Internal friction network piezoelectric hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention provides an internal friction network piezoelectric hydrogel microsphere as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the preparation method, supramolecular engineering and a microfluidic strategy are utilized, and a hydrogel microsphere system of an internal friction network is constructed through cooperative assembly of piezoelectric bismuth ferrite nanoparticles (BFs) and slip ring functionalized methacrylated hyaluronic acid (HAMA), so that physiological electroadaptation in degeneration tissues is realized. The BFs convert mechanical stimuli into electrical signals, while a stress-dependent internal friction network modulates energy dissipation. Under low stress, the slip ring moves to generate low friction, so that electric signal generation is enhanced; under high stress, the main chain is straightened, so that friction is increased, excessive signals are inhibited, and physiological electric adaptation is recovered. The microspheres can generate a stable electric field under dynamic loading, mitochondrial autophagy is promoted by activating a PINK1 / Parkin pathway, stable mitochondrial membrane potential is maintained, and nucleus pulposus cell apoptosis is reduced by 75%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an internally triboelectric network piezoelectric hydrogel microsphere, its preparation method, and its application. Background Technology

[0002] Mitophagy dysfunction is a core driver of the progression of degenerative tissue pathology. As a central mechanism of cellular quality control, mitophagy maintains cellular metabolic homeostasis and viability by selectively recognizing and degrading dysfunctional mitochondria. Specifically, functional mitophagy maintains cellular homeostasis primarily through the following pathways: First, timely removal of damaged mitochondria to prevent excessive accumulation of reactive oxygen species (ROS) and oxidative stress damage; second, maintenance of the functional integrity of the mitochondrial population to ensure efficient ATP synthesis to meet energy demands; third, stabilization of mitochondrial membrane potential to maintain intracellular calcium homeostasis and normal signal transduction; and finally, inhibition of mitochondrial-mediated apoptosis pathways by preventing the release of pro-apoptotic factors (such as cytochrome c and AIF) from mitochondria. Its molecular regulatory mechanism mainly relies on the PINK1 / Parkin-mediated signaling pathway. When mitochondria are dysfunctional, PINK1 accumulates stably on the depolarized outer mitochondrial membrane, activating its E3 ubiquitin ligase activity through phosphorylated ubiquitin and Parkin protein, ultimately guiding damaged mitochondria to be phagocytosed by autophagosomes and transported to lysosomes for degradation. However, in various degenerative tissues, such as degenerated cartilage and intervertebral disc degeneration (IVDD), the activity of the PINK1 / Parkin pathway is significantly suppressed. Damaged mitochondria cannot be cleared in time, leading to ROS accumulation, reduced ATP production, loss of mitochondrial membrane potential, and activation of the caspase cascade, which in turn induces apoptosis and extracellular matrix degradation, ultimately resulting in irreversible progression of tissue degeneration and functional loss. Therefore, restoring mitophagy function has become an important target for the treatment of degenerative diseases, but how to effectively activate this endogenous repair mechanism remains a major challenge.

[0003] Electromodulation of mitophagy offers a novel therapeutic opportunity for the repair of degenerated tissues because it can directly regulate cell membrane potential, ion channel activity, and intracellular calcium ion concentration, showing a unique advantage in activating mitophagy. Studies have shown that physiologically responsive electrical stimulation can activate AMPK by modulating the calcium ion signaling pathway, thereby initiating the autophagy process, and specifically activate the mitophagy pathway by regulating mitochondrial membrane potential, affecting PINK1 stability and Parkin recruitment. The core of physiological electroadaptation lies in precisely adjusting the electrical signal threshold, ensuring that the intensity of electrical stimulation is within a range that triggers biological effects without causing cell damage. Research indicates that electrical stimulation has a significant "threshold effect" in regulating mitophagy; insufficient stimulation fails to activate the necessary signaling pathways, while excessive stimulation may lead to cell damage. JaeHeeShin et al. demonstrated in a cell model of degenerated intervertebral disc annulus fibrosus that micro-electrical stimulation of 150 mV / mm significantly inhibited the secretion of inflammatory factors and reduced the activity of matrix-degrading enzymes, showing therapeutic potential. However, when electrical stimulation is too strong, nanoscale pores form on the cell membrane, leading to increased intracellular Ca²⁺ levels, loss of mitochondrial membrane potential, and accumulation of activated caspase proteins, thereby inducing apoptosis.

[0004] For example, Wang et al. reported that electrical stimulation significantly reduced ROS levels in IVDD patients (from 31.8±5.7 ng / L before treatment to 17.5±2.7 ng / L after treatment), suggesting that electrical stimulation may improve cell function by reducing oxidative stress. Poillot et al. demonstrated that piezoelectric effects in the intervertebral disc can generate local potentials, possibly by regulating Ca2+. 2+ Channels influence cell behavior and mechanotransduction. However, to achieve such precise control of electrical signal thresholds (50-150 mV / mm), traditional electrostimulation systems typically rely on external devices, wired connections, and invasive electrodes. This is not only highly invasive and biocompatible, but also difficult to achieve dynamic adaptive modulation in complex mechanical environments. Therefore, developing an electro-signal modulation system with endogenous physiological electrical adaptation capabilities has become a key scientific problem in overcoming existing therapeutic bottlenecks.

[0005] Piezoelectric materials convert the mechanical energy of tissue movement into electrical energy through their mechanoelectric conversion properties, enabling in-situ, non-invasive electrical stimulation without external electrodes. Previous studies have reported that mechanoelectric conversion hydrogel microspheres (Piezo@CR MPs) embedded with barium titanate nanoparticles and stem cell recruiting peptides generate electrical signals upon ultrasound activation, promoting stem cell colonization, chondrocyte differentiation, and chondrocyte repair. Notably, the electrical signal output threshold can be modulated by adjusting the energy loss during the mechanoelectric conversion process. Normal nucleus pulposus tissue possesses an inherent piezoelectric effect, maintaining the local electrical environment through mechanoelectric conversion; however, with degeneration, this piezoelectric effect is lost, and the electrical signal level drops below the physiological threshold. Furthermore, Song et al. reported significantly reduced PINK1 expression in nucleus pulposus cells of IVDD patients, accompanied by mitochondrial damage accumulation and elevated ROS levels, fully demonstrating the central role of mitophagy inhibition in IVDD. Therefore, IVDD provides an ideal disease model for piezoelectric materials to regulate mitophagy.

[0006] However, traditional piezoelectric materials still face the challenge of precisely controlling the threshold of electrical signal output in biomedical applications. Therefore, designing a piezoelectric material with strain response to achieve precise control of electrical signal output and accurate regulation of energy loss during mechano-electric conversion, providing precise threshold signals for cellular responses within the intervertebral disc, and precisely regulating mitochondrial function, has become an urgent technical problem to be solved. Summary of the Invention

[0007] This invention aims to solve the aforementioned technical problems by providing an internally triboelectric network piezoelectric hydrogel microsphere, its preparation method, and its applications. The technical objective of this invention is to provide a strain-dependent internally triboelectric network energy dissipation structure, successfully constructing an internally triboelectric network piezoelectric hydrogel microsphere. This aims to convert dynamic stress within the intervertebral disc into an electrical signal within the cellular response threshold, precisely regulating mitochondrial autophagy function and providing a new solution for the physiological electroadaptation of degenerated tissues.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing internally triboelectric network piezoelectric hydrogel microspheres, comprising the following steps: (1) Piezoelectric bismuth ferrite nanoparticles were prepared using bismuth salts and iron salts; (2) Synthesizing polyrotaxane crosslinking agent using α-cyclodextrin and polyethylene glycol; (3) Prepare methacrylated hyaluronic acid using hyaluronic acid and methacrylic anhydride; (4) Methacrylamide hyaluronic acid, piezoelectric bismuth ferrite nanoparticles, polyrotaxane crosslinking agent and photoinitiator were dissolved in water to prepare an aqueous phase, and paraffin oil and Span-80 were used to prepare an oil phase. Droplets were prepared by microfluidic device and photocrosslinked under ultraviolet light to prepare internally triboelectric network piezoelectric hydrogel microspheres.

[0009] Mitochondrial autophagy imbalance is a key factor driving apoptosis and tissue degeneration, and regulating it through physiological electroadaptation to reach cellular thresholds is crucial. However, degenerated tissues often lack endogenous electrical signals, leading to impaired cellular energy transfer. To address this technical challenge, this invention provides an innovative solution based on a slip-ring structure-based internally triboelectric hydrogel network. The slip-ring structure polyrotaxane is formed by cyclic molecules (such as α-cyclodextrin) interwoven with a linear polymer chain, exhibiting significant strain-dependent dynamic characteristics. Introducing slip-ring structures as dynamic crosslinking points into a piezoelectric flexible hydrogel network allows for the construction of a stress-responsive internally triboelectric network, enabling precise regulation of energy loss during mechanoelectric conversion. Under low strain conditions, the slip-ring structures in the internally triboelectric network disperse stress through free sliding, reducing energy loss and increasing the lower threshold for electrical signal generation. Under high strain conditions, stronger tensile coupling occurs between the slip-rings and the flexible backbone, increasing molecular friction within the internally triboelectric network and thus increasing energy loss, thereby lowering the upper threshold for electrical signal output.

[0010] The method provided by this invention successfully constructs a piezoelectric hydrogel system with physiological electroadaptive properties to achieve electrical signal output within the cellular response threshold range in degenerated tissues. This system successfully constructs a slip-ring polyrotaxane crosslinking agent through supramolecular self-assembly between cyclic molecules (α-cyclodextrin) and linear polymers (polyethylene glycol). These slip-ring structures synergistically assemble with flexible HAMA segments to form a stress-responsive internal friction network, endowing the system with adaptive energy dissipation regulation capabilities. Subsequently, bismuth ferrite nanoparticles (BF) with excellent piezoelectric properties were synthesized using a hydrothermal method, and then uniformly loaded onto HAMA hydrogel microspheres with polyrotaxane as dynamic crosslinking points using microfluidic technology to construct the internal friction network, thereby generating a piezoelectric system that synergistically regulates mechanoelectric conversion performance and energy dissipation behavior. Under intervertebral disc dynamic stress, the internal friction network effectively controls the electrical signal output level through strain-dependent dynamic energy dissipation characteristics, keeping it stably within the physiological response threshold range.

[0011] This invention first systematically evaluated the mechanical properties and electromechanical conversion characteristics of BF-PR-HA under different strain conditions, verifying its excellent mechanical stability and efficient electromechanical response. To explore its regulatory role in nucleus pulposus (NPC) apoptosis and specific metabolite expression, BF-PR-HA was subsequently co-cultured with NPCs under dynamic compression. This further elucidated the potential molecular mechanism by which BF-PR-HA regulates extracellular matrix synthesis and apoptosis in NPCs based on the internal friction network. Given that intervertebral disc degeneration involves the regulation of the dynamic mechanical microenvironment, this study further validated the therapeutic potential of BF-PR-HA at the whole-tissue level by implanting it into a rat caudal intervertebral disc degeneration model, and evaluated its repair effect on degenerated intervertebral discs using an RNA sequencing system. This strategy based on the internal friction network provides a controllable and efficient novel intervention method for treating degenerated tissues.

[0012] Furthermore, the piezoelectric bismuth ferrite nanoparticles described in step (1) are prepared in a high-pressure reactor lined with polytetrafluoroethylene, with a reaction temperature of 180°C and a reaction time of 8-12 hours.

[0013] Furthermore, the piezoelectric bismuth ferrite nanoparticles mentioned in step (1) were obtained by calcining at 650°C for 3 hours, with a heating rate of 5°C·min. -1 .

[0014] Furthermore, the molar ratio of bismuth salt to iron salt in step (1) is 1.05:1.

[0015] Furthermore, the specific operation of step (2) is as follows: α-cyclodextrin and polyethylene glycol are dissolved in DMSO and incubated overnight at 4°C. Then, the mixture is filtered through a filter membrane under vacuum and dried to obtain a white pseudo-polyrotaxane intermediate. The intermediate is then dissolved with maleic anhydride in DMSO and refluxed at 60°C for 6 h. Acetone is added to the reaction solution to precipitate the product. The precipitate is collected by filtration through a filter membrane and dried under vacuum to obtain the polyrotaxane crosslinking agent.

[0016] Furthermore, the specific operation of step (3) is as follows: In a carbonate buffer solution with pH=9.0, 10 wt.% hyaluronic acid and methacrylic anhydride are reacted at room temperature for 3 hours to obtain methacrylamide hyaluronic acid containing photocrosslinked double bonds. After the reaction, the product is dialyzed through a 3.5 kDa molecular weight cutoff dialysis bag and then freeze-dried to obtain the final product.

[0017] Furthermore, in step (4), the weight ratio of methacrylamide hyaluronic acid, piezoelectric bismuth ferrite nanoparticles, polyrotaxane crosslinking agent and photoinitiator is 100:10:20:1.

[0018] Furthermore, in step (4), the flow rate ratio of the water phase to the oil phase in the microfluidic device is controlled to be 1:6.

[0019] The second objective of this invention is to provide internally triboelectric network piezoelectric hydrogel microspheres prepared by the method described above.

[0020] A third objective of this invention is to provide the application of the internally triboelectric network piezoelectric hydrogel microspheres described above in the preparation of drugs for treating degenerative tissue diseases.

[0021] The beneficial effects of this invention are as follows: This invention utilizes supramolecular engineering and microfluidic strategies to construct an internally frictional network hydrogel microsphere system through the synergistic assembly of piezoelectric bismuth ferrite nanoparticles (BFs) and slip-ring functionalized methacrylamide hyaluronic acid (HAMA), achieving physiological electrofitting in degenerated tissues. BFs convert mechanical stimulation into electrical signals, while the stress-dependent internally frictional network regulates energy dissipation. Under low stress, slip-ring motion generates low friction, with a mechanoelectric conversion loss of 61.5 kJ / m³, thereby enhancing electrical signal generation. Under high stress, main chain straightening leads to an increase in friction to 78.3 kJ / m³, thus suppressing excessive signal and restoring physiological electrofitting. Under dynamic loading, the microspheres can generate a stable electric field (95-110 mV / mm), promoting mitophagy by activating the PINK1 / Parkin pathway, maintaining a stable mitochondrial membrane potential (JC-1 ratio increased by 49.2%), and reducing nucleus pulposus cell apoptosis by 75%. In vivo experiments showed that microsphere implantation restored the physiological electrical environment, enhanced mitochondrial autophagy, inhibited apoptosis, and slowed the progression of intervertebral disc degeneration, providing new insights into treating degenerated tissues by restoring electrical adaptation. Attached Figure Description

[0022] Figure 1Synthesis and characterization of BF-PR-HA composite piezoelectric hydrogels with internal triboelectric networks; A) Schematic diagram of the chemical formula, slip ring architecture, and HAMA structure of bismuth ferrite; B) Scanning electron microscope (SEM) micrographs of bismuth ferrite nanoparticles; C, D) Transmission electron microscope (TEM) images and corresponding energy-dispersive X-ray spectroscopy (EDS) elemental analysis of bismuth ferrite; E) Dynamic light scattering (DLS) particle size distribution analysis of bismuth ferrite; F, G) Atomic force microscopy (AFM) morphology images, piezoelectric hysteresis loops, and characteristic butterfly curves of bismuth ferrite; H) X-ray photoelectron spectroscopy (XPS) detection of bismuth ferrite; I) Fourier transform infrared spectroscopy (FTIR) of bismuth ferrite; J) X-ray diffraction (XRD) of bismuth ferrite; K) Optical microscopy and SEM characterization of BF-PR-HA composite microspheres; L) Particle size distribution analysis of BF-PR-HA microspheres by DLS measurement; M) BF-PR-HA EDS elemental spectrum and compositional analysis of the microspheres; time-lapse optical microscopy observation of the degradation kinetics of BF-PR-HA microspheres (N).

[0023] Figure 2 Evaluation of the electromechanical conversion performance of internally triboelectric piezoelectric hydrogels; A) Schematic diagram of the experimental setup used for mechanical property characterization and generator signal output detection of hydrogel samples; BD) Representative tensile stress-strain curves and corresponding Young's modulus determination of hydrogel compositions; EG) Energy dissipation curves of hydrogels under low-amplitude cyclic deformation; HJ) Energy dissipation characteristics of hydrogels under high-strain mechanical loading conditions; KM) Tensile and fatigue test curves showing mechanical behavior under different strain amplitudes; NQ) Quantitative analysis of open-circuit voltage and short-circuit current generation in hydrogel samples under dynamic mechanical stimulation.

[0024] Figure 3 To assess biocompatibility and its effects on the proliferation and apoptosis of nucleus pulposus cells treated with internally triboelectric network (MTN) piezoelectric hydrogel; A) Schematic diagram of the co-culture system of human nucleus pulposus cells with the MTN piezoelectric hydrogel construct; B, C) Live / dead fluorescence staining assessment after co-culture with MTN piezoelectric hydrogel; D) Cell viability assessment and corresponding quantitative analysis by CCK-8 colorimetric assay after treatment with MTN piezoelectric hydrogel, mechanical stimulation (MS), and osmotic stimulation (OS), respectively; E, F) Apoptosis analysis by flow cytometry using Annexin V-FITC / propidium iodide (PI) double staining, and statistical assessment after treatment with MTN piezoelectric hydrogel, MS, and OS alone and in combination; G, H) Quantitative assessment of mitochondrial membrane potential by JC-1 fluorescent probe and flow cytometry after intervention with MTN piezoelectric hydrogel, MS, and OS alone and in combination (n = 3, *p < 0.05, **p < 0.01).

[0025] Figure 4The effects of internally triboelectric network (ITN) piezoelectric hydrogel microspheres on the expression of extracellular matrix (ECM) and apoptosis markers in nucleus pulposus cells were investigated. The results included: (AD) Immunofluorescence microscopy observation of aggrecan and matrix metalloproteinase-13 (MMP13) expression in human nucleus pulposus cells after co-culturing with ITN piezoelectric hydrogel microspheres, accompanied by quantitative fluorescence intensity analysis; (EK) Real-time quantitative polymerase chain reaction (RT-qPCR) assessment of ECM-related and apoptosis-related gene expression profiles in nucleus pulposus cells after co-culturing with ITN piezoelectric hydrogel microspheres; (LR) Western blot immunoassay and corresponding optical density quantification of ECM-related and apoptosis-related protein markers in nucleus pulposus cells after exposure to ITN piezoelectric hydrogel microspheres. (n = 3, *p < 0.05, **p < 0.01).

[0026] Figure 5 To investigate the effects of internally triboelectric network piezoelectric hydrogels on the regulation of mitophagy to improve nucleus pulposus cell degeneration; A, B) Western blot immunoassay and corresponding optical density quantification of extracellular matrix (ECM)-related and mitophagy-related protein markers; C) Tandem fluorescent GFP-RFP-LC3 co-labeling for autophagy flux assessment; D, E) Enzymatic activity assays of cathepsin B (CTSB) and cathepsin D (CTSD) proteolytic functions; F, G) Confocal microscopy-based colocalization analysis of microtubule-associated protein 1A / 1B-light chain 3 (LC3) and lysosome-associated membrane protein 1 (LAMP1); H, I.) Immunofluorescence colocalization assessment of mitochondrial outer membrane translocase 20 (TOM20) and LAMP1 for mitophagy assessment; (n = 3, *p < 0.05, **p < 0.01).

[0027] Figure 6 Imaging analysis of intervertebral disc degeneration treated with internal friction network hydrogel microspheres: A) Schematic diagram of rat intervertebral disc compression loading experimental model; B) Serial X-ray images of all experimental groups at 1, 4 and 8 weeks after intervention; C) Magnetic resonance imaging (MRI) findings of all experimental groups at 1, 4 and 8 weeks after treatment; D) Percentage change in intervertebral disc height index (DHI) at each time point for each experimental group; E) Quantitative analysis of the average gray value of MRI assessments of all groups within the specified time interval; F) Pfirrmann classification of the severity of intervertebral disc degeneration in all experimental groups at 1, 4 and 8 weeks after treatment.

[0028] Figure 7Histological analysis of the use of internal friction network hydrogel microspheres for the treatment of intervertebral disc degeneration: A) Hematoxylin and eosin (H&E) staining results of all experimental groups at 1, 4 and 8 weeks after treatment, showing morphological changes in the nucleus pulposus (NP) and annulus fibrosus (AF) architecture; B) Safranin O-Fix Green (SO) staining of all experimental groups at 1, 4 and 8 weeks after intervention, illustrating the composition of the nucleus pulposus matrix and the distribution pattern of collagen fibers; C) Semi-quantitative histological scoring heatmap assessment: NP morphology, NP cell structure, NP-AF interface integrity, AF structural tissue and AF cell density.

[0029] Figure 8 Immunohistochemical and immunofluorescence analyses were performed to evaluate the use of internal friction network hydrogel microspheres for the treatment of intervertebral disc degeneration. The results included: A) Aggrecan immunohistochemical staining in all experimental groups at 1, 4, and 8 weeks post-treatment; B) Type II collagen (Col2) immunofluorescence staining in all experimental groups at 1, 4, and 8 weeks post-intervention; C) Quantitative analysis of the percentage of Col2-positive area in each experimental group at their respective time points; D) Quantitative assessment of the percentage of Aggrecan-positive area in all groups within specified time intervals; E) Comparative histological grading scores of all experimental groups at different time points. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

[0030] Figure 9 To explore the potential mechanisms of biomaterial action through bioinformatics analysis: A) Principal component analysis (PCA) scatter plot visualization; B) Volcano plot depicting differentially expressed genes across experimental conditions; C) Hierarchical clustering heatmap illustrating the expression profiles of the top 50 upregulated and downregulated genes; D) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis presented as bar charts; E, F) Comparison of Gene Set Enrichment Analysis (GSEA) results between the biomaterial treatment group and the control group; G) Box plot analysis showcasing differential expression patterns among key genomes involved in mitophagy and apoptosis activation pathways.

[0031] Figure 10 Schematic diagram of internally frictional hydrogel microspheres: preparation, load response reconstruction, and intradiscal action; A) Microfluidic droplet generation and UV crosslinking produce monodisperse internally frictional hydrogel microspheres; B) Under compression / cyclic loading, network sliding and dynamic bond exchange regulate internal friction and dissipate energy, generating piezoelectric / ionic signals; C) After intradiscal injection, the microspheres restore local support and activate PINK1–Parkin-mediated mitochondrial autophagy to alleviate IVDD.

[0032] Figure 11 For polyrotaxane in heavy water (D2O) 1 Characterization by 1H NMR (hydrogen nuclear magnetic resonance spectroscopy).

[0033] Figure 12 Characterization of the morphology and piezoelectric properties of the "internal friction network" piezoelectric hydrogel microspheres; A) Fluorescence microscopy image of the microspheres; B) Three-dimensional piezoelectric force microscopy (PFM) morphology of the microsphere surface; C) PFM amplitude mapping of the microsphere surface.

[0034] Figure 13 XPS elemental analysis and high-resolution energy dispersive spectroscopy of microspheres; A) O1s high-resolution scan spectrum; B) C1s high-resolution scan spectrum; C) Bi4f high-resolution scan spectrum; D) Fe2p high-resolution scan spectrum; E) EDS energy dispersive spectroscopy of microspheres.

[0035] Figure 14 The following are SEM-EDS elemental distribution mappings of the microspheres: A) Nitrogen elemental distribution (green); B) Oxygen elemental distribution (blue); C) Sulfur elemental distribution (cyan); D) EDS energy spectrum showing elemental composition.

[0036] Figure 15 JC-1 fluorescence detection of the regulatory effect of "internal friction network" piezoelectric hydrogel on mitochondrial membrane potential; JC-1 staining to assess changes in mitochondrial membrane potential in nucleus pulposus cells under different treatment conditions; aggregates (red fluorescence): healthy mitochondria with high membrane potential; monomers (green fluorescence): damaged mitochondria with low membrane potential; Merge: overlay image.

[0037] Figure 16 Transmission electron microscopy (TEM) observation of mitochondrial autophagy after intervention with piezoelectric hydrogels for the "internal friction network". Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0039] Example 1

[0040] I. Experimental Section 1. Preparation and characterization of internally triboelectric network piezoelectric hydrogel microspheres (1) Synthesis of piezoelectric bismuth ferrite nanoparticles BiFeO3 (BFO) nanoparticles were synthesized via a modified hydrothermal method. Specifically, bismuth and iron salts were completely dissolved in an organic solvent system. Citric acid was added as a multidentate chelating agent to stabilize the metal complex, and the system was adjusted to weakly acidic conditions using dilute acid to prevent premature precipitation of the precursors and optimize particle size distribution. The mixed solution was thoroughly stirred at a medium temperature to ensure homogeneous complexation, and then transferred to a polytetrafluoroethylene-lined high-pressure reactor. The reaction was carried out at 180 °C for 8–12 hours to promote crystal nucleation and phase transformation. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, and the product was collected by centrifugation and repeatedly washed with deionized water to remove residual ions and organic compounds. Subsequently, it was subjected to air at 5 °C·min. -1 The powder was calcined at 650°C for 3 hours to obtain pure-phase rhombohedral (R3c) BFO powder. This powder can provide a stable piezoelectric response when subsequently incorporated into hydrogel composites.

[0041] (2) Synthesis of polyrotaxane crosslinking agent (PEG-α-CD-IA) Polyrotaxane crosslinking agents were synthesized via inclusion complexation between α-cyclodextrin (α-CD) and polyethylene glycol (PEG). The specific procedures were as follows: α-CD and PEG were dissolved in deionized water and incubated overnight at 4°C to promote host-guest inclusion. The mixture was then vacuum filtered through a 0.45 μm membrane for 1.5 h to remove unincluded components and dried at 60°C for 6 h to obtain a white pseudopolyrotaxane intermediate. The intermediate was dissolved with maleic anhydride in anhydrous DMSO and refluxed at 60°C for approximately 6 h to achieve esterification of the α-CD hydroxyl groups and introduce active double bonds. Acetone was added to the reaction solution to precipitate the product. The precipitate was collected by filtration through a 0.45 μm membrane, washed three times with acetone, and vacuum dried at 60°C for 18 h to obtain the PEG-α-CD-IA crosslinking agent. 1 H-NMR analysis confirmed the product structure, with a focus on characteristic chemical shift changes caused by α-CD inclusion and vinyl-related signals introduced by esterification.

[0042] (3) Construction of internally triboelectric network piezoelectric hydrogel microspheres In a carbonate buffer solution at pH 9.0, 10 wt.% hyaluronic acid (Sigma, USA) was reacted with methacrylic anhydride (Sigma, USA) at room temperature for approximately 3 hours to obtain HAMA (methacryloyl hyaluronic acid) containing photocrosslinked double bonds. After the reaction, the product was placed in a 3.5 kDa molecular weight cutoff dialysis bag and dialyzed against deionized water for 2 days (with frequent water changes to remove unreacted monomers and small molecule byproducts), and then freeze-dried to obtain HAMA powder.

[0043] A homogeneous aqueous phase was prepared by dissolving HAMA, BFO nanoparticles (BF), polyrotaxane crosslinking agent (PR), and photoinitiator LAP in deionized water at a predetermined ratio; an oil phase was prepared using paraffin oil and the nonionic surfactant Span-80. Both phases were injected separately into a microfluidic device, and uniformly sized droplets were generated at the junction by precisely controlling the water / oil flow rate ratio. Ultraviolet irradiation at 60°C initiated crosslinking, allowing the HAMA molecular chains to rapidly photopolymerize and solidify, resulting in compact and uniformly dispersed composite hydrogel microspheres. Using the same precursor system, bulk hydrogels were prepared by injecting the solution into a mold and curing it under ultraviolet light for in vitro culture experiments involving the co-loading of cells and hydrogels under pressure.

[0044] To differentiate the roles of each component, piezoelectric hydrogel microspheres / blocks containing only BF (BF group) and hydrogel microspheres / blocks containing only PR (PR group) were prepared. Prior to biological experiments, all samples were sterilized according to standard procedures: soaked in 75% ethanol for 30 minutes, washed three times with PBS to remove residual alcohol, and irradiated with ultraviolet light for 30 minutes to ensure sterility and minimize endotoxin interference.

[0045] (4) Material characterization methods To systematically evaluate the material's structure and function, a variety of characterization techniques were employed: XRD (Rigaku Ultima IV, Japan) confirmed the BFO crystal structure and phase purity; SEM (Hitachi S-4800, Japan) and TEM-EDS (FEITalos F200X, USA) observed morphology, particle dispersion, and elemental distribution; DLS determined particle size distribution and dispersibility; XPS (Thermo K-Alpha, USA) analyzed surface elements and chemical states; FTIR (Nicolet iS 10, USA) identified organic functional groups and network structure; and PFM (Bruker Dimension ICON, Germany) was performed on ITO conductive glass (1×1 cm⁻¹) using the DART-PFM mode. 2 Single-point spectral measurements were performed on the device to obtain amplitude-voltage and phase-voltage curves for quantitative evaluation of nanoscale piezoelectric response.

[0046] 2. Evaluation of the electromechanical properties of materials (1) Mechanical performance testing To evaluate the deformation tolerance, toughness, and energy dissipation properties of the hydrogel under external loads, samples were cut to uniform dimensions and subjected to tensile and cyclic loading tests using a universal testing machine (CMT6103, China) under controlled temperature and humidity conditions. Unless otherwise specified, the tensile rate was kept constant at 100 mm / min. -1 Young's modulus is calculated based on the slope of the initial linear region of the stress-strain curve; toughness is determined as the area under the curve before fracture. Hysteresis is determined by cyclic loading-unloading: it is characterized as the ratio of the area enclosed by the unloading curve and the horizontal axis to the area enclosed by the loading curve and the horizontal axis (hysteresis = 100% minus this value).

[0047] The rheological properties were characterized by small-amplitude frequency scanning at 25 °C using a rotational rheometer (HAAKE MARS60, Germany) to obtain the storage modulus G′ and loss modulus G″ as a function of frequency. Stress relaxation tests (10% strain, 1 Hz frequency) were performed at room temperature to elucidate the network viscoelastic synergy and the internal friction energy dissipation mechanism.

[0048] (2) Piezoelectric output performance test To quantify the stress-electric coupling efficiency, the bulk conductivity of the sample was first measured using a digital potentiometer. Short-circuit voltage and open-circuit current were recorded using a digital oscilloscope (Tektronix TBS1202C, USA) under both ultrasonic and mechanical loading modes. The mechanical mode employed periodic compression / bending excitation, while the ultrasonic mode utilized an external field with a fixed frequency and power density. Steady-state output voltage and current were measured under various load resistances, power was calculated, and PR curves were constructed to compare the effects of different components and compositions on energy conversion efficiency.

[0049] 3. Cell culture and biological assessment (1) Cell culture Human nucleus pulposus cells (HNPC, ScienCell) were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and ITS additives at 37°C under a 5% CO2 atmosphere. The passage ratio was typically maintained at 1:2, with experiments preferably performed during the logarithmic growth phase to ensure cell viability and reproducibility.

[0050] (2) Biocompatibility assessment Human nucleus pulposus cells (HNPCs) were cultured according to ISO 10993-5 guidelines to assess the cell compatibility of the bulk hydrogel. Sterile hydrogel discs (approximately 5 mm thick) were placed at the bottom of a 24-well culture plate, and 1×10⁻⁶ smears were applied to the hydrogel surface. 4HNPCs were directly seeded at a standard density of cells / cm². Live / dead cell staining (Calcein AM / PI) was performed after 1, 3, and 5 days of culture, followed by fluorescence microscopy imaging and live-cell ratio quantification. Proliferation capacity was assessed using a CCK-8 assay by measuring absorbance at 450 nm. Blank control materials were included to correct for potential background interference.

[0051] (3) Compressed loading process To simulate the periodic compression within the intervertebral disc microenvironment, a cell loading model was established based on literature. At 37℃ and 5% CO2, cells were subjected to sinusoidal periodic loading of 0.2 MPa, 0.5 MPa, or 1.0 MPa (0.5 Hz) to achieve co-loading of the hydrogel and cells during loading; the control group did not receive loading but maintained the same other conditions.

[0052] 4. Molecular biological detection (1) Flow cytometry analysis Quantitative analysis of early / late apoptosis was performed using Annexin V-FITC / PI double staining. After treatment, cells were washed twice with PBS, stained according to the kit protocol, and incubated at room temperature in the dark for 10–15 minutes, followed by flow cytometry analysis. Mitochondrial membrane potential was assessed using the JC-1 probe, with an increased green / red fluorescence ratio indicating a decreased ΔΨm.

[0053] (2) Western Blot Cells were lysed using RIPA buffer, and total protein concentration was quantified using the BCA method, with an equal loading volume. After SDS-PAGE separation, proteins were transferred to a PVDF membrane and blocked with TBST containing 5% skim milk powder for 1 hour. Primary antibodies against aggrecan, COL-II, cleaved-caspase-3, MMP-3, MMP-13, Bcl-2, Bax, PINK1, Parkin, and p62 were added and incubated overnight at 4°C; HRP-conjugated secondary antibodies were incubated at room temperature for 1 hour. After ECL staining, semi-quantitative analysis of band density was performed using ImageJ.

[0054] (3) Real-time quantitative PCR (RT-qPCR) Total RNA was extracted using the TRIzol method, and cDNA was synthesized by reverse transcription after purity and concentration assessment. Amplification was performed using the SYBR Green system: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. GAPDH was used as an internal control. -ΔΔCtThe relative expression levels were calculated using the method; each group included at least three biological replicates. Primers for the target gene and the housekeeping gene GAPDH were designed using NCBI Primer-BLAST (primer sequences are shown in Table 1).

[0055] Table 1 Primer sequence listing

[0056] (4) Immunofluorescence detection Cells on coverslips were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour. Primary antibodies against COL-II, MMP-13, Tom20, LC3, and LAMP1 were added, and the cells were incubated overnight at 4°C. The next day, the cells were incubated with fluorescent secondary antibodies at room temperature in the dark for 1 hour, and the cell nuclei were counterstained with DAPI. Images were captured using a laser scanning confocal microscope, and fluorescence intensity and subcellular localization analyses were performed.

[0057] (5) Autophagy Flow Detection HNPCs were infected with the mRFP-GFP-LC3 adenovirus vector, and the number and colocalization of red / green fluorescent spots were observed 48 hours post-transfection. Since GFP was quenched in an acidic environment while mRFP remained stable, only the increase in red spots indicated the formation of autophagosome-lysosome fusion and autolysosomes; the Bafilomycin A1 group was included as an autophagy flow blocking control to distinguish between "autophagy activation" and "autophagy blockade".

[0058] (6) Lysosomal function assessment CTSB and CTSD activities were measured using specific fluorescent substrates, and the results were normalized to protein content to ensure comparability.

[0059] 5. In vitro intervertebral disc compression model A rat caudal intervertebral disc compression injury model was established according to the method of Masuda et al. (2005). Under anesthesia and aseptic conditions, adjacent caudal vertebrae (e.g., Co9 / Co11) were selected and fixed, and a constant axial compression load of approximately 1.0 MPa was applied using a pre-defined spring for a specified duration; the sham-operated group underwent only fixation without loading. After completion, specimens were collected and processed according to standard protocols: fixation with 4% paraformaldehyde, decalcification with EDTA, dehydration with graded ethanol, paraffin embedding, and cutting into 4 μm serial sections. Histologically, H&E staining was used to observe tissue structure and cell morphology, and Safranin O-Fixed Green staining was used to assess changes in glycosaminoglycan content. Pathological scoring was performed according to established criteria. Immunohistochemistry and immunofluorescence were used to detect the expression and distribution of COL-II and proteoglycans to evaluate the effect of material intervention on intervertebral disc degeneration.

[0060] II. Experimental Results and Discussion 1. Synthesis and Characterization of Internal Tribological Network Piezoelectric Hydrogels To develop an implantable device for intervertebral disc repair, BF-PR hydrogel microspheres not only need to possess excellent biocompatibility and piezoelectric properties, but also should effectively achieve physiological electrical adaptation to match and mimic the natural electrical environment of tissues, promoting cellular response and tissue regeneration. BF, with its excellent piezoelectric properties and biocompatibility, can facilitate the generation of electrical signals similar to those under physiological conditions through force-to-electric conversion, thereby achieving local tissue electrical stimulation and modulation (Figure 1A). We synthesized BF nanoparticles with a rhombohedral perovskite structure (R3c space group) using a hydrothermal method. Scanning electron microscopy (SEM; Figure 1B) and transmission electron microscopy (TEM; Figure 1C, D) showed that the BF particles were semi-hexagonal to spherical, with an average particle size of approximately 180 nm. Figure 1 The presence of PFM (E) facilitates uniform distribution within the microspheres and ensures a uniform electrical signal output. X-ray diffraction (XRD; J in Figure 1) verified the crystal structure of the BF particles, with diffraction peaks consistent with the JCPDS no. 86-1518 standard card, indicating the absence of impurities and second phase formation, thus ensuring the stability of the piezoelectric effect and maintaining reliable physiological electrical signal release. Figure 1 The F and G diagrams show that under an alternating electric field, BF particles exhibit significant amplitude changes, indicating the presence of piezoelectric domains with a piezoelectric response. The phase diagram clearly reveals a 180° phase difference region, indicating the presence of ferroelectric domains with different orientations. Furthermore, the butterfly curve and hysteresis loop tests clearly show a 180° phase angle reversal and a wide hysteresis loop, with a piezoelectric constant (d)... 33 Approximately 10.97 pm·V -1These results not only demonstrate the piezoelectric and ferroelectric properties of BF, but also show that it can generate perceptible physiological electrical signals through mechanical loading, thereby enabling it to adapt to the natural electrical environment of intervertebral disc tissue and promote cellular sensing and repair.

[0061] We selected polyrotaxane, which possesses a molecular sliding mechanism, as a crosslinking agent to optimize the mechanical conductivity of the hydrogel and establish an internal friction network structure. We synthesized PR using polyethylene glycol and α-cyclodextrin as raw materials and introduced polymerizable double bonds via maleic anhydride esterification. Figure 11 Subsequently, BF nanoparticles were incorporated into HAMA hydrogel with PR as a crosslinking agent, forming a network architecture centered on intramolecular friction of slip rings. This internal friction network can adaptively adjust energy dissipation according to stress magnitude, thereby achieving precise regulation of the BF piezoelectric output. Fourier transform infrared spectroscopy (FTIR; I in Figure 1) shows the characteristic peak of PR (methoxy vibration of ether bond, 1086 cm⁻¹). -1 X-ray photoelectron spectroscopy (XPS) detected Fe and Bi (H in Figure 1). Figure 12 This confirms the successful preparation of the composite material. SEM images show that the microspheres have a porous spherical structure and a uniform distribution of BF particles (K in Figure 1). Figure 13 This provides a suitable spatial structural basis for the formation of the internal friction network. Energy dispersive spectroscopy (EDS) confirmed the uniform distribution of Bi and Fe (M in Figure 1). Figure 14 The presence of BF indicates that there was no aggregation within the hydrogel microspheres. Dynamic light scattering (DLS; L in Figure 1) showed that the average microsphere diameter was approximately 200 µm. Biodegradation experiments showed that on day 0, the microsphere surface had no obvious pores or cracks, and the internal hydrated colloids were highly cross-linked, with no degradation or swelling yet observed. By day 7, increased internal hydration and relaxation of the colloidal network structure were observed. By day 14, irregular light and dark distributions began to appear on the surface of the spheres, indicating that the hydrogel network was undergoing slow hydration and swelling, initial chain scission, and enzymatic or hydrolytic processes. By day 21, the overall shape of the spheres was still recognizable, but no longer intact. By day 28, the originally complete circular outline had significantly collapsed, with increased indentation, and the spheres were approaching the critical point of structural disintegration. By day 49, almost no trace of the original intact spheres could be found, and the remaining parts appeared as a pale yellow or nearly transparent "flocculated" state. The entire region lost its original spherical shape and was in a state of fragmentation, dissolution, or complete release, indicating that the hydrogel colloidal backbone had been almost completely enzymatically or hydrolyzed (N in Figure 1). The dissolution process of the hydrogel microspheres demonstrates its good biocompatibility.

[0062] 2. Evaluation of the electromechanical conversion performance of internally triboelectric network piezoelectric hydrogels We evaluated the mechanical properties of the hydrogel using a universal testing machine (AD in Figure 2). The stress-strain curves showed that the mechanical properties of the material were slightly enhanced after adding BF compared to pure HAMA hydrogel. Further addition of PR as a crosslinking agent significantly enhanced the mechanical properties of the hydrogel through an internal friction network based on a slip ring structure, achieving a compressive modulus of 163 kPa and a strength of 32.6 kJ / m³. Subsequently, we tested the stress-strain behavior of BF-PR at different compression rates. The nearly fourfold increase in compression rate from 50 mm / min to 200 mm / min did not significantly affect the gel properties; the modulus and strength remained largely unchanged, indicating that the internal friction network can maintain the stability of the force-electric conversion under different mechanical conditions, achieving good physiological-electrical adaptation.

[0063] To investigate the energy dissipation behavior of hydrogels under compression, cyclic compression tests were conducted using a universal tensile testing machine. The compression rate was set to 10 mm / min, and the cylindrical compression sample had a diameter of 12 mm and a height of 20 mm. First, the hydrogel was cyclically compressed at small strains of 2%, 4%, 6%, 8%, and 10% (EG in Figure 2). The integral area enclosed by the loading curve and the coordinate axes was defined as the total energy, and the integral area enclosed by the loading and unloading curves was defined as the dissipated energy. As the strain increased from 2% to 10%, the total energy and dissipated energy increased from 6.8 and 1.6 kJ / m³ to 126.6 and 61.5 kJ / m³, respectively, both increasing with increasing strain. The ratio of dissipated energy to total energy was defined as the energy dissipation rate. At 2%, the energy dissipation rate was 23.5%, and at 10%, it was 48.6%. At lower strains, the energy dissipation rate decreased and increased slowly. This finding indicates that under low strain conditions, the slip rings in the internal friction network exhibit low motion resistance and minimal mechanical energy loss. The low energy dissipation and high recovery under low strain help the microspheres maintain stable force-electric output during repeated loading, reducing electrical signal fluctuations and enabling them to adapt to the natural electrical environment of the intervertebral disc.

[0064] For comparison, we also investigated the cyclic compression behavior of the hydrogel under high strain. Unlike the previous small strain (2%–10%), the high strain ranged from 20% to 70% (HJ in Figure 2). With increasing strain, the hysteresis effect of the hydrogel became increasingly pronounced, with total energy and dissipated energy increasing from 425.2 and 201.8 J / m³ to 11510 and 7830 J / m³, respectively. Both increased with increasing strain. The nearly three-fold increase in strain resulted in a nearly 25-fold increase in dissipated energy, demonstrating the increasingly enhanced energy dissipation capability of the internal friction network under high strain, with the energy dissipation rate also increasing from 48% to 68%. Although the stronger energy dissipation capability under high strain is detrimental to electroconversion efficiency, this indicates that the material can withstand extreme loads and continuously release electrical signals, which helps maintain a certain level of physiological electrical stimulation in severely damaged areas.

[0065] Unlike previous individual compression tests at different strains, we performed repeated cyclic tests on the same sample at different strains (small and large strains) (KM in Figure 2). As the strain increased, the hysteresis loop in the cyclic curve became larger and larger. We calculated the average energy dissipation rates at small and large strains to be 31.6% and 63.5%, respectively. Therefore, the gel exhibits strong energy dissipation capacity at large strains, while its energy dissipation capacity is weak at small strains.

[0066] These results are attributed to the stress response characteristics of the slip ring structure in the internal friction network constructed by PR. Under small strain, slip ring molecules can slide freely on the polymer chain to uniformly distribute stress, resulting in low internal friction resistance, which is beneficial for efficient force-to-electric conversion and stable physiological electrical signal output. Under large strain, the slip ring generates a larger normal force due to chain tension, which hinders molecular sliding, increases the friction coefficient in the internal friction network, and leads to significant hysteresis and energy loss, reducing the force-to-electric conversion efficiency and affecting fine electrical signal adaptation.

[0067] To evaluate the electromechanical coupling efficiency of the BF-PR device, we measured the short-circuit current and open-circuit voltage of the microspheres under pressure. No significant electrical signal was detected in the pure HAMA hydrogel device (N, O in Figure 2). The BF-PR device exhibited an open-circuit voltage of 190 mV and a short-circuit current of 10.11 µA under 0.3 MPa (low force) and 0.5 Hz conditions, which were 1.4 times that of BF. Under 1.0 MPa (high force) and 0.5 Hz conditions, the open-circuit voltage of BF was 429 mV and the short-circuit current was 12.34 µA, while the open-circuit voltage of BF-PR was 221 mV. This finding indicates that higher pressures prevent the single BF hydrogel from effectively providing stable voltage and current. Under periodic constant force, both voltage and current increase with increasing mechanical load. Excessively high voltages are detrimental to cell repair and may even accelerate cell death; therefore, maintaining appropriate electrical signals within physiological ranges is crucial.

[0068] Therefore, we further examined the voltage and current of the BF-PR under different strains. Although the voltage and current of the BF-PR increased with increasing mechanical load under periodic constant force, the BF-PR could maintain an appropriate level due to the adaptive adjustment mechanism of the internal friction network, thereby generating a stable physiological electrical signal that better matches the tissue electrical environment and achieves physiological electrofitting (P, Q in Figure 2). The BF-PR significantly improved the force-to-electric conversion efficiency under small strains, thanks to the synergistic effect of the high voltage of the BF and the low mechanical loss of the internal friction network. The slip ring structure reduced mechanical energy loss, while the BF achieved efficient electromechanical conversion. Under large strains, the increased friction caused by the increased normal force led to increased energy loss in the internal friction network, resulting in reduced mechanical energy loss and force-to-electric conversion efficiency, indicating that adjusting the strain range helps achieve the optimal physiological electrofitting effect.

[0069] 3. Biocompatibility of internally triboelectric network piezoelectric hydrogels and their effects on nucleus pulposus cell proliferation and apoptosis. We first co-cultured NPC with BF, PR, or BF-PR without applying any stress. Live / dead staining and cytotoxicity tests (Figure 3, B, C, D) showed no significant toxicity to NPC in any group (Figure 3, D), indicating that the material itself can coexist well with cells under no external force conditions, laying the foundation for subsequent physiological electroadaptation via force-to-electric conversion. Subsequently, dynamic compression was used to simulate a mechanical stress environment, with moderate stress (MS) set at 0.3 MPa and 0.5 Hz, and overload stress (OS) set at 1.0 MPa and 0.5 Hz. These conditions were combined with BF, PR, and BF-PR composites for NPC co-culture (experimental design shown in Figure 3, A). This design aimed to simulate the response behavior of the internal friction network in intervertebral disc tissue under different mechanical loads and the corresponding changes in the electrical environment, thereby testing the potential of this material in physiological electroadaptation.

[0070] CCK-8 assay (Figure 3, I) showed that the MS, MS-BF, MS-PR, and MS-BF-PR groups slightly promoted NPC proliferation, but the differences were not statistically significant (p > 0.05). In contrast, OS significantly inhibited NPC proliferation (p < 0.01), while OS-BF, OS-PR, and OS-BF-PR all alleviated this inhibition. Among them, OS-BF-PR was more effective than OS-BF and OS-PR (p < 0.05), while there was no significant difference between OS-BF and OS-PR (p > 0.05). These results indicate that under high-intensity mechanical loading, the internal friction network in BF-PR can effectively regulate the force-to-electricity conversion process, output appropriate physiological electrical signals, and help alleviate stress damage and maintain cell function.

[0071] Flow cytometry analysis (EH in Figure 3) further revealed that MS and its combination had minimal effect on NPC apoptosis (p > 0.05), while OS significantly increased the apoptosis rate (p < 0.01). OS-BF, OS-PR, and OS-BF-PR all reversed this effect, with OS-BF-PR exhibiting the strongest anti-apoptotic effect, significantly different from OS-BF and OS-PR (p < 0.05), while OS-BF and OS-PR showed similar effects (p > 0.05). These findings suggest that under pathomechanical conditions, the slip-ring-based internal friction network in the BF-PR composite material can adaptively regulate energy dissipation to ensure efficient and stable force-to-electric conversion output of physiological electrical stimulation, thereby achieving physiological electrical adaptation and effectively protecting cells from apoptosis.

[0072] These results demonstrate that HAMA hydrogel exhibits excellent biocompatibility as a carrier. The internal friction network structure formed after integration with BF and PR did not introduce toxicity. While maintaining good cell viability, the material responds to external mechanical stress by generating appropriate physiological electrical signals that match the natural tissue electrical environment, thus achieving physiological electrical adaptation, consistent with the characteristics of the natural intervertebral disc ECM. Under OS conditions, the inhibition of NPC proliferation and the increase in apoptosis reflect OS-induced cell damage, consistent with the pathological mechanism of IVDD. BF-PR performed well in alleviating OS-induced damage, stemming from the synergistic effect of the BF piezoelectric response and the PR internal friction network. The internal friction network shares and alleviates mechanical stress through a stress-responsive slip ring motion mechanism, making the electrical stimulation generated by BF more uniform and closer to physiological levels, thereby achieving good physiological electrical adaptation and inhibiting apoptosis. The BF-PR combination provides more comprehensive protection under pathological conditions but has no significant advantage under MS conditions, indicating that the internal friction network mainly plays a regulatory role under mechanical overload conditions, meeting the clinical need for tissue repair through restoring the physiological electrical environment in IVDD treatment. Given the beneficial effects of MS on intervertebral disc nucleus pulposus cells, our subsequent experiments focused on OS and its hydrogel composite, aiming to reveal how the internal friction network achieves physiological electrofitting through adaptive regulation under pathological mechanical loads, thereby regulating cell state and promoting tissue repair.

[0073] 4. Effects of internally triboelectric network piezoelectric hydrogel microspheres on ECM and apoptosis marker expression in nucleus pulposus cells To elucidate the protective mechanisms of BF, PR, and BF-PR at the molecular level, we used immunofluorescence (AD in Figure 4), RT-qPCR (… Figure 4 EK and Western blotting (LR in Figure 4) were used to analyze changes in the expression of ECM and apoptosis-related markers. Immunofluorescence staining showed that OS significantly reduced COL2A1 expression in NPC but increased MMP13 levels (p < 0.01). BF, PR, and BF-PR all reversed these changes, with BF-PR showing better effects in restoring COL2A1 and inhibiting MMP13 (p < 0.05). This phenomenon suggests that the slip-ring-based internal friction network in BF-PR can adaptively regulate mechanical stress transmission, ensuring that BF provides stable and appropriate physiological electrical signals, achieving adaptation to the native tissue electrical environment, thereby maintaining matrix metabolic homeostasis and inhibiting the abnormal activation of degradation-related enzymes.

[0074] RT-qPCR and Western blotting further confirmed that OS downregulated the expression of COL2A1, aggrecan, and Bcl-2, while upregulating the levels of ADAMTS5, MMP3, MMP13, Bax, and cleaved-caspase-3 (p < 0.01 compared to control group). BF, PR, and BF-PR treatments significantly reversed these changes, upregulating COL2A1, aggrecan, and Bcl-2, while downregulating ADAMTS5, MMP3, MMP13, Bax, and cleaved-caspase-3 (p < 0.01 compared to OS). This reversal effect suggests that under pathological high load, the internal friction network regulates energy dissipation through a stress-response mechanism, enabling BF-PR to output appropriate physiological electrical signals through optimized mechano-electric conversion, improving the local electrical environment, achieving physiological electrical adaptation, thereby enhancing ECM synthesis, inhibiting matrix degradation, and reducing apoptosis.

[0075] Most importantly, BF-PR was superior to BF or PR alone in enhancing the expression of protective markers (COL2A1, aggrecan, and Bcl-2) and inhibiting the expression of degenerative and apoptosis markers (ADAMTS5, MMP3, MMP13, Bax, and cleaved-caspase-3) (p < 0.05). This synergistic effect further confirms the physiological electroadaptation capability mediated by the internal friction network: the internal friction network constructed by PR improves the distribution of mechanical energy consumption, balances mechanical stress transmission, and utilizes the efficient piezoelectric properties of BF to generate uniform and appropriate electrical signals, regulating signaling pathways and maintaining matrix and cell survival.

[0076] OS-induced ECM degradation (reduction of COL2A1 and aggrecan) and upregulation of ADAMTS5, MMP3, and MMP13 reflect the loss of NPC function, while increased Bax and cleaved-caspase-3 indicate apoptosis. The significant regulatory effect of BF-PR on Bcl-2 and cleaved-caspase-3 further supports its ability to inhibit apoptosis. Through the internal friction network, achieving a high degree of adaptation to the natural electrical environment of intervertebral disc tissue, BF-PR not only maintains ECM homeostasis but also protects cell viability. This physiological electroadaptation mechanism based on the internal friction network provides a solid molecular basis for its potential in IVDD therapy.

[0077] Overall, BF-PR reconstructs the electrical environment under pathological load through a stress response mechanism involving the internal friction network, achieving physiological electrical adaptation, maintaining nucleus pulposus cell activity and matrix homeostasis, and effectively delaying the progression of IVDD.

[0078] 5. Internally triboelectric network piezoelectric hydrogel modulates mitochondrial autophagy to improve nucleus pulposus cell degeneration. To further explore the protective mechanism of BF-PR, we investigated its regulatory role in OS-induced mitophagy. When moderately activated, mitophagy can clear damaged mitochondria and maintain energy metabolism and cell survival, playing a crucial role in the regulation of the bioelectroadaptive microenvironment. However, when over-activated or inhibited, autophagy exacerbates apoptosis and disrupts tissue homeostasis. Western blotting results (Figure 5, A, C) showed that OS significantly upregulated the expression of MMP3, MMP13, and p62, while downregulating the expression of collagen-2, aggrecan, PINK1, Parkin, and LC3-II / I (p < 0.05), indicating that OS inhibited PINK1 / Parkin pathway-mediated mitophagy, thereby disrupting the cell's self-protective capacity. Since mitochondria are important electrochemical signaling centers, mitochondrial dysfunction and autophagy impairment disrupt bioelectroadaptation, leading to local electroenvironment dysregulation and exacerbating apoptosis.

[0079] mRFP-GFP-LC3 transfection experiment, JC-1, electron microscopy scanning (Figure 5, B). Figure 15 , Figure 16 Further analysis revealed a significant increase in yellow granules in the OS group, indicating inhibition of autophagic flux. We then validated the mechanism by which compression-induced mitophagic flux blockade was induced. CTSB and CTSD, key proteases in lysosomes that degrade captured cargo, were used to assess lysosomal degradation capacity. The results showed that the activity levels of both enzymes were significantly reduced in the OS group (D, E in Figure 5). The LC3-LAMP1 colocalization assay further showed that OS significantly reduced mitophagosome-lysosome fusion (F, H in Figure 5). Similarly, the colocalization of Tom20 and LAMP1 was also significantly reduced in the OS group (G, I in Figure 5), indicating that in OS-treated human nucleus pulposus cells, damaged mitochondria cannot be effectively phagocytosed and degraded, leading to persistent mitochondrial dysfunction. This dysfunction not only disrupts cellular metabolism but also interferes with physiological electroadaptation, leading to local electrical environment disturbances and exacerbating apoptosis.

[0080] Following BF-PR treatment, the adaptive regulatory mechanism of the internal friction network provided stable physiological electrical stimulation. The expression of the PINK1 / Parkin pathway and downstream proteins, mRFP-GFP-LC3 transfection signaling, and lysosomal protease activity all recovered to near-normal levels. Simultaneously, it promoted the co-localization of LAMP1 with LC3 and Tom20, significantly improving mitophagic flux and lysosomal function. These results indicate that the internal friction network in BF-PR cells ensures stable electrical signal output under OS through stress-responsive energy dissipation regulation, thereby restoring mitochondrial-lysosomal pathway function, promoting the clearance of damaged mitochondria, and rebuilding energy metabolism and electrochemical balance, thus achieving physiological electroadaptation and protecting cells. Notably, when Parkin siRNA interference was added concurrently with BF-PR treatment, its protective effect was significantly weakened, further confirming the crucial role of the PINK1 / Parkin pathway.

[0081] In summary, these findings indicate that BF-PR based on the internal friction network not only provides exogenous physiological electrical signals through mechanical support and force-to-electricity conversion, but also ensures precise electrical signal regulation through the adaptive properties of the internal friction network. By activating PINK1 / Parkin-mediated mitophagy to repair endogenous metabolism and the electrical environment, it achieves physiological-electrical adaptation at the cellular level, alleviates excessive stress damage, and promotes nucleus pulposus cell survival and matrix homeostasis.

[0082] 6. Evaluation of the in vivo therapeutic effect of internally triboelectric network piezoelectric hydrogel microspheres on caudal intervertebral disc degeneration in rats under OS To validate the clinical relevance of in vitro experimental results, we used a rat caudal intraepithelial dysplasia (IVDD) model to evaluate the therapeutic effects of BF, PR, and BF-PR hydrogel microspheres on OS-induced IVDD. Starting from day 1 post-modeling, hydrogel microspheres were locally injected via microinjector (the control group received PBS), and the degree of degeneration was assessed by X-ray and MR imaging at weeks 1, 4, and 8 (AE in Figure 6). The disc height index (DHI) and nucleus pulposus (NP) water content in the OS group decreased significantly over time. These changes indicate that pathological load disrupts the mechanical and electrical environment of the intervertebral disc, leading to physiological electrical adaptation imbalance and tissue degeneration. At week 1, DHI and NP hydration were mildly impaired in the OS group, while the BF, PR, and BF-PR treatment groups showed significant improvement (p < 0.05). At week 4, the DHI and NP water contents in the OS group decreased significantly; however, the BF and PR groups showed limited repair effects, while the BF-PR group based on the internal friction network showed a more significant repair effect (p < 0.01). At week 8, the effects of the BF and PR groups further weakened, while the BF-PR group maintained a good level through the continuous regulatory effect of the internal friction network (p < 0.01). Modified Pfirrmann grading (F in Figure 6) showed that the degree of degeneration in the BF-PR group was significantly lower than that in the OS group (p < 0.01), while the BF and PR groups showed only limited improvement at week 1. These results indicate that BF-PR can maintain the stability of the tissue's physiological electrical environment through long-term stable mechanoelectric conversion mediated by the internal friction network, achieving physiological electrical adaptation and thus delaying the degeneration process.

[0083] H&E and Safranin O-Fixed Green staining (AC in Figure 7) showed severe intervertebral disc structural damage and a significantly elevated histological degeneration score in the OS group (p < 0.01). In week 1, degeneration slowed in the BF, PR, and BF-PR groups; in week 4, histological degeneration significantly worsened in the BF and PR groups, while in the BF-PR group, only mild degeneration occurred due to the adaptive regulation of the internal friction network; in week 8, significant degeneration appeared in the BF and PR groups, while the BF-PR group remained in a state of mild degeneration under the continued protection of the internal friction network. Immunofluorescence and immunohistochemical staining (AE in Figure 8) showed that at week 1, the expression of collagen-2 and aggrecan in the BF, PR, and BF–PR groups was higher than that in the OS group (p < 0.05). At weeks 4 and 8, the expression levels in the BF and PR groups decreased significantly, while the BF–PR group remained stable under the stable regulation of the internal friction network and was significantly higher than other groups (p < 0.05). These long-term protective effects further support the idea that the physiological electroadaptation achieved through the internal friction network in BF–PR can maintain the stability of the tissue electrical environment, stable ECM synthesis, and NP hydration, thereby delaying the IVDD process.

[0084] Overall, in vivo results were consistent with in vitro data, demonstrating significant and durable therapeutic effects in alleviating OS-induced IVDD. Its advantages primarily stem from the synergistic effect of the piezoelectric properties of BF and the internal friction network constructed by PR: the highly efficient piezoelectric properties of BF generate continuous and appropriate electrical signals under mechanical loading, while the internal friction network unloads stress and optimizes energy dissipation distribution through a stress-responsive slip ring motion mechanism, making these electrical signals more uniform and stable. This achieves high adaptability to the tissue electrical environment and protects cells and the ECM. Due to the adaptive regulation of its internal friction network, BF-PR excels in maintaining NP water content and extracellular matrix protein levels, indicating that BF-PR not only maintains the biomechanical properties of the intervertebral disc but also stabilizes the electrical environment long-term through the continuous action of the internal friction network, significantly delaying the degenerative process. In contrast, the short-term improvement effects of using BF or PR alone diminish over time, possibly because a single mechanism cannot fully restore the mechano-electric balance and is insufficient to cope with the complex pathological environment of IVDD.

[0085] Transcriptome sequencing analysis revealed that the BF–PR hydrogel based on the internal friction network significantly modulated molecular pathways related to mitochondrial function, cell metabolism, and apoptosis in the OS-induced IVDD model, thereby maintaining physiological electrofitting and cellular homeostasis. PCA (Figure 9, A) showed a clear separation of gene expression patterns between the OS and OS-BF-PR groups. Volcano plots (Figure 9, B) and heatmaps (Figure 9, C) revealed a large number of differentially expressed genes, with protective genes upregulated and damage-related genes downregulated in the BF-PR group. GO enrichment analysis (Figure 9, D) revealed regulatory processes involved in multiple key biological processes, including mitochondrial localization, ATPase activity, protein kinase activity, and autophagy. GSEA (Figure 9, E, F) showed that BF-PR significantly restored the function of oxidative phosphorylation and the mitochondrial proton transport ATP synthase complex, indicating that physiological electrosignals regulated by the internal friction network effectively improved mitochondrial metabolic activity. The expression levels of key genes (G in Figure 9) further confirmed that BF-PR upregulated the expression of PINK1, BNIP3, and TFDP1, while inhibiting the expression of CASP3 and TIMOC1. This indicates that its physiological electroadaptation is achieved through an internal friction network, which activates PINK1 / Parkin-mediated mitophagy, inhibits apoptosis, and restores energy metabolism to achieve a protective effect. In summary, BF-PR reconstructs mitochondrial function and electrical microenvironment under pathological loading through the stress response mechanism of the internal friction network, achieving physiological electroadaptation, maintaining nucleus pulposus cell viability and matrix homeostasis, and effectively delaying the progression of IVDD. The durable protective effect mediated by the internal friction network in BF-PR highlights its great potential in clinical translation.

[0086] III. Conclusion This invention develops a piezoelectric hydrogel microsphere based on an internal friction network, which achieves physiological electroadaptation through a mechanical stress dissipation mechanism, thereby promoting intervertebral disc tissue repair. In this system, a stress-responsive molecular internal friction network synergistically constructed by slip ring structures and flexible HAMA segments acts as a key regulator of energy dissipation: it generates a stable electric field (95-110 mV / mm) under both low and high stress loading conditions, activates the mitochondrial autophagy pathway, and significantly inhibits nucleus pulposus cell apoptosis, thus providing an effective treatment strategy for intervertebral disc degenerative diseases. Figure 10 In summary, this invention establishes an innovative therapeutic approach for the regeneration and repair of various degenerated tissues.

Claims

1. A method for preparing internally triboelectric network piezoelectric hydrogel microspheres, characterized in that, Includes the following steps: (1) Piezoelectric bismuth ferrite nanoparticles were prepared using bismuth salts and iron salts; (2) Synthesizing polyrotaxane crosslinking agent using α-cyclodextrin and polyethylene glycol; (3) Prepare methacrylated hyaluronic acid using hyaluronic acid and methacrylic anhydride; (4) Methacrylamide hyaluronic acid, piezoelectric bismuth ferrite nanoparticles, polyrotaxane crosslinking agent and photoinitiator were dissolved in water to prepare an aqueous phase, and paraffin oil and Span-80 were used to prepare an oil phase. Droplets were prepared by microfluidic device and photocrosslinked under ultraviolet light to prepare internally triboelectric network piezoelectric hydrogel microspheres.

2. The method according to claim 1, characterized in that, The piezoelectric bismuth ferrite nanoparticles described in step (1) were prepared in a high-pressure reactor lined with polytetrafluoroethylene, at a reaction temperature of 180°C and a reaction time of 8-12 hours.

3. The method according to claim 1, characterized in that, The piezoelectric bismuth ferrite nanoparticles mentioned in step (1) were obtained by calcining at 650°C for 3 hours, with a heating rate of 5°C·min. -1 .

4. The method according to claim 1, characterized in that, The molar ratio of bismuth salt to iron salt in step (1) is 1.05:

1.

5. The method according to claim 1, characterized in that, The specific operation of step (2) is as follows: α-cyclodextrin and polyethylene glycol are dissolved in DMSO and incubated overnight at 4°C. Then, the mixture is filtered through a filter membrane under vacuum and dried to obtain a white pseudo-polyrotaxane intermediate. The intermediate is then dissolved with maleic anhydride in DMSO and refluxed at 60°C for 6 h. Acetone is added to the reaction solution to precipitate the product. The precipitate is collected by filtration through a filter membrane and dried under vacuum to obtain the polyrotaxane crosslinking agent.

6. The method according to claim 1, characterized in that, The specific operation of step (3) is as follows: In a carbonate buffer solution with pH=9.0, 10 wt.% hyaluronic acid and methacrylic anhydride are reacted at room temperature for 3 hours to obtain methacrylamide hyaluronic acid containing photocrosslinked double bonds. After the reaction, the product is dialyzed through a 3.5 kDa molecular weight cutoff dialysis bag and then freeze-dried to obtain the final product.

7. The method according to claim 1, characterized in that, In step (4), the weight ratio of methacrylamide hyaluronic acid, piezoelectric bismuth ferrite nanoparticles, polyrotaxane crosslinking agent and photoinitiator is 100:10:20:

1.

8. The method according to claim 1, characterized in that, In step (4), the flow rate ratio of the water phase to the oil phase in the microfluidic device is controlled to be 1:

6.

9. Internally triboelectric network piezoelectric hydrogel microspheres prepared by the method according to any one of claims 1-8.

10. The use of the internally triboelectric network piezoelectric hydrogel microspheres of claim 9 in the preparation of drugs for treating degenerative tissue diseases.

Images