Piezoelectric hydrogel for improving stem cell delivery survival rate and preparation method and application thereof

Abstract

The invention provides piezoelectric hydrogel for improving the delivery survival rate of stem cells as well as a preparation method and application of the piezoelectric hydrogel, and belongs to the technical field of biological medicines. The invention provides an electric protection strategy, the plasma membrane of the stem cell is protected in the transplantation process, and an endogenous repair mechanism after stress-induced damage is started. Wherein barium titanate (BTO) nanoparticles with piezoelectric characteristics are encapsulated in RGD polypeptide modified oxidized sodium alginate / adipic dihydrazide modified hyaluronic acid hydrogel, and the injectable hydrogel with an electric protection effect is constructed and is used for delivering the BMSCs. By increasing the concentration of free calcium in cells and starting endogenous membrane repair to re-close damaged membranes, the resistance of stem cells to abnormal stress is enhanced, and instant anti-damage protection is provided for cells. The survival rate of the stem cells in the transfer process is remarkably improved by the piezoelectric hydrogel with electric protection, and the piezoelectric hydrogel has a relatively great application prospect in treatment of intervertebral disc degeneration diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a piezoelectric hydrogel for improving the delivery survival rate of stem cells, a preparation method thereof, and an application thereof. Background Art

[0002] Stem cells have the ability of self-renewal and multi-potent differentiation, and have been proven to have important potential in the treatment of degenerative diseases. However, the low survival rate of stem cells during transplantation is the main limitation of their therapeutic efficacy. Most studies mainly focus on improving the host microenvironment to increase the survival rate of transplanted cells, while often ignoring the impact of abnormal mechanical stress during cell delivery. Research shows that the survival rate of stem cells delivered by needle injection of cell suspension is only 30%. High cell mortality weakens the treatment outcome, can trigger local immune responses, and exacerbate the damage to the injured tissue environment. In addition, the high mortality increases the number of stem cells required for transplantation, thus increasing the treatment cost for patients. Therefore, improving cell survival rate during delivery is an urgent challenge for stem cell therapy.

[0003] During the injection process, stem cells experience abnormal shear stress and fluid stretching, resulting in deformation and damage of the cell membrane. Mild membrane damage can trigger the apoptotic signaling pathway within the cell, significantly reducing its viability. After continuous severe and irreversible damage, the cell cannot maintain normal ion gradients and osmotic pressure, leading to acute necrosis and local inflammatory responses. During stem cell injection, the abnormal stress between the cell membrane and the surrounding culture medium is the main cause of cell death and low transplantation efficiency. Traditional strategies for limiting stress exposure focus on optimizing delivery parameters such as needle diameter, injection speed, cell concentration, and medium viscosity, while ignoring the key role of the endogenous cell repair mechanism. Therefore, it is crucial to understand the mechanical microenvironment and biological mechanisms that regulate apoptosis and necrosis during stem cell transplantation. Activating the endogenous membrane repair process and enhancing the resistance of stem cells to shear stress and fluid stretching can reduce abnormal stress damage and potentially improve the efficiency of stem cell transplantation.

[0004] Intracellular free Ca 2+ is a key signaling molecule during the resealing process of the plasma membrane after cell damage. Free Ca 2+ interacts with a variety of calcium sensors, including synaptotagmin VII (Syt VII), annexin, and apoptosis-related gene 2 (alg2), and promotes the rapid repair of the membrane through mechanisms such as the "lipid patch" model, the "endocytic clearance" model, and the "large vesicle shedding" model. In addition, the rapid increase in intracellular Ca 2+ triggers actin remodeling (CaAR), resulting in the formation of Ca 2+a perinuclear actin ring that depends on INF2, and stimulates microfilament polymerization in the endoplasmic reticulum. The CaAR mechanism alters the mechanical properties of cells, allowing them to respond rapidly to acute signals and stress, and enhancing resistance to external damage.

[0005] Previous studies have shown that piezoelectric ceramics are key channels for Ca 2+ influx and exhibit voltage-gated properties. Under electrical stimulation, the piezoelectric channels promote Ca 2+ influx into the plasma membrane and also trigger the release of endogenous calcium stored within the cell, including the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, and nucleus, resulting in a rapid increase in cytoplasmic free Ca 2+ concentration. This influx rapidly activates endogenous repair mechanisms, altering the mechanical properties of the cell and effectively reducing membrane damage caused by shear stress and fluid stretch. Therefore, during stem cell delivery, electrical stimulation of the piezoelectric channels can rapidly increase intracellular Ca 2+ levels, thereby enhancing cell survival through an "electrical protection" strategy.

[0006] Traditional active electrical stimulation involves bulky equipment, wired connections, and invasive or implantable electrodes, which complicates the surgical procedure. Additionally, the mechanical stresses experienced by stem cells during treatment are dynamic, and external power sources cannot adaptively match these different forces to provide targeted electrical stimulation. Abnormal stresses generated by shear forces and fluid stretch can disrupt the integrity of the plasma membrane. The activation of endogenous membrane repair mechanisms is slow, significantly reducing the survival rate of transplanted stem cells. Therefore, a stem cell carrier capable of providing mechanically stressed electrical stimulation must be developed as a trigger for the cell's endogenous repair mechanism to mitigate stress-induced damage during stem cell therapy.

[0007] Studies have shown that piezoelectric materials can generate charges in response to external mechanical stress and can generate electrical stimulation on their own without the need for an external power source. Through piezoelectric transducers, the abnormal stresses experienced by cells during injection can be converted into mechanically triggered piezoelectric potentials, providing precisely matched electrical stimulation to the mechanical stress. Compared with traditional electrical stimulation methods, piezoelectric stimulation has obvious advantages such as non-invasiveness, wireless control, self-power supply, and high spatiotemporal resolution. Therefore, using piezoelectric materials to convert abnormal stress into electrical stimulation can effectively trigger endogenous stem cell repair. This "electrical protection" strategy accelerates the repair of the cell membrane and enhances the cell's resistance to damage induced by external stress.

[0008] Therefore, how to provide a piezoelectric hydrogel material that can provide sufficient "electrical protection" for stem cells, thereby obtaining an enhanced stem cell delivery effect, improving the resistance of stem cells to abnormal stress, enhancing the vitality of bone marrow mesenchymal stem cells, promoting differentiation into nucleus pulposus cells, and being better used for the treatment of intervertebral disc degeneration has become a technical problem to be solved urgently. Summary of the Invention

[0009] The present invention is to solve the above technical problems, and thus provides a piezoelectric hydrogel for improving the delivery survival rate of stem cells, its preparation method and application in repairing intervertebral disc degeneration diseases. The technical object of the present invention is, on the one hand, to solve the problem that the existing piezoelectric hydrogel materials cannot provide sufficient "electrical protection" for stem cells and cannot be used to enhance the traditional effect of stem cells; on the other hand, to solve the problem that the existing piezoelectric hydrogel materials have insufficient curative effect in intervertebral disc degeneration diseases and still need to be further improved, so as to provide a piezoelectric hydrogel that can improve the delivery survival rate of stem cells to better repair intervertebral disc degeneration.

[0010] In order to achieve the above technical object, the technical scheme adopted by the present invention is as follows:

[0011] The present invention first provides a preparation method of a piezoelectric hydrogel for improving the delivery survival rate of stem cells, including the following steps:

[0012] (1) React sodium alginate with sodium periodate to obtain oxidized sodium alginate, then graft it with RGD peptide solution, and prepare RGD peptide-modified oxidized sodium alginate after freeze-drying.

[0013] (2) Activate the carboxyl group of hyaluronic acid with EDC and NHS, then add adipic dihydrazide to the mixture and stir to react. The obtained product is dialyzed and freeze-dried to prepare hyaluronic acid-adipic dihydrazide.

[0014] (3) Mix the RGD peptide-modified oxidized sodium alginate obtained in step (1) with the hyaluronic acid-adipic dihydrazide obtained in step (2) for crosslinking to prepare a hydrogel precursor solution, and add barium titanate nanoparticles to the above hydrogel precursor solution to prepare a piezoelectric hydrogel.

[0015] The above method of the present invention provides an "electrical protection" strategy to enhance the delivery of stem cells. It develops a bone marrow mesenchymal injection hydrogel using RGD peptide-modified oxidized sodium alginate (RGD-OSA) and hydrazide-modified hyaluronic acid (HA-ADH), and forms an injectable hydrogel through a Schiff base reaction. The modification of RGD peptide enhances biocompatibility, promotes cell adhesion, and enhances cell activity. Encapsulating barium titanate (BTO) nanoparticles with piezoelectric properties in the hydrogel forms a BTO / RGD-OSA / HA-ADH piezoelectric hydrogel.

[0016] The above-mentioned piezoelectric hydrogel provided by the present invention has the following characteristics: under mechanical stress, the hydrogel deforms, stores mechanical energy, and applies pressure to BTO. When compressed, BTO nanoparticles generate an internal electric field by replacing the positive and negative charge centers in the crystal structure, and convert mechanical energy into electrical energy. This electrical signal affects the stem cells in the hydrogel. The abnormal stress during the injection process is a "trigger signal" for endogenous cell repair, and the piezoelectric effect provides instantaneous electrical stimulation during stress-induced damage processes. Piezoelectric activation of Ca 2+ ion channels increases the intracellular free Ca 2+ concentration, promotes rapid membrane repair through multiple mechanisms, enhances cell stiffness through cortical actin polymerization, thereby reducing stress-induced deformation, and improving the resistance of stem cells to abnormal stress. Intervertebral disc degeneration (IVDD) is characterized by a decrease in the density of nucleus pulposus cells and impaired extracellular matrix secretion; therefore, exogenous stem cell supplementation is an effective treatment option. In a rat needle puncture IVDD model, the piezoelectric hydrogel with electrical protection significantly improved the survival rate of BMSCs during delivery. Once injected into the degenerated nucleus pulposus tissue, continuous electrical stimulation can enhance the viability of bone marrow mesenchymal stem cells, promote differentiation into nucleus pulposus cells, and stimulate the synthesis of extracellular matrix (ECM), thereby effectively slowing down the progression of intervertebral disc degeneration. This study shows that the piezoelectric hydrogel with electrical protection can significantly improve the delivery efficiency of stem cells. This innovative delivery strategy provides new possibilities for cell therapy in regenerative medicine and tissue engineering, and has good potential in the treatment of degenerative diseases.

[0017] Furthermore, the molar ratio of sodium alginate to sodium periodate in step (1) is 1:1 to 3; sodium alginate and sodium periodate are respectively dissolved in deionized water, and stirred and reacted for 4 hours under dark conditions at 20-25 °C to obtain the oxidized sodium alginate.

[0018] Furthermore, the concentration of the RGD peptide in step (1) is 1 mg / mL, and the weight ratio of the oxidized sodium alginate to the RGD peptide is 25:1.

[0019] Furthermore, the molar ratio of hyaluronic acid, EDC, and NHS in step (2) is 1:1.2:1.2.

[0020] Furthermore, the molar ratio of hyaluronic acid to adipic dihydrazide in step (2) is 1:10.

[0021] Furthermore, the molecular weight cut-off for dialysis in step (2) is 12-14 kDa.

[0022] Furthermore, the concentration of the hydrogel precursor solution in step (3) is 0.1-1% w / v.

[0023] The second object of the present invention is to provide a piezoelectric hydrogel prepared by the method as described above for improving the survival rate of stem cell delivery.

[0024] The third object of the present invention is to provide the application of the piezoelectric hydrogel as described above in the preparation of a drug for treating intervertebral disc degeneration. Specifically, the piezoelectric hydrogel can be loaded with bone marrow mesenchymal stem cells and formulated into an injectable solution as an injectable therapeutic drug for intervertebral disc degeneration.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The present invention proposes an "electrical protection" strategy to protect the stem cell plasma membrane during transplantation and initiate the endogenous repair mechanism after stress-induced injury. Piezoelectric barium titanate nanoparticles (BTO) are encapsulated in RGD-oxidized sodium alginate / hyaluronic acid amine (RGD-OSA / HA-ADH) hydrogel by RGD-oxidized sodium alginate. Piezoelectric BTO activates the piezoelectric channel, increases the intracellular free calcium concentration, and initiates endogenous membrane repair to reseal the damaged membrane, providing immediate anti-injury protection for cells. Calcium triggers the 2+ calcium-triggered actin remodeling (CaAR) mechanism, which regulates the stiffness of cells by remodeling the actin distribution, thereby reducing stress-induced deformation and enhancing the resistance of stem cells to abnormal stress. The RGD-OSA / HA-ADH hydrogel can adapt to changes in the mechanical microenvironment, regulate the mechanical properties through Schiff base reaction, and exhibit shear-thinning behavior, thereby reducing the viscosity during injection and fluid-induced membrane damage. In a rat needle-punctured intervertebral disc degeneration model, the BTO / RGD-OSA / HA-ADH hydrogel with electrical protection continuously stimulates electrically, enhances the proliferation of stem cells and their differentiation into nucleus pulposus cells, and delays intervertebral disc degeneration. The piezoelectric hydrogel with "electrical protection" significantly improves the survival rate of cells during delivery and shows excellent therapeutic potential based on cell therapy. Brief Description of the Drawings

[0027] Figure 1 It is a schematic diagram of the hydrogel of the present invention; (A) Schematic diagram of the piezoelectric BTO / RGD-OSA / HA-ADH hydrogel for bone marrow mesenchymal stem cell delivery; (B) The piezoelectric injectable hydrogel delivers bone marrow mesenchymal stem cells by generating electrical stimulation in response to mechanical stress. During the delivery of bone marrow mesenchymal stem cells, the piezoelectric stimulation activates the piezoelectric channel, rapidly increasing the intracellular free Ca 2+ level; this triggers the "lipid patch", "endocytic removal" and "macropinocytosis shedding" mechanisms to promote the rapid repair of the damaged site of the plasma membrane. The Ca 2+ influx induces Ca 2+The formation of the perinuclear actin ring is dependent, thereby changing the mechanical strength of the cell and enhancing the resistance of stem cells to abnormal stress.

[0028] Figure 2 Properties of the piezoelectric BTO / RGD-OSA / HA-ADH hydrogel; (A) Fourier transform infrared spectrum of HA-ADH shows characteristic absorption peaks; (B) Nuclear magnetic resonance spectrum of HA-ADH further confirms the success of the crosslinking reaction; (C) FTIR analysis of the crosslinking of RGD with oxidized sodium alginate shows changes in functional groups; (D) i: FTIR analysis of the RGD-OSA and HA-ADH crosslinked hydrogel sample; ii: Schematic diagram of the Schiff base crosslinking between RGD-OSA and HA-ADH; (E) Amplitude sweep curves of hydrogels with different concentrations of RGD-OSA / HA-ADH; (F) Stress recovery performance of the RGD-OSA / HA-ADH hydrogel under 1% low strain and 500% high strain conditions; (G) Viscosity-shear rate curve of the RGD-OSA / HA-ADH hydrogel; (H) Visual demonstration of the injectability and self-healing properties of the RGD-OSA / HA-ADH hydrogel, evaluated by the needle injectability test and the self-healing experiment; (I) Scanning electron microscope image and energy-dispersive x-ray of BTO nanoparticles; (J) Raman spectroscopic analysis of BTO nanoparticles; (K) Phase-voltage curve of BTO nanoparticles measured with a piezoelectric force microscope (PFM); (L) Amplitude-voltage curve of BTO nanoparticles measured using PFM; (M) Phase and amplitude images of BTO nanoparticles by atomic force microscope; (N) Representative scanning electron microscope images of hydrogel samples with different BTO concentrations; (O) Energy-dispersive spectrogram showing the elemental distribution in the 0.5% BTO / RGD-OSA / HA-ADH piezoelectric hydrogel; (P) Open-circuit voltage (i) and short-circuit current (ii) of the piezoelectric hydrogel under ultrasonic stimulation (red 0, yellow 0.5 W·cm -2 , blue 1 W·cm -2 , green 1.5 W·cm -2 ).

[0029] Figure 3For calcium-mediated membrane repair and cell survival; (A) Survival of bone marrow mesenchymal stem cells stained with DMEM / F12 liquid carrier (DMEM / F12), RGD-OSA / HA-ADH hydrogel (hydrogel), or BTO / RGD-OSA / HA-ADH hydrogel (HA+ hydrogel) (green, live cells) and PI (red, dead cells); (B) Quantitative analysis of the survival rate of bone marrow mesenchymal stem cells (n = 3); (C) Fluo-4, AM fluorescent dye, used to detect free calcium ions in bone marrow mesenchymal stem cells after injection, and representative fluorescence images showing changes in calcium ion concentration; (D) Flow cytometry analysis of intracellular free calcium ions in bone marrow mesenchymal stem cells after injection of DMEM / F12 liquid carrier (DMEM / F12), RGD-OSA / HA-ADH hydrogel (hydrogel), or BTO / RGD-OSA / HA-ADAH hydrogel (piezoelectric + hydrogel); (E) Quantitative analysis of Fluo-4, AM fluorescence imaging (n = 3); (F) Plasma membrane damage of bone marrow mesenchymal stem cells after injection, labeled with FM 1-43, and the integrity of the membrane was evaluated by confocal microscopy; (G) Quantitative analysis of the average fluorescence intensity of FM 1-43 in panel F (n = 3); (H) Schematic diagram of three calcium-mediated membrane repair mechanisms under piezoelectric stimulation. All values are expressed as mean ± SD; *P ≤ 0.05, **P ≤ 0.01; P ≥ 0.05 is considered not significant (ns). Figure 4 For the change in cell mechanical strength induced by electrical stimulation through the CaAR mechanism; (A) Schematic diagram describing how changes in calcium ion concentration lead to the polymerization of f-actin around the nucleus, thereby regulating cell mechanical strength; (B) Analysis of f-actin polymerization around the nucleus at different time points (0, 5, 30 min) after ultrasonic electrical stimulation, and changes in the fluorescence intensity of perinuclear f-actin; (C) Representative atomic force microscopy (AFM) topography images showing the detailed cell surface morphology; (D) AFM Young's modulus image corresponding to C, illustrating changes in the local Young's modulus to evaluate changes in cell mechanical strength; (E) Statistical analysis of the Young's modulus in the perinuclear and central nuclear regions; All values are expressed as mean ± SD; *P ≤ 0.05; P ≥ 0.05 is considered not significant (ns).

[0030] Figure 5To perform RNA-seq analysis on cells delivered by RGD-OSA / HA-ADH (normal group) and BTO / RGD-OSA / HA-ADH (piezoelectric group) hydrogels; (A) Volcano plot showing differences in gene expression between the piezoelectric group and the normal group, with red dots indicating significantly upregulated genes and blue dots indicating significantly downregulated genes; (B) Heatmap of DEGs showing gene expression levels in different samples, with color representing the expression level; (C) Radar chart comparing the expression of several key genes between the piezoelectric group and the normal group; (D) Oxidative graphene enrichment analysis classifying DEGs into different oxidative graphene categories (biological process, molecular function, cellular component); (E) Chord diagram illustrating the relationship between d and multiple GO terms, highlighting the potential roles of these genes in cell functions and mechanisms; (F) The top 10 GO terms with the highest enrichment scores among the upregulated DEGs; (G) The top 10 KEGG pathways enriched and upregulated, indicating that key signaling pathways in the piezoelectric group were significantly affected.

[0031] Figure 6 For ultrasound-activated piezoelectric materials to generate electrical signals to regulate the proliferation, migration, and differentiation of bone marrow mesenchymal stem cells; (A) Schematic diagram illustrating how ultrasound activates piezoelectric materials to generate electrical signals, thereby regulating the proliferation, migration, and differentiation of bone marrow mesenchymal stem cells; The proliferation of bone marrow mesenchymal stem cells in different treatment groups (day 3) (B) was detected by EdU staining, and the proliferating cells were labeled with green fluorescence; (C) The viability of bone marrow mesenchymal stem cells under different treatment conditions was compared using live / dead staining; (D) Migration of bone marrow mesenchymal stem cells, showing the migration area of different groups within a fixed time; (E) Quantitative analysis of the migration area of bone marrow mesenchymal stem cells; (F) Quantitative analysis of the survival rate of bone marrow mesenchymal stem cells under different treatment conditions; (G) Alcian blue staining showing glycosaminoglycans after culturing with different hydrogels for 7 days; (H) Immunofluorescence staining showing type II collagen (red) and cell nuclei (blue); (I) Quantitative analysis of type II collagen immunofluorescence staining; (J) Relative gene expression of COL2A1, ACAN, and SOX9 in bone marrow mesenchymal stem cells after culturing with piezoelectric hydrogels for 2 days; All values are expressed as mean ± SD; *P≤0.05, **P≤0.01, ***P≤0.001, ****P<0.0001; P≥0.05 was considered not significant (ns).

[0032] Figure 7Imaging evaluation of the rat intervertebral disc degeneration model; (A) shows a schematic diagram of the establishment of a degeneration model induced by puncturing the caudal intervertebral disc of rats, as well as imaging, in vivo tracking, and histological evaluation; the experimental workflow includes model construction, imaging evaluation, and cell labeling and tracking; (B) X-ray images of the intervertebral disc at 4 and 8 weeks after surgery; (C) MRI images showing the water content of the intervertebral disc at 4 and 8 weeks after surgery; (D) MRI T2 sequence pseudo-color images, providing a visual comparison of water distribution; (E) in vivo imaging of dir-labeled bone marrow mesenchymal stem cells at 2 and 4 weeks after surgery, showing the retention and migration of transplanted cells in the intervertebral disc; (F) changes in the disc height index (DHI) at 4 and 8 weeks after surgery (n = 6); (G) changes in the MRI signal intensity reflecting the water content at 4 and 8 weeks after surgery (n = 6); all values are expressed as mean ± SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001; P ≥ 0.05 is considered not significant (ns).

[0033] Figure 8 Histological analysis of animal experiments; (A) H&E staining of the intervertebral disc at 4 weeks after surgery, showing changes in the tissue structure of the intervertebral disc and evaluating the morphology of the nucleus pulposus and annulus fibrosus; (B) safranin O / fast green staining of the intervertebral disc at 4 weeks after surgery; (C) immunohistochemical staining of type II collagen at 4 weeks after surgery; (D) H&E staining of the intervertebral disc at 8 weeks after surgery; (E) O / safranin / fast green staining of the intervertebral disc at 8 weeks after surgery; (F) immunohistochemical staining of type II collagen at 8 weeks after surgery; (G) histological scores based on 5 classification criteria at 4 weeks after surgery (n = 6); (H) histological scores based on 5 classification criteria at 8 weeks after surgery (n = 6); (I) the proportion of type II collagen-positive cells in the nucleus pulposus tissue at 4 and 8 weeks after surgery (n = 6); all values are expressed as mean ± SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001; P ≥ 0.05 is considered not significant (ns). Detailed implementation manners

[0034] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still fall within the protection scope of the present invention.

[0035] Example 1

[0036] I. Experimental materials and methods

[0037] 1. Preparation of RGD-OSA / HA-ADH hydrogel

[0038] (1) Synthesis of RGD-modified oxidized sodium alginate

[0039] First, weigh 5 g of sodium alginate (SA, China, Aladdin), dissolve it in 250 mL of deionized water, and continuously stir it on a magnetic stirrer until completely dissolved. Then, dissolve 3.24 g of sodium periodate in 5 mL of deionized water, and slowly add the resulting solution to the sodium alginate solution. Stir for 4 hours at room temperature (20 - 25 °C) in the dark to promote oxidation. After the oxidation reaction is completed, add 2 mL of ethylene glycol and stir for 30 min to terminate the reaction. After the reaction stops, add 500 mL of ethanol to precipitate the product, and collect it as a white precipitate by vacuum filtration. Then redissolve the precipitate in 50 mL of distilled water, and dialyze it for 3 days using a dialysis bag with a molecular weight cut-off of 7000 Da, changing the dialysis solution 3 times a day. After dialysis, lyophilize the product to obtain oxidized sodium alginate (OSA). Then, dissolve 0.5 g of oxidized sodium alginate in 5 mL of distilled water, and add 20 mL of RGD peptide solution (1 mg / mL, Shanghai A+ Biotechnology Co., Ltd., China). Stir at room temperature for 36 h for grafting reaction. Finally, freeze-dry the reaction solution to obtain RGD peptide-modified oxidized sodium alginate (RGD-OSA).

[0040] (2) Synthesis of hyaluronic acid - adipic dihydrazide

[0041] Dissolve hyaluronic acid (HA) (Aladdin, China) in MES buffer with a pH of 6.0 at a concentration of 2 mg / mL. To activate the carboxyl group of HA, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (Aladdin, China) with a molar ratio of 1:1.2:1.2 (HA:EDC:NHS). Stir the mixture at room temperature for 30 min, add adipic dihydrazide (ADH) in a 10-fold molar amount, and continue to stir at room temperature (20 - 25 °C) for 4 hours. After the reaction is completed, remove the unreacted EDC, NHS, and ADH by dialysis or ultrafiltration. Dialyze in deionized water for 3 days using a dialysis bag with a molecular weight cut-off of 12 - 14 kDa, changing the dialysis solution 3 times a day. Finally, lyophilize the dialysis solution to obtain hyaluronic acid - adipic dihydrazide (HA-ADH) powder.

[0042] (3) FTIR spectral analysis of chemical modification characterization

[0043] Fourier transform infrared (FTIR) spectroscopy confirmed the successful synthesis and modification of the compound by analyzing key functional groups and their corresponding absorption bands. The operating procedure is as follows: Weigh 2 mg of freeze-dried SA, OSA, and RGD-OSA powder samples separately and thoroughly mix them with 200 mg of pre-dried potassium bromide powder in an agate mortar. Then, press these mixtures into microspheres using a tablet press. Place the prepared microspheres on the sample holder of a Fourier transform infrared spectrometer (IRSpirit, Shimadzu, Japan), and record the spectrum in the wavenumber range of 4000 - 500 cm -1 The FTIR analysis method of hyaluronic acid - adipic dihydrazide (HA-ADH) is the same as that of RGD-OSA. In addition, the FTIR spectrum of the RGD-OSA / HA-ADH hydrogel was also obtained to verify the changes in the chemical bonds of the hydrogel.

[0044] (4) 1 Nuclear magnetic resonance spectroscopy analysis of the H molecular structure

[0045] The synthesized HA-ADH sample was thoroughly dried to ensure complete removal of residual solvents. Dissolve an appropriate amount of the dried sample completely in deuterium oxide (D 2 2O). Then transfer the prepared sample solution to a nuclear magnetic resonance tube and adjust the liquid level to optimize nuclear magnetic resonance signal acquisition. Proton nuclear magnetic resonance ( 1 1H NMR) analysis was performed using a Bruker Ascend TM 600 MHz spectrometer (Germany) and 16 scans were carried out to improve the signal-to-noise ratio. The nuclear magnetic resonance tube was carefully aligned in the magnetic field to maximize the signal intensity. After data acquisition, baseline and phase correction were performed on the obtained 1 1H NMR spectrum, and characteristic peaks were identified and labeled.

[0046] 2. Physical property evaluation of injectable and self-heating hydrogels

[0047] Solutions of RGD-OSA and HA-ADH with concentrations of 1%, 2%, and 3% (w / v) were prepared. To form a 1% RGD-OSA / HA-ADH hydrogel, 1% RGD-OSA and 1% HA-ADH solutions were mixed in a 1:1 volume ratio. Similarly, 2% RGD and 3% RGD-OSA / HA-ADH hydrogels were prepared. The injectability of the hydrogels was semi-quantitatively evaluated by extruding through a 26G syringe needle under pressure. To evaluate the self-healing properties of the RGD-OSA / HA-ADH hydrogel, the slices were put together and its self-healing ability was evaluated using a semi-quantitative method.

[0048] The gel-liquid transition shear strain of the RGD-OSA / HA-ADH hydrogel was measured using a rotational rheometer (TA Instruments, USA). The experimental procedure involved placing the hydrogel sample on the rheometer and performing a strain sweep by gradually increasing the shear strain from a low level to a high level while recording the changes in the storage modulus (G') and loss modulus (G"). The self-healing performance of the 2% RGD-OSA / HA-ADH hydrogel was quantitatively evaluated using the rheometer. The storage modulus (G') and loss modulus (G") were measured at a low strain (1%) to determine the linear viscoelastic region. Then, a high strain (500%) was applied to disrupt the hydrogel network, and then it was restored to a low strain (1%), and the recovery of G' and G" was observed to evaluate the self-healing ability of the hydrogel.

[0049] 3. Swelling and Degradation Properties of RGD-OSA / HA-ADH Hydrogel

[0050] This step was used to evaluate the swelling performance of the RGD-OSA / HA-ADH hydrogel. First, 100 mg of the hydrogel sample was immersed in phosphate-buffered saline (PBS, pH 7.4). The initial weight of the freeze-dried hydrogel was recorded as W 0 , and the weight of the hydrogel (W 1 ) was measured at specific time points. The swelling ratio was calculated using the formula: This process was repeated three times until the weight of the hydrogel reached equilibrium.

[0051] (W 1 - W 0 ) / W 0 × 100%.

[0052] In the degradation study, a fully swollen RGD-OSA / HA-ADH hydrogel with an initial weight of 1000 mg was placed in a 15 mL test tube. Subsequently, 2 mL of PBS (pH 7.4) was added, and it was incubated at 37 °C while gently stirred at 80 rpm. At predetermined time points, the supernatant was removed, and the excess water was blotted with filter paper. The hydrogel was weighed, and its weight was recorded as W t . The degradation rate was calculated using the formula: This experiment was repeated three times until the weight of the hydrogel stabilized.

[0053] (W 0 - W t ) / W 0 × 100%.

[0054] 4. Synthesis and Structural Characterization of Barium Titanate Nanoparticles

[0055] (1) Synthesis of Barium Titanate Nanoparticles

[0056] Synthesize tetragonal barium titanate nanoparticles (BTO NPs) by solvothermal method. In this experiment, barium hydroxide (Ba(OH) 2 ·8H 2 O) (Aladdin, China) and titanium butoxide (Ti[O(CH 2 ) 3 CH 3 4 )(Macklin, China) are used as the main precursors. First, dissolve 17.018 g (50 mM) of titanium butoxide in 20 mL of ethanol, add 7 mL of ammonia solution (NH 3 ·H 2 O, 25%), and stir until homogeneous. Dissolve 23.660 g (75 mM) of barium hydroxide in 25 mL of deionized water respectively to obtain a transparent solution. While stirring gently, add the barium hydroxide solution to the titanium precursor mixture. Transfer the obtained suspension to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene (PTFE) and heat it to 200 °C for 48 hours. After cooling to room temperature (20 - 25 °C), wash it repeatedly with deionized water and high-purity ethanol, and then dry it in a vacuum oven at 80 °C for 24 hours to obtain cubic barium titanate nanoparticles (c-BTO NPs). Place the c-BTO NPs in an alumina crucible, heat it at a rate of 5 °C per minute to 90 °C, and hold at this temperature for 4 hours. Then increase the temperature to 800 °C at a rate of 2 °C per minute and anneal for 4 hours. Finally, naturally cool the product to room temperature to obtain BTO NPs.

[0057] (2) Characterization of BTO NPs

[0058] Perform X-ray diffraction (XRD) analysis on the BTO NPs sample using a Kα-ray diffraction system (Rigaku, Japan) (λ = 1.5406 Å). The measurement is carried out in the 2θ range from 10° to 80°, and the working voltage and current are set to 10 kV and 10 μA respectively. Scanning electron microscope (SEM) images are obtained using a field emission SEM (S-4800, Hitachi, Japan). Raman spectra are recorded using a laser Raman spectrometer (Thermo Scientific DXR, USA) with an excitation wavelength of 532 nm.

[0059] ​The microstructure and piezoelectric properties of the BTO NPs samples were investigated using an atomic force microscope (AFM) (Bruker Dimension Icon, Germany). The AFM operates in contact mode, using a conductive probe to image the surface of the BTO samples and record their morphology and microstructure. Piezoresponse force microscopy (PFM) measurements were also carried out using a conductive probe in contact mode to obtain PFM images and amplitude butterfly loops of the BTO samples. By applying a voltage while scanning the sample surface and measuring the corresponding piezoelectric response, an amplitude-butterfly loop was plotted to reflect the ferroelectric properties of the material. AFM imaging provided detailed information about the surface microstructure of the BTO samples, including particle size, shape, and distribution. PFM measurements revealed the distribution of piezoelectric responses, and the analysis of the response images allowed the observation of the formation and evolution of piezoelectric domains.

[0060] 5. Synthesis and Functional Characterization of Piezoelectric Hydrogels

[0061] BTO nanoparticles were added to RGD-OSA / HA-ADH hydrogel precursor solutions with concentrations of 0.1% (w / v), 0.5% (w / v), and 1% (w / v), respectively, and were labeled as 0.1%BTO / RGD-OSA / AHA-ADH, 0.5%BTO / RGD-OSA / HA-ADH, and 1%BTO / RGD-OSA / HA-ADH. After freeze-drying, the morphology of the piezoelectric hydrogels was characterized using a field emission scanning electron microscope (Zeiss, Germany). Elemental mapping of Ca, P, Ba, and Ti in the piezoelectric hydrogels was performed using energy-dispersive spectroscopy (Ultim-Max 40, Oxford, UK). The crystal structure, phase composition, and changes in grain size of the piezoelectric hydrogels were analyzed using x-ray diffraction (XRD, Rigaku, Japan).

[0062] The output voltage and current of the piezoelectric hydrogels were measured using a digital storage oscilloscope (RTM3000, Rohde & Schwarz, Germany) as follows: The piezoelectric hydrogel samples were fixed on a stable platform, and conductive electrodes were fixed on the sample surface to ensure good electrical contact. The oscilloscope was turned on, calibrated, and connected to the electrodes through the input channels. Appropriate time bases and voltage ranges were set, and a current probe with the correct range was used to measure the current. Ultrasonic stimuli with different intensities (0.5 W·cm-2, 1 W·cm-2, and 1.5 W·cm-2) were applied to the piezoelectric hydrogels, and the voltage and current signals generated by the hydrogels under each condition were collected using the oscilloscope. The stability and reproducibility of the waveforms were monitored during data acquisition.

[0063] 6. Isolation of Bone Marrow-Derived Mesenchymal Stem Cells and In Vitro Experimental Cultivation

[0064] Animal experiments were approved by the Ethics Committee of North Sichuan Medical College Affiliated Hospital (2023 - 115). According to the above method, carbon dioxide asphyxiation was used to euthanize SD rats. The hind limbs of the subjects were disinfected with 70% ethanol under sterile conditions. The bilateral femurs and tibias were aseptically excised using sterile scissors and forceps, and rinsed with normal saline to remove soft tissues. The ends of the bones were incised with sterile scissors or a scalpel to expose the bone marrow cavity. Then, pre-cooled medium was slowly injected into the cavity using a syringe to wash the bone marrow cell suspension. The collected suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min to form cell spheres, and then the supernatant was discarded. The cell spheres were resuspended in the medium and then treated with 0.25% trypsin or collagenase to further separate cell clumps. After another round of centrifugation and removal of the supernatant, the cell spheres were resuspended in complete medium and seeded in a culture dish. The cells were cultured at 37 °C in a 5% CO 2 incubator, and the medium was changed every 2 - 3 days to remove non-adherent cells. When the cell concentration reached 70 - 80%, the cells were digested with 0.25% trypsin and passaged for further culture.

[0065] 7. Manual syringe injection and cell viability assay

[0066] When the cells reached 70 - 80% confluence, the cells were digested with trypsin and collected. 1×10 5 bone marrow mesenchymal stem cells were mixed with 300 μL of DMEM / F12, RGD - OSA / HA - ADH hydrogel precursor solution, BTO / RGD - OSA / HA - ADH hydrogel precursor solution, or BTO / RGD - OSA / HA - ADH + GsMT×4 hydrogel precursor solution. Using the manual injection method, 50 μL of the mixture was injected in about 1 s. A live / dead staining solution was prepared by suspending 1 μM calcein AM and 2 μM propidium iodide (Beyotime, China) in PBS. In the cell viability assay, the samples were immediately treated with the live / dead staining solution after injection. The samples were incubated at 37 °C, 5% CO 2 and 95% humidity for 30 min. After incubation, confocal image stacks were obtained using an Olympus SpinSR10 super-resolution spinning disk confocal microscope at 20× magnification, with wavelengths of 488 nm and 561 nm respectively, a capture depth of 5600 - 6000 μm, and a z-spacing of 2 μm. The number of live and dead cells was quantified using a cell counter plugin. All experiments were repeated three times.

[0067] 8. Ca 2+ Fluorescence intensity monitoring of cellular calcium dynamics

[0068] Fluo-4, AM is widely used in cell biology experiments to detect the dynamic changes in intracellular calcium ion concentration. Fluo-4, AM penetrates the cell membrane and is hydrolyzed by intracellular esterase to form Fluo-4, which remains inside the cell. Free Fluo-4 exhibits low fluorescence intensity and is independent of calcium ion concentration. However, when Fluo-4 binds to intracellular calcium ions, its fluorescence intensity increases significantly. Mix 1×10 5 Bone marrow mesenchymal stem cells with 300 μL of DMEM / F12, RGD-OSA / HA-ADH hydrogel precursor solution, BTO / RGD-OSA / HA-ADH hydrogel precursor solution, or BTO / RGD-OSA / HA-ADH+GsMTx4 hydrogel precursor solution. Fluo-4 (5 μM; American Medical Chemical Express) was loaded into the cells at 37 °C for 15 min. Immediately after injection, calcium ion-dependent fluorescence signals were captured using an Olympus SpinSR10 super-resolution spinning disk confocal microscope. The acquisition depth of the image z-stack was 5620 - 5680 μm, the z-spacing was 2 μm, and the wavelength was 488 nm. The fluorescence intensity in the captured images was quantified using ImageJ software. Flow cytometry was also used to quantify calcium ion-dependent fluorescence signals through appropriate channels. All experiments were repeated three times.

[0069] 9. Evaluation of membrane integrity by FM 1-43 fluorescence intensity

[0070] The styryl dye FM 1-43 (Invitrogen, Thermo Fisher Scientific, USA) was added to the hydrogel at a concentration of 5 μg / mL to detect the fluorescence signal of plasma membrane damage caused by shear stress. The dye was introduced immediately before mixing with the cells. Bone marrow mesenchymal stem cells were digested with trypsin and centrifuged at 1000 rpm for 5 min. Mix 1×10 5 Cells with 300 μL of DMEM / F12, RGD-OSA / HA-ADH hydrogel precursor solution, BTO / RGD-OSA / HA-ADH hydrogel precursor solution, or BTO / RGD-OSA / HA-ADH+GsMTx4 hydrogel precursor solution. After injecting the hydrogel and crosslinking, the samples containing FM 1-43 were fixed with 4% paraformaldehyde for 15 minutes and then washed with DPBS for confocal imaging. The capture depth of the image z-stack was 5620 - 5680 μm, the z-spacing was 566 nm, and the wavelength was 2 μm. Post-imaging analysis was performed using ImageJ software to quantify the FM 1-43 fluorescence intensity of each cell and evaluate the degree of membrane damage. All experiments were repeated three times.

[0071] 10. Measurement of cell mechanical properties

[0072] (1) Dynamic rearrangement of the f-actin network in cell morphology and function

[0073] Bone marrow mesenchymal stem cells were seeded in glass-bottomed microwell plates (BioSharp, China) at a density of 1×10 5 cells per well and cultured at 37 °C in a 5% CO 2 incubator for 24 h. Then the cells were covered with the BTO / RGD-OSA / HA-ADH hydrogel precursor solution or the BTO / RGD-OSA / HA-ADH+GsMTx4 hydrogel precursor solution. After hydrogel formation, low-intensity pulsed ultrasound (1 W·cm -2 ; 1 MHz) was applied for 10 min to generate electrical stimulation. At 0, 10, and 30 min after stimulation, the cells were fixed with 4% paraformaldehyde. After fixation, the cells were permeabilized with 0.1% TritonX-100 (Time in China) for 10 min at room temperature and then washed three times with PBS. The cells were blocked with a rapid blocking solution (Beyotime, China) for 10 min at room temperature and then stained with phalloidin-tracker red (Beyotime, China) for 20 min for f-actin. After staining, the cells were washed three times with PBS, stained with DAPI for 10 min, and the cell nuclei were observed. Finally, the dynamic rearrangement of intracellular f-actin filaments was observed and recorded using an Olympus SpinSR10 super-resolution spinning disk confocal microscope. Random fields were selected for image acquisition, and the experiments were independently repeated 3 times to ensure the reliability of the results.

[0074] (2) Measuring the mechanical properties of cells with AFM

[0075] Bone marrow mesenchymal stem cells were seeded on the surface of the BTO / RGD-OSA / HA-ADH hydrogel to observe the effect of electrical stimulation generated by low-intensity pulsed ultrasound (1 W·cm-2; 1 MHz) on bone marrow mesenchymal stem cells. The peak force QNM mode (Nanoscope v9.3) of a Bioscope Resolve atomic force microscope (AFM, Bruker, USA) was used to characterize the cell morphology and nano-mechanical properties before and after electrical stimulation. Imaging was performed using a PFQNM-LC-CAL probe (Bruker, USA). In this experiment, the AFM tip was modeled as a rigid conical indenter with a spherical tip, and the Young's modulus (E) of the cells was fitted to the force curve using a cone-sphere model. The original force curve background was subtracted before fitting to eliminate the hysteresis effect caused by hydrodynamic damping in the liquid medium. The baseline of the force curve was calibrated using the 10-50% baseline segment, and the fitting range was set between 30-90% to ensure accuracy and reproducibility. Electrical stimulation was applied for 10 min by low-intensity pulsed ultrasound (1 W·cm -2 ; 1 MHz). Subsequently, the cells were mounted in the AFM sample chamber and imaged in serum-free medium. Changes in the Young's modulus of the cells were observed at 0, 10, and 30 min after stimulation.

[0076] 11. Viability assay of cells cultured in hydrogel envelopes

[0077] First, 1×10 5 bone marrow mesenchymal stem cells were mixed with 300 μL of RGD-OSA / HA-ADH hydrogel precursor solution or BTO / RGD-OSA / HA-ADH hydrogel precursor solution and seeded in glass-bottomed microwell plates (Biosharp, China). Low-intensity pulsed ultrasound (LIPUS) was applied daily for 10 min to generate electrical stimulation, while the control group received no stimulation. At 2 and 4 days, live / dead cell staining was performed using 1 μM calcein and 2 μM propidium iodide staining solution (Time, China) to evaluate the effect of LIPUS-induced electrical stimulation on cell viability, thereby evaluating the biocompatibility of the composite piezoelectric hydrogel and its effect on the proliferation of bone marrow mesenchymal stem cells. Imaging was performed using an Olympus SpinSR10 super-resolution spinning disk confocal microscope at 10× and 20× magnifications to obtain an overlay of confocal images at wavelengths of 488 nm and 561 nm, with a coverage depth of 5600 - 6000 μm and a z-spacing of 2 μm. After image acquisition, the cell counter plugin in ImageJ software was used to quantify live and dead cells to determine cell viability under different experimental conditions and further evaluate the effect of the hydrogel on cell proliferation. All experiments were repeated three times.

[0078] 12. Cell migration

[0079] Bone marrow mesenchymal stem cells were seeded in glass-bottomed microwell plates (Bio Sharp, China) at a density of 1×10 5 cells per well and cultured in a 5% CO 2 incubator at 37 °C until the cells reached 90% confluence. Then, a scratch was created on the cell monolayer using a 1000 μL pipette tip to simulate a wound model. After removing the culture medium, the cells were washed 3 times with 1×PBS to completely remove floating cells in the scratched area. Subsequently, 300 μL of RGD-OSA / HA-ADH hydrogel precursor solution or BTO / RGD-OSA / HA-ADH hydrogel precursor solution was added, and the hydrogel was allowed to solidify for 20 min, followed by the addition of fresh culture medium. Low-intensity pulsed ultrasound (1 W·cm -2; 1 MHz) was applied for 10 min to generate electrical stimulation, while the control group received no stimulation. After 24 h, the cells were stained with 1 μM calcein AM to observe cell migration and evaluate the repair and migration abilities of the cells under different conditions. Imaging was performed using an Olympus SpinSR10 super-resolution spinning disk confocal microscope, and a random field was selected for image acquisition. Using ImageJ software, the area of cell migration was quantified by outlining the boundary of the scratched area and calculating the difference in area before and after migration to evaluate the migration ability of the cells under different conditions.

[0080] 13. Detection of cell proliferation by EdU

[0081] First, 1×10 5 bone marrow mesenchymal stem cells were mixed with 300 μL of RGD-OSA / HA-ADH hydrogel precursor solution or BTO / RGD-OSA / HA-ADH hydrogel precursor solution and seeded in glass-bottomed microwell plates (Biosharp, China). Low-intensity pulsed ultrasound (1 W·cm -2 ; 1 MHz) was applied daily for 10 min to generate electrical stimulation, while the control group received no stimulation. On the 3rd day, EdU (5-ethynyl-2'-deoxyuridine) staining (Beyotime, China) was used to evaluate DNA synthesis activity. EdU staining was performed according to the manufacturer's instructions: during cell incubation, EdU was added to the culture medium to label cells undergoing DNA synthesis. After EdU labeling, the cells were fixed, washed, and permeabilized. Then, the click chemistry method was used to conjugate EdU with FITC fluorescent dye to label cells actively synthesizing DNA. After staining, the cell nuclei were counterstained with DAPI. We used an Olympus SpinSR10 super-resolution spinning disk confocal microscope for imaging, and a random field was selected to observe the proliferation status of the cells in the hydrogel and evaluate cell viability and DNA synthesis.

[0082] 14. Alcian blue staining of the hydrogel

[0083] First, 1×10 5 bone marrow mesenchymal stem cells were mixed with 300 μL of RGD-OSA / HA-ADH hydrogel precursor solution or BTO / RGD-OSA / HA-ADH hydrogel precursor solution and seeded into 96-well plates (Biosharp, China). Low-intensity pulsed ultrasound (1 W·cm -2; 1 MHz), for 10 min to generate electrical stimulation, while the control group received no stimulation. The treatment lasted for 7 days. At the end of the 7th day, the samples were washed 3 times with PBS and fixed with 4% paraformaldehyde for 3 h. The hydrogel scaffolds were embedded in OCT compound, and 5-μm cryosections were prepared using a cryostat. The sections were stained with Alcian blue for 15 min and then washed with deionized water. The sections were stained with Nuclear Fast Red for 3 min and then rinsed with tap water. Similarly, 1 mL of Nuclear Fast Red was added to each well for 3 min and then washed with distilled water. The sections were dehydrated in absolute ethanol I, II, and III for 5 min, and then cleared in xylene I and II for 5 min each. Finally, the sections were mounted with neutral balsam. Images of the stained sections were randomly captured under a microscope for further analysis.

[0084] 15. Immunofluorescence staining of COL2A1 hydrogel

[0085] First, 1×10 5 bone marrow mesenchymal stem cells were mixed with 300 μL of RGD-OSA / HA-ADH hydrogel precursor solution or BTO / RGD-OSA / HA-ADH hydrogel precursor solution and seeded into 96-well plates (Biosharp, China). Low-intensity pulsed ultrasound (1 W·cm -2 ; 1 MHz) was applied daily for 10 min to generate electrical stimulation, while the control group received no stimulation. The treatment lasted for 7 days. At the end of the 7th day, the samples were washed 3 times with PBS, embedded in OCT compound, and then sectioned into 5-μm thick slices using a cryostat. Then the cryosections were dried in an oven at 37 °C for 10 min to remove moisture. After drying, the sections were fixed with fixative for 30 min and then washed 3 times with PBS (pH 7.4) on a shaker for 5 min each. After gently drying the sections, a hydrophobic barrier was drawn around the tissue with a PAP pen, and the sections were blocked with BSA for 30 min. After blocking, the sections were incubated with the prepared anti-COL2A1 primary antibody overnight in a humid chamber at 4 °C. The next day, they were rinsed 3 times with PBS (pH 7.4) for 5 min each. Then the appropriate fluorescent secondary antibody was applied, and the slides were incubated in the dark at room temperature (20 - 25 °C) for 50 min. They were washed 3 times with PBS (pH 7.4) for 5 min each. DAPI staining solution was added, and the sections were incubated in the dark at room temperature for 10 min. Finally, they were covered with an anti-fading mounting medium. After mounting, fluorescent images of the sections were randomly obtained using a fluorescence microscope. The fluorescence intensity was further analyzed using ImageJ software and data quantification was performed.

[0086] 16. Isolation of RNA and real-time fluorescence quantitative PCR

[0087] With or without low-intensity pulsed ultrasound (1 W·cm -2After treatment with 1 MHz for 1 day, electrical stimulation was applied for 10 min every day, and the samples were washed 3 times with PBS. Total RNA was extracted from bone marrow mesenchymal stem cells using Trizol reagent (Vazyme, China), and reverse transcription was performed using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). Then, RT-qPCR was performed using an ABI 7300 Real-Time PCR System (ABI, USA). Chondrogenic differentiation-related genes analyzed included collagen type II (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX-9), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an internal control. The relative gene expression levels were calculated using the 2-ΔΔCt method. The gene sequences of COL2A1, ACAN, SOX-9, and GAPDH are shown in Table S1. The cycle threshold (Ct) values were compared using the 2-ΔΔCt method to quantify the relative mRNA expression levels. All experiments were repeated three times.

[0088] 17. Construction of in vivo animal model

[0089] Three rats were housed in each cage and had free access to water, and the environmental temperature was maintained at 22 ± 2 °C. After 1 month of acclimation, 18 three-month-old Sprague-Dawley rats (body weight 300 - 350 g) were randomly selected for in vivo experiments. According to the above method, a rat intervertebral disc degeneration (IVDD) model was established. Under isoflurane inhalation anesthesia, the Co 7 / 8, Co 8 / 9, and Co 9 / 10 intervertebral discs were punctured with an 18G needle, and the Co 6 / 7 intervertebral disc was used as the uninjured control group (Sham). The needle was inserted into the center of the nucleus pulposus, rotated 360°, and removed after holding for 30 s. After puncture, 10 μL of different treatments were injected into each intervertebral disc using a 26G needle: 10 μL of PBS was injected into Co 7 / 8, 10 μL of DMEM / F12 + bone marrow mesenchymal stem cells was injected into Co 8 / 9, 10 μL of RGD-OSA / HA-ADH hydrogel + bone marrow mesenchymal stem cells was injected into Co 8 / 9, and 10 μL of BTO / RGD-OSA / HA-ADH hydrogel + bone marrow mesenchymal stem cells was injected into Co 9 / 10. The healthy Co 6 / 7 intervertebral disc was used as the control group (Sham). Six rats (n = 6) were used in each group at each time point. All procedures were performed under sterile conditions.

[0090] 18. In vivo fluorescence imaging for monitoring the viability of bone marrow mesenchymal stem cells

[0091] Before bone marrow mesenchymal stem cell transplantation, the cells were labeled with DiI (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) dye (China) following the protocol described in a previous study. On days 14 and 28 after injection, the rats were anesthetized as described above. After pre-cooling the camera of the imaging system, the anesthetized rats were sequentially placed in the detection chamber of the in vivo imaging system (IVIS), ensuring that the tail was centered on the imaging panel. The DiR fluorescence intensity in the injection area was detected using the IVIS Lumina III system (Xenogen, USA) with an excitation wavelength of 587 nm and an emission wavelength of 610 nm. The captured fluorescence signal was superimposed on the grayscale image of the rat, and the fluorescence intensity within the region of interest (ROI) was quantified using Living Image software to evaluate the change in fluorescence signal over time.

[0092] 19. Radiological examination of intervertebral disc degeneration

[0093] At 4 and 8 weeks after surgery, the rats were anesthetized, and X-ray and MRI images were recorded. The intervertebral disc height was measured using an X-ray imaging system, and the disc height index (DHI) was calculated as described above. In addition, the rat caudal intervertebral disc was scanned in the sagittal T2-weighted plane using a 7.0T MRI scanner (7.0T MRI Biospec GmbH, PharmaScan7016, Germany) to evaluate the water content of the intervertebral disc. The gray value of the MRI image was analyzed using DICOM Viewer to evaluate the change in water content within the disc.

[0094] 20. Histological analysis of tissue regeneration

[0095] Specimens were collected at 4 and 8 weeks after surgery. After disc excision, the disc tissue was fixed with 4% paraformaldehyde at 4 °C for 1 week and then decalcified with EDTA for 4 weeks. The samples were embedded in paraffin, sectioned into 5-μm sections, and stained with hematoxylin and eosin (H&E) and safranin O / fast green. The sections were observed under a bright-field microscope and given a histological score. Then, immunohistochemistry was used to quantify the expression of type II collagen and Piezo1 protein in the nucleus pulposus.

[0096] 21. RNA-seq for differential gene expression analysis

[0097] Total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The purity and quantification of RNA were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The integrity of RNA was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Then, according to the manufacturer's instructions, a library was constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit. Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. (Shanghai, China). Differential expression analysis was performed using DESeq2. A Q value < 0.05 and a fold change > 1.5 or fold change < 0.5 were used as the thresholds for significantly differentially expressed genes (DEGs). The list of differentially expressed genes was further subjected to gene ontology (GO) and KEGG pathway enrichment analysis. Using the clusterProfiler software package, significantly enriched biological processes and signaling pathways were identified by hypergeometric detection. The Benjamini-Hochberg method was used to adjust the p-values for multiple comparisons.

[0098] 22. Statistical Analysis

[0099] All data were expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 10.1.1 software, and statistical differences between data were analyzed using one-way analysis of variance (ANOVA) and t-tests. P < 0.05 was considered statistically significant. Statistical significance was defined as *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001. P ≥ 0.05 was not significantly different (ns).

[0100] II. Experimental Results and Discussion

[0101] 1. Properties of the Piezoelectric RGD-OSA / HA-ADH Hydrogel

[0102] The present invention developed an injectable hydrogel crosslinked by Schiff base reaction. The hydrogel was composed of RGD peptide-modified oxidized sodium alginate (RGD-OSA) and hydrazine-modified hyaluronic acid (HA-ADH) (as Figure 1 ), including barium titanate (BTO) nanoparticles and bone marrow mesenchymal stem cells (BMSCs). This hydrogel provided an electrically stimulated microenvironment to protect bone marrow mesenchymal stem cells and promote functional recovery. First, hyaluronic acid (HA) was crosslinked with adipic dihydrazide (ADH) to generate HA-ADH, as Figure 2 shown. Fourier transform infrared (FTIR) spectroscopy showed a redshift of the absorption peak from 3420 cm-1 to 3270 cm-1 and a new peak at 1602 cm-1, confirming the formation of amide bonds and the successful amidation of HA ( Figure 2in A). The results of nuclear magnetic resonance (NMR) spectroscopy further verified this conclusion ( Figure 2 in B). The synthesis of RGD-OSA is as Figure 1 shown in A. The oxidation of sodium alginate produced an aldehyde C=O absorption peak at 1734 cm-1, indicating successful oxidation ( Figure 2 in C). Although there was no significant change in the characteristic peaks after crosslinking of RGD peptide with oxidized sodium alginate, two-dimensional correlation spectroscopy (2D-COS) showed obvious hydroxyl signals at 3565 cm-1 and 3140 cm-1, which was due to the introduction of -OH and N-H groups by RGD. In addition, three spin peaks were observed at 1600 cm-1, 1560 cm-1 and 1400 cm-1, corresponding to asymmetric C=O stretching, N-H bending and symmetric C=O stretching vibrations respectively, confirming the successful synthesis of RGD-OSA. The crosslinking process between RGD-OSA and HA-ADH is as Figure 2 shown in H. The infrared characteristics of the crosslinked sample are as Figure 2 shown in D(i), indicating that the amino group on amidated HA reacted with the aldehyde group of oxidized sodium alginate through Schiff base reaction to form a C=N bond ( Figure 2 in D(ii)). The RGD modification of OSA provides specific adhesion sites for cells, significantly improving the bioactivity of the material and making it an effective strategy to promote cell adhesion, proliferation, differentiation and tissue regeneration.

[0103] First, RGD-OSA / HA-ADH hydrogels with different concentrations (1%, 2% and 3%) were prepared, and the gelation time was determined by the vial tilting test method. The results showed that all samples gelled within 30 s, and the gelation rate increased with the increase in the concentration of RGD-OSA or HA-HADH. When injected into the target site, the hydrogel quickly formed a stable structure, preventing local leakage within the intervertebral disc (IVD). The swelling rate of the hydrogel in PBS solution at 37 °C over time was studied, and the results showed that all hydrogels reached equilibrium swelling after about 10 hours. As Figure 2 shown in H, the hydrogel had good injectability. Rheological tests showed that in the amplitude sweep test (0.1 - 300% strain range), the storage modulus (G') remained stable, the loss modulus (G") decreased slightly with the increase in strain, and the loss factor (G' / G") gradually decreased. At all frequencies, G' was always higher than G", and the loss coefficient remained below 0.1, indicating good elastic properties. By adjusting the component ratio, the rheological parameters of 2% RGD-OSA / HA-ADH were matched with the mechanical properties of the IVD nucleus pulposus, confirming its potential as a biomimetic material for nucleus pulposus tissue. Figure 2In (E). Different from traditional covalently crosslinked hydrogels that are prone to wear under external mechanical forces, the RGD-OSA / HA-ADH hydrogel exhibits strong self-healing ability due to multiple dynamic bonds. Strain tests showed that at 500% strain (G‘ < G”), the hydrogel network collapsed, but it quickly returned to its original state at low strain (1%) and maintained this behavior over three cycles, demonstrating superior self-healing ability. Figure 2 In (F). To observe the self-healing process, hydrogels stained with yellow and blue dyes were cut in half and reconnected. As Figure 2 shown in (H), the separated hydrogels transformed into a cohesive whole within 5 min. Rheological analysis showed that the BTO / RGD-OSA / HA-ADH hydrogel had shear-thinning behavior. At low shear rates (~1 s-1), the viscosity was high (~800 Pa·s), while at high shear rates (~6 - 7 s-1), the viscosity dropped to nearly 0 Pa·s. Figure 2 In (G). This shear-dilution property reduces the viscosity during injection, minimizes the tensile stress caused by the fluid, and alleviates the mechanical pressure on cells; combined with electrical stimulation, this helps reduce cell damage and maintain viability. The degradation rate of the hydrogel was evaluated by placing the samples in PBS solution at 37 °C while stirring at 80 rpm. Over time, the breakage of dynamic bonds led to almost complete degradation of all hydrogels within 2 weeks. In summary, the 2% RGD-OSA / HA-ADH hydrogel has good injectability and self-healing properties. Rapid gelation minimizes leakage and improves the success rate of stem cell implantation and colonization, making it a promising candidate for treating IVD degeneration.

[0104] Then, orthorhombic barium titanate nanoparticles (BTO NPs) with piezoelectric properties were synthesized by a hydrothermal method. Scanning electron microscopy (SEM) images showed that the BTO NPs exhibited a nearly tetragonal morphology with a uniform particle size distribution. Figure 2 In (I). Energy-dispersive X-ray (EDX) elemental mapping showed the uniform distribution of Ba, Ti, and O elements with an atomic ratio of 1:1:3. Figure 2 In (I). In the X-ray diffraction (XRD) pattern of the BTO NPs, two split peaks at 2θ = 45° confirmed the characteristic structure of perovskite. Raman spectroscopy further verified the non-centrosymmetric structure caused by tetragonal distortion and the B1 + E (308 cm-1) scattering mode generated by the asymmetric vibration of [TiO 6 octahedra. Figure 2 In (J). Piezoresponse force microscopy (PFM) measurements confirmed the piezoelectric response of the BTO NPs. Figure 2Among K, L, and M). Under the applied voltage bias, the bright and dark regions in the amplitude and phase diagrams correspond, indicating piezoelectricity. The amplitude curve and the rhombus shape of the 180° phase hysteresis loop further demonstrate the local polarization switching behavior under the voltage bias condition. The observation of the ferroelectric hysteresis further confirms the electromechanical conversion ability of the BTO NPs.

[0105] Piezoelectric BTO NPs were added to the RGD-OSA / HA-ADH hydrogel matrix to successfully prepare a piezoelectric hydrogel. Under mechanical stress or other physical stimuli, the hydrogel deforms, stores mechanical energy, and exerts a "squeezing" force on the BTO NPs through mechanical transmission. When the BTO NPs are compressed, the positive and negative charge centers of their crystal structures move relative to each other, generating an internal electric field. This process converts the mechanical energy absorbed by the hydrogel into electrical energy, which then acts on the stem cells in the form of bioelectric stimulation. At 1 Hz, the rheological behaviors of the NP tissue were G' and G", approximately equal to 320 and 85 Pa, respectively. According to the mechanical property analysis, 2% RGD-OSA / HA-ADH hydrogel was selected as the most matched with the mechanical properties of the NP tissue and experiments were carried out. Piezoelectric hydrogels were prepared using different concentrations of BTO NPs, 0.1%, 0.5%, w.5%, and 1%, and were named 0.1% BTO / RGD-OSA / A / HA-ADH, 0.5% BTO / RGD-OSA / HA-ADH, and 1% BTO / RGD-OSA / HA-ADH, respectively. The scanning electron microscopy observation results showed that the surface of the RGD-OSA / HA-ADH hydrogel was smooth and BTO NPs were added, introducing nanoparticle characteristics. With the increase in the concentration of BTO NPs, the surface roughness of the hydrogel also increased, while its porosity decreased ( Figure 2 Among O). X-ray diffraction (XRD) analysis further confirmed the presence of BTO NPs. As shown in Figure S7, in the XRD spectrum of the BTO np-bound hydrogel, characteristic peaks were observed at 2θ = 31.5°, 38.5°, and 45.4°. It is worth noting that the split peak at ~45.4° corresponds to the tetragonal phase of the BTO NPs, indicating their ferroelectric and piezoelectric properties. With the increase in the BTO NP content, the intensity of the diffraction peak also increased, and the SEM analysis further confirmed the uniform distribution of the nanoparticles in the hydrogel and their piezoelectric effect ( Figure 2Under continuous ultrasonic stimulation, the piezoelectric hydrogel exhibits typical sinusoidal electrical signal output, and the output increases with the response to higher ultrasonic power. This indicates that the abnormal stress experienced by cells during injection can be converted into mechanically triggered piezoelectric potential, and the output voltage corresponds precisely to the applied mechanical stress, providing accurate and timely electrical stimulation to cells. The injection simulation experiment evaluated the protective effect of hydrogels with different BTO NP concentrations on stem cells. The results showed that the 0.5% (w / v) bto-doped RGD-OSA / HA-ADH hydrogel had the most effective cell protection. Based on these findings, subsequent experiments will explore the mechanism by which the 0.5% (w / v) BTO / RGD-OSA / HA-ADH hydrogel protects stem cells and helps restore their function.

[0106] 2. Mechanical protection against shear force to protect cell viability

[0107] First, live / dead cell staining was used to evaluate the viability of different bone marrow mesenchymal stem cells under drug delivery conditions, and cell damage was analyzed in real time, with a focus on the damage suffered when passing through a 26G needle at a flow rate of 1000 μL / min. The results showed that in the traditional delivery system, using cell culture medium as the injection medium would cause more than 20% of the cells to be damaged or die during injection. In contrast, when bone marrow mesenchymal stem cells were encapsulated in a conventional hydrogel, cell viability increased, and 90% of the cells remained viable immediately after injection. Notably, when bone marrow mesenchymal stem cells were embedded in the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel, cell viability further increased to over 95%, and the cells maintained a uniform three-dimensional (3D) distribution after injection ( Figure 3 in Figures A and B). These results indicate that the shear stress and fluid-induced tensile force of the traditional liquid carrier can deform or damage cells during injection. In contrast, the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel can effectively relieve the abnormal damage induced by cell stress, activate the endogenous repair mechanism of cells, and enhance their resistance to shear stress. After injection, the viscosity of the hydrogel increases rapidly, maintaining the three-dimensional distribution of cells and further improving cell viability and functional performance. A new friction force model was used to simulate the abnormal stress during injection, and the mechanical protection of the piezoelectric hydrogel under more extreme conditions was tested. Bidirectional sliding was performed between glass plates to generate lateral shear force. After 30 s of bidirectional friction at a speed of 5 mm / s, more than 25% of the bone marrow mesenchymal stem cells in the conventional delivery medium were damaged, while more than 95% of the cells in the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel survived. These findings further demonstrate that the piezoelectric hydrogel can effectively protect cells from high shear force during transportation, thereby improving the therapeutic efficiency of bone marrow mesenchymal stem cells.

[0108] We also investigated the relationship between intracellular calcium ion levels and cell viability. The concentration of free calcium ions in bone marrow mesenchymal stem cells was measured using the fluorescent dye Fluo-4, AM to evaluate the effect of different delivery media on intracellular calcium levels. It was found that when the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel was used as the stem cell delivery medium, the concentration of free calcium ions in bone marrow mesenchymal stem cells increased significantly. However, after using the piezoelectric channel inhibitor GsMTx4, the intracellular calcium ion concentration decreased significantly ( Figure 3 in C and E). Flow cytometry further confirmed this finding ( Figure 3 in D). To monitor plasma membrane damage during injection, we used the lipid bilayer-binding fluorescent dye FM 1-43 to observe membrane integrity. The results showed that the increased stress during conventional injection led to enhanced FM 1-43 uptake and increased intracellular fluorescence, indicating cell membrane damage. In contrast, when the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel was used as the delivery medium, the degree of membrane damage was significantly reduced ( Figure 3 in F and G).

[0109] In summary, the results of this study indicate that the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel, as a stem cell delivery medium, can effectively convert abnormal stress during injection into a "trigger signal" for endogenous cell repair. When cells experience shear stress and fluid-induced tensile forces, this stress directly acts on the hydrogel and rapidly induces the piezoelectric effect, providing immediate electrical stimulation to activate piezoelectric channels. The activation of piezoelectric channels subsequently triggers a rapid increase in the intracellular free calcium ion concentration. Calcium ions act on various calcium sensors, including the TRPML1 channel, synaptotagmin-7 (Syt-7), dysferlin, and apoptosis-linked gene-2 (alg-2), thereby promoting rapid membrane repair at the damaged site through mechanisms such as endocytic removal, lipid patching, and large vesicle shedding ( Figure 3In the endocytosis removal model, calcium ions promote the release of acid sphingomyelinase, which catalyzes the production of ceramide. Ceramide promotes the elimination of damaged membrane regions through endocytosis. In the lipid patch model, calcium influx activates the lysosomal transient receptor potential mucolipin 1 (TRPML1) membrane channel, triggering lysosomal remodeling of the local lipid membrane by secreting acid sphingomyelinase. The endocytosis removal and lipid patch models are mainly considered as mechanisms to address moderate membrane damage. In the large vesicle shedding model, the damaged membrane forms outer vesicles at the time of injury. This process is driven by a calcium ion surge, enabling alg2 to accumulate at the injury site. The accumulation of alg2 promotes the assembly of apoptosis-related gene 2 interacting protein X (ALIX) and ESCRT-III, which is crucial for membrane repair, especially in membrane breakage and vesicle shedding. ESCRT-mediated cleavage of severely damaged regions is the final line of defense for plasma membrane repair. Depending on the cell type, injury size, and nature of the injury, one or more of these repair mechanisms may be recruited. The "endocytosis removal", "lipid patch", and "large vesicle shedding" models coordinately regulate plasma membrane repair, and all three processes strictly depend on Ca 2+ as the central sensor.

[0110] 3. Mechanism Study on Piezoelectric Stimulation Enhancing Cell Resistance

[0111] Figure 4 Part A in [X] shows the changes in nuclear-peripheral f-actin aggregation after calcium ion concentration fluctuations and its effect on cell mechanical strength. To further investigate the effect of piezoelectric stimulation on the cytoskeleton, we activated the piezoelectric material using low-intensity ultrasound and then generated electrical signals. Fluorescence imaging technology was used to observe the state of f-actin in bone marrow mesenchymal stem cells at different time points ( Figure 4 Part B in [X]). The results showed that after piezoelectric stimulation, f-actin filaments stained with phalloidin rapidly accumulated in the perinuclear region, with a wider distribution than in unstimulated cells. In addition to the transient accumulation of actin in the perinuclear region, obvious peripheral structures formed near the cell nucleus under the action of local mechanical forces. The perinuclear f-actin aggregation began to decrease after 5 min and returned to the baseline level after 30 min. However, no similar f-actin accumulation was observed in cells treated with the piezoelectric channel inhibitor GsMTx4. To further evaluate the effect of the piezoelectric hydrogel on cell mechanical properties, we monitored the dynamic changes in cell mechanics under piezoelectric stimulation using atomic force microscopy (AFM) ( Figure 4Parts C, D, and E). Young's modulus is a key parameter of the mechanical properties of mammalian cells. It reflects the stiffness and elasticity of cells and is closely related to the physiological state. By fitting the cone-sphere model to the force-distance curve, the Young's modulus of the cells was calculated. Experiments showed that after applying the electrical signals generated by low-intensity ultrasound to the cells, the Young's modulus in the perinuclear region increased by 50% (from 3.22 ± 0.56 kPa to 5.68 ± 0.65 kPa). This phenomenon indicates that electrical stimulation increases the stiffness of the cells, thereby enhancing the cells' ability to resist external mechanical stress. Over time, the f-actin filaments gradually depolymerize and return to the initial level after 30 minutes.

[0112] The cytoskeleton is a fibrous scaffold structure widely distributed within cells. It ensures the integrity of the structure and the stability of the function and plays a key role in determining the mechanical properties of cells. The f-actin network, as a major cytoskeletal component, participates in the cell's response to external stimuli through dynamic rearrangement and indirectly regulates the mechanical properties of cells. In a high stress-transfer environment, this increased stiffness helps reduce cell deformation, thereby minimizing the rupture or leakage of the plasma membrane caused by mechanical damage. This enhances the integrity of the structure and reduces the risk of damage. Calcium ion-mediated actin reorganization (CaAR) links Ca 2+ signal transduction, cell mechanics, and actin dynamics. CaAR enables cells to rapidly adjust their mechanical properties to adapt to changes in pressure, thereby enhancing their resistance to damage induced by exogenous stress. This process helps cells maintain homeostasis in a high-stress environment and improves their ability to adapt to external damage stimuli.

[0113] To clarify the protective mechanism of BTO / RGD-OSA / HA-ADH piezoelectric hydrogels on bone marrow mesenchymal stem cells and the possible molecular pathways involved, we performed RNA-seq analysis on the delivered cells using RGD-OSA / HA-ADH (normal group) and BTO / RGD-OSA / HA-ADH (piezoelectric group) hydrogels. By comparing the gene expression changes between the two groups, differentially expressed genes (DEGs) were identified according to fold change > 1.5 and adjusted p-value < 0.05. We generated a volcano plot to visually represent the trend of gene expression changes ( Figure 5 Part A in). The x-axis of the volcano plot represents the fold change in gene expression (log2 fold change), and the y-axis represents the significance of gene expression (-log10 adjusted p-value). Red dots in the figure represent significantly upregulated genes, and blue dots represent significantly downregulated genes. We generated a heatmap ( Figure 5Part B) to display the expression levels of deg in the whole sample. The color gradient in the heatmap, from blue (low expression) to red (high expression), indicates the differences in gene expression under different treatment conditions. We also created a radar chart to compare the expression differences of several key genes between the piezoelectric group and the normal group ( Figure 5 Part C).

[0114] Gene Ontology (GO) enrichment analysis is often used to explore enrichments in the BP (biological process), CC (cellular component), and MF (molecular function) categories. Figure 5 Part D shows the results of the graphene oxide enrichment analysis of the top 15 graphene oxide terms, revealing the enrichment of deg in different graphene oxide categories. Some graphene oxide terms are related to calcium ions in the BP category, such as "calcium entry for storage operation", indicating that DEGs are involved in calcium signal transduction and homeostasis regulation. In the CC category, multiple genes are enriched in specific cellular components, such as "voltage-gated calcium channel complex" and "L-type voltage-gated calcium channel complex". These genes are closely related to the assembly of calcium ion channels and their localization in specific membrane structures. In the MF category, the enriched genes focus on regulating molecular functions, especially "voltage-gated calcium channel activity" and "calmodulin binding", indicating that these genes play a key role in regulating calcium channel function and calmodulin binding. Deg has important functions in calcium signal transduction and transmembrane transport, especially in regulating voltage-gated calcium channel complexes and related molecular functions. This indicates that electrical stimulation changes the membrane potential (depolarization), which in turn activates voltage-gated calcium channels (VGCCs), allowing calcium ions to flow into the cell from the extracellular environment and rapidly increasing the intracellular calcium level.

[0115] Figure 5 The chord diagram in Part E illustrates the relationship between the upregulated deg and various GO terms, indicating that these genes are involved in specific biological processes or molecular functions. For example, the CAMK1 gene is closely related to the "calmodulin binding" function and the "voltage-gated calcium channel complex". Figure 5 Part F shows the top 10 GO terms of the enrichment scores of the upregulated DEGs, including "calcium ion transport" and "voltage-gated calcium channel activity", further emphasizing the core role of calcium signaling in regulating cell functions. Figure 5Part G of the figure shows the KEGG pathway enrichment analysis results of up-regulated DEGs, evaluating their enrichment in cellular signaling pathways. The bubble plot lists the top 10 significantly enriched KEGG pathways, among which the "calcium signaling pathway", "MAPK signaling pathway" and "cAMP signaling pathway" are the most significantly enriched. This indicates that calcium signaling, as a second messenger, plays a key role in activating various cellular functions by activating members of the MAPK family (ERK, JNK, p38 MAPK), regulating gene expression through phosphorylation of specific transcription factors, and participating in membrane repair, cell survival and stress responses. In addition, it promotes cytoskeletal reorganization and facilitates vesicle trafficking and fusion, which helps repair damaged cell membranes. The MAPK pathway also interacts with other signaling pathways (such as PI3K-Akt and PKC) to form a complex signaling network that jointly regulates the membrane repair process and ensures rapid recovery of cell function after injury. In addition, activation of the cAMP signaling pathway regulates energy metabolism and intracellular signal transduction. Up-regulation of the mTOR signaling pathway may indicate enhanced cell growth, metabolic activity and autophagy, thereby protecting bone marrow mesenchymal stem cells from apoptosis. Up-regulation of the Wnt signaling pathway may also be related to the promotion of the differentiation of bone marrow mesenchymal stem cells into the nucleus pulposus lineage by piezoelectric stimulation.

[0116] The calcium ion-mediated plasma membrane repair mechanism is ubiquitous in all types of cells. This study found that electrical stimulation increased the intracellular free calcium ion concentration, triggering the intrinsic plasma membrane repair and Ca 2+ -triggered actin remodeling (CaAR) injury adaptation mechanism. This universal and effective mechanism makes it applicable to any cell type. It can also be activated during the transfer of adipose-derived stem cells, embryonic stem cells and organoids. 4. Piezoelectric hydrogels promote the proliferation, migration and nucleus pulposus differentiation of bone marrow mesenchymal stem cells in vitro

[0117] Low-intensity ultrasound activates piezoelectric materials to generate electrical signals, simulating piezoelectric stimulation of intervertebral disc tissue under load conditions. We observed the effects of these electrical signals on stem cell behavior, especially in terms of proliferation, migration and differentiation, to evaluate their potential to promote the regeneration and repair of the nucleus pulposus and other related tissues. This process helps to explore the application of piezoelectric signals in regulating cell behavior, as Figure 6 shown in Part A of the figure. This study first used EdU staining to evaluate cell proliferation in different treatment groups ( Figure 6 Part B of the figure). The results showed that the piezoelectric + US group exhibited the strongest fluorescence signal in the EdU staining assay, indicating increased DNA synthesis under piezoelectric and ultrasonic stimulation. The cell proliferation rate of the piezoelectric + US group was higher than that of other groups, indicating that piezoelectric stimulation can effectively promote cell proliferation. Figure 6Section C compared the cell viability under different treatment conditions, including non-piezoelectric, piezoelectric, non-piezoelectric + ultrasound, and piezoelectric + ultrasound groups. The results showed that the cell density in the piezoelectric + US group was higher than that in other groups, suggesting that the combined action of piezoelectric stimulation and ultrasound could improve cell proliferation and viability. In contrast, the cell viability in the non-piezoelectric group was less but lower. Over time, the piezoelectric + ultrasound group consistently showed the highest cell survival rate, and the number of live cells in all groups increased( Figure 6 Sections C and F). In the cell migration experiment, bone marrow mesenchymal stem cells were seeded in glass-bottom culture dishes. Once the cells were confluent, a scratch was made in the cell layer with a pipette to create an in vitro scratch model. Piezoelectric hydrogels and non-piezoelectric hydrogels were used to fill the scratched area. Fluorescent images of cell distribution at 0 h and 24 h after treatment are shown in Figure 6 Sections D and E. The results showed that the migrated bone marrow mesenchymal stem cells covered most of the scratched area, while there were still obvious gaps in the scratched areas of other groups. This indicated that the piezoelectric charge stimulation accelerated the migration of stem cells.

[0118] Studies have shown that electrical stimulation can effectively promote the generation of chondrocyte extracellular matrix (ECM) by inducing stem cell migration and enhancing the release of TGF-β1. TGF-β1 promotes the proliferation of nucleus pulposus cells and the production of ECM. The two main proteins in the nucleus pulposus matrix are glycosaminoglycans (GAGs) and type II collagen, which play key roles in the nucleus pulposus tissue. GAGs help the tissue resist compressive loads and participate in cell signaling, while type II collagen provides elasticity and mechanical strength to the nucleus pulposus. As shown in Figure 6 Section G, Alcian blue staining showed that the highest amount of GAGs was synthesized by implanting bone marrow mesenchymal stem cells into the piezoelectric hydrogel and activating the electrical stimulation with ultrasound. Figure 6 Sections H and I showed the results of immunofluorescent staining of type II collagen among different groups, and the level of type II collagen synthesized in the piezoelectric + US combination was the highest. The gene and protein expression levels of type II collagen (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9) reflect the matrix production function and are usually used as markers for nucleus pulposus cells (NPCs) and chondrocytes.

[0119] To evaluate the ability of bone marrow mesenchymal stem cells to differentiate into NPCs, we analyzed the key marker genes for nucleus pulposus formation, including COL2A1, ACAN, and SOX9. As shown in Figure 6As shown in part J of the figure, after 7 days of electrical stimulation, the expression levels of COL2A1, ACAN, and SOX9 in the Piezo+US group were higher than those in other groups, increasing by 3 - 6 times. In contrast, other treatment groups (including non-Piezo groups with or without ultrasound and Piezo groups without ultrasound) showed only partial upregulation of individual genes (SOX9, ACAN, or COL2A1), but did not match the Piezo+US group in the expression of all three genes. Although mechanical stimulation or ultrasound activation enhanced the ability of bone marrow mesenchymal stem cells to differentiate into NPCs to some extent, their effects were far less significant than those of Piezo hydrogel stimulation.

[0120] The Piezo+US group enhanced plasma membrane repair by increasing intracellular free Ca 2+ concentration and activating the MAPK and Wnt / β-catenin signaling pathways, and had a better differentiation efficiency towards NPCs and ECM expression compared to the control groups lacking Piezoelectric effect or ultrasound activation. These results indicate that under ultrasound activation conditions, the local piezoelectric charges generated by the BTO / RGD-OSA / HA-ADH piezoelectric hydrogel play a key role in inducing the differentiation of bone marrow mesenchymal stem cells into NPCs. Recent studies have shown that the piezoelectric effect enhances the differentiation potential of bone marrow mesenchymal stem cells and accelerates the regeneration of the nucleus pulposus tissue by regulating multiple intracellular signaling pathways and matrix synthesis processes.

[0121] 5. Therapeutic effect in vivo

[0122] To further investigate the efficacy of stem cell piezoelectric injection hydrogel in promoting nucleus pulposus regeneration, a rat intervertebral disc degeneration model ( Figure 7 part A in the figure) was used; the Co 7 / 8, Co 8 / 9, and Co 9 / 10 intervertebral discs were punctured with an 18G needle to establish an intervertebral disc degeneration model. Subsequently, 10 μL of different treatment components were injected with a 26G syringe: 10 μL of PBS was injected into the Co 7 / 8 group (puncture), 10 μL of DMEM / F12 + bone marrow mesenchymal stem cells was injected into the Co 8 / 9 group, 10 μL of RGD-OSA / HA-A-ADH hydrogel + bone marrow mesenchymal stem cells was injected into another Co 8 / 9 group, and 10 μL of BTO / RGD / OSA / HA-ADH hydrogel + bone marrow mesenchymal stem cells was injected into the Co 9 / 10 group. The healthy Co 6 / 7 disc was used as a sham control group. The IVIS Lumina imaging system was used to visualize and detect the survival of iodinated bone marrow mesenchymal stem cells transplanted into the intervertebral disc. The results showed that compared with other groups, the DiR iodination signal attenuation in the piezoelectric hydrogel stem cell group was the slowest, and detectable signals were still present 4 weeks after surgery ( Figure 7 part E in the figure). In contrast, the signals in the group with only moderately bone marrow mesenchymal stem cells almost completely disappeared at 2 weeks. The disc height index (DHI) reflects the change in the intervertebral disc space and was evaluated using imaging techniques at 4 weeks and 8 weeks after surgery, such asFigure 7 as shown in part A. X-ray imaging showed that the DHI of the piezoelectric injection hydrogel stem cell group was closest to that of the Sham control group ( Figure 7 in parts B and F). At 8 weeks postoperatively, the piezoelectric hydrogel stem cell group maintained the highest DHI value, while the disc spaces of the other treatment groups rapidly collapsed. Since the intervertebral disc contains 70-80% water, we used MRI to measure the change in the water content of the intervertebral disc and evaluate the degree of degeneration. The results showed that although the disc signal intensity decreased in all treatment groups at 4 and 8 weeks postoperatively, the water content in the BTO / RGD-OSA / HA-ADH group remained the highest ( Figure 7 in parts C, D, and G). These results indicate that the combination of piezoelectric hydrogel and electroprotection strategy can effectively improve the survival and regeneration potential of stem cell delivery, thereby promoting nucleus pulposus regeneration, maintaining the integrity of the intervertebral disc structure, and reducing water loss.

[0123] Histological analysis of the rat intervertebral disc was performed at 4 and 8 weeks postoperatively. Hematoxylin and eosin (H&E) staining showed that in the piezoelectric hydrogel stem cell group, the intervertebral disc structure remained intact, and the boundary between the nucleus pulposus (NP) and the annulus fibrosus (AF) was still clearly visible at 4 and 8 weeks ( Figure 8 in parts A and D). In addition, the NP area in this group was the largest, while the AF-NP boundary in the other treatment groups was significantly disrupted. Safranin O / Fast Green staining reflected proteoglycan expression ( Figure 8 in parts B and E), and although the staining intensity decreased at 8 weeks postoperatively, the piezoelectric hydrogel stem cell group maintained the highest level of type II collagen at each time point ( Figure 8 in parts C, F, and I).

[0124] Based on the previously described histological scoring criteria, we evaluated the tissue using a five-category degeneration scoring system, with higher scores indicating more severe degeneration. The results showed that the histological scores of the piezoelectric hydrogel stem cell group were lower than those of the other treatment groups at 4 and 8 weeks postoperatively ( Figure 8In parts G and H, it was shown that piezoelectric hydrogel stem cell therapy effectively delayed intervertebral disc degeneration. Immunohistochemistry further confirmed that at 8 weeks after surgery, the piezoelectric hydrogel stem cell group showed the most significant expression in Piezo1, approaching the level of the Sham control group. This indicates that the piezoelectric hydrogel in degenerated intervertebral disc tissue can generate piezoelectric signals in response to mechanical loading, thereby upregulating the expression of Piezo1. This enhances the cell's adaptation to mechanical stress by reducing the damage and degradation of NP under excessive pressure. In addition, electrical stimulation can not only protect stem cells from shear stress damage during injection but also activate piezoelectric ceramics, which reduce apoptosis and oxidative stress by regulating the expression of anti-apoptotic factors (Bcl-2 family proteins) and antioxidant pathways (Nrf2 pathway). This process increases the survival rate of stem cells in a harsh environment, thereby slowing down intervertebral disc degeneration.

[0125] This study observed the protective effect of piezoelectric BTO / RGD-OSA / HA-ADH injectable hydrogel on bone marrow mesenchymal stem cells and focused on exploring the basic mechanism of electrical stimulation-induced membrane repair. In the future, it is necessary to study the clinical application potential of this method, with a focus on long-term observation, large animal / human trials, and improvement of the drug delivery method. Specifically, the long-term effects of electrical stimulation on different cell types must be evaluated, the delivery system for clinical application optimized, the treatment outcomes observed in large animal models, and in vivo studies conducted to evaluate the safety and effectiveness of the proposed treatment strategy. In addition, further exploration is needed in new disease contexts, such as bone defects, osteoarthritis, and chronic wounds, as well as in other situations where mechanical energy can be converted into electrical energy, thus expanding the applicability of this treatment system.

[0126] III. Conclusion

[0127] The present invention proposes an "electrical protection" strategy adopted during stem cell delivery, aiming to improve the delivery efficiency by initiating endogenous membrane repair and enhancing the resistance of stem cells to exogenous stress. The BTO / RGD-OSA / HA-ADH piezoelectric injectable hydrogel was developed to deliver bone marrow mesenchymal stem cells by providing electrical stimulation to mechanical stress. During the delivery of bone marrow mesenchymal stem cells, piezoelectric stimulation activates piezoelectric channels, rapidly increasing the intracellular free Ca 2+ concentration. This triggers mechanisms such as "lipid patch repair", "endocytic clearance", and "Merkel vesicle shedding" to promote the rapid repair of damaged sites on the plasma membrane. Calcium influx can also induce the production of Ca 2+The formation of the perinuclear actin ring that is dependent changes the mechanical strength of cells and enhances the resistance of stem cells to abnormal stress. In degenerated IVD tissues, the voltage generated by continuous mechanical loading can activate the MAPK and Wnt / β-catenin signaling pathways, promoting the proliferation of bone marrow mesenchymal stem cells and their differentiation into nucleus pulposus cells. Both in vitro and in vivo experiments have shown that the "electroprotection" strategy effectively improves the survival rate of stem cell delivery, prolongs the in vivo cell retention time, and enhances the results of tissue repair. In summary, bioelectric materials provide a necessary protective effect during stem cell delivery and continuously promote the proliferation and differentiation of stem cells in vivo, highlighting their application potential in tissue regeneration.

Claims

1. A method for preparing a piezoelectric hydrogel for improving the survival rate of stem cell delivery, characterized in that: The following steps are involved: (1) reacting sodium alginate with sodium periodate to obtain oxidized sodium alginate, which is then grafted with an RGD peptide solution, and then freeze-dried to obtain RGD peptide-modified oxidized sodium alginate; (2) activating the carboxyl group of hyaluronic acid with EDC and NHS, then adding adipic acid hydrazide to the mixture, stirring and reacting, dialyzing the obtained product and freeze-drying it to prepare hyaluronic acid-adipic acid hydrazide; (3) The RGD peptide-modified oxidized sodium alginate obtained in step (1) and the hyaluronic acid-adipic dihydrazide obtained in step (2) are mixed and cross-linked to prepare a hydrogel precursor solution, and barium titanate nanoparticles are added to the hydrogel precursor solution to prepare a piezoelectric hydrogel.

2. The method according to claim 1, characterized in that: In step (1), the molar ratio of sodium alginate to sodium periodate is 1:1-3; sodium alginate and sodium periodate are dissolved in deionized water respectively, and stirred for reaction at 20-25° C. in the dark for 4 hours to obtain the oxidized sodium alginate.

3. The method according to claim 1, characterized in that The concentration of the RGD peptide in step (1) is 1 mg / mL, and the weight ratio of the oxidized sodium alginate to the RGD peptide is 25:

1.

4. The method according to claim 1, characterized in that: The molar ratio of hyaluronic acid, EDC and NHS in step (2) is 1:1.2:1.

2.

5. The method according to claim 1, characterized in that The molar ratio of hyaluronic acid to adipic acid dihydrazide in step (2) is 1:

10.

6. The method according to claim 1, characterized in that The molecular weight cutoff of the dialysis in step (2) is 12-14 kDa.

7. The method according to claim 1, characterized in that The concentration of the hydrogel precursor solution in step (3) is 0.1-1% w / v.

8. Piezoelectric hydrogel for improving the survival rate of stem cell delivery prepared by the method according to any one of claims 1 to 7.

9. Use of the piezoelectric hydrogel for improving the survival rate of stem cell delivery as described in claim 8 in the preparation of a drug for treating intervertebral disc degeneration.

10. The use according to claim 9, characterized in that: The piezoelectric hydrogel is loaded with bone marrow mesenchymal stem cells and prepared into an injectable solution as an injectable therapeutic drug for intervertebral disc degeneration.

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