Nano-composite hydrogel for diabetic bone repair and preparation method of nano-composite hydrogel

Abstract

The invention belongs to the technical field of biomedical engineering materials, and discloses a nano composite hydrogel for diabetic bone repair and a preparation method thereof.The preparation method comprises the steps that oxidized hyaluronic acid is prepared and modified through phenylboronic acid to obtain OHA-PBA; then preparing a halloysite nanotube loaded with bulbus lilii glycoside B; and finally, uniformly mixing an OHA-PBA aqueous solution with the RB-HNT powder, adding a polyvinyl alcohol aqueous solution, and constructing a dynamic cross-linked network through reversible borate bonds formed by phenylboronic acid and hydroxyl groups and hydrogen bonds formed by the hydroxyl groups on the surface of the RB-HNT and the polymer. The nano-composite hydrogel disclosed by the invention has diabetes pathology microenvironment responsiveness, can be controllably degraded under high glucose, ROS or acidic conditions, slowly releases RB and silicon ions, and synergistically exerts antioxidant, anti-inflammatory and osteogenesis promoting effects; meanwhile, excellent injectability and self-healing ability are achieved, the material can be attached to irregular bone defect parts, and minimally invasive drug delivery is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering materials technology, and particularly relates to a nanocomposite hydrogel for bone repair in diabetes and its preparation method. Background Technology

[0002] Bone defects are a common orthopedic disease in clinical practice, and their repair is affected by various factors such as trauma, tumor resection, and congenital diseases. Diabetes mellitus, as a chronic metabolic disorder, is characterized by hyperglycemia and persistent inflammation, which significantly increases the risk of bone disease and delays the bone defect repair process. In the microenvironment of diabetic bone defects, hyperglycemia directly disrupts bone and immune homeostasis, inhibiting the osteogenic differentiation capacity of osteoblast-related cells; at the same time, it triggers immune cell dysfunction, leading to the continuous activation of macrophage M1 type and the release of a large number of pro-inflammatory factors, exacerbating the local inflammatory response; in addition, the high-glucose environment accelerates the generation and accumulation of reactive oxygen species (ROS), leading to oxidative stress and mitochondrial dysfunction, further inhibiting bone regeneration activity, forming a vicious cycle of "hyperglycemia-oxidative stress-chronic inflammation," which seriously hinders bone healing.

[0003] Currently, commonly used bone repair materials in clinical practice mainly include inorganic scaffolds such as metals, ceramics, and bioactive glass. While these materials possess good strength and osteoconductivity, they suffer from poor compliance, require intraoperative shaping, lack inherent adhesion, and cannot regulate the pathological microenvironment of diabetes, thus failing to meet the needs of bone defect repair in diabetic patients. Novel injectable self-healing hydrogels have attracted considerable attention in bone tissue repair due to their ability to mimic the natural extracellular matrix, high porosity, and biodegradability, as well as their potential as delivery carriers for bioactive substances. However, single hydrogels suffer from drawbacks such as low strength, uncontrollable degradation rates, and a lack of targeted bioactivity.

[0004] Therefore, developing a composite hydrogel that combines pathological microenvironment responsiveness, good mechanical properties, injectability, and self-healing ability is of great significance for promoting the clinical treatment of diabetic bone defects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a nanocomposite hydrogel for bone repair in diabetes and its preparation method. A dual dynamic cross-linking network is constructed using phenylboronic acid-modified oxidized hyaluronic acid (OHA-PBA) and polyvinyl alcohol (PVA), and then loaded with rutin B / haloite nanotubes (RB-HNT) composite nanoparticles. This hydrogel can achieve controlled degradation in response to the pathological microenvironment of diabetes (hyperglycemia, ROS, acidic pH), simultaneously exerting functions such as ROS scavenging, inflammation reduction, and osteogenic promotion. Furthermore, it possesses excellent injectability and self-healing capabilities.

[0006] To achieve the above objectives, the present invention provides a method for preparing a nanocomposite hydrogel for bone repair in diabetes, comprising the following steps: 1) Preparation of oxidized hyaluronic acid: Sodium hyaluronate was added to PBS buffer solution and stirred to dissolve. Sodium periodate was added and stirred under light-protected conditions. Ethylene glycol was added and stirring was continued. The resulting solution was dialyzed and lyophilized to obtain solid OHA. 2) Preparation of phenylboronic acid modified oxidized hyaluronic acid: Add water to the OHA solid obtained in step 1), stir to dissolve, add 3-aminophenylboronic acid, continue stirring, filter, dialyze, and freeze dry to obtain OHA-PBA solid; 3) Preparation of halloysite nanotubes loaded with limonene B: Limonene B solution was prepared with ethanol, halloysite nanotubes were added to the solution, stirred and sonicated to obtain a mixture; then the mixture was placed in a vacuum environment and allowed to stand, and freeze-dried to obtain RB-HNT powder. 4) Preparation of nanocomposite hydrogel: Dissolve the OHA-PBA solid obtained in step 2), add RB-HNT powder, stir, add PVA solution, and continue stirring to obtain nanocomposite hydrogel.

[0007] Further, in step 1), the pH of the PBS buffer solution was 5, and the amount added was 30 mL; the amount of sodium hyaluronate added was 1.0 g; the amount of sodium periodate added was 0.401 g; the reaction time under light-protected conditions was 18 h; the amount of ethylene glycol added was 1 mL, and the stirring time continued for 20 min after addition; dialysis was performed using a dialysis bag with a molecular weight cutoff of 3500 D, the dialysis medium was ultrapure water, the dialysis time was 48 h, and the dialysis medium was replaced every 24 h; the lyophilization conditions were -100~-50℃, 5~50 Pa, and the lyophilization time was 20~24 h.

[0008] Further, in step 2), the water is ultrapure water, and the addition amount is 30 mL; the addition amount of 3-aminophenylboronic acid is 680 mg, and the stirring time is continued for 45 min; dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 D, the dialysis medium is ultrapure water, the dialysis time is 24 h, and the dialysis medium is replaced every 12 h; the lyophilization conditions are -100~-50℃, 5~50 Pa, and the lyophilization time is 20~24 h.

[0009] Further, in step 3), the ethanol is a 50% ethanol solution by volume, the volume of the prepared Wangbaihe glycoside B solution is 8 mL, and the concentration is 250 μg / mL; the amount of halloysite nanotubes added is 1 g; the stirring time is 2 h; the ultrasonic treatment power is 120~200 W, the ultrasonic frequency is 40 Hz, and the ultrasonic time is 1 h; the standing time under vacuum is 12 h; the freeze-drying temperature is -100~-50℃, and the drying time is 24 h.

[0010] Further, in step 4), the concentration of OHA-PBA solid after dissolving in ultrapure water is 40 mg / mL; the amount of RB-HNT powder added is 1%~2% of the mass of OHA-PBA; the concentration of PVA solution is 80 mg / mL, and the volume ratio of OHA-PBA solution to PVA solution is 1:3; the stirring time after adding RB-HNT is 30 min; and the stirring time after adding PVA solution is 2 min.

[0011] A nanocomposite hydrogel for bone repair in diabetes is also provided, which is prepared according to the preparation method for a nanocomposite hydrogel for bone repair in diabetes.

[0012] Furthermore, the nanocomposite hydrogel possesses injectability and self-healing capabilities.

[0013] It also provides an application of nanocomposite hydrogels in the preparation of biomaterials for repairing bone defects in diabetic patients.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: This invention prepares a composite hydrogel with dual responsiveness to reactive oxygen species (ROS) and glucose. It is mainly composed of hyaluronic acid and polyvinyl alcohol (PVA), and loaded with halloysite nanotubes containing linaloside B. The amino groups on the phenylboronic acid-modified oxidized hyaluronic acid form reversible covalent Schiff base bonds with the residual aldehyde groups, which then crosslink with the hydroxyl-rich PVA, constructing a doubly dynamically covalently crosslinked hydrogel network. Furthermore, the abundant hydroxyl groups on the surface of the halloysite nanotubes can form hydrogen bonds with the hydroxyl groups of the phenylboronic acid-modified oxidized hyaluronic acid and PVA, thereby further enhancing the mechanical properties of the hydrogel. Simultaneously, the numerous hydrogen bonds endow the hydrogel with good self-healing and injectability. In the pathological microenvironment of high ROS and high glucose in diabetic patients, the hydrogel undergoes responsive degradation in the presence of phenylboronic acid, exposing the halloysite nanotubes and achieving sustained release of linaloside B. Its aim is to scavenge ROS in the microenvironment, alleviate excessive inflammatory responses, and reverse mitochondrial dysfunction in metabolically disordered bone marrow mesenchymal stem cells. Furthermore, the silicon ions released by the degradation of halloysite nanotubes can further promote osteogenic differentiation of bone marrow mesenchymal stem cells, providing a feasible treatment strategy and theoretical support for the repair of diabetic bone defects.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the preparation and application of HHPP hydrogels. Figure 2 The images are transmission electron microscope (TEM) images of HNT and RB-HNT, where A represents HNT and B represents RB-HNT. Figure 3 Scanning electron microscope (SEM) images of the lyophilized cross sections of HPP, 1%HHPP, and 2%HHPP hydrogels (scale bar: 1µm). In the figure, A represents HPP, B represents 1%HHPP, and C represents 2%HHPP. Figure 4 The images show the energy dispersive spectroscopy (EDS) surface scans of HPP, 1%HHPP, and 2%HHPP hydrogels (scale bar: 5µm). In the figures, A represents HPP, B represents 1%HHPP, C represents 2%HHPP, D represents the distribution of Al in HPP, E represents the distribution of Si in HPP, F represents the distribution of Al in 1%HHPP, G represents the distribution of Si in 1%HHPP, H represents the distribution of Al in 2%HHPP, and I represents the distribution of Si in 2%HHPP. Figure 5 The graph shows the total antioxidant activity and H2O2 scavenging rate of RB-HNT. In the graph, A represents the total antioxidant activity of RB-HNT, and B represents the H2O2 scavenging rate of RB-HNT. represent P <0.001; Figure 6 Infrared spectra of HA and OHA; Figure 7 The figures show the 1H NMR spectra of HA, OHA, and OHA-PBA. In the figure, A represents the 1H NMR spectrum of HA, B represents the 1H NMR spectrum of OHA, and C represents the 1H NMR spectrum of OHA-PBA. Figure 8 This is a schematic diagram of the experimental results of injectability, moldability and self-healing ability of hydrogel HPP. In the figure, A represents the schematic diagram of the hydrogel injectability experimental results, B represents the schematic diagram of the hydrogel moldability experimental results, C represents the schematic diagram of the hydrogel self-healing ability experimental results, and D represents the schematic diagram of the internal bonding of the hydrogel. Figure 9 Rheological tests were performed on HPP, 1%HHPP, and 2%HHPP hydrogels. Figure A shows the frequency scan test results of the hydrogels at a constant frequency of 10 rad / s and a strain range of 0.1% to 1000%. Figure B shows the frequency scan results of the elastic modulus and loss modulus of the hydrogels measured under 5% strain in frequency scan mode. Figure C shows the results of the sequential step strain test of the hydrogels cyclically between small strain (1%, 60 seconds) and large strain (300%, 60 seconds). Figure 10The figures show the hydrogel degradation rates under different reactive oxygen species (ROS) concentrations (0 mM H2O2 or 1 mM H2O2), different pH conditions (pH 7.0 or pH 5.5), and different glucose concentrations (0 mM or 25 mM glucose). In the figure, A represents different ROS concentrations (0 mM H2O2 or 1 mM H2O2), B represents different pH conditions (pH 7.0 or pH 5.5), and C represents different glucose concentrations (0 mM or 25 mM). Figure 11 Swelling rate curves for HPP, 1%HHPP, and 2%HHPP hydrogels; Figure 12 The cumulative release curves of RB and silicon in 1%HHPP and 2%HHPP hydrogels are shown in the figure. A is the cumulative release curve of RB and B is the cumulative release curve of silicon. Figure 13 To detect the cell viability of RAW264.7 cells and bone marrow mesenchymal stem cells after culturing in different hydrogel extracts for 1, 3, and 5 days using the MTT assay, A in the figure represents RAW264.7 cells, and B represents bone marrow mesenchymal stem cells. represent P <0.001; Figure 14 This image shows microscopic images (scale bar: 100µm) of bone marrow mesenchymal stem cells and RAW264.7 cells stained with the DCFH probe under reactive oxygen species (ROS) conditions, along with corresponding semi-quantitative analysis results. Figure A shows a microscopic image of bone marrow mesenchymal stem cells stained with the DCFH probe; B shows the semi-quantitative analysis results of bone marrow mesenchymal stem cells; C shows a microscopic image of RAW264.7 cells stained with the DCFH probe; and D shows the semi-quantitative analysis results of RAW264.7 cells. represent P <0.01, represent P <0.001; Figure 15 Images (scale bar: 100µm) of mitochondrial membrane potential detected using JC-1 staining and their semi-quantitative analysis are shown in the figure. Figure A shows the image of mitochondrial membrane potential detected using JC-1 staining, and figure B shows the semi-quantitative analysis. represent P <0.01, represent P <0.001; Figure 16Immunofluorescence staining and semi-quantitative analysis of macrophage polarization markers CD86 and Arg-1 are shown (scale bar: 100 μm). In the figure, A is the immunofluorescence staining of CD86, B is the semi-quantitative analysis of CD86, C is the immunofluorescence staining of Arg-1, and D is the semi-quantitative analysis of ARG1. represent P <0.05, represent P <0.01; Figure 17 This figure shows the results of quantitative analysis of gene expression of pro-inflammatory factors (TNF-α and IL-1β) and anti-inflammatory factors (Arg-1 and IL-10). In the figure, A represents TNF-α, B represents IL-1β, C represents Arg-1, and D represents IL-10. represent P <0.05, represent P <0.01, represent P <0.001; Figure 18 Images and quantitative analysis diagrams of alkaline phosphatase (ALP) staining are shown. In the figure, A is the ALP staining image, B is the quantitative analysis diagram at 3 days, and C is the quantitative analysis diagram at 7 days. represent P <0.05, represent P <0.001; Figure 19 Images and quantitative analysis diagrams of alizarin red stained mineralized nodules are shown. In the figure, A is an image of alizarin red stained mineralized nodules, B is the quantitative analysis at 14 days, and C is the quantitative analysis at 21 days. represent P <0.001; Figure 20 Images show BMP-2 immunofluorescence staining (scale bar: 100 μm) and quantitative analysis. In the image, A is the BMP-2 immunofluorescence staining image, and B is the BMP-2 quantitative analysis image. represent P <0.01, represent P <0.001; Figure 21This is a graph showing the quantitative analysis of RUNX2, COL-I, BMP-2, and ALP gene expression. In the graph, A represents RUNX2, B represents COL-I, C represents BMP-2, and D represents ALP. represent P <0.01, represent P <0.001; Figure 22 Microscopic CT scan images of femoral defect healing; Figure 23 Figure 1 shows quantitative analysis charts for bone volume fraction, bone mineral density, trabecular separation, and trabecular thickness. Chart A represents the quantitative analysis chart for bone volume fraction, chart B for bone mineral density, chart C for trabecular separation, and chart D for trabecular thickness. represent P <0.05, represent P <0.01, represent P <0.001; Figure 24 Images of hematoxylin-eosin and Masson trichrome staining of femoral tissue 4 weeks postoperatively are shown. In the figure, A is the hematoxylin-eosin staining image and B is the Masson trichrome staining image. Figure 25 Immunohistochemical staining images of CD86, CD206, OCN, and BMP-2; Figure 26 This figure shows the semi-quantitative analysis results of immunohistochemical staining for CD86, CD206, OCN, and BMP-2. In the figure, A represents CD86, B represents CD206, C represents OCN, and D represents BMP-2. represent P <0.01, represent P <0.001. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0019] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0020] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0021] The RAW264.7 cells used in this invention were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). They were cultured in high-glucose DMEM medium containing 1% (v / v) penicillin / streptomycin solution and 10% (v / v) fetal bovine serum. The cells were cultured in a cell culture incubator at 37°C with 5% CO2.

[0022] The BMSCs used in this invention are primary BMSCs isolated from the medullary cavities of the femur and tibia of SD rats: After anesthetizing and euthanizing the SD rats, the surface was disinfected by soaking in 75% alcohol for 15 minutes, and the femur and tibia were removed under aseptic conditions; the bones were placed in a sterile culture dish containing low-glucose DMEM medium containing 1% (v / v) penicillin / streptomycin solution and 10% (v / v) fetal bovine serum, and the medullary cavity of the bone was opened with scissors. An appropriate amount of culture medium was drawn up with a syringe and the needle was used to repeatedly flush the medullary cavity until all the medullary cavities turned white; the culture medium containing bone marrow was carefully and repeatedly blown with a syringe to fully disperse the cells and form a uniform suspension; the mixture was transferred to a cell culture flask with a syringe, cultured under standard conditions for 12 hours, and then the medium was changed and labeled as P0 generation BMSCs cells.

[0023] Example 1 Preparation of nanocomposite hydrogels.

[0024] 1) Preparation of oxidized hyaluronic acid (OHA).

[0025] Add 30 mL of PBS buffer solution (pH=5) and 1.0 g of sodium hyaluronate to a round-bottom flask and stir until completely dissolved at room temperature. Add 0.401 g (1.87 mmol) of sodium periodate and continue stirring for 18 hours in the dark. After the reaction is complete, add 1 mL of ethylene glycol and stir for 20 minutes to terminate the reaction. Transfer the resulting solution to a dialysis bag with a molecular weight cutoff of 3500 D and dialyze with ultrapure water for 48 hours, changing the dialysis medium every 24 hours. After dialysis, freeze-dry the solution at -50°C and 30 Pa for 24 hours to obtain a white, spongy OHA solid.

[0026] 2) Preparation of phenylboronic acid-modified oxidized hyaluronic acid (OHA-PBA).

[0027] Add 30 mL of ultrapure water to the OHA solid obtained in step 1.1 and stir at room temperature until completely dissolved; add 680 mg of 3-aminophenylboronic acid and continue stirring for 45 minutes; after filtering to remove insoluble impurities, put the filtrate into a dialysis bag (3500D) and dialyze with ultrapure water for 24 hours, changing the dialysis medium every 12 hours; after dialysis, freeze dry (under the same conditions as step 1) to obtain brown spongy OHA-PBA solid.

[0028] 3) Preparation of halloysite nanotubes loaded with lily glycoside B (RB-HNT).

[0029] Prepare 8 mL of a 250 μg / mL solution of rubigin B (Shanghai Yuanye Biotechnology Co., Ltd.) using 50% (volume fraction) ethanol; add 1 g halloysite nanotubes (HNT) to the solution and stir at 200 r / min for 2 hours at room temperature to ensure uniform dispersion of HNT; then sonicate at 160 W and 40 kHz for 1 hour to enhance the loading efficiency of rubigin B; then place the mixture under vacuum and let it stand for 12 hours to remove the hollow structure of HNT and the air between the particles; finally freeze-dry at -80℃ for 24 hours to obtain RB-HNT powder.

[0030] 4) Preparation of hydrogels (HPP) and nanocomposite hydrogels (HHPP).

[0031] The OHA-PBA solid obtained in step 2) was dissolved in ultrapure water to prepare a 40 mg / mL OHA-PBA solution. RB-HNT powder obtained in step 3) was weighed at 1%–2% of the OHA-PBA mass and added to the OHA-PBA solution. The mixture was stirred at room temperature for 120 minutes until uniformly dispersed. An 80 mg / mL PVA solution was added to the mixture, with a volume ratio of OHA-PBA solution to PVA solution of 1:3. The mixture was stirred for another 2 minutes to allow the phenylboronic acid in OHA-PBA to fully crosslink with the hydroxyl groups in PVA, while simultaneously forming hydrogen bonds between the hydroxyl groups on the surface of RB-HNT and the polymer chains, resulting in nanocomposite hydrogels, named 1% HHPP (1% RB-HNT mass fraction) and 2% HHPP (2% RB-HNT mass fraction), respectively. The preparation and application of this HHPP hydrogel are described below. Figure 1 As shown.

[0032] Hydrogel (HPP): The OHA-PBA solid obtained in step 2) was dissolved in ultrapure water to prepare a 40 mg / mL OHA-PBA solution; 80 mg / mL PVA solution was added to the above solution, with a volume ratio of OHA-PBA solution to PVA solution of 1:3, and the mixture was stirred for 2 minutes to allow the phenylboronic acid in OHA-PBA to fully crosslink with the hydroxyl groups in PVA, thus obtaining the hydrogel (HPP).

[0033] Example 2 I. Characterization Tests.

[0034] TEM characterization: The surface morphology of the material was characterized using TEM at 100 kV. For example... Figure 2 As shown, RB-HNT retains the typical hollow tubular structure of halloysite nanotubes (HNT), indicating that the structure was not destroyed after loading with lily glycoside B.

[0035] SEM characterization: Micromorphology and elemental distribution were observed using SEM at 15 kV. Results are as follows: Figure 3 As shown in the scanning electron microscope images, HPP, 1%HHPP and 2%HHPP hydrogels all exhibit typical porous structures, which are crucial for the morphological basis required to construct ideal biomaterial carriers and drug delivery systems.

[0036] Energy spectral scanning results show (e.g.) Figure 4As shown in the figure, Al and Si elements are uniformly distributed in 1% HHPP and 2% HHPP hydrogels, and their distribution is positively correlated with the doping concentration, confirming the uniform dispersion of RB-HNTs within the hydrogel. With increasing RB-HNT doping concentration, the average pore size of the hydrogel tends to decrease. Simultaneously, the degree of pore wall collapse is significantly reduced in hydrogels with higher RB-HNT concentrations, resulting in a more three-dimensional and regular overall structure. This structural change may be attributed to the crucial physical support role of RB-HNTs within the gel network, effectively enhancing the stability of the network framework. Specifically, as nanoscale particles, RB-HNTs can embed into the gaps in the network formed by flexible polymer chains to reduce porosity, i.e., fill the free volume between polymer chains, making the pore size smaller and the structure denser, thereby restricting chain segment movement. These embedded nanotubes act as physical barriers to the surrounding polymer chain segments, limiting their fluidity and oscillation ability. This restricted movement also makes the network macroscopically denser and increases its rigidity. Furthermore, the positively charged Al and Si on the surface of halloysite nanotubes can form stable coordination bonds with the lone pairs of electrons provided by the carboxyl groups and hydroxyl groups of hyaluronic acid, thereby enhancing the overall mechanical properties of the hydrogel. Simultaneously, the abundant hydroxyl groups on the surface of halloysite nanotubes can form a broad hydrogen bond network with the polar groups on the hydrogel polymer chains, further strengthening the mechanical properties of the hydrogel network. The resulting smaller pore size also facilitates sustained drug release.

[0037] Antioxidant activity: The total antioxidant capacity was determined using a total antioxidant capacity assay kit for EG-OHA@μCMP hydrogel PBS dispersions at different concentrations (0.5, 1, 2, 3, 4 mg / mL) and PBS dispersions of different materials (concentration uniformly 1 mg / mL). According to the kit instructions, FRAP working solution and FeSO4 standard solutions of different concentrations were prepared in advance for standard curve determination. 180 μL of FRAP working solution was added to each well of a 96-well plate, followed by 5 μL of PBS in the blank control wells, 5 μL of FeSO4 standard solutions of different concentrations in the standard curve wells, and 5 μL of each material's PBS dispersion in the sample wells. After gentle mixing, the mixture was incubated at 37°C for 5 min. The absorbance of the solutions at 593 nm was measured using a microplate reader, with three replicates per group (n=3). The total antioxidant capacity was calculated based on the standard curve. Results are as follows: Figure 5 As shown, the total antioxidant capacity and hydrogen peroxide scavenging rate of RB-HNT are concentration-dependent.

[0038] Fourier Transform Infrared (FTIR) Characterization: Weigh 50 mg each of OHA and HA, mix them thoroughly with 5 g of pure potassium bromide, grind them completely in a grinder, and then dry them for later use. Take a small amount of the sample mixture and fill it into a mold to press it into a sample sheet. Prepare a background sheet using pure potassium bromide. Then, perform infrared spectroscopy detection on the pressed sheet, setting the scanning range to 500–4000 cm⁻¹. -1 The result is as follows Figure 6 As shown, the spectrum of oxidized hyaluronic acid is at 1740 cm⁻¹. -1 The appearance of a new characteristic peak indicates that the aldehyde group has been successfully introduced into the hyaluronic acid molecule.

[0039] 1H NMR spectrum ( 1 Characterization by 1H-NMR: Weigh 7.5 mg each of dried OHA, HA, and OHA-PBA into NMR tubes, add 0.5 mL of deuterated water (D2O) reagent to each, cap the tubes, shake to dissolve completely, and let stand upright overnight. Analyze the chemical structure of each sample using a 1H NMR spectrometer. Figure 7 As shown, in the oxidation of hyaluronic acid 1 In the 1H NMR spectroscopy analysis, three new chemical shifts appeared at 4.94, 5.05, and 5.14 ppm, corresponding to hemiacetal protons originating from adjacent hydroxyl and aldehyde groups, respectively. Therefore, 1 The H NMR spectroscopy results also confirmed the successful preparation of oxidized hyaluronic acid.

[0040] II. Research on self-healing ability, injectability and moldability.

[0041] To observe the injectability and plasticity of HPP hydrogel, the hydrogel was loaded into a 2 mL syringe and then squeezed through a syringe into a beaker containing PBS solution.

[0042] Using HPP hydrogel as an example, the self-healing ability of hydrogels was demonstrated. First, two spherical hydrogels with a diameter of 10 mm were prepared, and one was stained with methylene blue for observation. Then, the two hydrogels of different colors were placed together and allowed to heal at room temperature without any external stimuli, and photographs of the self-healing adhesion were taken.

[0043] like Figure 8 As shown in Figure A, the hydrogel can be squeezed into PBS using a syringe. Furthermore, as... Figure 8 As shown in Figure B, writing can be done in a petri dish using the hydrogel extruded from a syringe. Self-healing ability is crucial for maintaining the structural and functional integrity of the hydrogel, helping to extend the lifespan of bioengineered materials.

[0044] To evaluate the self-healing properties, two pieces of hydrogel were placed in contact, one of which was stained dark blue. Figure 8The results from the C-cell assay showed that after standing at room temperature for 15 minutes, the two hydrogels spontaneously fused, and their boundaries disappeared. Figure 8 As shown in Figure D, this self-healing ability is attributed to the dynamic hydrogen bond network formed between phenylboronic acid in oxidized hyaluronic acid and the hydroxyl-rich surface of polyvinyl alcohol and RB-HNT.

[0045] III. Hydrogel Rheological Testing.

[0046] The rheological properties of the hydrogel (HPP) were determined using a rheometer (TA, DHR-2) with parallel plates (20 mm). The storage modulus (G') and loss modulus (G'') of the hydrogel (HPP) were measured at a frequency of 10 rad / s over a strain range of 0.1% to 1000%. The rheological properties of HPP, 1% HHPP, and 2% HHPP hydrogels were evaluated using a rheometer. Figure 9 As shown in Figure A, frequency scanning test results indicate that within the linear viscoelastic region, the storage modulus (G') of all hydrogels is significantly higher than the loss modulus (G''), proving that all three materials successfully formed a stable three-dimensional network structure dominated by elastic solid behavior, which is a fundamental prerequisite for their use as scaffold materials in bone tissue engineering. After the addition of RB-HNT, the linear viscoelastic region of the nanocomposite hydrogel is significantly expanded compared to the pure HPP hydrogel, and this range shows a positive correlation with the amount of RB-HNT incorporated. The expansion of the linear viscoelastic region means that the nanocomposite hydrogel network can withstand greater strain while maintaining structural integrity, indicating its higher structural stability.

[0047] Based on the linear viscoelastic region, small-amplitude frequency sweeps were performed to test the elastic modulus and loss modulus at 5% strain. Figure 9 As shown in Figure B, the frequency scanning results indicate that the modulus of the nanocomposite hydrogel increases with the addition of RB-HNT, directly demonstrating that the incorporation of HNT enhances the rigidity and strength of the hydrogel. This is partly because RB-HNT can act as physical crosslinking points, uniformly dispersed in the hydrogel network, effectively sharing and transferring stress; and partly because the abundant hydroxyl groups on the HNT surface can form numerous hydrogen bonds with the functional groups in the hydrogel, enhancing the cohesion of the hydrogel network and thus improving its mechanical properties. This is consistent with... Figure 3 The results show that the hydrogel microstructure is consistent.

[0048] The self-healing capability was determined by conducting continuous step strain experiments under cyclic small strain (1%, 60 s) and large strain (300%, 60 s). Figure 9 As shown in Figure C, the step strain scan verifies the excellent self-healing properties of the hydrogel, which can quickly recover its original mechanical properties from structural damage caused by large strain. This proves that there are a large number of dynamically reversible non-covalent bonds in its internal cross-linking network, thus giving it good mechanical properties and self-healing ability.

[0049] IV. In vitro degradation performance test.

[0050] Based on the oxidative stress and hyperglycemic microenvironment of diabetic bone defects, the degradation behavior of hydrogels in different solutions was investigated: PBS buffer at pH 7 or 5.5, PBS buffer containing 100 μm H2O2 (0 mM H2O2 or 1 mM H2O2), and PBS buffer containing glucose (0 mM or 25 mM glucose). Specifically, hydrogels with a radius of 5 mm and a height of 5 mm were placed in 50 ml centrifuge tubes at room temperature, and the above solutions were added. The remaining hydrogels were collected on days 1, 4, 7, 14, and 21, and then freeze-dried. The weight of each sample was measured. The amount of remaining hydrogel was determined using the following formula: .

[0051] in, The weight of the original components. This represents the weight of the residual hydrogel.

[0052] The results are as follows Figure 10 As shown, in pure PBS, the composite hydrogel lost approximately 43% of its weight after 21 days of degradation at room temperature; however, under high reactive oxygen species (ROS) concentrations, the hydrogel exhibited a faster degradation rate, with a weight loss of approximately 80%. Similarly, hydrogels cultured in low pH environments showed similar degradation behavior, with residual amounts of 57% and 20% in neutral and low pH environments, respectively. High sugar environments also promoted hydrogel degradation, with residual amounts of 57% and 45% in sugar-free and high sugar environments, respectively. These results indicate that the hydrogel possesses a responsiveness to pathological microenvironments, attributed to the abundance of phenylboronic acid within the hydrogel. High ROS oxidize and break these sensitive bonds, leading to rapid dissociation of the polymer network, thereby achieving ROS-responsive degradation. A slightly acidic environment provides a large amount of H₂O. + Ions attack and protonate the oxygen atom on the boron atom, significantly weakening the coordination bond between boron and oxygen atoms. This leads to a highly unstable ester bond structure, ultimately resulting in rapid hydrolysis and breakage. Furthermore, in a high-sugar environment, the hydroxyl groups on glucose compete with hydroxyl groups from other components for reversible binding sites on phenylboronic acid, further accelerating degradation.

[0053] V. Swelling performance test.

[0054] Equal volumes of freeze-dried gel samples were immersed in distilled water at 37°C to evaluate swelling capacity. The unswollen gel samples were weighed and recorded as follows: After 2, 4, 8, 12, and 24 hours, excess surface moisture was removed using filter paper. The swollen hydrogel (HPP) samples were weighed and recorded as follows. The formula for calculating the degree of swelling is: .

[0055] The results are as follows Figure 11 As shown, the lyophilized hydrogel swelled rapidly in PBS buffer for the first two hours, followed by a slower but continuous weight gain. After 24 hours of soaking, the swelling ratios of HPP, 1% HHPP, and 2% HHPP hydrogels were 476.06±32.5%, 374.14±29.03%, and 362.15±46.87%, respectively. Compared to HPP, the RB-HNT introduced in 1% HHPP and 2% HHPP significantly reduced the swelling ratio of the hydrogel. This is because the hydroxyl groups on the surface of RB-HNT can also participate in the formation of phenylboronic acid ester bonds, thereby increasing the crosslinking density of the polymer and reducing the swelling ratio of the hydrogel. Furthermore, this result is consistent with data obtained from scanning electron microscopy and rheological measurements.

[0056] VI. Ion and drug release from hydrogels.

[0057] A hydrogel sample with a height and diameter of 5 mm was immersed in 30 mL of PBS solution at 37 °C. 5 mL of supernatant was collected on days 1, 4, 7, 14, and 21. An equal volume of fresh PBS was then added to the leaching solution. The Si concentration in the leaching solution was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES).

[0058] Similarly, the release of royal glycoside B at 325 nm was detected using a micro-ultraviolet spectrophotometer.

[0059] The results are as follows Figure 12 As shown, RB is released rapidly in the first week, followed by a gradual decrease and stabilization of the release rate. This release kinetics of RB is beneficial for clearing reactive oxygen species and regulating the inflammatory microenvironment in the early stages of bone tissue repair, laying the foundation for subsequent new bone tissue regeneration. In contrast, bioactive Si maintains a relatively stable and slow release trend over 21 days. Notably, bioactive Si is released relatively quickly in the first 7 days, after which the release rate stabilizes over time. This pattern may be attributed to the easier degradation of HNTs located on the hydrogel surface, leading to an initial rapid release of silicon ions; subsequently, HNTs are slowly exposed to the liquid as the hydrogel degrades, resulting in a long-term, slow, and continuous release of silicon ions.

[0060] Example 3 Biocompatibility assessment of nanocomposite hydrogels.

[0061] Good cell compatibility is a necessary prerequisite for the application of hydrogels in bone tissue engineering. To evaluate the biocompatibility of the composite hydrogel and explore its optimal RB-HNT doping concentration, MTT assays and live / dead staining analyses were performed on RAW264.7 cells and bone marrow mesenchymal stem cells treated with different concentrations of HHPP hydrogel extract.

[0062] Five hydrogels with RB-HNT and HPP mass percentages of 0, 1, 2, 4, and 8% were prepared and named HPP, 1%, 2%, 4%, and 8%, respectively. The extracts from each group were then collected for subsequent experiments to culture RAW264.7 cells and bone marrow mesenchymal stem cells (BMSCs).

[0063] RAW264.7 and BMSC were compared at a ratio of 1×10⁻⁶. 4 / cm -2 The cells were initially seeded at a density of [missing information] into 48-well plates and cultured for a period of time to reach 90% confluence. Subsequently, the culture medium was replaced with 1 mL of HPP, 1% HHPP, 2% HHPP, 4% HHPP, and 8% HHPP hydrogel extract, respectively. After 24 hours, the cell culture medium was replaced with [missing information] supplemented with MTT (0.5 mg / mL). -1 1 mL of serum-free culture medium was added to each well. The plate was then incubated at 37°C for another 4 hours. After removing the medium containing formazan crystals (MTT), 1 mL of dimethyl sulfoxide was added to each well to dissolve the MTT. Finally, the absorbance of the blue solution was measured at 490 nm using an automated microplate reader. Relative cell viability was calculated as follows: .

[0064] in, and The absorbance values ​​are those of the well containing hydrogel and the control well (without hydrogel), respectively.

[0065] The results are as follows Figure 13 As shown, after RAW264.7 cells were cultured with the extract for 1, 3, and 5 days, the 1% HHPP and 2% HHPP groups exhibited good cell viability compared to the HPP group, while the cell survival rate of the 4% HHPP and 8% HHPP groups decreased significantly, demonstrating cytotoxicity. Meanwhile, the experimental results for bone marrow mesenchymal stem cells showed a similar trend to those for RAW264.7 cells.

[0066] Example 4 Study on the role of nanocomposite hydrogels in scavenging reactive oxygen species and regulating inflammation in vitro.

[0067] 200 μM hydrogen peroxide and 30 mM glucose were added to DMEM medium to simulate the high reactive oxygen species environment of diabetes, and used for in vitro culture of RAW264.7 cells and bone marrow mesenchymal stem cells.

[0068] In vitro study - intracellular ROS detection: After 3 days of culture, the reactive oxygen species (ROS) levels in bone marrow mesenchymal stem cells and RAW264.7 cells under high glucose and high ROS microenvironment were observed using the reactive oxygen species fluorescent probe DCFH-DA (cells were incubated with 500 μL DCFH-DA staining solution at 37°C for 30 minutes, then washed twice with PBS, and the samples were then analyzed by fluorescence microscopy).

[0069] The results are as follows Figure 14 As shown, both RAW264.7 cells and bone marrow mesenchymal stem cells exhibited strong green fluorescence signals in the HPP hydrogel group, indicating a high level of reactive oxygen species (ROS) accumulation in these cells. In contrast, the fluorescence intensity of cells in the 1% HHPP and 2% HHPP composite hydrogel groups was significantly reduced, and the difference was statistically significant, indicating that the 1% HHPP and 2% HHPP composite hydrogel groups could effectively scavenge ROS from the environment and within cells. The semi-quantitative analysis results of fluorescence microscopy were consistent with the qualitative observations.

[0070] Detection of the fluorescent probe JC-1: When the mitochondrial membrane potential is normal, JC-1 forms aggregates, while when the membrane potential is decreased, it exists in monomeric form. Results are as follows: Figure 15 As shown, under simulated diabetic pathological microenvironment conditions, the HPP group exhibited more green fluorescence, while the green fluorescence of the 1%HHPP and 2%HHPP groups was significantly reduced, and the red fluorescence intensity showed the opposite trend. Semi-quantitative analysis revealed that, compared with the HPP group, the 1%HHPP and 2%HHPP groups effectively increased the mitochondrial membrane potential level of bone marrow mesenchymal stem cells, with the 2%HHPP group showing a particularly significant effect. This indicates that the 1%HHPP and 2%HHPP groups can effectively enhance the mitochondrial membrane potential level in the pathological microenvironment, reverse mitochondrial damage in bone marrow mesenchymal stem cells, restore their normal function, and lay the foundation for further promoting their osteogenic differentiation.

[0071] Immunofluorescence analysis: After 3 days of culture, cells were fixed in 4% paraformaldehyde for 20 minutes and incubated in 0.2% Triton 100X for 15 minutes. Subsequently, the samples were blocked with 5% BSA for 1 hour. The relevant primary antibodies, including CD86, Arg-1, and BMP-2 antibodies, were added to the samples and incubated overnight at 4°C. The next day, FITC 488 and DAPI were incubated with the samples at 37°C for 1 hour. Before each step, the samples were washed three times with PBS for 5 minutes each time. Finally, images were captured using a fluorescence microscope. Immunofluorescence results are shown below. Figure 16As shown, compared with the HPP control group, the staining of the M1 marker CD86 was weakened in the 1%HHPP and 2%HHPP groups, while the staining of the M2 marker Arg-1 was significantly enhanced, indicating that the material can effectively promote the activation of M2 macrophages.

[0072] In vitro inflammation study: qRT-PCR was used to detect the expression of inflammatory genes, with triglyceride dehydrogenase (GAPDH) as an internal control. Inflammatory genes detected included TNF-α, Arg-1, IL-10, and IL-1β. Results are as follows: Figure 17 As shown, compared with the HPP group, the expression of pro-inflammatory genes was downregulated and the expression of anti-inflammatory genes was significantly upregulated in the 1% HHPP and 2% HHPP groups. Therefore, the 1% HHPP and 2% HHPP groups can effectively promote macrophage polarization to the M2 phenotype while inhibiting the M1 phenotype, thereby alleviating the pro-inflammatory response in the chronic inflammatory state of diabetes and maintaining the homeostasis of the immune microenvironment, which is of great significance for the bone regeneration process.

[0073] Example 5 Tests on the effect of nanocomposite hydrogels on promoting osteogenic differentiation of bone marrow mesenchymal stem cells under diabetic conditions.

[0074] In vitro osteogenic differentiation of BMSCs: The in vitro osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) was assessed using an alkaline phosphatase (ALP) staining kit, an ALP activity assay kit, and a commercially available osteoblast mineralization nodule staining solution (Alizarin Red staining).

[0075] like Figure 18 As shown in the figure, the results indicate that the alkaline phosphatase expression levels in the 1%HHPP and 2%HHPP groups were significantly higher than those in the HPP group.

[0076] like Figure 19 As shown, after 14 and 21 days of culture, the alizarin red staining results showed the same trend, and quantitative analysis of alkaline phosphatase and alizarin red also confirmed the statistical differences between groups. The expression level of bone morphogenetic protein 2 (BMP-2) was assessed by immunofluorescence staining. Figure 20 As shown, compared with the control group, the expression of bone morphogenetic protein 2 was significantly increased in the 1% HHPP and 2% HHPP groups, and semi-quantitative analysis also confirmed the difference between the groups.

[0077] In vitro osteogenic study: The expression of osteogenic genes was detected by qRT-PCR, with triglyceride dehydrogenase (GAPDH) as an internal reference.

[0078] like Figure 21As shown, quantitative polymerase chain reaction (PCR) analysis was used to assess the expression of osteogenic-related genes in bone marrow mesenchymal stem cells (BMSCs). Compared with the control group, the expression levels of alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) were significantly upregulated in the 1% HHPP and 2% HHPP groups. This indicates that the material can effectively alleviate the inhibitory effect of high glucose culture on osteogenic differentiation while promoting osteogenic differentiation. Therefore, the RB-HNT composite nanoparticles release lily glycoside B, which synergistically enhances the osteogenic differentiation capacity of BMSCs with bioactive silicon.

[0079] Example 6 Tests on the acceleration of bone regeneration in diabetic patients using nanocomposite hydrogels.

[0080] In vivo studies – Establishment of a diabetic rat model: Thirty healthy, 4-week-old male SD rats of similar size were used. Twenty-four of these rats were randomly selected and fed a high-fat diet (HFD) for two months to establish an insulin-resistant rat model. A low-dose streptozotocin solution dissolved in citrate buffer (pH 4.5) was then injected intraperitoneally to induce diabetes. Successful modeling of diabetes was confirmed by monitoring non-fasting blood glucose levels (>16.7 mM) and observing stable hyperglycemia for one week.

[0081] In vivo study - Construction of a rat femoral condyle defect model: The in vivo bone regeneration capacity of the composite hydrogel was evaluated using a diabetic rat femoral condyle defect model. The control group consisted of 6 healthy SD rats. The 24 diabetic rats were randomly divided into 4 groups: DM (streptozotocin group, n=6), DM+HPP group (n=6), DM+1%HHPP group (n=6), and DM+2%HHPP group (n=6). 2% sodium pentobarbital solution (50 mg / kg) was used. -1 Abdominal anesthesia was administered. The surgical site was carefully disinfected and covered with sterile drapes. After incision, the femoral condyle was carefully dissected and exposed through the intermuscular spaces. Cortical bone from the medial and lateral aspects of the femoral condyle was drilled using a 3mm diameter drill bit. After rinsing the bone defect area with saline, hydrogel was implanted into the area for each group, while the control group and the DM group received saline injections. Finally, the wound was meticulously sutured layer by layer, and penicillin was administered intramuscularly three days postoperatively to prevent infection.

[0082] In vivo studies - imaging analysis: Rats were euthanized 4 weeks post-surgery. Femoral tissue was then harvested and preserved in 4% paraformaldehyde solution for subsequent analysis. Scans were performed using a micro-CT scanner. The cylindrical region at the center of the 3 mm diameter bone defect was analyzed using DataView and CT Analyzer software to determine parameters such as BV / TV, BMD, Tb.th, and Tb.Sp (43n=3). 3D reconstruction was performed using Mimics software.

[0083] The results are as follows Figure 22 As shown, the new bone formation capacity in the streptozotocin group was weaker than that in the control group. Compared with the streptozotocin group and the control group, the HPP, 1%HHPP, and 2%HHPP groups showed better osteogenic effects, which was attributed to the superior osteoconductivity of the implant material. Compared with the HPP group, the 1%HHPP and 2%HHPP groups showed a significant increase in bone formation. Furthermore, as... Figure 23 As shown, quantitative analysis of the microstructural parameters of the defect area further confirms that the 1% HHPP and 2% HHPP groups have a greater advantage in promoting bone regeneration.

[0084] In vivo studies - histological analysis: The femoral specimen was CT scanned, then decalcified with 10% EDTA, dehydrated, and embedded in paraffin. The sagittal plane of the femoral condyle was obtained with a thickness of 6 μm. Subsequently, H&E and Masson staining were performed to assess bone regeneration in the surgical area.

[0085] The results are as follows Figure 24 As shown, the femoral defect sites in all groups were partially filled with new bone and connective tissue, with new bone formation mainly occurring at the edges of the defect area. Compared to the other three groups, the control group and the streptozotocin group had the least amount of bone tissue and collagen fibers in the defect sites, possibly due to the lack of necessary cell adhesion and attachment interfaces. The 2% HHPP group had the most new bone and collagen fibers, followed by the 1% HHPP group and the HPP group, which is consistent with the microCT results. These results confirm that nanocomposite hydrogels can effectively promote bone regeneration in vivo.

[0086] Immunohistochemical staining: Immunofluorescence staining was used to detect CD86, CD206, and OCN markers to evaluate the immunomodulatory and osteogenic capabilities of the implanted biomaterial. Four weeks post-operation, histological sections and H&E staining (OCN, BMP-2, CD86, and CD206 staining) were performed on the five groups of tissue sections.

[0087] Immunohistochemical staining images such as Figure 25 As shown, the semi-quantitative analysis results are as follows: Figure 26 As shown, the expression levels of OCN and BMP-2 proteins in the 1% HHPP and 2% HHPP groups were significantly higher than those in the control and streptozotocin groups, consistent with in vitro experiments and microCT results. Simultaneously, the expression level of the M1 macrophage marker CD86 in the control and streptozotocin groups was significantly higher than that in the 1% HHPP and 2% HHPP groups; while the expression of the M2 marker CD206 showed the opposite trend. These results indicate that the material can promote macrophage polarization towards the M2 phenotype in vivo, consistent with in vitro observations.

[0088] In summary, the nanocomposite hydrogel of this invention has the effects of reducing tissue inflammation in diabetic rats, promoting the transformation of macrophages to an anti-inflammatory phenotype, and promoting osteogenic formation. Overall, the developed hydrogel can promote the transformation of healing-promoting macrophage phenotypes, alleviate local inflammation, and promote bone formation in diabetic rats.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a nanocomposite hydrogel for bone repair in diabetes, characterized in that, Includes the following steps: 1) Preparation of oxidized hyaluronic acid: Sodium hyaluronate was added to PBS buffer solution and stirred to dissolve. Sodium periodate was added and stirred under light-protected conditions. Ethylene glycol was added and stirring was continued. The resulting solution was dialyzed and lyophilized to obtain solid OHA. 2) Preparation of phenylboronic acid modified oxidized hyaluronic acid: Add water to the OHA solid obtained in step 1), stir to dissolve, add 3-aminophenylboronic acid, continue stirring, filter, dialyze, and freeze dry to obtain OHA-PBA solid; 3) Preparation of halloysite nanotubes loaded with limonene B: Limonene B solution was prepared with ethanol, halloysite nanotubes were added to the solution, stirred and sonicated to obtain a mixture; then the mixture was placed in a vacuum environment and allowed to stand, and freeze-dried to obtain RB-HNT powder. 4) Preparation of nanocomposite hydrogel: Dissolve the OHA-PBA solid obtained in step 2), add RB-HNT powder, stir, add PVA solution, and continue stirring to obtain nanocomposite hydrogel.

2. The preparation method according to claim 1, characterized in that, In step 1), the pH of the PBS buffer solution was 5, and the addition volume was 30 mL; the addition volume of sodium hyaluronate was 1.0 g; the addition volume of sodium periodate was 0.401 g; the reaction time under light-protected conditions was 18 h; the addition volume of ethylene glycol was 1 mL, and the stirring time was continued for 20 min after addition; dialysis was performed using a dialysis bag with a molecular weight cutoff of 3500 D, the dialysis medium was ultrapure water, the dialysis time was 48 h, and the dialysis medium was changed every 24 h; the lyophilization conditions were -100~-50℃, 5~50 Pa, and the lyophilization time was 20~24 h.

3. The preparation method according to claim 1, characterized in that, The water mentioned in step 2) is ultrapure water, and the addition amount is 30 mL; the addition amount of 3-aminophenylboronic acid is 680 mg, and the stirring time is continued for 45 min; dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 D, the dialysis medium is ultrapure water, the dialysis time is 24 h, and the dialysis medium is replaced every 12 h; the lyophilization conditions are -100~-50℃, 5~50 Pa, and the lyophilization time is 20~24 h.

4. The preparation method according to claim 1, characterized in that, In step 3), the ethanol is a 50% ethanol solution by volume, the volume of the prepared Wangbaihe glycoside B solution is 8 mL, and the concentration is 250 μg / mL; the amount of halloysite nanotubes added is 1 g; the stirring time is 2 h; the ultrasonic treatment power is 120~200 W, the ultrasonic frequency is 40 Hz, and the ultrasonic time is 1 h; the standing time under vacuum is 12 h; the freeze-drying temperature is -100~-50℃, and the drying time is 24 h.

5. The preparation method according to claim 1, characterized in that, In step 4), the concentration of the OHA-PBA solid dissolved in ultrapure water is 40 mg / mL; the amount of RB-HNT powder added is 1%~2% of the mass of OHA-PBA; the concentration of the PVA solution is 80 mg / mL, and the volume ratio of OHA-PBA solution to PVA solution is 1:3; the stirring time after adding RB-HNT is 30 min; and the stirring time after adding PVA solution is 2 min.

6. A nanocomposite hydrogel for bone repair in diabetes, characterized in that, It is prepared according to the preparation method according to any one of claims 1 to 5.

7. The nanocomposite hydrogel according to claim 6, characterized in that, The nanocomposite hydrogel is injectable and has self-healing capabilities.

8. The application of the nanocomposite hydrogel as described in claim 6 in the preparation of biomaterials for repairing bone defects in diabetic patients.

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