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

Abstract

A cerium-doped mesoporous bioactive glass nanoparticle, its preparation method, hydrogel and application. The Ce-MBG@L-Car composite nanoparticle is synthesized by the surfactant template method to obtain MBG with a uniform mesoporous structure and good particle size distribution, providing ideal drug carrier properties. By doping cerium element through ion replacement, the antioxidant performance of the material is significantly improved, and the stability and loading capacity of L-carnosine are enhanced through coordination chelation. All the characterization and in vitro and in vivo experimental results show that the Ce-MBG@L-Car composite nanoparticle not only has a high drug loading capacity, but also has potential dual anti-glycation and antioxidant effects in the diabetic microenvironment, providing a solid material basis for subsequent bone defect treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a cerium-doped mesoporous bioactive glass nanoparticle, a preparation method thereof, a hydrogel and an application. Background Art

[0002] Diabetes is a chronic systemic disease characterized by metabolic imbalance, hyperglycemia, and destruction of pancreatic islet B cells or insulin resistance. Various complications caused by it can affect multiple organs and systems such as the heart, brain, blood vessels, kidneys, and skin, bringing a serious burden to 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 damage the osteogenic potential of osteoblasts and disrupt the bone remodeling process.

[0003] Oxidative stress is a state in which the balance between oxidation and antioxidant effects in the body is imbalanced and tends to oxidize, which is a negative impact on the body caused by free radicals generated in the body. Reactive oxygen species (ROS) is a complex term that mainly describes the single-electron reduction substances of oxygen in the body, including hydrogen peroxide (H2O2), superoxide anion (O2 - ), and hydroxyl radical (•OH). In patients with poor long-term blood glucose control before and after diabetes diagnosis, abnormally elevated blood glucose in the body can induce high levels of ROS, which are generated in different parts of cells through multiple mechanisms, including but not limited to the activation of the polyol pathway, the activation of protein kinase C isoforms, protein glycosylation under diabetic conditions, glucose auto-oxidation, and mitochondrial overproduction of superoxide. An appropriate level of ROS contributes to normal cell homeostasis and function. However, under diabetic conditions, the production of ROS increases, and the balance between its quantity and antioxidant defense is disrupted, leading to the occurrence of oxidative stress. Excessive intracellular ROS can directly react with DNA, proteins, and lipids, generating multiple chain reactions, forming new free radicals, and spreading through bone tissue, ultimately causing more oxidative damage. ROS may activate oxidative stress-related signaling pathways, resulting in 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(NO3)3·6H2O) as a cerium source doped into 58S bioactive glass (MBG), thereby obtaining cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG), and then placing the cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) in a carbonate aqueous solution of L-carnosine to combine with L-carnosine, thereby obtaining 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 carnosine by two methods: chelation coordination bonds and physical adsorption.

[0009] Preferably, the element ratio of the 58S bioactive glass is 60SiO2: (36 - x)CaO: 4P2O5, 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 preparation method of mesoporous bioactive glass nanoparticles loaded with carnosine and doped with cerium, which comprises the following steps:

[0012] (1) Synthesis of cerium-doped mesoporous bioactive glass: Weigh cetylpyridinium bromide (CPB) and urea and add them to a flask. Add deionized water and ultrasonically treat to completely dissolve CPB and urea, and the solution turns milky white. Under magnetic stirring, add cyclohexane and isopropanol to this solution in sequence, and continuously stir the solution at room temperature. Introduce tetraethyl orthosilicate (TEOs), stir the reaction vigorously, and at the same time preheat and raise the temperature in a constant temperature water bath. Place the flask in a water bath and continue stirring. Add triethyl phosphate (TEP) for reaction. The element ratio of the 58S bioactive glass is 60SiO2: (36 - x)CaO: 4P2O5, where x is the molar percentage of doped cationic metal cerium. Therefore, according to the molar ratio relationship, x% molar ratio of cerium element is doped, and the corresponding molar ratio of calcium element is 36 - x%. Calculate and weigh the corresponding masses according to the relative molecular masses of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and cerium nitrate hexahydrate (Ce(NO3)3·6H2O). Dissolve Ca(NO3)2·4H2O and Ce(NO3)3·6H2O in water respectively. At the same time, drop 2 ml solutions of Ca(NO3)2·4H2O and Ce(NO3)3·6H2O into the above mixed solution at a speed of 100 μl / min to make these two solutions fully mixed and react with the above mixed solution. Continue stirring to make the mixed solution fully react. After the reaction, collect the suspension, centrifuge at 9500 - 10000 rpm for 15 min, collect the precipitate, wash the precipitate with acetone, 75% ethanol, and deionized water in sequence, and collect the precipitate after each washing. Freeze-dry the pure precipitate until the precipitate is freeze-dried into a loose structure. Place the collected precipitate in a tubular muffle furnace and calcine it in a nitrogen atmosphere to obtain Ce-MBG black powder;

[0013] (2)Preparation of L-carnosine-loaded cerium-doped bioactive glass nanoparticles (Ce-MBG@L-Car): Weigh the freeze-dried powder of L-carnosine and dissolve it in carbonate buffer (pH = 8.3) to reach its isoelectric point. Put 10% molar ratio of Ce-MBG into the above L-carnosine carbonate buffer solution, and stir magnetically under constant temperature conditions. Then, centrifuge to collect the precipitate and freeze-dry the precipitate until it becomes a loose structure. The harvested Ce-MBG@L-Car is stored at -20°C.

[0014] Preferably, the molar percentage of doped cationic metal cerium in step (1) is 10%.

[0015] Preferably, the freeze-drying of the pure precipitate in step (1) is specifically carried out by placing it in a -20°C refrigerator for 2 h and then in an -80°C refrigerator for 4 h.

[0016] Preferably, the calcination step in step (1) is to set the heating rate at 1°C / min, calcine the dry powder to 650°C, with a calcination time of 10 hours, and then cool it to room temperature at 5°C / min to obtain the Ce-MBG black powder.

[0017] A composite hydrogel loaded with L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles, and the composite hydrogel is prepared by the following steps:

[0018] Synthesis of Gelma / Ce-MBG@L-Car composite hydrogel: Take the freeze-dried Gelma hydrogel and put it into deionized water. Under the condition of constant temperature at 40 - 50°C, prepare a 10% Gelma solution, add 2 photoinitiator I2959, making its proportion to the dry weight of the Gelma freeze-dried product 5%. Wrap the glass bottle containing the mixed solution with tin foil to achieve the effect of light shielding, and continue to stir at 50°C hydrothermally to make the photoinitiator and Gelma solution mix evenly. After mixing evenly, filter it aseptically with a filter membrane, and then add the Ce-MBG@L-Car nanoparticles sterilized by ultraviolet light into the above solution, making the proportion of Ce-MBG@L-Car nanoparticles to the dry weight of the Gelma freeze-dried product 10%. After mixing the Gelma solution with the added photoinitiator and the nanoparticles evenly, a Gelma / Ce-MBG@L-Car composite hydrogel is formed.

[0019] Application of a composite hydrogel loaded with L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles in the preparation of a repair material for diabetic bone defects.

[0020] The beneficial effects of the present invention are as follows: A cerium-doped mesoporous bioactive glass nanoparticle, its preparation method, hydrogel and application. The Ce-MBG@L-Car composite nanoparticle is synthesized by the surfactant template method to obtain MBG with a uniform mesoporous structure and good particle size distribution, providing ideal drug carrier properties. By doping cerium element through ion replacement, the antioxidant performance of the material is significantly improved, and the stability and loading capacity of L-carnosine are enhanced through coordination chelation. All the characterization and in vitro and in vivo experimental results show that the Ce-MBG@L-Car composite nanoparticle not only has a high drug loading capacity, but also has potential dual anti-glycation and antioxidant effects in the diabetic microenvironment, providing a solid material basis for subsequent bone defect treatment. Description of the Drawings

[0021] Figure 1 SEM characterization of Ce-MBG with different doping molar ratios.

[0022] Figure 2 TEM and EDS characterization of 10Ce-MBG.

[0023] Figure 3 XRD characterization of MBG and 10Ce-MBG.

[0024] Figure 4 Antioxidant performance characterization of Ce-MBG with different doping molar ratios; A: ABTs+; B: DPPH.

[0025] Figure 5 TGA analysis of 10Ce-MBG, L-Car, and 10Ce-MBG@L-Car.

[0026] 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 diagram of 10Ce-MBG C. Nitrogen adsorption and desorption curve of 10Ce-MBG@L-Car D. Pore size distribution diagram of 10Ce-MBG@L-Car.

[0027] 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 respectively.

[0028] Figure 8 XPS analysis. A. Total energy spectrum of 10Ce-MBG; B. Ce energy spectrum; C. Si energy spectrum; D. O energy spectrum.

[0029] Figure 9 For Ce 3+Molecular simulation of chelation with L-Car. A. Analysis of the positive (blue) and negative (red) electrostatic potential of L-carnosine; B. Analysis of the positive (red) and negative (blue) electrostatic potential of 10Ce-MBGs@L-Car; C. Schematic diagram of the HOMO / LUMO and energy gap of 10Ce-MBGs@L-Car in the ground state.

[0030] Figure 10 It is for the CCK8 cytotoxicity experiment in vitro. A. CCK8 cytotoxicity experiment with different concentrations of 10Ce-MBG particle suspensions after 48 h; B. CCK8 cytotoxicity experiment with different concentrations of L-carnosine solution after 48 h.

[0031] Figure 11 It is for the CCK8 cytotoxicity experiment in vitro. A. CCK8 cytotoxicity experiment with different concentrations of 10Ce-MBG particle suspensions in different concentrations of L-carnosine drugs after 24 h; B. CCK8 cytotoxicity experiment with different concentrations of 10Ce-MBG particle suspensions in different concentrations of L-carnosine drugs after 48 h.

[0032] Figure 12 It is for the antioxidant and anti-glycation experiments in vitro. A: DCFH-DA experiment; B: RAGE immunofluorescence staining experiment; C: Semi-quantitative analysis of ROS fluorescence intensity; D: Semi-quantitative analysis of RAGE immunofluorescence staining fluorescence intensity.

[0033] Figure 13 It is for the ALP experiment of in vitro high-glucose modeling. A. ALP staining experimental diagram at different high-glucose concentrations; B. Semi-quantitative analysis of the optical density related to ALP staining.

[0034] Figure 14 It is for the in 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 the optical density of ALP in different materials; D. Quantitative analysis of the enzyme activity unit (DEA) of ALP protein in different materials; E. Semi-quantitative analysis of the optical density of ARS in different materials; F-H. Relative gene expression levels of ALP, Runx2, and OPN in different materials, respectively.

[0035] Figure 15 It is for the in vivo Micro CT analysis of skull defects. A. Schematic diagram of osteogenesis of skull defects in different modeling groups; B. Bone mineral density (BMD) of skull defects in different modeling groups; C. Bone volume fraction (BV / TV) of skull defects in different modeling groups; D. Trabecular bone thickness (Tb.Th) of skull defects in different modeling groups; E. Trabecular bone number (Tb.N) of skull defects in different modeling groups.

[0036] Figure 16For in vivo skull defect HE & Masson staining analysis. A: HE staining; B: Masson staining

[0037] N: New bone; F: Fibrous tissue; G: Gelma hydrogel.

[0038] Figure 17 For in vivo skull defect immunohistochemical staining. A. Immunohistochemical staining of OCN, TNF-a, RAGE; B. Semi-quantitative statistical analysis of the area of the immunohistochemical positive region of RAGE; C. Semi-quantitative statistical analysis of the area of the immunohistochemical positive region of TNF-a; D. Semi-quantitative statistical analysis of the area of the immunohistochemical positive region of OCN.

[0039] Figure 18 For the experimental flow chart of the preparation of Ce-MBG@L-Car. Specific implementation manners

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Example 1 Preparation and characterization of cerium-doped mesoporous bioactive glass nanoparticles

[0042] Synthesis of cerium-doped mesoporous bioactive glass

[0043] Weigh 2.5 g of CPB (cetylpyridinium bromide) and 1.5 g of urea and add them to a 250 ml flask. Add 75 ml of deionized water and ultrasonically treat for 10 min to completely dissolve CPB and urea, and the solution turns milky white. Under magnetic stirring, sequentially add 75 ml of cyclohexane and 2.3 g of isopropanol to this solution, and continuously stir the solution at room temperature for 2 h. Introduce 6.75 ml of tetraethyl orthosilicate (TEOs), vigorously stir and react for 30 min, and at the same time turn on the constant temperature water bath to preheat and raise the temperature to 70 °C. Place the flask in the water bath and continue stirring for 7 h. Add 300 μl of triethyl phosphate (TEP) and react for 30 min. The elemental ratios of the bioactive glass at 58 s are 60SiO2: (36 - x)CaO: 4P2O5, where x is the molar percentage of other doped cationic metals. Therefore, according to the molar ratio relationship, when doping cerium elements with molar ratios of 2%, 5%, 10%, 12%, 15%, and 20%, the corresponding molar ratios of calcium elements are 34%, 31%, 26%, 24%, 21%, and 16% respectively. Calculate and weigh the corresponding masses according to the relative molecular masses of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and cerium nitrate hexahydrate (Ce(NO3)3·6H2O). Dissolve Ca(NO3)2·4H2O and Ce(NO3)3·6H2O separately in 2 mL of water, and at the same time use two syringes to drip the 2 ml solutions of Ca(NO3)2·4H2O and Ce(NO3)3·6H2O into the above mixed solution at a speed of 100 μl / min to make these two solutions fully mixed and react with the above mixed solution. Continue stirring for 16 h to make the mixed solution fully react. After the reaction, collect the suspension, centrifuge at 9500 - 10000 rpm for 15 min, collect the precipitate, wash the precipitate with acetone 3 times, 75% ethanol 2 times, and deionized water 3 times. Each time after washing, collect the precipitate at 10000 rpm for 15 min. Put the pure precipitate into a -20 °C refrigerator for 2 h, and then put it into an -80 °C refrigerator and freeze for 4 h. Turn on the freeze dryer, pre-treat for about 15 min, and dry for about 2 days until the precipitate is freeze-dried into a loose structure. Put the collected precipitate into a tubular muffle furnace under a nitrogen atmosphere, set the heating rate to 1 °C / min, calcine the dried powder to 650 °C, the calcination time is 10 h, and cool to room temperature at 5 °C / min to obtain Ce-MBG black powder.

[0044] L-Carnosine Loading on Cerium-Doped Bioactive Glass Nanoparticles (Ce-MBG@L-Car)

[0045] Weigh 50 mg of freeze-dried L-carnosine powder and dissolve it in 5 ml of carbonate buffer (pH = 8.3) to bring L-carnosine to its isoelectric point. Put 50 mg of Ce-MBG with a 10% molar ratio into the above 5 ml of L-carnosine carbonate buffer and magnetically stir for 24 h at a constant temperature of 50 °C. Then centrifuge at 3000 rpm for 15 min to collect the precipitate, place the precipitate in a -20 °C refrigerator for 2 h, and then freeze it in an -80 °C refrigerator for 4 h. Turn on the freeze-dryer, pre-treat for about 15 min, and dry for about 2 days until the precipitate is freeze-dried into a loose structure. The harvested Ce-MBG@L-Car is stored in a -20 °C refrigerator.

[0046] Structures and antioxidant characteristics of Ce-MBG nanoparticles with different doping molar ratios

[0047] There are many synthesis methods for MBG. Different synthesis techniques will affect its structure, pore characteristics, and biological activity, and thus determine its application effects 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 been gradually proposed and widely used, such as chemical vapor deposition method, microwave-assisted synthesis method, solvothermal method, and solvent-free method, etc. In recent years, as an emerging synthesis strategy, the template-guided method has gradually attracted attention. This method can precisely control the pore size, morphology, and particle size of the glass, thus significantly enhancing its application potential in the biomedical field. When synthesizing using the CPB template-guided method, the uniform doping of cerium elements further improves the antioxidant performance and surface chemical characteristics of bioactive glass: as a rare earth element with good antioxidant properties, cerium can regulate the redox state in the reaction environment through the reversible transformation between its Ce +3 and Ce +4 valence states

[38] ; the surface of cerium has abundant oxygen vacancies and active sites, which can form a chelation effect 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 enhances the antioxidant and anti-glycation effects bidirectionally, thus breaking the glycation-oxidation vicious cycle in the diabetic bone healing microenvironment.

[0048] As Figure 1 shown by the scanning electron microscopy results in

[0049] The doping of 12% and 15% cerium causes severe aggregation of the particles, uneven particle size distribution, more irregular morphology, and it can be seen that the surface pore structure collapses. This indicates that the incorporation of a high concentration of cerium leads to the aggregation of the particles, affecting the morphological uniformity and pore structure of the material. Therefore, in the following article, the present invention mainly uses MBG, 2%, 5%, and 10% molar ratio Ce-MBG for material characterization.

[0050] According to Figure 2 the TEM images, the morphological characteristics of the 10Ce-MBG sample can be observed. The HAADF (High Angle Annular Dark Field Imaging) in the figure shows the overall morphology of the particles. The particles exhibit a spherical structure, with a size of approximately 200 - 250 nm, a smooth surface, and a uniform pore structure. This indicates 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). The spectrum shows the presence of elements such as calcium (Ca), cerium (Ce), silicon (Si), and phosphorus (P). The distribution of cerium element is represented in red, indicating its relatively uniform distribution in the particles. Calcium and silicon elements are shown in green and blue respectively, showing their rich distribution in the particles. Due to the increase in the cerium doping molar ratio, to a certain extent, the doping of calcium element is reduced (the molar doping ratio of calcium element is reduced to 26%). Therefore, the signal intensity of calcium element is relatively weak in terms of elemental distribution. According to the atomic fraction and mass fraction data of the elements, the atomic proportion of cerium element in the particles is 0.25%, showing a moderate doping ratio. Overall, the results of this TEM and EDS analysis confirm the successful synthesis of 10Ce-MBG and demonstrate its good morphology and uniform elemental distribution.

[0051] Figure 3 The XRD image in shows that the 10Ce-MBG particles exhibit a typical inorganic glass doughnut peak structure and no other peaks appear. Overall, this indicates that the synthesis process of doping Ce into 10Ce-MBG particles is successful. The intensity of the glass doughnut peak structure of 10Ce-MBG is not as large 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 °C, nitrogen is passed through during the overall sintering process, resulting in a relatively reduced oxygen content, and the Si-O bonds in the inorganic crystals are relatively reduced compared to MBG sintered in an air atmosphere.

[0052] Subsequently, we characterized the antioxidant properties of Ce-MBG with different doping molar ratios. As Figure 4As shown in (A), at 30 min, Ce-MBG with different doping molar ratios and the blank control (Blank) both had a certain scavenging effect on oxidation free radicals. Among them, the Abs absorbance curves of MBG, 2%, and 5% doped Ce-MBG overlapped. Perhaps because the original doping ratio of Ce was relatively small and did not achieve an obvious oxygen free radical scavenging effect. The decrease in the absorbance curve of MBG without Ce doping might be due to its rich mesoporous structure adsorbing the liquid fuel, thus making the ABTs + become lighter in blue, while the Abs curve of 10Ce-MBG was the lowest, indicating that as the doping molar ratio of Ce increased, the antioxidant performance was enhanced. Similarly, as shown in Figure 4 (B), it can also be observed that as the doping molar ratio of Ce increased, the ability of the particles to capture oxygen free radicals, that is, the antioxidant function, was enhanced. Therefore, through SEM, TEM, XRD, ABTs + and DPPH antioxidant characteristic experiments, it was verified that 10Ce-MBG was successfully doped, had a uniform particle size distribution and pore distribution, and at the same time had good antioxidant performance. Therefore, 10Ce-MBG was selected for the following drug loading and other material characterization experiments.

[0053] Physicochemical characterization of the synthesized particles

[0054] Thermogravimetric analyzer (TGA)

[0055] According to Figure 5 the thermogravimetric analysis graph (TGA), in the case of passing nitrogen to prevent the mass increase caused by oxidation, the mass changes of different materials during the heating process can be observed, and the mass percentage of drug loading can be known according to the curve. L-carnosine showed the most significant mass change. Especially when the temperature rose to 300 °C, the mass decreased rapidly, and finally the mass dropped to nearly zero at 600 °C. 10Ce-MBG had almost no mass loss during the heating process. When the temperature rose from room temperature to 600 °C, the mass remained basically stable, indicating its good thermal stability. 10Ce-MBG@L-Car showed obvious mass loss. Especially when the temperature rose to about 300 °C, the mass decreased 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 nanoparticle 10Ce-MBG does not decompose, so it can be roughly estimated that the mass of L-carnosine in the total material 10Ce-MBG@L-Car is about 19%.

[0056] Specific surface area and porosity analyzer (BET)

[0057] 10Ce-MBG: The N2 isothermal adsorption-desorption curve belongs to type IV in the IUPAC classification, with an H4 hysteresis loop, indicating that the adsorbent has narrow mesoporous slits and the pore size distribution peaks at 4 nm. The "narrow" hysteresis loop proves that the pore size is small, the pressure for capillary condensation is low, and there is a greater tendency for micropore filling. The "steep" hysteresis loop proves that the capillary condensation section is steeper and the mesopore distribution is relatively uniform; the nitrogen adsorption capacity ranges from 100 to 500 cm 2 / g, indicating well-developed pores and a large specific surface area. In addition, the desorption curve of 10Ce-MBG almost coincides with the adsorption curve, indicating that the material has strong adsorption and a low hysteresis effect. The steep rise of the adsorption curve indicates that the material can rapidly adsorb gas at low relative pressures, making it suitable for application scenarios that require rapid adsorption.

[0058] 10Ce-MBG@Car: The N2 isothermal adsorption-desorption curve belongs to type IV in the IUPAC classification, with an H3 hysteresis loop, indicating that the adsorbent has slit-shaped mesopores similar to clay with a lamellar structure, suggesting successful loading of L-carnosine. There is a certain hysteresis phenomenon between its adsorption curve and desorption curve, indicating that the gas release is slower after the adsorption process. Compared with 10Ce-MBG, after adding L-carnosine, the pore structure of the material changes, resulting in a slower gas diffusion rate, thus affecting its gas desorption process.

[0059] Table BET specific surface area, total pore volume, and average pore size of 10Ce-MBG and 10Ce-MBG@L-Car

[0060]

[0061] According to the BET analysis in the table, the successful loading of L-carnosine can be seen from three aspects. 1) BET surface area: The BET surface area of 10Ce-MBG is 933.8999 m² / g, while that of 10Ce-MBG@L-Car significantly decreases to 2.5414 m² / g. This difference indicates that the addition of L-Car greatly reduces the surface area of the material, probably because the presence of L-Car fills some pores, reducing the available surface area, indirectly proving the successful loading of L-carnosine. 2) Total pore volume: The total pore volume of 10Ce-MBG is 0.61736 cm³ / g, while that of 10Ce-MBG@L-Car is lower, at 0.009853 cm³ / g. This data 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 that 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 pore size distribution.

[0062] Fourier transform infrared spectrometer (FTIR)

[0063] The Fourier transform infrared spectra (FTIR) of L-carnosine, 10Ce-MBG, and their complex are as Figure 7 shown. The characteristic peaks of L-carnosine show that the broad peak at 2988 cm -1 is attributed to the stretching vibration of C=N (histidine imidazole ring); the strong absorption peaks at 1648 cm -1 and 1561 cm -1 correspond to amide I band (C=O stretching vibration) and amide II band (N-H bending vibration) respectively, which indicates the complete structure of the peptide bond. When L-carnosine chelates with 10Ce-MBG, the infrared spectrum changes significantly: the peak width in the amino-hydroxy region (2988 cm -1 ) increases and shifts to a lower wavenumber of 2977 cm -1 , which indicates that the -NH2 or -COO⁻ group of L-carnosine binds to Ce on the surface of Ce-MBG through a coordination bond; at the same time, the characteristic peak of the carboxylate group (1268 cm -1 ) shifts to a lower wavenumber (1235 cm -1 ) in the chelating ligand, presumably due to the coordination effect of the carboxylate oxygen atom with Ce³⁺ resulting in a change in the vibration mode; more importantly, the amide I band and II band shift to lower wavenumbers of 1640 cm -1 and 1558 cm -1, indicating that the C=O and N-H of the peptide bond may participate in surface adsorption, and the redistribution of electron cloud density further confirms the strong interaction between the two. The above results show that L-carnosine is anchored on the surface of Ce-MBG through multi-site coordination of carboxylate, amino group and peptide bond, forming a stable complex structure, demonstrating its coordination with cations. These results indicate the successful synthesis of 10Ce-MBG@L-Car.

[0064] X-ray photoelectron spectroscopy (XPS)

[0065] In order to further verify the synthesis results of 10Ce-MBG, X-ray photoelectron spectroscopy (XPS) analysis was carried out on it. The full spectrum of X-ray photoelectron spectroscopy (XPS) of 10Ce-MBG Figure 8 A confirmed the coexistence of Si, O, and Ce elements in the 10Ce-MBG chelating material. Further detailed peak fitting was carried out to characterize the chemical states of the elements on 10Ce-MBG. As Figure 8 shown in B, cerium elements are loaded on the surface of mesoporous bioactive glass in the form of mixed valence states (Ce³⁺ / Ce 4 ⁺). Through the peak fitting of the Ce 3d orbitals, it was observed that the characteristic peaks of the Ce 4 ⁺ main peaks (881.7 eV and 900.0 eV) coexist with the Ce³⁺ main peaks (885.4 eV and 904.2 eV). The analysis of the O 1s orbitals ( Figure 8 D) shows that at 532.2 eV and 529.8 eV, which are attributed to the O-Si of SiO2 and the bridging bond of Si-O-Ce respectively. The existence of the bridging bond further reveals the interfacial interaction between cerium and mesoporous bioactive glass. At the same time, the formation of the Si-O-C bond (101.25 eV) was observed in the Si 2p spectrum, and 103.1 eV is attributed to the Si-O bond of SiO2, ensuring the integrity of the glass body structure. In summary, this composite structure provides active sites for cerium species while maintaining the stability of the SiO2 substrate, enhancing its redox performance.

[0066] Chelate molecular simulation

[0067] L-carnosine, as a natural dipeptide, contains amino, carboxyl, and imidazole groups in its molecular structure, endowing it with multi-dentate coordination ability and possibly forming stable chelates with metal ions through N / O atoms

[42] . To deeply analyze the coordination driving force, we calculated the electrostatic potential (ESP) of the L-carnosine molecule using density functional theory (DFT / B3LYP / 6-311++G(d,p)). As Figure 9As shown in Figure 9 A, B, the π - electron - rich region of the imidazole ring and the oxygen atoms of the carboxylate group in L - carnosine are potential nucleophilic active sites, which preferentially undergo electrostatic attraction with the positively charged Ce³⁺. Meanwhile, 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) maps of free L - carnosine and its Ce³⁺ chelate ( Figure 9 C shows the lowest unoccupied molecular orbital (LUMO) and the 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 HOMO is delocalized over the entire conjugated main chain. The calculated energy gap between the HOMO and LUMO is 3.20 eV, which may be due to the hybridization of the 4f orbit of Ce³⁺ with the π orbit of the imidazole ring, resulting in a ligand - to - metal charge - transfer band. The energy levels and spatial distributions of the HOMO - LUMO orbitals after chelation significantly improve the electron - transfer efficiency, endowing it with bifunctional antioxidant activity.

[0068] Example 2: In vitro cell experiments

[0069] CCK - 8 cytotoxicity experiment to screen the application concentration of nanoparticle suspension

[0070] Before exploring the safe concentration range of 10Ce - MBG@L - Car for biological use, 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. Figure 10 As shown in A, after treating BMSCs with 10Ce - MBG solutions at different concentrations for 48 h, the cell viability showed a certain concentration - dependence. When the concentration of the 10Ce - MBG solution was low, that is, less than 15.625 μg / mL, the cell viability basically remained at a level close to 100%, and there was no significant difference from the control group. However, when the concentration reached a certain level, such as above 31.25 μg / mL, the cell viability decreased significantly, showing an obvious inhibitory effect compared with the control group. This indicates that the 10Ce - MBG solution has an inhibitory effect on the cell viability of BMSCs at higher concentrations, suggesting that the bioactive glass doped with cerium elements may have a certain toxic effect on cells.

[0071] Figure 10Group B shows the effects of different concentrations of L-carnosine on the viability of BMSCs after 48 h. It can be seen from the data that L-carnosine can significantly promote the 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. Especially at a concentration of 31.25 μg / mL, the cell viability reached the highest level, indicating that L-carnosine has a good stimulating effect on BMSCs. However, when the concentration further increased to 1 mg / mL and above, the cell viability began to decline, and the difference was not statistically significant. This result indicates that L-carnosine can promote the proliferation of BMSCs within a certain concentration range. Although it does not promote cell proliferation at too high concentrations, its safe concentration range has exceeded the μg / mL order of magnitude. Therefore, when applying 10Ce-MBG nanoparticles loaded with L-carnosine at a concentration in the μg / mL order of magnitude, the released concentration of L-carnosine can also be determined to be within the safe concentration range for use.

[0072] According to the principle of "using the highest concentration within the biosafe concentration of the material to ensure both biosafety and significant function" discussed in Figure - 11, three high-concentration 10Ce-MBG particle suspensions: 15.625 μg / mL, 7.813 μg / mL, 3.906 μg / mL (the final concentration when co-incubating with cells) were selected and added to L-carnosine drugs at different concentrations to complete the preparation of the final material 10Ce-MBG@L-Car. Although in terms of single factors, that is, the concentration of 10Ce-MBG particle suspension and the concentration of L-carnosine drug are both selected within the safe concentration, whether the final product of their combination has cytotoxicity is still unknown. Figure 11 A and B respectively show the effects on cell viability when the final material 10Ce-MBG@L-Car is co-incubated with BMSCs after 24 h and 48 h. Figure 11 There were no significant differences between groups and within groups after 24 h in A; Figure 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 an obvious effect of promoting the proliferation of BMSCs. Especially when the final concentration of 10Ce-MBG is 7.813 μg / mL and the concentration of the L-carnosine-loaded solution is 2.5 mg / ml, the effect of promoting the proliferation of BMSCs is the most obvious. Therefore, the concentration of 10Ce-MBG@L-Car particles prepared with 10Ce-MBG at a final concentration of 7.813 μg / ml and an L-carnosine concentration solution of 2.5 mg / ml was selected as the experimental group material for cell and animal experiments.

[0073] Antioxidant & Anti-glycation Cell Experiments

[0074] High oxidative stress can significantly induce the transformation of BMSCs cells into an adverse state, increasing cell damage and dysfunction. Through DCFH-DA staining, it was observed that the ROS level in BMSCs cells in the HG group was significantly increased, indicating that oxidative stress exacerbated the oxidative damage of the cells. However, in the HG+10Ce-MBG group and the HG+10Ce-MBG@L-Car group, the ROS level decreased significantly, indicating that these two treatments could effectively alleviate the effects of oxidative stress. Oxygen is the product of the cellular antioxidant system's response to ROS. In the HG group, the cellular antioxidant system was inhibited, resulting in insufficient oxygen generation and the inability to effectively eliminate oxidative reactions, so a large number of positive expressions were observed. In the HG+10Ce-MBG group and the HG+10Ce-MBG@L-Car group, by inducing the repair of the endogenous antioxidant system in BMSCs cells, where the Ce 3+ / Ce 4+ -like SOD / CAT enzyme function can react with ROS to generate harmless oxygen and water, thereby reducing the accumulation of ROS. And L-carnosine itself, in addition to its antiglycation effect, also has a certain antioxidant stress effect. After the formation of a chelate between Ce and L-carnosine, the antioxidant effect in the cells 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, the Ru(dpp)3Cl2 fluorescent probe was quenched, showing less positive expression, further proving the effective inhibitory effect on oxidative stress. Although the fluorescence in the HG+MBG group also decreased to some extent, the difference was not obvious. ( Figure 12 A, Figure 12 C)

[0075] RAGE (receptor for advanced glycation end products) is a transmembrane protein that participates in regulating processes such as inflammation, oxidative stress, and apoptosis. Extracellularly, under hyperglycemic conditions, excessive glucose reacts with proteins on the surface of osteocytes to form stable AGEs. The binding of AGE-RAGE activates downstream signaling pathways. Intracellularly, a large amount of excessive glucose metabolism cannot undergo glycolysis in time, activating six metabolic branches. Among them, glycation also causes a large accumulation of AGEs in cells. The accumulated intracellular AGEs in turn promote the expression of RAGE. Therefore, the expression of RAGE is closely related to the content of AGEs and can be used as a detection index for testing the concentration level of AGEs in the bone microenvironment. Through RAGE immunofluorescence staining, it was observed that the level of RAGE in BMSCs cells in the HG group increased significantly, indicating that AGEs also accumulated to a considerable level in the hyperglycemic environment. However, in the HG+10Ce-MBG@L-Car group, the level of RAGE 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 antiglycation effect of the drug L-carnosine, which can scavenge aldoses inside and outside cells, thereby reducing the generation of AGEs. After the chelate formed by Ce and L-carnosine, the antiglycation effect inside cells 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, since it can scavenge the promoting effect of oxidative stress on the generation of AGEs to a certain extent, indirectly slowing down glycation, but the antiglycation effect is not significant. ( Figure 12 B, Figure 12 D)

[0076] Osteogenic differentiation cell experiment

[0077] In cell experiments, a stable high glucose concentration must first be determined to ensure that high glucose damage is caused to cells during the experiment. Alkaline phosphatase (ALP) is mainly used in the mineralization process of the bone matrix in osteoblasts. It promotes the accumulation of inorganic phosphate through dephosphorylation reactions, thereby promoting bone formation and mineralization, and is an important marker enzyme for bone formation. Therefore, the ALP staining index is used to reflect the damage of BMSCs osteogenic function. Such as Figure 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.

[0078] 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 ( Figure 14 A, B). The results of the ALP protein quantification experiment further verified this conclusion ( Figure 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. ( Figure 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.

[0079] qRT-PCR further demonstrated that the nanoparticle material could restore the osteogenic function of BMSCs ( Figure 14 F-H). Runx2 and ALP are early osteogenic markers. OPN affects bone metabolism by regulating the functions of osteoblasts and osteoclasts during osteogenesis. Under HG conditions, the osteogenesis-related indicators ALP and Runx2 were significantly downregulated. At the gene level, 10Ce-MBG and 10Ce-MBG@L-Car could not only restore the osteogenic differentiation function of BMSCs to the normal level, but also showed better osteogenic effects than BMSCs in the healthy state. It is worth noting that even in the HG environment, OPN still has the ability of high expression. In some studies, OPN was considered to have a certain protective effect. The tissue damage caused by high glucose may promote the upregulation of OPN expression, so as to reduce the damage of high glucose 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 the tissue damage caused by high glucose. Among the upregulation of OPN expression, the OPN expression was the most significant after adding 10Ce-MBG@L-Car, which may be attributed to its regulation of intracellular oxidative stress and glucose metabolism, indirectly enhancing the expression of bone repair-related genes.

[0080] Example 3: In vivo animal experiment

[0081] In vivo animal Micro CT analysis

[0082] The 5 mm defect constructed in this experiment belongs to the category of critical size, and this model prevents diabetic rats from self-healing in a short time. As Figure 15 shown in A, at 8 weeks after surgery, the DM group hardly healed and the defect area basically remained. In the group implanted with Gelma hydrogel, new bone of varying degrees could be observed at the edge of the host bone, but all were immature. In the Gelma / 10Ce-MBG@L-Car composite hydrogel group at the injury site, not only was the amount of new bone formation around the host bone significant, but the area of the new bone was relatively dense, and almost the entire defect area had been filled, indicating that the 10Ce-MBG@L-Car composite hydrogel had better effects on inducing bone regeneration and bone repair than other experimental groups.

[0083] Micro-CT based semi-quantitative analysis further confirmed the osteogenic effect of Gelma / 10Ce-MBG@L-Car composite hydrogel. Bone Mineral Density (BMD) refers to the amount of minerals contained in bone per unit area or volume, reflecting the strength and hardness of bone, and is measured by the X-ray Attenuation Coefficient (AC). Bone Volume to Total Volume (BV / TV) is an important index for evaluating bone density and bone quality, and together with BMD, it is an important index 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. With the generation of new bone, the thickness of trabecular bone should gradually recover, and a higher trabecular number usually means a denser bone structure. At 8 weeks, the BMD of DM and the composite hydrogel was 0.72±0.01, much higher than 0.41±0.03 in the DM group and 0.47±0.062 in the DM and Gelma group (p<0.01), indicating that the bones in the experimental group tended to be in a normal healthy state; while the BV / TV of the DM and composite hydrogel group was 14.47±2.66, the Tb.Th was 20.77±4.01, and the Tb.N was 0.007±0.0013. Compared with the DM group and the DM+Gelma group, the differences were statistically significant (p<0.05), indicating that the trabecular bone structure in the experimental group was denser and the bone mass was more mature. Micro-CT results showed that the implantation of Gelma / 10Ce-MBG@L-Car had an obvious promoting effect on osteogenic regeneration in bone defects, and this result will be further verified by histological and osteogenic immunohistochemical experiments in subsequent experiments.

[0084] In vivo histological staining experiment

[0085] (1)HE&Masson staining

[0086] Figure 16A shows the H&E staining results of bone defects and surrounding tissues in each group 8 weeks after surgery. The staining results showed that no new bone formation was found in the bone defect area of the DM group, and only a certain amount of fibrous tissue was found near the host bone. In the DM+Gelma group, due to the slow degradation of Gelma, it could promote some cells in the bone microenvironment to crawl to the central area, and a little new bone was produced at the central fibrous tissue. In the DM+Gelma / 10Ce-MBG@L-Car composite hydrogel group, not only new bones of different sizes were produced at the boundary of the host bone, but obvious hydrogel material residues were also visible. A large amount of new bone was produced in the center of the defect area at the lower end to form an osteal bridge, connecting the host bones on both sides. In addition, the implanted hydrogel had good biocompatibility with tissues, and no large-scale inflammation occurred around it. It is worth noting that in the composite hydrogel group, new bone was generated along the fiber edge at the lower end of its coverage, and the shape of the new bone began to be similar to that of the host bone. Mature osteocytes could be seen in the magnified view. Due to the doping of solid particles, the degradation rate of Gelma was slower, so a relatively complete material contour could still be found in all composite hydrogel groups 8 weeks after surgery. There are research reports that the biodegradation rate of Gelma is related to the concentration of cross-linking agent added, the mass ratio of doped solid particles, the implantation site, and the metabolic rate in the organism. And due to incomplete degradation, it also played a role as a certain barrier membrane. It can be seen that the upper surface of the composite hydrogel was covered with fibrous tissue, protecting the osteogenic reaction at its lower end from being disturbed by the faster-growing fibrotic tissue. Therefore, doping solid particles also had certain advantages in terms of degradation rate.

[0087] The maturity of bone quality was analyzed by the different colors stained by Masson. The collagen in bone was mainly type I collagen. During the process of new bone becoming more mature, a red-blue alternating situation would appear on its surface. The maturity of collagen in bone tissue directly affected the Masson staining. As Figure 16 shown in B, almost no new bone tissue could be found in the DM group 8 weeks after surgery, which was consistent with the conclusion obtained from the above H&E staining. In the composite hydrogel group, the proportion of red-stained new bone mass was relatively large 8 weeks after surgery, indicating a high maturity of collagen in bone tissue at this time. In the Gelma group, the proportion of red fibers of bone collagen in new bone was small and the proportion of blue new bone was large, which was similar to the above H&E results.

[0088] (2)Immunohistochemical staining results of OCN, TNF-a, and RAGE

[0089] Osteocalcin (OCN) is a non-collagen protein secreted by osteoblasts, mainly involved in the bone mineralization process, and is a marker of bone maturity. At 8 weeks after surgery, the higher the mature bone mass, the stronger the bone repair ability. To investigate the in vivo osteogenic induction potential and the effect of promoting osteogenic differentiation of the DM+Gelma / 10Ce-MBG@L-Car composite hydrogel group, we performed OCN immunohistochemical staining on the samples of each group at 8 weeks after surgery. In the experimental results, a positive staining area of OCN with a certain area appeared in each experimental group ( Figure 17 B). The large area of positive OCN staining in the composite hydrogel group indicates that the mature bone in the new bone area is the most, suggesting obvious mineralization. The composite hydrogel has the best potential for osteogenic induction and the effect of promoting osteogenic differentiation.

[0090] In the bone microenvironment of diabetes, the high expression of TNF-α promotes the chronic inflammatory response. By activating the inflammatory pathway and oxidative stress, the function of osteoblasts is inhibited, further deteriorating bone metabolism. At 8 weeks after surgery, the persistent inflammation in the DM group led to non-union of the bone. 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 decrease in the 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 vitro cell experiments. Both directions jointly illustrate that the composite hydrogel has a good antioxidant stress effect. There are experimental records that the Gelma material itself also has a certain antioxidant effect, and the TNF-a in the Gelma hydrogel group of this article also has a certain alleviating effect.

[0091] At 8 weeks after surgery, although there were certain differential expressions in the semi-quantitative positive area analysis between DM and DM+Gelma, the staining expression of the RAGE positive area in Gelma was also as high as 38%. The decrease in RAGE expression in the Gelma group may be related to its antioxidant properties. With the reduction of oxidative stress, its promoting effect on the generation of AGEs is weakened, resulting in a decrease in RAGE expression. The expression of the RAGE positive area in the DM+Gelma / 10Ce-MBG@L-Car composite hydrogel group decreased to less than 10%, indicating that the anti-AGEs effect of the material has a significant difference, which is consistent with the RAGE fluorescence staining in vitro cell experiments.

[0092] Conclusion

[0093] The present invention adopts the sol-gel method in combination with an organic template agent CPB, and later dopes cerium elements through an ion exchange technique, and loads L-carnosine by methods such as cation chelation and physical adsorption, and successfully prepares mesoporous bioactive glass nanoparticles (10Ce-MBG@L-Car) loaded with L-carnosine and doped with cerium. Through research such as SEM and antioxidant performance, it is determined that applying Ce-MBG doped with 10 molar ratios can maintain good morphology and the best antioxidant characteristics, and it is determined to apply Ce-MBG doped with 10 molar ratios for application research, and TEM, EDS, and XRD are used to prove that the cerium element doping of 10Ce-MBG is successful. After the L-carnosine drug is loaded, its mass accounts for about 19% of the weight of 10Ce-MBG@L-Car. Through a series of physicochemical property tests, we deeply explored the physicochemical characteristics of 10Ce-MBG and its complex with L-carnosine (10Ce-MBG@L-Car): 1) 10Ce-MBG has narrow mesoporous fissures, while the 10Ce-MBG@L-Car composite material has a lamellar structure, which meets the expectation after L-carnosine is loaded. 2) In the Fourier transform infrared spectroscopy (FTIR) analysis, L-carnosine is successfully combined with the surface of Ce-MBG through multi-site coordination to form a stable composite structure. 3) X-ray photoelectron spectroscopy (XPS) analysis further confirms that the cerium element exists in the form of a mixed valence state (Ce³⁺ / Ce 4+ ), mainly in the form of Ce³⁺. 4) The results of density functional theory (DFT) show that the oxygen atoms of the imidazole ring and carboxylate group of L-carnosine are nucleophilic active sites and are prone to electrostatic attraction with Ce³⁺ to form a coordination bond.

[0094] In summary, through a series of physicochemical property tests, the present invention proves the successful synthesis of 10Ce-MBG and its L-carnosine complex, and reveals its excellent pore structure, surface chemical characteristics, and effective loading performance. The potential of MBG in the biomedical field has been further verified, especially its application prospects in aspects such as drug loading and element doping.

[0095] 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 on clearing the etiology in organisms, and study its potential for bone defect repair in a diabetic environment.

[0096] The application concentration of nanoparticles within the biosafety range was obtained through the CCK8 cytotoxicity experiment. To ensure that this material would 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 the combinations 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 loaded L-carnosine concentration was 2.5 mg / mL, the composite material exhibited the most significant cell proliferation effect. This concentration combination demonstrated the optimal bioactivity in the CCK-8 experiment of BMSCs and had no significant toxic reaction, so it was selected as the experimental group material for further cell and animal experiments.

[0097] In this invention, the antioxidant stress and anti-glycation dual effects of the 10Ce-MBG@L-Car composite hydrogel on BMSCs cells in a high glucose (HG) environment were evaluated 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 the HG environment, leading to cell damage and dysfunction, while the 10Ce-MBG and 10Ce-MBG@L-Car composite hydrogels could significantly alleviate this effect. This effect may be related to Ce 3+ / Ce 4+is related to the activities of superoxide dismutase (SOD) and catalase (CAT) in the class. The redox ability of Ce can scavenge ROS, generating harmless oxygen and water. In addition, in addition to its antiglycation effect, L-carnosine also exhibits an antioxidant stress effect. The chelation of the two enhances the antioxidant capacity of cells, making 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 good bone immunomodulatory effects; in the HG group, the level of RAGE (receptor for advanced glycation end products) increased significantly, which indicates that under high glucose conditions, the accumulation of AGEs promoted the expression of RAGE, further exacerbating oxidative stress and inflammatory responses. 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 antiglycation effect of L-carnosine. L-carnosine inhibits the generation of AGEs by scavenging aldoses inside and outside cells, and chelation with cerium further enhances the antiglycation effect of the material. The results of in vivo animal experiments were consistent: the expression of RAGE in the 10Ce-MBG@L-Car composite hydrogel group was significantly lower than that of other groups, showing that the composite material has a more significant effect in the aspect of anti-AGEs.

[0098] Through in vitro cell experiments and animal experiments, the injury modeling concentration of BMSCs (bone marrow mesenchymal stem cells) under high glucose conditions and the effects of different materials on bone regeneration were explored. First, cell experiments confirmed that at a high glucose concentration of 100 mM, the osteogenic function of BMSCs was significantly inhibited, manifested as a decrease in alkaline phosphatase (ALP) activity and calcium nodule formation, and a lower concentration of high glucose (such as 25 mM) did not cause obvious damage to cells. Further experiments showed that under high glucose conditions, the treatment containing 10Ce-MBG (calcium- and phosphorus-doped glass material) and 10Ce-MBG@L-Car (L-carnosine) nanoparticles could significantly promote osteogenic differentiation and restore the bone formation 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 antiglycation characteristics, and can slow down the effects of oxidative stress and abnormal glucose metabolism on cells. In in vivo animal experiments, a bone defect model of diabetic rats was used to evaluate the osteogenic effect of the composite hydrogel. At 8 weeks after surgery, the Gelma / 10Ce-MBG@L-Car composite hydrogel group not only significantly promoted new bone formation, but also had a higher maturity of new bone and obvious bone mineralization effect. Through Micro-CT and histological analysis, the bone mineral density (BMD) and bone volume ratio (BV / TV) of the composite hydrogel group were significantly higher than those of other groups, showing the superior effect of the material in bone repair.

[0099] In summary, this study demonstrated through in vitro and in vivo experiments that the 10Ce-MBG@L-Car nanoparticles and their composite hydrogels with Gelma have significant bone repair effects and can play an important role in bone injuries caused by diabetes. By means of antioxidant, anti-glycation, and promotion of osteogenic function recovery, they provide new ideas and strategies for the treatment of diabetes-related bone defects.

Claims

1. A kind of mesoporous bioactive glass nanoparticles loaded with L-carnosine and doped with cerium, characterized in that, The cerium-doped mesoporous bioactive glass nanoparticles are prepared by using cerium nitrate hexahydrate (Ce(NO3)3·6H2O) as a cerium source doped into 58S bioactive glass (MBG) to obtain cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG). Then, the cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) are placed in an aqueous carbonate solution of L-carnosine to combine with L-carnosine, obtaining bioactive glass nanoparticles loaded with L-carnosine and doped with cerium (Ce-MBG@L-Car). The cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) combine with L-carnosine through two methods: chelation coordination bonds and physical adsorption. The element ratio of the 58S bioactive glass is 60SiO2: (36 - x)CaO: 4P2O5, where x is the molar percentage of doped cerium. The molar ratio of cerium doping in the cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBG) is 10%.

2. A preparation method of the load of L-carnosine combined with cerium-doped mesoporous bioactive glass nanoparticles according to claim 1, characterized in that, It includes the following steps: (1) Synthesis of cerium-doped mesoporous bioactive glass: Weigh cetylpyridinium bromide (CPB) and urea and add them 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 this solution in sequence and continuously stir the solution at room temperature. Introduce tetraethyl orthosilicate (TEOs), stir the reaction vigorously, and at the same time preheat and raise the temperature in a constant-temperature water bath. Place the flask in a water bath and continue stirring. Add triethyl phosphate (TEP) for reaction. The element ratio of the 58S bioactive glass is 60SiO2: (36 - x)CaO: 4P2O5, where x is the molar percentage of doped cationic metal cerium. Therefore, according to the molar ratio relationship, when doping x% molar ratio of cerium element, the corresponding molar ratio of calcium element is 36 - x%, and calculate and weigh the corresponding masses according to the relative molecular masses of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and cerium nitrate hexahydrate (Ce(NO3)3·6H2O). Dissolve Ca(NO3)2·4H2O and Ce(NO3)3·6H2O in water respectively. At the same time, drop 2 ml solutions of Ca(NO3)2·4H2O and Ce(NO3)3·6H2O into the above mixed solution at a speed of 100 μl / min respectively to make these two solutions fully mix and react with the above mixed solution. Continue stirring to make the mixed solution react fully. After the reaction, collect the suspension, centrifuge at 9500 - 10000 rpm for 15 min, collect the precipitate, wash the precipitate with acetone, 75% ethanol, and deionized water in sequence, and collect the precipitate after each washing. Freeze-dry the precipitate until it is freeze-dried into a loose structure. Place the collected precipitate in a tubular muffle furnace and calcine it in a nitrogen atmosphere to obtain Ce-MBG black powder; (2)Preparation of L-carnosine-loaded cerium-doped bioactive glass nanoparticles (Ce-MBG@L-Car): Weigh the freeze-dried powder of L-carnosine and dissolve it in a carbonate buffer solution with a pH of 8.3 to reach its isoelectric point. Put Ce-MBG with a cerium doping molar ratio of 10% into the above L-carnosine carbonate buffer solution and stir magnetically under constant temperature conditions. Then, centrifuge to collect the precipitate and freeze-dry the precipitate until it has a loose structure. The harvested Ce-MBG@L-Car is stored at -20°C.

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

4. The preparation method according to claim 2, characterized in that In step (1), the calcination step is to set the heating rate at 1°C / min, calcine the dried powder to 650°C for 10 h, and cool it to room temperature at 5°C / min to obtain the Ce-MBG black powder.

5. A composite hydrogel comprising the loaded L-carnosine and cerium-doped mesoporous bioactive glass nanoparticles as described in claim 1, characterized in that, The composite hydrogel is prepared by the following steps: Synthesis of Gelma / Ce-MBG@L-Car composite hydrogel: Take the freeze-dried Gelma hydrogel and put it into deionized water. Under the condition of constant temperature at 40 - 50°C, prepare a 10% Gelma solution, add the photoinitiator I2959 so that its mass ratio to the freeze-dried Gelma is 5%. Wrap the glass bottle containing the mixture with tin foil to achieve the effect of light shielding, and continue to stir hydrothermally at 50°C to make the photoinitiator and the Gelma solution mix evenly. After mixing evenly, filter it aseptically with a filter membrane, and then add the Ce-MBG@L-Car nanoparticles sterilized by ultraviolet light into the above solution so that the mass ratio of Ce-MBG@L-Car nanoparticles to the freeze-dried Gelma is 10%. After mixing the Gelma solution with the added photoinitiator and the nanoparticles evenly, a Gelma / Ce-MBG@L-Car composite hydrogel is formed.

6. Application of the composite hydrogel loaded with L-carnosine and doped with cerium in mesoporous bioactive glass nanoparticles as claimed in claim 5 in the preparation of a repair material for diabetic bone defects.

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