Hydrogel materials, methods of making and uses thereof

Abstract

This invention relates to medical, oral, or dental hygiene devices. It discloses a hydrogel material, its preparation method, and its applications. The preparation method of the hydrogel material includes: preparing a ZnSe / rGO heterojunction by hydrothermal reaction of ZnSe and rGO, wherein ZnSe is the semiconductor material zinc selenide and rGO is the conductor material reduced graphene oxide; and uniformly dispersing the ZnSe / rGO heterojunction in a GelMA matrix, loading and immobilizing the ZnSe / rGO heterojunction in GelMA to obtain the hydrogel material Z / r@G, where Z refers to ZnSe, r refers to rGO, G refers to GelMA, and GelMA is methacrylamide gelatin. This invention develops a preventative strategy that can delay or reduce cellular senescence, avoiding excessive removal of senescent cells that could affect later bone repair, while also exerting a broad anti-aging effect.

Description

Technical Field

[0001] This invention belongs to the field of medical new materials technology, and more specifically, relates to a hydrogel material, its preparation method and application. Background Technology

[0002] Bacterial infection-related bone defects are a common complication in orthopedic and dental clinics, characterized by persistent infection and delayed healing of the bone defect. With the increasing global burden of chronic diseases, the clinical challenges of infected bone defects are becoming increasingly severe. Notably, diabetes mellitus, one of the most common chronic diseases, has a significantly higher incidence of infection at bone defect sites compared to non-diabetic patients. Therefore, exploring a more comprehensive platform designed to achieve glycemic control is crucial for managing implant-related infections and diabetic skeletal integration.

[0003] Hyperglycemia in diabetic patients not only impairs vascular function but also directly promotes bacterial adhesion and colonization, as well as biofilm formation, significantly weakening their antibacterial capacity and bone repair potential. Furthermore, hyperglycemia and persistent inflammatory responses in diabetic patients exacerbate cellular oxidative stress, driving cellular senescence. Senescent cells can directly lead to functional impairments in the proliferation, differentiation, and biomineralization of bone stem cells and osteoblasts. Moreover, the senescence-associated secretory phenotype (SASP) they secrete can promote chronic inflammatory responses, forming a pathological cycle of "inflammation-oxidative stress-cellular senescence-inflammation," further affecting the healing of diabetic infectious bone defects. Current clinical treatments primarily employ surgical debridement, antibiotic therapy, and bone grafting, focusing mainly on controlling infection and providing structural support. However, these approaches face multiple challenges, including antibiotic overuse and increased resistance, limited donor bone sources, secondary damage to the bone harvesting site, and immune rejection. Moreover, they struggle to effectively intervene in the core pathological process of cellular senescence. Summary of the Invention

[0004] One objective of this invention is to overcome the shortcomings of the existing technology and provide a preventative strategy that can delay or reduce cellular senescence, avoiding excessive removal of senescent cells that could negatively impact later bone repair, while simultaneously exerting a broad anti-aging effect. Specifically, it provides a novel repair material for diabetic infectious bone defects that can control infection, regulate cellular senescence, and promote osteogenesis.

[0005] According to one aspect of the present invention, a method for preparing a hydrogel material is provided, the method comprising: preparing a ZnSe / rGO heterojunction by hydrothermal method, wherein ZnSe is zinc selenide, a semiconductor material, and rGO is reduced graphene oxide, a conductor material; and uniformly dispersing the ZnSe / rGO heterojunction in a GelMA matrix, loading and fixing the ZnSe / rGO heterojunction in GelMA to obtain a hydrogel material Z / r@G, wherein Z refers to ZnSe, r refers to rGO, G refers to GelMA, and GelMA is methacrylamide gelatin.

[0006] In this embodiment, the hydrothermal temperature of the hydrothermal method is controlled at 175–190°C for 10–14 hours.

[0007] In an embodiment, the step of preparing ZnSe / rGO heterojunction by hydrothermal method includes: dispersing graphene oxide and zinc nitrate heptahydrate in a mixed solution of ethanol and water to obtain mixed solution a; adding selenium powder and sodium hydroxide to deionized water, stirring at room temperature, then adding hydrazine hydrate, and continuing stirring to obtain a reaction solution containing an active selenium source; and adding the mixed solution a to the reaction solution containing the active selenium source, adding polyvinylpyrrolidone, stirring, and performing a hydrothermal reaction to obtain the reaction product.

[0008] In the embodiments, the amount of selenium powder and the amount of zinc nitrate heptahydrate satisfy the relationship between Se and Zn. 2+ The molar ratio is controlled between 0.5 and 1.5; the mass ratio of the amount of graphene oxide to the zinc nitrate heptahydrate is controlled between 1:20 and 1:10; and the amount of polyvinylpyrrolidone is 200 to 250 mg.

[0009] In the embodiment, the reaction product was washed sequentially with anhydrous ethanol and deionized water, and then the precipitate was collected by filtration; the precipitate was dried under vacuum freeze-drying conditions to obtain ZnSe / rGO heterojunction powder.

[0010] In this embodiment, the steps of uniformly dispersing ZnSe / rGO heterojunctions in a GelMA matrix, loading and immobilizing the ZnSe / rGO heterojunctions in GelMA to obtain the hydrogel material Z / r@G include: dissolving GelMA in phosphate buffer and stirring to obtain a GelMA solution; adding photoinitiator LAP and ZnSe / rGO heterojunctions to the GelMA solution and mixing thoroughly to obtain a mixed solution, wherein the concentration of ZnSe / rGO added is 0.25–2.5 mg / mL; and pouring the mixed solution into a mold and crosslinking and curing it under ultraviolet light to form a ZnSe / rGO composite GelMA hydrogel, thereby obtaining the Z / r@G.

[0011] According to another aspect of the present invention, a hydrogel material is provided, the hydrogel material being Z / r@G, wherein Z refers to ZnSe, r refers to rGO, G refers to GelMA, GelMA is methacrylamide gelatin, ZnSe is zinc selenide, a semiconductor material, and rGO is reduced graphene oxide, a conductor material; ZnSe serves as a source of H2Se gas, enabling the controlled release of H2Se in an acidic microenvironment accompanied by photothermal heating; rGO serves as a photothermal conversion unit, providing a heating effect for the release of H2Se from ZnSe under acidic and photothermal synergistic conditions at the infection site; and GelMA serves as an injectable three-dimensional carrier for loading and immobilizing the ZnSe / rGO heterojunction.

[0012] According to another aspect of the present invention, the hydrogel material prepared by the preparation method described above or the hydrogel material described above is provided for use as a material for preparing repair of infected bone defects.

[0013] In this embodiment, the infectious bone defect repair material is used for the repair of infectious bone defects in diabetic patients.

[0014] In this embodiment, Z / r@G is a controlled-release H2Se hydrogel that can controllably release H2Se at the site of bone defect.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention prepares a controlled-release H2Se hydrogel composed of ZnSe / rGO heterostructure and GelMA, which can controllably release H2Se at the site of bone defect. ZnSe can achieve controlled release of H2Se under acidic thermal response conditions. The microenvironment of the bacterial infection site is acidic, but ZnSe lacks a heating effect. By constructing a ZnSe / rGO bioheterostructure, the high photothermal conversion rate of rGO provides the conditions for the controlled release of H2Se.

[0016] (2) This invention can achieve rapid sterilization without relying on the use of antibiotics, by utilizing photothermal effect and H2Se synergistic therapy, thus avoiding the generation of drug-resistant bacteria. Under irradiation with 808 nm near-infrared light, the ZnSe / rGO bioheterostructure in the Z / r@G hydrogel exhibits excellent photothermal properties, enabling rapid sterilization in a short period of time; and the generated H2Se, after entering the bacterial cells, can interfere with the sulfur metabolism pathway of bacteria, enhancing the antibacterial effect.

[0017] (3) This invention can effectively inhibit cell senescence by constructing ZnSe / rGO bioheterostructures to integrate the antioxidant function of glutathione peroxidase activity and enhance the ROS scavenging ability of rGO, thereby reducing the oxidative stress level of cells under high oxidative stress culture conditions of hydrogen peroxide; at the same time, the generated H2Se can alleviate DNA damage in cells under high oxidative stress culture conditions of hydrogen peroxide, stabilize the morphology and function of mitochondria, and alleviate cell senescence.

[0018] (4) Zn produced by decomposition in this invention 2+ It can effectively promote osteogenic repair through Zn 2+ In synergy with anti-aging effects, Z / r@G hydrogel can guide the maturation and differentiation of both normal osteoblast precursor cells and hydrogen peroxide-induced senescent osteoblast precursor cells, and enhance their alkaline phosphatase activity and biomineralization function. Attached Figure Description

[0019] The accompanying drawings illustrate exemplary embodiments of the inventive concept and, together with the description, serve to explain the principles of the inventive concept. The drawings are included to provide a further understanding of the inventive concept and are incorporated in and form a part of this specification.

[0020] Figure 1 Characterization diagrams of ZnSe / rGO heterojunctions and Z / r@G hydrogels according to exemplary embodiments of the present invention are shown. Figure 1 The image shows scanning electron microscope images of the morphology of rGO, ZnSe, and ZnSe / rGO. Figure 1 d is the elemental diagram of the energy dispersive spectrum; Figure 1 'e' is a transmission electron microscope image; Figure 1 f is a high-resolution transmission electron microscope image; Figure 1 The image of g is a SEM image of the Z / r@G hydrogel.

[0021] Figure 2 The diagram illustrates the results of photothermal performance and H2Se gas release performance tests of the Z / r@G hydrogel, and the antioxidant performance test of the ZnSe / rGO heterostructure, according to an exemplary embodiment of the present invention. Figure 2 Figure a shows the photothermal heating curves of ZnSe, ZnSe / rGO solutions, and Z@G and Z / r@G hydrogels. Figure 2 b represents the fluorescence intensity of each group detected using the 2,1,3-benzoselenidazole fluorescence method for H2Se release. Figure 2 c represents the H2Se release concentration of each group detected using an H2Se gas detector. Figure 2 d represents the absorbance spectrum for detecting ·OH levels. Figure 2 e is for detecting O2 - Horizontal absorbance spectrum, Figure 2 f is the absorbance spectrum for detecting DPPH free radical levels. Figure 2 g is the absorbance spectrum for detecting PTIO free radicals.

[0022] Figure 3 The results of the antibacterial performance test are shown. Among them, Figure 3 'a' represents the colony count of MRSA under pH=7.4 and pH=5.0 conditions; Figure 3 b represents the planktonic bacterial morphology and biofilm morphology of MRSA under pH=7.4 and pH=5.0 conditions.

[0023] Figure 4 The results of the anti-cellular aging performance test are shown. Among them, Figure 4 Image 'a' shows fluorescence images used to detect intracellular ROS levels in each group. Figure 4 b is a fluorescence image used to detect intracellular DNA damage in each group; Figure 4 c is a staining image used to detect the SA-β-Gal expression level in cells of each group.

[0024] Figure 5 A schematic diagram of the biocompatibility assessment results is shown. Among them, Figure 5 'a' represents the cell morphology recorded by SEM in different groups of cultured cells; Figure 5 b represents the CCK-8 detection results for each group of cells.

[0025] Figure 6 The osteogenic performance test diagram is shown. Among them, Figure 6 'a' represents the ALP activity of each group after 7 days of inducing osteogenic differentiation of normal cells. Figure 6 b is a graph showing ALP activity in each group after 7 days of induced osteogenic differentiation of senescent cells. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0027] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] In this document, the terms “containing” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0029] In this document, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately in this document. For example, “A and / or B” covers “A”, “A and B”, and “B”. For example, “A, B and / or C” covers “A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and “A and B and C”.

[0030] The purpose of this invention is to develop preventative strategies that can delay or reduce cellular senescence, avoiding the excessive removal of senescent cells that could negatively impact later bone repair, while simultaneously exerting a broad anti-aging effect. This invention provides a controlled-release H2Se hydrogel for the repair of diabetic infectious bone defects. The hydrogel is composed of photothermally responsive ZnSe / rGO heterojunctions uniformly dispersed in an injectable methacrylamide gelatin (GelMA) matrix. The ZnSe / rGO heterojunction is composed of a semiconductor material, zinc selenide (ZnSe), and a conductor material, reduced graphene oxide (rGO). ZnSe serves as the H2Se gas source, enabling controlled release of H2Se in an acidic microenvironment accompanied by photothermal heating. rGO acts as a photothermal conversion unit, providing a heating effect for the release of H2Se from ZnSe under the synergistic acidic and photothermal conditions at the infection site. The GelMA hydrogel serves as an injectable three-dimensional carrier for loading and immobilizing the ZnSe / rGO heterojunction.

[0031] This invention also provides a method for preparing the above-mentioned controlled-release H2Se hydrogel, including the preparation of ZnSe / rGO heterojunctions and the synthesis of GelMA by sonochemical-assisted hydrothermal method, specifically the following steps: S1, Preparation of GO / Zn 2+ Precursor dispersion system Graphene oxide (GO) and zinc nitrate heptahydrate (Zn(NO3)2·7H2O) were dispersed in a mixed solution of ethanol and water to obtain mixed solution a; wherein, the amount of Zn(NO3)2·7H2O was 594.98 mg, and the mass ratio of GO to Zn(NO3)2·7H2O was controlled at 1:20~1:10. (If the GO content is relatively high, although there are sufficient carrier sites, the ZnSe content per unit mass of product decreases, and the upper limit of acid-triggered hydrogen selenide release is reduced; moreover, excessive GO will increase the viscosity of the system, thereby inhibiting the effective nucleation and loading of ZnSe. If the GO content is relatively low, the rGO framework and anchoring sites are insufficient, and ZnSe is more likely to nucleate and aggregate in the solution phase, resulting in uneven local distribution, an increased proportion of free state, and a decrease in photothermal efficiency, leading to instability in the acid-triggered release process.) The volume ratio of ethanol to water is 1:1, and the total volume of the mixed solution is 20-25 mL; the mixed solution is ultrasonically treated for 1-2 h to obtain mixed solution a for later use.

[0032] S2. Preparation of active selenium source solution Add selenium powder and 4g sodium hydroxide to 20mL of deionized water and stir at room temperature for 10-15min; wherein the amount of selenium powder used is such that the molar ratio of Se to Zn2+ is controlled between 0.5 and 1.5, as described in step 1 for Zn(NO3)2·7H2O. 2+ Controlling the value between 0.5 and 1.5 ensures sufficient Zn²⁺ selenization while mitigating losses during Se activation and conversion. When the Se:Zn ratio is... 2+ Below 0.5, insufficient selenium source easily leads to incomplete reaction and a tendency for zinc-enriched secondary phases, resulting in poor crystallization and uneven loading; when Se:Zn 2+ Above 1.5, elemental Se residue or coating / byproduct formation is likely to occur, affecting product purity and interfacial properties. Subsequently, 2 mL of hydrazine hydrate was added to the system, and stirring was continued for 10–15 min to obtain a reaction solution containing the active selenium source.

[0033] S3, Hydrothermal Synthesis of ZnSe / rGO Heterojunctions Add the mixed solution a from step S1 to the reaction solution obtained in step S2, and add polyvinylpyrrolidone (PVP) in an amount of 200-250 mg. Here, PVP, by adsorbing onto crystal nuclei / crystal faces and providing steric hindrance, can suppress fused crystal agglomeration and achieve confined growth, thereby facilitating the acquisition of ZnSe / rGO heterostructures with more uniform particle size and loading. After stirring for 10-15 min, transfer the mixture to a stainless steel high-pressure reactor with a polytetrafluoroethylene liner and seal it. Perform a hydrothermal reaction at 170-190 °C for 10-14 h to obtain the reaction product. Here, the hydrothermal temperature of 170-190 °C and the time of 10-14 h are used to ensure sufficient crystallization of ZnSe and stable loading on rGO: too low a temperature / time will lead to insufficient crystallization and weak interfacial bonding, while too high a temperature or too long a time will lead to grain growth, enhanced agglomeration, and reduced specific surface area, and may also exacerbate rGO agglomeration, affecting the uniformity of the heterostructure.

[0034] Washing and drying of S4, ZnSe / rGO heterojunctions The reaction product obtained in step S3 was washed three times each with anhydrous ethanol and deionized water, and then the precipitate was collected by filtration. The precipitate was dried under vacuum freeze-drying conditions for 24 h to obtain ZnSe / rGO heterojunction powder.

[0035] S5, preparation of GelMA and preparation of ZnSe / rGO composite GelMA hydrogel (Z / r@G) Synthesis of S5.1 and GelMA 10g of porcine skin gelatin was added to 100mL of Duchenne phosphate buffer (DPBS) and stirred at 50°C for 30min to fully dissolve the gelatin, obtaining a gelatin solution. Subsequently, methacrylic anhydride was added dropwise to the gelatin solution at 5% (v / v) of the gelatin solution volume, and the reaction was continued with stirring for 3h to induce methacrylation. After the reaction was complete, an equal volume of DPBS, preheated to 40°C, was added to the reaction system to terminate the methacrylation reaction. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed against deionized water at 40°C for 5 days, with the deionized water changed 3 times daily. After dialysis, the solution was filtered through a 0.22μm pore size filter and freeze-dried to obtain GelMA. Store at 20°C.

[0036] Preparation of S5.2, Z / r@G hydrogel The GelMA described in step S5.1 was dissolved in phosphate-buffered saline (PBS) and stirred at 60°C in the dark for 30 min to form a 5% (w / w) GelMA solution. The photoinitiator LAP and ZnSe / rGO heterojunction were added to the GelMA solution and mixed thoroughly to obtain a mixed solution. The amount of LAP was 50 mg, and the concentration of ZnSe / rGO was 0.25–2.5 mg / mL. The mixed solution was poured into a mold and crosslinked and cured for 60 s under ultraviolet light irradiation at a wavelength of 405 nm to form a ZnSe / rGO composite GelMA hydrogel (Z / r@G).

[0037] Preparation points Control of GO and Zn(NO3)2·7H2O dosage: The mass ratio of GO to Zn(NO3)2·7H2O is controlled within the range of 1:20 to 1:10. Under the premise of ensuring the continuity and dispersion stability of the rGO skeleton, the system has a high ZnSe generation / loading level, thereby increasing the "effective selenium source reserve" that can release selenium-containing gas under acidic conditions. The rGO content is still sufficient to provide a good light absorption and heat conduction channel, ensuring that the local temperature rise is faster and more uniform under light irradiation, thereby improving the photothermal antibacterial efficiency. The anchoring loading of ZnSe on the rGO surface helps to avoid severe aggregation of functional phases, making the photothermal effect and acid-triggered release more controllable and repeatable.

[0038] Se source system control: Insufficient or excessive Se source can lead to incomplete reaction, residual impurities, or agglomeration and coarsening.

[0039] PVP dispersion control: Too little PVP results in insufficient ability to suppress aggregation; too much PVP forms organic coatings, affecting interfacial transfer and increasing washing difficulty.

[0040] Hydrothermal temperature / time control: The hydrothermal temperature of the heterojunction is controlled at 175–190℃ for 10–14 h. Too low a temperature / time will result in insufficient crystallization and weak interfacial bonding; too high a temperature / time will result in grain growth, rGO agglomeration, and decreased heterojunction uniformity.

[0041] Washing and Drying: The adequacy of washing and drying directly affects product purity, repeatability, and subsequent composite molding stability. Alternating ethanol / water washing can remove inorganic salts, alkalis, unreacted precursors, and some organic residues; insufficient washing can lead to residual ions or PVP, affecting purity and performance consistency. Freeze-drying can avoid hard agglomeration and structural collapse caused by conventional drying, improving redispersibility. Insufficient drying time can easily lead to residual solvents and unstable dispersion, while excessive time reduces efficiency.

[0042] To better understand the exemplary embodiments of the present invention described above, further explanation is provided below with reference to specific examples and comparative examples.

[0043] Example 1 This example provides a method for preparing a controlled-release H2Se hydrogel for repairing infectious bone defects in diabetic patients. The hydrogel is composed of photothermally responsive ZnSe / rGO heterostructures uniformly dispersed in a GelMA matrix, and includes the following steps: 1. Preparation of GO / Zn 2+ Precursor dispersion system Graphene oxide (GO) and zinc nitrate heptahydrate (Zn(NO3)2·7H2O) were dispersed in a mixed solution of ethanol and water to obtain mixed solution a; wherein the amount of GO was 40 mg, the amount of Zn(NO3)2·7H2O was 595 mg, the volume ratio of ethanol to water was 1:1, and the total volume of the mixed solution was 20 mL; the mixed solution was ultrasonically treated for 1 h to obtain mixed solution a for later use.

[0044] 2. Preparation of active selenium source solution Selenium powder and 4g sodium hydroxide were added to 20mL of deionized water and stirred at room temperature for 10-15min. The amount of selenium powder used was 157.9mg, and its molar ratio with Zn(NO3)2·7H2O in step 1 was 1. Then, 2mL of hydrazine hydrate was added to the system, and stirring was continued for 15min to obtain a reaction solution containing an active selenium source.

[0045] 3. Hydrothermal synthesis of ZnSe / rGO heterojunctions Add the mixed solution a from step 1 to the reaction solution obtained in step 2, and add 225 mg of polyvinylpyrrolidone (PVP). Stir for 15 min, then transfer to a stainless steel high-pressure reactor with a polytetrafluoroethylene liner and seal. Perform hydrothermal reaction at 180 °C for 12 h to obtain the reaction product.

[0046] 4. Washing and drying of ZnSe / rGO heterojunctions The reaction product obtained in step 3 was washed three times each with anhydrous ethanol and deionized water, and then the precipitate was collected by filtration. The precipitate was dried under vacuum freeze-drying conditions for 24 h to obtain ZnSe / rGO heterojunction powder.

[0047] 5. Preparation of GelMA and ZnSe / rGO composite GelMA hydrogel (Z / r@G) 5.1. Synthesis of GelMA 10g of porcine skin gelatin was added to 100mL of Duchenne phosphate buffer (DPBS) and stirred at 50℃ for 30min to fully dissolve the gelatin, obtaining a gelatin solution. Then, methacrylic anhydride was added dropwise to the gelatin solution at 5% (v / v) relative to the volume of the gelatin solution, and the reaction was continued with stirring for 3h to induce methacrylation. After the reaction was complete, an equal volume of DPBS, preheated to 40℃, was added to the reaction system to terminate the methacrylation reaction. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed against deionized water at 40℃ for 5 days, with the deionized water changed 3 times daily. After dialysis, the solution was filtered through a 0.22μm pore size filter and freeze-dried to obtain GelMA. Store at 20°C.

[0048] 5.2. Preparation of Z / r@G hydrogel Dissolve the GelMA from step 5.1 in phosphate-buffered saline (PBS) and stir for 30 min at 60 °C in the dark to form a 5% (w / w) GelMA solution. Add the photoinitiator LAP and ZnSe / rGO heterojunction to the GelMA solution and mix thoroughly to obtain a mixed solution. The amount of LAP is 50 mg and the concentration of ZnSe / rGO is 0.25–2.5 mg / mL. Pour the mixed solution into a mold and crosslink and cure for 60 s under ultraviolet light at a wavelength of 405 nm to form a ZnSe / rGO composite GelMA hydrogel (Z / r@G).

[0049] Results: The obtained ZnSe / rGO heterojunctions and Z / r@G hydrogels were characterized and showed good photothermal properties, antioxidant, antibacterial and anti-aging effects.

[0050] Example 2: This embodiment provides a method for preparing controlled-release H2Se hydrogel for repairing diabetic infectious bone defects. The hydrogel is composed of photothermally responsive ZnSe / rGO heterostructures uniformly dispersed in a GelMA matrix, and includes the following steps: 1. Preparation of GO / Zn 2+ Precursor dispersion system Graphene oxide (GO) and zinc nitrate heptahydrate (Zn(NO3)2·7H2O) were dispersed in a mixed solution of ethanol and water to obtain mixed solution a; wherein the amount of GO was 30 mg, the amount of Zn(NO3)2·7H2O was 595 mg, the volume ratio of ethanol to water was 1:1, and the total volume of the mixed solution was 20 mL; the mixed solution was ultrasonically treated for 1 h to obtain mixed solution a for later use.

[0051] 2. Preparation of active selenium source solution Selenium powder and 4g sodium hydroxide were added to 20mL of deionized water and stirred at room temperature for 10-15min. The amount of selenium powder used was 74.5mg, and its molar ratio with Zn(NO3)2·7H2O in step 1 was 0.5. Then, 2mL of hydrazine hydrate was added to the system, and stirring was continued for 15min to obtain a reaction solution containing an active selenium source.

[0052] 3. Hydrothermal synthesis of ZnSe / rGO heterojunctions Add the mixed solution a from step 1 to the reaction solution obtained in step 2, and add 200 mg of polyvinylpyrrolidone (PVP). Stir for 15 min, then transfer to a stainless steel high-pressure reactor with a polytetrafluoroethylene liner and seal. Perform hydrothermal reaction at 170 °C for 10 h to obtain the reaction product.

[0053] 4. Washing and drying of ZnSe / rGO heterojunctions The reaction product obtained in step 3 was washed three times each with anhydrous ethanol and deionized water, and then the precipitate was collected by filtration. The precipitate was dried under vacuum freeze-drying conditions for 24 h to obtain ZnSe / rGO heterojunction powder.

[0054] 5. Preparation of GelMA and ZnSe / rGO composite GelMA hydrogel (Z / r@G) 5.1. Synthesis of GelMA 10g of porcine skin gelatin was added to 100mL of Duchenne phosphate buffer (DPBS) and stirred at 50℃ for 30min to fully dissolve the gelatin, obtaining a gelatin solution. Then, methacrylic anhydride was added dropwise to the gelatin solution at 5% (v / v) relative to the volume of the gelatin solution, and the reaction was continued with stirring for 3h to induce methacrylation. After the reaction was complete, an equal volume of DPBS, preheated to 40℃, was added to the reaction system to terminate the methacrylation reaction. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed against deionized water at 40℃ for 5 days, with the deionized water changed 3 times daily. After dialysis, the solution was filtered through a 0.22μm pore size filter and freeze-dried to obtain GelMA. Store at 20°C.

[0055] 5.2. Preparation of Z / r@G hydrogel Dissolve the GelMA from step 5.1 in phosphate-buffered saline (PBS) and stir for 30 min at 60 °C in the dark to form a 5% (w / w) GelMA solution. Add the photoinitiator LAP and ZnSe / rGO heterojunction to the GelMA solution and mix thoroughly to obtain a mixed solution. The amount of LAP is 50 mg and the concentration of ZnSe / rGO is 0.25–2.5 mg / mL. Pour the mixed solution into a mold and crosslink and cure for 60 s under ultraviolet light at a wavelength of 405 nm to form a ZnSe / rGO composite GelMA hydrogel (Z / r@G).

[0056] Results: The crystallinity of the obtained ZnSe / rGO heterojunction was slightly lower, and its photothermal properties, antibacterial, antioxidant and anti-aging effects were slightly lower than those of Example 1, but still within an acceptable range, proving the feasibility of the lower end of the parameter range.

[0057] Example 3: This embodiment provides a method for preparing controlled-release H2Se hydrogel for repairing diabetic infectious bone defects. The hydrogel is composed of photothermally responsive ZnSe / rGO heterostructures uniformly dispersed in a GelMA matrix, and includes the following steps: 1. Preparation of GO / Zn 2+ Precursor dispersion system Graphene oxide (GO) and zinc nitrate heptahydrate (Zn(NO3)2·7H2O) were dispersed in a mixed solution of ethanol and water to obtain mixed solution a; wherein the amount of GO was 60 mg, the amount of Zn(NO3)2·7H2O was 595 mg, the volume ratio of ethanol to water was 1:1, and the total volume of the mixed solution was 20 mL; the mixed solution was ultrasonically treated for 1 h to obtain mixed solution a for later use.

[0058] 2. Preparation of active selenium source solution Selenium powder and 4g sodium hydroxide were added to 20mL of deionized water and stirred at room temperature for 10-15min. The amount of selenium powder used was 223.4mg, and its molar ratio with Zn(NO3)2·7H2O in step 1 was 1.5. Then, 2mL of hydrazine hydrate was added to the system, and stirring was continued for 15min to obtain a reaction solution containing an active selenium source.

[0059] 3. Hydrothermal synthesis of ZnSe / rGO heterojunctions Add the mixed solution a from step 1 to the reaction solution obtained in step 2, and add 250 mg of polyvinylpyrrolidone (PVP). Stir for 15 min, then transfer to a stainless steel high-pressure reactor with a polytetrafluoroethylene liner and seal. Perform hydrothermal reaction at 190 °C for 14 h to obtain the reaction product.

[0060] 4. Washing and drying of ZnSe / rGO heterojunctions The reaction product obtained in step 3 was washed three times each with anhydrous ethanol and deionized water, and then the precipitate was collected by filtration. The precipitate was dried under vacuum freeze-drying conditions for 24 h to obtain ZnSe / rGO heterojunction powder. S2: Preparation of GelMA hydrogel: 5. Preparation of GelMA and ZnSe / rGO composite GelMA hydrogel (Z / r@G) 5.1. Synthesis of GelMA 10g of porcine skin gelatin was added to 100mL of Duchenne phosphate buffer (DPBS) and stirred at 50℃ for 30min to fully dissolve the gelatin, obtaining a gelatin solution. Then, methacrylic anhydride was added dropwise to the gelatin solution at 5% (v / v) relative to the volume of the gelatin solution, and the reaction was continued with stirring for 3h to induce methacrylation. After the reaction was complete, an equal volume of DPBS, preheated to 40℃, was added to the reaction system to terminate the methacrylation reaction. The resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed against deionized water at 40℃ for 5 days, with the deionized water changed 3 times daily. After dialysis, the solution was filtered through a 0.22μm pore size filter and freeze-dried to obtain GelMA. Store at 20°C.

[0061] 5.2. Preparation of Z / r@G hydrogel Dissolve the GelMA from step 5.1 in phosphate-buffered saline (PBS) and stir for 30 min at 60 °C in the dark to form a 5% (w / w) GelMA solution. Add the photoinitiator LAP and ZnSe / rGO heterojunction to the GelMA solution and mix thoroughly to obtain a mixed solution. The amount of LAP is 50 mg and the concentration of ZnSe / rGO is 0.25–2.5 mg / mL. Pour the mixed solution into a mold and crosslink and cure for 60 s under ultraviolet light at a wavelength of 405 nm to form a ZnSe / rGO composite GelMA hydrogel (Z / r@G).

[0062] Results: The obtained ZnSe / rGO heterojunction has more ZnSe agglomeration. Its photothermal properties, antioxidant, antibacterial and anti-aging effects are slightly lower than those of Example 1, but still within the acceptable range, proving the feasibility of the high-end parameter range.

[0063] Comparative Example 1: Hydrothermal temperature too low Preparation process: basically the same as Example 1, but in step 3, the hydrothermal reaction temperature of the heterojunction is set to 130℃.

[0064] Results: The final ZnSe / rGO heterojunction interface was loosely bonded, resulting in low charge transfer efficiency. Its photothermal performance and antioxidant effect were significantly lower than those of Example 1.

[0065] Comparative Example 2: Hydrothermal temperature too high Preparation process: basically the same as Example 1, but in step 3, the hydrothermal reaction temperature of the heterojunction is set to 230℃.

[0066] Results: In the heterojunction product, large / agglomerated ZnSe particles were unevenly distributed on the rGO surface, with some areas exposed. Due to the excessively large particle size, the effective heterojunction interface ratio was reduced, and the final photothermal properties and antioxidant effect of the material were far lower than those of Example 1.

[0067] Comparative Example 3: Excessive PVP Addition Preparation process: basically the same as Example 1, but in step 3, 500 mg of polyvinylpyrrolidone is added.

[0068] Results: The ZnSe surface is more fully coated by PVP, and a polymer isolation layer may appear between the particles and rGO, resulting in the particles exhibiting dot-like adhesion and a less compact heterojunction interface. Some ZnSe particles are more likely to exist in a free state, and the particle density and uniformity loaded on the rGO sheets decrease. This leads to the inability to form an effective ZnSe / rGO heterojunction, resulting in the functional failure of the entire material system. Ultimately, the photothermal performance and antioxidant effect of the material are far lower than those of Example 1.

[0069] Test Example 1 Preparation and characterization of ZnSe / rGO heterostructures and Z / r@G hydrogels Methods: ZnSe / rGO heterojunctions were observed using scanning electron microscopy and transmission electron microscopy; Z / r@G hydrogels were observed using scanning electron microscopy.

[0070] Result: As Figure 1 As shown in Figure ac, scanning electron microscopy observation of the ZnSe / rGO morphology revealed that ZnSe nanoparticles (approximately 10–100 nm) were uniformly loaded / stacked on the surface of the wrinkled graphene sheet. Energy dispersive spectroscopy elemental mapping (EDS) Figure 1 d) The distribution areas of Zn and Se elements are consistent with the ZnSe particle regions, confirming the successful loading of ZnSe on the rGO surface. Transmission electron microscopy (TEM) Figure 1 e) This further demonstrates that ZnSe particles smaller than 100 nm are tightly attached to the sheet-like graphene; high-resolution transmission electron microscopy image ( Figure 1f) It can be seen that the ZnSe and graphene interfaces are in close contact. The ZnSe lattice spacing of approximately 3.2 Å corresponds to its (111) crystal plane, while the graphene exhibits clear lattice fringes, indicating that GO is well reduced during the hydrothermal process and forms a ZnSe / rGO heterojunction structure. SEM of Z / r@G hydrogel ( Figure 1 g) shows that the hydrogels all have a continuous porous structure with relatively smooth pore walls, and ZnSe nanoparticles were observed in the pore walls.

[0071] Conclusion: ZnSe / rGO heterojunctions and Z / r@G hydrogels were successfully prepared and characterized.

[0072] Test Example 2 Photothermal performance and H2Se gas release performance test method: a. ZnSe and ZnSe / rGO were both prepared into 1 mg / mL solutions using ultrapure water, and Z@G and Z / r@G hydrogels were prepared accordingly. 200 μL of the solution / hydrogel precursor solution was added to each well of a 48-well plate using a pipette. The hydrogels were crosslinked and cured for 60 s under UV irradiation at 405 nm using 1.5 W / cm² light. 2 Each hole was illuminated for 10 minutes with NIR light of 808 nm wavelength at high power. During this period, an infrared imager was used to take pictures and record the temperature every 1 minute to create a temperature rise-time curve.

[0073] b. ZnSe / rGO was dispersed in PBS at concentrations of 0.25, 0.5, 0.75, and 1 mg / mL, respectively, at pH 7.4, 6, and 5. While magnetically stirring, the NIR irradiation group was treated with 1.5 W / cm² light. 2 Irradiate the solution with NIR light at 808 nm for 10 min while continuously purging nitrogen gas above it. The gas produced in the reaction is then passed into a PBS solution (10x, pH=7.4) containing 20 μm of 2,1,3-benzoselenidazole, and the fluorescence intensity of the resulting solution is measured (excitation wavelength λex=245 nm, emission wavelength λem=430 nm).

[0074] c. ZnSe and ZnSe / rGO were dispersed in PBS at concentrations of 0.25, 0.5, 0.75, and 1 mg / mL, respectively, at pH 5. The NIR light irradiation group was treated with 1.5 W / cm² light. 2 The H2Se gas was qualitatively detected by irradiating it with NIR light of 808 nm wavelength for 10 minutes using a commercially available H2Se detector.

[0075] Results: The test results are as follows Figure 2 As shown in ac, Figure 2a shows the photothermal heating curves of ZnSe, ZnSe / rGO solutions, and Z@G and Z / r@G hydrogels. Figure 2 b represents the fluorescence intensity of each group detected using the 2,1,3-benzoselenidazole fluorescence method for H2Se release. Figure 2 c represents the H2Se release concentration of each group detected using an H2Se gas detector.

[0076] Conclusion: A 1 mg / mL ZnSe / rGO solution can raise the temperature to 50.47 °C within 10 min, with a photothermal conversion efficiency 5.69 times higher than that of a 1 mg / mL ZnSe solution. Furthermore, the Z / r@GO hydrogel also exhibits a highly efficient photothermal effect, with no significant difference in heating performance compared to the ZnSe / rGO solution of the same concentration, while the PTT effect of the Z@G hydrogel remains poor. Additionally, under acidic conditions, combined with the photothermal heating characteristics assisted by NIR light, the 1 mg / mL ZnSe / rGO heterojunction can release more H2Se compared to ZnSe.

[0077] Test Example 3 Antioxidant performance test of ZnSe / rGO heterojunction method: a. Hydroxyl radical (·OH) scavenging experiment: First, based on the Fenton reaction, a ·OH working solution was prepared by mixing 600 μL FeCl2·4H2O (5 mM) with 400 μL H2O2 (5 mM). After 5 min, ZnSe / rGO (0.25, 0.5, 0.75, 1 mg / mL) and MB (1 mM) were added sequentially and stored at room temperature for 50 min. The final concentrations of MB, FeCl2·4H2O, and H2O2 were 15 μM, 1.5 mM, and 1.0 mM, respectively. Finally, the absorbance curves of the pure MB solution, the MB solution with added ·OH, and the mixed solution of MB, ·OH, and ZnSe / rGO were measured using UV-Vis, as well as the absorbance value at 664 nm. Each group was measured in triplicate.

[0078] b. Superoxide anion radical (·O2) -Scavenging experiment: 776 mg of methionine was added to 40 mL of deionized water to prepare a methionine solution (130 mM); 3.76 mg of riboflavin was added to 50 mL of deionized water to prepare a riboflavin solution (200 μM); 30.66 mg of NBT was added to 50 mL of deionized water to prepare an NBT solution (750 μM). 0.3 mL of each of the methionine, riboflavin, and NBT solutions were mixed. 1.1 mL of ZnSe / rGO solutions of different concentrations (0.25, 0.5, 0.75, and 1 mg / mL) were added to the mixture, and the mixture was irradiated with ultraviolet light for 5 min. Subsequently, the supernatant was extracted, and the absorbance curve and absorbance value at 560 nm were detected using UV-Vis. Each group was repeated three times.

[0079] c. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging experiment: 1 mg DPPH was added to 20 mL of anhydrous ethanol and sonicated for 5 min to obtain a DPPH solution. The absorbance curves and absorbance values ​​at 519 nm were measured using UV-Vis for a mixture of 2 mL DPPH solution and 1 mL anhydrous ethanol, and for a mixture of 2 mL DPPH test solution and 1 mL of ZnSe / rGO solutions of different concentrations (0.25, 0.5, 0.75, 1 mg / mL). Each group was repeated three times.

[0080] d. 2-Phenyl-4,4,5,5-Tetramethylimidazoline-1-oxygen (PTIO) radical scavenging experiment: 3 mg of PTIO was dissolved in 20 mL of deionized water to prepare the PTIO test solution. 1 mL of ZnSe / rGO solutions of different concentrations (0.25, 0.5, 0.75, 1 mg / mL) were mixed with 1.4 mL of the PTIO test solution and placed in a 37℃ drying oven for 30 min. Subsequently, the supernatant was extracted, and the absorbance curve and absorbance value at 557 nm were detected using UV-Vis. Each group was repeated three times.

[0081] The results are as follows Figure 2 As shown in dg, Figure 2 d is the absorbance spectrum for detecting ·OH levels. Figure 2 e is for detecting O2 - Horizontal absorbance spectrum, Figure 2 f is the absorbance spectrum for detecting DPPH free radical levels. Figure 2 g represents the absorbance spectrum for detecting PTIO free radicals.

[0082] Conclusion: ZnSe / rGO heterojunction pairs ·OH, ·O2 - DPPH and PTIO have highly efficient free radical scavenging capabilities and are significantly superior to rGO at the same concentration.

[0083] Test Example 4 Antibacterial performance test Methods: The antibacterial properties of Z@G and Z / r@G hydrogels were tested under physiological conditions (pH=7.4) and acidic conditions (pH=5.0). Under acidic conditions, the pH of LB liquid medium and PBS was adjusted to 5.0 before the experiment.

[0084] a. Plating method: Bacteria treated with sterile PBS were used as a control group. First, MRSA was further diluted to 10⁻⁶ with sterile PBS. 5 CFU / mL, each 200 μL of PBS or hydrogel containing 0.25, 0.5, 0.75, or 1 mg / mL ZnSe or ZnSe / rGO solution was placed in a 48-well microplate and incubated with 1 mL of bacterial suspension for 24 h. Then, the NIR group was treated with an NIR laser (808 nm, 1.5 W / cm²). 2 Irradiate for 10 min; incubate in the dark group at room temperature for 10 min. Then, take 50 μL of the treated bacterial solution and spread it evenly on LB agar plates, and incubate in a bacterial incubator for 24 h.

[0085] b. Detection of airborne bacteria and biofilm morphology: SEM was used to observe the morphology and structure of bacteria after different treatments. For the airborne bacteria group, 400 μL of PBS or hydrogel containing 0.25, 0.5, 0.75, or 1 mg / mL ZnSe or ZnSe / rGO solution was placed in a 24-well plate pre-filled with sterile cell smears, along with 2 mL of MRSA bacterial suspension (2 × 10⁻⁶). 6 The biofilm group was co-incubated with MRSA bacterial suspension (2 mL, 2 × 10⁻⁶ CFU / mL) for 24 h. 6 CFU / mL was added to well plates containing sterile cell crawling sheets and cultured in a bacterial incubator for one week, with the LB broth (pH=7.4) medium being changed every 24 h to allow biofilm formation on the crawling sheets. After biofilm formation, the medium was removed, and the cells were rinsed three times with 1 mL of sterile water to remove airborne bacteria. The biofilm was then co-incubated with 400 μL of PBS or a hydrogel containing 0.25, 0.5, 0.75, or 1 mg / mL ZnSe or ZnSe / rGO solutions for 24 h. Subsequently, the airborne bacteria and biofilm groups were compared with the hydrogel NIR group using a NIR laser (808 nm, 1.5 W / cm²). 2 Irradiate the bacteria in the well plate for 10 min. Then wash the slides three times with sterile PBS, immerse them in 2.5% glutaraldehyde fixative for 1 h, and then perform gradient dehydration with ethanol (volume fractions of 30%, 50%, 70%, 80%, 90%, and 100% respectively), with each dehydration treatment lasting 10 min. Finally, spray the surface of the slides with gold and observe them by SEM.

[0086] The results are as follows Figure 3 As shown, Figure 3 a represents the colony count of MRSA under pH=7.4 and pH=5.0 conditions; Figure 3 b represents the morphology of planktonic bacteria and biofilm in MRSA under pH=7.4 and pH=5.0 conditions.

[0087] Conclusion: Under light irradiation, 1 mg / mL Z / r@G hydrogel showed the best antibacterial effect against MRSA. It killed the bacteria by damaging the cell membrane through photothermal effect and H2Se entering the bacterial cell, thus disrupting cell integrity.

[0088] Test Example 5 Anti-cellular aging performance test method: a. Mouse embryonic osteoblast precursor cells (MC3T3-E1) were seeded in well plates and cultured for 24 h. The control group was given 400 μm H2O2 and ordinary cell culture medium. The GelMA, Z@G and Z / r@G groups were placed in a medium containing 400 μm H2O2. GelMA hydrogel extract or hydrogel extract containing 0.75, 1.0 or 2.5 mg / mL Z@G or Z / r@G (extraction concentration of 0.02 g / mL) was added to the medium. In the Z@G + H2Se and Z / r@G + H2Se groups, cell chambers containing 0.02 g / mL aluminum selenide solution were placed in culture plates after adding 400 μm H2O2 and hydrogel extract containing 0.75, 1, and 2.5 mg / mL Z@G or Z / r@G solution, respectively. The cells were then removed after being placed in the culture plates. This simulated the concentration of H2Se produced under acidic heating conditions of Z@G and Z / r@G. The cells in each group were cultured in a cell culture incubator for 24 h to construct a cell senescence model.

[0089] b. Intracellular ROS detection: After treating each group of cells according to step (4)a, wash the well plate with PBS 3 times, add DCFH-DA solution (10μm) to each well to completely immerse the cells, incubate in the cell culture incubator in the dark for 30 min, wash the well plate with PBS 3 times, and then collect and analyze the images under an inverted fluorescence microscope.

[0090] c. γ-H2AX immunofluorescence staining: Cells were seeded in 24-well plates containing cell crawling smears and cultured for 12 h. After treating each group of cells according to step (4)a, the well plates were washed with PBS buffer, fixative was added, and the plates were incubated at room temperature for 15 min. After removing the fixative, the well plates were washed three times (3 min each time) with special washing solution. Immunoblocking buffer was injected and the plates were treated at room temperature for 20 min. After discarding the blocking solution, γ-H2AX-specific rabbit primary antibody was added and the plates were incubated at room temperature for 1 h. The working solution of the primary antibody was removed, and the plates were washed three times (5 min each time) with washing solution. Fluorescently labeled anti-rabbit secondary antibody was added and the plates were incubated at room temperature in the dark for 1 h. After removing the working solution of the secondary antibody, the plates were washed twice (5 min each time). Nuclear staining reagent was added and the plates were stained at room temperature in the dark for 5 min. After completing the nuclear staining, the plates were washed three times (3 min each time) and then observed under a laser confocal microscope.

[0091] d. Staining of aging-related β-galactosidase (SA-β-Gal): After treating each group of cells according to step (4)a, wash the well plate once with PBS, add 300 μL of β-galactosidase staining fixative, and fix at room temperature for 15 min; wash the well plate three times with PBS, add 300 μL of staining working solution; seal the well plate with sealing film, incubate at 37℃ for 24 h, and then collect and analyze images under a microscope.

[0092] The results are as follows Figure 4 As shown, Figure 4 a shows fluorescence images of intracellular ROS levels in each group; Figure 4 b shows fluorescence images used to detect intracellular DNA damage in each group; Figure 4 c is a staining image used to detect the SA-β-Gal expression level in cells of each group.

[0093] Conclusion: The Z / r@G + H2Se group can significantly reduce ROS levels, alleviate cellular DNA damage, and significantly improve the cellular senescence state.

[0094] Test Example 6 Biocompatibility assessment method: a. Cells were cultured in α-MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin mixture. Cells were placed in a 37°C, 5% CO2 incubator. When the adherent cell density approached 80%, trypsin was used for digestion. After digestion, the decanted cells were resuspended in fresh medium, and the cell density was controlled at 2 × 10⁻⁶ cells / year by counting. 4 Cells / mL. Subsequently, 100 μL of cell suspension was seeded into 48-well plates and 400 mL of cell culture medium was added to prepare for subsequent cell experiments.

[0095] b. Cell morphology observation: Cells were seeded in 48-well plates containing cell slides and cultured for 12 h. The original culture medium was then discarded, and 500 μL of hydrogel extract containing GelMA, or hydrogel extract containing 0.75, 1.0, or 2.5 mg / mL Z@G or Z / r@G (extraction concentration 0.02 g / mL) was added. The medium was changed every 2 days. On the second day, the plates were washed three times with PBS. Pre-cooled 2.5% glutaraldehyde fixative (4°C) was slowly added along the container wall, and the plates were stored overnight at 4°C. After fixation, the slides were washed three times with PBS, followed by a gradient dehydration process with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol for 10 min each time. After drying, the slides were sputter-coated with gold and observed using SEM.

[0096] c. CCK-8 method: After seeding cells in 48-well plates and culturing for 12 h, the original culture medium was discarded, and 500 μL of hydrogel extraction medium containing GelMA was added, or hydrogel extraction medium containing 0.75, 1.0, or 2.5 mg / mL Z@G or Z / r@G (extraction concentration of 0.02 g / mL) was added. The medium was changed every 2 days using the extraction medium. On days 1, 3, and 5, the wells were washed three times with PBS. The CCK-8 reagent and culture medium were mixed at a ratio of 1:10 to prepare the working solution. 500 μL of the working solution was added to each well, and after incubation for 1 h, the absorbance value at 450 nm was measured using a microplate reader.

[0097] The results are as follows Figure 5 As shown, Figure 5 a represents the cell morphology recorded by SEM in different groups of cultured cells; Figure 5 b represents the CCK-8 detection results for each group of cells.

[0098] Conclusion: GelMA, Z@G, and Z / r@G hydrogels exhibit excellent biocompatibility, and cells can grow on nanofiber membranes with good morphology.

[0099] Test Example 7 Osteogenic performance test method: a. Normal cell culture: MC3T3-E1 cells were seeded at 2×10⁵ cells / well in 24-well plates. The GelMA, Z@G, and Z / r@G groups were treated with culture medium containing hydrogel extract (0.02 g / mL). The Z@G + H₂Se and Z / r@G + H₂Se groups were treated with culture medium containing hydrogel extract (0.02 g / mL), and cell chambers containing 0.025 mg / mL and 0.1 mg / mL aluminum selenide solution, respectively, were placed in the culture plate for 10 min and then removed. The control group received no treatment. All cells were cultured in a cell culture incubator for 24 h.

[0100] b. Construction of osteoblast senescence model: The MC3T3-E1 cell culture procedure is the same as that in test case 5a.

[0101] c. An osteogenic induction culture system was constructed by adding 50 μg / mL ascorbic acid, 10 nM dexamethasone, and 10 nM β-glycerophosphate to the basal medium (containing serum). For the hydrogel group, water was added to the gel extraction buffer (0.02 g / mL) during the preparation of the osteogenic induction medium. For both test cases 5a and b, osteogenic induction medium was used for subsequent medium changes every 2-3 days.

[0102] d. Cells were induced and cultured in osteogenic medium for 7 days, and the ALP activity of cells in each group was detected using an alkaline phosphatase (ALP) assay kit.

[0103] The results are as follows Figure 6 As shown, Figure 6 a) To detect ALP activity in each group 7 days after inducing osteogenic differentiation of normal cells; Figure 6 b represents the detection of ALP activity in each group 7 days after inducing osteogenic differentiation of senescent cells.

[0104] Conclusion: Z / r@G hydrogel significantly increased ALP activity in MC3T3-E1 cells, and the Z / r@G + H2Se group could alleviate the inhibitory effect on osteoblast differentiation under senescence.

[0105] Comparative Test Example 1: rGO was prepared using the same method as in Example 1 (without adding Zn(NO3)2·7H2O and selenium powder).

[0106] like Figure 2 As shown in dg, the antioxidant capacity of this material is significantly reduced compared to Z / r@G.

[0107] Comparative Test Example 2: ZnSe and ZnSe-containing composite GelMA hydrogels (Z@G) were prepared using the same method as in Example 1 (without adding GO).

[0108] like Figure 3 As shown in a and b, the antibacterial efficiency of this material under near-infrared light irradiation is significantly lower than that of Z / r@G.

[0109] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel material, characterized in that, The hydrogel material is used as a drug for repairing diabetic infectious bone defects, and the preparation method includes: ZnSe / rGO heterojunctions were prepared by a hydrothermal method using ZnSe and rGO, where ZnSe is zinc selenide, a semiconductor material, and rGO is reduced graphene oxide, a conductor material. ZnSe / rGO heterostructures were uniformly dispersed in a GelMA matrix, and the ZnSe / rGO heterostructures were loaded and fixed in the GelMA to obtain the hydrogel material Z / r@G, where Z refers to ZnSe, r refers to rGO, G refers to GelMA, and GelMA is methacryloyl gelatin. The step of preparing ZnSe / rGO heterojunction by hydrothermal method includes: Graphene oxide and zinc nitrate heptahydrate were dispersed in a mixed solution of ethanol and water to obtain mixed solution a; Selenium powder and sodium hydroxide were added to deionized water and stirred at room temperature. Then hydrazine hydrate was added, and stirring continued to yield a reaction solution containing an active selenium source. Add the mixed solution a to the reaction solution containing the active selenium source, add polyvinylpyrrolidone, stir, and perform a hydrothermal reaction to obtain the reaction product; The amount of selenium powder and the amount of zinc nitrate heptahydrate satisfy the relationship between Se and Zn. 2+ The molar ratio is controlled between 0.5 and 1.5; the mass ratio of the amount of graphene oxide to the zinc nitrate heptahydrate is controlled between 1:20 and 1:10; and the amount of polyvinylpyrrolidone is 200 to 250 mg.

2. The method for preparing the hydrogel material according to claim 1, characterized in that, The hydrothermal method is performed with the hydrothermal temperature controlled at 175–190°C for 10–14 hours.

3. The method for preparing the hydrogel material according to claim 1, characterized in that, The reaction product was washed sequentially with anhydrous ethanol and deionized water, and then the precipitate was collected by filtration. The precipitate was dried under vacuum freeze-drying conditions to obtain ZnSe / rGO heterojunction powder.

4. The method for preparing the hydrogel material according to claim 1, characterized in that, The steps for uniformly dispersing ZnSe / rGO heterostructures in a GelMA matrix, loading and immobilizing the ZnSe / rGO heterostructures in the GelMA matrix to obtain the hydrogel material Z / r@G include: Dissolve GelMA in phosphate buffer and stir to obtain a GelMA solution; Photoinitiator LAP and a ZnSe / rGO heterojunction were added to a GelMA solution and mixed thoroughly to obtain a mixed solution, wherein the concentration of ZnSe / rGO was 0.25–2.5 mg / mL; and The mixed solution was poured into a mold and cross-linked and cured under ultraviolet light to form a ZnSe / rGO composite GelMA hydrogel, thus obtaining the Z / r@G.

5. A hydrogel material, characterized in that, A hydrogel material is obtained by the preparation method described in any one of claims 1-4. The hydrogel material is Z / r@G, where Z refers to ZnSe, r refers to rGO, and G refers to GelMA. GelMA is methacrylamide gelatin, ZnSe is zinc selenide, a semiconductor material, and rGO is reduced graphene oxide, a conductor material. ZnSe serves as a source of H2Se gas and can achieve controlled release of H2Se in an acidic microenvironment accompanied by photothermal heating. rGO serves as a photothermal conversion unit and provides a heating effect for the release of H2Se from ZnSe under acidic and photothermal synergistic conditions at the infection site. GelMA serves as an injectable three-dimensional carrier for loading and immobilizing the ZnSe / rGO heterojunction.

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