxia (70% O2) D but not (1% O2) I. Consis3T cells , J and KOA4 ΔIDR L, whereanormoxia M. Taken ells see  section,of NCOA4 .Figure 6via NCOA4-mediated macroautophagy, promoting the release of stored iron -induced NCOA4 condensation , which are the central regulators of iron metabolism in Saccharomyces cerevisiae (A), the sensing mechanism may play an important role in maintaining iron homeostasis in various organisms. A recent study reported that NCOA4-dependent ferritinophagy prioritizes the supply of iron to Fe–S cluster synthesis to maintain pancreatic cancer proliferation . Thus, in cells under high oxygen tension, it seems necessary to deliver available iron via ferritin autophagy, and to simultaneously protect cells against iron toxicity and the associated oxidative stress by increasing the amount of ferritin, a process that is achieved by coordinated regulation of the FBXL5–IRP system and the NCOA4–ferritin axis. In our experiments, the threshold of oxygen tension varied between cell lines, which might be attributable to differences in the intracellular reductive milieu, destruction of Fe–S clusters, and/or the generation of ferric iron.Iron is often utilized as an electron carrier, especially in enzymatic reactions catalyzing oxidation or reduction in organisms . As mentP system , A and Bvia ferritinophagy in some pathological settings. Further studies are required to understand the relationships between ferroptosis and NCOA4-mediated ferritinophagy in various oxygen level conditions in vivo.The amount of available iron is a critical component determining the sensitivity of ferroptosis , 7, and NCOA4 was cloned by RT–PCR using mRNA isolated from HEK293 cells. The following variants were generated: C404S, C410S, C416S, C418S, C422S, and 4CS . The human HERC2 complementary DNA was a gift from Dr Tomohiko Ohta (St Marianna University School of Medicine). The complementary DNAs were ligated to the appropriate epitope-tag sequences and then cloned into the pcDNA3.2, pMX IRES puro FLAG, and pGEX6p-1 vectors. An sgRNA targeting NCOA4 (5′-AAAAGGGTCATTACCTCTCC-3′) was cloned into the PX458 plasmid (Addgene) to produce NCOA4 KO cells.The open reading frame of human The following antibodies were used in this study: anti-ferritin , anti-FTH1 , anti-NCOA4 , anti-NCOA4 , anti-HERC2 , anti-TAX1BP1 , anti-IRP2 , anti-IRP2 , anti-FBXL5 , anti-TfR1 , anti-ISCU , anti-HIF1 , anti-FECH , anti-β-actin , anti-tubulin , anti-FLAG , anti-DDDDK , anti-myc , anti-H2B , anti-laminB1 , horseradish peroxidase–linked anti-mouse immunoglobulin G , and horseradish peroxidase–linked anti-rabbit immunoglobulin G .The following reagents were used in this study: FAC , deferoxamine mesilate , E64d , and pepstatin A .2 in humidified air. A multigas incubator  was used to culture cells under 1% O2 conditions. For culture under hyperoxia, cell culture plates or dishes were placed into a plastic bag , and the bag was sealed completely with a clip. The air was then flushed out, and the bag was refilled with a high oxygen gas mixture . Subsequently, the gas mixture was flushed out, and the step was repeated twice. Finally, the bag was filled with the high oxygen gas mixture and placed in a 37 °C incubator.MEFs and the HepG2, U2OS, HeLa, HEK293T, RD4, UOK111, and MCF7 cell lines were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin, and 100 μg/ml streptomycin and were maintained at 37 °C under 7.5% COAppropriate pcDNA3.2 plasmids were transfected into HEK293T cells using PEI MAX (Polysciences). The siRNAs were transfected into cells using Lipofectamine RNAiMAX. Plasmid DNA and siRNAs were cotransfected into cells using Lipofectamine 2000.HEK293T cells were transfected with the PE2 plasmid, epegRNA plasmid, sgRNA plasmid (for PE3), and pcDNA3.2 GFP plasmid (for sorting) using Lipofectamine 2000. After 24 h, GFP-positive cells were selected using a FACS Aria III cell sorter. Fifty cells were seeded into a 10 cm dish, and single clones were obtained using cloning rings. The C410A mutation was confirmed by sequencing of PCR products generated from genomic DNA.NCOA4 was transfected into HEK293T cells using PEI MAX. After 24 h, GFP-expressing cells were isolated using an FACS Aria III sorter (BD Biosciences), and single clones were obtained using cloning rings.To generate NCOA4 KO cells, a PX458 plasmid encoding a sgRNA targeting Appropriate pMXs plasmids and the pVSV-G plasmid were transfected into GP2-293 packaging cells (Takara Bio) using PEI MAX. After 48 h, retrovirus in the culture medium was collected and passed through a 0.45 μm filter. NCOA4 KO cells were infected with the retrovirus in the presence of polybrene (10 μg/ml) for 16 h. The infected cells were selected using puromycin.For preparation of total cell lysates, cells were lysed in 1× sample buffer . The cell lysate was sonicated to shear DNA and then boiled at 95 °C for 10 min.g for 20 min at 4 °C. After the addition of 4× sample buffer, the sample was boiled at 95 °C for 5 min. The insoluble pellet was washed once with Triton buffer and resuspended in 1× sample buffer. The insoluble sample was then sonicated and boiled at 95 °C for 10 min.To prepare soluble and insoluble samples, cells were lysed in Triton buffer  supplemented with 2 mM PMSF and protease inhibitor cocktail (Roche). After incubation on ice for 20 min, the soluble extract was collected after centrifugation at 20,000Samples were resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking in Tris-buffered saline containing 0.1% Tween-20 and 5% (w/v) nonfat dry milk, membranes were incubated with the appropriate primary antibodies, followed by the corresponding secondary antibodies. Bands were visualized by enhanced chemiluminescence, and the signals were detected on a LAS4000 mini-instrument .To detect the interaction between NCOA4 and HERC2, an anti-FLAG antibody was incubated with the Triton X-100 soluble cell lysate for 90 min at 4 °C. After the addition of Protein A beads, the sample was incubated with rotation for 60 min at 4 °C, followed by four washes with Triton buffer and two washes with 20 mM Tris–HCl (pH 8.0). Immunoprecipitated proteins were denatured by the addition of 2× sample buffer and boiling at 95 °C for 5 min.An anti-ferritin antibody was used to isolate endogenous ferritin from Triton X-100 soluble cell lysates. An anti-FLAG antibody was used as a control. Dynabeads Protein A (Invitrogen) conjugated with the antibodies were added to the soluble lysates, and the samples were incubated with rotation for 60 min at 4 °C, followed by four washes with Triton buffer. Immunoprecipitated proteins were denatured by the addition of 2× sample buffer and boiling at 95 °C for 5 min.Escherichia coli strain BL21-CodonPlus (DE3)-RIPL. When WT GST-NCOA4 (amino acids 383–522), and its mutants were purified, bacteria were grown in LB medium supplemented with 121 μg/ml Cys, 25 μg/ml FAC, and 50 μg/ml ampicillin. The bacteria were grown to an absorbance o 0.6 at 600 nm for 30 °C before induction with 100 μM IPTG at 16 °C for 18 h. Next, the bacteria were collected by centrifugation and lysed by sonication in GST lysis buffer  supplemented with 2 mM PMSF and protease inhibitor cocktail. The sample was centrifuged at 23,000g for 30 min at 4 °C, and the supernatant was mixed with Glutathione Sepharose 4 Fast Flow  for 1 h at 4 °C. The beads were washed with GST lysis buffer, and GST protein was eluted with glutathione buffer . The eluted sample was desalted using a PD-10 column and GST lysis buffer.Proteins were expressed in and purified from g for 30 min at 4 °C, and the supernatant was mixed with nickel–nitrilotriacetic acid beads (QIAGEN) for 1 h at 4 °C. The beads were then washed three times with wash buffer 1  and twice with wash buffer 2 . The His-tagged protein was eluted with His elution buffer , and the eluted sample was desalted using a PD-10 column and a buffer comprising 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 1 mM DTT.When His-HERC2 (amino acids 2540–2700) was purified, bacteria were grown in LB medium supplemented with 50 μg/ml ampicillin. The bacteria were grown to an absorbance of 0.6 at 600 nm at 30 °C before induction with 100 μM IPTG at 16 °C for 18 h. Next, the bacteria were collected by centrifugation and lysed by sonication in His lysis buffer  supplemented with 2 mM PMSF, 2 mM 2-ME, and protease inhibitor cocktail. The sample was centrifuged at 23,000UV–visible absorption spectra were measured at a range of 250 to 800 nm using a V-750 UV–Visible Spectrophotometer (JASCO) at room temperature. The protein samples (50 μl of 0.8–2.0 mg/ml) were in buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 1 mM DTT.GST or GST-tagged proteins (2 μg) and His-HERC2 (amino acids 2540–2700) (2.8 μg) were mixed in 800 μl of binding buffer . After the addition of 10 μl of Glutathione Sepharose 4 Fast Flow beads, the samples were incubated for 1 h at 4 °C with rotation. The beads were then washed four times with binding buffer and twice with PBS containing 1 mM DTT. Proteins were eluted with 2× sample buffer and boiled at 95 °C for 5 min.Cells were seeded onto collagen-coated cover glasses at least 24 h before use. The cells were then fixed with 4% formaldehyde in PBS for 20 min at room temperature. The fixed cells were washed three times with PBS and then permeabilized for 3 min with 0.1% Triton X-100 in PBS. After washing three times with PBS, the cells were incubated with blocking buffer  for 1 h at room temperature. The samples were then incubated with the primary antibodies in blocking buffer for 1 h at room temperature or overnight at 4 °C, washed three times in PBS, and incubated with the secondary antibodies in blocking buffer for 1 h at room temperature. Subsequently, the samples were washed again with PBS and then stained with 0.1 μg/ml 4′,6-diamidino-2-phenylindole in PBS for 5 min at room temperature. Finally, the samples were washed three times with PBS and mounted with Fluoro-KEEPER Antifade Reagent . Confocal fluorescence images were acquired with an IX81 inverted microscope (Olympus) equipped with an FV1000 confocal imaging system (Olympus) and a 60×/1.42 numerical aperture oil objective lens . Images were analyzed and quantified using the ImageJ (NIH) and R software packages (R Core Team).Cells were seeded into collagen-coated glass-bottomed dishes and treated with FAC as indicated. After washing with PBS, the cells were incubated with 5 μM Mito-FerroGreen (Dojindo) in Hanks’ balanced salt solution at 37 °C for 30 min and then washed twice with Hanks’ balanced salt solution. Confocal fluorescence images were acquired as mentioned previously. Images were analyzed and quantified using the ImageJ and R software packages.t test, Welch’s two-sample t test, one-way ANOVA with Dunnett post hoc test, or Kruskal–Wallis ANOVA with Dunn’s multiple comparison test, as indicated in each figure legend. Data visualization and statistical analyses were performed using R software packages. All experiments were performed at least twice, and consistent results were obtained.All bar graphs show the mean ± SD. In boxplots, bold lines indicate the median, boxes indicate the interquartile range (25–75th percentile), and whiskers indicate 1.5 times the interquartile range. Statistical significance was analyzed by one-sample This study includes no data deposited in external repositories.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children’s quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality.Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function.Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol  were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing.Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues. The impairment of both the central and peripheral nervous system is a major cause of mortality and disability . It variCongenital neuropathic motility disorders of the gut are the reason behind rare and typically severe diseases in pediatric patients. These diseases vary from a local hypo- or aganglionosis of the terminal colon to infestation of the total gut. Whereas the typical disease in this field, Hirschsprung’s disease, affects one in 5000 live births , a complThe enteric nervous system (ENS) harbors a potent and easily accessible autologous source of enteric neural stem cells  that are, where flash-freezing is applied.In general, cryopreservation strategies usually require a slow and continuous decrease of temperature during the freezing process to avoid structural damage caused by the formation of ice crystals throughout physical changes . The twoIn this context slow programmable freezing describes the gradual freezing with a controlled stepwise reduction of temperature . Here, lIn comparison, the process of vitrification (flash-freezing in the range of mega kelvins per second) describes a separate physical phenomenon that occurs within a small range of temperature described as the “glass transition temperature” around −80 to −130°C .In vitrification, samples change from liquid to solid state without ice crystal formation . For theAs an alternative to solutions containing DMSO, the cryopreservative agent StemCell Keep™ has been developed by Matsumura et al. . The addIn this study we aimed to find a method that minimizes harmful freezing effects with minimal influence on cell properties and at the same time allows cryopreservation of a large number of cells for future transplantation. Hence, we compared slow and rapid cryopreservation methods with different cryopreservation media for the storage of ENS-derived neurospheres (ENSdN). Furthermore, we investigated the impact of different freezing and thawing protocols upon enteric neural stem cell survival, protein and gene expression. Finally, using calcium microfluorimetry we demonstrated that cell functionality remained unaffected by the freezing procedure.Pregnant Sprague Dawley rats were obtained from JANVIER LABS and newborn pups used for experiments. This study was carried out in strict accordance with the recommendations for the care and use of laboratory animals of the German animal protection law. Animal experiments were approved by the internal Veterinary Inspection Office in Mannheim. Authorization number: I-29/23 R.6 cells/25 cm2 flask). After 2 days of culture in vitro, the first floating neurospheres were seen, while neurons and radiating glial cells expanded at the bottom (Newborn rat pups (age 4–6 days) were decapitated, dissected and the whole intestine removed and immediately stored on ice in MEM  containing antibiotics as previously described . Then ste bottom . Cells wENSdN were either frozen at a controlled rate of slow freezing or based on a flash-freezing process. We used four different media with/without DMSO and maintained ENSdN for 2 weeks at −180°C .• Condition 1: M1 = 90% Fetal Calf Serum (FCS) + 10% DMSO.• Condition 2: M2 = 20% FCS +70% culture medium , supplemented with 2% B27- , 1% albumin (Sigma-Aldrich), 0.25% 2-mercaptoethanol , 0.12% glutamine , 1% penicillin/streptomycin (PAA Laboratories)) + 10% DMSO.• Condition 3: M3 = 90% culture medium +10% DMSO.• Condition 4: M4 = StemCell Keep™ (BioVerde) without DMSO, serum or protein.In preparation for freezing ENSdN were centrifuged, the supernatant removed and the cell pellet was resuspended in the corresponding freezing medium.6 cells in DMSO containing cryopreservation medium and the slow-freezing method with a cooling rate of 1 °C/min in a freezing container.For conditions 1–3 we used approximately 1.5 × 10. We used 1.5 × 106 cells in five cryovials, containing 200 µL cryopreservation medium (M4) each, that were immediately frozen by superfast cooling in liquid nitrogen. Cells were stored in liquid nitrogen for 2 weeks before using them for in vitro experiments.Condition 4 combines a DMSO and FCS-free cryopreservation medium  with the process of flash-freezing (vitrification)Condition 1–3:ENSdN were thawed in the cryovial in a water bath at 37 °C and transferred in culture medium.Condition 4:For thawing of cells treated under condition M4, 1 mL of culture medium (37 °C) was added to each cryovial, resuspended and then transferred to 4 mL culture medium.After 3 days of proliferative culture, the floating ENSdN (fresh or thawed) were harvested and utilized either for differentiation directly or dissociated in accutase (PAA Laboratories GmbH) for 20 min and triturated using consecutively 23 and 27 gauge needles, 3 times each. To avoid shear stress, aspiration of the tissues was performed slowly. After dissociation, the cells were centrifuged , the supernatant removed and the pellet resuspended in 1 mL medium. The intact or dissociated ENSdN were plated on poly-L-lysine-laminin coated coverslips in 24 or 12 well plates (Becton Dickinson) in culture medium for differentiation  supplemented with 2% B27+ , 1% albumin (Sigma-Aldrich), 0.25% 2-mercaptoethanol , 0.12% glutamine , GDNF , neuturin (10 ng/mL Tebue), gentamycin and metronidazole (100 μg/mL) and 1% penicillin/streptomycin (PAA Laboratories GmbH)). In case of functional imaging, 10% of FCS (Gibco) was added to the medium in order to help cell attachment.2. Then the staining solution was removed and the samples were washed two times with PBS. Samples were fixed with 4% formaldehyde for 10 min. The cultures were washed twice with PBS and then incubated with DAPI , which was used as nuclear stain.For cell survival analysis, the cells were stained with propidium iodide  for 10 min at 37°C with 5% COTo quantify the cells, PI stained (dead cells) cell numbers were assessed and related to the total cell number, based on nuclear DAPI stains .Total cellular RNA from freshly isolated myenteric plexus was extracted directly (d0) or after cultivation in proliferation medium for 3 days (d3) using an RNA Micro Isolation Kit (Bioline) following the manufacturer’s protocol. RNA concentration was determined spectrophotometrically with the infinite M200 micro plate reader (Tecan) and RNA quality assessment was tested with RNA 6000 Nano Kit (Agilent Technologies) and the Agilent Bioanalyzer 2100 (Agilent Technologies). BioScript TM was used to generate cDNAs (Bioline). For real time PCR the SensiMixSYBR Low-Rox Kit (Bioline) was used on a MX3005 (Stratagene). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18 sRNA primers were used as an internal standard. The PCR conditions were as follows: initial denaturation 10 min, 95°C, 40 cycles of denaturation, 30 s, 95°C; annealing 30 s, 58°C; 30 s, 72°C. Primer sequences were used as listed below . Data we® 488 anti-mouse-IgG, Alexa® 488 anti-rabbit-IgG, Alexa® 488 anti-goat-IgG for 4 h at room temperature (RT). 4′, 6-diamidino-2-phenylindole (DAPI)  was used as nuclear stain. Cell numbers were related to an overall cell count based on DAPI stainings.Differentiated cultures were fixed with formaldehyde, followed by 45 min permeabilization in 0.5% triton X-100 and then blocked in 10% normal goat serum (DAKO) for 1 h. The corresponding primary antibodies for Nestin , Nanog , Sox2 , Oct4 , ß-Tubulin III , GFAP , S100 , PGP9.5  and p75  were added and incubated over night at 4 °C. Samples were washed three times with PBS and incubated with the corresponding secondary antibodies  and the analysis software BZ-II Analyzer (Keyence). Image processing was performed with the open source program Gimp.2+ measurements of ENSdN were performed after the differentiation of unfrozen or frozen neurospheres treated with condition M1 (ENSdN_frM1). For differentiation, cells were plated on PLL  and ECM  coated coverslips for better adhesion in 12-well plates until density was about 1/3 of the coverslip. For measurements of [Ca2+]i, cells were loaded with 3 µL FURA-2AM  and the same amount (in µl) of Pluronic F-12775  for 45–60 min in Tyrode’s solution . After washing with Tyrode’s solution for 30 min, cells were mounted on the stage of an inverted microscope  in an open bath chamber  and superfused by Tyrode’s solution (1–3 mL/min) at room temperature (22°C–24 °C). Fluorescent signals of the cells were evoked by alternating illumination (340/380 nm wavelength) and respective fluorescent signals (510 nm) detected by an ORCA-R2 camera (Hamamatsu Photonics). The ratio of emission for 340/380 nm excitation was used as relative change in [Ca2+]i. For analysis, cells were marked as regions of interest (ROIs) and changes in [Ca2+]i during the period of stimulus application were calculated as difference between maximum value and basal value of corresponding cells. Cells were assorted to subgroups according to responsiveness to different stimuli , K+ solution (140 mM)).Single-cell intracellular Cap-values <0.05 were considered significant .Results are demonstrated as mean ± standard error of the mean (SEM). Data were visually evaluated for normal distribution using quantile-quantile (Q-Q) plots and tested for statistical significance using unpaired Student’s t-test for unequal variances (two-tailed). The influence of freezing on the survival of enteric nervous system derived neurospheres (ENSdN) was assessed using a live-dead-assay, based on combined propidium iodide (PI) and DAPI staining. Number of ENSdN before  and after freezing  was analyzed ._fr) up to 87.2 (±16.8) or 88.2 (±10.5) respectively. After freezing with M3 the outcome was even worse and delivered only 60.0 (±12.9) surviving spheres. M4 protocol nearly led to a complete loss of tissue with a number of only 7.3 (±0.7) surviving ENSdN_fr .Our results show that the process of freezing significantly reduced the number of surviving ENSdN. The average number of living intact ENSdN was 219.5 (±47.1) before freezing. Both M1 and M2 freezing protocols resulted in a similar reduction of spheres (ENSdN_diss) the number of surviving cells after freezing with protocol M1-M4 was also reduced significantly. We counted a total number of 5760.5 (±416.3) living cells per coverslips for unfrozen ENSdN_diss, compared to 2635.2 (±625.4), 2795.3 (±298.5) and 3425.0 (±516.3) cells for ENSdN_fr+dissM1, M2, and M3, respectively . Cells frozen following the M4 procedure did not survive at all after dissociation  in both intact and dissociated neurospheres. Overall survival was slightly higher when cells were cultured dissociated.DMSO is known to induce differentiation in embryonic stem cells and initiates downregulation of stem cell markers such as Nanog, Nestin, Sox-2 and Oct-4 in a concentration-dependent manner . In ordeIn a first step, change in RNA expression during the process of cell cultivation was investigated. RNA expression profile from freshly isolated myenteric plexus (d0) was compared to cells that were cultured in proliferation medium for 3 days d3; .p = 0.0027 and p = 0.0049; SEM). GFAP expression increased from 1.23 (±0.31) to 6.98 (±1.39) and the expression of the two stem cell marker Oct4 and Sox2 was increased from 0.76 (±0.16) to 2.93 (±0.34) and 1.57 (±0.32) to 2.97 (±0.24) .We could observe that growing cells in proliferation culture promoted relative mRNA expression of PGP9.5 and ß-TubulinIII from 0.60 (±0.14) to 1.48 (±0.14) and from 0.88 (±0.08) to 2.68 (±0.31), respectively (_fr) were compared to the expression profile of intact ENSdN after 3 days of proliferation culture. For ENSdN_frM1 and M2 we observed a significant elevation of Sox2 mRNA levels for M1 (p = 0.0237) and ß-TubulinIII for M2  to 5.08 (±0.38) and from 2.68 (±0.31) to 9.92 (±1.43); p = 0.0017 and 0.0177. Glial marker expression was also promoted from 6.98 (±1.39) to 23.22 (±4.44) for GFAP and from 1.30 (±0.22) to 2.63 (±0.35) for S100; p = 0.0404 and 0.0325. Expression of stem cell markers was significantly increased from 1.46 (±0.24) to 3.88 (±0.35) for p75, 1.82 (±0.05) to 4.76 (±0.45) for Nestin and 2.97 (±0.24) to 18.86 (±4.11) for Sox2, p = 0.0026, 0.0093 and 0.0403. When the M4 freezing condition was used, similar mRNA expression profiles as for condition M3 could be observed with a significant increase in stem cell marker p75 (p = 0.0012). However, the glial and neuronal marker expression missed the level of significance.In order to investigate the additional effect of different freezing methods on mRNA expression of ENSdN, mRNA expression profiles of frozen ENSdN  and compared to frozen neurospheres (ENSdN_fr) or cells (ENSdN_fr+diss)  was assessed using immunohistochemical stainings. ENSdN were cultured and differentiated either as intact neurospheres (ENSdN) or after dissociation as single cells (ENSdNfr+diss) ._frM1) did not alter protein expression at all, while M2 protocol led to a significant upregulation of Nanog (66.29% (±2.03%), p = 0.0142). ENSdN_frM3 displayed a significant upregulation of Oct4 (88.84% (±2.76%), p = 0.0398), combined with a significant upregulation of S100 (76.13% (±2.94%), p = 0.0194; _frM4, no protein profiles could be assessed for this condition.For ENSdN we could observe the following protein distribution: 37.48% (±2.42%) of the cells were PGP9.5-positive, 55.43% (±3.20%) displayed a ß-TubulinIII staining, 65.01% (±5.85%) were GFAP-positive and 61.35% (±4.06%) expressed the S100 protein. Concerning neural stem cell markers we found 47.32% (±5.22%) of the cells p75-positive, 74.56% (±10.82%) displayed expression of nestin, while 52.29% (±3.92%), 96.41% (±0.67%) and 84.84% (±5.22%) presented Nanog, Oct4 and Sox2, respectively. Freezing of ENSdN with condition M1 (ENSdN_diss).To investigate the impact of dissociation of neurospheres prior to differentiation, we likewise investigated the protein patterns of dissociated ENSdN .When we compared protein expression of ENSdN and ENSdN_fr+dissM1, M2, M3) and unfrozen cultures of dissociated cells (ENSdN_diss), except for ENSdN_fr+dissM1, where Sox2 was significantly upregulated to (71.59% (±8.44%), p = 0.0231, data not shown).In contrast to protein expression patterns in intact ENSdN cultures, there were no significant differences between frozen  were plated, cultivated and differentiated on glass coverslips for 3 days, until they formed a layer and networks of neurons and glia in ganglion-like structures that were visible under differential interference contrast (DIC) microscopy.To investigate the effect of freezing and thawing on cell function, we compared intracellular calcium responses using calcium imaging. Since freezing condition M1 showed good survival rates and did not significantly alter gene as well as protein expression patterns in differentiated ENSdN in our setting, we chose ENSdN_frM1 were tested for differential calcium responses to nicotine (50 µM), acetylcholine (50 µM), ATP (100 µM) and high K+ (140 mM) for 60 s sequentially  ROIs/coverslip for ENSdN vs n = 8 coverslips; 57.6 (±5.6) ROIs/coverslip for ENSdN_frM1). However, subsequent application of nicotine, acetylcholine, K+ and ATP revealed different response patterns of cells. According to the response pattern of each single cell, cells were assorted to four different groups  also displayed a rise in [Ca2+]i when challenged with high K+ and ATP subsequently. Group III: Cells responding to high K+ (without prior response to nicotine and acetylcholine) also displayed a rise in [Ca2+]i when challenged with ATP subsequently. Group IV: Cells responding to ATP only.Group I: Cells responding to nicotine also displayed a rise in [Ca2+]i (quantified as change in ratio 340/380 [510 nm]) for ENSdN and ENSdN_frM1  for ENSdN and 48.7 (±4.8%) for ENSdN_frM1—a difference that was considered significant (p = 0.007). A rise in [Ca2+]i following stimulation with acetylcholine in cells that did not respond to a prior application of nicotine—representing cell group II—could be observed in 23.4 (±4.9%) cells for ENSdN and in 6.3 (±1.2%) cells for M1 group (p = 0.006).Regarding the average number of cells in each group we could observe differences for ENSdN and ENSdN_frM1 . With specific modifications survival could be improved significantly . In currUnder the slow freezing conditions used in our study, we achieved survival rates between 27% and 40% for intact neurospheres and 45%–60% in dissociated cells, demonstrating that—in terms of cell survival—these methods seem to be an appropriate alternative to the cryopreservation of ENSdN. Supporting our observations, similar results have been shown by Imaizumi et al., who also observed higher survival rates for human pluripotent stem cells by slow freezing with DMSO ._fr. The quantity of mRNA of stem cell, neural and glial markers was analyzed by RT-PCR to assess changes in mRNA expression during the process of proliferation and as a second step after freezing of ENSdN. Interestingly, we observed a promotion of distinct progenitor, glial and neuronal markers during the process of cell proliferation, as well as alterations in mRNA expression profiles for the genes of interest after freezing with condition M1, M2 and M3.However, it has been reported that DMSO might initiate a coordinated differentiation program in various cell types . But obvOct4 has been described as a specific marker for undifferentiated cells  and AdleTo evaluate the effect of freezing on differentiated cultures of ENSdN we assessed expression of neuronal, glial, and progenitor markers using immunohistochemical stainings._frM1) did not alter protein expression at all in our setting. However, ENSdN_frM2 and ENSdN_frM3 resulted in an overexpression of Nanog, Oct4 and S100.Freezing of differentiated ENSdN with condition M1 (ENSdN_diss). We could observe a significant lower expression of glial and stem cell markers  in dissociated cells, suggesting an effect of the dissociation process on cell differentiation and expression patterns. However, the process of freezing did not additionally alter protein expression in dissociated cells (data not shown).Since we achieved slightly better surviving rates in cultures of dissociated ENSdN, we additionally compared the protein expression of unfrozen differentiated spheres and dissociated cells  of ENSdN and ENSdN_frM1. We could identify different functional cell subgroups, that might match cell subtypes involved in ENS networks  and 40% contain neuronal nitric oxide synthase (NOS) , a rate It has been shown that the expression of neuronal or glial markers changes during different developmental stages of cells dependent on days of cultivation and/or the use of specific growth factors , a fact Cryopreservation of rodent ENSdN is possible and survival rates are sufficient to generate neurons and glial cells after freezing, but choosing the appropriate freezing mode and medium is crucial to prevent influence on cell gene and protein expression patterns. We believe that slow freezing of ENSdN with freezing condition M1 is the most appropriate method. It provided good cell survival, did not alter protein expression of differentiated spheres and did not impair neuronal function in live cell imaging. However, the distribution of neuronal cell subtypes might be affected by cryopreservation of cells.


The BSFCux  crystallized in a cubic perovskite structure (−1 (x = 0.15) at 500 °C. The ASR value as an indicator of ORR activity decreased by 72.6% from 0.135 Ω cm2 (x = 0) to 0.037 Ω cm2 (x = 0.15) at 700 °C. The Cu doping increased oxygen vacancy concentration and electron concentration in the valence band to promote electron exchange with adsorbed oxygen, thereby improving ORR activity.The oxygen reduction reaction (ORR) activity of a Cu-doped Ba To solve this problem, cobalt-based oxides with mixed ionic-electronic conductibility (MIEC) have been studied as potential cathode materials for IT-SOFCs [One of the important challenges for the commercialization of solid oxide fuel cells (SOFCs) is to lower the operating temperature to an intermediate temperature (IT) range of 500–800 °C . The intIT-SOFCs ,3,4. HowIT-SOFCs . Therefo3−δ (BFO) materials exhibit good oxygen–electron mixed conduction and excellent chemical and thermal stabilities compared with those of cobalt-based materials [0.5Sr0.5FeO3−δ (BSF) achieved high electronic conductivity [0.5Sr0.5FeO3−δ can be improved by substituting Fe with other transition metals to promote oxygen inflow and oxygen ion transport [Among the iron-based cathode materials, the cubic BaFeOaterials . The cubaterials . For stauctivity ,10,11. Iransport .3+/4+ cations can form more oxygen vacancies, which can enhance the ORR activity. For this purpose, some researchers have doped Ni, Gd, etc., at the Fe site, and perovskite cubic materials substituted with B-sites such as BaFe0.75Ni0.25O3−δ and BaF1−xGdxO3−δ provided high ORR activity [0.5Sr0.5Fe0.8Cu0.2O3−δ and Nd0.5Sr0.5Fe0.8Cu0.2O3−δ by doping Cu into the Fe site [1−xCuxO3−δ series, and LaNi0.5Cu0.5O3−δ showed a low polarization resistance of 0.056 Ω cm2 at 800 °C and a relatively high power density output of 870 mW cm−2 at 900 °C [1−xSrxNi1−yCuyO3−δ and achieved the maximum power value of 450 mW cm−2 at 650 °C [+, Cu2+, Cu3+), similar to Co, Fe, and Mn, it is reasonable to use perovskite materials containing Cu as the SOFC cathode [Replacing cations with lower valences (2+/3+) for Feactivity ,14. Yin  Fe site ,16. In at 900 °C . Jakub et 650 °C . Conside cathode .The mechanism of ORR at the SOFC cathode surface involves four steps, including diffusion and adsorption of oxygen molecules, dissociation of the oxygen molecules to form oxygen atoms, lattice mixing of oxygen ions, and movement of the oxygen ions to the electrode/electrolyte interface . However0.5Sr0.5Fe1−xCuxO3−δ perovskite cathode materials were investigated to confirm the relationship between the change in the valence band structure according to Cu doping and ORR activity. The change in oxygen vacancy concentration of BSFCux according to Cu doping was confirmed through thermogravimetric analysis (TGA) and X-ray photoelectron analysis (XPS). In addition, the oxidation state and the valence band structure were analyzed using XPS and the polarization resistance of BSFCux was calculated as an indication for change in ORR activity via the impedance spectrum.In this study, Ba0.5Sr0.5Fe1−xCuxO3−δ  powders were synthesized by a solid-state reaction using BaCO3 , SrCO3 , Fe2O3 , and CuO  as starting materials. After mixing each material according to the stoichiometric ratio, it was ball milled together in a polyethylene container with ethanol and zirconia balls for 24 h and dried at 100 °C for 12 h. The green bodies were prepared using dried powder by uniaxial compression under hydraulic pressure of 20 MPa; the green body of BSF was calcined at 1100 °C for 10 h, and the green bodies of BSFCux  were calcined at 950 °C for 10 h. These calcined green bodies were ground using a mortar and sieved using a mesh of 250 μm. Finally, BSFCux powder was synthesized by repeating the calcining, grounding, and sieving process twice.Ba−1) at room temperature. The structure parameters were obtained by analyzing XRD data with Rietveld refinement ). The line shapes of the diffraction peaks were generated by a pseudo-Voigt function and the background refined to a 4th degree polynomial.The crystal structure of the synthesized powders was analyzed using powder X-ray diffraction  in the 2θ range of 20–80° with a step scan procedure  for the powder samples were calculated using the following equation [0 is the oxygen non-stoichiometry at room temperature, M0 is the molar mass of the samples with non-stoichiometry δ0, 15.99 is the atomic weight of oxygen atoms, m is the final weight of the samples under various temperatures, and m0 is the initial weight of the samples. The δ0 was calculated by electroneutrality condition with X-ray photoelectron spectroscopy  data [In order to determine the temperature of oxygen vacancy formation and weight reduction of BSFCux, thermogravimetric analysis (TGA) was carried out on a thermal analyzer (NETSCH STA 409 PC/PG) with about 100 mg of BSFCux powders measured in a nitrogen atmosphere at a heating rate of 5 °C minequation :(1)δ=δ0+SA) data ,26,27,283. A direct current of 50 mA was supplied from a current source , and the corresponding voltage drop was collected using a multimeter .The electrical conductivity of BSFCux was evaluated using the 4-probe DC technique in the range of 300–900 °C, and Pt wires were wrapped around the sintered bars with dimensions of 5 × 3 × 30 mm2. After drying, the symmetric cells were calcined at 900 °C for 2 h in air to improve the adhesion between the electrolyte and the electrode. Electrochemical impedance spectra (EIS) were obtained using Iviumstat  equipment, and a 10 mV excitation voltage was applied in the frequency range of 106–10−2 Hz at 700 °C. The obtained EIS data were fitted using Zview software version 3.0.The microstructure of the BSFCu0.15|SDC|BSFCu0.15 symmetric cell was observed using a scanning electron microscope . The area-specific resistance (ASR) of symmetrical cells of BSFCux electrodes was measured by AC impedance spectroscopy. For an impedance analysis, a dense disk of SDC  was used as an electrolyte. BSFCux powder was mixed with a vehicle  to prepare BSFCux pastes using a three-roll mill, and these pastes were screen-printed on both sides of the SDC pellets with an area of 0.2826 cm+ (0.77 Å) and Cu2+ (0.73 Å) than those of Fe3+ (0.645 Å) and Fe4+ (0.585 Å). The XRD patterns show that BSFCux crystallizes into a cubic perovskite structure with a space group 3 (x = 0) to 61.4428 Å3 (x = 0.15) with the Cu content.up Pm3¯m . The str0) from the XPS analysis. In order to more clearly observe the oxygen vacancy formation according to the Cu content, it was measured in an inert atmosphere using nitrogen gas. There were weight losses of less than 1% during heating before the slope changing point, which is due to the desorption of physically and chemically adsorbed water and other gases [Thermogravimetric analysis was carried out to confirm the effect of Cu doping on oxygen vacancy formation. er gases . The sloer gases .3/2, Fe 2p3/2, and O 1s XPS spectra for each BSFCux composition and the area ratios of peaks are summarized in 3/2 peak was deconvoluted into two peaks at approximately 932.3 eV (Cu+) and 934.1 eV (Cu2+), as shown in 3/2 peak was deconvoluted into 709.1 eV (Fe3+) and 710.8 eV (Fe4+) peaks, and a weak satellite shake-up peak was observed in the range of 715 eV to 720 eV, as shown in + for Ba and Sr, the room-temperature oxygen non-stoichiometry (δ0) was calculated according to the electroneutrality condition, and the oxygen non-stoichiometry increased from 0.21 (x = 0) to 0.39 (x = 0.15) as the Cu doping increased (0 indicates the amount of oxygen vacancies in the lattice.The X-ray photoelectron spectroscopy (XPS) analysis was performed to characterize the surface chemical bonding state of the BSFCux samples. ncreased . It is tlat), adsorbed oxygen (Oads), and surface moisture (Omoi), respectively [asd to Olat (Oads/Olat) can be used as a criterion to evaluate the relative content of oxygen vacancies in materials [ads/Olat were 1.54 (x = 0), 1.60 (x = 0.05), 1.62 (x = 0.10), and 1.65 (x = 0.15), which increased with the amount of Cu doping. It can be interpreted that BSFCu0.15 has the highest oxygen vacancy concentration among the BSFCux  in the intermediate temperature region, which corresponds with the thermogravimetric results  spectra of BSFCux are shown in −1 (x = 0.15). This semiconducting behavior can be explained by a thermally activated small polaron hopping mechanism [4+ (Equation (2)), which is in good agreement with the thermogravimetric results [4+ and Fe3+, Fe-based perovskite oxides show mixed ionic-electronic conductivity (MIEC) because of the simultaneous presence of oxygen vacancies and electron holes as charge carriers. However, since the electrical conductivity is about two orders of magnitude higher than the ionic conductivity, the conductivity data of echanism . The dec results .(2)2FeFσ) and temperature follows the Arrhenius equation [A, aE, k, and T are the pre-exponential constant, activation energy, Boltzman constant, and temperature, respectively. According to the slope of linear fit over the temperature of 300–500 °C, the activation energy (Ea) was 0.26 eV (x = 0), 0.24 eV (x = 0.05), 0.23 eV (x = 0.10), and 0.21 eV (x = 0.15).equation :(3)σ=AexThe low activation energy value helps to improve the hopping of the charge carrier, thereby increasing the electrical conductivity.HF//CPEHF)-(RMF//CPEMF)-(RLF//CPELF), which is inserted in The cross-section view of the BSFCu0.15|SDC|BSFCu0.15 symmetrical cell shows that the interface contact is tightly formed between the porous electrode and the dense electrolyte a. The ORMF decreased the most, by about 81%, from 0.285 Ω (x = 0) to 0.054 Ω (x = 0.15), and it is thought that this is because the oxygen surface exchange reaction was accelerated by increasing the electron concentration of VB with Cu doping.These impedance spectra consist of high-, medium-, and low-frequency arcs. In general, the high-frequency (HF) arc is related to the process of diffusion of oxygen ions from the triple-phase boundaries into the electrolyte, the medium-frequency (MF) arc is associated with the oxygen surface exchange, and the low-frequency (LF) arc is attributed to the gas diffusion process and oxygen adsorption–dissociation process . In all ASR) can be calculated by measuring the distance between the intercepts of Z′ or by using the equation as follows:pR = RHF + RMF + RLF and the active area of the cathode is 0.2826 cm2. The ASR of the BSFCux was 0.135 Ω cm2 (x = 0), 0.085 Ω cm2 (x = 0.05), 0.045 Ω cm2 (x = 0.10), and 0.037 Ω cm2 (x = 0.15). The BSFCu0.15 showed a 72.6% smaller value compared to that of BSF, which can be explained by higher oxygen vacancy concentration and electrical conductivity. A comparison of the ASRs for different iron-based electrodes is listed in The area-specific resistance  perovskite cathode was investigated. BSFCux was crystallized into a cubic perovskite structure with a −1 (x = 0.15) at 500 °C. The ASR of BSFCu0.15 was 72.6% smaller compared to that of BSF, which can be explained by higher oxygen vacancy concentration and electrical conductivity. The ORR activity of BSFCux increased with Cu doping due to an increase in oxygen vacancy concentration and electron concentration in the valence band, which promotes electron exchange between the cathode surface and adsorbed oxygen.The oxygen reduction reaction (ORR) activity of a Ba