Cerium-doped mesoporous bioactive glass nanoparticles, preparation method, hydrogel and application

Abstract

The invention relates to cerium-doped mesoporous bioactive glass nanoparticles, a preparation method thereof, hydrogel and application of the cerium-doped mesoporous bioactive glass nanoparticles, the Ce-MBG-coated L-Car composite nanoparticles are synthesized into MBG through a surfactant template method, the MBG has a uniform mesoporous structure and good particle size distribution, and ideal drug carrier characteristics are provided. The Ce-MBG (at) L-Car composite nano-particles are doped with the cerium element through ion replacement, the oxidation resistance of the material is remarkably improved, the stability and the loading capacity of the L-carnosine are enhanced through coordination chelation, and all characterization and in-vivo and in-vitro experiment results show that the Ce-MBG (at) L-Car composite nano-particles not only have high drug loading capacity, but also have good anti-oxidation performance. Meanwhile, potential anti-saccharification and anti-oxidation dual effects are achieved in a diabetes mellitus microenvironment, and a solid material basis is provided for subsequent bone defect treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to cerium-doped mesoporous bioactive glass nanoparticles, a preparation method and a hydrogel, and applications thereof. Background Art

[0002] Diabetes is a chronic systemic disease characterized by metabolic imbalance, hyperglycemia, and pancreatic B cell destruction or insulin resistance. The various complications caused by diabetes can affect multiple organs and systems such as the heart, brain, blood vessels, kidneys, and skin, placing a serious burden on the medical system and patients. The negative impact of diabetes on the skeletal system has received increasing attention, and insufficient bone regeneration has become a common and serious complication of diabetes. These complications stem from hyperglycemia-induced oxidative stress, chronic inflammation, and the accumulation of advanced glycation end products (AGEs), which together impair the osteogenic potential of bone-forming cells and disrupt the bone remodeling process.

[0003] Oxidative stress is a state in which the body's oxidative and antioxidant effects are unbalanced in favor of oxidation, which is a negative impact of free radicals produced in the body. Reactive oxygen species (ROS) is a complex term that mainly describes single-electron reduced species of oxygen in the body, including hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 - ) and hydroxyl radicals (•OH). In patients with long-term poor glycemic control before and after the diagnosis of diabetes, abnormally elevated blood glucose in the body can induce high levels of ROS, which are produced in different parts of the cell through multiple mechanisms, including but not limited to activation of the polyol pathway, activation of protein kinase C isoforms, protein glycosylation under diabetic conditions, glucose autooxidation, and excessive mitochondrial superoxide production. Appropriate levels of ROS contribute to normal cellular homeostasis and function. However, under diabetic conditions, the production of ROS increases, and the balance between their number and antioxidant defense is disrupted, leading to the occurrence of oxidative stress. Excessive intracellular ROS can directly react with DNA, proteins, and lipids, produce multiple chain reactions, form new free radicals, and spread through bone tissue, ultimately causing more oxidative damage. ROS may activate signaling pathways related to oxidative stress, leading to increased damage.

[0004] Sustained high blood sugar levels can increase non-enzymatic glycosylation of proteins, leading to the production of extracellular AGEs, which can bind to AGE receptors (Receptor for Advanced Glycation Endproducts, RAGE) on osteoblasts and inhibit the synthesis of osteocalcitonin (OCN) and type I collagen. This may impair the mineralization function of osteoblasts and reduce osteogenesis. Chronic high blood sugar levels increase the potential for non-enzymatic glycosylation of glucose and other reducing sugar molecules with protein, nucleic acid and lipid biomolecules, accumulating AGEs. At the same time, glycated collagen causes collagen to lose its elasticity, providing poor cell adhesion sites for osteoblasts and osteoclasts, making it more difficult to absorb, thereby hindering the entire bone remodeling stage.

[18] AGEs are also responsible for the increased production of pro-inflammatory cytokines and ROS. Collectively, all of these changes inhibit the bone remodeling process, leading to impaired bone repair.

[0005] When a large amount of glucose enters the cell, there is no time for glycolysis, 3-phospho-glyceraldehyde metabolism is inhibited, 6 metabolic branches are activated, methylglyoxal is generated, and it undergoes glycation with proteins, etc., leading to the generation of reactive oxygen species in the cell. Reactive oxygen species in multiple parts of the cell, such as mitochondria, in turn activate the formation pathway of advanced glycation end products AGEs. Therefore, a vicious cycle of AGE-ROS can be formed in the bone microenvironment inside and outside the cell, and the accumulation of AGEs in the cell also promotes the expression of RAGE receptors on the cell surface. Summary of the invention

[0006] In order to develop a composite material with dual anti-sugar and anti-oxidation effects to inhibit the AGE-ROS vicious cycle in the diabetic bone microenvironment, the present invention provides a cerium-doped mesoporous bioactive glass nanoparticle, a preparation method, a hydrogel and an application thereof.

[0007] The technical solution adopted by the present invention is: a mesoporous bioactive glass nanoparticle loaded with L-carnosine and doped with cerium, wherein the cerium-doped mesoporous bioactive glass nanoparticle is cerium nitrate hexahydrate (Ce(NO 3 ) 3 6H 2 O) was doped into 58S bioactive glass (MBG) as a cerium source to obtain cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG). The cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) were then placed in a carbonate aqueous solution of L-carnosine to combine with L-carnosine to obtain L-carnosine-loaded and cerium-doped bioactive glass nanoparticles (Ce-MBG@L-Car).

[0008] Preferably, the cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) are combined with L-carnosine through chelation coordination bonds and physical adsorption.

[0009] Preferably, the ratio of each element in the 58s bioactive glass is 60SiO 2 : (36-x)CaO: 4P 2 O 5 , where x is the molar percentage of doped cerium.

[0010] Preferably, the molar ratio of cerium doping in the cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) is 10%.

[0011] A method for preparing mesoporous bioactive glass nanoparticles loaded with L-carnosine and doped with cerium, comprising the following steps: (1) Synthesis of cerium-doped mesoporous bioactive glass: Cetylpyridinium bromide (CPB) and urea were weighed and added to a flask, and then added to deionized water. Ultrasonic treatment was performed to completely dissolve CPB and urea, and the solution turned milky white. Cyclohexane and isopropanol were added to the solution in sequence under magnetic stirring. The solution was continuously stirred at room temperature. Tetraethyl orthosilicate (TEOs) was introduced and stirred vigorously to react. At the same time, a constant temperature water bath was preheated to raise the temperature. The flask was placed in a water bath and stirred continuously. Triethyl phosphate (TEP) was added to react. The ratio of each element in the 58s bioactive glass was 60SiO 2 : (36-x)CaO: 4P 2 O 5 , where x is the molar percentage of the doped cationic metal cerium. Therefore, according to the molar ratio relationship, the molar ratio of calcium element corresponding to the doping of x% cerium element is 36-x%, and according to calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ·4H 2 O), cerium nitrate hexahydrate (Ce(NO 3 ) 3 6H 2 O) and weigh the corresponding mass; 3 ) 2 ·4H2O、Ce(NO 3 ) 3 6H 2 O are dissolved in water, and Ca(NO 3 ) 2 ·4H 2 O、Ce(NO 3 ) 3 6H 22 ml of O solution was dripped into the mixed solution at a speed of 100 μl / min, so that the two solutions were fully mixed and reacted with the mixed solution; stirring was continued to allow the mixed solution to fully react; after the reaction, the suspension was collected, centrifuged at 9500-10000 rpm for 15 min, and the precipitate was collected. The precipitate was washed with acetone, 75% ethanol, and deionized water in turn, and the precipitate was collected after each washing; the pure precipitate was freeze-dried until the precipitate was freeze-dried to a loose structure; the collected precipitate was placed in a tubular muffle furnace and calcined under a nitrogen atmosphere to obtain Ce-MBG black powder; (2) Preparation of Ce-MBG@L-Car loaded with cerium-doped bioactive glass nanoparticles: Weigh the freeze-dried powder of L-carnosine and dissolve it in carbonate buffer (pH=8.3) to make L-carnosine reach its isoelectric point; put 10% molar ratio of Ce-MBG into the above-mentioned L-carnosine carbonate buffer and stir it magnetically at a constant temperature; then collect the precipitate by centrifugation and freeze-dry it until the precipitate is freeze-dried to a loose structure; the harvested Ce-MBG@L-Car is stored at -20°C.

[0012] Preferably, the molar percentage of the doped cationic metal cerium in step (1) is 10%.

[0013] Preferably, the pure precipitate freeze-dried in step (1) is placed in a -20°C refrigerator for 2 hours, and then placed in a -80°C refrigerator for 4 hours.

[0014] Preferably, the calcination step in step (1) is to set the heating rate to 1°C / min, calcine the dried powder to 650°C, the calcination time is 10 hours, and then cool to room temperature at 5°C / min to obtain Ce-MBG black powder.

[0015] A composite hydrogel loaded with L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles, wherein the composite hydrogel is prepared by the following steps: Synthesis of Gelma / Ce-MBG@L-Car composite hydrogel: Take the Gelma hydrogel freeze-dried product and put it into deionized water, prepare a Gelma solution with a concentration of 10% at a constant temperature of 40~50°C, add 2 photoinitiator I2959, so that it accounts for 5% of the dry weight of the Gelma freeze-dried product, wrap the glass bottle of the mixed solution with tin foil to achieve the effect of light protection, continue hydrothermal stirring at 50°C, so that the photoinitiator and the Gelma solution are evenly mixed; after the mixture is evenly mixed, filter it with a filter membrane to sterilize it, and then add Ce-MBG@L-Car nanoparticles sterilized by ultraviolet light to the above solution, so that the Ce-MBG@L-Car nanoparticles account for 10% of the dry weight of the Gelma freeze-dried product, and the Gelma solution to which the photoinitiator has been added and the nanoparticles are evenly mixed to form a Gelma / Ce-MBG@L-Car composite hydrogel.

[0016] Application of a composite hydrogel loaded with L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles in the preparation of diabetic bone defect repair materials.

[0017] The beneficial effects of the present invention are: a cerium-doped mesoporous bioactive glass nanoparticle, a preparation method, a hydrogel and an application. The MBG synthesized by the surfactant template method in the Ce-MBG@L-Car composite nanoparticle has a uniform mesoporous structure and a good particle size distribution, providing ideal drug carrier characteristics. The antioxidant properties of the material are significantly improved by doping the cerium element by ion replacement, and the stability and loading capacity of L-carnosine are enhanced by coordination chelation. All characterization and in vitro and in vivo experimental results show that the Ce-MBG@L-Car composite nanoparticles not only have a high drug loading capacity, but also have potential anti-glycation and antioxidant dual effects in the diabetic microenvironment, providing a solid material foundation for subsequent bone defect treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 SEM characterization of Ce-MBG with different doping molar ratios.

[0019] Figure 2 TEM and EDS characterization of 10Ce-MBG.

[0020] Figure 3 XRD characterization of MBG and 10Ce-MBG.

[0021] Figure 4 Characterization of the antioxidant properties of Ce-MBG with different doping molar ratios; A: ABTs+; B: DPPH.

[0022] Figure 5 TGA analysis of 10Ce-MBG, L-Car, and 10Ce-MBG@L-Car.

[0023] Figure 6 BET analysis of 10Ce-MBG and 10Ce-MBG@L-Car. A. Nitrogen adsorption and desorption curve of 10Ce-MBG B. Pore size distribution of 10Ce-MBG C. Nitrogen adsorption and desorption curve of 10Ce-MBG@L-Car D. Pore size distribution of 10Ce-MBG.

[0024] Figure 7 FTIR analysis of L-Car, MBG, 10Ce-MBG, and 10Ce-MBG@L-Car. The obvious functional group characteristic peaks are marked with arrows.

[0025] Figure 8 XPS analysis. A. 10Ce-MBG total energy spectrum; B. Ce energy spectrum; C. Si energy spectrum; D. O energy spectrum.

[0026] Fig. 9 For Ce 3+ Simulation of chelating molecules with L-Car. A. Analysis of positive (blue) and negative (red) electrostatic potential of L-carnosine; B. Analysis of positive (red) and negative (blue) electrostatic potential of 10Ce-MBGs@L-Car; C. Schematic diagram of HOMO / LUMO and energy gap of 10Ce-MBGs@L-Car in the ground state.

[0027] Fig.10 In vitro CCK8 cytotoxicity experiments. A. CCK8 cytotoxicity experiments of different concentrations of 10Ce-MBG particle suspension after 48 hours. B. CCK8 cytotoxicity experiments of different concentrations of L-carnosine solution after 48 hours.

[0028] Fig.11 In vitro CCK8 cytotoxicity experiments. A. 24 h later, different concentrations of 10Ce-MBG particle suspensions were placed in different concentrations of L-carnosine drugs for CCK8 cytotoxicity experiments. B. 48 h later, different concentrations of 10Ce-MBG particle suspensions were placed in different concentrations of L-carnosine drugs for CCK8 cytotoxicity experiments.

[0029] Fig.12 In vitro antioxidant and anti-glycation experiments. A: DCFH-DA experiment. B: RAGE immunofluorescence staining experiment. C: Semi-quantitative analysis of ROS fluorescence intensity. D: Semi-quantitative analysis of RAGE immunofluorescence intensity.

[0030] Fig.13 This is an in vitro high-glucose modeling ALP experiment. A. ALP staining experiment under different high-glucose concentrations. B. Semi-quantitative analysis of ALP staining related optical density.

[0031] Fig.14In vitro BMSCs osteogenic differentiation experiment. A. ALP staining experiment of different material groups; B. ARS staining of different material groups; C. Semi-quantitative analysis of ALP optical density of different materials; D. Quantitative analysis of enzyme activity units (DEA) of ALP protein of different materials; E. Semi-quantitative analysis of ARS optical density of different materials; FH. The expression levels of related genes of ALP, Runx2 and OPN in different materials.

[0032] Fig.15 Micro CT analysis of skull defects in vivo. A. Schematic diagram of skull defect osteogenesis in different modeling groups. B. Bone mineralization density (BMD) of skull defects in different modeling groups. C. Bone volume fraction (BV / TV) of skull defects in different modeling groups. D. Trabecular thickness (Tb.Th) of skull defects in different modeling groups. E. Trabecular number (Tb.N) of skull defects in different modeling groups.

[0033] Fig.16 HE & Masson staining analysis of skull defect in vivo. A: HE staining; B: Masson staining N: new bone; F: fibrous tissue; G: Gelma hydrogel.

[0034] Fig.17 Immunohistochemical staining of skull defects in vivo. A. Immunohistochemical staining of OCN, TNF-a, and RAGE; B. Semi-quantitative statistical analysis of the area of ​​RAGE immunohistochemical positive region; C. Semi-quantitative statistical analysis of the area of ​​TNF-a immunohistochemical positive region; D. Semi-quantitative statistical analysis of the area of ​​OCN immunohistochemical positive region.

[0035] Fig.18 Experimental flow chart for the preparation of Ce-MBG@L-Car. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1 Preparation and Characterization of Cerium-doped Mesoporous Bioactive Glass Nanoparticles Synthesis of Ce-doped Mesoporous Bioactive Glass Weigh 2.5g CPB (cetylpyridinium bromide) and 1.5g urea into a 250ml flask, add 75ml deionized water, and ultrasonically treat for 10min to completely dissolve CPB and urea, and the solution turns milky white; under magnetic stirring, add 75ml cyclohexane and 2.3g isopropanol to the solution in sequence, and stir the solution continuously for 2h at room temperature; introduce 6.75ml tetraethyl orthosilicate (TEOs), stir vigorously for 30min to react, and at the same time, preheat the water bath to 70°C, put the flask into the water bath, and continue stirring for 7h; add 300μl triethyl phosphate (TEP), react for 30min; the ratio of each element in 58s bioactive glass is 60SiO 2 : (36-x)CaO: 4P 2 O 5 , where x is the molar percentage of other cationic metals doped. Therefore, according to the molar ratio, 2%, 5%, 10%, 12%, 15%, and 20% of cerium doped with cerium have corresponding molar ratios of 34%, 31%, 26%, 24%, 21%, and 16%, respectively. 3 ) 2 ·4H 2 O), cerium nitrate hexahydrate (Ce(NO 3 ) 3 6H 2 O) and weigh the corresponding mass; 3 ) 2 ·4H2O、Ce(NO 3 ) 3 6H 2 O were dissolved in 2 mL of water, and Ca(NO 3 ) 2 ·4H 2 O、Ce(NO 3 ) 3 6H 22ml solution of O was dripped into the above mixed solution at a speed of 100μl / min, so that the two solutions were fully mixed and reacted with the above mixed solution; stirring was continued for 16h to allow the mixed solution to fully react; after the reaction, the suspension was collected, centrifuged at 9500~10000rpm, 15min, and the precipitate was collected. The precipitate was washed with acetone 3 times, 75% ethanol 2 times, and deionized water 3 times in sequence. After each washing, the precipitate was collected at 10000rpm, 15min; the pure precipitate was placed in a -20℃ refrigerator for 2h, and then placed in a -80℃ refrigerator for 4h; the freeze dryer was turned on, pretreated for about 15min, and dried for about 2 days until the precipitate was freeze-dried to a loose structure; the collected precipitate was placed in a tubular muffle furnace under a nitrogen atmosphere, the heating rate was set to 1°C / min, the dried powder was calcined to 650°C, the calcination time was 10 hours, and the Ce-MBG black powder was obtained by cooling to room temperature at 5°C / min.

[0038] L-Carnosine loading on cerium-doped bioactive glass nanoparticles (Ce-MBG@L-Car) Weigh 50 mg of L-carnosine freeze-dried powder and dissolve it in 5 ml of carbonate buffer (PH=8.3) to make L-carnosine reach its isoelectric point; put 50 mg of 10% molar ratio Ce-MBG into the above 5 ml L-carnosine carbonate buffer, and stir magnetically for 24 hours at a constant temperature of 50°C; then centrifuge at 3000 rpm for 15 minutes to collect the precipitate, and put the precipitate into a -20°C refrigerator for 2 hours, and then put it into a -80°C refrigerator for 4 hours; turn on the freeze dryer, pretreat for about 15 minutes, and dry for about 2 days until the precipitate is freeze-dried to a loose structure; the harvested Ce-MBG@L-Car is stored in a -20°C refrigerator.

[0039] Structure and antioxidant characteristics of Ce-MBG nanoparticles with different doping molar ratios There are many methods for synthesizing MBG. Different synthesis techniques will affect its structure, pore characteristics and biological activity, and thus determine its application effect in the fields of tissue engineering and bone repair. Since the initial sol-gel method and high-temperature melting method, many new synthesis methods have gradually been proposed and widely used, such as vapor deposition method, microwave-assisted synthesis method, solvothermal method and solvent-free method. In recent years, the template-guided method has gradually attracted attention as an emerging synthesis strategy. This method can accurately control the pore size, morphology and particle size of the glass, thereby significantly enhancing its application potential in the biomedical field. When synthesizing using the CPB template-guided method, the uniform doping of cerium further improves the antioxidant properties and surface chemical properties of bioactive glass: cerium, as a rare earth element with good antioxidant properties, can be synthesized through its Ce +3 and Ce+4 Reversible transformation between valence states, regulating the redox state in the reaction environment

[38] The cerium surface has abundant oxygen vacancies and active sites, which can form chelation with drug molecules, such as L-carnosine. This effect not only improves the drug loading capacity and release stability, but also the Ce-L-carnosine chelate bidirectionally enhances the antioxidant and anti-glycation effects, thereby breaking the glycation-oxidation vicious cycle in the diabetic bone healing microenvironment.

[0040] like Figure 1 The results of scanning electron microscopy show that the morphology of MBG particles changes significantly with the increase of the molar ratio of cerium doping. MBG particles without cerium doping show a uniform spherical structure with uniform pore distribution. After adding 2%, 5%, and 10% molar ratio of cerium, the morphology of the particles does not change much, but slight aggregation occurs.

[0041] 12% and 15% cerium doping caused serious aggregation of particles, uneven particle size distribution, more irregular morphology, and visible surface pore structure collapse. This shows that the incorporation of high concentrations of cerium leads to particle aggregation, affecting the morphological uniformity and pore structure of the material. Therefore, in the following articles, the present invention mainly uses MBG, 2%, 5%, and 10% molar ratio Ce-MBG for material characterization.

[0042] according to Figure 2 TEM images show the morphological characteristics of the 10Ce-MBG sample. The HAADF (high-angle annular dark field imaging) in the figure shows the overall morphology of the particles. The particles show a spherical structure, with a size of about 200~250 nm, a smooth surface and a uniform pore structure. This shows that 10Ce-MBG maintains good morphological stability during the synthesis process. The elemental composition of the sample was analyzed in detail by EDS (energy dispersive X-ray spectroscopy), and the spectrum showed the presence of elements such as calcium (Ca), cerium (Ce), silicon (Si) and phosphorus (P). The distribution of cerium is represented by red, indicating that its distribution in the particles is relatively uniform. Calcium and silicon are shown in green and blue, respectively, showing that they are richly distributed in the particles. Due to the increase in the molar ratio of cerium doping, the doping of calcium is reduced to a certain extent (the molar doping ratio of calcium is reduced to 26%), so the signal intensity of calcium in the element distribution is relatively weak. According to the atomic fraction and mass fraction data of the element, the atomic proportion of cerium in the particles is 0.25%, showing a moderate doping ratio. Overall, the TEM and EDS analysis results confirmed the successful synthesis of 10Ce-MBG and demonstrated its good morphology and uniform distribution of elements.

[0043] Figure 3The XRD images in the figure show that the 10Ce-MBG particles present a typical inorganic glass bun peak structure, and no other peaks appear. In summary, the Ce-doped process of 10Ce-MBG particles was successfully synthesized. The glass bun peak structure strength of 10Ce-MBG is not as strong as that of MBG. The possible reason is that in order to prevent the doped Ce from oxidizing and precipitating to form cerium dioxide crystals during the sintering process at 650℃, nitrogen was passed through the entire sintering process, and the oxygen content was relatively reduced. The Si-O bonds in the inorganic crystals were relatively reduced compared to MBG sintered in an air atmosphere.

[0044] Subsequently, we characterized the antioxidant properties of Ce-MBG with different doping molar ratios. Figure 4 As shown in (A), at 30 minutes, Ce-MBG with different doping molar ratios and the blank control (Blank) have a certain scavenging effect on oxidative free radicals; the Abs absorbance curves of MBG, 2%, and 5% Ce-MBG doping ratios overlap, which may be because the original Ce doping ratio is too low and no obvious oxygen free radical scavenging effect can be achieved. The decrease in the absorbance curve of MBG without Ce doping may be due to its rich mesoporous structure adsorbing liquid fuel, thereby ABTs + The blue color becomes lighter, and the 10Ce-MBG absorbance Abs curve is the lowest, indicating that the antioxidant performance is enhanced as the molar ratio of Ce element doping increases. Figure 4 (B) It can also be observed that as the molar ratio of Ce element doping increases, the ability of particles to capture oxygen free radicals increases, that is, the antioxidant function increases. Therefore, through SEM, TEM, XRD, ABTs + The DPPH antioxidant characteristics experiment verified that 10Ce-MBG was successfully doped and had uniform particle size distribution and pore distribution, as well as good antioxidant properties. Therefore, 10Ce-MBG was selected for the following drug loading and other material characterization experiments.

[0045] Physicochemical characterization of synthesized particles Thermogravimetric Analyzer (TGA) according to Figure 5Thermogravimetric analysis (TGA) was performed under nitrogen to prevent the increase in mass caused by oxidation. The changes in mass of different materials during the heating process can be observed, and the mass proportion of drug loading can be known from the curve. L-carnosine showed the most significant mass change, especially when the temperature rose to 300°C, the mass dropped rapidly, and finally dropped to near zero at 600°C. 10Ce-MBG had almost no mass loss during the heating process, and the mass remained basically stable when the temperature rose from room temperature to 600°C. This shows that it has good thermal stability. 10Ce-MBG@L-Car showed obvious mass loss. Especially when the temperature rose to about 300°C, the mass dropped sharply, and when the temperature was further raised to 600°C, the mass loss reached about 19%. Because L-carnosine can be thermally decomposed at 300~600°C, while the nanoparticles 10Ce-MBG do not decompose, it can be roughly estimated that L-carnosine accounts for about 19% of the total material 10Ce-MBG@L-Car.

[0046] Specific surface area and porosity analyzer (BET) 10Ce-MBG: N2 isothermal adsorption-desorption curve belongs to type IV in IUPAC classification, with H4 hysteresis loop, reflecting that the adsorbent has narrow mesoporous cracks, and the pore size distribution has a peak at 4 nm. The "narrow" hysteresis loop proves that the pore size is small, the pressure of capillary condensation is small, and there is a tendency to fill micropores. The "steep" hysteresis loop proves that the steeper the capillary condensation section, the more uniform the mesopore distribution; the nitrogen adsorption range is 100~500 cm 2 / g, indicating that the pores are well developed and the specific surface area is large. In addition, the desorption curve of 10Ce-MBG almost coincides with the adsorption curve, indicating that the material has strong adsorption and low hysteresis effect. The steep rise of the adsorption curve indicates that the material can quickly adsorb gas at low relative pressure, which is suitable for application scenarios that require rapid adsorption.

[0047] 10Ce-MBG@Car: The N2 isothermal adsorption-desorption curve belongs to type IV in the IUPAC classification, with an H3 hysteresis loop, reflecting that the adsorbent has a lamellar structure of slit mesoporous clay, indicating that L-carnosine loading is successful. Its adsorption curve and desorption curve show a certain hysteresis phenomenon, which indicates that the material releases gas slowly after the adsorption process. Compared with 10Ce-MBG, after adding L-carnosine, the pore structure of the material changes, resulting in a slower diffusion rate of the gas, which affects the desorption process of the gas.

[0048] Table 10BET specific surface area, total pore volume and average pore size of Ce-MBG and 10Ce-MBG@L-Car

[0049] According to the BET analysis in the table, it can be seen from three aspects that L-carnosine loading is successful. 1) Specific surface area (BET surface area): The specific surface area of ​​10Ce-MBG is 933.8999 m² / g, while the specific surface area of ​​10Ce-MBG@L-Car drops significantly to 2.5414 m² / g. This difference shows that the addition of L-Car greatly reduces the surface area of ​​the material, probably because the presence of L-Car fills part of the pores and reduces the available surface area, which indirectly proves that L-carnosine loading is successful. 2) Total pore volume (Total pore volume): The total pore volume of 10Ce-MBG is 0.61736 cm³ / g, while the total pore volume of 10Ce-MBG@L-Car is lower, at 0.009853 cm³ / g. This data once again shows that the addition of L-carnosine significantly reduces the pore volume of the material. 3) Average pore size: The average pore size of 10Ce-MBG is 7.0914 nm, while the average pore size of 10Ce-MBG@L-Car increases to 16.4743 nm. This indicates that the addition of L-Car not only reduces the pore volume, but also may affect the uniformity of the pore structure and the distribution of pore size.

[0050] Fourier Transform Infrared Spectrometer (FTIR) Fourier transform infrared spectra (FTIR) of L-carnosine, 10Ce-MBG and their complexes are shown in Figure 2. Figure 7 The characteristic peak of L-carnosine is shown at 2988 cm -1 The broad peak at 1648 cm is attributed to the stretching vibration of C=N (histidine imidazole ring); -1 and 1561 cm -1 The strong absorption peaks at 2988 cm-1 and 2997 cm-2 correspond to the amide I band (C=O stretching vibration) and amide II band (NH bending vibration), which indicates the complete structure of the peptide bond. When L-carnosine is chelated with 10Ce-MBG, the infrared spectrum changes significantly: the amino-hydroxyl region (2988 cm-1) -1 ) The peak width increases and shifts to a lower wave number of 2977 cm -1 , which indicates that -NH 2 or -COO⁻ groups bind to Ce on the surface of Ce-MBG through coordination bonds; at the same time, the characteristic peak of carboxylate (1268 cm -1 ) shifts to a lower wavenumber (1235 cm -1 ), it is speculated that the coordination between the carboxylic acid oxygen atom and Ce³⁺ causes the vibration mode to change; more importantly, the amide I and II bands move to low wavenumbers to 1640 cm -1 and 1558 cm -1, indicating that the C=O and NH of the peptide bond may participate in the surface adsorption, and the electron cloud density is redistributed, further confirming the strong interaction between the two. The above results show that L-carnosine is anchored on the Ce-MBG surface through multi-site coordination of carboxylate, amino group and peptide bond to form a stable complex structure, proving its coordination effect with cations. These results indicate that the synthesis of 10Ce-MBG@L-Car is successful.

[0051] X-ray Photoelectron Spectroscopy (XPS) In order to further verify the synthesis results of 10Ce-MBG, the present invention conducted an X-ray photoelectron spectroscopy (XPS) analysis on it. Full spectrum of X-ray photoelectron spectroscopy (XPS) of 10Ce-MBG Figure 8 A confirms the coexistence of Si, O, and Ce elements in the 10Ce-MBG chelate material. Detailed peak fitting is further performed to characterize the chemical state of the elements on 10Ce-MBG. Figure 8 As shown in B, cerium is in a mixed valence state (Ce³⁺ / Ce 4 ⁺) was loaded on the surface of mesoporous bioactive glass. Through Ce 3d orbital peak fitting, it was observed that Ce 4 The main peaks of ⁺ (881.7 eV and 900.0 eV) coexist with the characteristic peaks of Ce³⁺ (885.4 eV and 904.2 eV). Figure 8 D) Analysis shows that the ions at 532.2 eV and 529.8 eV are attributed to SiO 2 The existence of bridging bonds further reveals the interfacial interaction between cerium and mesoporous bioactive glass. At the same time, the generation of Si-OC bonds (101.25 eV) was observed in the Si 2p spectrum, and 103.1 eV was attributed to SiO 2 The Si-O bond ensures that the glass structure remains intact. 2 While improving substrate stability, it also provides active sites for cerium species and enhances their redox properties.

[0052] Chelate molecular simulation L-Carnosine is a natural dipeptide. Its molecular structure contains amino, carboxyl and imidazole groups, which give it multi-dentate coordination ability and may form stable chelates with metal ions through N / O atoms.

[42] To further analyze the driving force of coordination, we used density functional theory (DFT / B3LYP / 6-311++G(d,p)) to calculate the electrostatic potential (ESP) of L-carnosine. Fig. 9As shown in A, the π electron-rich region of the imidazole ring of L-carnosine and the oxygen atom of the carboxylate are potential nucleophilic active sites, which preferentially electrostatically attract the positively charged Ce³⁺. At the same time, the formation of the chelate significantly changes the molecular electrostatic potential distribution of L-carnosine, and its charge redistribution behavior directly reflects the electron transfer characteristics of the coordination bond. By comparing the electrostatic potential (ESP) diagram of free L-carnosine and Ce³⁺ chelate ( Fig. 9 A, B) It can be seen that the potential values ​​of the strong negative potential areas in the original L-carnosine molecule (such as the imidazole ring π system and the carboxylate oxygen atom) increased significantly after chelation, indicating that Ce³⁺ obtains electrons from these areas through coordination bonds, resulting in a decrease in the local electron cloud density. Fig. 9 C shows the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) of 10Ce-MBGs@L-Car. The results show that the LUMO state is mainly localized on the Ce atom, while the HUMO is delocalized on the entire conjugated main chain. The energy gap between HOMO and LUMO is calculated to be 3.20 eV, which may be due to the hybridization of the 4f orbital of Ce³⁺ and the π orbital of the imidazole ring to produce a charge transfer band from ligand to metal. The chelated HOMO-LUMO orbital energy level and spatial distribution significantly improve the electron transfer efficiency and give it dual-functional antioxidant activity.

[0053] Example 2: In vitro cell experiment CCK-8 cytotoxicity assay to screen the concentration of nanoparticle suspension Before exploring the safe use concentration range of 10Ce-MBG@L-Car, the safe concentrations of single factors that may cause cytotoxicity should be verified one by one, such as the rare earth element Ce doped in the material design and the concentration of the drug L-carnosine. Fig.10 A shows that after being treated with 10Ce-MBG solutions of different concentrations for 48 h, the cell viability of BMSCs showed a certain concentration dependence. When the concentration of 10Ce-MBG solution was low, that is, the concentration was less than 15.625 μg / mL, the cell viability was basically maintained at a level close to 100%, and there was no significant difference between the control group and the control group. However, when the concentration reached a certain level, such as above 31.25 μg / mL, the cell viability decreased significantly, and showed a significant inhibitory effect compared with the control group. This shows that the 10Ce-MBG solution inhibited the cell viability of BMSCs at a higher concentration, suggesting that the bioactive glass doped with cerium may have a certain toxic effect on cells.

[0054] Fig.10Figure B shows the effect of different concentrations of L-carnosine on BMSCs cell viability after 48 h. It can be seen from the data that L-carnosine can significantly promote the cell viability of BMSCs at low to medium concentrations (0.488-31.25 μg / mL), showing a significant increase in viability compared with the control group. In particular, at a concentration of 31.25 μg / mL, cell viability reached the highest level, indicating that L-carnosine has a good stimulating effect on BMSCs. However, when the concentration was further increased to 1 mg / mL and above, cell viability began to decline, and the difference was not statistically significant. This result shows that L-carnosine can promote BMSCs proliferation within a certain concentration range. Although it does not promote cell proliferation at too high a concentration, its safe concentration range has exceeded the μg / ml order of magnitude. Therefore, when 10Ce-MBG nanoparticles with a concentration of μg / ml are used to load L-carnosine, the L-carnosine release concentration is also determined to be within the safe concentration range used.

[0055] According to the principle of "materials should use the highest concentration within the biosafety concentration to ensure both biosafety and significant functions" as discussed in Figure 11, three high-concentration 10Ce-MBG particle suspensions were selected: 15.625 μg / mL, 7.813 μg / mL, and 3.906 μg / mL (the final concentration when incubated with cells) and added to different concentrations of L-carnosine drugs to complete the preparation of the final material 10Ce-MBG@L-Car. Although the single factor, that is, the concentration of 10Ce-MBG particle suspension and the concentration of L-carnosine drug were both selected at safe concentrations, it is still unknown whether the final product of the combination of the two is cytotoxic. Fig.11 A and B show the effects of the final material 10Ce-MBG@L-Car on cell viability when co-incubated with BMSCs after 24 h and 48 h, respectively. Fig.11 In A, there were no significant differences between or within the groups after 24 h; Fig.11 B shows that the final material 10Ce-MBG@L-Car prepared in a low concentration solution of L-carnosine (2.5 mg / ml, 1 mg / ml) has a significant effect on promoting the proliferation of BMSCs, especially when the final concentration of 10Ce-MBG is 7.813 μg / mL and the concentration of L-carnosine loaded is 2.5 mg / ml, the BMSCs proliferation promoting effect is the most obvious. Therefore, the 10Ce-MBG@L-Car particle concentration prepared with a final concentration of 7.813 μg / ml 10Ce-MBG and a 2.5 mg / ml L-carnosine concentration solution was selected as the experimental group material for cell and animal experiments.

[0056] Antioxidant & Anti-glycation Cell Experiment High oxidative stress can significantly induce BMSCs cells to shift to an unfavorable state, increasing cell damage and dysfunction. By DCFH-DA staining, it was observed that the level of ROS in BMSCs cells in the HG group was significantly increased, indicating that oxidative stress aggravated the oxidative damage of the cells. However, in the HG+10Ce-MBG group and the HG+10Ce-MBG@L-Car group, the level of ROS decreased significantly, indicating that these two treatments can effectively alleviate the effects of oxidative stress. Oxygen is the product of the cellular antioxidant system in response to ROS. In the HG group, the cellular antioxidant system was inhibited, resulting in insufficient oxygen production, and the oxidation reaction could not be effectively eliminated, so a large amount of positive expression was observed. In the HG+10Ce-MBG group and the HG+10Ce-MBG@L-Car group, by inducing the repair of the endogenous antioxidant system of BMSCs cells, Ce 3+ / Ce 4+ The SOD / CAT enzyme-like effect can react with ROS to generate harmless oxygen and water, thereby reducing the accumulation of ROS. In addition to its anti-sugar effect, L-carnosine itself also has a certain anti-oxidative stress effect. After Ce and L-carnosine form a chelate, the antioxidant effect in the cell is stronger, and the overall fluorescence intensity is close to that of the control group, that is, the cell state has recovered well. After exposure to oxygen, Ru(dpp) 3 Cl 2 The fluorescence probe was quenched, showing less positive expression, which further proved the effective inhibition of oxidative stress. Although the fluorescence of the HG+MBG group was also slightly weakened, the difference was not obvious. Fig.12 A, Fig.12 C) RAGE (receptor for advanced glycation end products) is a transmembrane protein involved in regulating processes such as inflammation, oxidative stress, and apoptosis. Extracellularly, under hyperglycemia conditions, excess glucose reacts with proteins on the surface of bone cells to form stable AGEs. AGE-RAGE binds and activates downstream signaling pathways. Intracellularly, excessive glucose metabolism does not have time for glycolysis, activating six metabolic branches, among which glycation also causes a large amount of AGEs to accumulate in cells, and the accumulated intracellular AGEs in turn promote the expression of RAGE. Therefore, RAGE expression is closely related to AGE content and can be used as a detection indicator to test the concentration level of AGEs in the bone microenvironment. By RAGE immunofluorescence staining, it was observed that the intracellular RAGE level of BMSCs in the HG group was significantly increased, indicating that AGEs also accumulated to a considerable level in a high-sugar environment. However, in the HG+10Ce-MBG@L-Car group, the RAGE level decreased significantly, indicating that this treatment can effectively slow down the generation of AGEs, thereby reducing the expression of RAGE membrane protein. This may be attributed to the anti-glycation effect of the drug L-carnosine, which can remove aldose inside and outside the cell, thereby reducing the production of AGEs. After Ce forms a chelate with L-carnosine, the anti-glycation effect in the cell is stronger, and the overall fluorescence intensity is close to that of the control group, that is, the cell state has recovered well. In the HG+10Ce-MBG group, because it can remove the effect of promoting AGEs production caused by oxidative stress to a certain extent, it indirectly slows down the glycation effect, but the anti-glycation effect is not significant. ( Fig.12 B, Fig.12 D) Osteogenic differentiation cell assay The cell experiment must first determine a stable high sugar concentration to ensure that the cells are not damaged by high sugar during the experiment. Alkaline phosphatase (ALP) is mainly used in the mineralization process of bone matrix in osteoblasts. It promotes the accumulation of inorganic phosphates through dephosphorylation reactions, thereby promoting bone formation and mineralization, and is an important marker enzyme for bone formation. Therefore, ALP staining is used to reflect the damage of BMSCs osteogenic function. Fig.13As shown in A and B, only at a high glucose concentration of 100 mM in vitro, cells have obvious ALP production function inhibition. This is inconsistent with the high glucose concentration in vivo in diabetic patients. In theory, 25 mM is the preferred concentration for in vitro simulation of high glucose concentration in vivo, and experiments have shown that 25 mM not only does not damage cells, but may also enhance function due to enhanced glucose metabolism. The possible reasons for the stability of the model using 100 mM are analyzed as follows: the long-term accumulated high-sugar microenvironment of diabetic patients cannot be completely simulated in vitro, and in order to improve the cell survival rate after BMSCs extraction, it is preferred to extract healthy bone marrow stem cells from normal SD rats rather than diabetic model rats, because after extraction, BMSCs are in a functional decompensation stage for a long time, the cell yield will decrease, and it is difficult to culture. Healthy bone marrow stem cells have good compensatory function. For a short period of time, such as ALP osteogenic differentiation induction time of only 7 days and ARS 14 days, BMSCs are still in the glucose metabolism compensation stage in a relatively high concentration and high sugar environment in the body, that is, above 25 mM, and their functional phenotype may not be affected. However, when the concentration of the high sugar environment reaches 100 mM, the cells can be in a state of glucose metabolism decompensation in a short period of time, thereby affecting the function and inhibiting the phenotype. Therefore, the subsequent cell experiments used 100 mM high sugar concentration as a cell high sugar model.

[0057] The osteogenic induction ability of 10Ce-MBG@L-Car nanoparticles under high glucose conditions was further evaluated by ALP staining and ARS staining of ECM calcium nodules. Under HG conditions, both 10Ce-MBG and 10Ce-MBG@L-Car showed good osteogenic effects, with high positive expression of ALP staining and more calcium nodules clearly observed in ARS staining ( Fig.14 A, B). The results of the ALP protein quantification experiment further verified this conclusion ( Fig.14 D). Although the overall ALP protein expression level was significantly suppressed under high glucose conditions, ALP expression was significantly increased in the 10Ce-MBG and 10Ce-MBG@L-Car treatment groups, demonstrating the potential of these nanoparticles in restoring cell osteogenic differentiation. ( Fig.14 C, E) The MBG group also has a certain trend of bone formation recovery, which may be directly related to the minerals released by MBG (containing silicon, calcium, phosphorus and other elements) in the cellular environment. The silicon, calcium and phosphorus elements of MBG are key components of bone mineralization. They can promote the proliferation and differentiation of osteocytes and enhance the formation of bone matrix, thereby compensating for the lack of bone formation under high glucose conditions to a certain extent. In addition, compared with simple 10Ce-MBG, 10Ce-MBG@L-Car nanoparticles showed a more significant bone formation promoting effect, which may be related to the role of Ce and L-carnosine chelate as an anti-sugar and antioxidant agent.

[0058] qRT-PCR further demonstrated that the nanoparticles could restore the osteogenic function of BMSCs ( Fig.14 FH). Runx2 and ALP are early osteogenic markers. OPN affects bone metabolism by regulating the functions of osteoblasts and osteoclasts during osteogenesis. Under HG conditions, osteogenesis-related indicators ALP and Runx2 were significantly downregulated. At the gene level, 10Ce-MBG and 10Ce-MBG@L-Car can not only restore the osteogenic differentiation function of BMSCs to normal levels, but also the results show that both have better osteogenesis than BMSCs in a healthy state. It is worth noting that even in the HG environment, OPN still has a high expression capacity. In some studies, OPN is considered to have a certain protective effect. Tissue damage caused by high sugar may promote the increase of OPN expression to reduce the damage of high sugar to cells through its regulatory effects on cell adhesion, migration and apoptosis. The high expression of OPN may be a cell self-protection mechanism to help repair tissue damage caused by high sugar. Among the up-regulated expressions of OPN, the most significant expression of OPN was observed after the addition of 10Ce-MBG@L-Car, which may be attributed to its regulation of intracellular oxidative stress and glucose metabolism, indirectly enhancing the expression of genes related to bone repair.

[0059] Example 3: In vivo animal experiment In vivo animal Micro CT analysis The 5 mm defect constructed in this experiment is within the critical size range, and this model makes it impossible for diabetic rats to achieve self-healing in a short period of time. Fig.15 A shows that 8 weeks after surgery, the DM group had almost no healing, and the defect area basically existed. In the group implanted with Gelma hydrogel, new bone of varying degrees could be observed at the edge of the host bone, but they were all immature. In the Gelma / 10Ce-MBG@L-Car composite hydrogel group, not only was the amount of new bone generated around the host bone significant at the site of injury, but the area of ​​the new bone was relatively dense, and almost the entire defect area had been filled, indicating that the effect of inducing bone regeneration and bone repair after implantation of 10Ce-MBG@L-Car composite hydrogel was better than that of other experimental groups.

[0060] Semi-quantitative analysis based on Micro CT further confirmed the osteogenesis of Gelma / 10Ce-MBG@L-Car composite hydrogel. Bone mineral density (BMD) refers to the amount of minerals contained in bones per unit area or unit volume, reflecting the strength and hardness of bones, and is measured by the X-ray attenuation coefficient (AC). Bone volume fraction (BV / TV) is an important indicator for evaluating bone density and bone quality, and is used together with BMD as an important indicator for evaluating bone health. Trabecular bone thickness (Tb.Th) and trabecular number (Tb.N) are often used to evaluate the quality and progress of bone healing. As new bone is generated, the thickness of trabeculae should gradually recover, and a higher number of trabeculae usually means a tighter bone structure. At 8 weeks, the BMD of DM and composite hydrogel was 0.72±0.01, which was much higher than 0.41±0.03 of DM group and 0.47±0.062 of DM and Gelma group (p<0.01), indicating that the bones of the experimental group tended to be in a normal healthy state; while the BV / TV of DM and composite hydrogel group was 14.47±2.66, Tb.Th was 20.77±4.01, and Tb.N was 0.007±0.0013, which were statistically significant compared with DM group and DM+Gelma group (p<0.05), indicating that the trabecular bone structure of the experimental group was tighter and the bone was more mature. Micro-CT results showed that the implantation of Gelma / 10Ce-MBG@L-Car significantly promoted the regeneration of bone in bone defects, and subsequent experiments will further verify this result through histology and osteogenic immunohistochemistry.

[0061] In vivo histological staining experiments (1) HE & Masson staining Fig.16A is the H&E staining results of bone defects and surrounding tissues in each group 8 weeks after surgery. The staining results show that no new bone formation was found in the bone defect area in the DM group, and only a certain amount of fibrous tissue was found near the host bone. In DM+Gelma, due to the slow degradation of Gelma, some cells in the bone microenvironment can be promoted to crawl to the central area, and a small amount of new bone can be seen in the central fibrous tissue. In the DM+Gelma / 10Ce-MBG@L-Car composite hydrogel group, not only can new bones of different sizes be seen at the boundary of the host bone, but also obvious hydrogel material residues can be seen. A large amount of new bone can be seen at the lower end of the defect area to form a bone bridge, connecting the host bones on both sides. In addition, the implanted hydrogel has good biocompatibility with the tissue, and there is no large-scale inflammation around it. It is worth noting that in the composite hydrogel group, new bone is generated along the fiber edge at the lower end of its coverage, and the new bone begins to become similar to the host bone morphology. Mature bone cells can be seen in the enlarged image. Since the addition of solid particles will make Gelma degrade more slowly, all composite hydrogel groups can still find a relatively complete material outline 8 weeks after surgery. Studies have reported that the biodegradation rate of Gelma is related to the concentration of the cross-linking agent added, the mass experience ratio of the solid particles added, the implantation site, and the speed of metabolism in the organism. And because it is not completely degraded, it also plays a certain role as a barrier membrane. It can be seen that the upper surface of the composite hydrogel is covered with fibrous tissue, which protects the osteogenic reaction at the lower end from being disturbed by the faster growing fibroblasts. Therefore, the addition of solid particles also has certain advantages in terms of degradation rate.

[0062] The degree of bone maturity can be analyzed by the different colors of Masson staining. The collagen in the bone is mainly type I collagen. As the new bone matures, its surface will show red and blue. The maturity of the collagen in the bone tissue directly affects the Masson staining. Fig.16 As shown in B, at 8 weeks after surgery, almost no new bone tissue was found in the DM group, which is consistent with the conclusion of the above H&E staining. At 8 weeks after surgery, the proportion of new bone in the composite hydrogel group was mostly red, indicating that the collagen in the bone tissue was highly mature at this time, while the proportion of red collagen fibers in the new bone in the Gelma group was less and the proportion of blue new bone was more, which is similar to the above H&E results.

[0063] (2) OCN, TNF-a, and RAGE immunohistochemical staining results Osteocalcin (OCN) is a non-collagenous protein secreted by osteoblasts. It is mainly involved in the process of bone mineralization and is a bone maturity marker. Eight weeks after surgery, the higher the quality of mature bone, the stronger the bone repair ability. To investigate the in vivo osteogenesis-inducing potential and osteogenic differentiation-promoting effect of the DM+Gelma / 10Ce-MBG@L-Car composite hydrogel group, we performed OCN immunohistochemical staining on samples from each group eight weeks after surgery. In the experimental results, each experimental group showed a certain area of ​​OCN positive staining area ( Fig.17 B). The large area of ​​OCN positive staining in the composite hydrogel group indicates that the mature bone in the new bone area is the largest, indicating that mineralization is obvious. The composite hydrogel has the best potential for inducing osteogenesis and promoting osteogenic differentiation.

[0064] In the diabetic bone microenvironment, high expression of TNF-α promotes chronic inflammatory response, inhibits the function of osteoblasts by activating inflammatory pathways and oxidative stress, and further deteriorates bone metabolism. Eight weeks after surgery, the continued presence of inflammation in the DM group led to bone nonunion. In the composite hydrogel group, the material significantly reduced the expression of inflammatory factors, indicating that the material has a certain bone immune regulation effect. The reduced expression of inflammatory factors reflects the reduction of oxidative stress in the bone microenvironment, which is consistent with the results of the DCFH-DA experiment in in vitro cell experiments. The two directions together show that the composite hydrogel has a good anti-oxidative stress effect. There are experimental records that the Gelma material itself also has a certain antioxidant effect. The TNF-a of the Gelma hydrogel group in this article also has a certain alleviating effect.

[0065] Eight weeks after surgery, although DM and DM+Gelma showed certain differential expression in semi-quantitative positive area analysis, the RAGE positive area staining expression of Gelma was as high as 38%. The decreased RAGE expression in the Gelma group may be related to its antioxidant properties. The reduced oxidative stress weakened its effect on promoting the generation of AGEs, thus reducing RAGE expression. The RAGE positive area expression of the DM+Gelma / 10Ce-MBG@L-Car composite hydrogel group dropped to less than 10%, indicating that the material had significant differences in anti-AGEs effects, which was consistent with the RAGE fluorescence staining of in vitro cell experiments.

[0066] in conclusion The present invention adopts a sol-gel method combined with an organic template CPB, and later uses ion replacement technology to dope cerium, cation chelation and physical adsorption to load L-carnosine, and successfully prepares mesoporous bioactive glass nanoparticles (10Ce-MBG@L-Car) loaded with L-carnosine and doped with cerium. Through SEM and antioxidant performance studies, it is determined that Ce-MBG doped with a 10 molar mass ratio can maintain a good morphology and the best antioxidant properties, and TEM, EDS, and XRD are used to prove that 10Ce-MBG is successfully doped with cerium. After L-carnosine drug loading, its mass accounts for about 19% of the weight of 10Ce-MBG@L-Car. Through a series of physical and chemical performance tests, we deeply explored the physical and chemical properties of 10Ce-MBG and its complex with L-carnosine (10Ce-MBG@L-Car): 1) 10Ce-MBG has narrow mesoporous cracks, while the 10Ce-MBG@L-Car composite material has a lamellar structure, which is in line with the expectations after L-carnosine loading. 2) In Fourier transform infrared spectroscopy (FTIR) analysis, L-carnosine successfully binds to the surface of Ce-MBG through multi-site coordination to form a stable complex structure. 3) X-ray photoelectron spectroscopy (XPS) analysis further confirmed that cerium is in a mixed valence state (Ce³⁺ / Ce 4+ ) forms, mainly Ce³⁺. 4) Density functional theory (DFT) results show that the oxygen atoms of the imidazole ring and carboxylate of L-carnosine are nucleophilic active sites, which are easily electrostatically attracted to Ce³⁺ to form coordination bonds.

[0067] In summary, the present invention has successfully synthesized 10Ce-MBG and its L-carnosine complex through a series of physical and chemical performance tests, and revealed its excellent pore structure, surface chemical properties and effective loading performance. The potential of MBG in the biomedical field has been further verified, especially in the application prospects of drug loading and element doping.

[0068] The present invention is divided into two parts: in vitro cell experiments and in vivo animal experiments, which jointly verify the anti-glycation and antioxidant effects of 10Ce-MBG@L-Car nanoparticles in vivo to functionally eliminate the cause of disease, and study their potential for repairing bone defects in a diabetic environment.

[0069] The application concentration of nanoparticles within the biosafety range was obtained through CCK8 cytotoxicity experiments. To ensure that the material does not cause toxicity or adverse reactions to cells when used in vivo, we verified its biosafety concentration range through cell co-culture experiments at different concentrations. By analyzing the single-factor effects of 10Ce-MBG particles and L-carnosine, we determined their respective safe concentration ranges. Based on these data, we finally selected a combination of 10Ce-MBG particles and L-carnosine within three concentration ranges to ensure the balance between the biosafety and functionality of the material. In particular, when the concentration of 10Ce-MBG particles was 7.813 μg / mL and the concentration of loaded L-carnosine was 2.5 mg / mL, the composite material showed the most significant cell proliferation effect. This concentration combination showed the best bioactivity in the CCK-8 experiment of BMSCs without significant toxicity, so it was selected as the experimental group material for further cell and animal experiments.

[0070] The present invention evaluated the dual effects of 10Ce-MBG@L-Car composite hydrogel on anti-oxidative stress and anti-glycation of BMSCs cells in a high glucose (HG) environment through in vitro cell experiments and in vivo animal experiments. The study found that high oxidative stress significantly increased the level of reactive oxygen species (ROS) in BMSCs cells in an HG environment, leading to cell damage and dysfunction, while 10Ce-MBG and 10Ce-MBG@L-Car composite hydrogels can significantly alleviate this effect. This effect may be related to Ce 3+ / Ce 4+The superoxide dismutase (SOD) and catalase (CAT) activities of Ce are related. The redox ability of Ce can remove ROS and generate harmless oxygen and water. In addition, in addition to its anti-sugar effect, L-carnosine also exhibits an anti-oxidative stress effect. The chelation of the two enhances the antioxidant capacity of cells and makes the fluorescence intensity of cells close to that of the control group, indicating that the cell state has been restored. The immunohistochemical results of in vivo animal experiments further verified these findings. The expression of TNF-α decreased significantly, indicating that the material has a good bone immunomodulatory effect; in the HG group, the level of RAGE (receptor for advanced glycation end products) increased significantly, indicating that in a high-sugar environment, the accumulation of AGEs promoted the expression of RAGE, further exacerbating oxidative stress and inflammatory response. In the HG+10Ce-MBG@L-Car group, the level of RAGE decreased significantly, indicating that the composite material can effectively slow down the generation of AGEs and reduce the expression of RAGE. This may be related to the anti-glycation effect of L-carnosine, which inhibits the formation of AGEs by removing aldose inside and outside the cells, and chelating with cerium further enhances the anti-glycation effect of the material. The results of in vivo animal experiments are consistent: the RAGE expression of the 10Ce-MBG@L-Car composite hydrogel group is significantly lower than that of other groups, indicating that the composite material has a more significant effect in anti-AGEs.

[0071] Through in vitro cell experiments and animal experiments, the damage modeling concentration of BMSCs (bone marrow mesenchymal stem cells) and the effects of different materials on bone regeneration under high glucose environment were explored. First, cell experiments confirmed that at a high glucose concentration of 100 mM, the osteogenic function of BMSCs was significantly inhibited, as shown by the reduction of alkaline phosphatase (ALP) activity and calcium nodule formation, and lower concentrations of high glucose (such as 25 mM) failed to cause significant damage to the cells. Further experiments showed that under high glucose conditions, treatment with nanoparticles containing 10Ce-MBG (glass material doped with calcium and phosphorus elements) and 10Ce-MBG@L-Car (L-carnosine) could significantly promote osteogenic differentiation and restore the osteogenic function of BMSCs. In particular, the 10Ce-MBG@L-Car group showed a stronger osteogenic promotion effect than the 10Ce-MBG group alone, which may be related to its antioxidant and anti-glycation properties, and can alleviate the effects of oxidative stress and abnormal glucose metabolism on cells. In in vivo animal experiments, the bone defect model of diabetic rats was used to evaluate the osteogenic effect of the composite hydrogel. Eight weeks after surgery, the Gelma / 10Ce-MBG@L-Car composite hydrogel group not only significantly promoted new bone formation, but also had a high degree of new bone maturity and obvious bone mineralization effect. Micro-CT and histological analysis showed that the bone density (BMD) and bone volume ratio (BV / TV) of the composite hydrogel group were significantly higher than those of the other groups, demonstrating the superior effect of this material in bone repair.

[0072] In summary, this study showed through in vitro and in vivo experiments that 10Ce-MBG@L-Car nanoparticles and their composite hydrogels with Gelma have significant bone repair effects and can play an important role in bone damage caused by diabetes. It provides new ideas and strategies for the treatment of diabetes-related bone defects through anti-oxidation, anti-glycation and promotion of osteogenesis function recovery.

Claims

1. A mesoporous bioactive glass nanoparticle loaded with L-carnosine and doped with cerium, characterized in that: The cerium-doped mesoporous bioactive glass nanoparticles are prepared by doping cerium nitrate hexahydrate (Ce(NO3)3·6H2O) as a cerium source into 58S bioactive glass (MBG) to obtain cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG). The cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) are then placed in a carbonate aqueous solution of L-carnosine to combine with L-carnosine to obtain L-carnosine-loaded and cerium-doped bioactive glass nanoparticles (Ce-MBG@L-Car).

2. The mesoporous bioactive glass nanoparticles loaded with L-carnosine and doped with cerium according to claim 1, characterized in that: The cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) are combined with L-carnosine through chelation coordination bonds and physical adsorption.

3. The mesoporous bioactive glass nanoparticles loaded with L-carnosine and doped with cerium according to claim 1, characterized in that: The ratio of each element in the 58s bioactive glass is 60SiO2: (36-x)CaO: 4P2O5, wherein x is the molar percentage of doped cerium.

4. The mesoporous bioactive glass nanoparticles loaded with L-carnosine and doped with cerium according to claim 3, characterized in that: The molar ratio of cerium doping in the cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) is 10%.

5. A method for preparing the mesoporous bioactive glass nanoparticles loaded with L-carnosine and doped with cerium according to claim 1, characterized in that: The method comprises the following steps: (1) Synthesis of cerium-doped mesoporous bioactive glass: Weigh cetylpyridinium bromide (CPB) and urea into a flask, add deionized water, and perform ultrasonic treatment to completely dissolve CPB and urea, and the solution turns milky white; under magnetic stirring, add cyclohexane and isopropanol to the solution in sequence, and stir the solution continuously at room temperature; introduce tetraethyl orthosilicate (TEOs), stir vigorously to react, and preheat the temperature in a constant temperature water bath, place the flask in a water bath, and continue stirring; add triethyl phosphate (TEP) to react; the ratio of each element in 58s bioactive glass is 60SiO2: (36-x)CaO: 4P2O5, where x is the molar percentage of the doped cationic metal cerium, so according to the molar ratio, doping x % molar ratio of cerium element, the corresponding molar ratio of calcium element is 36-x%, and the corresponding mass is calculated and weighed according to the relative molecular mass of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and cerium nitrate hexahydrate (Ce(NO3)3·6H2O); Ca(NO3)2·4H2O and Ce(NO3)3·6H2O are dissolved in water respectively, and 2ml solution of Ca(NO3)2·4H2O and Ce(NO3)3·6H2O are dripped into the above mixed solution at a speed of 100μl / min respectively, so that the two solutions are fully mixed and reacted with the above mixed solution; stirring is continued to make the mixed solution fully react; after the reaction, the suspension is collected and centrifuged at 9500~10000rpm , 15min, collect the precipitate, wash the precipitate with acetone, 75% ethanol, and deionized water in turn, and collect the precipitate after each washing; freeze-dry the pure precipitate until the precipitate is freeze-dried to a loose structure; put the collected precipitate into a tubular muffle furnace under a nitrogen atmosphere and calcine to obtain Ce-MBG black powder; (2) Preparation of Ce-MBG@L-Car loaded with cerium-doped bioactive glass nanoparticles: Weigh the freeze-dried powder of L-carnosine and dissolve it in carbonate buffer (pH=8.3) to make L-carnosine reach its isoelectric point; put 10% molar ratio of Ce-MBG into the above-mentioned L-carnosine carbonate buffer and stir it magnetically at a constant temperature; then collect the precipitate by centrifugation and freeze-dry it until the precipitate is freeze-dried to a loose structure; the harvested Ce-MBG@L-Car is stored at -20°C.

6. The preparation method according to claim 5, characterized in that: The molar percentage of the doped cationic metal cerium in the step (1) is 10%.

7. The preparation method according to claim 5, characterized in that In the step (1), the pure precipitate is freeze-dried by placing it in a -20°C refrigerator for 2 hours and then freezing it in a -80°C refrigerator for 4 hours.

8. The preparation method according to claim 5, characterized in that: The calcination step in step (1) is to set the heating rate to 1°C / min, calcine the dried powder to 650°C, the calcination time is 10 hours, and then cool to room temperature at 5°C / min to obtain Ce-MBG black powder.

9. A composite hydrogel loaded with L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles, characterized in that: The composite hydrogel is prepared by the following steps: Synthesis of Gelma / Ce-MBG@L-Car composite hydrogel: Take the Gelma hydrogel freeze-dried product and put it into deionized water, prepare a Gelma solution with a concentration of 10% at a constant temperature of 40~50°C, add 2 photoinitiator I2959, so that it accounts for 5% of the dry weight of the Gelma freeze-dried product, wrap the glass bottle of the mixed solution with tin foil to achieve the effect of light protection, continue hydrothermal stirring at 50°C, so that the photoinitiator and the Gelma solution are evenly mixed; after the mixture is evenly mixed, filter it with a filter membrane to sterilize it, and then add Ce-MBG@L-Car nanoparticles sterilized by ultraviolet light to the above solution, so that the Ce-MBG@L-Car nanoparticles account for 10% of the dry weight of the Gelma freeze-dried product, and the Gelma solution to which the photoinitiator has been added and the nanoparticles are evenly mixed to form a Gelma / Ce-MBG@L-Car composite hydrogel.

10. Use of the composite hydrogel loaded with L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles as claimed in claim 9 in preparing diabetic bone defect repair materials.

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