Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration

Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration Lizhen Wang, Kang Song, Xuezheng Geng, Huan Yin, Yanzhu Shi, Jiawei Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6497438/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Piezoelectric stimulation regulates cellular metabolism and enhances bone repair. However, the overproduction of reactive oxygen species (ROS) and hypoxia-induced oxidative stress reduce the efficacy of electrical stimulation and hinder regeneration. To address these challenges, a platinum-decorated graphdiyne oxide (GDYO@Pt) multifunctional piezoelectric semiconductor was engineered for the first time to eliminate ROS and oxygen self-supply while enabling electrical stimulation. In this system, the interface dipole drives a built-in electric field, triggering charge redistribution in GDYO and breaking symmetry to amplify piezoelectricity. Ultrasound-triggered polarized charges at the Schottky junction lower the barrier and promote GDYO→Pt electron transfer for hydrogen production, where the generated H₂ neutralizes cytotoxic •OH radicals, while the holes/nanozyme-driven H₂O₂→O₂ conversion​, synergistically alleviating oxidative stress. In vitro and vivo studies demonstrate that ultrasound-activated GDYO@Pt accelerates cranial defect repair via osteogenesis, angiogenesis, and immunomodulation. This work establishes the inaugural paradigm of piezoelectric-catalytic synergy bone regeneration, where the GDYO@Pt heterointerface uniquely integrates energy conversion with biological regulation through its precisely engineered asymmetric structure. Physical sciences/Materials science/Biomaterials/Biomedical materials Physical sciences/Materials science/Materials for energy and catalysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Piezoelectric semiconductor-based electrical stimulation offers a non-invasive alternative by converting mechanical stress into electrical energy under frequency- and intensity-controlled ultrasound (US) irradiation, generating adjustable electric fields for therapeutic intervention 1 , 2 , 3 . The majority of piezoelectric semiconductors including BaTiO 3 , ZnO, and Bi 2 WO 6 for bone repair exhibit excellent piezoelectric responses but have wide bandgaps (> 2.0 eV) 4 , 5 , 6 , 7 , 8 . The piezoelectric potential generation is accompanied by the generation of carriers that undergo redox reactions with water, resulting in ROS which cause oxidative damage to healthy tissues 9 , 10 . The overproduction of endogenous ROS and the presence of a hypoxic microenvironment at a bone-defect site severely impede healing, prolonging the course of electrical stimulation 11 , 12 . So it is essential to develop narrow-bandgap piezoelectric materials delivering efficient electrical stimulation and regulating oxidative stress simultaneously. The absence of a centrosymmetric structure is a fundamental characteristic for achieving piezoelectric properties 13 , 14 , 15 . Strategies including interlayer stacking, elemental doping, and electric field stimulation would reduce or break the inversion symmetry to induce piezoelectric responses 15 , 16 , 17 , 18 , 19 . Monolayer graphene exhibits no intrinsic piezoelectricity but multilayer stacking configurations disrupt its overall inversion symmetry, enabling piezoelectric effects through interlayer coupling and intralayer electron transition competition 16 , 17 , 20 . Oxygen as a dopant into graphene oxide further breaks the intralayer inversion symmetry via "clamp–release" structural distortions, enhancing the piezoelectric effect 15 , 21 . Charge redistribution in graphene nanostructures is driven by adjacent charged molecules, permanent dipoles, or built-in electric fields. This interaction can disrupt the inversion symmetry to amplify the piezoelectric performance 22 , 23 , 24 . Built-in electric fields, which arise from band bending or chemical potential gradients at heterojunction interfaces, have been shown to universally induce polar structures and substantial piezoelectric effects in semiconductors when interfaced with noble metals, as demonstrated by Yang et al. 25 , 18 . These findings underscore the feasibility of engineering nonpiezoelectric 2D carbon materials into high-performance piezoelectric systems through strategic modifications. Piezoelectric effect-driven electrical stimulation not only directly modulates cellular electrophysiological activity, but also its accompanying piezocatalytic reactions can directionally generate redox-active species (e.g., ROS, H₂, or O₂) through ultrasound-induced charge separation, dynamically regulating local oxidative stress levels 26 , 27 , 28 , 29 , 30 . The underlying mechanism lies in the fact that the valence/conduction band positions of piezoelectric semiconductors determine interfacial redox potentials (e.g., the conduction band ought to be positioned below the H⁺/H₂ redox potential to drive hydrogen production, while the valence band should exceed the H₂O/•OH and H₂O₂/O₂ potentials to trigger hydroxyl radical generation and hypoxia-alleviating oxygen supply) 28 , 29 , 31 . This provides a solution for precisely regulating oxidative stress via band engineering. During oxidative stress modulation, molecular hydrogen (H₂) serves as both a safe therapeutic agent and an anti-inflammatory mediator, which selectively neutralizes highly toxic •OH radicals through its antioxidant activity 32 , 33 , 34 . The exceptional tissue permeability of H 2 allows its effective diffusion into ROS-generating organelles (e.g., mitochondria and nuclei) 33 . However, hypoxia is known to upregulate hypoxia-inducible factor 1α (HIF-1α) in infiltrating immune cells, thereby promoting ROS production 35 , 36 , 37 . Concurrently addressing the issue of hypoxia during ROS scavenging may offer a more efficient strategy for managing oxidative stress and electrical stimulation therapy. Graphdiyne (GDY), a carbon-based two-dimensional material, exhibits exceptional structural stability and functional tunability due to its unique configuration of sp-/sp²-hybridized carbon atoms 38 , 39 . The sp-hybridized carbon (C ≡ C) bonds endow GDY with superior reducibility and electron transfer capability 40 , 41 , while its narrow bandgap (~ 1.22 eV, comparable to silicon's 1.11 eV) further establishes it as an ideal platform for synergistic regulation of piezoelectric effects and catalytic reactions 42 , 43 . Herein, this study focuses on the development of a narrow-bandgap piezoelectric semiconductor heterojunction (GDYO@Pt) as a dual-function platform for piezoelectric stimulation and oxidative stress regulation (Fig. 1 ). In this system, the charge distribution in the graphdiyne oxide (GDYO) nanosheets is reconfigured by the interfacial dipole-induced built-in electric field within the depletion region. This is proposed to promote breaking of the structural inversion symmetry and the formation of asymmetric polar configurations, aiming to significantly enhance the piezoelectric performance. Positively polarized charges are generated at the Schottky junction interface under ultrasound, which should induce downward band bending and barrier reduction. It is expected to facilitate electron transfer from GDYO to Pt, and promote sustained H 2 production. Concurrently, it is proposed that the holes will oxidize H 2 O 2 to generate O 2 , while the GDYO@Pt will act as a nanozyme to catalytically decompose H 2 O 2 into O 2 . This strategy effectively alleviates hypoxia and scavenges ROS, overcoming the limitations of oxidative stress on piezoelectric stimulation. Cellular validation confirms the GDYO@Pt system's modulation of BMSCs membrane potential, calcium influx, H₂/O₂ levels, and ROS clearance, verifying piezoelectric–catalytic synergy. Finally, GDYO@Pt-loaded thermoresponsive hydrogels are applied under ultrasound activation to promote defect repair via osteogenic differentiation, angiogenesis, and immunomodulation. Results Materials preparation and characterization GDY was synthesized according to a previously reported method 44 , 45 . As shown in Fig. 1 , GDYO nanosheets were prepared via a modified Hummer's method using H 2 O 2 /H 2 SO 4 as oxidants. GDYO@Pt was subsequently fabricated by in situ reduction of K 2 PtCl 4 with ascorbic acid. During this process, the strong affinity between the C ≡ C bonds of GDYO and the Pt 2+ species enabled efficient capture and reduction of the Pt ions. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed nanosheet structures for both GDYO and GDYO@Pt, with lateral dimensions of ~ 500 nm and thicknesses of ~ 3 and ~ 7 nm, respectively (Figs. 2 A, 2 E and 2 F). Pt nanoparticles (~ 3 nm in diameter) were uniformly distributed on the GDYO surface (Supplementary Fig. 1). Energy-dispersive X-ray spectroscopy (EDS) mapping confirmed the homogeneous distribution of C, O, and Pt in the prepared GDYO@Pt (Fig. 2 B). Raman spectroscopy yielded characteristic peaks at 1950 and 2159 cm − 1 (Fig. 2 C), while Fourier-transform infrared (FTIR) spectroscopy gave a single peak at 2050 cm − 1 (Supplementary Fig. 2), thereby confirming the retention of C ≡ C bonds in GDYO and GDYO@Pt. The X-ray diffractometry (XRD) results showed that GDYO@Pt exhibited diffraction peaks corresponding to the Pt standard card (PDF#040802), consistent with the lattice spacing observed in the high-resolution TEM image (Figs. 2 D, Supplementary Fig. 3), and confirming the presence of metallic Pt. X-ray photoelectron spectroscopy (XPS, Figs. 2 G, 2 H and Supplementary Fig. 4) was employed to further elucidate the chemical states of the prepared GDYO@Pt. The distinct Pt 4f peaks were observed, corresponding to metallic Pt 0 (Pt 4f 7/2 : 71.12 eV; Pt 4f 5/2 : 74.38 eV) and Pt 2+ (Pt 4f 7/2 : 72.54 eV; Pt 4f 5/2 : 76.16 eV), indicating the predominant reduction of Pt 2+ to Pt 0 , but with some residual surface oxidation. The high-resolution C 1s spectra recorded for both GDYO and GDYO@Pt displayed characteristic peaks corresponding to C = C, C ≡ C, C = O, and C–O bonds, confirming the structural integrity of GDYO following the incorporation of Pt (Figs. 2 G, 2 H). All C 1s peaks in GDYO@Pt shifted to higher binding energies than those in GDYO, suggesting electron transfer from GDYO to Pt during GDYO@Pt formation. Poloxamer 407 (P407), a PEO-PPO-PEO triblock copolymer (PEO: poly(ethylene oxide); PPO: poly(propylene oxide)), requires concentrations ≥ 18% (w/v) to form thermoresponsive hydrogels 46 , 47 . While higher P407 concentrations (20–24%) lead to increased mechanical strengths, they reduce the sol–gel transition temperature from 26.03 ± 0.21 to 22.07 ± 0.21°C, rendering the resulting hydrogels unsuitable for injectable applications 46 , 48 . Thus, a P407 concentration of 18% was selected for the purpose of the current study. Tannic acid (TA), which is a natural polyphenol with bioadhesive properties, was incorporated at varying concentrations (0–2%) to optimize crosslinking 48 , 49 . As shown in Supplementary Figs. 5 to 6 and Fig. 2 Q, TA concentrations ≥ 1% prevented gelation at 37°C, although the incorporation of GDYO@Pt (0.5 mg/mL) maintained the thermoresponsive nature of the hydrogel. Thus, a TA concentration of 0.7% was selected for subsequent experiments. Scanning electron microscopy (SEM) of the lyophilized gels revealed porous network structures for both the gel (P407/TA) and GDYO@Pt gel (Fig. 2 I, i). The gel exhibited uniformly distributed surface micropores, whereas the GDYO@Pt gel showed markedly reduced porosity, indicating homogeneous GDYO@Pt dispersion within the matrix (Fig. 2 I, ii和iii). This spatial uniformity was confirmed by EDS mapping, revealing consistent distribution of C, O, and Pt elements across the composite (Fig. 2 J). The FTIR spectra confirmed successful modification, with peaks corresponding to the C = O (1730.5 cm − 1 ) stretching vibrations of TA, and the C–O (1110.8 cm − 1 ) and C–H (2889.5 cm − 1 ) vibrations of P407 dominating the spectra (Fig. 2 K). Rheological studies using the GDYO@Pt gel demonstrated its shear-thinning behavior (Fig. 2 L), self-healing capability under step strain/shear tests (Figs. 2 L and 2 M), and optimal viscoelastic properties during strain/frequency sweep tests (Figs. 2 N and Supplementary Fig. 7). Time-dependent viscosity measurements and tube inversion tests confirmed a sol–gel transition time of 5 s and full gelation within 3 min at 37°C (Figs. 2 O and Supplementary Fig. 8). Temperature ramps revealed a phase transition temperature of 26.3°C, enabling liquid-state storage at room temperature and rapid solidification upon injection into the tissues (Fig. 2 P). The visual observations supported this temperature-triggered phase transition, thereby confirming successful fabrication of the GDYO@Pt gel (Fig. 2 Q). Piezoelectric response characterization of the nanosheets The piezoelectric properties of the nanosheets were investigated using piezoresponse force microscopy (PFM), a technique that is widely employed for the high-resolution characterization of piezoelectric materials. The vertical piezoresponse amplitudes and phase images of the GDYO and GDYO@Pt nanosheets were shown in Figs. 3 A, 3 B, 3 E and 3 F. Both materials exhibited distinct contrast variations, indicative of their piezoelectric activities, with GDYO@Pt demonstrating an enhanced responsiveness. Under a ± 6 V or ± 10 V ramped voltage, distinct butterfly-shaped amplitude loops were observed, demonstrating that a consistent strain variation was induced by the applied electric field. The corresponding local piezoelectric hysteresis loops revealed an ~ 180° phase switching behavior, confirming the intrinsic piezoelectric nature of these materials (Figs. 3 C, 3 G). Quantitative analysis of the amplitude loop slopes demonstrated that Pt incorporation significantly enhanced the piezoelectric response of GDYO (Fig. 3 D). Kelvin probe force microscopy (KPFM) measurements showed a surface potential of 41 mV for GDYO@Pt, which was markedly higher than that of GDYO (22 mV), attributed to piezoelectricity-induced electrical polarization effects (Figs. 3 I– 3 K). COMSOL simulations performed under a 10 8 Pa mechanical stress revealed piezoelectric potentials of 5 V for GDYO and 9 V for GDYO@Pt, confirming the critical role of Pt nanoparticle modification in amplifying the piezoelectric performance of GDYO (Figs. 3 M, 3 N). Density functional theory (DFT) and finite element analysis were employed to elucidate the mechanism of the enhanced piezoelectric effect. The differential charge density diagram clearly revealed the out-of-plane charge transfer behavior between the Pt nanoparticles and GDYO (Fig. 3 H), indicating the formation of a strong interfacial electric field through electron delocalization during the tight bonding process between the Pt and GDYO. The process of charge transfer was also one of charge redistribution. As shown in Fig. 3 O, the contact interface between the GDYO nanosheets and the Pt metal particle exhibited a potential difference of ~ 3.3 mV, with adjacent areas of the nanosheets showing polarization-affected potentials, which may be attributed to charge redistribution. Figure 3 L showed that along the a- and b-axes, the unit cell dipole moments of GDYO@Pt significantly increased to 82.03 and − 176.59 Debye (D), respectively, which were 1.4-fold greater than those of GDYO (i.e., 57.67 and − 132.24 D). These values confirmed the substantial enhancement in the degree of polarization of GDYO following the loading of Pt nanoparticles. As shown in Supplementary Fig. 9, the increased relative dielectric constant of GDYO@Pt quantitatively reflected the enhanced polar intensity, consistent with the aforementioned results. These results indicate that in the Pt-GDYO Schottky junction, interface charge transfer triggered by work function differences forms a dipole layer, and the resulting built-in electric field further induces charge redistribution within GDYO nanosheets. This process disrupts the central symmetry of the semiconductor, enabling more efficient separation of polarization charges under stress and thereby significantly enhancing the material’s piezoelectric performance (Fig. 3 P). Piezocatalytic properties of the GDYO@Pt nanosheets Considering the excellent piezoelectric properties of the GDYO@Pt nanosheets, the piezocatalytic reactions induced by the US-activated nanosheets were evaluated. Using electron spin resonance (ESR) spectroscopy to detect the generation of ROS (Supplementary Fig. 10), it was found that no ROS production occurred under US stimulation, perhaps due to the insufficient redox potential of the band structure for ROS generation. The piezocatalytic H 2 production performance of the nanosheets was evaluated using a methylene blue (MB) probe to detect the catalytic H 2 generation (Figs. 4 A, 4 B, and Supplementary Fig. 11). The GDYO@Pt solution containing the MB probe exhibited a time-dependent decrease in absorbance at 664 nm, with a more significant reduction observed compared to the other groups, indicating that US-activated H 2 generation occurred in the GDYO@Pt system. Gas chromatography (GC) was used to quantify H 2 production (Fig. 4 C), and it was found that the GDYO@Pt + US group exhibited the highest H 2 yield. Since Pt nanoparticles are known to catalyze the generation of O 2 from H 2 O 2 , the O 2 generation capacity was also monitored (Fig. 4 D). It was found that GDYO@Pt acted as a nanozyme, with the GDYO@Pt + H 2 O 2 + US group exhibiting significantly enhanced O 2 production compared to the GDYO + H 2 O 2 + US and Pt + H 2 O 2 + US groups. These phenomena were attributed to Schottky junction formation between the Pt metal and GDYO, which enhanced the piezoelectric performance and carrier separation efficiency, further promoting the catalytic performance in both H 2 and O 2 generation. The energy band structures of GDYO and GDYO@Pt were analyzed by Mott–Schottky measurements and ultraviolet-visible diffuse reflectance spectroscopy (UV-vis DRS) (Figs. 4 H– 4 K and Supplementary Figs. 12 to 13). It was determined that GDYO and GDYO@Pt exhibited n-type semiconductor characteristics (positive slopes), with flat band potentials (E fb ) of − 0.83 and − 1.03 V (vs. Ag/AgCl) at pH 7, and corresponding to − 0.63 and − 0.83 V vs. NHE (pH 7), respectively. After calibration to pH 0, the CB potentials were determined to be − 0.22 and − 0.42 V (vs. NHE, pH 0), both of which were more negative than the H⁺/H 2 redox potential (i.e., 0 V vs. NHE, pH 0), indicating efficient electron-driven H 2 evolution. The bandgaps of GDYO and GDYO@Pt were 1.36 and 1.49 eV, respectively. The calculated VB potentials were determined to be 1.14 and 1.07 V (vs. NHE, pH 0), which exceeded‌‌ the oxidation potential of H 2 O 2 /O 2 (0.69 V vs. NHE, pH 0), satisfying the thermodynamic requirements for the hole-mediated generation of O 2 from H 2 O 2 . Collectively, the band positions of GDYO and GDYO@Pt theoretically satisfied both H 2 and O 2 evolution criteria. The mechanism of piezocatalysis was further investigated. Ultraviolet photoelectron spectroscopy (UPS) was employed to determine the work functions (W) of GDYO, Pt, and GDYO@Pt (Figs. 4 E– 4 G). The cutoff energies (E cutoff ) for GDYO, Pt, and GDYO@Pt were determined to be 17.93, 16.80, and 17.52 eV, respectively. Using the equation W = 21.22 − |E cutoff – E F |, the calculated W values for GDYO, Pt, and GDYO@Pt were obtained, i.e., 3.29, 4.42, and 3.70 eV (relative to the vacuum level), respectively. The Pt nanoparticles exhibited a higher W value than GDYO, indicating that when GDYO was in intimate contact with the Pt nanoparticles, electrons transferred‌‌ from GDYO to Pt until the interfacial Fermi level reached equilibrium. The space-charge region formed on the GDYO side caused upward bending of the energy bands, establishing a Schottky barrier (Figs. 4 H and 4 I). Upon ultrasonic excitation, the valence band (VB) electrons of GDYO were excited to the conduction band (CB) and transferred to the Fermi level of the Pt nanoparticles through the Schottky barrier, whereas holes remained in the VB of GDYO (Fig. 4 J). The E fb and CB potentials of ultrasound-activated GDYO@Pt were − 0.85 and − 0.24 V (vs. NHE, pH 0), respectively, which were positively shifted by 0.18 V compared to those obtained under static conditions (Figs. 4 K, 4 L). This suggested that the piezoelectric effect in GDYO@Pt induced downward band bending and lowered the Schottky barrier height (SBH), thereby facilitating electron migration from GDYO to Pt and enhancing the H 2 and O 2 production (Figs. 4 I and 4 J). DFT calculations were performed to validate the electron transfer process (Figs. 4 M and 4 N). As shown in Fig. 4 M, electrons predominantly accumulate on the Pt side (yellow regions), whereas the green peripheral regions of GDYO indicate electron depletion. Corresponding to the differential charge distribution, charge displacement (Δρ) in the GDYO@Pt system was quantitatively calculated (Fig. 4 N). The negative and positive Δρ values observed for the GDYO and Pt regions, respectively, confirmed that the primary electron migration pathway was from GDYO to Pt, which was consistent with the above results (Fig. 2 H). Electrochemical impedance spectroscopy (EIS) revealed significantly reduced arc diameters for GDYO@Pt (Supplementary Fig. 14), indicating lower impedance and improved carrier separation efficiency compared to GDYO. As shown in Supplementary Fig. 15, DFT simulations were used to visualize the top and side views of the H 2 O adsorption configurations on different materials. Compared to GDYO (− 0.508 eV) and Pt (− 0.613 eV), GDYO@Pt (− 0.847 eV) was found to exhibit a stronger adsorption energy, indicating an enhanced surface affinity for water molecules and more abundant reaction sites (Fig. 4 O). The Gibbs free energy of H 2 adsorption (|ΔG H* |) serves as a critical descriptor for determining the H 2 precipitation activity of a system, with values closer to zero indicating a greater reaction probability. The calculated |ΔG H* | value for GDYO@Pt was 0.15 eV, which was significantly lower compared to GDYO (0.40 eV) and Pt (0.28 eV) (Figs. 4 P and Supplementary Fig. 16), thus the Schottky junction formed by GDYO and Pt effectively lowered the activation energy for H 2 precipitation. The superior H 2 O adsorption capacity of GDYO@Pt along with its reduced H* activation energy led to more abundant surface reduction reactions, ensuring efficient piezocatalytic H 2 evolution. Ultrasound-activated GDYO@Pt nanosheets promote cell proliferation The cytocompatibility of the GDYO and GDYO@Pt nanosheets was subsequently evaluated using the Cell Counting Kit-8 (CCK-8) assay. The obtained results demonstrated that neither GDYO nor GDYO@Pt exhibited observable toxicity toward BMSCs, even at a high concentration of 500 µg/mL (Supplementary Fig. 17). Live/dead cell staining confirmed the excellent cytocompatibility of the nanosheets (Supplementary Fig. 18), consistent with the aforementioned results. To simulate a damaged microenvironment, oxidative stress injury was induced in BMSCs through the addition of H 2 O 2 (0.2 mM). The oxidative stress-injured BMSCs were exposed to US at different power densities (0.3, 0.5, 0.7, 1, and 1.5 W/cm 2 ) for 10 min to determine the optimal conditions. Compared to the control group, the BMSCs viability significantly decreased by day 3 in the 1.0 and 1.5 W/cm 2 groups, whereas the other groups remained unaffected. However, good cell viability was maintained when US was applied at a power density of 0.7 W/cm 2 for 10 min, indicating that this was a suitable power density and duration for subsequent experiments (Figs. 5 A and Supplementary Fig. 19). The effects of the ultrasound-activated nanosheets on BMSCs proliferation were further assessed using CCK-8 assays and live/dead cell staining. Compared to the GDYO@Pt and GDYO + US groups, the GDYO@Pt + US group exhibited significantly enhanced proliferation levels on days 3, 5, and 9 (Figs. 5 B and Supplementary Fig. 20). This enhancement was attributed to the synergistic effects of the Schottky junction-induced piezoelectric stimulation and oxidative stress regulation in GDYO@Pt. The GDYO@Pt group showed a slight improvement in cell viability compared with the control group. This was likely due to the nanozyme activity of GDYO@Pt, which consumes overexpressed H 2 O 2 in damaged cells to generate O 2 , thereby enhancing the cell viability. Piezoelectric stimulation of BMSCs The piezoelectric properties and cycle stabilities of the nanosheets were systematically evaluated in physiological environments through ultrasound activation in a physiological medium. As shown in Figs. 5 C and 5 D, compared to a neutral pH environment (pH 7.4), a pH of 4.5 (simulating the bone-defect microenvironment) significantly enhanced piezoelectric current generation in the nanosheets. During five on-off cycling tests, the GDYO@Pt nanosheets exhibited stable current–output characteristics. The current intensity of the GDYO@Pt system reached 1.7 µA (pH 4.5), which was ~ 2.1-fold higher than that of the GDYO nanosheets (0.8 µA), consistent with the piezoelectric characterization results (Fig. 3 ). Further evaluations revealed that the piezoelectric current of US-activated GDYO@Pt displayed significant power-dependent characteristics (0–0.7 W/cm 2 ) (Fig. 5 E). In bone electrophysiology, it has previously been reported that microcurrents in the 0.1–10 µA range effectively promote bone tissue regeneration 50 , 51 . This study confirmed that the piezoelectric current intensity generated by GDYO@Pt fell precisely within this range. Furthermore, the observed environmental adaptability and output stability of this system met the requirements for bone-defect repair applications, highlighting its significant potential in bone regeneration therapy. After evaluating the piezoelectric properties, the effects of piezoelectric stimulation on the electrophysiological activity of BMSCs were investigated. For this purpose, a spatially separated experimental design was adopted, wherein BMSCs were seeded in the central area (1.0 × 1.0 cm 2 ) of the sterilized conductive glass, while the nanosheet-coated region was fixed at the bottom. To precisely assess the direct impact of the piezoelectric effect of the material on the cellular electrophysiology, the nanosheet-loaded electrode area was submerged in a physiological medium (pH 4.5), whereas the BMSCs-seeded electrode area remained exposed (Fig. 5 F). The voltage-sensitive fluorescent probe Di-8-ANEPPS was used to monitor real-time changes in the cell membrane potential induced by US-activated piezoelectric stimulation (Figs. 5 F– 5 H). This probe operates via an electrochromic mechanism, wherein its excitation/emission spectra shift in response to changes in the membrane potential 52 , 53 . The slow internalization properties of Di-8-ANEPPS ensured specific localization on the cell membrane surface, enabling the accurate detection of membrane dipole potential alterations 54 , 55 . As shown in Figs. 5 G and 5 H, the GDYO@Pt group exhibited greater fluorescence intensity changes than the GDYO group, with a statistically significant increase in the fluorescence intensity ratio observed after US stimulation. This phenomenon directly correlated with the enhanced piezoelectric current output of GDYO@Pt (1.7 vs. 0.8 µA, as shown above), confirming that US-activated GDYO@Pt can modulate the electrophysiological activity of BMSCs by altering the cell membrane potential through superior piezoelectric stimulation. As a secondary messenger that regulates osteogenesis, intracellular calcium ion (Ca 2+ ) signaling is closely linked to the electrophysiological activity 56 , 57 . To investigate the possible activation of calcium signaling pathways under the influence of GDYO@Pt piezoelectric stimulation, a Fluo-4 fluorescent probe was employed with the same spatially separated design approach described above (Figs. 5 F, 5 I and 5 J). The experimental results revealed that the GDYO@Pt group exhibited a significantly enhanced Ca 2+ fluorescence signal intensity under US stimulation, with a 1.6-fold increase in the fluorescence ratio (post-/pre-stimulation) compared to the control group. This indicated that the piezoelectric electric field generated by the material effectively induced intracellular Ca 2+ enrichment in BMSCs. Combined with the membrane potential results (Figs. 5 G and 5 H), this result suggested that the corresponding mechanism likely involves piezoelectric stimulation-induced membrane depolarization, which regulated the voltage-gated calcium channel (VGCC) activity to promote extracellular Ca 2+ influx. The specific activation of Ca 2+ signaling pathways provided a critical ionic environment for BMSCs proliferation, osteogenic differentiation, and extracellular matrix mineralization. Evaluation of oxidative stress levels in vitro The effects of nanosheet-based piezocatalysis on the oxidative stress levels in BMSCs were evaluated. Initially, the GC and MB probe were used to analyze intracellular H 2 release. As shown in Fig. 6 A, US-activated GDYO@Pt-treated BMSCs exhibited a significant time-dependent increase in H₂ generation, with a hydrogen evolution rate far exceeding that of other groups. Further validation via the MB probe (Fig. 6 B) revealed near-complete fading (loss of blue color) in the GDYO@Pt + US group, whereas other groups retained distinct blue coloration, visually confirming its superior hydrogen production activity. These results demonstrated the universal mechanism by which the Schottky heterojunction (GDYO@Pt) enhanced piezocatalytic hydrogen production across diverse environments, spanning from in vitro systems to cellular contexts. Although H 2 is beneficial for scavenging highly toxic hydroxyl radicals (i.e., ROS) in damaged cells, a hypoxic environment can lead to the upregulated expression of HIF-1α in the infiltrating immune cells, further inducing ROS production and creating a vicious cycle of oxidative stress. Alleviating hypoxia while scavenging ROS may lead to the more efficient resolution of oxidative stress in a bone defect microenvironment. As shown in Fig. 6 C, the hypoxia-sensitive fluorescent probe Ru(dpp) 3 Cl 2 was employed to evaluate nanosheet-catalyzed O 2 generation in the BMSCs. Compared with the ROS group (oxidative stress model group), hypoxia was significantly alleviated in the GDYO@Pt, GDYO + US, and GDYO@Pt + US groups, with the GDYO@Pt + US group exhibiting the strongest hypoxia mitigation capability. This was attributed to the synergistic catalytic decomposition of H 2 O 2 into O 2 by the GDYO@Pt nanosheets and holes, thereby providing a safeguard for ROS regulation. The indicator 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used to investigate the effects of different treatments on the intracellular ROS levels. DCFH from DCFH-DA is initially hydrolyzed by cellular esterases, and is subsequently oxidized by ROS to generate the strongly green-fluorescent 2′,7′-dichlorofluorescein (DCF). As shown in Fig. 6 D, compared to the ROS group, the GDYO@Pt group exhibited weakened green fluorescence, indicating its intrinsic ROS-scavenging capability owing to the ability of the GDYO@Pt nanozyme to decompose H 2 O 2 . More importantly, DCF fluorescence was almost absent in the GDYO@Pt + US group, demonstrating a significantly enhanced ROS depletion level that far exceeded that in the GDYO + US group. This result stems from the synergistic effects of piezocatalytic H 2 and O 2 production during the continuous ROS-scavenging process. Promotion of osteogenic differentiation To evaluate the effects of US-activated GDYO@Pt on the osteogenic differentiation of BMSCs, assessments of the alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining were conducted. ALP is a critical intracellular enzyme that serves as a marker of early osteogenic differentiation. As shown in Fig. 7 A, compared with the GDYO@Pt and GDYO + US groups, the GDYO@Pt + US group exhibited a larger ALP-stained area after 7 d, correlating with the ALP activity values presented in Fig. 7 B. Furthermore, the late-stage (14 d) mineralization of BMSCs was assessed using Alizarin Red S (ARS) staining. As shown in Figs. 7 A and 7 C, the GDYO@Pt + US group displayed the highest number of mineralized nodules and cellular calcium deposits, indicating superior mineralization compared with the other groups. These results further confirmed that US-triggered GDYO@Pt facilitated the osteogenic differentiation of BMSCs. The molecular mechanisms underlying this enhanced osteogenic differentiation were elucidated by analyzing gene expression levels using the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Key osteogenic markers including ALP, osteocalcin (OCN), osteopontin (OPN), type I collagen (COL-1), and runt-related transcription factor 2 (RUNX-2) were examined. As shown in Figs. 7 D– 7 H, the GDYO@Pt + US group exhibited significantly higher expression levels of these osteogenic genes than the other groups after 14 d, indicating a markedly augmented osteogenic differentiation capacity. This can be attributed to the synergistic effects of the enhanced piezoelectric stimulation and down-regulated oxidative stress at the Schottky junction. In vivo skull regeneration and repair To systematically evaluate the osteoinductive potential of US-activated GDYO@Pt nanosheets in vivo, a nanocomposite hydrogel system was developed by encapsulating the nanosheets within a TA-modified poloxamer thermosensitive gel to enhance their retention at bone-defect sites. This composite gel exhibited excellent biosafety and viscoelasticity, with the optimized formulation enabling liquid-state behavior at room temperature (25°C) and semi-solid-state behavior at physiological temperature (37°C) (Figs. 2 L– 2 Q; Supplementary Figs. 7, 8 and 21). According to the therapeutic protocol shown in Fig. 8 A, a 3 mm diameter cranial defect model was created in the parietal bone of mice using a precision drill. Subsequently, an aliquot (300 µL) of pre-chilled (4°C) gel, GDYO gel, or GDYO@Pt gel was precisely injected into the defect cavity via a micro syringe and maintained at 37°C for 10 min to ensure in situ gelation. The surgical incisions were closed in layers, followed by postoperative analgesia and anti-infection treatments. Twenty-four mice were randomized into six groups (control, gel + US, GDYO gel, GDYO@Pt gel, GDYO gel + US, and GDYO@Pt gel + US; n = 4 per group) across two timepoints (6 and 12 weeks). The US parameters (1 MHz, 0.7 W/cm 2 , 10 min/d) matched those used in the in vitro studies, and treatment was applied for 4 weeks. Micro-computed tomography (micro-CT) imaging and reconstruction revealed that the GDYO@Pt gel + US group achieved the highest degree of bone regeneration and repair at both 6 and 12 weeks compared to the other groups (Fig. 8 B). Semi-quantitative analysis (Figs. 8 C– 8 E) indicated poor bone regeneration in the control, gel + US, and GDYO gel groups, with low new bone area ratios (%), bone volume/total volume ratios (BV/TV), and bone mineral densities (BMD) observed in each case. In contrast, the GDYO@Pt gel + US group exhibited superior repair metrics, reaching a new bone area of 75.37%, a BV/TV ratio of 69.00%, and a BMD of 0.658 g/cm 3 at 12 weeks, significantly surpassing the other groups. Histological analysis using hematoxylin and eosin (H&E) and Masson’s trichrome staining confirmed these findings. More specifically, from H&E staining, the GDYO@Pt gel + US group displayed a continuous, well-organized neobone with abundant vascularization (Fig. 8 F), whereas Masson’s staining highlighted mature osteoid deposition within the defect (Fig. 8 G); the other groups showed limited discontinuous bone formation. Immunohistochemical (IHC) staining for osteogenic markers (OCN and RUNX-2; Figs. 8 H and 8 I) showed the strongest degree of staining in the GDYO@Pt gel + US group, consistent with the micro-CT and histological results. CD31 immunostaining (angiogenesis marker) revealed superior vascular protein expression in this group (Fig. 8 J). These results were attributed to the synergistic effects of Schottky junction-enhanced piezoelectric stimulation and oxidative stress modulation. Immunofluorescence (IF) staining was subsequently performed to investigate the effects of the different treatment conditions on the inflammatory microenvironment at the defect site. M1 (CD86) and M2 (CD206) macrophages, representing pro-inflammatory and anti-inflammatory phenotypes respectively, play critical roles in regulating the osteogenic microenvironment. Although short-term inflammation facilitates the recruitment of endogenous stem cells, prolonged inflammation impedes bone repair. As shown in Fig. 8 K, compared to the other groups, the GDYO@Pt gel + US group exhibited a significantly higher proportion of M2-type (CD206, red) macrophages and a lower proportion of M1-type (CD86, green) macrophages at the cranial defect site. This polarization toward an anti-inflammatory phenotype is conducive to bone tissue repair, thereby indicating that the US-triggered GDYO@Pt gel effectively orchestrated the osteogenic, angiogenic, and immune microenvironments to promote bone regeneration. Assessment of oxidative stress levels in bone defect tissues Considering the aforementioned in vivo bone repair outcomes, the mechanism underlying the US-activated GDYO@Pt-mediated remodeling of redox homeostasis in the bone-defect microenvironment was further elucidated. Initially, a dihydroethidium (DHE) fluorescent probe was used to evaluate the ROS levels at defect sites. As shown in Fig. 9 A, the GDYO@Pt gel + US group displayed substantially attenuated red fluorescence compared with the other groups, confirming its superior ROS-scavenging capacity. HIF-1α immunofluorescence analysis demonstrated significantly reduced HIF-1α expression in this group (Fig. 9 B), indicating that the GDYO@Pt system-mediated elevation of the O 2 partial pressure alleviated tissue hypoxia, thereby disrupting the "hypoxia → HIF-1α activation → ROS accumulation" cycle. To further explore the regulatory pathways associated with oxidative stress, immunohistochemical staining was performed to analyze the expression of nuclear factor erythroid 2-related factor 2 (NRF2). As shown in Fig. 9 C, the GDYO@Pt gel + US group exhibited a significantly higher proportion of NRF2-positive cells in the newly formed bone tissue than the other groups. These findings demonstrated‌‌ that this multidimensional antioxidant strategy not only effectively reduced local ROS levels, but also enhanced endogenous antioxidant defenses via the NRF2 pathway, establishing a redox-balanced microenvironment conducive to electrical stimulation therapy for bone defect repair. H&E staining of tissue sections from all treatment groups post-therapy demonstrated a preserved structural integrity in each case, in addition to the absence of pathological abnormalities in the heart, liver, spleen, lungs, and kidneys (Supplementary Fig. 21). These results indicated the favorable biocompatibility of GDYO@Pt gel at therapeutic doses in vivo. Discussion Piezoelectric stimulation, a non-invasive therapeutic approach, modulates cellular metabolism and promotes bone regeneration. However, excessive ROS generation and hypoxia-induced oxidative stress within the bone defect microenvironment compromise the therapeutic efficacy of electrical stimulation. Therefore, the development of narrow-bandgap piezoelectric semiconductors integrated with dual-functional piezoelectric stimulation and ROS scavenging capabilities is imperative. In this study, a bone-repair platform that integrates efficient piezoelectric stimulation and multimodal antioxidant stress functions is developed based on a Schottky heterojunction formed between GDYO nanosheets and Pt nanoparticles. The interfacial dipole effect at the heterojunction induces a built-in electric field, driving charge rearrangement in GDYO nanosheets to break the semiconductor’s inversion symmetry, enhance polarity, and significantly boost piezoelectric performance. In addition, the US-triggered Schottky interfaces are found to generate positive polarization charges that induce downward band bending, lowering the energy barriers, and promoting electron migration from GDYO to Pt for enhanced H 2 evolution. Concurrently, the hole-mediated oxidation of H 2 O 2 generates O 2 , synergizing with the nanozyme activity of GDYO@Pt to achieve the multimodal scavenging of ROS. In vitro and vivo experiments demonstrate that the US-activated GDYO@Pt nanosheets promote osteogenic differentiation, angiogenesis, and immunomodulation by remodeling the electrophysiological microenvironments and downregulating oxidative stress, ultimately achieving a superior cranial bone defect repair efficacy. This piezoelectric-catalytic dual-function platform innovatively synchronizes efficient electrical stimulation with oxidative stress regulation, offering a transformative solution for bone defect repair. Through band engineering, it precisely controls interfacial redox kinetics during piezoelectric stimulation, avoiding toxic ROS generation while directionally producing therapeutic H₂ and O₂ to establish a low-oxidative-stress microenvironment conducive to osteogenesis. This mechanism can be extended to diverse regenerative applications, including articular cartilage repair, neural axon regeneration, and skin wound healing, demonstrating universal applicability. Thus, elucidating the spatiotemporal regulatory principles of electro-chemical coupling on cell fate and establishing quantitative structure-activity relationships among band structures, interfacial reaction kinetics, and biological responses will critically guide the development of next-generation intelligent bone repair materials. Materials and Methods Materials Graphite was sourced from Beijing Gaoke New Materials (China). Potassium tetrachloroplatinate (II) (K 2 PtCl 4 ), H 2 O 2 (30%), H 2 SO 4 (98%) and ethylene glycol (EG, 99%) were purchased from Shanghai Chemical Reagent (China). Kolliphor® P 407 was obtained from BASF (Ludwigshafen, Germany). Tannic acid (USP, ≥ 99%) and Methylene blue (≥ 98%) were obtained from Macklin (China). The water in the experiments was deionized. All reagents were used directly without further purification. Synthesis of GDYO According to previously reported methods 44 , 45 , graphdiyne (GDY) was first synthesized on a copper surface via cross-coupling reactions using hexaethynylbenzene as the precursor. Subsequently, GDY powder (50 mg) was homogenized with H 2 SO 4 (2.5 mL) under ice-bath conditions, followed by slow dropwise addition of 30% hydrogen peroxide solution (1 mL) under vigorous stirring for 2 hours. Distilled water (20 mL) was then added to the mixture, and the suspension was ultrasonicated for 1 hour, followed by centrifugation (8,000 rpm, 10 min) and repeated washing with deionized water until the supernatant reached neutral pH. To further exfoliate stacked GDYO, concentrated hydrochloric acid (20 µL) was added to 20 mL of GDYO dispersion (0.5 mg/mL), followed by ice-bath ultrasonication for 6 hours (150 W power, 40 kHz frequency). The dispersion was then centrifuged (8,000 rpm, 10 min) and washed twice with deionized water. Finally, unexfoliated residues and large aggregated nanosheets were removed via low-speed centrifugation (3,000 rpm, 5 min), yielding a stable exfoliated GDYO nanosheet dispersion, which was stored at 4 ℃ for subsequent experiments. Fabrication of GDYO@Pt The heterojunction GDYO@Pt nanosheets were synthesized via an in situ reduction method. First, GDYO (60.0 mg) and K₂PtCl₄ (30.0 mg) were dispersed in deionized water (120 mL) under ultrasonication. The mixture was then stirred for 30 minutes at 600 rpm. Subsequently, 12 mL of ascorbic acid solution (0.1 M) was added dropwise to the above solution, followed by stirring at 60°C for 3 hours. The precipitate was collected by centrifugation (8,000 rpm, 10 min) and washed three times with deionized water. Finally, the GDYO@Pt nanosheets were obtained as the final product through freeze-drying. Fabrication of GDYO@Pt gel First, the blank thermosensitive gel was prepared via a low-temperature swelling method. Specifically, 0.07 g of TA (tannic acid) was added to 10 mL of deionized water and stirred continuously at room temperature until fully dissolved. Subsequently,1.8 g of P407 (poloxamer 407) was slowly added to the TA solution, followed by stirring for 30 minutes. The mixture was then transferred to a 4 ℃ refrigerator and allowed to stand for 24 hours to obtain the blank thermosensitive gel solution. Next, GDYO@Pt nanosheets were incorporated into the blank gel and thoroughly mixed under ice-bath conditions to prepare the thermosensitive GDYO@Pt gel (0.5 mg/mL). Characterization The comprehensive characterization of the materials was systematically conducted using multiple analytical techniques. Morphological features were examined by transmission electron microscopy (TEM, JEOL JEM-2100F, Japan), scanning electron microscopy (SEM, ZEISS MERLIN Compact, Germany), and atomic force microscopy (AFM, Bruker Dimension ICON, Germany). Crystallographic and chemical analyses were performed via X-ray diffraction (XRD, Bruker D8 Advance, Germany), X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA), and Fourier transform infrared spectroscopy (FT-IR, Thermo Scientific Nicolet iS50, USA). Optical properties were assessed through ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy (Shimadzu UV-1900 and Hitachi UH4150, Japan), while reactive oxygen species (ROS) signals were detected via electron spin resonance (ESR, Bruker EMXplus, Germany). Functional evaluations included rheological measurements (Anton Paar MCR 702e, Austria), piezoelectric/piezocatalytic activation using an ultrasonic therapeutic device (Chattanooga Intelect 2776, USA), and quantitative hydrogen gas analysis via gas chromatography (Agilent GC7890, USA). The finite element simulations were carried out using COMSOL Multiphysics 6.2 software, which has the main advantage of adapting to multiphysics field coupled simulations, making it easier to perform iterative calculations between different physics fields. Specifically, the simulations were carried out through the solid mechanics module and the electrostatics module in COMSOL, and the use of the stress-charge form was determined according to the material and the requirements. The imposed boundary conditions were a fixed constraint on the left side and a constant stress of 100 MPa on the right side. Details of the DFT calculations were provided in Supplementary Note 1. Gelation and rheological properties of the thermosensitive gel The gelation behavior of various hydrogel formulations was assessed via the tube inversion method. Specifically, glass vials containing gel-1 (18% P407, 0% TA), gel-2 (18% P407, 0.5% TA), gel-3 (18% P407, 0.7% TA), gel-4 (18% P407, 1% TA), gel-5 (18% P407, 2% TA), and GDYO@Pt gel formulations (0.5 mg/mL GDYO@Pt with 18% P407 and 0%, 0.5%, 0.7%, 1%, or 2% TA) were immersed in a 37 ℃ water bath for 10 minutes. After thermal equilibration, the vials were inverted to confirm the sol-gel transition. Additionally, rheological experiments were conducted using a parallel plate (40 mm diameter, 1 mm gap) on a rheometer to characterize GDYO@Pt gel. The storage modulus (G′) and loss modulus (G″) of GDYO@Pt gel were quantified under varying conditions. Oscillatory strain sweeps were performed at 37°C and 1 Hz to measure G′ and G″ as a function of strain amplitude. Furthermore, time- and frequency-dependent measurements of G′ and G″ were carried out at 37°C with a fixed strain amplitude of 0.5%. Step-strain measurements were conducted under high (45%) and low (0.1%) strain conditions at 37°C and 1 Hz. Steady-state shear viscosity was analyzed at 37°C by applying shear rate sweeps, while step-shear measurements employed low (1 s⁻¹) and high (100 s⁻¹) shear rates. In vitro hydrogen detection GDYO and GDYO@Pt were uniformly dispersed in PBS-containing headspace vials (0.5 mg/mL) and sealed. The samples were then exposed to ultrasound (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density) in the dark. At different time points (0, 10, 20, 30, and 40 minutes), 1 mL of gas from the vial headspace was extracted and analyzed for hydrogen using a gas chromatograph (GC) equipped with a thermal conductivity detector (TCD) and nitrogen as the carrier gas. Under the catalytic action of platinum (Pt) nanoparticles, H₂ reacts with methylene blue (MB) to form colorless reduced leucomethylene blue (MBH₂). Thus, MB can also serve as an indicator for H₂ detection. Experimental details are as follows: 2 mL of PBS, GDYO, or GDYO@Pt dispersion (200 µg/mL) was added to quartz Petri dishes (3 cm diameter), followed by 8 µL of MB solution (1 mg/mL). Equal amounts of Pt nanoparticles were added to the GDYO and PBS groups as catalysts. To eliminate interference from •OH radicals, methanol was added as a quencher to all groups. Ultrasound treatment (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density) was applied in the dark at fixed intervals. The dynamic variations in MB levels were tracked by ultraviolet-visible spectrophotometry through absorbance detection at 664 nm. The normalized absorbance was calculated as: A = A t min /A 0 min × 100% where A 0 min and A t min represent the absorbance values at 664 nm before and after ultrasound treatment, respectively. In vitro oxygen detection Oxygen production was monitored using a dissolved oxygen meter (OHAUS ST 300D, USA). Specifically, GDYO, Pt, and GDYO@Pt (200 µg/mL) were dispersed in 30 mL of 10 mM hydrogen peroxide (H₂O₂) solution. Ultrasound stimulation was then applied to each group, and dissolved oxygen levels were recorded every 1 second. Piezoelectric and electrochemical characterization Piezoelectric current measurements were conducted using an electrochemical workstation (Metrohm VIONIC, Switzerland) with a standard two-electrode system, where indium tin oxide (ITO) conductive glass (1.0 × 4.0 cm²) served as both counter and working electrodes. The working electrode was modified with the material as follows: 100 µL of sample dispersion (1.5 mg/mL in H 2 O) was drop-casted onto one end of the ITO glass (1.0 × 1.0 cm²) and dried at 60°C for 24 hours. The piezoelectric behavior of the material was evaluated by real-time monitoring of current responses under varying ultrasound power densities (0, 0.3, 0.5, and 0.7 W/cm²) and five on/off cycles in PBS electrolytes at pH 4.5 and pH 7.4. Mott-Schottky measurements were performed in a 0.5 M Na₂SO₄ electrolyte using a three-electrode system. The working electrode (prepared as described above), a Pt plate counter electrode, and an Ag/AgCl reference electrode were employed. Scanning potentials from 1 V to 0.1 V were applied at frequencies of 1000 Hz, 1316 Hz, and 1732 Hz to obtain Mott-Schottky curves before and after ultrasound stimulation. Electrochemical impedance was measured in a mixed electrolyte containing 1 mM potassium ferricyanide (K₃[Fe(CN)₆]), 1 mM potassium ferrocyanide (K₄[Fe(CN)₆]), and 0.5 M KCl. The working electrode was prepared by drop-casting 10 µL of sample dispersion (1.0 mg/mL) onto a glassy carbon electrode. The counter and reference electrodes were identical to those used in the Mott-Schottky analysis. AC impedance spectra of different samples were acquired using an electrochemical workstation within a selected frequency range. Cell culture BMSCs were purchased from Yuchi Biotechnology Co., Ltd. (Shanghai, China) and cultured in complete medium (Gibco, Thermo Fisher Scientific, USA; composition: α-MEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin). Cells were maintained in a humidified incubator (Thermo Fisher Scientific, USA) at 37°C with 5% CO₂. Cell viability assessment with nanosheets at varying concentrations Cells were seeded in 24-well plates at a density of 5 × 10³ cells per well and co-cultured with nanosheets at varying concentrations (n = 4). After the designated incubation period, the medium (with or without nanosheets) was removed, and 10% CCK-8 solution (Beyotime Biotechnology, China) was added. The plates were then incubated at 37°C for 1 hour. Subsequently, 100 µL of solution from each well was transferred to a 96-well plate, and absorbance values were recorded at 450 nm. The relative cell viability was calculated as follows: Cell viability (%) = (Absorption value of different treatment groups)/(Mean absorption value of control) × 100% Effects of ultrasound-activated nanosheets on cell viability First, oxidative stress injury was induced in BMSCs using H₂O₂. Specifically, BMSCs (2 × 10⁴ cells/well) were seeded in 6-well plates and cultured for 24 hours. The medium was then replaced with H₂O₂-containing medium (0.2 mM) for 6 hours, followed by fresh complete medium. To evaluate the impact of ultrasound treatment on cell viability, Oxidative stress-injured BMSCs (2 × 10⁴ cells/well in 6-well plates) were exposed to ultrasound at varying power densities (0.3, 0.5, 0.7, 1.0, and 1.5 W/cm²) and durations (0, 5, 10, 15, and 20 minutes) to determine the threshold of ultrasound parameters affecting cell survival (n = 4). Then, nanosheets (200 µg/mL) were incubated with oxidative stress-damaged BMSCs, ultrasound stimulation (1.0 MHz frequency, 0.7 W/cm² power density, 50% duty cycle, 10 min per session) was applied or withheld on days 1, 3, and 7. Cell viability was assessed for all groups on days 3, 5, and 9 (n = 4). Effects of ultrasound-induced piezoelectricity on cell membrane potential The materials were spin-coated and immobilized on one end of the ITO conductive glass as described in Section 2.9. BMSCs were seeded at a density of 1 × 10⁴ cells onto the central region of sterilized ITO glass (seeding area: 1.0 × 1.0 cm²) and incubated in complete medium for 24 hours. The medium was then replaced with complete medium containing Di-8-ANEPPS (2 µM), followed by 30 minutes of incubation in the dark. Cells were gently rinsed three times with dye-free medium to remove excess probe. Subsequently, the material-coated end of the ITO glass was immersed in a PBS-filled electrochemical cell (pH 4.5). After stabilizing the dual-electrode system in the electrolyte for 30 seconds, ultrasound (0.7 W/cm², 5 minutes) was applied to activate the piezoelectric signals of the material. Fluorescence microscopy (Olympus CKX41SF, Japan) was employed to detect cellular fluorescence on the ITO glass (excitation: 488 nm, emission: 605 nm, red fluorescence). Effects of US-induced piezoelectricity on intracellular calcium levels Following the aforementioned protocol, 1 × 10⁴ BMSCs were seeded onto the central region of sterilized ITO conductive glass pre-coated with materials (seeding area: 1.0 × 1.0 cm 2 ) and incubated in complete medium for 24 hours. The medium was then replaced with Hanks' Balanced Salt Solution (HBSS) containing Fluo-4 (4 µM), followed by 30 minutes of incubation in the dark. Cells were gently rinsed three times with HEPES-buffered saline to remove excess dye. The material-coated end of the ITO glass was subsequently immersed in a PBS-filled electrochemical cell (pH 4.5). After stabilizing the dual-electrode system in the electrolyte for 30 seconds, ultrasound (0.7 W/cm², 5 minutes) was applied to activate the piezoelectric signals of the material. Intracellular calcium dynamics were monitored via fluorescence microscopy by detecting green fluorescence signals (excitation: 494 nm; emission: 516 nm) from Fluo-4-loaded cells. In vitro detection of intracellular hydrogen Adherent BMSCs were co-cultured with GDYO and GDYO@Pt (200 µg/mL) for 12 hours. Following trypsin digestion, PBS was used to prepare cell suspensions (density: 1 × 10 6 cells/mL). 1 mL of suspension was transferred into sealed headspace vials under gas-tight conditions. After ultrasonic irradiation for 0, 10, 20, 30, and 40 min respectively, 1 mL of headspace gas was collected for gas chromatography quantification of H₂ production. BMSCs were seeded in 6-well plates at a density of 2 × 10⁴ cells per well and cultured for 24 hours. Subsequently, the cells were incubated with GDYO and GDYO@Pt (200 µg/mL) for an additional 12 hours. Equal amounts of Pt nanoparticles were added to the GDYO and PBS groups as catalysts. After incubation, the cells were gently rinsed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes. The fixed cells were then co-mixed with methylene blue (MB, 100 µM) in PBS for 20 minutes. Afterwards, the medium was removed and the cells were washed three times with PBS. Ultrasound irradiation was applied to the cells with or without the following parameters: 1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density, and irradiation durations of 10 min. Bright-field images were acquired using fluorescence microscopy to analyze changes in the blue intensity of MB. In vitro detection of intracellular oxygen The Ru(dpp)₃Cl₂ (RDPP) probe was employed to monitor O₂ generation. RDPP is a red-fluorescent oxygen-sensitive indicator (excitation/emission wavelengths: 488 nm/620 nm), whose fluorescence is quenched upon interaction with O₂ via energy transfer. Thus, changes in RDPP fluorescence intensity inversely correlate with intracellular oxygen levels. Specifically, BMSCs were seeded in 6-well plates at a density of 2 × 10⁴ cells/well and allowed to adhere. The cells were then incubated with complete medium containing GDYO or GDYO@Pt (200 µg/mL) for 24 hours. Subsequently, the cells were incubated in complete medium supplemented with Ru(dpp)₃Cl₂ (30 µM) in the dark for 40 minutes. After incubation, the medium was removed, and the cells were gently washed three times with PBS. Ultrasound irradiation (parameters: 1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density, 5-minute duration) was applied or withheld. Fluorescence microscopy was used to capture images and analyze changes in the red fluorescence intensity of Ru(bpy)₃Cl₂. In vitro detection of intracellular ROS Intracellular ROS levels were measured using a ROS assay kit (DCFH-DA fluorescent probe; Beyotime Biotechnology, China). Briefly, BMSCs were seeded in 6-well plates at a density of 2 × 10⁴ cells per well and cultured for 24 hours. Experimental groups were then co-incubated with 200 µg/mL GDYO@Pt or GDYO nanosheets for 24 hours, followed by ultrasound treatment (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density, 5-minute irradiation) or no treatment. After washing with PBS, all groups were stained with the DCFH-DA probe under dark conditions for 30 minutes and washed again with PBS to remove unbound probes. DCF fluorescence signals (excitation: blue light ; emission: green light ) in BMSCs were detected using an inverted fluorescence microscope. ALP staining assay BMSCs were cultured at a density of 2 × 10⁴ cells per well in medium containing GDYO (200 µg/mL) or GDYO@Pt (200 µg/mL) nanosheets for 7 days, during which ultrasound was applied/not applied every two days (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density, irradiation time of 10 min), and control cells were incubated in medium without nanosheets for the same time. Subsequently, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 30 min, and an appropriate amount of ALP staining solution (Beyotime Biotechnology, China) was added to cover the cells uniformly, and then stained for 30 min at room temperature and protected from light. After the staining was completed, the cells were washed thoroughly with PBS, and the ALP-active areas were observed and photographed under a microscope to record the ALP activity areas to assess the early osteogenic differentiation ability of BMSCs. For quantitative analysis, ALP activity was measured using an ALP assay kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions (n = 3). Alizarin Red S staining assay BMSCs were cultured at a cell density of 2 × 10⁴ BMSCs per well in medium containing GDYO (200 µg/mL) or GDYO@Pt (200 µg/mL) nanosheets for 14 days, during which ultrasound was applied/not applied every two days (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm² power density, irradiation time of 10 min, respectively), and control cells were incubated in medium without nanosheets for the same time. After incubation, cells were washed with PBS and fixed with 4% PFA for 30 minutes. A 0.2% Alizarin Red S solution (Solarbio Biotechnology, China) was added to completely cover the cells, followed by 30 minutes of staining at room temperature. Unbound dye was removed by thorough PBS rinsing. Calcium nodule formation, indicated by orange-red mineralized deposits, was visualized and photographed under a microscope to evaluate late-stage osteogenic differentiation and mineralization capacity. For quantitative analysis, calcium deposits were dissolved in 2% cetylpyridinium chloride (Sigma-Aldrich, USA), and absorbance was measured at 550 nm using a microplate reader (BioTek Cytation 3, USA) (n = 3). RT-qPCR analysis of osteogenic differentiation genes in BMSCs To further investigate the osteogenic effects of ultrasound-activated nanosheets, total mRNA was extracted from all experimental groups using Trizol reagent (Invitrogen, USA). First-strand cDNA was synthesized with the PrimeScript RT reagent kit (TaKaRa, Japan) following the manufacturer's protocol. Real-time quantitative PCR amplification was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA) using SYBR Green fluorescent dye (Roche, USA). Gene-specific primers for osteogenic markers – alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), type I collagen (COL-1), and Runt-related transcription factor 2 (RUNX-2) – were designed (primer sequences provided in Supplementary Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference gene for data normalization. Relative mRNA expression levels were calculated using the 2 − ΔΔ Ct method to quantify ultrasound-enhanced osteogenic differentiation (n = 3). In vivo experimental study All experimental procedures were conducted in strict compliance with the National Standards for the Care and Use of Laboratory Animals in China. Eight-week-old wild-type C57BL/6 mice (purchased from the National Institutes for Food and Drug Control, NIFDC, China) were housed in specific pathogen-free (SPF) facilities pre- and post-operatively, with ad libitum access to water and a standard rodent diet. Surgical protocols and perioperative management followed the guidelines approved by the Animal Care and Use Committee of Beihang University (License No. BM201900084). Establishment of cranial defect model and in situ injection of Gel Mice were group-housed for one week preoperatively for environmental acclimatization. Anesthesia was induced and maintained via isoflurane inhalation. After incising the scalp, a 3.0 mm diameter full-thickness circular bone defect was created in the parietal bone using a dental trephine drill. The defect area was irrigated with saline, followed by in situ injection of thermosensitive gel formulations into the defect cavity according to experimental groups. After complete gelation (37°C, 10 minutes), the wound was closed in layers. Postoperatively, ibuprofen (10 mg/kg, Macklin, China) was administered in drinking water for 24 hours to alleviate pain. To prevent infection, sulfamethoxazole (15 mg/kg) and trimethoprim (30 mg/kg) (Macklin, China) were added to drinking water for one week. Therapeutic procedure for murine cranial defect repair Cranial defect-bearing mice were randomly allocated into six experimental groups (n = 4): (1) Control (no treatment), (2) gel + US (thermosensitive gel with ultrasound stimulation), (3) GDYO gel (gel containing 0.5 mg/mL GDYO nanosheets without ultrasound), (4) GDYO@Pt gel (gel containing 0.5 mg/mL GDYO@Pt nanosheets without ultrasound), (5) GDYO gel + US (GDYO-loaded gel with ultrasound), and (6) GDYO@Pt gel + US (GDYO@Pt-loaded gel with ultrasound). All interventions were standardized to ensure uniform nanosheet concentrations and treatment protocols across groups. Ultrasound stimulation (1.0 MHz, 50% duty cycle, 0.7 W/cm²) was administered once daily (10 minutes per session), for 4 consecutive weeks. Cranial bone samples were harvested at 6 and 12 weeks post-treatment and analyzed for bone regeneration outcomes using micro-CT and histological assessments. Micro-CT Analysis The extracted cranial bone samples post-treatment were scanned using a Micro-CT system (Skyscan 1276, Bruker, Belgium) under 55 kV voltage and 200 µA current. Three-dimensional image reconstruction and analysis of the bone defect region were performed using NRecon and CTvox software. Bone regeneration parameters, including BV/TV and BMD, were quantified with CTAn software. Histological Evaluation Following micro-CT analysis, samples from the 12-week treatment groups were decalcified in 15% ethylenediaminetetraacetic acid (EDTA) solution and subsequently embedded in paraffin. Tissue sections (4 µm thickness) were prepared, deparaffinized with xylene, and rehydrated through a graded ethanol series (70–100%). Each sample underwent hematoxylin and eosin (H&E) staining and Masson’s trichrome staining. Histomorphological structures were examined using a digital tissue section scanner (Pannoramic MIDI, Hungary). IHC and IF Staining Protocol IHC Staining: Paraffin-embedded sections were dewaxed and blocked, then incubated overnight at 4°C with the following primary antibodies: anti-OCN antibody, anti-RUNX-2 antibody, anti-CD31 antibody and anti-NRF2 antibody. All primary antibodies were diluted at 1:200 using 3% (w/v) bovine serum albumin (BSA, Sigma-Aldrich, USA). After PBS washing, samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Staining signals were visualized using the digital tissue section scanner. IF Staining: Dewaxed paraffin sections were blocked with 3% BSA to prevent nonspecific binding, followed by overnight incubation at 4°C with primary antibody (HIF-1α antibody). 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Supplementary Files SupplementaryInformation.docx Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6497438","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":449582861,"identity":"0053bcdb-d4f8-4109-baff-8fd3fef66e06","order_by":0,"name":"Lizhen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACPmYGNiiT+RiUkYBfCxtCC1sakVoY4Fp4zIjUws7+7MHHHYflzfnXfHvMU2PHwM+eY8Dwcwdeh6Ubzjxz2HDnjLfbjXmOJTNI9rwxYOw9g1fLMWnettuMG26c3SbNA+Qa3MgxYGZsw6eFsU36b9tt+w03zjyT5vlXz2BPWAszmzRj2+3EDed72IDWHWYwkCCohY1Nsrftf/KGG2zmhnP7jvNInHlWcLAXjxZ+/uPPJH62pdluOH/42YM336rl+NuTNz74iUcLAkgkgCkeEHGAGA1A+4hUNwpGwSgYBSMPAAAQeEvj+jt/EgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9658-659X","institution":"School of Biological Science and Medical Engineering, Beihang University","correspondingAuthor":true,"prefix":"","firstName":"Lizhen","middleName":"","lastName":"Wang","suffix":""},{"id":449582862,"identity":"3b78502a-0c07-4bf9-8072-ac6544dcf0a2","order_by":1,"name":"Kang Song","email":"","orcid":"","institution":"School of Biological Science and Medical Engineering, Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Song","suffix":""},{"id":449582863,"identity":"55f11626-f844-48ad-bae4-5b499edbdaa2","order_by":2,"name":"Xuezheng Geng","email":"","orcid":"","institution":"School of Biological Science and Medical Engineering, Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Xuezheng","middleName":"","lastName":"Geng","suffix":""},{"id":449582864,"identity":"d5b2b2c7-9f68-4c5b-b593-c2b3f2d79bb2","order_by":3,"name":"Huan Yin","email":"","orcid":"","institution":"Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Yin","suffix":""},{"id":449582865,"identity":"a821b6b2-ea1b-4a60-adba-a13d1296d51a","order_by":4,"name":"Yanzhu Shi","email":"","orcid":"","institution":"Beijing Institute of Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Yanzhu","middleName":"","lastName":"Shi","suffix":""},{"id":449582866,"identity":"eef5e811-f2a1-4c35-a64a-e5a11ce62656","order_by":5,"name":"Jiawei Wang","email":"","orcid":"","institution":"School of Biological Science and Medical Engineering, Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Jiawei","middleName":"","lastName":"Wang","suffix":""},{"id":449582867,"identity":"790cc055-a14b-45f2-aa86-ad50e30966ce","order_by":6,"name":"Jiayu Yu","email":"","orcid":"","institution":"School of Biological Science and Medical Engineering, Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Jiayu","middleName":"","lastName":"Yu","suffix":""},{"id":449582868,"identity":"d470e9f4-2572-4f32-b137-2167a14ab067","order_by":7,"name":"Mateng Bai","email":"","orcid":"","institution":"School of Biological Science and Medical Engineering, Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Mateng","middleName":"","lastName":"Bai","suffix":""},{"id":449582869,"identity":"ba4d09ec-079c-4252-92de-bcb213734c34","order_by":8,"name":"Yurui Xue","email":"","orcid":"https://orcid.org/0000-0002-9783-1753","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yurui","middleName":"","lastName":"Xue","suffix":""},{"id":449582870,"identity":"f6899d79-797d-4284-a0a7-25f994e2e4ac","order_by":9,"name":"Chunli Song","email":"","orcid":"https://orcid.org/0000-0002-3690-9457","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunli","middleName":"","lastName":"Song","suffix":""},{"id":449582871,"identity":"5971a12b-c990-4802-8d2a-8b720e92316c","order_by":10,"name":"Yubo Fan","email":"","orcid":"https://orcid.org/0000-0002-3480-4395","institution":"Beihang University","correspondingAuthor":false,"prefix":"","firstName":"Yubo","middleName":"","lastName":"Fan","suffix":""}],"badges":[],"createdAt":"2025-04-21 15:40:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6497438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6497438/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-63550-8","type":"published","date":"2025-09-26T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81666690,"identity":"22eac73a-8225-4fa5-8713-0ab8be6f27d3","added_by":"auto","created_at":"2025-04-30 03:33:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":834834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of GDYO@Pt synthesis and synergistic therapy for bone defects. \u003c/strong\u003eFabrication protocol of GDYO@Pt nanosheets and their hydrogel composite. Enhancement mechanism and workflow of piezoelectric activation and catalytic therapy.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/f6b55faba526e116c602c080.png"},{"id":81666691,"identity":"4b8c3cb7-c162-4c5f-a916-30adfb4657ce","added_by":"auto","created_at":"2025-04-30 03:33:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":971560,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of nanosheets and thermoresponsive gels.\u003c/strong\u003e (A) TEM image of GDYO and GDYO@Pt. (B) The elements scanning mapping of GDYO@Pt. (C) The raman spectra of GDYO and GDYO@Pt. (D) High-resolution TEM of GDYO@Pt. (E) AFM image (3D topography) of GDYO and GDYO@Pt. (F) AFM image (2D) and size distribution curve of GDYO@Pt nanosheets (inset). High-resolution XPS spectra of (G) Pt and (H) C. (I) SEM images at magnifications from (i) low to (iii) high, (J) elements scanning mapping, (K) FT-IR spectra and rheological testing including (L) shear-thinning (orange line) and step-shear (cyan line), (M) step-strain-sweep, (N) oscillatory strain-sweep, (O) oscillatory time-sweep, (P) temperature-dependent-sweep of GDYO@Pt gel. (Q) Digital photographs of GDYO@Pt gel at room temperature (25°C) and physiological temperature (37°C).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/264c6bd4eccf16237ce3a880.png"},{"id":81666693,"identity":"2bc9e07a-ff2c-49fc-99b5-ca1da476b439","added_by":"auto","created_at":"2025-04-30 03:33:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":759114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePiezoelectric and mechanistic of nanosheets. \u003c/strong\u003ePFM including (A, E) Amplitude, (B, F) phase mapping, (C, G) the butterfly amplitude loop and phase curve and (D) the slope of the hysteresis loop of GDYO and GDYO@Pt. (H) The charge difference distribution (2D slice) of GDYO@Pt interface, red and blue indicate charge accumulation and depletion, respectively. (I, J) The potential maps in KPFM mode and (K) their corresponding potential amplitude of GDYO and GDYO@Pt. (L) The calculated dipole moment of Pt, GDYO and GDYO@Pt. The simulated potential distribution of (M) GDYO and (N) GDYO@Pt. (O) The potential data extracted from the potential distribution between GDYO and Pt. (P) Schematic representation of the polarization mechanism of GDYO@Pt nanosheets.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/67a56f0e33ade0d96db4870b.png"},{"id":81666695,"identity":"7b205a9f-6fe4-44eb-938c-b32d4e7aa3da","added_by":"auto","created_at":"2025-04-30 03:33:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":711091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePiezocatalytic behavior and mechanisms of GDYO@Pt Nanosheets.\u003c/strong\u003e (A) UV–vis–NIR spectra of the mixture of GDYO@Pt and MB under US stimulation at different times. (B) The ratio of absorbance at 664 nm of GDYO or GDYO@Pt mixed with MB under US stimulation at different time points. (C) The amount of H\u003csub\u003e2\u003c/sub\u003e produced in each group of solutions was measured by GC. (D) Oxygen generation by GDYO, Pt and GDYO@Pt under H₂O₂ and ultrasonic treatment (On: 5 min; Off: 5 min, repeated for 5 cycles). Work function of (E) GDYO nanosheets, (F) GDYO@Pt nanosheets and (G) Pt nanoparticles. (H-J) ‌Schematic diagram of schottky junction construction, charge transfer, and piezocatalytic mechanism in GDYO@Pt. (K, L) ‌Mott-Schottky curves of GDYO@Pt nanosheets tested with/without Ultrasonic Vibration. (M) The electron density distribution at the GDYO/Pt interface (yellow: electron-enriched regions; blue: electron-depleted zones). (N) Laterally averaged electron density difference along the z-direction. (O) Adsorption energy of water and (P) Gibbs free energy diagram of hydrogen evolution for Pt, GDYO, and GDYO@Pt.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/dc0ec3e487f8781b6b1552a9.png"},{"id":81667141,"identity":"fb60e5e1-d23a-4f4e-b208-e71905ff0f11","added_by":"auto","created_at":"2025-04-30 03:41:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1814789,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePiezoelectric stimulation of BMSCs in physiological media.\u003c/strong\u003e (A) Viability of BMSCs cultured for 3 days under varied ultrasonic power (1.0 MHz, 50% duty cycle). (B) The cellular survival rates of various treatment groups, including control, ROS, US, GDYO, GDYO@Pt, GDYO + US and GDYO@Pt + US (US: 0.7 W cm\u003csup\u003e−2\u003c/sup\u003e, 10 min) (mean ± SD, n = 4). (C, D) The current stability of ultrasound-triggered samples in PBS with varying pH levels was systematically assessed over five repeated ultrasound irradiation on/off cycles. (E) The current generated by GDYO@Pt under ultrasound irradiation at varying power levels (5-minute excitation duration) were rigorously quantified. (F) Schematic illustration of the decoupled experimental setup for detecting electrical signals generated by ultrasound-activated materials and applying electrical stimulation to cells. Fluorescence images and semi-quantitative statistics (F/F\u003csub\u003e0\u003c/sub\u003e represents the ratio of post-ultrasound fluorescence intensity to pre-ultrasound fluorescence intensity, with the inset displaying the statistical analysis of fluorescence intensities across experimental groups) of BMSCs stained with (G, H) Di-8-ANEPPS (red) and (I, J) Fluo-4 (green) after different treatments. Statistical differences were calculated using independent samples T-test (***P \u0026lt; 0.001, **P \u0026lt; 0.01 and *P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/3cd8feac32b2abbf127cc466.png"},{"id":81667188,"identity":"2dded741-fd1c-4d2a-8f32-e3775a43f6ca","added_by":"auto","created_at":"2025-04-30 03:49:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1592089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of oxidative stress levels at the cellular level.\u003c/strong\u003e (A) Quantitative assessment of ultrasound-activated GDYO@Pt for H₂ production in BMSCs and (B) qualitative evaluation of H₂ production using MB staining in BMSCs under different treatments including control, US, GDYO, GDYO@Pt, GDYO + US, GDYO@Pt + US. (C) Fluorescence images of BMSCs stained with [Ru(dpp)\u003csub\u003e3\u003c/sub\u003e]Cl\u003csub\u003e2\u003c/sub\u003e (red) and DAPI (blue) after different treatments. (D) Fluorescence microscopy images of BMSCs stained with DCFH-DA (green) and DAPI (blue) after different treatments. (US: 1.0 MHz, 50% duty cycle, 0.7 W cm\u003csup\u003e−2\u003c/sup\u003e, 5 min)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/9c75b6ba1695d39a79f4544a.png"},{"id":81667144,"identity":"b56f95e1-ab8f-4a3d-9ffe-0a13fa216c89","added_by":"auto","created_at":"2025-04-30 03:41:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":673688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsteogenic differentiation at the cellular level.\u003c/strong\u003e (A, B) ALP (7 days) and (A, C) ARS (14 days) staining images and semi-quantitative analysis of BMSCs in different treatments including control, ROS, GDYO, GDYO@Pt, GDYO + US, GDYO@Pt + US. Relative mRNA expression levels of osteogenic genes from BMSCs after different treatments, including (D) ALP, (E) OCN, (F) OPN, (G) COL-1 and (H) RUNX-2. (US: 1.0 MHz, 50% duty cycle, 0.7 W cm\u003csup\u003e−2\u003c/sup\u003e, 10 min). Statistical differences were calculated using independent samples T-test (***P \u0026lt; 0.001, **P \u0026lt; 0.01 and *P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/d9a1f2df54b2e8578216e9b8.png"},{"id":81666700,"identity":"878daade-dd0a-4f9f-bb73-c25f4b4fa485","added_by":"auto","created_at":"2025-04-30 03:33:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1458445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e bone regeneration assessment. \u003c/strong\u003e(A) Therapeutic protocol for calvarial defect repair in C57BL/6 mice. (B) Representative microscopic CT images of the cranial defect site at 6 and 12 weeks after implantation in different treatment groups. Scale bars: 0.5 mm. White circles represent defect boundaries. (C) The ratio of new bone to total area, (D) BMD and (E) BV/TV of cranial defect region in control, gel + US, GDYO gel, GDYO@Pt gel, GDYO gel + US and GDYO@Pt gel + US at different times. Representative (F) HE and (G) Masson’s trichrome staining images of the cranial defect areas across various groups at the 12-week post-implantation period (CT: connective tissues; NB: new bone). Typical immunohistochemical staining patterns demonstrating (H) OCN, (I) RUNX-2, and (J) CD31 expression in neoformed calvarial regions at 12 weeks post-implantation (black arrows: positive staining regions). (K) Immunofluorescence micrographs showing CD206 (red) and CD86 (green) expression in the defect region. (US: 1.0 MHz, 50% duty cycle, 0.7 W cm\u003csup\u003e−2\u003c/sup\u003e, 10 min). Statistical differences were calculated using independent samples T-test (***P \u0026lt; 0.001, **P \u0026lt; 0.01 and *P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/3739f6280f572cc24c2ca6f1.png"},{"id":81666697,"identity":"a5c6beb1-20db-458c-bf52-b2000e38d8eb","added_by":"auto","created_at":"2025-04-30 03:33:06","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":979035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress levels \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) Fluorescent photographs of ROS levels in different treatment groups. (B) Immunofluorescence staining analysis of HIF-1α in bone defect area. (C) Characteristic immunohistochemical staining profiles of NRF2 following various therapeutic interventions at 12 weeks post-treatment. (US: 1.0 MHz, 50% duty cycle, 0.7 W cm\u003csup\u003e−2\u003c/sup\u003e, 10 min).\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/9139d666f3a5a7e6ad33413b.png"},{"id":92305124,"identity":"4ebf15a4-c005-407b-83be-dc1f2e5a6b8b","added_by":"auto","created_at":"2025-09-27 07:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11132249,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/5f95142d-9357-4f5e-ab40-d517c2464493.pdf"},{"id":81667147,"identity":"824b1eb9-6798-4917-a4df-cc65e08c50db","added_by":"auto","created_at":"2025-04-30 03:41:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6144065,"visible":true,"origin":"","legend":"Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6497438/v1/2174ec63ddb7de7dbbc3693f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePiezoelectric semiconductor-based electrical stimulation offers a non-invasive alternative by converting mechanical stress into electrical energy under frequency- and intensity-controlled ultrasound (US) irradiation, generating adjustable electric fields for therapeutic intervention\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The majority of piezoelectric semiconductors including BaTiO\u003csub\u003e3\u003c/sub\u003e, ZnO, and Bi\u003csub\u003e2\u003c/sub\u003eWO\u003csub\u003e6\u003c/sub\u003e for bone repair exhibit excellent piezoelectric responses but have wide bandgaps (\u0026gt;\u0026thinsp;2.0 eV)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The piezoelectric potential generation is accompanied by the generation of carriers that undergo redox reactions with water, resulting in ROS which cause oxidative damage to healthy tissues\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The overproduction of endogenous ROS and the presence of a hypoxic microenvironment at a bone-defect site severely impede healing, prolonging the course of electrical stimulation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. So it is essential to develop narrow-bandgap piezoelectric materials delivering efficient electrical stimulation and regulating oxidative stress simultaneously.\u003c/p\u003e \u003cp\u003eThe absence of a centrosymmetric structure is a fundamental characteristic for achieving piezoelectric properties\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Strategies including interlayer stacking, elemental doping, and electric field stimulation would reduce or break the inversion symmetry to induce piezoelectric responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Monolayer graphene exhibits no intrinsic piezoelectricity but multilayer stacking configurations disrupt its overall inversion symmetry, enabling piezoelectric effects through interlayer coupling and intralayer electron transition competition\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Oxygen as a dopant into graphene oxide further breaks the intralayer inversion symmetry via \"clamp\u0026ndash;release\" structural distortions, enhancing the piezoelectric effect\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Charge redistribution in graphene nanostructures is driven by adjacent charged molecules, permanent dipoles, or built-in electric fields. This interaction can disrupt the inversion symmetry to amplify the piezoelectric performance\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Built-in electric fields, which arise from band bending or chemical potential gradients at heterojunction interfaces, have been shown to universally induce polar structures and substantial piezoelectric effects in semiconductors when interfaced with noble metals, as demonstrated by Yang et al.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These findings underscore the feasibility of engineering nonpiezoelectric 2D carbon materials into high-performance piezoelectric systems through strategic modifications.\u003c/p\u003e \u003cp\u003ePiezoelectric effect-driven electrical stimulation not only directly modulates cellular electrophysiological activity, but also its accompanying piezocatalytic reactions can directionally generate redox-active species (e.g., ROS, H₂, or O₂) through ultrasound-induced charge separation, dynamically regulating local oxidative stress levels\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The underlying mechanism lies in the fact that the valence/conduction band positions of piezoelectric semiconductors determine interfacial redox potentials (e.g., the conduction band ought to be positioned below the H⁺/H₂ redox potential to drive hydrogen production, while the valence band should exceed the H₂O/\u0026bull;OH and H₂O₂/O₂ potentials to trigger hydroxyl radical generation and hypoxia-alleviating oxygen supply)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This provides a solution for precisely regulating oxidative stress via band engineering. During oxidative stress modulation, molecular hydrogen (H₂) serves as both a safe therapeutic agent and an anti-inflammatory mediator, which selectively neutralizes highly toxic \u0026bull;OH radicals through its antioxidant activity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The exceptional tissue permeability of H\u003csub\u003e2\u003c/sub\u003e allows its effective diffusion into ROS-generating organelles (e.g., mitochondria and nuclei)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, hypoxia is known to upregulate hypoxia-inducible factor 1α (HIF-1α) in infiltrating immune cells, thereby promoting ROS production\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Concurrently addressing the issue of hypoxia during ROS scavenging may offer a more efficient strategy for managing oxidative stress and electrical stimulation therapy.\u003c/p\u003e \u003cp\u003eGraphdiyne (GDY), a carbon-based two-dimensional material, exhibits exceptional structural stability and functional tunability due to its unique configuration of sp-/sp\u0026sup2;-hybridized carbon atoms\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The sp-hybridized carbon (C\u0026thinsp;\u0026equiv;\u0026thinsp;C) bonds endow GDY with superior reducibility and electron transfer capability\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, while its narrow bandgap (~\u0026thinsp;1.22 eV, comparable to silicon's 1.11 eV) further establishes it as an ideal platform for synergistic regulation of piezoelectric effects and catalytic reactions\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Herein, this study focuses on the development of a narrow-bandgap piezoelectric semiconductor heterojunction (GDYO@Pt) as a dual-function platform for piezoelectric stimulation and oxidative stress regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In this system, the charge distribution in the graphdiyne oxide (GDYO) nanosheets is reconfigured by the interfacial dipole-induced built-in electric field within the depletion region. This is proposed to promote breaking of the structural inversion symmetry and the formation of asymmetric polar configurations, aiming to significantly enhance the piezoelectric performance. Positively polarized charges are generated at the Schottky junction interface under ultrasound, which should induce downward band bending and barrier reduction. It is expected to facilitate electron transfer from GDYO to Pt, and promote sustained H\u003csub\u003e2\u003c/sub\u003e production. Concurrently, it is proposed that the holes will oxidize H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate O\u003csub\u003e2\u003c/sub\u003e, while the GDYO@Pt will act as a nanozyme to catalytically decompose H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into O\u003csub\u003e2\u003c/sub\u003e. This strategy effectively alleviates hypoxia and scavenges ROS, overcoming the limitations of oxidative stress on piezoelectric stimulation. Cellular validation confirms the GDYO@Pt system's modulation of BMSCs membrane potential, calcium influx, H₂/O₂ levels, and ROS clearance, verifying piezoelectric\u0026ndash;catalytic synergy. Finally, GDYO@Pt-loaded thermoresponsive hydrogels are applied under ultrasound activation to promote defect repair via osteogenic differentiation, angiogenesis, and immunomodulation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials preparation and characterization\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGDY was synthesized according to a previously reported method\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, GDYO nanosheets were prepared via a modified Hummer's method using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as oxidants. GDYO@Pt was subsequently fabricated by in situ reduction of K\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e4\u003c/sub\u003e with ascorbic acid. During this process, the strong affinity between the C\u0026thinsp;\u0026equiv;\u0026thinsp;C bonds of GDYO and the Pt\u003csup\u003e2+\u003c/sup\u003e species enabled efficient capture and reduction of the Pt ions. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed nanosheet structures for both GDYO and GDYO@Pt, with lateral dimensions of ~\u0026thinsp;500 nm and thicknesses of ~\u0026thinsp;3 and ~\u0026thinsp;7 nm, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Pt nanoparticles (~\u0026thinsp;3 nm in diameter) were uniformly distributed on the GDYO surface (Supplementary Fig.\u0026nbsp;1). Energy-dispersive X-ray spectroscopy (EDS) mapping confirmed the homogeneous distribution of C, O, and Pt in the prepared GDYO@Pt (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Raman spectroscopy yielded characteristic peaks at 1950 and 2159 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), while Fourier-transform infrared (FTIR) spectroscopy gave a single peak at 2050 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2), thereby confirming the retention of C\u0026thinsp;\u0026equiv;\u0026thinsp;C bonds in GDYO and GDYO@Pt. The X-ray diffractometry (XRD) results showed that GDYO@Pt exhibited diffraction peaks corresponding to the Pt standard card (PDF#040802), consistent with the lattice spacing observed in the high-resolution TEM image (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Supplementary Fig.\u0026nbsp;3), and confirming the presence of metallic Pt. X-ray photoelectron spectroscopy (XPS, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and Supplementary Fig.\u0026nbsp;4) was employed to further elucidate the chemical states of the prepared GDYO@Pt. The distinct Pt 4f peaks were observed, corresponding to metallic Pt\u003csup\u003e0\u003c/sup\u003e (Pt 4f\u003csub\u003e7/2\u003c/sub\u003e: 71.12 eV; Pt 4f\u003csub\u003e5/2\u003c/sub\u003e: 74.38 eV) and Pt\u003csup\u003e2+\u003c/sup\u003e (Pt 4f\u003csub\u003e7/2\u003c/sub\u003e: 72.54 eV; Pt 4f\u003csub\u003e5/2\u003c/sub\u003e: 76.16 eV), indicating the predominant reduction of Pt\u003csup\u003e2+\u003c/sup\u003e to Pt\u003csup\u003e0\u003c/sup\u003e, but with some residual surface oxidation. The high-resolution C 1s spectra recorded for both GDYO and GDYO@Pt displayed characteristic peaks corresponding to C\u0026thinsp;=\u0026thinsp;C, C\u0026thinsp;\u0026equiv;\u0026thinsp;C, C\u0026thinsp;=\u0026thinsp;O, and C\u0026ndash;O bonds, confirming the structural integrity of GDYO following the incorporation of Pt (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). All C 1s peaks in GDYO@Pt shifted to higher binding energies than those in GDYO, suggesting electron transfer from GDYO to Pt during GDYO@Pt formation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePoloxamer 407 (P407), a PEO-PPO-PEO triblock copolymer (PEO: poly(ethylene oxide); PPO: poly(propylene oxide)), requires concentrations\u0026thinsp;\u0026ge;\u0026thinsp;18% (w/v) to form thermoresponsive hydrogels\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. While higher P407 concentrations (20\u0026ndash;24%) lead to increased mechanical strengths, they reduce the sol\u0026ndash;gel transition temperature from 26.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 to 22.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u0026deg;C, rendering the resulting hydrogels unsuitable for injectable applications\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Thus, a P407 concentration of 18% was selected for the purpose of the current study. Tannic acid (TA), which is a natural polyphenol with bioadhesive properties, was incorporated at varying concentrations (0\u0026ndash;2%) to optimize crosslinking\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. As shown in Supplementary Figs.\u0026nbsp;5 to 6 and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ, TA concentrations\u0026thinsp;\u0026ge;\u0026thinsp;1% prevented gelation at 37\u0026deg;C, although the incorporation of GDYO@Pt (0.5 mg/mL) maintained the thermoresponsive nature of the hydrogel. Thus, a TA concentration of 0.7% was selected for subsequent experiments.\u003c/p\u003e \u003cp\u003eScanning electron microscopy (SEM) of the lyophilized gels revealed porous network structures for both the gel (P407/TA) and GDYO@Pt gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, i). The gel exhibited uniformly distributed surface micropores, whereas the GDYO@Pt gel showed markedly reduced porosity, indicating homogeneous GDYO@Pt dispersion within the matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, ii和iii). This spatial uniformity was confirmed by EDS mapping, revealing consistent distribution of C, O, and Pt elements across the composite (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). The FTIR spectra confirmed successful modification, with peaks corresponding to the C\u0026thinsp;=\u0026thinsp;O (1730.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) stretching vibrations of TA, and the C\u0026ndash;O (1110.8 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and C\u0026ndash;H (2889.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) vibrations of P407 dominating the spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Rheological studies using the GDYO@Pt gel demonstrated its shear-thinning behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL), self-healing capability under step strain/shear tests (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM), and optimal viscoelastic properties during strain/frequency sweep tests (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN and Supplementary Fig.\u0026nbsp;7). Time-dependent viscosity measurements and tube inversion tests confirmed a sol\u0026ndash;gel transition time of 5 s and full gelation within 3 min at 37\u0026deg;C (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO and Supplementary Fig.\u0026nbsp;8). Temperature ramps revealed a phase transition temperature of 26.3\u0026deg;C, enabling liquid-state storage at room temperature and rapid solidification upon injection into the tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP). The visual observations supported this temperature-triggered phase transition, thereby confirming successful fabrication of the GDYO@Pt gel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePiezoelectric response characterization of the nanosheets\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe piezoelectric properties of the nanosheets were investigated using piezoresponse force microscopy (PFM), a technique that is widely employed for the high-resolution characterization of piezoelectric materials. The vertical piezoresponse amplitudes and phase images of the GDYO and GDYO@Pt nanosheets were shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF. Both materials exhibited distinct contrast variations, indicative of their piezoelectric activities, with GDYO@Pt demonstrating an enhanced responsiveness. Under a\u0026thinsp;\u0026plusmn;\u0026thinsp;6 V or \u0026plusmn;\u0026thinsp;10 V ramped voltage, distinct butterfly-shaped amplitude loops were observed, demonstrating that a consistent strain variation was induced by the applied electric field. The corresponding local piezoelectric hysteresis loops revealed an ~\u0026thinsp;180\u0026deg; phase switching behavior, confirming the intrinsic piezoelectric nature of these materials (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Quantitative analysis of the amplitude loop slopes demonstrated that Pt incorporation significantly enhanced the piezoelectric response of GDYO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Kelvin probe force microscopy (KPFM) measurements showed a surface potential of 41 mV for GDYO@Pt, which was markedly higher than that of GDYO (22 mV), attributed to piezoelectricity-induced electrical polarization effects (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). COMSOL simulations performed under a 10\u003csup\u003e8\u003c/sup\u003e Pa mechanical stress revealed piezoelectric potentials of 5 V for GDYO and 9 V for GDYO@Pt, confirming the critical role of Pt nanoparticle modification in amplifying the piezoelectric performance of GDYO (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDensity functional theory (DFT) and finite element analysis were employed to elucidate the mechanism of the enhanced piezoelectric effect. The differential charge density diagram clearly revealed the out-of-plane charge transfer behavior between the Pt nanoparticles and GDYO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), indicating the formation of a strong interfacial electric field through electron delocalization during the tight bonding process between the Pt and GDYO. The process of charge transfer was also one of charge redistribution. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO, the contact interface between the GDYO nanosheets and the Pt metal particle exhibited a potential difference of ~\u0026thinsp;3.3 mV, with adjacent areas of the nanosheets showing polarization-affected potentials, which may be attributed to charge redistribution. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL showed that along the a- and b-axes, the unit cell dipole moments of GDYO@Pt significantly increased to 82.03 and \u0026minus;\u0026thinsp;176.59 Debye (D), respectively, which were 1.4-fold greater than those of GDYO (i.e., 57.67 and \u0026minus;\u0026thinsp;132.24 D). These values confirmed the substantial enhancement in the degree of polarization of GDYO following the loading of Pt nanoparticles. As shown in Supplementary Fig.\u0026nbsp;9, the increased relative dielectric constant of GDYO@Pt quantitatively reflected the enhanced polar intensity, consistent with the aforementioned results. These results indicate that in the Pt-GDYO Schottky junction, interface charge transfer triggered by work function differences forms a dipole layer, and the resulting built-in electric field further induces charge redistribution within GDYO nanosheets. This process disrupts the central symmetry of the semiconductor, enabling more efficient separation of polarization charges under stress and thereby significantly enhancing the material\u0026rsquo;s piezoelectric performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP).\u003c/p\u003e\n\u003ch3\u003ePiezocatalytic properties of the GDYO@Pt nanosheets\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eConsidering the excellent piezoelectric properties of the GDYO@Pt nanosheets, the piezocatalytic reactions induced by the US-activated nanosheets were evaluated. Using electron spin resonance (ESR) spectroscopy to detect the generation of ROS (Supplementary Fig.\u0026nbsp;10), it was found that no ROS production occurred under US stimulation, perhaps due to the insufficient redox potential of the band structure for ROS generation. The piezocatalytic H\u003csub\u003e2\u003c/sub\u003e production performance of the nanosheets was evaluated using a methylene blue (MB) probe to detect the catalytic H\u003csub\u003e2\u003c/sub\u003e generation (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, and Supplementary Fig.\u0026nbsp;11). The GDYO@Pt solution containing the MB probe exhibited a time-dependent decrease in absorbance at 664 nm, with a more significant reduction observed compared to the other groups, indicating that US-activated H\u003csub\u003e2\u003c/sub\u003e generation occurred in the GDYO@Pt system. Gas chromatography (GC) was used to quantify H\u003csub\u003e2\u003c/sub\u003e production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and it was found that the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group exhibited the highest H\u003csub\u003e2\u003c/sub\u003e yield. Since Pt nanoparticles are known to catalyze the generation of O\u003csub\u003e2\u003c/sub\u003e from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the O\u003csub\u003e2\u003c/sub\u003e generation capacity was also monitored (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). It was found that GDYO@Pt acted as a nanozyme, with the GDYO@Pt\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;US group exhibiting significantly enhanced O\u003csub\u003e2\u003c/sub\u003e production compared to the GDYO\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;US and Pt\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;US groups. These phenomena were attributed to Schottky junction formation between the Pt metal and GDYO, which enhanced the piezoelectric performance and carrier separation efficiency, further promoting the catalytic performance in both H\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e generation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe energy band structures of GDYO and GDYO@Pt were analyzed by Mott\u0026ndash;Schottky measurements and ultraviolet-visible diffuse reflectance spectroscopy (UV-vis DRS) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK and Supplementary Figs.\u0026nbsp;12 to 13). It was determined that GDYO and GDYO@Pt exhibited n-type semiconductor characteristics (positive slopes), with flat band potentials (E\u003csub\u003efb\u003c/sub\u003e) of \u0026minus;\u0026thinsp;0.83 and \u0026minus;\u0026thinsp;1.03 V (vs. Ag/AgCl) at pH 7, and corresponding to \u0026minus;\u0026thinsp;0.63 and \u0026minus;\u0026thinsp;0.83 V vs. NHE (pH 7), respectively. After calibration to pH 0, the CB potentials were determined to be \u0026minus;\u0026thinsp;0.22 and \u0026minus;\u0026thinsp;0.42 V (vs. NHE, pH 0), both of which were more negative than the H⁺/H\u003csub\u003e2\u003c/sub\u003e redox potential (i.e., 0 V vs. NHE, pH 0), indicating efficient electron-driven H\u003csub\u003e2\u003c/sub\u003e evolution. The bandgaps of GDYO and GDYO@Pt were 1.36 and 1.49 eV, respectively. The calculated VB potentials were determined to be 1.14 and 1.07 V (vs. NHE, pH 0), which exceeded\u0026zwnj;\u0026zwnj; the oxidation potential of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/O\u003csub\u003e2\u003c/sub\u003e (0.69 V vs. NHE, pH 0), satisfying the thermodynamic requirements for the hole-mediated generation of O\u003csub\u003e2\u003c/sub\u003e from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Collectively, the band positions of GDYO and GDYO@Pt theoretically satisfied both H\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e evolution criteria.\u003c/p\u003e \u003cp\u003eThe mechanism of piezocatalysis was further investigated. Ultraviolet photoelectron spectroscopy (UPS) was employed to determine the work functions (W) of GDYO, Pt, and GDYO@Pt (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). The cutoff energies (E\u003csub\u003ecutoff\u003c/sub\u003e) for GDYO, Pt, and GDYO@Pt were determined to be 17.93, 16.80, and 17.52 eV, respectively. Using the equation W\u0026thinsp;=\u0026thinsp;21.22 \u0026minus; |E\u003csub\u003ecutoff\u003c/sub\u003e \u0026ndash; E\u003csub\u003eF\u003c/sub\u003e|, the calculated W values for GDYO, Pt, and GDYO@Pt were obtained, i.e., 3.29, 4.42, and 3.70 eV (relative to the vacuum level), respectively. The Pt nanoparticles exhibited a higher W value than GDYO, indicating that when GDYO was in intimate contact with the Pt nanoparticles, electrons transferred\u0026zwnj;\u0026zwnj; from GDYO to Pt until the interfacial Fermi level reached equilibrium. The space-charge region formed on the GDYO side caused upward bending of the energy bands, establishing a Schottky barrier (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Upon ultrasonic excitation, the valence band (VB) electrons of GDYO were excited to the conduction band (CB) and transferred to the Fermi level of the Pt nanoparticles through the Schottky barrier, whereas holes remained in the VB of GDYO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). The E\u003csub\u003efb\u003c/sub\u003e and CB potentials of ultrasound-activated GDYO@Pt were \u0026minus;\u0026thinsp;0.85 and \u0026minus;\u0026thinsp;0.24 V (vs. NHE, pH 0), respectively, which were positively shifted by 0.18 V compared to those obtained under static conditions (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). This suggested that the piezoelectric effect in GDYO@Pt induced downward band bending and lowered the Schottky barrier height (SBH), thereby facilitating electron migration from GDYO to Pt and enhancing the H\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e production (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eDFT calculations were performed to validate the electron transfer process (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM, electrons predominantly accumulate on the Pt side (yellow regions), whereas the green peripheral regions of GDYO indicate electron depletion. Corresponding to the differential charge distribution, charge displacement (Δρ) in the GDYO@Pt system was quantitatively calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). The negative and positive Δρ values observed for the GDYO and Pt regions, respectively, confirmed that the primary electron migration pathway was from GDYO to Pt, which was consistent with the above results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Electrochemical impedance spectroscopy (EIS) revealed significantly reduced arc diameters for GDYO@Pt (Supplementary Fig.\u0026nbsp;14), indicating lower impedance and improved carrier separation efficiency compared to GDYO.\u003c/p\u003e \u003cp\u003eAs shown in Supplementary Fig.\u0026nbsp;15, DFT simulations were used to visualize the top and side views of the H\u003csub\u003e2\u003c/sub\u003eO adsorption configurations on different materials. Compared to GDYO (\u0026minus;\u0026thinsp;0.508 eV) and Pt (\u0026minus;\u0026thinsp;0.613 eV), GDYO@Pt (\u0026minus;\u0026thinsp;0.847 eV) was found to exhibit a stronger adsorption energy, indicating an enhanced surface affinity for water molecules and more abundant reaction sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). The Gibbs free energy of H\u003csub\u003e2\u003c/sub\u003e adsorption (|ΔG\u003csub\u003eH*\u003c/sub\u003e|) serves as a critical descriptor for determining the H\u003csub\u003e2\u003c/sub\u003e precipitation activity of a system, with values closer to zero indicating a greater reaction probability. The calculated |ΔG\u003csub\u003eH*\u003c/sub\u003e| value for GDYO@Pt was 0.15 eV, which was significantly lower compared to GDYO (0.40 eV) and Pt (0.28 eV) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP and Supplementary Fig.\u0026nbsp;16), thus the Schottky junction formed by GDYO and Pt effectively lowered the activation energy for H\u003csub\u003e2\u003c/sub\u003e precipitation. The superior H\u003csub\u003e2\u003c/sub\u003eO adsorption capacity of GDYO@Pt along with its reduced H* activation energy led to more abundant surface reduction reactions, ensuring efficient piezocatalytic H\u003csub\u003e2\u003c/sub\u003e evolution.\u003c/p\u003e\n\u003ch3\u003eUltrasound-activated GDYO@Pt nanosheets promote cell proliferation\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cytocompatibility of the GDYO and GDYO@Pt nanosheets was subsequently evaluated using the Cell Counting Kit-8 (CCK-8) assay. The obtained results demonstrated that neither GDYO nor GDYO@Pt exhibited observable toxicity toward BMSCs, even at a high concentration of 500 \u0026micro;g/mL (Supplementary Fig.\u0026nbsp;17). Live/dead cell staining confirmed the excellent cytocompatibility of the nanosheets (Supplementary Fig.\u0026nbsp;18), consistent with the aforementioned results.\u003c/p\u003e \u003cp\u003eTo simulate a damaged microenvironment, oxidative stress injury was induced in BMSCs through the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (0.2 mM). The oxidative stress-injured BMSCs were exposed to US at different power densities (0.3, 0.5, 0.7, 1, and 1.5 W/cm\u003csup\u003e2\u003c/sup\u003e) for 10 min to determine the optimal conditions. Compared to the control group, the BMSCs viability significantly decreased by day 3 in the 1.0 and 1.5 W/cm\u003csup\u003e2\u003c/sup\u003e groups, whereas the other groups remained unaffected. However, good cell viability was maintained when US was applied at a power density of 0.7 W/cm\u003csup\u003e2\u003c/sup\u003e for 10 min, indicating that this was a suitable power density and duration for subsequent experiments (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;19).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects of the ultrasound-activated nanosheets on BMSCs proliferation were further assessed using CCK-8 assays and live/dead cell staining. Compared to the GDYO@Pt and GDYO\u0026thinsp;+\u0026thinsp;US groups, the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group exhibited significantly enhanced proliferation levels on days 3, 5, and 9 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;20). This enhancement was attributed to the synergistic effects of the Schottky junction-induced piezoelectric stimulation and oxidative stress regulation in GDYO@Pt. The GDYO@Pt group showed a slight improvement in cell viability compared with the control group. This was likely due to the nanozyme activity of GDYO@Pt, which consumes overexpressed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in damaged cells to generate O\u003csub\u003e2\u003c/sub\u003e, thereby enhancing the cell viability.\u003c/p\u003e\n\u003ch3\u003ePiezoelectric stimulation of BMSCs\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe piezoelectric properties and cycle stabilities of the nanosheets were systematically evaluated in physiological environments through ultrasound activation in a physiological medium. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, compared to a neutral pH environment (pH 7.4), a pH of 4.5 (simulating the bone-defect microenvironment) significantly enhanced piezoelectric current generation in the nanosheets. During five on-off cycling tests, the GDYO@Pt nanosheets exhibited stable current\u0026ndash;output characteristics. The current intensity of the GDYO@Pt system reached 1.7 \u0026micro;A (pH 4.5), which was ~\u0026thinsp;2.1-fold higher than that of the GDYO nanosheets (0.8 \u0026micro;A), consistent with the piezoelectric characterization results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Further evaluations revealed that the piezoelectric current of US-activated GDYO@Pt displayed significant power-dependent characteristics (0\u0026ndash;0.7 W/cm\u003csup\u003e2\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In bone electrophysiology, it has previously been reported that microcurrents in the 0.1\u0026ndash;10 \u0026micro;A range effectively promote bone tissue regeneration\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. This study confirmed that the piezoelectric current intensity generated by GDYO@Pt fell precisely within this range. Furthermore, the observed environmental adaptability and output stability of this system met the requirements for bone-defect repair applications, highlighting its significant potential in bone regeneration therapy.\u003c/p\u003e \u003cp\u003eAfter evaluating the piezoelectric properties, the effects of piezoelectric stimulation on the electrophysiological activity of BMSCs were investigated. For this purpose, a spatially separated experimental design was adopted, wherein BMSCs were seeded in the central area (1.0 \u0026times; 1.0 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) of the sterilized conductive glass, while the nanosheet-coated region was fixed at the bottom. To precisely assess the direct impact of the piezoelectric effect of the material on the cellular electrophysiology, the nanosheet-loaded electrode area was submerged in a physiological medium (pH 4.5), whereas the BMSCs-seeded electrode area remained exposed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The voltage-sensitive fluorescent probe Di-8-ANEPPS was used to monitor real-time changes in the cell membrane potential induced by US-activated piezoelectric stimulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). This probe operates via an electrochromic mechanism, wherein its excitation/emission spectra shift in response to changes in the membrane potential\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The slow internalization properties of Di-8-ANEPPS ensured specific localization on the cell membrane surface, enabling the accurate detection of membrane dipole potential alterations\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, the GDYO@Pt group exhibited greater fluorescence intensity changes than the GDYO group, with a statistically significant increase in the fluorescence intensity ratio observed after US stimulation. This phenomenon directly correlated with the enhanced piezoelectric current output of GDYO@Pt (1.7 vs. 0.8 \u0026micro;A, as shown above), confirming that US-activated GDYO@Pt can modulate the electrophysiological activity of BMSCs by altering the cell membrane potential through superior piezoelectric stimulation.\u003c/p\u003e \u003cp\u003eAs a secondary messenger that regulates osteogenesis, intracellular calcium ion (Ca\u003csup\u003e2+\u003c/sup\u003e) signaling is closely linked to the electrophysiological activity\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. To investigate the possible activation of calcium signaling pathways under the influence of GDYO@Pt piezoelectric stimulation, a Fluo-4 fluorescent probe was employed with the same spatially separated design approach described above (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). The experimental results revealed that the GDYO@Pt group exhibited a significantly enhanced Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence signal intensity under US stimulation, with a 1.6-fold increase in the fluorescence ratio (post-/pre-stimulation) compared to the control group. This indicated that the piezoelectric electric field generated by the material effectively induced intracellular Ca\u003csup\u003e2+\u003c/sup\u003e enrichment in BMSCs. Combined with the membrane potential results (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), this result suggested that the corresponding mechanism likely involves piezoelectric stimulation-induced membrane depolarization, which regulated the voltage-gated calcium channel (VGCC) activity to promote extracellular Ca\u003csup\u003e2+\u003c/sup\u003e influx. The specific activation of Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathways provided a critical ionic environment for BMSCs proliferation, osteogenic differentiation, and extracellular matrix mineralization.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of oxidative stress levels in vitro\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe effects of nanosheet-based piezocatalysis on the oxidative stress levels in BMSCs were evaluated. Initially, the GC and MB probe were used to analyze intracellular H\u003csub\u003e2\u003c/sub\u003e release. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, US-activated GDYO@Pt-treated BMSCs exhibited a significant time-dependent increase in H₂ generation, with a hydrogen evolution rate far exceeding that of other groups. Further validation via the MB probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) revealed near-complete fading (loss of blue color) in the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group, whereas other groups retained distinct blue coloration, visually confirming its superior hydrogen production activity. These results demonstrated the universal mechanism by which the Schottky heterojunction (GDYO@Pt) enhanced piezocatalytic hydrogen production across diverse environments, spanning from in vitro systems to cellular contexts.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough H\u003csub\u003e2\u003c/sub\u003e is beneficial for scavenging highly toxic hydroxyl radicals (i.e., ROS) in damaged cells, a hypoxic environment can lead to the upregulated expression of HIF-1α in the infiltrating immune cells, further inducing ROS production and creating a vicious cycle of oxidative stress. Alleviating hypoxia while scavenging ROS may lead to the more efficient resolution of oxidative stress in a bone defect microenvironment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, the hypoxia-sensitive fluorescent probe Ru(dpp)\u003csub\u003e3\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e was employed to evaluate nanosheet-catalyzed O\u003csub\u003e2\u003c/sub\u003e generation in the BMSCs. Compared with the ROS group (oxidative stress model group), hypoxia was significantly alleviated in the GDYO@Pt, GDYO\u0026thinsp;+\u0026thinsp;US, and GDYO@Pt\u0026thinsp;+\u0026thinsp;US groups, with the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group exhibiting the strongest hypoxia mitigation capability. This was attributed to the synergistic catalytic decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into O\u003csub\u003e2\u003c/sub\u003e by the GDYO@Pt nanosheets and holes, thereby providing a safeguard for ROS regulation.\u003c/p\u003e \u003cp\u003eThe indicator 2\u0026prime;,7\u0026prime;-dichlorofluorescin diacetate (DCFH-DA) was used to investigate the effects of different treatments on the intracellular ROS levels. DCFH from DCFH-DA is initially hydrolyzed by cellular esterases, and is subsequently oxidized by ROS to generate the strongly green-fluorescent 2\u0026prime;,7\u0026prime;-dichlorofluorescein (DCF). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, compared to the ROS group, the GDYO@Pt group exhibited weakened green fluorescence, indicating its intrinsic ROS-scavenging capability owing to the ability of the GDYO@Pt nanozyme to decompose H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. More importantly, DCF fluorescence was almost absent in the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group, demonstrating a significantly enhanced ROS depletion level that far exceeded that in the GDYO\u0026thinsp;+\u0026thinsp;US group. This result stems from the synergistic effects of piezocatalytic H\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e production during the continuous ROS-scavenging process.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePromotion of osteogenic differentiation\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate the effects of US-activated GDYO@Pt on the osteogenic differentiation of BMSCs, assessments of the alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining were conducted. ALP is a critical intracellular enzyme that serves as a marker of early osteogenic differentiation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, compared with the GDYO@Pt and GDYO\u0026thinsp;+\u0026thinsp;US groups, the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group exhibited a larger ALP-stained area after 7 d, correlating with the ALP activity values presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. Furthermore, the late-stage (14 d) mineralization of BMSCs was assessed using Alizarin Red S (ARS) staining. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group displayed the highest number of mineralized nodules and cellular calcium deposits, indicating superior mineralization compared with the other groups. These results further confirmed that US-triggered GDYO@Pt facilitated the osteogenic differentiation of BMSCs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe molecular mechanisms underlying this enhanced osteogenic differentiation were elucidated by analyzing gene expression levels using the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Key osteogenic markers including ALP, osteocalcin (OCN), osteopontin (OPN), type I collagen (COL-1), and runt-related transcription factor 2 (RUNX-2) were examined. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH, the GDYO@Pt\u0026thinsp;+\u0026thinsp;US group exhibited significantly higher expression levels of these osteogenic genes than the other groups after 14 d, indicating a markedly augmented osteogenic differentiation capacity. This can be attributed to the synergistic effects of the enhanced piezoelectric stimulation and down-regulated oxidative stress at the Schottky junction.\u003c/p\u003e\n\u003ch3\u003eIn vivo skull regeneration and repair\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo systematically evaluate the osteoinductive potential of US-activated GDYO@Pt nanosheets in vivo, a nanocomposite hydrogel system was developed by encapsulating the nanosheets within a TA-modified poloxamer thermosensitive gel to enhance their retention at bone-defect sites. This composite gel exhibited excellent biosafety and viscoelasticity, with the optimized formulation enabling liquid-state behavior at room temperature (25\u0026deg;C) and semi-solid-state behavior at physiological temperature (37\u0026deg;C) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eQ; Supplementary Figs.\u0026nbsp;7, 8 and 21). According to the therapeutic protocol shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, a \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e mm diameter cranial defect model was created in the parietal bone of mice using a precision drill. Subsequently, an aliquot (300 \u0026micro;L) of pre-chilled (4\u0026deg;C) gel, GDYO gel, or GDYO@Pt gel was precisely injected into the defect cavity via a micro syringe and maintained at 37\u0026deg;C for 10 min to ensure in situ gelation. The surgical incisions were closed in layers, followed by postoperative analgesia and anti-infection treatments. Twenty-four mice were randomized into six groups (control, gel\u0026thinsp;+\u0026thinsp;US, GDYO gel, GDYO@Pt gel, GDYO gel\u0026thinsp;+\u0026thinsp;US, and GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group) across two timepoints (6 and 12 weeks). The US parameters (1 MHz, 0.7 W/cm\u003csup\u003e2\u003c/sup\u003e, 10 min/d) matched those used in the in vitro studies, and treatment was applied for 4 weeks.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicro-computed tomography (micro-CT) imaging and reconstruction revealed that the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group achieved the highest degree of bone regeneration and repair at both 6 and 12 weeks compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Semi-quantitative analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) indicated poor bone regeneration in the control, gel\u0026thinsp;+\u0026thinsp;US, and GDYO gel groups, with low new bone area ratios (%), bone volume/total volume ratios (BV/TV), and bone mineral densities (BMD) observed in each case. In contrast, the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group exhibited superior repair metrics, reaching a new bone area of 75.37%, a BV/TV ratio of 69.00%, and a BMD of 0.658 g/cm\u003csup\u003e3\u003c/sup\u003e at 12 weeks, significantly surpassing the other groups. Histological analysis using hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining confirmed these findings. More specifically, from H\u0026amp;E staining, the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group displayed a continuous, well-organized neobone with abundant vascularization (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF), whereas Masson\u0026rsquo;s staining highlighted mature osteoid deposition within the defect (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG); the other groups showed limited discontinuous bone formation. Immunohistochemical (IHC) staining for osteogenic markers (OCN and RUNX-2; Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI) showed the strongest degree of staining in the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group, consistent with the micro-CT and histological results. CD31 immunostaining (angiogenesis marker) revealed superior vascular protein expression in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). These results were attributed to the synergistic effects of Schottky junction-enhanced piezoelectric stimulation and oxidative stress modulation.\u003c/p\u003e \u003cp\u003eImmunofluorescence (IF) staining was subsequently performed to investigate the effects of the different treatment conditions on the inflammatory microenvironment at the defect site. M1 (CD86) and M2 (CD206) macrophages, representing pro-inflammatory and anti-inflammatory phenotypes respectively, play critical roles in regulating the osteogenic microenvironment. Although short-term inflammation facilitates the recruitment of endogenous stem cells, prolonged inflammation impedes bone repair. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK, compared to the other groups, the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group exhibited a significantly higher proportion of M2-type (CD206, red) macrophages and a lower proportion of M1-type (CD86, green) macrophages at the cranial defect site. This polarization toward an anti-inflammatory phenotype is conducive to bone tissue repair, thereby indicating that the US-triggered GDYO@Pt gel effectively orchestrated the osteogenic, angiogenic, and immune microenvironments to promote bone regeneration.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of oxidative stress levels in bone defect tissues\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eConsidering the aforementioned in vivo bone repair outcomes, the mechanism underlying the US-activated GDYO@Pt-mediated remodeling of redox homeostasis in the bone-defect microenvironment was further elucidated. Initially, a dihydroethidium (DHE) fluorescent probe was used to evaluate the ROS levels at defect sites. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group displayed substantially attenuated red fluorescence compared with the other groups, confirming its superior ROS-scavenging capacity. HIF-1α immunofluorescence analysis demonstrated significantly reduced HIF-1α expression in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), indicating that the GDYO@Pt system-mediated elevation of the O\u003csub\u003e2\u003c/sub\u003e partial pressure alleviated tissue hypoxia, thereby disrupting the \"hypoxia \u0026rarr; HIF-1α activation \u0026rarr; ROS accumulation\" cycle.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the regulatory pathways associated with oxidative stress, immunohistochemical staining was performed to analyze the expression of nuclear factor erythroid 2-related factor 2 (NRF2). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC, the GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US group exhibited a significantly higher proportion of NRF2-positive cells in the newly formed bone tissue than the other groups. These findings demonstrated\u0026zwnj;\u0026zwnj; that this multidimensional antioxidant strategy not only effectively reduced local ROS levels, but also enhanced endogenous antioxidant defenses via the NRF2 pathway, establishing a redox-balanced microenvironment conducive to electrical stimulation therapy for bone defect repair.\u003c/p\u003e \u003cp\u003eH\u0026amp;E staining of tissue sections from all treatment groups post-therapy demonstrated a preserved structural integrity in each case, in addition to the absence of pathological abnormalities in the heart, liver, spleen, lungs, and kidneys (Supplementary Fig.\u0026nbsp;21). These results indicated the favorable biocompatibility of GDYO@Pt gel at therapeutic doses in vivo.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePiezoelectric stimulation, a non-invasive therapeutic approach, modulates cellular metabolism and promotes bone regeneration. However, excessive ROS generation and hypoxia-induced oxidative stress within the bone defect microenvironment compromise the therapeutic efficacy of electrical stimulation. Therefore, the development of narrow-bandgap piezoelectric semiconductors integrated with dual-functional piezoelectric stimulation and ROS scavenging capabilities is imperative.\u003c/p\u003e \u003cp\u003eIn this study, a bone-repair platform that integrates efficient piezoelectric stimulation and multimodal antioxidant stress functions is developed based on a Schottky heterojunction formed between GDYO nanosheets and Pt nanoparticles. The interfacial dipole effect at the heterojunction induces a built-in electric field, driving charge rearrangement in GDYO nanosheets to break the semiconductor\u0026rsquo;s inversion symmetry, enhance polarity, and significantly boost piezoelectric performance. In addition, the US-triggered Schottky interfaces are found to generate positive polarization charges that induce downward band bending, lowering the energy barriers, and promoting electron migration from GDYO to Pt for enhanced H\u003csub\u003e2\u003c/sub\u003e evolution. Concurrently, the hole-mediated oxidation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generates O\u003csub\u003e2\u003c/sub\u003e, synergizing with the nanozyme activity of GDYO@Pt to achieve the multimodal scavenging of ROS. In vitro and vivo experiments demonstrate that the US-activated GDYO@Pt nanosheets promote osteogenic differentiation, angiogenesis, and immunomodulation by remodeling the electrophysiological microenvironments and downregulating oxidative stress, ultimately achieving a superior cranial bone defect repair efficacy. This piezoelectric-catalytic dual-function platform innovatively synchronizes efficient electrical stimulation with oxidative stress regulation, offering a transformative solution for bone defect repair. Through band engineering, it precisely controls interfacial redox kinetics during piezoelectric stimulation, avoiding toxic ROS generation while directionally producing therapeutic H₂ and O₂ to establish a low-oxidative-stress microenvironment conducive to osteogenesis.\u003c/p\u003e \u003cp\u003eThis mechanism can be extended to diverse regenerative applications, including articular cartilage repair, neural axon regeneration, and skin wound healing, demonstrating universal applicability. Thus, elucidating the spatiotemporal regulatory principles of electro-chemical coupling on cell fate and establishing quantitative structure-activity relationships among band structures, interfacial reaction kinetics, and biological responses will critically guide the development of next-generation intelligent bone repair materials.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eGraphite was sourced from Beijing Gaoke New Materials (China). Potassium tetrachloroplatinate (II) (K\u003csub\u003e2\u003c/sub\u003ePtCl\u003csub\u003e4\u003c/sub\u003e), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (30%), H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (98%) and ethylene glycol (EG, 99%) were purchased from Shanghai Chemical Reagent (China). Kolliphor\u0026reg; P 407 was obtained from BASF (Ludwigshafen, Germany). Tannic acid (USP, \u0026ge;\u0026thinsp;99%) and Methylene blue (\u0026ge;\u0026thinsp;98%) were obtained from Macklin (China). The water in the experiments was deionized. All reagents were used directly without further purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of GDYO\u003c/h2\u003e \u003cp\u003eAccording to previously reported methods\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, graphdiyne (GDY) was first synthesized on a copper surface via cross-coupling reactions using hexaethynylbenzene as the precursor. Subsequently, GDY powder (50 mg) was homogenized with H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (2.5 mL) under ice-bath conditions, followed by slow dropwise addition of 30% hydrogen peroxide solution (1 mL) under vigorous stirring for 2 hours. Distilled water (20 mL) was then added to the mixture, and the suspension was ultrasonicated for 1 hour, followed by centrifugation (8,000 rpm, 10 min) and repeated washing with deionized water until the supernatant reached neutral pH.\u003c/p\u003e \u003cp\u003e To further exfoliate stacked GDYO, concentrated hydrochloric acid (20 \u0026micro;L) was added to 20 mL of GDYO dispersion (0.5 mg/mL), followed by ice-bath ultrasonication for 6 hours (150 W power, 40 kHz frequency). The dispersion was then centrifuged (8,000 rpm, 10 min) and washed twice with deionized water. Finally, unexfoliated residues and large aggregated nanosheets were removed via low-speed centrifugation (3,000 rpm, 5 min), yielding a stable exfoliated GDYO nanosheet dispersion, which was stored at 4 ℃ for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFabrication of GDYO@Pt\u003c/h2\u003e \u003cp\u003eThe heterojunction GDYO@Pt nanosheets were synthesized via an in situ reduction method. First, GDYO (60.0 mg) and K₂PtCl₄ (30.0 mg) were dispersed in deionized water (120 mL) under ultrasonication. The mixture was then stirred for 30 minutes at 600 rpm. Subsequently, 12 mL of ascorbic acid solution (0.1 M) was added dropwise to the above solution, followed by stirring at 60\u0026deg;C for 3 hours. The precipitate was collected by centrifugation (8,000 rpm, 10 min) and washed three times with deionized water. Finally, the GDYO@Pt nanosheets were obtained as the final product through freeze-drying.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFabrication of GDYO@Pt gel\u003c/h2\u003e \u003cp\u003eFirst, the blank thermosensitive gel was prepared via a low-temperature swelling method. Specifically, 0.07 g of TA (tannic acid) was added to 10 mL of deionized water and stirred continuously at room temperature until fully dissolved. Subsequently,1.8 g of P407 (poloxamer 407) was slowly added to the TA solution, followed by stirring for 30 minutes. The mixture was then transferred to a 4 ℃ refrigerator and allowed to stand for 24 hours to obtain the blank thermosensitive gel solution. Next, GDYO@Pt nanosheets were incorporated into the blank gel and thoroughly mixed under ice-bath conditions to prepare the thermosensitive GDYO@Pt gel (0.5 mg/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization\u003c/h2\u003e \u003cp\u003eThe comprehensive characterization of the materials was systematically conducted using multiple analytical techniques. Morphological features were examined by transmission electron microscopy (TEM, JEOL JEM-2100F, Japan), scanning electron microscopy (SEM, ZEISS MERLIN Compact, Germany), and atomic force microscopy (AFM, Bruker Dimension ICON, Germany). Crystallographic and chemical analyses were performed via X-ray diffraction (XRD, Bruker D8 Advance, Germany), X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA), and Fourier transform infrared spectroscopy (FT-IR, Thermo Scientific Nicolet iS50, USA). Optical properties were assessed through ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy (Shimadzu UV-1900 and Hitachi UH4150, Japan), while reactive oxygen species (ROS) signals were detected via electron spin resonance (ESR, Bruker EMXplus, Germany). Functional evaluations included rheological measurements (Anton Paar MCR 702e, Austria), piezoelectric/piezocatalytic activation using an ultrasonic therapeutic device (Chattanooga Intelect 2776, USA), and quantitative hydrogen gas analysis via gas chromatography (Agilent GC7890, USA). The finite element simulations were carried out using COMSOL Multiphysics 6.2 software, which has the main advantage of adapting to multiphysics field coupled simulations, making it easier to perform iterative calculations between different physics fields. Specifically, the simulations were carried out through the solid mechanics module and the electrostatics module in COMSOL, and the use of the stress-charge form was determined according to the material and the requirements. The imposed boundary conditions were a fixed constraint on the left side and a constant stress of 100 MPa on the right side. Details of the DFT calculations were provided in Supplementary Note 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGelation and rheological properties of the thermosensitive gel\u003c/h2\u003e \u003cp\u003eThe gelation behavior of various hydrogel formulations was assessed via the tube inversion method. Specifically, glass vials containing gel-1 (18% P407, 0% TA), gel-2 (18% P407, 0.5% TA), gel-3 (18% P407, 0.7% TA), gel-4 (18% P407, 1% TA), gel-5 (18% P407, 2% TA), and GDYO@Pt gel formulations (0.5 mg/mL GDYO@Pt with 18% P407 and 0%, 0.5%, 0.7%, 1%, or 2% TA) were immersed in a 37 ℃ water bath for 10 minutes. After thermal equilibration, the vials were inverted to confirm the sol-gel transition.\u003c/p\u003e \u003cp\u003eAdditionally, rheological experiments were conducted using a parallel plate (40 mm diameter, 1 mm gap) on a rheometer to characterize GDYO@Pt gel. The storage modulus (G\u0026prime;) and loss modulus (G\u0026Prime;) of GDYO@Pt gel were quantified under varying conditions. Oscillatory strain sweeps were performed at 37\u0026deg;C and 1 Hz to measure G\u0026prime; and G\u0026Prime; as a function of strain amplitude. Furthermore, time- and frequency-dependent measurements of G\u0026prime; and G\u0026Prime; were carried out at 37\u0026deg;C with a fixed strain amplitude of 0.5%. Step-strain measurements were conducted under high (45%) and low (0.1%) strain conditions at 37\u0026deg;C and 1 Hz. Steady-state shear viscosity was analyzed at 37\u0026deg;C by applying shear rate sweeps, while step-shear measurements employed low (1 s⁻\u0026sup1;) and high (100 s⁻\u0026sup1;) shear rates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro hydrogen detection\u003c/h2\u003e \u003cp\u003eGDYO and GDYO@Pt were uniformly dispersed in PBS-containing headspace vials (0.5 mg/mL) and sealed. The samples were then exposed to ultrasound (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density) in the dark. At different time points (0, 10, 20, 30, and 40 minutes), 1 mL of gas from the vial headspace was extracted and analyzed for hydrogen using a gas chromatograph (GC) equipped with a thermal conductivity detector (TCD) and nitrogen as the carrier gas. Under the catalytic action of platinum (Pt) nanoparticles, H₂ reacts with methylene blue (MB) to form colorless reduced leucomethylene blue (MBH₂). Thus, MB can also serve as an indicator for H₂ detection. Experimental details are as follows: 2 mL of PBS, GDYO, or GDYO@Pt dispersion (200 \u0026micro;g/mL) was added to quartz Petri dishes (3 cm diameter), followed by 8 \u0026micro;L of MB solution (1 mg/mL). Equal amounts of Pt nanoparticles were added to the GDYO and PBS groups as catalysts. To eliminate interference from \u0026bull;OH radicals, methanol was added as a quencher to all groups. Ultrasound treatment (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density) was applied in the dark at fixed intervals. The dynamic variations in MB levels were tracked by ultraviolet-visible spectrophotometry through absorbance detection at 664 nm. The normalized absorbance was calculated as:\u003c/p\u003e \u003cp\u003eA\u0026thinsp;=\u0026thinsp;A\u003csub\u003et min\u003c/sub\u003e/A\u003csub\u003e0 min\u003c/sub\u003e \u0026times; 100%\u003c/p\u003e \u003cp\u003ewhere A\u003csub\u003e0 min\u003c/sub\u003e and A\u003csub\u003et min\u003c/sub\u003e represent the absorbance values at 664 nm before and after ultrasound treatment, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro oxygen detection\u003c/h2\u003e \u003cp\u003eOxygen production was monitored using a dissolved oxygen meter (OHAUS ST 300D, USA). Specifically, GDYO, Pt, and GDYO@Pt (200 \u0026micro;g/mL) were dispersed in 30 mL of 10 mM hydrogen peroxide (H₂O₂) solution. Ultrasound stimulation was then applied to each group, and dissolved oxygen levels were recorded every 1 second.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePiezoelectric and electrochemical characterization\u003c/h2\u003e \u003cp\u003ePiezoelectric current measurements were conducted using an electrochemical workstation (Metrohm VIONIC, Switzerland) with a standard two-electrode system, where indium tin oxide (ITO) conductive glass (1.0 \u0026times; 4.0 cm\u0026sup2;) served as both counter and working electrodes. The working electrode was modified with the material as follows: 100 \u0026micro;L of sample dispersion (1.5 mg/mL in H\u003csub\u003e2\u003c/sub\u003eO) was drop-casted onto one end of the ITO glass (1.0 \u0026times; 1.0 cm\u0026sup2;) and dried at 60\u0026deg;C for 24 hours. The piezoelectric behavior of the material was evaluated by real-time monitoring of current responses under varying ultrasound power densities (0, 0.3, 0.5, and 0.7 W/cm\u0026sup2;) and five on/off cycles in PBS electrolytes at pH 4.5 and pH 7.4.\u003c/p\u003e \u003cp\u003eMott-Schottky measurements were performed in a 0.5 M Na₂SO₄ electrolyte using a three-electrode system. The working electrode (prepared as described above), a Pt plate counter electrode, and an Ag/AgCl reference electrode were employed. Scanning potentials from 1 V to 0.1 V were applied at frequencies of 1000 Hz, 1316 Hz, and 1732 Hz to obtain Mott-Schottky curves before and after ultrasound stimulation.\u003c/p\u003e \u003cp\u003eElectrochemical impedance was measured in a mixed electrolyte containing 1 mM potassium ferricyanide (K₃[Fe(CN)₆]), 1 mM potassium ferrocyanide (K₄[Fe(CN)₆]), and 0.5 M KCl. The working electrode was prepared by drop-casting 10 \u0026micro;L of sample dispersion (1.0 mg/mL) onto a glassy carbon electrode. The counter and reference electrodes were identical to those used in the Mott-Schottky analysis. AC impedance spectra of different samples were acquired using an electrochemical workstation within a selected frequency range.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eBMSCs were purchased from Yuchi Biotechnology Co., Ltd. (Shanghai, China) and cultured in complete medium (Gibco, Thermo Fisher Scientific, USA; composition: α-MEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 \u0026micro;g/mL streptomycin). Cells were maintained in a humidified incubator (Thermo Fisher Scientific, USA) at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assessment with nanosheets at varying concentrations\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates at a density of 5 \u0026times; 10\u0026sup3; cells per well and co-cultured with nanosheets at varying concentrations (n\u0026thinsp;=\u0026thinsp;4). After the designated incubation period, the medium (with or without nanosheets) was removed, and 10% CCK-8 solution (Beyotime Biotechnology, China) was added. The plates were then incubated at 37\u0026deg;C for 1 hour. Subsequently, 100 \u0026micro;L of solution from each well was transferred to a 96-well plate, and absorbance values were recorded at 450 nm. The relative cell viability was calculated as follows:\u003c/p\u003e \u003cp\u003eCell viability (%) = (Absorption value of different treatment groups)/(Mean absorption value of control) \u0026times; 100%\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eEffects of ultrasound-activated nanosheets on cell viability\u003c/h2\u003e \u003cp\u003eFirst, oxidative stress injury was induced in BMSCs using H₂O₂. Specifically, BMSCs (2 \u0026times; 10⁴ cells/well) were seeded in 6-well plates and cultured for 24 hours. The medium was then replaced with H₂O₂-containing medium (0.2 mM) for 6 hours, followed by fresh complete medium.\u003c/p\u003e \u003cp\u003eTo evaluate the impact of ultrasound treatment on cell viability, Oxidative stress-injured BMSCs (2 \u0026times; 10⁴ cells/well in 6-well plates) were exposed to ultrasound at varying power densities (0.3, 0.5, 0.7, 1.0, and 1.5 W/cm\u0026sup2;) and durations (0, 5, 10, 15, and 20 minutes) to determine the threshold of ultrasound parameters affecting cell survival (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003eThen, nanosheets (200 \u0026micro;g/mL) were incubated with oxidative stress-damaged BMSCs, ultrasound stimulation (1.0 MHz frequency, 0.7 W/cm\u0026sup2; power density, 50% duty cycle, 10 min per session) was applied or withheld on days 1, 3, and 7. Cell viability was assessed for all groups on days 3, 5, and 9 (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEffects of ultrasound-induced piezoelectricity on cell membrane potential\u003c/h2\u003e \u003cp\u003eThe materials were spin-coated and immobilized on one end of the ITO conductive glass as described in Section 2.9. BMSCs were seeded at a density of 1 \u0026times; 10⁴ cells onto the central region of sterilized ITO glass (seeding area: 1.0 \u0026times; 1.0 cm\u0026sup2;) and incubated in complete medium for 24 hours. The medium was then replaced with complete medium containing Di-8-ANEPPS (2 \u0026micro;M), followed by 30 minutes of incubation in the dark. Cells were gently rinsed three times with dye-free medium to remove excess probe.\u003c/p\u003e \u003cp\u003eSubsequently, the material-coated end of the ITO glass was immersed in a PBS-filled electrochemical cell (pH 4.5). After stabilizing the dual-electrode system in the electrolyte for 30 seconds, ultrasound (0.7 W/cm\u0026sup2;, 5 minutes) was applied to activate the piezoelectric signals of the material. Fluorescence microscopy (Olympus CKX41SF, Japan) was employed to detect cellular fluorescence on the ITO glass (excitation: 488 nm, emission: 605 nm, red fluorescence).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eEffects of US-induced piezoelectricity on intracellular calcium levels\u003c/h2\u003e \u003cp\u003eFollowing the aforementioned protocol, 1 \u0026times; 10⁴ BMSCs were seeded onto the central region of sterilized ITO conductive glass pre-coated with materials (seeding area: 1.0 \u0026times; 1.0 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) and incubated in complete medium for 24 hours. The medium was then replaced with Hanks' Balanced Salt Solution (HBSS) containing Fluo-4 (4 \u0026micro;M), followed by 30 minutes of incubation in the dark. Cells were gently rinsed three times with HEPES-buffered saline to remove excess dye.\u003c/p\u003e \u003cp\u003eThe material-coated end of the ITO glass was subsequently immersed in a PBS-filled electrochemical cell (pH 4.5). After stabilizing the dual-electrode system in the electrolyte for 30 seconds, ultrasound (0.7 W/cm\u0026sup2;, 5 minutes) was applied to activate the piezoelectric signals of the material. Intracellular calcium dynamics were monitored via fluorescence microscopy by detecting green fluorescence signals (excitation: 494 nm; emission: 516 nm) from Fluo-4-loaded cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro detection of intracellular hydrogen\u003c/h2\u003e \u003cp\u003eAdherent BMSCs were co-cultured with GDYO and GDYO@Pt (200 \u0026micro;g/mL) for 12 hours. Following trypsin digestion, PBS was used to prepare cell suspensions (density: 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL). 1 mL of suspension was transferred into sealed headspace vials under gas-tight conditions. After ultrasonic irradiation for 0, 10, 20, 30, and 40 min respectively, 1 mL of headspace gas was collected for gas chromatography quantification of H₂ production.\u003c/p\u003e \u003cp\u003eBMSCs were seeded in 6-well plates at a density of 2 \u0026times; 10⁴ cells per well and cultured for 24 hours. Subsequently, the cells were incubated with GDYO and GDYO@Pt (200 \u0026micro;g/mL) for an additional 12 hours. Equal amounts of Pt nanoparticles were added to the GDYO and PBS groups as catalysts. After incubation, the cells were gently rinsed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes. The fixed cells were then co-mixed with methylene blue (MB, 100 \u0026micro;M) in PBS for 20 minutes. Afterwards, the medium was removed and the cells were washed three times with PBS. Ultrasound irradiation was applied to the cells with or without the following parameters: 1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density, and irradiation durations of 10 min. Bright-field images were acquired using fluorescence microscopy to analyze changes in the blue intensity of MB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro detection of intracellular oxygen\u003c/h2\u003e \u003cp\u003eThe Ru(dpp)₃Cl₂ (RDPP) probe was employed to monitor O₂ generation. RDPP is a red-fluorescent oxygen-sensitive indicator (excitation/emission wavelengths: 488 nm/620 nm), whose fluorescence is quenched upon interaction with O₂ via energy transfer. Thus, changes in RDPP fluorescence intensity inversely correlate with intracellular oxygen levels.\u003c/p\u003e \u003cp\u003eSpecifically, BMSCs were seeded in 6-well plates at a density of 2 \u0026times; 10⁴ cells/well and allowed to adhere. The cells were then incubated with complete medium containing GDYO or GDYO@Pt (200 \u0026micro;g/mL) for 24 hours. Subsequently, the cells were incubated in complete medium supplemented with Ru(dpp)₃Cl₂ (30 \u0026micro;M) in the dark for 40 minutes. After incubation, the medium was removed, and the cells were gently washed three times with PBS. Ultrasound irradiation (parameters: 1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density, 5-minute duration) was applied or withheld. Fluorescence microscopy was used to capture images and analyze changes in the red fluorescence intensity of Ru(bpy)₃Cl₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro detection of intracellular ROS\u003c/h3\u003e\n\u003cp\u003eIntracellular ROS levels were measured using a ROS assay kit (DCFH-DA fluorescent probe; Beyotime Biotechnology, China). Briefly, BMSCs were seeded in 6-well plates at a density of 2 \u0026times; 10⁴ cells per well and cultured for 24 hours. Experimental groups were then co-incubated with 200 \u0026micro;g/mL GDYO@Pt or GDYO nanosheets for 24 hours, followed by ultrasound treatment (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density, 5-minute irradiation) or no treatment.\u003c/p\u003e \u003cp\u003eAfter washing with PBS, all groups were stained with the DCFH-DA probe under dark conditions for 30 minutes and washed again with PBS to remove unbound probes. DCF fluorescence signals (excitation: blue light ; emission: green light ) in BMSCs were detected using an inverted fluorescence microscope.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eALP staining assay\u003c/h2\u003e \u003cp\u003eBMSCs were cultured at a density of 2 \u0026times; 10⁴ cells per well in medium containing GDYO (200 \u0026micro;g/mL) or GDYO@Pt (200 \u0026micro;g/mL) nanosheets for 7 days, during which ultrasound was applied/not applied every two days (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density, irradiation time of 10 min), and control cells were incubated in medium without nanosheets for the same time. Subsequently, the cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 30 min, and an appropriate amount of ALP staining solution (Beyotime Biotechnology, China) was added to cover the cells uniformly, and then stained for 30 min at room temperature and protected from light. After the staining was completed, the cells were washed thoroughly with PBS, and the ALP-active areas were observed and photographed under a microscope to record the ALP activity areas to assess the early osteogenic differentiation ability of BMSCs. For quantitative analysis, ALP activity was measured using an ALP assay kit (Beyotime Biotechnology, China) according to the manufacturer\u0026rsquo;s instructions (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eAlizarin Red S staining assay\u003c/h2\u003e \u003cp\u003eBMSCs were cultured at a cell density of 2 \u0026times; 10⁴ BMSCs per well in medium containing GDYO (200 \u0026micro;g/mL) or GDYO@Pt (200 \u0026micro;g/mL) nanosheets for 14 days, during which ultrasound was applied/not applied every two days (1.0 MHz frequency, 50% duty cycle, 0.7 W/cm\u0026sup2; power density, irradiation time of 10 min, respectively), and control cells were incubated in medium without nanosheets for the same time. After incubation, cells were washed with PBS and fixed with 4% PFA for 30 minutes. A 0.2% Alizarin Red S solution (Solarbio Biotechnology, China) was added to completely cover the cells, followed by 30 minutes of staining at room temperature. Unbound dye was removed by thorough PBS rinsing. Calcium nodule formation, indicated by orange-red mineralized deposits, was visualized and photographed under a microscope to evaluate late-stage osteogenic differentiation and mineralization capacity. For quantitative analysis, calcium deposits were dissolved in 2% cetylpyridinium chloride (Sigma-Aldrich, USA), and absorbance was measured at 550 nm using a microplate reader (BioTek Cytation 3, USA) (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eRT-qPCR analysis of osteogenic differentiation genes in BMSCs\u003c/h2\u003e \u003cp\u003eTo further investigate the osteogenic effects of ultrasound-activated nanosheets, total mRNA was extracted from all experimental groups using Trizol reagent (Invitrogen, USA). First-strand cDNA was synthesized with the PrimeScript RT reagent kit (TaKaRa, Japan) following the manufacturer's protocol. Real-time quantitative PCR amplification was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA) using SYBR Green fluorescent dye (Roche, USA). Gene-specific primers for osteogenic markers \u0026ndash; alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), type I collagen (COL-1), and Runt-related transcription factor 2 (RUNX-2) \u0026ndash; were designed (primer sequences provided in Supplementary Table\u0026nbsp;1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference gene for data normalization. Relative mRNA expression levels were calculated using the 2\u003csup\u003e\u0026minus;\u003cem\u003eΔΔ\u003c/em\u003eCt\u003c/sup\u003e method to quantify ultrasound-enhanced osteogenic differentiation (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eIn vivo experimental study\u003c/h2\u003e \u003cp\u003e All experimental procedures were conducted in strict compliance with the National Standards for the Care and Use of Laboratory Animals in China. Eight-week-old wild-type C57BL/6 mice (purchased from the National Institutes for Food and Drug Control, NIFDC, China) were housed in specific pathogen-free (SPF) facilities pre- and post-operatively, with ad libitum access to water and a standard rodent diet. Surgical protocols and perioperative management followed the guidelines approved by the Animal Care and Use Committee of Beihang University (License No. BM201900084).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eEstablishment of cranial defect model and in situ injection of Gel\u003c/h3\u003e\n\u003cp\u003eMice were group-housed for one week preoperatively for environmental acclimatization. Anesthesia was induced and maintained via isoflurane inhalation. After incising the scalp, a 3.0 mm diameter full-thickness circular bone defect was created in the parietal bone using a dental trephine drill. The defect area was irrigated with saline, followed by in situ injection of thermosensitive gel formulations into the defect cavity according to experimental groups. After complete gelation (37\u0026deg;C, 10 minutes), the wound was closed in layers. Postoperatively, ibuprofen (10 mg/kg, Macklin, China) was administered in drinking water for 24 hours to alleviate pain. To prevent infection, sulfamethoxazole (15 mg/kg) and trimethoprim (30 mg/kg) (Macklin, China) were added to drinking water for one week.\u003c/p\u003e\n\u003ch3\u003eTherapeutic procedure for murine cranial defect repair\u003c/h3\u003e\n\u003cp\u003eCranial defect-bearing mice were randomly allocated into six experimental groups (n\u0026thinsp;=\u0026thinsp;4): (1) Control (no treatment), (2) gel\u0026thinsp;+\u0026thinsp;US (thermosensitive gel with ultrasound stimulation), (3) GDYO gel (gel containing 0.5 mg/mL GDYO nanosheets without ultrasound), (4) GDYO@Pt gel (gel containing 0.5 mg/mL GDYO@Pt nanosheets without ultrasound), (5) GDYO gel\u0026thinsp;+\u0026thinsp;US (GDYO-loaded gel with ultrasound), and (6) GDYO@Pt gel\u0026thinsp;+\u0026thinsp;US (GDYO@Pt-loaded gel with ultrasound). All interventions were standardized to ensure uniform nanosheet concentrations and treatment protocols across groups. Ultrasound stimulation (1.0 MHz, 50% duty cycle, 0.7 W/cm\u0026sup2;) was administered once daily (10 minutes per session), for 4 consecutive weeks. Cranial bone samples were harvested at 6 and 12 weeks post-treatment and analyzed for bone regeneration outcomes using micro-CT and histological assessments.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eMicro-CT Analysis\u003c/h2\u003e \u003cp\u003eThe extracted cranial bone samples post-treatment were scanned using a Micro-CT system (Skyscan 1276, Bruker, Belgium) under 55 kV voltage and 200 \u0026micro;A current. Three-dimensional image reconstruction and analysis of the bone defect region were performed using NRecon and CTvox software. Bone regeneration parameters, including BV/TV and BMD, were quantified with CTAn software.\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eHistological Evaluation\u003c/h2\u003e \u003cp\u003eFollowing micro-CT analysis, samples from the 12-week treatment groups were decalcified in 15% ethylenediaminetetraacetic acid (EDTA) solution and subsequently embedded in paraffin. Tissue sections (4 \u0026micro;m thickness) were prepared, deparaffinized with xylene, and rehydrated through a graded ethanol series (70\u0026ndash;100%). Each sample underwent hematoxylin and eosin (H\u0026amp;E) staining and Masson\u0026rsquo;s trichrome staining. Histomorphological structures were examined using a digital tissue section scanner (Pannoramic MIDI, Hungary).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eIHC and IF Staining Protocol\u003c/h2\u003e \u003cp\u003eIHC Staining: Paraffin-embedded sections were dewaxed and blocked, then incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-OCN antibody, anti-RUNX-2 antibody, anti-CD31 antibody and anti-NRF2 antibody. All primary antibodies were diluted at 1:200 using 3% (w/v) bovine serum albumin (BSA, Sigma-Aldrich, USA). After PBS washing, samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Staining signals were visualized using the digital tissue section scanner.\u003c/p\u003e \u003cp\u003eIF Staining: Dewaxed paraffin sections were blocked with 3% BSA to prevent nonspecific binding, followed by overnight incubation at 4\u0026deg;C with primary antibody (HIF-1α antibody). After PBS washing, sections were incubated with Alexa Fluor 488-labeled secondary antibody at room temperature for 1 hour, then counterstained with DAPI for nuclear visualization. Final images were captured using the digital tissue section scanner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical differences were calculated using independent samples T-test in IBM SPSS Statistics 25 software. Significance levels were denoted as follows: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Replicate experimental data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang L, Li R-W (2024) A more biofriendly piezoelectric material. Science 383:1416\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S et al (2023) Piezocatalytic medicine: an emerging frontier using piezoelectric materials for biomedical applications. Adv Mater 35:2208256\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandez-Yague MA et al (2021) A self-powered piezo‐bioelectric device regulates tendon repair‐associated signaling pathways through modulation of mechanosensitive ion channels. Adv Mater 33:2008788\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y et al (2025) Piezoelectricity in Half-Heusler narrow-bandgap semiconductors. 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Phys Rev Lett 77:3865\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6497438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6497438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePiezoelectric stimulation regulates cellular metabolism and enhances bone repair. However, the overproduction of reactive oxygen species (ROS) and hypoxia-induced oxidative stress reduce the efficacy of electrical stimulation and hinder regeneration. To address these challenges, a platinum-decorated graphdiyne oxide (GDYO@Pt) multifunctional piezoelectric semiconductor was engineered for the first time to eliminate ROS and oxygen self-supply while enabling electrical stimulation. In this system, the interface dipole drives a built-in electric field, triggering charge redistribution in GDYO and breaking symmetry to amplify piezoelectricity. Ultrasound-triggered polarized charges at the Schottky junction lower the barrier and promote GDYO\u0026rarr;Pt electron transfer for hydrogen production, where the generated H₂ neutralizes cytotoxic \u0026bull;OH radicals, while the holes/nanozyme-driven H₂O₂\u0026rarr;O₂ conversion​, synergistically alleviating oxidative stress. In vitro and vivo studies demonstrate that ultrasound-activated GDYO@Pt accelerates cranial defect repair via osteogenesis, angiogenesis, and immunomodulation. This work establishes the inaugural paradigm of piezoelectric-catalytic synergy bone regeneration, where the GDYO@Pt heterointerface uniquely integrates energy conversion with biological regulation through its precisely engineered asymmetric structure.\u003c/p\u003e","manuscriptTitle":"Schottky engineering of GDYO@Pt to boost piezoelectric and oxidative stress modulation for accelerated cranial regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 03:33:01","doi":"10.21203/rs.3.rs-6497438/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aba4eb2a-68e4-42dd-be0d-bcd2a038b5c7","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47839871,"name":"Physical sciences/Materials science/Biomaterials/Biomedical materials"},{"id":47839872,"name":"Physical sciences/Materials science/Materials for energy and catalysis"}],"tags":[],"updatedAt":"2025-09-27T07:11:36+00:00","versionOfRecord":{"articleIdentity":"rs-6497438","link":"https://doi.org/10.1038/s41467-025-63550-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-09-26 04:00:00","publishedOnDateReadable":"September 26th, 2025"},"versionCreatedAt":"2025-04-30 03:33:01","video":"","vorDoi":"10.1038/s41467-025-63550-8","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63550-8","workflowStages":[]},"version":"v1","identity":"rs-6497438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6497438","identity":"rs-6497438","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}