Application of multifunctional manganese-based hydrogel in adjustment of energy metabolism and promotion of bone regeneration

Abstract

The invention relates to the technical field of biology, in particular to application of multifunctional manganese-based hydrogel to adjustment of energy metabolism and promotion of bone regeneration. The invention provides hydrogel, which comprises chitosan-coated dexamethasone-containing manganese dioxide nanoparticles, and in-vitro experiment results show that the hydrogel can effectively remove excessive ROS, recover mitochondrial functions, reduce the level of inflammatory factors, reduce glycolysis by inhibiting ERK / HIF-1alpha / GLUT1 pathway, improve the activity of the hydrogel, and improve the activity of the hydrogel. And M1 macrophages are driven to be converted into M2 phenotype to remodel anti-inflammatory and regeneration-promoting ecological niche. Results of an in-vivo experiment and an in-vitro experiment are consistent, the ROS level in a rat body can be reduced, a local immune microenvironment is remodeled, and bone regeneration is continuously promoted.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of multifunctional manganese-based hydrogel in regulating energy metabolism and promoting bone regeneration. Background Art

[0002] Bone defects caused by various reasons affect millions of patients psychologically and physiologically and significantly reduce their quality of life. More and more studies have shown that bone healing is an elaborate process involving the close interaction of multiple systems including the skeletal system and the immune system. Immune cells secrete factors to establish a microenvironment that regulates osteoblasts and osteoclasts, playing a crucial regulatory role in bone resorption and bone formation. Various biomaterials have been studied with an emphasis on developing bone substitutes with ideal physicochemical, mechanical and osteogenic properties related to bone formation. However, ignoring the regulatory role of the immune system in the biomaterial-mediated bone microenvironment may lead to adverse outcomes. After implantation in vivo, the interaction between biomaterials and immune cells without regulatory functions can induce an inflammatory response. Uncontrolled inflammatory responses can disrupt bone homeostasis and delay wound healing and bone tissue regeneration. In contrast, a beneficial anti-inflammatory microenvironment regulated by biomaterials can ensure osteoblast differentiation, angiogenesis and tissue repair.

[0003] Macrophages are an important component of the innate immune system and are highly plastic. They can be transformed into classically activated macrophages (M1) or selectively activated macrophages (M2) according to the molecular mediators in the microenvironment. M2 macrophages usually express high levels of interleukin-10 (IL-10), arginase-1 (Arg-1), bone morphogenetic protein 2 (BMP-2), transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), and their functions are to inhibit inflammation, recruit progenitor cells, activate angiogenesis, extracellular matrix remodeling and bone regeneration. The dynamic changes of this phenotype are closely related to their mitochondrial metabolic state. M1 macrophages meet the high energy requirements required for proinflammatory responses through glycolysis and the pentose phosphate pathway (PPP), while M2 macrophages mainly rely on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). The use of the glycolysis inhibitor 2-DG or knockdown of key glycolytic enzymes can inhibit the proinflammatory response of M1 macrophages. Reprogramming macrophages to an M2 phenotype by protecting mitochondrial function and regulating their energy metabolism appears to be a potential strategy to promote bone regeneration. In addition, bone marrow mesenchymal stem cells (BMSCs) are an important cell source for bone formation. During bone healing, BMSCs migrate to the bone defect area, proliferate and differentiate into osteoblasts, increase the formation of trabeculae, and ultimately promote bone regeneration. However, mitochondrial dysfunction after bone defect occurs disrupts the redox balance of BMSCs, resulting in impaired osteogenic differentiation of BMSCs and delayed bone healing. Therefore, new therapeutic approaches can be designed to achieve bone healing by regulating mitochondrial function to reshape the immune microenvironment and promote osteogenic differentiation.

[0004] Mitochondria are the main site for the production of intracellular reactive oxygen species (ROS). Under pathological conditions of fracture, mitochondrial dysfunction, abnormal activation of energy metabolism, and inhibition of the intracellular antioxidant system can lead to excessive ROS production, which has been shown to aggravate inflammatory responses and hinder bone repair. Similarly, excessive ROS and the transformation of macrophage phenotypes promote each other. Excessive M1 polarization affects mitochondria and promotes more ROS production, while a large amount of ROS produced by mitochondrial dysfunction promotes macrophages to be in a "pseudo-hypoxia" state and leads to changes in energy metabolism pathways, aggravating inflammatory responses and tissue damage, forming a vicious cycle. It can be seen that clearing ROS in the bone microenvironment and cellular mitochondria is crucial for protecting mitochondria and promoting bone repair.

[0005] However, traditional antioxidants may face challenges such as poor stability, excessive dosage and low bioavailability. With the development of nanoscience, some nanomaterials with inherent catalytic properties have shown great advantages. Manganese nanoparticles (MnNPs) have ideal multi-enzyme-like activities, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), etc. Manganese dioxide (MnO2) can directly catalyze H2O2 to generate O2 and Mn 2+ . Mn 2+ Can simulate the activity of SOD, and •O2 − The reaction produces H2O2. In addition, Mn 2+ It also plays an important role in muscle and bone metabolism. Yang et al. designed a hollow mesoporous manganese dioxide (H-MnO2) nanoparticle to achieve drug delivery and controlled release through the hollow nanostructure of H-MnO2. Shen et al. used H-MnO2 to catalyze H2O2 to generate O2 in local areas, promote lactate depletion, and alleviate the nucleus pulposus microenvironment of lactate accumulation. These characteristics make H-MnO2 one of the most promising nanozymes and drug delivery platforms. Chitosan (CS) is a biodegradable natural cationic polysaccharide with excellent biocompatibility and is widely used in biomedical implants, scaffolds, drug delivery systems, and biosensors. Recently, CS has also been used in pharmaceutical nanotechnology as a surface coating for nanocarriers. The presence of CS coating not only enhances the extracellular transport of NPs, but also promotes their cellular uptake. In addition, the positive charge carried by CS-coated NPs can increase their internalization through interactions with negatively charged protein domains or phospholipid head groups on the cell surface. Coating the positively charged CS on the negatively charged H-MnO2 NPs surface through electrostatic self-assembly can promote their contact with the cell membrane and uptake, so that the NPs can fully exert their efficacy in the cells. Although NPs have high catalytic efficiency in scavenging ROS, considering the diversity of the pathological mechanisms of bone defects, it is necessary to combine the scavenging of ROS and the promotion of osteogenic differentiation of BMSCs to further promote the regeneration of bone tissue.

[0006] Biomolecules, including growth factors and osteogenic small molecules, are important components for improving osteogenic activity in bone tissue engineering. Compared with the high cost, instability, immunogenicity and structural integrity requirements of growth factors, osteogenic small molecules have obvious advantages. Among these osteogenic small molecules, dexamethasone (Dex) is representative. Many studies have shown that Dex can replace growth factors and promote bone regeneration. It binds to glucocorticoid receptors, induces upregulation of bone-related genes such as alkaline phosphatase (ALP) and osteocalcin (OCN), and promotes the differentiation of stem cells into osteoblasts. Although excessive doses of Dex may cause side effects such as osteoporosis and non-traumatic osteonecrosis that are not conducive to bone remodeling. However, lower concentrations of Dex (10~100 nM) have been widely used to promote osteogenic differentiation of stem cells, enhance bone mineralization and upregulate the expression of osteogenic-related genes. In addition, lower concentrations of Dex can also be used as anti-inflammatory drugs for immunomodulation. Webber et al. used peptide nanofiber gels to controllably release Dex to regulate inflammatory responses. Xie et al. functionalized Dex on the implant surface, and in the first few weeks after implantation, the stable and controlled release of low doses inhibited inflammation and stimulated bone formation, effectively avoiding its side effects and promoting the smooth progress of the osteogenesis process. Alginate is a polymer biomaterial derived from the ocean and has been widely used in biomedical and biotechnological fields such as tissue engineering, wound repair, and drug delivery. Alginate hydrogels can be formed by in situ ionic crosslinking with multivalent cations under physiological conditions. However, ionic crosslinking can lead to poor mechanical properties and heterogeneous structures, which hinders the application of alginate hydrogels in biomedical materials. Summary of the invention

[0007] In view of this, the present invention provides an application of a multifunctional manganese-based hydrogel in regulating energy metabolism to promote bone regeneration, which not only develops a multifunctional drug delivery system for bone defect repair, but also reveals the significance and feasibility of reverse reprogramming by regulating the energy metabolism of macrophages during bone regeneration.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing a hydrogel, comprising the following steps:

[0010] S1. Under the action of ultrasound, potassium permanganate solution was added dropwise to the silica nanoparticle suspension, and the ultrasonic treatment was continued for 1 h. The suspension was stirred overnight, and the supernatant was discarded to obtain silica nanoparticles coated with manganese dioxide.

[0011] S2, mixing the manganese dioxide-coated silica nanoparticles with a sodium carbonate solution, etching at 60° C., removing the supernatant, and washing to obtain hollow mesoporous manganese dioxide;

[0012] S3, mixing the hollow mesoporous manganese dioxide with a dexamethasone alcohol solution, ultrasonically shaking, removing the supernatant, and obtaining manganese dioxide nanoparticles containing dexamethasone;

[0013] S4, mixing the chitosan solution with the manganese dioxide nanoparticles containing dexamethasone, and stirring to obtain positively charged manganese dioxide nanoparticles containing dexamethasone;

[0014] S5. Mixing the AlgMA solution with the positively charged manganese dioxide nanoparticles containing dexamethasone, and photocrosslinking to obtain a hydrogel.

[0015] In some specific embodiments of the present invention, the concentration of the potassium permanganate solution in the above preparation method is 600 mg / 20 mL.

[0016] In some specific embodiments of the present invention, the concentration of the sodium carbonate solution in the above preparation method is 2 mol / L.

[0017] In some specific embodiments of the present invention, the dexamethasone alcohol solution in the above preparation method is a dexamethasone ethanol solution.

[0018] In some specific embodiments of the present invention, the chitosan content of the chitosan solution in the above preparation method is 1 mg / mL, and the solvent is 1% acetic acid.

[0019] In some specific embodiments of the present invention, the AlgMA solution in the above preparation method contains 500 mg AlgMA and 0.025 g I2595 photoinitiator, and the solvent is 10 mL PBS.

[0020] The invention also provides the hydrogel prepared by the preparation method.

[0021] The present invention also provides the use of the above hydrogel in any of the following items:

[0022] (i) Preparation of products for scavenging ROS during bone regeneration;

[0023] (ii) preparing products for improving mitochondrial function;

[0024] (iii) preparing a product of reprogrammed macrophages;

[0025] (iv) preparing products for promoting osteogenic differentiation of mesenchymal stem cells;

[0026] The product includes a reagent or a drug.

[0027] The present invention also provides the use of the above hydrogel in preparing products for regulating energy metabolism, improving local immune microenvironment and / or promoting bone regeneration;

[0028] The product includes a reagent or a drug.

[0029] The present invention also provides a medicine, comprising the above hydrogel and acceptable auxiliary materials or adjuvants.

[0030] The present invention also provides a method for treating orthopedic diseases, comprising administering the above hydrogel;

[0031] The orthopedic disease may be nonunion of fracture, delayed union of fracture, bone defect, osteoporotic fracture or osteonecrosis.

[0032] The present invention has the following effects:

[0033] Hollow mesoporous manganese dioxide with good ROS scavenging and drug loading capacity was used, and dexamethasone was loaded and then electrostatically self-assembled through chitosan coating and incorporated into photo-crosslinkable sodium alginate hydrogel to obtain nanogel. The results of in vitro experiments showed that nanozymes can effectively remove excess ROS, restore mitochondrial function, and reduce the level of inflammatory factors. It down-regulates glycolysis by inhibiting the ERK / HIF-1α / GLUT1 pathway and drives M1 macrophages to convert to M2 phenotype to reshape the anti-inflammatory and pro-regenerative niche. The results of in vivo experiments are consistent with those of in vitro experiments. Nanozymes can reduce the level of ROS in rats, reshape the local immune microenvironment, and continuously promote bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0035] Figure 1 Schematic diagram of the preparation and application process of hydrogel, wherein A is a schematic diagram of the preparation of multifunctional manganese-based hydrogel, B is a schematic diagram of the SOD-like and CAT activities of hollow mesoporous manganese dioxide nanoparticles, C is a schematic diagram of the multifunctional manganese-based hydrogel transforming the phenotype of macrophages and reducing the oxidative stress of BMSCs, and D is a schematic diagram of the hydrogel establishing a suitable immune microenvironment in vitro and promoting bone regeneration;

[0036] Figure 2 The preparation of nanoparticles and their physical and chemical properties are shown, wherein A shows the preparation process of AlgMA, B shows the characterization of AlgMA, C~E show SEM images of hollow mesoporous manganese dioxide, F shows the HAADF-STEM image of hollow mesoporous manganese dioxide, G shows the XRD analysis of hollow mesoporous manganese dioxide, H shows the XPS analysis of hollow mesoporous manganese dioxide, I shows the cell viability of hollow mesoporous manganese dioxide at different concentrations, J shows the CAT-like activity of hollow mesoporous manganese dioxide at different concentrations, K shows the SOD-like activity of hollow mesoporous manganese dioxide at different concentrations, and L shows the ABTS capacity of hollow mesoporous manganese dioxide at different concentrations;

[0037] Figure 3: shows the characterization of the multifunctional composite hydrogel, wherein A shows the SEM images of different manganese dioxide nanoparticles, B shows the TEM images of different manganese dioxide nanoparticles, C shows the SEM images of different hydrogels, D shows the zeta potential of different manganese dioxide nanoparticles, E shows the FTIR of nanoparticles, F shows the rheological analysis of different hydrogels, G shows the manganese release curves of different hydrogels, H shows the Dex drug release curves of different hydrogels, and I shows the swelling ratios of different hydrogels;

[0038] Figure 4 Nanogels reprogram macrophages and regulate mitochondrial function, wherein A-B show fluorescence images and semi-quantitative analysis of mitochondrial membrane potential, C-D show iNOS and CD206 expression and semi-quantitative analysis of macrophages treated with different groups of hydrogels, E shows iNOS and Arg-1 protein expression in macrophages treated with different groups of hydrogels, F shows flow cytometry analysis of macrophages, G-H show OCR levels and semi-quantitative analysis of macrophages treated with different groups of hydrogels, I-J show ECAR levels and semi-quantitative analysis of macrophages treated with different groups of hydrogels;

[0039] Figure 5 The multifunctional hydrogel reprogramming macrophages promotes osteogenic differentiation of mesenchymal stem cells, wherein A shows the preparation of macrophage conditioned medium, B~E show the ELISA test results of osteogenic related active factors TNF-α, IL-10, TGF-β and BMP-2, F~G show ALP staining and semi-quantitative analysis of different material groups, H~I show ARS staining and semi-quantitative analysis of different material groups, J~K show RUNX2 and OPN immunofluorescence staining and semi-quantitative analysis of different material groups, L~M show RUNX2 and OPN protein level expression and semi-quantitative analysis of different material groups, N~Q show the expression levels of osteogenic related genes in different material groups;

[0040] Figure 6 Figure 4 shows the evaluation of the ability of hydrogel to directly promote osteogenic differentiation, where A-B show BMSCs live-dead staining and live cell staining fluorescence quantitative analysis, C-D show CCK-8 detection of cell proliferation, E-H show RUNX2 and OPN immunofluorescence staining, and I-J show RUNX2 and OPN protein expression levels;

[0041] Figure 7 The in vitro immunomodulatory ability of the hydrogel is shown, wherein A-B show the level of reactive oxygen species in vitro and semi-quantitative analysis, C shows iNOS fluorescence staining, and D shows Arg-1 fluorescence staining semi-quantitative analysis;

[0042] Figure 8The hydrogel promoted bone regeneration in rats in vitro, where A shows CT images and three-dimensional reconstructions at 2 and 4 weeks, B-E show bone regeneration indexes evaluated by CT at 2 and 4 weeks, F-G show HE and MASSON sections of each group, and H-I show OPN and OCN fluorescence expressions of each group. DETAILED DESCRIPTION

[0043] The present invention discloses the application of multifunctional manganese-based hydrogel in regulating energy metabolism and promoting bone regeneration. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] In order to solve the problems of the background technology, the present application provides a photo-crosslinking technology for gelation of alginate aqueous solution. Methacrylic anhydride-modified alginate can be photocured into gel under the action of a photoinitiator, and compared with ion-crosslinked hydrogels, it has better mechanical properties, structural stability and adaptability to different clinical scenarios. Due to its in situ photo-crosslinking characteristics, methacrylated sodium alginate hydrogel (AlgMA) can adapt well to bone defect areas of different shapes and confine NPs to the fracture site to effectively play the role of clearing ROS, regulating inflammation and promoting osteogenesis. Inspired by this, Dex was loaded into the cavity of CS-coated H-MnO2 and added to AlgMA hydrogel to achieve low-dose stable and controllable drug release, which can regulate macrophage polarization and promote bone defect repair.

[0045] This application constructs a multifunctional nano drug delivery platform that can quickly remove excess ROS, exert anti-inflammatory effects, and continuously promote the osteogenic differentiation of BMSCs. First, this application uses the "shell-core" method to synthesize H-MnO2 and explores its antioxidant properties and drug loading capacity. Dex is loaded into H-MnO2 to promote bone regeneration by stable and sustained release at low doses. Subsequently, CS is used to encapsulate NPs to improve sustained release and cellular uptake capacity. CS-coated NPs are internalized by macrophages during the acute inflammatory period, promoting the transition of macrophages to the M2 phenotype. The combined analysis of transcriptomics and metabolomics further explored the potential mechanism by which NPs affect changes in macrophage function. In addition, the effect of macrophages on the osteogenic differentiation of BMSCs through the secretory pathway and the stable and continuous promotion of osteogenesis by NPs through drug release were also studied, proving the role of NPs in improving the immune microenvironment and promoting bone healing after bone injury. Finally, NPs were incorporated into AlgMA hydrogel and implanted into the femoral condyle defect model via in situ photocrosslinking to evaluate the immunomodulatory and osteogenic differentiation promoting effects of the nanomaterials in vivo.

[0046] 1. Preparation of methacrylated alginate (AlgMA)

[0047] 1 g of Alg powder was added to deionized water and stirred at 60 °C to obtain a 1% (w / v) Alg solution. 16 mL of MA solution was slowly added to 100 mL of the mixture and stirred continuously at 50 °C for 4 h. After the reaction was completed, the product was collected and dialyzed against deionized water (molecular weight cutoff 12-14 kDa) for 72 h. The dialyzed water was replaced twice a day to remove excess MA. The remaining purified solution was frozen at -80 °C for 12 h and freeze-dried for 48 h to obtain the synthetic product AlgMA.

[0048] 2. Characterization of AlgMA

[0049] The spectra of Alg and AlgMA hydrogels were recorded on a Bruker AVANCE III 500 (500 MHz) spectrometer (Bruker, Germany). 1 H NMR spectroscopy was used to identify the changes in the newly formed peaks of the reaction. Briefly, Alg and AlgMA were dissolved in D2O (2% w / v) and examined.

[0050] 3. Preparation of hollow mesoporous manganese dioxide (H-MnO2NPs) and loading of dexamethasone

[0051] The improved "shell-core" method was used for synthesis. First, solid silica nanoparticles were synthesized by the reverse microemulsion method, and the prepared silica was immediately dispersed in 10 mL of deionized water. Then, 20 mL of aqueous solution containing KMnO4 (600 mg) was added dropwise under ultrasonication, and ultrasonication was continued for 1 h, and stirred at room temperature overnight. The reactants were centrifuged at 14800 rpm for 10 min, and the supernatant was removed to obtain MnO2-coated SiO2 nanoparticles. The nanoparticles were then dispersed in 2 M Na2CO3 solution and etched at 60 °C for 12 h. The next day, the reactants were centrifuged at 14800 rpm for 10 min and washed several times with deionized water to obtain H-MnO2NPs.

[0052] The suspension was sonicated for 20 min and slowly shaken in an oscillator at 37°C for 24 h. The suspension was then centrifuged at 14800 rpm for 10 min to obtain manganese dioxide nanoparticles (MD) containing dexamethasone.

[0053] 4. Preparation of Chitosan-coated MnO2 Nanoparticles

[0054] Chitosan solution (1 mg / mL dissolved in 1% acetic acid) was pumped into the H-MnO2 solution at a rate of 0.5 mL / min and stirred for 30 min to obtain positively charged nanoparticles.

[0055] 5. Preparation of multifunctional composite hydrogels

[0056] 500 mg AlgMA was dissolved in 10 mL PBS (containing 0.025 g I2595 Photoinitiator) to prepare a 5% AlgMA solution, which was then photocrosslinked using 405 nm UV light to obtain AlgMA hydrogel. 1 mg / mL MD@CS was added to the AlgMA solution and photocrosslinked to obtain Alg-MD@CS hydrogel.

[0057] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0058] The present invention will be further described below in conjunction with the embodiments.

[0059] Example 1: Preparation of multifunctional manganese-based nanohydrogel

[0060] 1. Preparation of hollow mesoporous manganese dioxide

[0061] The improved "shell-core" method was used for synthesis. First, solid silica nanoparticles were synthesized by the reverse microemulsion method, and the prepared silica was immediately dispersed in 10 mL of deionized water. Then, 20 mL of aqueous solution containing KMnO4 (600 mg) was added dropwise under ultrasonication, and ultrasonication was continued for 1 h, and stirred at room temperature overnight. The reactants were centrifuged at 14800 rpm for 10 min, and the supernatant was removed to obtain MnO2-coated SiO2 nanoparticles. The nanoparticles were then dispersed in 2 M Na2CO3 solution and etched at 60 °C for 12 h. The next day, the reactants were centrifuged at 14800 rpm for 10 min and washed several times with deionized water to obtain H-MnO2NPs.

[0062] 2. Dexamethasone loading in hollow mesoporous manganese dioxide

[0063] H-MnO2 suspensions were prepared in Dex-Ethanol solutions with different concentrations, including 1.25 wt%, 2.5 wt%, 5 wt%, 10 wt% and 20 wt% Dex. The suspensions were sonicated for 20 min and slowly shaken in an oscillator at 37 °C for 24 h. The suspensions were then centrifuged at 14800 rpm for 10 min to obtain MD. The encapsulation efficiency was calculated by measuring the absorbance of Dex in the supernatant at 5 wt% concentration at 240 nm using a UV spectrophotometer (Agilent, USA).

[0064] 3. Chitosan-coated hollow mesoporous manganese dioxide loaded with dexamethasone

[0065] Chitosan solution (1 mg / mL dissolved in 1% acetic acid) was pumped into the H-MnO2 solution at a rate of 0.5 mL / min and stirred for 30 min to obtain positively charged nanoparticles MD@CS.

[0066] 4. Preparation of Nanohydrogel

[0067] 500 mg AlgMA was dissolved in 10 mL PBS (containing 0.025 g I2595 Photoinitiator) to prepare a 5% AlgMA solution, which was then photocrosslinked using 405 nm UV light to obtain AlgMA hydrogel. 1 mg / mL MD@CS was added to the AlgMA solution and photocrosslinked to obtain Alg-MD@CS hydrogel.

[0068] Example 2: Characterization of physical and chemical properties of manganese dioxide and concentration screening

[0069] 1. Cell viability and proliferation assay

[0070] The cells were seeded in 24-well plates (1 × 10 4cells) were co-cultured with each hydrogel. After 3 days of co-culture, the cell viability was calculated by live / dead cell staining. Live / dead cell working staining solution (Solarbio, China) was added to the double-stained cells and incubated at 37°C in the dark according to the instructions. Live / dead cell images were taken using an inverted fluorescence microscope (Carl Zeiss, Germany). CCK-8 reagent (Vazyme, China) was added to the cell culture plate at a ratio of 1:10 to detect cell proliferation on days 1, 3, and 5. 100 μL of supernatant was transferred to a 96-well plate and the absorbance was measured at 450 nm using a microplate reader (Agilent, USA) after incubation for 4 h.

[0071] 2. Determination of SOD-like activity

[0072] The activity was calculated by nitro blue tetrazolium (NBT) assay. Different concentrations of H-MnO2NPs (0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL and 1 mg / mL) were mixed with riboflavin (20 μM), methionine (12.5 mM) and NBT (75 μM) under constant light intensity for 10 min. The full wavenumber scan curve of the solution was measured, and the absorbance of the solution at 560 nm was recorded.

[0073] 3. CAT-like activity assay

[0074] The CAT activity of H-MnO2NPs with different concentrations (0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL and 1 mg / mL) was detected using a CAT activity detection kit (Beyotime, China). The operation was carried out according to the instructions, the full wave number scan curve of the solution was measured, and the absorbance of the solution at 520 nm was recorded.

[0075] 4. Total Antioxidant Capacity Determination

[0076] The total antioxidant capacity was evaluated by ABTS detection kit (Beyotime Biotechnology, China). According to the manufacturer's instructions, the total antioxidant capacity of the above-mentioned H-MnO2NPs at different concentrations was tested, the full wave number scanning curve of the solution was measured, and the absorbance of the solution at 405 nm was recorded. The scavenging rates of SOD, CAT and ABTS were calculated by the following formula:

[0077] Scavenging Efficiency (%) = ;

[0078] Where A0 is the absorbance without H-MnO2NPs, and A is the absorbance with H-MnO2NPs.

[0079] Example 3: Characterization of the physicochemical properties of nanogels

[0080] 1. Scanning Electron Microscopy (SEM)

[0081] The sample was pasted on the SEM sample stage with conductive glue. After the gold spraying process was completed using an ion sputtering coater, the fiber membrane morphology was observed and pictures were taken using SEM with an accelerating voltage of 10 kV.

[0082] 2. Transmission electron microscopy (TEM)

[0083] The internal structure of the hydrogel was observed by transmission electron microscopy at 120 kV.

[0084] 3. X-ray Photoelectron Spectroscopy (XPS)

[0085] Different groups of nanoparticles were used to study the chemical element composition on the surface of the fiber bundle using an X-ray photoelectron spectroscopy analyzer.

[0086] 4. Mechanical compression test

[0087] To evaluate the mechanical properties of the hydrogels, a universal mechanical tester (Hengyi, HY-1080, China) was used for testing. The hydrogels were made into corresponding shapes for compression tests. Then a cylinder (d = 10 mm, h = 5 mm) was prepared as a compression specimen and the pressure was adjusted at 5 mm min -1 The hydrogel is compressed at a rate of , and the compression modulus is determined by calculating the slope of the stress-strain curve passing through the origin.

[0088] 5. Fourier Transform Infrared Spectroscopy (FTIR)

[0089] Each sample was ground with potassium bromide to prepare a sheet, which was then scanned 128 times at a resolution of 4 cm -1 , ranging from 500 to 4000 cm -1 , detect the changes in different groups of hydrogels.

[0090] 6. Rheological testing

[0091] The rheological properties of the hydrogel samples were evaluated using a rheometer (Thermo Scientific, HAAKE RheoStress 6000). During the test, the hydrogel samples (d = 25 mm, h = 2 mm) were placed on the sample stage and the frequency and strain tests were performed at 37 °C using a 25 mm diameter plate apparatus. The storage modulus (G′) and loss modulus (G″) of the hydrogel were measured at oscillation frequencies of 0.1–10 Hz.

[0092] 7. Dexamethasone and manganese release assay

[0093] 1 mL of hydrogel was immersed in 10 mL of PBS in a 50 mL centrifuge tube. The tube was placed in a thermostatic shaker at 37 °C and 100 cycles / min. At time points (0.5 d, 3 d, 5 d, 7 d, 14 d, 21 d, and 28 d), the buffer was collected from the centrifuge tube, and the Dex concentration in the buffer was measured by measuring the absorbance using a UV spectrophotometer (Agilent, USA).

[0094] 1 mL of hydrogel was added to 10 mL of PBS, which was replaced at regular intervals and the resulting supernatant was collected. The concentration of elemental manganese in the supernatant collected at each time point (1 d, 3 d, 5 d, 7 d, 14 d, 21 d, and 28 d) was measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS, PerkinElmer, USA).

[0095] Example 4: In vitro study of multifunctional nanogels

[0096] 1. Preparation of cells for research

[0097] Bone marrow stromal cells (BMSCs) were extracted from rat bone marrow (6 weeks, 200 g) and cultured in DMEM-α (Gibco, USA) medium containing 10% fetal bovine serum (Gibco, USA) and 1% Pencillin-Streptomycin (Gibco, USA) at 37°C in a 5% CO2 incubator, with the medium replaced every 2-3 days.

[0098] RAW 264.7 cell line was purchased from Wuhan Procell Company and cultured in high-glucose DMEM medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), which was replaced every 2-3 days.

[0099] 2. Cell live / dead fluorescence staining detection

[0100] The cells were seeded in 24-well plates (1 × 10 4 cells) were co-cultured with each hydrogel. After 3 days of co-culture, the cell viability was calculated by live / dead cell staining. Live / dead cell working staining solution (Solarbio, China) was added to the double-stained cells and incubated at 37°C in the dark according to the instructions of the manufacturer. Live / dead cell images were taken using an inverted fluorescence microscope (Carl Zeiss, Germany).

[0101] 3. CCK-8 cell proliferation assay

[0102] CCK-8 reagent (Vazyme, China) was added to the cell culture plate at a ratio of 1:10, and the cell proliferation was detected on days 1, 3, and 5. 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader (Agilent, USA) after incubation for 4 h.

[0103] 4. Immunofluorescence staining experiment

[0104] All samples were fixed in 4% paraformaldehyde (PFA, Biosharp, China) for 30 min, treated with 0.3% TritonX-100 (Sigma-Aldrich, USA) for 15 min, and blocked with 5% BSA solution at 4°C overnight. In addition, the samples were incubated with primary antibodies at 4°C overnight. The samples were incubated with secondary antibodies at 37°C for 2 h. These were incubated with Phalloidin (Yeasen, China) at 37°C for 1 h and DAPI (Yeasen, China) at room temperature for 10 min in the dark. The samples were observed using an inverted fluorescence microscope (Carl Zeiss, Germany).

[0105] 5. Mitochondrial membrane potential measurement

[0106] The JC-1 MMP detection kit (Beyotime, China) was used to measure the MMP of cells. Briefly, cells were incubated with JC-1 staining working solution at 37°C for 20 min and then washed twice with PBS. Images were captured using a fluorescence microscope (Carl Zeiss, Germany). The red-green fluorescence intensity ratio was calculated using ImageJ software.

[0107] 6. Determination of Intracellular Reactive Oxygen Species

[0108] According to the manufacturer’s instructions, 2″,7″-Dichlorodihydrofluorescein Diacetate (DCFH-DA, MCE, USA) and Mitosox Red (MCE, USA) were used to detect the levels of ROS in the cytoplasm and mitochondria, respectively. The working solution was incubated with the cells at 37°C and then washed with PBS. Images were captured using a confocal laser microscope (Carl Zeiss, Germany) and an inverted fluorescence microscope (Carl Zeiss, Germany).

[0109] 7. Evaluation of Osteogenic Differentiation Ability in Vitro

[0110] Rat BMSCs were cultured with different macrophage-conditioned medium, and the medium was refreshed every other day. After a 7-day culture period, each BMSC group was stained and ALP activity was quantified using an ALP staining kit (Beyotime, China) and an ALP activity quantification kit (Jiancheng, China). After a 7-day and 14-day culture period, different BMSC groups were immunofluorescently stained using RUNX2 primary antibody (Abcam, USA) and OPN primary antibody (Abcam, USA), respectively, following the previously described immunofluorescent staining procedure. After 21 days of co-culture, BMSCs were stained using an Alizarin Red Calcium Staining Kit (Cyagen, China) to observe calcium nodules. Calcium nodules were subsequently observed and photographed using an optical microscope. The calcium nodules were then dissolved in hydrochloric acid, and their absorbance was measured at 420 nm using a microplate reader. Total RNA was extracted from osteoblasts at different time points for qRT-PCR. BMSCs were co-cultured for 7 and 14 days, and then total RNA was extracted using Trizol (Invitrogen, USA) reagent. qRT-PCR was performed as described previously (to evaluate the gene expression levels of ALP and RUNX2 at 7 days, and the expression levels of OPN and OCN at 14 days).

[0111] Example 5: In vivo evaluation of multifunctional nanogels

[0112] 1. Construction of rat femoral condyle defect model

[0113] The experimental rats were male SD rats weighing 300-350 g. Rats were purchased from the Experimental Animal Center of Soochow University. All surgical procedures and postoperative treatments were approved by the Ethics Committee of Soochow University (SUDA20231113A01). A rat femoral condyle defect model was established to evaluate the therapeutic effect of hydrogel on femoral condyle defect. First, 2% sodium pentobarbital was used for intraperitoneal injection of 2.5 mL / kg for anesthesia. Then the surgical area was exposed, and the femoral condyle was exposed through blunt separation of the muscle gap. During the operation, attention was paid to protecting the surrounding blood vessels and nerves. A 3 mm diameter drill was used to drill through the double cortex at a low speed. During the operation, physiological saline was dripped into the surgical area to prevent excessive local temperature. Finally, different hydrogel groups were implanted into the fracture area, and no implanted material was used as a blank control. Penicillin was given intramuscularly for 3 days after surgery to prevent infection, and the rats were placed in a warm and ventilated place until they woke up naturally.

[0114] 2. In vivo imaging of reactive oxygen species

[0115] ROS Brite 700 (AAT Bioquest, USA) was used to detect the ROS level in rats 3 days after femoral condyle defect. The rats were first anesthetized with isoflurane and ROS Brite 700 (100×10 -6 M in Hanks with 20×10 -3 M HHBS) were injected into the injury site, and bioluminescence imaging was performed using an in vivo imaging system (Spectral Instruments Imaging, USA).

[0116] 3. Sample Collection and CT Analysis

[0117] At 2 and 4 weeks after surgery, the rats were euthanized, and the femoral tissues were collected and fixed with 4% paraformaldehyde. The samples were scanned with Micro-CT (SkyScan, Belgium) at 65 kV and 385 mA. Three-dimensional reconstruction was performed using Mimics software. For the cylindrical defect area, the following data were analyzed by the software: BV / TV, BMD, TP.th, and TP.Sp (n = 3).

[0118] 4. Histological Analysis of Femur

[0119] All specimens were decalcified after micro-CT scanning. After decalcification, the samples were trimmed, gradient dehydrated, and embedded in paraffin. The femoral condyle wax blocks were cut in the sagittal plane, approximately 5 μm thick, for hematoxylin and eosin (H&E) and Masson staining. Immunofluorescence staining was then performed. The samples collected 1 week after surgery were stained with Arg-1 (Servicebio, China) and iNOS (Servicebio, China). The remaining specimens were stained with OCN and OPN antibodies (Servicebio, China) to evaluate the polarization of macrophages in the surgical area and fracture healing (n = 3).

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel, characterized in that: The following steps are involved: S1. Add potassium permanganate solution to the suspension of silica nanoparticles under ultrasonic vibration, continue ultrasonic treatment for 1 h, stir overnight, discard the supernatant, and obtain silica nanoparticles coated with manganese dioxide; S2, mixing the manganese dioxide-coated silicon dioxide nanoparticles with a sodium carbonate solution, etching, removing the supernatant, and washing to obtain hollow mesoporous manganese dioxide; S3, mixing the hollow mesoporous manganese dioxide with a dexamethasone alcohol solution, ultrasonically shaking, removing the supernatant, and obtaining manganese dioxide nanoparticles containing dexamethasone; S4, mixing the chitosan solution with the manganese dioxide nanoparticles containing dexamethasone, and stirring to obtain positively charged manganese dioxide nanoparticles containing dexamethasone; S5. Mixing the AlgMA solution with the positively charged manganese dioxide nanoparticles containing dexamethasone, and photocrosslinking them to obtain a hydrogel.

2. The preparation method according to claim 1, characterized in that The concentration of the potassium permanganate solution is 600 mg / 20 mL.

3. The preparation method according to claim 1 or 2, characterized in that: The concentration of the sodium carbonate solution is 2 mol / L.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The dexamethasone alcohol solution is a dexamethasone ethanol solution.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The chitosan content of the chitosan solution is 1 mg / mL, and the solvent is 1% acetic acid.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The AlgMA solution contained 500 mg AlgMA and 0.025 g I2595 photoinitiator, and the solvent was 10 mL PBS.

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

8. Use of the hydrogel according to claim 7 in any of the following items: (i) Preparation of products for scavenging ROS during bone regeneration; (ii) preparing products for improving mitochondrial function; (iii) preparing a product of reprogrammed macrophages; (iv) preparing products for promoting osteogenic differentiation of mesenchymal stem cells; The product includes a reagent or a drug.

9. Use of the hydrogel according to claim 7 in preparing a product for regulating energy metabolism, improving local immune microenvironment and / or promoting bone regeneration; The product includes a reagent or a drug.

10. A drug, characterized in that The invention comprises the hydrogel according to claim 7 and acceptable auxiliary materials or additives.

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