Hydrogel scaffold material for sustained release of hydrogen and magnesium ions and preparation method and application thereof

Abstract

The application belongs to the technical field of bone defect repair materials, and particularly relates to a hydrogel scaffold material capable of continuously releasing hydrogen and magnesium ions, a preparation method and application. The preparation method of the hydrogel scaffold material provided by the application combines gas foaming and photocuring technologies on the basis of synthesizing magnesium hydride microspheres coated with polylactic acid-glycolic acid copolymer (PLGA) and methacryl gelatin to obtain a novel hydrogel scaffold material. Tests prove that the scaffold material prepared by the application can reduce the generation of ROS, promote the transformation of macrophages from M1 type to M2 type, enhance cell migration, improve local inflammation and oxidative stress, and jointly promote the angiogenesis and bone regeneration of diabetic bone defects. Therefore, the scaffold material of the application can provide a new treatment strategy and research direction for diabetic bone defects, and has a wide application prospect in the treatment of diabetic bone defect patients.

Description

Technical Field

[0001] This invention belongs to the field of bone defect repair materials technology, specifically relating to a hydrogel scaffold material that continuously releases hydrogen and magnesium ions, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease that seriously threatens public health. Treating bone defects caused by trauma, tumors, infections, or other factors in diabetic patients presents a significant clinical challenge. Compared to non-diabetic individuals, the diabetic environment, characterized by hyperglycemia, not only disrupts the inflammatory response but also promotes the production of reactive oxygen species (ROS) and microvascular complications, all of which prolong the healing process of bone defects. These factors contribute to a higher incidence of nonunion in diabetic patients. Although several clinical interventions exist for treating bone defects, the unique complexity of the diabetic environment significantly limits their efficacy. Therefore, developing biomaterials that can modulate the inflammatory environment, reduce oxidative stress, and promote angiogenesis to enhance the regeneration and repair of bone defects in the diabetic environment is a pressing technical challenge that needs to be addressed in the medical field.

[0003] Currently, clinical synthetic materials used for bone defect reconstruction, such as bone cement, certain bioceramics, and specific metals, exhibit limitations in terms of vascularization, cell migration, degradation kinetics, and biocompatibility. Hydrogels, as an emerging material that mimics the natural extracellular matrix (ECM), are promising scaffold materials due to their excellent injectability, extensibility, and compatibility with various bioactive components.

[0004] Hydrogen (H2), as an antioxidant, selectively reduces reactive oxygen species (ROS) and converts them into water without disrupting their physiological functions or other biomolecules. In vitro studies have shown that hydrogen can inhibit rankl-mediated NF-κB pathway activation and suppress osteoclast differentiation, representing a promising approach to addressing bone resorption. However, the low solubility and rapid dissipation of hydrogen pose challenges to maintaining consistent therapeutic concentrations for local treatment, particularly in diabetic bone defects. Meanwhile, magnesium ions have been used to enhance bone stability and promote an anti-inflammatory environment. However, magnesium hydroxide generated during the application of magnesium materials physiologically hinders the release of therapeutic magnesium ions, further limiting its effectiveness.

[0005] Therefore, designing a biomaterial that can regulate inflammatory responses, clear ROS, promote angiogenesis, and provide mechanical support is of great significance for advancing the treatment of bone defects in diabetic patients. Summary of the Invention

[0006] To address the aforementioned problems, one objective of this invention is to provide a method for preparing a hydrogel scaffold material that continuously releases hydrogen and magnesium ions. The scaffold material prepared using this method can reduce ROS, promote M2 macrophage polarization, improve cell migration, and alleviate local inflammation, thereby promoting angiogenesis and bone regeneration, and providing a new and feasible strategy for the treatment of diabetic bone defects.

[0007] The second objective of this invention is to provide a hydrogel scaffold material that continuously releases hydrogen and magnesium ions, prepared by the above-mentioned method.

[0008] A third objective of this invention is to provide the application of the aforementioned hydrogel scaffold material that continuously releases hydrogen and magnesium ions.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a hydrogel scaffold material that continuously releases hydrogen and magnesium ions includes the following steps:

[0011] (1) Dissolve polylactic acid-glycolic acid copolymer in a solvent, then add magnesium hydride and mix to obtain a PLGA+MgH2 mixed solution; add the PLGA+MgH2 mixed solution to a polyethylene glycol solution and stir to mix, then evaporate the solvent, centrifuge, take the precipitate and dry to obtain magnesium hydride microspheres coated with polylactic acid-glycolic acid copolymer, denoted as MgH2@PLGA microspheres;

[0012] Separately, gelatin was dissolved in water to swell, then methacrylic anhydride was added and mixed, followed by dialysis, centrifugation, and freeze-drying to obtain methacryloyl gelatin; the methacryloyl gelatin was dissolved to obtain a methacryloyl gelatin solution, denoted as GelMA solution;

[0013] (2) The MgH2@PLGA microspheres and GelMA solution obtained in step (1) are mixed with air to foam the foamed material. The foamed material is then injected into a mold and cross-linked and cured under ultraviolet conditions to obtain the hydrogel scaffold material that continuously releases hydrogen and magnesium ions, denoted as MgH2@PLGA / F-GM.

[0014] As a further preferred embodiment, in step (1), the solvent is one or more selected from 1,4-dioxane, dimethyl sulfoxide, chloroform, and dichloromethane. More preferably, the solvent is 1,4-dioxane.

[0015] As a further preferred embodiment, the ratio of the polylactic acid-glycolic acid copolymer, solvent, and magnesium hydride is (8-12) mg : (0.8-1.2) mL : (8-12) mg. More preferably, the ratio of the polylactic acid-glycolic acid copolymer, solvent, and magnesium hydride is 10 mg : 1 mL : 10 mg.

[0016] As a further preferred embodiment, in step (1), the polyethylene glycol solution is a dimethyl sulfoxide solution of polyethylene glycol; the concentration of polyethylene glycol in the polyethylene glycol solution is 2-10 mg / mL. More preferably, the concentration of polyethylene glycol in the polyethylene glycol solution is 2 mg / mL.

[0017] As a further preferred embodiment, the mass ratio of polyethylene glycol, polylactic acid-glycolic acid copolymer, and magnesium hydride is 1–3:1–5:1–5. More preferably, the mass ratio of polyethylene glycol, polylactic acid-glycolic acid copolymer, and magnesium hydride is 3:5:5.

[0018] As a further preferred embodiment, in step (1), the ratio of gelatin to methacrylic anhydride is 8-12 g : 5-7 mL. More preferably, the ratio of gelatin to methacrylic anhydride is 10 g : 6 mL.

[0019] As a further preferred option, in step (1), the dialysis is performed using a dialysis bag with a capacity of 8-14 kDa.

[0020] As a further preferred embodiment, in step (1), the dissolution is to dissolve methacryloyl gelatin in a lithium phenyl (2,4,6-trimethylbenzoyl)phosphonate solution to obtain a methacryloyl gelatin solution.

[0021] As a further preferred option, in step (2), the foaming is carried out using a three-way valve connected to at least two syringes. The GelMA solution and MgH2@PLGA microspheres are mixed by the reciprocating push of different syringes, so that air is evenly distributed in the mixed hydrogel to form a foamed material.

[0022] As a further preferred option, in step (2), the wavelength of the ultraviolet light used for crosslinking curing is 400-410 nm.

[0023] A hydrogel scaffold material that continuously releases hydrogen and magnesium ions was prepared by the preparation method described above.

[0024] As a further preferred option, the hydrogel scaffold material is an injectable scaffold.

[0025] Application of a hydrogel scaffold material that continuously releases hydrogen and magnesium ions as described above in the preparation of scaffold materials for repairing bone defects.

[0026] As a further preferred option, the bone defect is a diabetic bone defect.

[0027] The special advantages and beneficial effects of the technical solution of this invention are as follows:

[0028] Currently, diabetes-related bone defects pose a significant challenge to healing, and this challenge is exacerbated by hyperglycemia-induced inflammation and oxidative stress. Current treatments are insufficient to address this complex environment, thus creating an urgent need for materials capable of modulating inflammation and promoting angiogenesis.

[0029] The present invention provides a method for preparing a hydrogel scaffold material that continuously releases hydrogen and magnesium ions. First, it involves the preparation of magnesium hydride microspheres (MgH2@PLGA) encapsulated with polylactic acid-glycolic acid copolymer and a methacryloyl gelatin (GelMA) solution. Then, based on the synthesis of MgH2@PLGA microspheres and GelMA, a novel hydrogel scaffold material (MgH2@PLGA / F-GM) is obtained using a combination of gas foaming and photocuring techniques. This preparation method is simple, easy to implement, and the resulting scaffold material, composed of foamed methacryloyl gelatin mixed with polylactic acid-glycolic acid copolymer-encapsulated magnesium hydride microspheres, is injectable.

[0030] Furthermore, the scaffold material prepared by the above method can provide certain mechanical support for bone defects and can also conform to complex bone defect shapes to form in-situ bone tissue engineering regeneration scaffolds, thus regulating the diabetic microenvironment. When the scaffold degrades, MgH2 generates magnesium hydroxide (Mg(OH)2), while PLGA degrades to release lactic acid and glycolic acid. The released acidic byproducts promote the dissolution of the Mg(OH)2 layer, thereby enhancing the therapeutic effect of magnesium ions (Mg... 2+ The release of Mg. 2+ It is key to enhancing macrophage phenotypic transformation, reducing inflammation, and promoting angiogenesis, and is crucial for the healing of diabetic bone defects. On the one hand, Mg 2+ It can induce macrophage phenotypic changes, alleviate the pro-inflammatory microenvironment, and regulate local inflammatory responses. On the other hand, Mg 2+ It helps reduce microvascular complications associated with hyperglycemia, promotes angiogenesis, and accelerates blood vessel formation to support ossification. Furthermore, the hydrogen (H2) released from the degradation of MgH2 in the scaffold material of this invention can combat oxidative stress by reducing the production of reactive oxygen species (ROS).

[0031] In summary, the scaffold material provided by this invention reduces ROS, promotes M2 macrophage polarization, improves cell migration, alleviates local inflammation, and promotes angiogenesis and bone regeneration. In particular, multiple in vitro and in vivo studies have demonstrated that the PLGA-coated MgH2 microspheres within the aerosol photocured hydrogel scaffold provided by this invention can reduce ROS production, promote the conversion of macrophages from M1 to M2 polarization, enhance cell migration, and improve local inflammation and oxidative stress. These effects promote angiogenesis and bone regeneration in diabetic bone defects. Therefore, the scaffold material provided by this invention can offer new treatment strategies and research directions for diabetic bone defects and has broad application prospects in the rehabilitation treatment of patients with diabetic bone defects. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the preparation route of magnesium hydride microspheres (MgH2@PLGA) encapsulated by polylactic acid-glycolic acid copolymer in step (1) of the present invention;

[0033] Figure 2 This is a schematic diagram of the preparation route of the hydrogel scaffold material (MgH2@PLGA / F-GM) that continuously releases hydrogen and magnesium ions in step (3) of the present invention;

[0034] Figure 3 The images show SEM images of the MgH2@PLGA microspheres obtained in step (1) of this invention (right image) and the MgH2 microspheres before encapsulation (left image);

[0035] Figure 4 The images show the XRD patterns of the MgH2@PLGA microspheres obtained in step (1) of this invention and the MgH2 microspheres before encapsulation.

[0036] Figure 5 The appearance and SEM spectra of the MgH2@PLGA / F-GM material of Example 1, the F-GM scaffold material of Comparative Example 1, and the MgH2 / F-GM scaffold material of Comparative Example 2 are shown.

[0037] Figure 6 The mechanical property test results are for the MgH2@PLGA / F-GM material of Example 1, the F-GM scaffold material of Comparative Example 1, and the MgH2 / F-GM scaffold material of Comparative Example 2 of the present invention.

[0038] Figure 7 The degradation rate of the MgH2@PLGA / F-GM material in Example 1 of this invention in pure water, PBS, and modified simulated body fluid (M-SBF);

[0039] Figure 8The MgH2@PLGA / F-GM material prepared in this invention and the MgH2 / F-GM material of Comparative Example 2 release H2 and Mg in vitro. 2+ The release curve;

[0040] Figure 9 The results show the effects of the MgH2@PLGA / F-GM material of Example 1, the F-GM scaffold material of Comparative Example 1, and the MgH2 / F-GM scaffold material of Comparative Example 2 on cytotoxicity and viability.

[0041] Figure 10 Fluorescence imaging of the effect of MgH2 powder, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention on ROS content after in vitro treatment of Raw264.7 cells;

[0042] Figure 11 The quantitative results of ROS content in Raw264.7 cells after in vitro treatment of MgH2 powder, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention;

[0043] Figure 12 SEM images of MgH2 powder, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention after in vitro treatment on Raw264.7 cells;

[0044] Figure 13 The results of in vitro treatment of Raw264.7 cells with MgH2 powder, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention on the content of biomarkers IL-6, IL-1β, and Arg-1 were obtained.

[0045] Figure 14 The results of cell migration and void closure after treatment with scratches using MgH2, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention are shown.

[0046] Figure 15 The results of cell angiogenesis after treatment with MgH2, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention are shown.

[0047] Figure 16 The quantitative results of the healing area, number of nodes, tube length, and vascular markers after treating cells with MgH2, MgH2 / F-GM material of Comparative Example 2, and MgH2@PLGA / F-GM material of Example 1 of this invention;

[0048] Figure 17 The results show the ratio of skull bone volume to tissue volume in diabetic mice from the control group, MgH2 group, MgH2 / F-GM group, and MgH2@PLGA / F-GM group.

[0049] Figure 18 The images show the results of osteogenic (OCN) and angiogenesis (CD31) measurements in the skulls of diabetic mice treated with the control group, MgH2 group, MgH2 / F-GM group, and MgH2@PLGA / F-GM group. Detailed Implementation

[0050] The present invention will be further described in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely illustrative of the invention and not intended to limit it. Unless otherwise specified, the reagents used in the following embodiments are commercially available.

[0051] In the following examples, the polylactic acid-glycolic acid copolymer (PLGA) used was from Sigma-Aldrich, with a molecular weight of 38,000 to 54,000 and catalog number 719900-5G; the polyethylene glycol was from Aladdin, with a molecular weight of 1,000 and catalog number P103719-500g; the methacrylic anhydride was from Sigma-Alorich, with a molar mass of 154.16 g / mol; and the gelatin had a molecular weight of 300,000 to 1,000,000 Da.

[0052] Example 1

[0053] This embodiment provides a hydrogel scaffold material that continuously releases hydrogen and magnesium ions and its preparation method, including the following steps:

[0054] (1) Preparation of magnesium hydride microspheres (MgH2@PLGA) encapsulated by polylactic acid-glycolic acid copolymer

[0055] The operation diagram for this step is as follows: Figure 1 As shown. Specifically: 10 mg of polylactic acid-glycolic acid copolymer (PLGA) was weighed and dissolved in 1 mL of 1,4-dioxane and stirred for 30 min. Then, 10 mg of magnesium hydride (MgH2) was added and stirring was continued to obtain a PLGA+MgH2 mixed solution.

[0056] In addition, 6 mg of polyethylene glycol (PEG) was weighed and dissolved in 3 mL of dimethyl sulfoxide (DMSO), and stirred until dissolved to obtain a PEG solution.

[0057] The obtained PLGA+MgH2 mixed solution was slowly added dropwise to a PEG solution under high-speed stirring. The organic solvent in the resulting mixed solution was then evaporated overnight (>6h). The next day, the mixed solution was centrifuged at room temperature in a high-speed centrifuge. The supernatant was discarded, and the precipitate was dried in a 40℃ drying oven to obtain magnesium hydride microspheres encapsulated by polylactic acid-glycolic acid copolymer, denoted as MgH2@PLGA.

[0058] (2) Preparation of methacryloyl gelatin (GelMA) solution

[0059] Add 10g of gelatin to 100mL of double-distilled water and let it swell at room temperature for 1 hour. Stir in a 60℃ water bath until the gelatin dissolves. Next, add 6mL of methacrylic anhydride (MA) to the gelatin solution at a rate of 1mL / min. Shake the resulting mixture at 50℃ for 3 hours. Then add 400mL of double-distilled water and dialyze the diluted solution into an 8-14kDa dialysis bag for one week. Change the dialysate every 4 hours. Centrifuge the solution at 3000rpm for 10 minutes, discard the precipitate, and freeze-dry at -80℃ to obtain methacryloyl gelatin (GelMA). Dissolve the freeze-dried GelMA in lithium phenyl (2,4,6-trimethylbenzoyl)phosphonate (LAP) solution to obtain a 10% GelMA solution (prepared by dissolving 100mg of GelMA in 1mL of LAP solution). The LAP solution had a mass concentration of 0.25% and was prepared by dissolving LAP powder in PBS.

[0060] (3) Preparation of MgH2@PLGA / F-GM hydrogel scaffold

[0061] The operation diagram for this step is as follows: Figure 2 As shown. The GelMA solution obtained in step (2) and the MgH2@PLGA microspheres obtained in step (1) were foamed using a medical three-way valve, and then cross-linked in a mold using a 405nm ultraviolet lamp to obtain the hydrogel scaffold material of Example 1 (cylindrical, 6mm in diameter, 2.5mm thick, the shape and size can be changed according to actual needs).

[0062] Specifically, the foaming device used in the foaming process is a medical three-way connector (Ming An Kang, China), which connects two syringes. One syringe contains the GelMA solution obtained in step (2) and air; the other syringe contains the MgH2@PLGA microsphere powder obtained in step (1) and air. During gas foaming, the two ends of the syringe are pushed about 100 times to mix the GelMA solution and MgH2@PLGA microsphere powder. The mechanical shear force generated by repeatedly pushing the two ends of the syringe makes the air evenly distributed in the hydrogel, forming a foaming material, denoted as F-GM.

[0063] F-GM was injected into a designated mold and crosslinked using 405nm blue light. Because the blue light penetrating power of crosslinked F-GM is weak, a large amount of F-GM could not be injected simultaneously during the process. Therefore, a layered injection method was used, injecting only a thin layer of F-GM at a time, followed by photocuring. The next layer was then injected until the entire scaffold was completed, resulting in the hydrogel scaffold material that continuously releases hydrogen and magnesium ions, denoted as MgH2@PLGA / F-GM.

[0064] Comparative Example 1

[0065] This comparative example provides a methacrylamide gelatin hydrogel scaffold, denoted as F-GM, which is prepared by the following method:

[0066] In this comparative example, the foaming process followed the method of Example 1, and the foaming device used was a medical three-way connector (Ming An Kang, China), which connected two syringes. The preparation process of F-GM was as follows: Take the 10% GelMA solution prepared in step (2) of Example 1, use one syringe to draw up the GelMA solution, and leave the other syringe with air. That is, the two syringes contain GM-LAP solution and air respectively. Push the two ends of the syringe alternately about 100 times. Through the mechanical shear force generated by pushing the two ends of the syringe repeatedly, the air is evenly distributed in the hydrogel to form F-GM. Inject F-GM into the designated mold and crosslink it using 405nm blue light. Since the blue light penetration of crosslinked F-GM is weak, a large amount of F-GM cannot be injected at the same time. Therefore, a layered injection method is used, injecting only a thin layer of F-GM each time, and then performing photocuring. Then inject the next layer until the entire stent is injected.

[0067] Comparative Example 2

[0068] This comparative example provides a magnesium hydroxide-methacryl gelatin hydrogel scaffold, denoted as MgH2 / F-GM, which is prepared by the following method:

[0069] In this comparative example, the foaming process followed the method of Example 1. The foaming device used was a medical three-way valve (Ming An Kang, China), which connected two syringes. The preparation process of MgH2 / F-GM was as follows: Take the 10% GelMA solution prepared in step (2) of Example 1, use one syringe to draw up the GelMA solution, denoted as GM, and leave air + MgH2 powder in the other syringe. That is, the two syringes contain GM-LAP solution and air + MgH2 powder respectively. Push the two ends of the syringe alternately about 100 times. Through the mechanical shear force generated by pushing the two ends of the syringe repeatedly, the air is evenly distributed in the hydrogel to form MgH2 / F-GM. Inject MgH2 / F-GM into the designated mold and crosslink it using 405nm blue light. The same layer injection method was used, injecting only a thin layer of MgH2 / F-GM each time, and then photocuring it. Then inject the next layer until the entire scaffold is injected.

[0070] Test case

[0071] In the following experimental examples, all data were collected in triplicate or in triplicate and are presented as mean and standard deviation. Except for the use of Image J to analyze scratch area and angiogenesis data, all data were evaluated using GraphPad. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 were considered statistically significant, and ns indicated no statistically significant difference.

[0072] Example 1: Characterization of MgH2@PLGA microspheres

[0073] In step (1) of Example 1 of this invention, MgH2 is encapsulated in PLGA to form microspheres, which protect it from water damage and achieve the reaction of H2 and Mg. 2+ The slow release of the microspheres was named MgH2@PLGA. In this experiment, the MgH2@PLGA microspheres obtained after drying in step (1) of Example 1 and the MgH2 before encapsulation were detected by scanning electron microscopy (ZEISS GeminiSEM 300, Germany) and XRD (Rigaku MiniFlex600, Japan).

[0074] The dried microspheres were photographed using a scanning electron microscope, and particle size analysis was performed using ImageJ software. The SEM image results are shown below. Figure 3 As shown. Figure 3 The results show that the spherical morphology of MgH2 (left figure) and MgH2@PLGA (right figure) is relatively uniform, and the average diameters of the two are 19.1 μm and 21.2 μm after particle size analysis.

[0075] Furthermore, this invention utilizes XRD to further verify the purity of MgH2 and MgH2@PLGA, with results as follows: Figure 4 As shown. Figure 4 The XRD results showed that MgH2 and MgH2@PLGA have similar peak structures, indicating that MgH2@PLGA microspheres were successfully synthesized.

[0076] Experimental Example 2: Morphological Characterization and Performance Testing of Scaffold Materials

[0077] 2.1 Morphological observation

[0078] The appearance and pore size of the MgH2@PLGA / F-GM scaffold material prepared in Example 1 of this invention, as well as the F-GM scaffold material of Comparative Example 1 and the MgH2 / F-GM scaffold material of Comparative Example 2, were observed and analyzed by the naked eye and scanning electron microscopy. Porosity was calculated using ImageJ software. Furthermore, the surface morphology and microstructure of the freeze-dried samples were observed using scanning electron microscopy (SEM, GeminiSEM 300, ZEISS). The appearance (top image) and corresponding SEM images (bottom image) of different scaffold materials are shown below. Figure 5 As shown.

[0079] Depend on Figure 5 Visually, the foamed and freeze-dried F-GM and MgH2 / F-GM appear white, while MgH2@PLGA / F-GM appears white with a slight yellow tinge. This is related to the addition of PLGA. Scanning electron microscopy revealed relatively uniform air bubbles within F-GM, MgH2 / F-GM, and MgH2@PLGA / F-GM. ImageJ software was used to analyze the porosity of F-GM, MgH2 / F-GM, and MgH2@PLGA / F-GM. Porosity tests showed no significant difference between MgH2 / F-GM and MgH2@PLGA / F-GM and pure F-GM; however, the larger pore structure was more conducive to oxygen and nutrient transport, blood vessel ingrowth, and cell migration.

[0080] 2.2 Evaluation of Mechanical Properties

[0081] Based on the previous synthesis of MgH2@PLGA microspheres, a combination of gas foaming and photocuring techniques was used to produce foamed F-GM, which provides mechanical support for bone defects. To further evaluate the performance of different F-GMs, mechanical and degradation tests were conducted. Compression tests were performed on the MgH2@PLGA / F-GM scaffold material prepared in Example 1, the F-GM scaffold material of Comparative Example 1, and the MgH2 / F-GM scaffold material of Comparative Example 2 using a mechanical testing machine (INSTRON-5542, China). Compression was carried out at a rate of 5 mm / s on the mechanical testing machine. All tests were conducted at room temperature, with each group repeated three times. The results are as follows: Figure 6 As shown.

[0082] Depend on Figure 6 It is known that foaming significantly increases the elasticity of the hydrogel. When the hydrogel is compressed to 70% of its original height, MgH2 / F-GM and MgH2@PLGA / F-GM have better elasticity than F-GM. In particular, the MgH2@PLGA / F-GM material of Example 1 of this invention exhibits significantly better elasticity than other materials, thus giving the scaffold material good injectability.

[0083] 2.3 In vitro degradation test

[0084] To evaluate the in vitro degradation of the MgH2@PLGA / F-GM material obtained in Example 1 of this invention, the scaffold (n=3) was immersed in PBS, pure water, and simulated body fluid for 30 days at room temperature. The initial weight (W0) of the hydrogel was weighed after vacuum drying. Then, after treatment in PBS, pure water, and simulated body fluid for 5, 10, 15, 20, 25, and 30 days, the remaining material was removed, vacuum dried, and weighed (W1). All experiments were repeated three times. The degradation rate was calculated using the following formula: D = W0 - W1 / W0 × 100%. The results are as follows: Figure 7 As shown.

[0085] Figure 7 In this study, the degradation rate of MgH2@PLGA / F-GM over 30 days was measured in pure water, PBS, and modified simulated body fluid (M-SBF). SBF is a metastable solution, a supersaturated solution of apatite containing calcium and phosphate ions, widely used for the evaluation of bioactive materials in vitro. Figure 7The degradation rate of MgH2@PLGA / F-GM was fastest in simulated body fluids. By day 30, the material was almost completely degraded, with the remaining hydrogel representing 5.61% of its original weight. In contrast, the degradation rates of MgH2@PLGA / F-GM in PBS and pure water by day 30 were 18.74% and 16.17%, respectively. This indicates that the degradation rate of MgH2@PLGA / F-GM matches the inflammatory phase following bone defect, allowing it to better leverage the role of hydrogen in reducing reactive oxygen species production and the role of magnesium ions in promoting the transformation of macrophages from pro-inflammatory to anti-inflammatory.

[0086] The above experimental results show that, compared with other materials used for diabetic skin wounds, the present invention, through the addition of PLGA microspheres and GelMA, has the advantages of strong injectability, easy dispersion, and promotion of magnesium ion and hydrogen release, which is more conducive to its role in the repair of diabetic bone defects.

[0087] Experimental Example 3: In vitro release of MgH2@PLGA / F-GM

[0088] After successfully preparing MgH2@PLGA, this invention added foamed gelatin methacrylic acid (F-GM) to investigate the H2 and Mg content in the MgH2 / F-GM material of Comparative Example 2 and the MgH2@PLGA / F-GM material of Example 1. 2+ Release curve in vitro.

[0089] To test the in vitro H2 release of MgH2 / F-GM and MgH2@PLGA / F-GM, MgH2 / F-GM and MgH2@PLGA / F-GM containing 3 mg of MgH2 were immediately sealed in 15 mL vials containing 10 mL of pure water (pH = 7.0). At different time points (0 h, 5 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 5 days, and 7 days), 1 mL of gas was collected, and the H2 content was determined using a gas chromatograph (Agilent 8890, USA). The hydrogen content in the vials was also measured at different time points (0 h, 5 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 5 days, 7 days, 9 days, 11 days, 13 days, and 15 days). The obtained data will be statistically analyzed.

[0090] To test the in vitro release of Mg from MgH2 / F-GM and MgH2@PLGA / F-GM. 2+ In this case, the Mg content in MgH2 / F-GM and MgH2@PLGA / F-GM was determined in pure water (pH=7.0). 2+The release curve was obtained. Specifically, MgH2 / F-GM and MgH2@PLGA / F-GM containing 3 mg MgH2 were immersed in 10 mL of pure water. 2 mL of liquid was collected at different time points (0 h, 5 min, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 5 days, and 7 days), and the MgH2 was quantitatively determined using ICP-OES (USA, Agilent 720ES (OES)). 2+ To determine the content, after each sample was taken, the liquid was replenished to 10 mL, and the cumulative release was measured. Results are as follows: Figure 8 As shown.

[0091] Figure 8 In section A, under the same experimental conditions, the present invention immersed MgH2 / F-GM and MgH2@PLGA / F-GM in pure water and investigated the effects of Mg... 2+ The release effect is determined by Figure 8 A observed that on day 7, MgH2@PLGA / F-GM produced Mg 2+ It is 1.46 times that of MgH2 / F-GM. This is because the Mg(OH)2 layer formed on the surface of MgH2 particles hinders the hydrolysis reaction of MgH2, while the acidic degradation products of PLGA in MgH2@PLGA / F-GM can dissolve the Mg(OH)2 layer, thereby accelerating the reaction of H2 and soluble Mg. 2+ The formation of [something]. This proves that the acidic environment created by the degradation of PLGA is characterized by H2 and Mg. 2+ The increased generation created opportunities. Additionally, on the seventh day, the H2 produced by MgH2@PLGA / F-GM in the collected air was 1.45 times that produced by MgH2 / F-GM. Figure 8 B). Simultaneously, the hydrogen content dissolved in the water was measured. On day 14, the H2 produced by MgH2@PLGA / F-GM was 1.86 times that of MgH2 / F-GM. Figure 8 C). The above results demonstrate that the presence of PLGA in the scaffold material of this invention creates an acidic environment, with H2 and soluble Mg... 2+ The generation will increase.

[0092] Experimental Example 4: Biocompatibility Testing

[0093] The biocompatibility of synthetic materials is crucial for tissue and bone regeneration. This invention then investigates the biocompatibility of the synthetic materials in vitro using cell viability assays and the CCK-8 assay, examining the effect of MgH2@PLGA / F-GM on cell biocompatibility in vitro.

[0094] 4.1 CCK8 Detection

[0095] In the concentration-dependent assay, bone marrow mesenchymal stem cells (BMSCs) were seeded into 96-well plates at a density of 5000 cells per well. After culturing the BMSCs in 100 μL of low-glucose DMEM for 12 hours, they were co-cultured for 3 days with different concentrations of MgH2 solution (dissolved in complete culture medium, 0, 0.5, 1, 1.5, 2, 2.5, and 3 μg / mL). On the third day, a solution containing 10% CCK8 was added to each well to allow interaction with the live cells. After incubating at 37°C for 2 hours, the absorbance of different groups in the 96-well plates was analyzed using a SpectraMAX iD3 microplate reader.

[0096] Then, MgH2 powder, MgH2 / F-GM material powder (Comparative Example 2), and MgH2@PLGA / F-GM material powder (Example 1) were soaked in 10 mL PBS for 3 days to obtain extracts for each group. Human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates at 3000 cells per well, divided into four groups: control group, MgH2 powder group, MgH2 / F-GM group, and MgH2@PLGA / F-GM group. After co-culturing cells with the extracts for 1, 3, and 7 days, cell viability was assessed using the CCK-8 assay following the same experimental procedures described above. Five replicates were performed for each group. The results of the CCK-8 assay for the effects of MgH2, MgH2 / F-GM, and MgH2@PLGA / F-GM on cytotoxicity and viability are shown below. Figure 9 As shown.

[0097] Figure 9 The test results showed that after 72 hours of treatment, there was no significant difference in the viability of bone marrow mesenchymal stem cells (BMSCs) between the MgH2@PLGA / F-GM group and the control group, indicating that MgH2@PLGA / F-GM has no cytotoxicity to bone marrow mesenchymal stem cells and does not affect cell proliferation. Figure 9 A). Simultaneously, the optimal concentration of MgH2 was determined to be 1 μg / mL. Therefore, unless otherwise specified, a 1 μg / mL MgH2 solution was used in the following experiments. Next, human umbilical vein endothelial cells (HUVECs) were treated with the extracts in each group for 1, 3, and 7 days. Over time, there was no significant difference between the MgH2, MgH2 / F-GM, and MgH2@PLGA / F-GM groups and the normal control group (control group), indicating that MgH2@PLGA / F-GM is not toxic to human umbilical vein endothelial cells and does not affect their normal growth. Figure 9 B). Therefore, the scaffold material of the present invention has good biocompatibility.

[0098] 4.2 Cell liveness / death staining

[0099] Human umbilical vein endothelial cells (HUVECs) were divided into 4 groups, with 1–2 × 10⁶ cells per dish. 6 Cells were seeded in 12-well plates. Cells from each group were co-cultured for 1 day, 3 days, and 7 days with normal cell culture medium (control group), MgH2 powder, MgH2 / F-GM, and MgH2@PLGA / F-GM extract, respectively. At the corresponding time points, the culture medium was aspirated, and 1 mL of Calcein-AM / PI assay solution was added to each culture dish. After incubation at 37°C and 5% CO2 for 15 min, the cells from each group were photographed using a fluorescence microscope (Leica SP5, Leica Camera AG, Germany).

[0100] Cell viability and death staining imaging results showed that F-GM prepared by gas foaming and photopolymerization techniques had no cytotoxicity and good biocompatibility. On the first and third days, there was no significant difference in cell viability among the MgH2, F-GM, MgH2 / F-GM, and MgH2@PLGA / F-GM groups, and no dead cells were observed. As time progressed, by the fifth day, only a few red dots (indicating dead cells) were observed on the surface. This indicates that MgH2@PLGA / F-GM has good biocompatibility.

[0101] Experimental Example 5: Reducing ROS Production in Vitro with MgH2@PLGA / F-GM

[0102] After verifying the effect of MgH2@PLGA / F-GM in prolonging H2 release, this invention investigated whether it could reduce ROS production in vitro. First, Raw264.7 cells were seeded in 6-well plates, and lipopolysaccharide (LPS) was added to induce excessive ROS production. Then, the cells were co-cultured with MgH2 powder (1 μg / mL), MgH2 / F-GM, an extract of MgH2@PLGA / F-GM, and normal cell culture medium (control group). After 24 h of co-culture, Raw264.7 cells were stained with ROS using DCFH-DA (green) and then imaged under a fluorescence microscope. The stained Raw264.7 cells were collected for flow cytometry analysis to quantify the ROS levels. The results are as follows: Figure 10 , 11 As shown.

[0103] Figure 10 Fluorescence imaging results showed that, compared with the untreated control group, the ROS of the MgH2@PLGA / F-GM extract was the most significantly reduced compared with the normal culture medium (Control).

[0104] To quantify the decrease in ROS production, stained Raw264.7 cells were collected for flow cytometry analysis. The data obtained from the flow cytometry were then quantified, such as... Figure 11 As shown. Figure 11 The results showed that, compared with the untreated group (Control group), the MgH2, MgH2 / F-GM, and MgH2@PLGA / F-GM extract groups reduced reactive oxygen species to 84.46%, 74.92%, and 40.10% of their original values, respectively, confirming that the MgH2@PLGA / F-GM material of the present invention is indeed highly advantageous in reducing reactive oxygen species levels.

[0105] Experimental Example 6: Promoting M2 polarization of macrophages in vitro using MgH2@P / F-GM

[0106] After verifying the advantages of hydrogen therapy for ROS, this invention further investigated the effect of MgH2@PLGA / F-GM on the release of Mg 2+ And its function in inducing macrophage phenotypic changes. To observe the morphological changes of macrophages, 1×10 5 Raw264.7 cells were seeded into 12-well plates containing sterile silica discs. After co-culturing with MgH2 solution (1 μg / mL), MgH2 / F-GM, MgH2@PLGA / F-GM extract, and normal cell culture medium for 24 h, the silica discs containing the Raw264.7 cells were removed from the plates and fixed with 2.5% glutaraldehyde for 30 min, followed by dehydration using an alcohol concentration gradient (30% for 5 min, 50% for 5 min, 70% for 10 min, 80% for 10 min, 95% for 15 min, and 100% for 15 min). The polarization of the Raw264.7 cells was then analyzed using a SEM (ZEISS GeminiSEM 300, Germany), and the results are shown in Figure 12.

[0107] Figure 12 SEM images showed that Raw264.7 cells treated with MgH2 solution (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM extract exhibited distinct morphological changes compared to cells treated with the control group (untreated). This indicates that MgH2 is a more effective morphological agent for the formation of healthy cells. 2+ Induces macrophage polarization.

[0108] To conduct a more detailed study, real-time quantitative PCR (RT-qPCR) analysis was performed on Raw264.7 cells treated with extracts for 3 days in each group. Untreated cells served as the blank group, and LPS-induced cells served as the control group. RT-qPCR was used to detect the expression levels of mRNA levels of M1 macrophage biomarkers (IL-6, IL-1β) and M2 macrophage biomarker (Arg-1). The results are as follows: Figure 13 As shown.

[0109] Figure 13 The results showed that IL-6 in the MgH2@PLGA / F-GM extract group ( Figure 13 B) and IL-1β( Figure 13 The expression of A) was statistically significantly reduced, indicating that M1 polarization was inhibited after treatment. However, the expression of Arg-1 was significantly increased in the MgH2@PLGA / F-GM extract group. Figure 13 C) indicates that M2 repolarization was simultaneously enhanced after treatment. Therefore, MgH2@PLGA / F-GM can promote the polarization of pro-inflammatory M1 macrophages into pro-healing M2 macrophages.

[0110] Experimental Example 7: The Angiogenic Effect of MgH2@P / F-GM in Vitro

[0111] In this study, cell migration ability was assessed in vitro using scratch assays and tube formation assays.

[0112] 7.1 Tube Formation Test

[0113] Pipette 50 μL of matrix gel into a 96-well plate and incubate at 37°C for 40-60 minutes, ensuring the surface is smooth and free of air bubbles. Then, seed human umbilical vein endothelial cells into the 96-well plate at a density of 2-3 × 10⁶ cells per well. 4 Cultured human umbilical vein endothelial cells were divided into groups and incubated with MgH2 powder, MgH2 / F-GM, and MgH2@PLGA / F-GM extracts. The cells were then incubated at 37°C for 4-6 hours, photographed under a microscope, and the tube length and node number were analyzed using ImageJ.

[0114] 7.2 Scratch Test

[0115] Endothelial cells were seeded in 6-well plates, with each well containing 1.2 × 10⁶ cells. 6 For each cell, use a pipette tip to draw a straight line along a sterile ruler. Then, gently rinse with PBS to remove any floating cells. Treat the cells with serum-free culture medium and extracts of MgH2 powder (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM. Take photos at the same location at 0h, 6h, 12h, and 24h to observe cell migration.

[0116] The above test results are as follows Figures 14-16 As shown.

[0117] The voids caused by scratches were treated using MgH2 (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM extracts, and the results are as follows: Figure 14 As shown. From Figure 14As can be seen, each group showed varying degrees of healing, but the MgH2@PLGA / F-GM material in Example 1 exhibited the best healing effect. Furthermore, the healing effect of MgH2@PLGA / F-GM became more pronounced over time, with even closure of the gaps occurring at the 24th hour of the observation period. In contrast, the control group still had wide gaps, indicating that MgH2@PLGA / F-GM has a significant advantage in promoting the migration of human umbilical vein endothelial cells in vitro.

[0118] Subsequently, to verify the pro-angiogenic effect of MgH2@PLGA / F-GM in vitro, an angiogenesis assay was performed. Human umbilical vein endothelial cells were incubated with normal cell culture medium (control group), MgH2 (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM extract for 4-6 hours, respectively. The results were photographed under a microscope. Figure 15 As shown. Figure 15 It is evident that, compared to the control group treated with normal culture medium, the tube formation effect of the MgH2 (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM extract groups was much better. Among them, the MgH2@PLGA / F-GM extract group had the best tube formation effect, which was significantly better than the other groups.

[0119] Figure 16 In the bar charts A through D, the bars represent the Control group, MgH2 group, MgH2 / F-GM group, and MgH2@PLGA / F-GM group, respectively. ImageJ was used to quantitatively analyze the healing area. The void closure rates of the MgH2 (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM extract groups were 1.03 times, 1.13 times, and 1.21 times higher than the control group, respectively. Figure 16 A). Subsequently, ImageJ software was used to quantify the number of nodes and tube lengths in different groups. The bar chart shows that after treatment with MgH2@PLGA / F-GM extract, compared with the control group, MgH2 group, and MgH2@PLGA group, the number of nodes and tube length increased by 2.43 times, 2.19 times, and 1.78 times, respectively. Figure 16 B. Figure 16C). Finally, the in vitro angiogenesis effect of MgH2@PLGA / F-GM was verified by real-time quantitative PCR. Vascular markers such as platelet-endothelial cell adhesion molecule-1 (PECAM-1 / CD31) and vascular endothelial growth factor (VEGF) showed that, compared with the blank control group, the expression of vascular markers in the MgH2 (1 μg / mL), MgH2 / F-GM, and MgH2@PLGA / F-GM extract treatment groups all increased to varying degrees, indicating that magnesium ions had a pro-angiogenic effect. Most importantly, the expression of vascular markers in the MgH2@PLGA / F-GM extract treatment group was the highest. Figure 16 D).

[0120] In summary, the advantages of MgH2@PLGA / F-GM in promoting endothelial cell migration and angiogenesis in vitro have been verified, and it is expected to promote angiogenesis and further promote bone regeneration in vivo.

[0121] Experimental Example 8: In vivo animal experiments to investigate the osteogenesis effect of MgH2@PLGA / F-GM in vivo.

[0122] In vitro, the biocompatibility, inflammation regulation, and pro-angiogenic effects of the material were verified. Next, we will verify the in vivo repair effect of MgH2@PLGA / F-GM on skull defects in diabetic mice.

[0123] 8.1 Miniature CT Scan Test

[0124] Experimental Materials: 6-8 week old male diabetic mice, Cas9-KO strain, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., were randomly divided into 4 groups of 3 mice each. After anesthetizing the diabetic mice, the hair on the heads of each mouse was shaved, and the external skin was cut to fully expose the skull. A skull defect model (3 mm in diameter, one defect per diabetic mouse) was created using a bone drill. The control group had only a defect created without treatment, the MgH2 group was sprinkled with powder, and the MgH2 / F-GM and MgH2@PLGA / F-GM groups had cylindrical material (3 mm in diameter, approximately 1 mm thick) implanted into the defect area. After the wound was sutured, antibiotics were applied to prevent infection. Skull samples were harvested at 4 and 12 weeks. Three-dimensional computed tomography (CT) scans of the skull were performed: 4 and 12 weeks after implantation, the diabetic mice were euthanized by cervical dislocation to obtain the entire skull, which was then scanned using a miniature CT scanner (Scanco, Switzerland).

[0125] In this experiment, the present invention mainly involved implanting the foamed material into the skull defects of diabetic mice for 4 and 12 weeks, and then observing the osteogenesis effect of the entire skull of the diabetic mice. Micro-CT scans were performed on the skulls to measure the formation of new bone. The results of the Micro-CT scans showed that at 4 weeks after modeling, the untreated control group showed almost no bone regeneration at the defect site. The treatment groups, including the MgH2 group, the MgH2 / F-GM group (Comparative Example 2), and the MgH2@PLGA / F-GM group (Example 1), were superior to the control group. More importantly, the MgH2@PLGA / F-GM group showed the best bone regeneration effect at 4 weeks. With the extension of implantation time, at 12 weeks, the regenerated bone area in the blank control group increased compared to 4 weeks, but the regeneration rate was slow. The bone regeneration effects of MgH2, MgH2 / F-GM, and MgH2@PLGA / F-GM were significantly better than the blank control group, and the bone repair effect of MgH2@PLGA / F-GM in Example 1 was significantly better than the other groups. Next, based on the MicroCT report, the statistical analysis results of the bone volume to tissue volume ratio of the skull of diabetic mice at 4 weeks and 8 weeks were calculated, and the results are as follows: Figure 17 As shown.

[0126] Figure 17 The statistical results were consistent with the Micro-CT results. At 4 weeks, the BV / TV ratio of regenerated bone in the MgH2@PLGA / F-GM group was significantly higher than that in the other three groups, being 2.04 times, 1.55 times, and 1.28 times higher than that in the control group, MgH2 group, and MgH2 / F-GM group, respectively. This trend remained consistent over time, reaching 2.49 times, 1.69 times, and 1.29 times higher than those in the control group, MgH2 group, and MgH2 / F-GM group at 12 weeks. These results indicate that MgH2@PLGA / F-GM can reduce the production of reactive oxygen species, alleviate oxidative stress, and promote angiogenesis in the hyperglycemic environment of diabetes, thereby promoting bone repair and regeneration.

[0127] 8.2 H&E staining, Masson staining, and immunofluorescence staining

[0128] Histological evaluation and immunohistochemical staining: The skulls of diabetic mice obtained above were fixed in 4% paraformaldehyde solution for at least 24 hours. Subsequently, the fixed tissues were immersed in 10% EDTA solution for decalcification for about one week. Then, the samples were paraffin-embedded, and a 5 μm cross-section of the defect was cut from the center of the defect for hematoxylin and eosin (H&E) staining and Masson's staining. Immunofluorescence staining was also performed for osteocalcin (OCN) and platelet endothelial cell adhesion molecule-1 (CD31).

[0129] Hematoxylin and eosin (H&E) staining and Masson's staining were performed on the defect areas at 4 and 12 weeks after implantation to assess the area of ​​regenerated tissue. Light red areas within the defect were considered regenerated bone. In HE staining, at 4 weeks post-implantation, all groups showed light red staining areas, with MgH2@PLGA / F-GM showing the largest staining area. As implantation time increased, the staining area in all groups increased, with MgH2@PLGA / F-GM showing a significant increase. In contrast, the blank control group showed only thin staining areas with large unrepaired regions.

[0130] In Masson staining, the results were similar to those of HE staining. Masson staining's reaction to collagen in bone tissue is related to the degree of collagen maturation. Bone tissue appears red, while uncalcified cartilage appears blue. The results of Masson staining are consistent with those of HE staining. Regardless of whether it is 4 weeks or 12 weeks, MgH2@PLGA / F-GM has a larger bone regeneration area.

[0131] Immunofluorescence staining was then performed on skull defect samples 4 and 12 weeks after implantation to detect the effects of osteogenic (OCN) and angiogenesis (CD31) in the defect area. The results are as follows: Figure 18 As shown in the figure. The bars, from left to right, represent the Control group, the MgH2 group, the MgH2 / F-GM group, and the MgH2@PLGA / F-GM group.

[0132] Depend on Figure 18 It was found that from 4 to 12 weeks, MgH2@PLGA / F-GM showed greater positive expression areas for the vascular marker CD31 and the osteogenic marker OCN, while the blank control group showed the least. The quantitative results were consistent with the immunofluorescence staining results mentioned above. This indicates that MgH2@PLGA / F-GM has a good pro-angiogenic effect, participates in the blood and nutrient supply for bone repair, lays a good foundation for later ossification, and thus promotes the regeneration and repair of bone defects in the diabetic environment.

[0133] In summary, this invention studies scaffold materials needed for diabetic bone defects. A foamed material combining PLGA-encapsulated magnesium hydride microspheres and a photocurable hydrogel was prepared using a combination of gas foaming and photocuring techniques. A model promoting the regeneration and repair of diabetic bone defects was established to verify the application effect of this scaffold material. In the scaffold material of this invention, the hydrogen gas and magnesium ions generated by magnesium hydride upon contact with water can reduce the production of reactive oxygen species, alleviate inflammatory responses, and promote angiogenesis, thereby achieving the goal of repairing diabetic bone defects. Furthermore, the acid produced by PLGA degradation promotes the release of magnesium ions from magnesium hydride, which enhances the angiogenesis effect. The foamed hydrogel is conducive to the growth of tissue blood vessels, thus providing oxygen and nutrients for bone repair. In conclusion, both in vitro and in vivo experimental results show that the scaffold material provided by this invention has the effects of reducing ROS, promoting M2 macrophage polarization, improving cell migration, alleviating local inflammation, and promoting angiogenesis and bone regeneration. Therefore, the injectable hydrogel MgH2@PLGA / F-GM material of the present invention has great potential in the regeneration and repair of bone defects in diabetic patients, and can provide new reference methods and feasible strategies for clinical bone repair.

[0134] The above embodiments and test examples are merely preferred embodiments of the present invention and are used only to explain the present invention, not to limit it. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel scaffold material that continuously releases hydrogen and magnesium ions, characterized in that, Includes the following steps: (1) Dissolve polylactic acid-glycolic acid copolymer in a solvent, then add magnesium hydride and mix to obtain a PLGA+MgH2 mixed solution; add the PLGA+MgH2 mixed solution to a polyethylene glycol solution and stir to mix, then evaporate the solvent, centrifuge, take the precipitate and dry to obtain magnesium hydride microspheres coated with polylactic acid-glycolic acid copolymer, denoted as MgH2@PLGA microspheres; Separately, gelatin was dissolved in water to swell, then methacrylic anhydride was added and mixed, followed by dialysis, centrifugation, and freeze-drying to obtain methacryloyl gelatin; the methacryloyl gelatin was dissolved to obtain a methacryloyl gelatin solution, denoted as GelMA solution; (2) The MgH2@PLGA microspheres and GelMA solution obtained in step (1) are mixed with air to foam the foamed material. The foamed material is then injected into a mold and cross-linked and cured under ultraviolet conditions to obtain the hydrogel scaffold material that continuously releases hydrogen and magnesium ions, denoted as MgH2@PLGA / F-GM.

2. The method for preparing the hydrogel scaffold material that continuously releases hydrogen and magnesium ions according to claim 1, characterized in that, In step (1), the solvent is one or more of 1,4-dioxane, dimethyl sulfoxide, chloroform, and dichloromethane; the ratio of polylactic acid-hydroxyacetic acid copolymer, solvent, and magnesium hydride is (8-12) mg: (0.8-1.2) mL: (8-12) mg.

3. The method for preparing the hydrogel scaffold material that continuously releases hydrogen and magnesium ions according to claim 1, characterized in that, In step (1), the polyethylene glycol solution is a dimethyl sulfoxide solution of polyethylene glycol; the concentration of polyethylene glycol in the polyethylene glycol solution is 2-10 mg / mL; the mass ratio of polyethylene glycol, polylactic acid-glycolic acid copolymer, and magnesium hydride is 1-3:1-5:1-5.

4. The method for preparing the hydrogel scaffold material that continuously releases hydrogen and magnesium ions according to claim 1, characterized in that, In step (1), the ratio of gelatin to methacrylic anhydride is 8-12 g: 5-7 mL.

5. The method for preparing the hydrogel scaffold material that continuously releases hydrogen and magnesium ions according to claim 1, characterized in that, In step (1), the dialysis is performed using a dialysis bag with a capacity of 8-14 kDa; the dissolution is performed by dissolving methacryloyl gelatin in a lithium phenyl (2,4,6-trimethylbenzoyl)phosphonate solution to obtain a methacryloyl gelatin solution.

6. The method for preparing the hydrogel scaffold material that continuously releases hydrogen and magnesium ions according to claim 1, characterized in that, In step (2), the foaming is carried out using a three-way valve connected to at least two syringes. The GelMA solution and MgH2@PLGA microspheres are mixed by the reciprocating push of different syringes, so that air is evenly distributed in the mixed hydrogel to form a foaming material. The wavelength of the ultraviolet light used for cross-linking and curing is 400-410nm.

7. A hydrogel scaffold material that continuously releases hydrogen and magnesium ions, prepared by the preparation method according to any one of claims 1 to 6.

8. The hydrogel scaffold material for continuously releasing hydrogen and magnesium ions according to claim 7, characterized in that, The hydrogel scaffold material is an injectable scaffold.

9. The application of a hydrogel scaffold material that continuously releases hydrogen and magnesium ions as described in claim 7 or 8, characterized in that, Application in the preparation of scaffold materials for repairing bone defects.

10. The application of the hydrogel scaffold material for continuously releasing hydrogen and magnesium ions as described in claim 9, characterized in that, The bone defect is diabetic bone defect.

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