An electrically stimulated LDH hydrogel, and a preparation method and application thereof

Abstract

The application relates to the technical field of biological materials, and discloses an electrically stimulated LDH hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving a zinc salt, a vanadium salt and urea in water under a nitrogen atmosphere to obtain a precursor solution; performing a hydrothermal reaction on the precursor solution to obtain ZnV-LDH; gradually adding methacrylic anhydride into a gelatin solution under stirring, heating and reacting; adding preheated water into the reaction solution to stop the reaction; performing dialysis purification, pre-freezing and freeze-drying on the reaction solution to obtain a hydrogel; and adding the ZnV-LDH into the hydrogel, ultrasonic mixing, and obtaining the electrically stimulated LDH hydrogel. Through the synergistic effect of the ZnV-LDH and the hydrogel, the electrically stimulated LDH hydrogel has electric activity, can respond to external stimulation to generate a potential change, can generate a concentration-dependent electric response under ultrasonic stimulation, and the response has periodicity and stability, so that the osteogenic differentiation and the cell mechanical properties can be promoted to the maximum extent.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, specifically to an electrically stimulated LDH hydrogel, its preparation method, and its application. Background Technology

[0002] Osteosarcoma is a highly aggressive malignant bone tumor that primarily occurs in children and adolescents; approximately 26,000 new cases are reported globally each year, with a high mortality rate. Recurrence and metastasis of osteosarcoma result in a 5-year overall survival rate of less than 20%, making treatment particularly challenging in clinical settings. Current treatments for osteosarcoma typically involve pre- and post-operative chemotherapy drugs such as methotrexate and cisplatin, which, while inhibiting tumor progression, suffer from significant side effects and a high risk of drug resistance. Post-operative repair often relies on materials such as bone cement, which have limitations in biocompatibility and osseointegration. Therefore, there is an urgent need to develop a drug that can modulate the post-operative inflammatory microenvironment, exert multimodal anti-cancer effects, and promote the regeneration of tumor-induced bone defects.

[0003] Hydrogels, as novel implants, can serve as delivery platforms for various substances, including small molecule drugs, biomacromolecules, and nanoparticles, and are used for the delivery of drugs to osteosarcoma. However, the low sensitivity of hydrogels to the tumor microenvironment limits the precise regulation of therapeutic drug release behavior, thus restricting the availability of hydrogel-based delivery systems for anti-tumor therapy. By rationally designing hydrogel networks to achieve precise release of therapeutic agents in response to tumor microenvironment stimuli, local drug concentrations can be increased and systemic side effects reduced. Furthermore, by endowing hydrogel implants with injectability and self-healing capabilities, they can fill and conform to complex bone defects, promoting bone defect repair.

[0004] Layered bimetallic hydroxides (LDHs), as typical layered nanomaterials, possess excellent biocompatibility. Their chemical composition and physical structure are tunable, demonstrating great potential in tumor diagnosis, cancer treatment, drug delivery, and tissue engineering. However, despite the promising prospects of LDH-based materials in bone regeneration and tumor therapy, research on their synergistic effects in osteosarcoma treatment and bone repair is relatively scarce. Furthermore, there is still significant room for improvement in the synergistic anti-tumor effects and osteogenic properties of LDH materials.

[0005] Based on this, the development of an LDH composite hydrogel material that combines significant osteosarcoma treatment efficacy with good osteogenic properties is of great significance for preventing postoperative recurrence of osteosarcoma and achieving bone reconstruction. Summary of the Invention

[0006] This application provides an electrically stimulated LDH hydrogel, its preparation method, and its application, aiming to solve the technical problems of poor treatment efficacy and osteogenic properties of existing osteosarcoma treatment drugs.

[0007] To achieve the above objectives, the present application adopts the following technical solution.

[0008] A first aspect of this application provides a method for preparing an electrically stimulated LDH hydrogel, comprising:

[0009] S1, under a nitrogen atmosphere, zinc salt, vanadium salt and urea are dissolved in water to obtain a precursor solution; the precursor solution is subjected to a hydrothermal reaction to obtain ZnV-LDH;

[0010] S2, under stirring, methacrylic anhydride was gradually added dropwise to the gelatin solution, and the reaction was carried out by heating; preheated hot water was added to the reaction solution to stop the reaction; the reaction solution was purified by dialysis, pre-frozen, and then freeze-dried to obtain a hydrogel;

[0011] S3. Add ZnV-LDH to the hydrogel and mix by ultrasound to obtain an electrically stimulated LDH hydrogel.

[0012] Preferably, the zinc salt includes any one of zinc nitrate, zinc sulfate, or zinc chloride;

[0013] The vanadium salt includes any one of vanadium nitrate, vanadium sulfate, or vanadium chloride.

[0014] The total concentration of zinc salt and vanadium salt in the precursor solution is 30-125 mmol / L; the concentration of urea is 40-80 mmol / L.

[0015] The molar ratio of zinc salt to vanadium salt is 3:(1~3).

[0016] Preferably, the hydrothermal reaction temperature is 60~150 ℃ and the reaction time is 12~48 h.

[0017] Preferably, the gelatin solution is an aqueous solution of gelatin or a PBS solution of gelatin;

[0018] The concentration of the gelatin solution is 0.05~0.15g / mL.

[0019] Preferably, the mass of the methacrylic anhydride is 0.6 to 1.2 times the mass of the gelatin.

[0020] Preferably, the reaction temperature in S2 is 45~65°C, the reaction time is 3~6 hours, and the pH of the reaction is 7.5~9.5.

[0021] Preferably, the reaction is stopped by adding preheated water to the reaction solution, wherein the temperature of the preheated water is 40~50°C and the volume of the water is 4~6 times the volume of the reaction solution;

[0022] The dialysis purification time is 3-7 days;

[0023] The pre-freezing temperature is -70 ~ -90℃.

[0024] Preferably, the mass of the ZnV-LDH is 1 to 4 wt% of the mass of the hydrogel.

[0025] A second aspect of this application provides an electrically stimulated LDH hydrogel prepared by the above-described preparation method.

[0026] A third aspect of this application provides the use of the above-mentioned electrically stimulated LDH hydrogel in the preparation of pharmaceuticals for the treatment of osteosarcoma, pharmaceuticals for the prevention and treatment of postoperative recurrence of osteosarcoma, or pharmaceuticals for the repair of bone defects.

[0027] Compared with the prior art, the beneficial effects of this application are as follows:

[0028] In the electrostimulated LDH hydrogel prepared in this application, ZnV-LDH acts as a sonosensitive agent, exhibiting excellent sonodynamic activity under ultrasonic stimulation, generating a large number of superoxide anions and hydroxyl radicals, thus possessing anti-osteosarcoma properties. The hydrogel, as a stable carrier of ZnV-LDH, endows the system with electroactivity, and can generate a concentration-dependent, periodic, and stable electrical response in response to ultrasonic stimulation. Through the synergistic effect of ZnV-LDH and hydrogel, the electrostimulated LDH hydrogel of this application is electroactive and can generate potential changes in response to external stimuli. Under ultrasonic stimulation, it can generate a concentration-dependent electrical response, and this response is periodic and stable, which can maximize the promotion of osteogenic differentiation and cell mechanical properties, achieving the dual function of "anti-osteosarcoma + bone repair".

[0029] The electrostimulated LDH hydrogel prepared in this application can be used as a medicine for treating osteosarcoma, preventing osteosarcoma recurrence and bone remodeling, and the medicine has excellent therapeutic effect on osteosarcoma. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The image shows the results of testing superoxide anions with the DHR-123 probe in ZnV-LDH of Example 1.

[0032] Figure 2 This is a graph showing the results of testing hydroxyl radicals using a TA probe on ZnV-LDH in Example 1;

[0033] Figure 3The images show the SEM and mapping of the ZnV-LDH hydrogel from Example 1.

[0034] Figure 4 The image shows the results of ultrasonic rheological testing of the ZnV-LDH hydrogel in Example 1.

[0035] Figure 5 The figure shows the tensile and compression test results of the ZnV-LDH hydrogel in Example 1;

[0036] Figure 6 The results of strain scanning test and gel yield strain test of ZnV-LDH hydrogel in Example 1 are shown in the figure.

[0037] Figure 7 The image shows the immunofluorescence staining results of osteocalcin OCN in the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0038] Figure 8 The graph shows the results of the osteogenic transcription factor RUX2 assay in the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0039] Figure 9 Figure 1 shows the results of type I collagen staining test on the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0040] Figure 10 The image shows the results of cell bone morphogenesis protein staining test of the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0041] Figure 11 Figure 1 shows the results of Vinculin staining test for the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0042] Figure 12 The image shows the results of cellular alkaline phosphatase (ALP) staining of the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0043] Figure 13 The image shows the results of Alizarin Red staining of cells in the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1.

[0044] Figure 14 The image shows the results of β-Catein staining of cells in the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1. Detailed Implementation

[0045] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0046] In the following description of this embodiment, the terms "including", "comprising", "having", and "containing" are all open-ended terms, meaning that they include but are not limited to.

[0047] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0048] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0049] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0050] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0051] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood as each intermediate value between the upper and lower limits of the specifically disclosed range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] A first aspect of this application provides a method for preparing an electrically stimulated LDH hydrogel, comprising:

[0054] S1, under a nitrogen atmosphere, zinc salt, vanadium salt and urea are dissolved in water to obtain a precursor solution; the precursor solution is subjected to a hydrothermal reaction to obtain ZnV-LDH;

[0055] In this application, the zinc salt is selected from any one of zinc nitrate, zinc sulfate, or zinc chloride; the vanadium salt is selected from any one of vanadium nitrate, vanadium sulfate, or vanadium chloride.

[0056] In the precursor solution, the total concentration of zinc salt and vanadium salt is preferably 30~125 mmol / L, more preferably 40 mmol / L; wherein the molar ratio of zinc salt and vanadium salt is preferably 3:(1~3), more preferably 3:1; and the concentration of urea is 40~80 mmol / L.

[0057] The preferred reaction temperature for the hydrothermal reaction is 60~150 ℃, more preferably 120 ℃; the preferred reaction time is 12~48 h, more preferably 12 h.

[0058] S2, under stirring, methacrylic anhydride was gradually added dropwise to the gelatin solution, and the reaction was carried out by heating; preheated hot water was added to the reaction solution to stop the reaction; the reaction solution was purified by dialysis, pre-frozen, and then freeze-dried to obtain a hydrogel;

[0059] In this application, the gelatin solution is an aqueous solution of gelatin or a PBS solution of gelatin, and the concentration of the gelatin solution is preferably 0.05~0.15 g / mL, more preferably 0.1 g / mL;

[0060] The mass of the methacrylic anhydride is preferably 0.6 to 1.2 times the mass of the gelatin, more preferably 0.8 times.

[0061] The reaction temperature is preferably 45~65°C, more preferably 50°C; the reaction time is preferably 3~6h, more preferably 4h; and the pH of the reaction is controlled at 7.5~9.5, more preferably pH 8.

[0062] In this application, the reaction is stopped by adding preheated hot water to dilute the reaction solution; the temperature of the preheated hot water is preferably 40~50°C, more preferably 45°C; the volume of the preheated hot water is preferably 4~6 times the volume of the reaction solution, more preferably 5 times.

[0063] In this application, the reaction solution is purified by dialysis in deionized water after being placed in a dialysis bag. The dialysis purification time is preferably 3-7 days, more preferably 5 days.

[0064] The dialyzed solution is pre-frozen and then freeze-dried to obtain a porous, sponge-like hydrogel solid. The pre-freezing temperature is preferably -70 to -90 °C, more preferably -80 °C.

[0065] S3. Add ZnV-LDH to the hydrogel and mix by ultrasound to obtain an electrically stimulated LDH hydrogel.

[0066] In this application, the mass of the ZnV-LDH is preferably 1 to 4 wt% of the mass of the hydrogel, more preferably 2%.

[0067] In the electrostimulated LDH hydrogel prepared in this application, ZnV-LDH acts as a sonosensitive agent, exhibiting excellent sonodynamic activity under ultrasonic stimulation, generating a large number of superoxide anions and hydroxyl radicals, thus possessing anti-osteosarcoma properties. The hydrogel, as a stable carrier of ZnV-LDH, endows the system with electroactivity, and can generate a concentration-dependent, periodic, and stable electrical response in response to ultrasonic stimulation. Through the synergistic effect of ZnV-LDH and hydrogel, the electrostimulated LDH hydrogel of this application is electroactive and can generate potential changes in response to external stimuli. Under ultrasonic stimulation, it can generate a concentration-dependent electrical response, and this response is periodic and stable, which can maximize the promotion of osteogenic differentiation and cell mechanical properties, achieving the dual function of "anti-osteosarcoma + bone repair".

[0068] The electrostimulated LDH hydrogel prepared in this application can be used as a drug for treating osteosarcoma, preventing osteosarcoma recurrence and bone reconstruction, or for preparing drugs for treating osteosarcoma, preparing drugs for preventing and treating postoperative recurrence of osteosarcoma, or drugs for repairing bone defects. The drugs have excellent therapeutic effects on osteosarcoma.

[0069] The present application will be further illustrated by the following examples.

[0070] Example 1

[0071] This embodiment provides a method for preparing an electrically stimulated LDH hydrogel, including:

[0072] S1, 2.4 mmol zinc chloride hexahydrate, 0.8 mmol vanadium trichloride and 0.3 g urea were dissolved in 80 mL of deionized water and stirred continuously for 30 min under nitrogen purging to maintain an anaerobic environment to obtain the precursor solution;

[0073] The precursor solution was transferred to a stainless steel polytetrafluoroethylene-lined autoclave and subjected to a hydrothermal reaction at 120 °C in a convection oven for 12 h. After natural cooling to room temperature, the precipitate was collected by centrifugation, washed three times with deionized water and anhydrous ethanol, and finally dried in a vacuum oven at 70 °C for 12 h to obtain ZnV-LDH.

[0074] S2, 10g of gelatin powder was added to 100mL of deionized water at 50°C and stirred until completely dissolved, yielding a 0.1g / mL gelatin solution. 8g of methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring. The pH was adjusted to 8 with carbonate buffer, and the reaction was carried out at 50°C for 4 hours. The reaction solution was then diluted with 500mL of preheated hot water at 45°C to terminate the reaction. The reaction solution was placed in a dialysis bag and dialyzed in deionized water at 40°C for 5 days to remove unreacted methacrylic anhydride (MA) and byproducts. The dialyzed solution was pre-frozen at -80°C and then freeze-dried to obtain a porous, sponge-like hydrogel.

[0075] S3, 0.2g of ZnV-LDH and 10g of hydrogel are mixed in an ultrasonic environment to obtain ZnV-LDH hydrogel, i.e., electrically stimulated LDH hydrogel.

[0076] Example 2

[0077] This embodiment provides a method for preparing an electrically stimulated LDH hydrogel, including:

[0078] S1, 4.8 mmol zinc chloride hexahydrate, 4.8 mmol vanadium trichloride and 0.3 g urea were dissolved in 80 mL deionized water and stirred continuously for 30 min under nitrogen purging to maintain an anaerobic environment to obtain the precursor solution.

[0079] The precursor solution was transferred to a stainless steel polytetrafluoroethylene-lined autoclave and subjected to a hydrothermal reaction at 120 °C in a convection oven for 12 h. After natural cooling to room temperature, the precipitate was collected by centrifugation, washed three times with deionized water and anhydrous ethanol, and finally dried in a vacuum oven at 70 °C for 12 h to obtain ZnV-LDH.

[0080] S2, 10g of gelatin powder was added to 100mL of deionized water at 50°C and stirred until completely dissolved, yielding a 0.1g / mL gelatin solution. 8g of methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring. The pH was adjusted to 8 with carbonate buffer, and the reaction was carried out at 50°C for 4 hours. The reaction solution was then diluted with 500mL of preheated hot water at 45°C to terminate the reaction. The reaction solution was placed in a dialysis bag and dialyzed in deionized water at 40°C for 5 days to remove unreacted methacrylic anhydride (MA) and byproducts. The dialyzed solution was pre-frozen at -80°C and then freeze-dried to obtain a porous, sponge-like hydrogel.

[0081] S3, 0.1g of ZnV-LDH and 10g of hydrogel are mixed in an ultrasonic environment to obtain ZnV-LDH hydrogel, i.e., electrically stimulated LDH hydrogel.

[0082] Example 3

[0083] S1, 4.8 mmol zinc chloride hexahydrate, 1.6 mmol vanadium trichloride and 0.3 g urea were dissolved in 80 mL deionized water and stirred continuously for 30 min under nitrogen purging to maintain an anaerobic environment to obtain the precursor solution;

[0084] The precursor solution was transferred to a stainless steel polytetrafluoroethylene-lined autoclave and subjected to a hydrothermal reaction at 120 °C in a convection oven for 12 h. After natural cooling to room temperature, the precipitate was collected by centrifugation, washed three times with deionized water and anhydrous ethanol, and finally dried in a vacuum oven at 70 °C for 12 h to obtain ZnV-LDH.

[0085] S2, 10g of gelatin powder was added to 100mL of deionized water at 50°C and stirred until completely dissolved, yielding a 0.1g / mL gelatin solution. 8g of methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring. The pH was adjusted to 8 with carbonate buffer, and the reaction was carried out at 50°C for 4 hours. The reaction solution was then diluted with 500mL of preheated hot water at 45°C to terminate the reaction. The reaction solution was placed in a dialysis bag and dialyzed in deionized water at 40°C for 5 days to remove unreacted methacrylic anhydride (MA) and byproducts. The dialyzed solution was pre-frozen at -80°C and then freeze-dried to obtain a porous, sponge-like hydrogel.

[0086] S3, 0.4g of ZnV-LDH and 10g of hydrogel were mixed in an ultrasonic environment to obtain ZnV-LDH hydrogel, i.e., electrically stimulated LDH hydrogel.

[0087] Comparative Example 1

[0088] 10g of gelatin powder was added to 100mL of deionized water at 50°C and stirred until completely dissolved, yielding a 0.1g / mL gelatin solution. 8g of methacrylic anhydride was slowly added dropwise to the gelatin solution while stirring. The pH was adjusted to 8 with carbonate buffer, and the reaction was carried out at 50°C for 4 hours. The reaction solution was then diluted with 500mL of preheated hot water at 45°C to terminate the reaction. The reaction solution was placed in a dialysis bag and dialyzed in deionized water at 40°C for 5 days to remove unreacted methacrylic anhydride (MA) and byproducts. The dialyzed solution was pre-frozen at -80°C and then freeze-dried to obtain a porous, sponge-like hydrogel.

[0089] The ZnV-LDH and ZnV-LDH hydrogels prepared in Example 1 were characterized as follows:

[0090] The ZnV-LDH prepared in Example 1 was tested for superoxide anions using a DHR-123 probe, and the results are as follows: Figure 1 As shown. From Figure 1 It is known that ZnV-LDH can generate abundant superoxide anions.

[0091] The ZnV-LDH prepared in Example 1 was tested for hydroxyl radicals using a TA probe, and the results are as follows: Figure 2 As shown. From Figure 2 It can be seen that ZnV-LDH can generate abundant hydroxyl radicals.

[0092] The morphology and elemental distribution of the ZnV-LDH hydrogel prepared in Example 1 are as follows: Figure 3 As shown. Among them, Figure 3 The leftmost image is a SEM image of the ZnV-LDH hydrogel, and the other images are mapping images of the ZnV-LDH hydrogel. Figure 3 It can be seen that the ZnV-LDH hydrogel has a porous / layered structure, which gives it a high specific surface area; the uniform distribution of Zn, V and O elements indicates that ZnV-LDH is well dispersed in the material.

[0093] The ZnV-LDH hydrogel prepared in Example 1 was subjected to ultrasonic rheological and tensile / compression tests, and the test results are as follows: Figure 4 and Figure 5 As shown. From Figure 4 and Figure 5 It is known that ZnV-LDH hydrogel has concentration-dependent rheological properties, and ultrasound can enhance its structural stability. The tensile strength of ZnV-LDH hydrogel increases with increasing concentration, and it exhibits strain hardening characteristics under compression. The higher the concentration, the stronger the resistance to compression. ZnV-LDH hydrogel is electroactive and can generate potential changes in response to external stimuli. Under ultrasound stimulation, it can generate a concentration-dependent electrical response, and the response is periodic and stable.

[0094] The ZnV-LDH hydrogel prepared in Example 1 was subjected to strain scanning test and gel yield strain test. The test results are as follows: Figure 6 As shown. From Figure 6 It is known that ZnV-LDH, as an inorganic filler, improves the deformation resistance of hydrogels and can significantly enhance their toughness and ductility.

[0095] The performance of the ZnV-LDH hydrogel of Example 1 and the hydrogel of Comparative Example 1 was evaluated, as follows:

[0096] test Figure 7-14 In the table, Control, Gel, LDH / Gel, Gel+Force, and LDH / Gel+Force represent the control group, hydrogel group, LDH hydrogel group, hydrogel + electrical stimulation group, and LDH hydrogel + electrical stimulation group, respectively.

[0097] 1. Immunofluorescence staining test of cellular osteocalcin OCN

[0098] The testing method was as follows: after fixing permeable cells / tissues and blocking non-specific binding sites, OCN-specific primary antibody and fluorescently labeled secondary antibody were incubated sequentially, followed by DAPI staining of the nuclei. Finally, the expression and distribution of OCN within the cells were observed using a fluorescence microscope. The test results are as follows: Figure 7 As shown. From Figure 7 It is known that the combination of ZnV-LDH hydrogel and electrical stimulation has a synergistic effect, which can maximize the promotion of osteogenic differentiation and cell mechanical properties.

[0099] 2. Osteoblastic transcription factor RUX2 assay

[0100] The testing method was as follows: After cell fixation, cells were permeabilized and blocked, then incubated sequentially with RUNX2-specific primary antibody and fluorescently labeled secondary antibody. Nuclei were then stained with DAPI. Finally, the expression and localization of RUNX2 within the cells (mostly the nucleus) were observed using a fluorescence microscope. The test results are as follows: Figure 8 As shown. From Figure 8 It is known that ZnV-LDH hydrogel combined with electrical stimulation can significantly upregulate RUNX2 expression and promote its nuclear localization, thus initiating osteogenic differentiation. ZnV-LDH hydrogel combined with electrical stimulation can synergistically promote osteogenic differentiation and cytoskeleton remodeling.

[0101] 3. Type I collagen staining test

[0102] The assay methods included: Cellular type I collagen immunofluorescence staining: After fixing cells / tissue (permeability testing could be omitted for extracellular collagen), the cells were blocked and sequentially incubated with type I collagen-specific primary antibody and fluorescent secondary antibody. Nuclear staining was performed using DAPI, and the expression and distribution of type I collagen in the extracellular matrix were observed under a fluorescence microscope. Cellular type I collagen Sirius red staining assay: After fixing cells / tissue, type I collagen secreted by cells in the extracellular matrix was stained with Sirius red. Deposition and content were observed using a conventional or polarized light microscope. The test results are as follows: Figure 9 As shown. From Figure 9 It is known that ZnV-LDH hydrogel combined with electrical stimulation can significantly upregulate Coll-I expression, promote the maturation of osteogenic differentiation, promote F-actin recombination, and enhance the mechanical properties of cells.

[0103] 4. Cellular bone morphogenetic protein staining test

[0104] The testing method is as follows: First, fix the cells / tissues and treat them according to the detection requirements (intracellular BMP needs to be permeable). After blocking, incubate sequentially with BMP-specific primary antibody and fluorescently labeled secondary antibody (immunofluorescence method) or enzyme-labeled secondary antibody (immunohistochemistry method). After nuclear staining, observe the expression sites and levels of BMP under a corresponding microscope. The test results are as follows: Figure 10 As shown. From Figure 10 It can be seen that the binding force stimulation of ZnV-LDH hydrogel can significantly upregulate BMP expression and enhance osteogenic induction.

[0105] 5. Vinculin staining test for cell adhesion plaques

[0106] The testing method is as follows: First, cells are fixed and permeabilized (because Vinculin is mostly distributed under the cell membrane). After blocking non-specific binding, cells are sequentially incubated with Vinculin-specific primary antibody and fluorescently labeled secondary antibody, often combined with phalloidin staining of actin filaments (to visualize the cytoskeleton). Finally, the expression and localization of Vinculin at cell adhesion focal sites are observed using a fluorescence microscope. The test results are as follows: Figure 11 As shown. From Figure 11 The results showed that Vinculin signal was significantly enhanced in the ZnV-LDH hydrogel group, suggesting that the combination of ZnV-LDH and hydrogel further enhanced cell adhesion. After electrical stimulation, the Vinculin signal was strongest and more concentrated, indicating the strongest cell adhesion in this group. This demonstrates that ZnV-LDH hydrogel combined with electrical stimulation can significantly upregulate Vinculin expression and enhance cell-matrix adhesion.

[0107] 6. Cellular alkaline phosphatase (ALP) staining test

[0108] The test method is as follows: After cell fixation, cells are incubated with ALP-specific substrates (such as naphthol AS-MX phosphate and azo salt), causing ALP to catalyze the formation of a colored precipitate (e.g., red or blue). The distribution and intensity of the precipitate are observed using an optical microscope to reflect ALP activity. The test results are as follows: Figure 12 As shown. From Figure 12 It is known that ZnV-LDH hydrogel combined with electrical stimulation can significantly enhance ALP activity and promote the early stage of osteogenic differentiation.

[0109] 7. Alizarin Red staining test for cells

[0110] The test method is as follows: After fixing the cells, they are incubated with Alizarin Red staining solution, allowing the staining solution to combine with the calcium nodules (mineralized matrix) secreted by the cells to form a red precipitate. The formation and quantity of the precipitate are observed using an optical microscope to assess the cell's mineralization capacity. The test results are as follows: Figure 13 As shown. From Figure 13 It is known that ZnV-LDH hydrogel combined with electrical stimulation can significantly enhance calcium deposition and promote the mature stage of osteogenic differentiation.

[0111] 8. Cellular β-Catein staining test

[0112] The testing method was as follows: After cell fixation and permeabilization, cells were blocked and sequentially incubated with β-Catenin-specific primary antibody and fluorescently labeled secondary antibody. The nuclei were then stained with DAPI, and the expression and localization of β-Catenin within the cells (normally the cell membrane, but after activation, it enters the nucleus) were observed using a fluorescence microscope. The test results are as follows: Figure 14 As shown. From Figure 14 It is known that the binding force stimulation of ZnV-LDH hydrogel can significantly upregulate β-Catenin expression and promote its nuclear localization, thereby promoting osteogenic differentiation.

[0113] Although this application has been described in detail in this specification with general descriptions and specific embodiments, some modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this application are all within the scope of protection claimed in this application.

Claims

1. A method for preparing an electrically stimulated LDH hydrogel, characterized in that, include: S1, under a nitrogen atmosphere, zinc salt, vanadium salt and urea are dissolved in water to obtain a precursor solution; the precursor solution is subjected to a hydrothermal reaction to obtain ZnV-LDH; The zinc salt includes any one of zinc nitrate, zinc sulfate, or zinc chloride; The vanadium salt includes any one of vanadium nitrate, vanadium sulfate, or vanadium chloride. The total concentration of zinc salt and vanadium salt in the precursor solution is 30-125 mmol / L; the concentration of urea is 40-80 mmol / L; and the molar ratio of zinc salt to vanadium salt is 3:(1-3). S2, under stirring, methacrylic anhydride was gradually added dropwise to the gelatin solution, and the reaction was carried out by heating; preheated hot water was added to the reaction solution to stop the reaction; the reaction solution was purified by dialysis, pre-frozen, and then freeze-dried to obtain a hydrogel; S3, ZnV-LDH is added to the hydrogel and mixed by ultrasound to obtain an electrically stimulated LDH hydrogel; The mass of the ZnV-LDH is 1 to 4 wt% of the mass of the hydrogel.

2. The preparation method according to claim 1, characterized in that, The hydrothermal reaction is carried out at a temperature of 60-150℃ for 12-48 hours.

3. The preparation method according to claim 1, characterized in that, The gelatin solution is an aqueous solution of gelatin or a PBS solution of gelatin; The concentration of the gelatin solution is 0.05~0.15g / mL.

4. The preparation method according to claim 1, characterized in that, The mass of the methacrylic anhydride is 0.6 to 1.2 times the mass of the gelatin.

5. The preparation method according to claim 1, characterized in that, The reaction temperature described in S2 is 45~65℃, the reaction time is 3~6 hours, and the pH of the reaction is 7.5~9.

5.

6. The preparation method according to claim 1, characterized in that, The reaction is stopped by adding preheated water to the reaction solution, wherein the temperature of the preheated water is 40~50℃ and the volume of the water is 4~6 times the volume of the reaction solution. The dialysis purification time is 3-7 days; The pre-freezing temperature is -70 ~ -90℃.

7. The electrically stimulated LDH hydrogel prepared by the preparation method according to any one of claims 1-6.

8. The use of the electrically stimulated LDH hydrogel according to claim 7 in the preparation of pharmaceuticals for bone defect repair.

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