Copper hydrogen phosphate loaded hydrogel with pH responsiveness and multi-enzyme activity as well as preparation method and application of copper hydrogen phosphate loaded hydrogel

Abstract

The invention relates to a pH-responsive copper hydrogen phosphate-loaded hydrogel with multiple enzyme-like activities and a preparation method and application thereof. The material mainly shows catalase-like activity in a neutral microenvironment, and can remove H2O2 and relieve oxidative stress; in a weakly acidic microenvironment, the compound mainly shows peroxidase-like activity and catalyzes H2O2 to generate. OH bacteria with higher toxicity; meanwhile, the inherent osteogenesis promoting performance of copper synergistically enhances bone regeneration, the biocompatibility, the oxidation resistance, the osteogenesis promoting capacity under the oxidative stress condition and the antibacterial efficiency under the weak acid condition of the SA / CuHP hydrogel are evaluated through in-vitro experiments, and a type II diabetes rat periodontitis model is established; the in-vivo antibacterial and osteogenesis promoting effects and the biological safety performance of the compound are verified, and a new strategy is provided for treatment of periodontitis with diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a copper hydrogen phosphate-loaded hydrogel with pH responsiveness and various enzyme activities, and a preparation method and application thereof. Background Art

[0002] The alveolar bone is an important periodontal tissue. During the development of periodontitis, irreversible resorption and destruction of the alveolar bone can lead to tooth loosening and even tooth loss. The excessive production of reactive oxygen species (ROS) and the resulting oxidative stress are one of the important mechanisms by which diabetes exacerbates alveolar bone destruction. ROS is a general term for oxygen-containing free radicals related to oxygen metabolism in organisms and peroxides that are prone to form free radicals, including superoxide radicals (O 2 - ), hydroxyl radicals (·OH), nitric oxide radicals (·NO), singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), etc. After being attacked by pathogenic bacteria, periodontal inflammatory tissues up-regulate the production of ROS to eliminate microorganisms. However, excessive ROS cannot be cleared in time, resulting in an imbalance between oxidation and antioxidant effects in periodontal tissues, that is, oxidative stress. Hyperglycemia induces the production of ROS through the polyol pathway, hexosamine pathway, protein kinase C pathway, and advanced glycation end product pathway, etc., exacerbating oxidative stress. In periodontal tissues, excessive ROS leads to increased apoptosis of osteoblast-related cells and reduced osteogenic differentiation through pathways such as lipid peroxidation, protein denaturation, and DNA damage, interfering with the synthesis and mineralization processes of bone matrix, damaging the quality and structure of the alveolar bone, and accelerating the resorption of the alveolar bone.

[0003] Diabetes exacerbates the difficulty of antibacterial treatment for periodontitis. Under normal circumstances, the buffering effect of saliva maintains the oral pH in the neutral range, which is very important for maintaining oral health; under periodontitis conditions, the formation of bacterial biofilms and dental plaques leads to a weakly acidic local microenvironment in the periodontium (pH 4.5–6.5). The glucose content in the gingival crevicular fluid of diabetic patients increases significantly, providing a rich nutrient source for subgingival microorganisms, changing the composition and structure of the microbial community, shifting it in the direction favorable for the growth of pathogenic bacteria, and promoting the occurrence and development of periodontitis. This local microecological imbalance causes diabetes-associated periodontitis to be more likely to progress to extensive lesions and involve the furcation area, reducing the cleaning efficiency of traditional mechanical debridement instruments for this area. Therefore, in the treatment of diabetes-associated periodontitis, controlling plaque microorganisms remains a challenge.

[0004] Therefore, for diabetes-associated periodontitis, there is an urgent need to develop treatment strategies with antibacterial, antioxidant, and bone repair-promoting functions to slow down the destruction of the alveolar bone.

[0005] At present, in the research on the treatment of diabetic periodontitis with nanozymes, the enzyme activity and function are single, and most of them focus on the single aspect of catalytically generating ROS or scavenging ROS. Therefore, based on the dual regulation requirements of ROS in the microenvironment of diabetic periodontitis, that is, achieving antibacterial effects through ROS generation and scavenging excessive ROS to relieve oxidative stress damage, designing novel multifunctional biomaterials is beneficial to improving the treatment effect of diabetic periodontitis. Summary of the Invention

[0006] To solve the technical defect of single enzyme activity and function in the existing research on the treatment of diabetic periodontitis with nanozymes, the present invention provides a copper hydrogen phosphate-loaded hydrogel with pH-responsive multiple enzyme activities, its preparation method and application.

[0007] The technical solution adopted by the present invention is: a copper hydrogen phosphate-loaded hydrogel with pH-responsive multiple enzyme activities, wherein the copper hydrogen phosphate-loaded hydrogel is constructed by loading copper hydrogen phosphate (CuHP) onto an injectable sodium alginate hydrogel (SA), and the copper hydrogen phosphate is a copper hydrogen phosphate microflower composed of nanosheets, and the mass fraction of copper hydrogen phosphate in the copper hydrogen phosphate-loaded hydrogel is 0.5-1 wt%.

[0008] Preferably, the mass fraction of copper hydrogen phosphate in the copper hydrogen phosphate-loaded hydrogel is 1 wt%.

[0009] A preparation method of a copper hydrogen phosphate-loaded hydrogel with pH-responsive multiple enzyme activities, characterized by including the following steps: (1) Preparation of CuHP microflowers: Dissolve copper sulfate powder in 10 mL of double-distilled water, then add this solution to 500 mL of PBS. Use copper sulfate (CuSO 4 ) powder to dissolve in double-distilled water, then add this solution to phosphate buffer solution (PBS), and oscillate the reaction to ensure uniform mixing and complete reaction. After the reaction is completed, centrifuge to separate the supernatant. Finally, after washing and freeze-drying, obtain CuHP microflowers; (2) Preparation of SA / CuHP composite hydrogel: Add SA to double-distilled water, stir magnetically until completely dissolved, sterilize by high temperature and high pressure, and cool to obtain an SA solution; then weigh calcium chloride and deionized water, stir to prepare a 1% calcium chloride solution for standby; then use a double-pass tube and a syringe to mix CuHP and SA sol evenly; finally, use a syringe to drop the mixed solution into CaCl 2 solution to form an SA / CuHP hydrogel.

[0010] Preferably, the concentration of SA in step (2) is 1.5 wt%.

[0011] Preferably, the sterilization conditions for high-temperature and high-pressure sterilization in step (2) are sterilization at 121 °C for 15 minutes.

[0012] Preferably, the mass fraction of CuHP in step (2) is 0.5 - 1 wt%.

[0013] Application of a copper hydrogen phosphate-loaded hydrogel with pH responsiveness and various enzyme activities in the preparation of a bone repair material for diabetic periodontitis.

[0014] The pH-responsive conditions of the bone repair material for diabetic periodontitis are as follows: it exhibits peroxidase (POD)-like enzyme activity under weak acid conditions, catalyzing the generation of reactive oxygen species (ROS); it exhibits catalase (CAT)-like enzyme activity under neutral conditions, scavenging ROS.

[0015] The weak acid condition is pH = 5, and the neutral condition is pH = 7.4.

[0016] The POD-like enzyme reaction process of the SA / CuHP hydrogel is shown in reaction formulas (1) and (2), and the CAT-like enzyme reaction process is shown in reaction formulas (3) and (4). The mechanism by which SA / CuHP exhibits POD-like and CAT-like enzyme activities under different pH conditions may be as follows: (1) Variable valence states of copper ions (Cu + / Cu 2+ ): Cu in CuHP is the core of the catalytic active center. Under acidic conditions, Cu 2+ tends to remain stable and catalyzes the heterolytic cleavage of H 2 O 2 to generate ·OH through a Fenton-like reaction, i.e., reaction (1); under neutral and weakly alkaline conditions, the concentration of OH - increases, Cu + tends to remain stable, and at the same time, H 2 O 2 tends to dissociate into HO 2 ·, promoting the homolytic cleavage of H 2 O 2 to generate O 2 . (2) Changes in the oxidizing property of H 2 O 2 : Under acidic conditions, H 2 O 2 has stronger oxidizing property, and H 2 O 2 is more easily reduced by Cu + to generate ·OH; under alkaline conditions, H 2 O 2 has stronger reducing property, and H 2 O 2 is more easily oxidized by Cu 2+ to finally generate O 2 .

[0017]

[0018] The beneficial effects of the present invention are as follows: A copper hydrogen phosphate-loaded hydrogel with pH-responsive multiple enzyme activities, its preparation method and application. This material mainly exhibits catalase-like activity in a neutral microenvironment to scavenge H 2 O 2 and relieve oxidative stress; in a weakly acidic microenvironment, it mainly shows peroxidase-like activity to catalyze H 2 O 2 to generate more toxic ·OH for antibacterial; at the same time, the inherent osteogenic property of copper synergistically enhances bone regeneration. The present invention evaluates the biocompatibility, antioxidant performance, osteogenic ability under oxidative stress conditions and antibacterial efficacy under weak acid conditions of the SA / CuHP hydrogel through in vitro experiments, and establishes a periodontitis model in type 2 diabetic rats to verify its in vivo antibacterial and osteogenic effects and biosafety performance, providing a new strategy for the treatment of diabetic periodontitis. Brief Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the preparation process of the SA / CuHP hydrogel and its application in the treatment of diabetic periodontitis.

[0020] Figure 2 It is the characterization of CuHP; (A) SEM image of CuHP (B) EDS map of Cu, P, and O in CuHP (C) XRD characteristic peak map of CuHP.

[0021] Figure 3 It is the macroscopic view (A) and SEM images (B) of SA / CuHP hydrogels with different concentrations.

[0022] Figure 4 It is the gel-forming property (A), injectability (B) and in-situ gel-forming property (C) of the SA / CuHP hydrogel.

[0023] Figure 5 It is the POD-like enzyme activity of the SA / CuHP hydrogel; (A) SA / CuHP hydrogels with different concentrations (B) different pH values.

[0024] Figure 6 It is the CAT-like enzyme activity of the SA / CuHP hydrogel under different pH conditions; (A) Generation of O2 bubbles (B) Scavenging rate of H 2 O 2

[0025] Figure 7 It is rat jaw BMSCs.

[0026] Figure 8 ​BMSCs were co-cultured with different concentrations of SA / CuHP for 1, 3, and 5 days, and cell proliferation was detected by CCK8 assay.

[0027] Figure 9 For BMSCs stimulated with 300 μM or 500 μM H 2 O 2 for 3 hours and then cultured for another 24 hours, cell viability was detected by CCK8 assay.

[0028] Figure 10 For (A) DCFH-DA fluorescence images of BMSCs after being stimulated with different treatments for 3 hours and then cultured for another 24 hours. (B) Semi-quantitative analysis of intracellular ROS levels.

[0029] Figure 11 For BMSCs stimulated with different treatments for 3 hours and then cultured for another 24 hours, cell viability was detected by CCK8 assay.

[0030] Figure 12 For (A) live / dead cell staining images of BMSCs after being stimulated with different treatments for 3 hours and then cultured for another 24 hours. (B) Semi-quantitative analysis of the viable cell rate.

[0031] Figure 13 For (A) scratch images of BMSCs at 0 hour and 24 hours after being stimulated with different treatments for 3 hours and then cultured for another 24 hours. (B) Semi-quantitative analysis of cell migration rate.

[0032] Figure 14 For (A) ALP staining images of BMSCs after being stimulated with different treatments for 3 hours and then osteogenic-induced cultured for 7 days. (B) Semi-quantitative analysis of ALP activity.

[0033] Figure 15 For (A) alizarin red staining images of BMSCs after being stimulated with different treatments for 3 hours and then osteogenic-induced cultured for 21 days. (B) Semi-quantitative analysis of calcium nodule formation.

[0034] Figure 16 For (A) RUNX2 immunofluorescence staining images of BMSCs after being stimulated with different treatments for 3 hours and then osteogenic-induced cultured for 4 days. (B) Quantitative analysis of RUNX2 fluorescence intensity.

[0035] Figure 17 For the expression levels of osteogenesis-related mRNAs of BMSCs after being stimulated with different treatments for 3 hours and then osteogenic-induced cultured for 4 days.

[0036] Figure 18 For the expression levels of osteogenesis-related proteins of BMSCs after being stimulated with different treatments for 3 hours and then osteogenic-induced cultured for 4 days.

[0037] Figure 19is (A) A.a Dot plate colony formation images after 24-hour culture with different treatments (B) Colony counting.

[0038] Figure 20 is A.a Live and dead bacteria staining images after 24-hour culture with different treatments.

[0039] Figure 21 is A.a SEM images after 24-hour culture with different treatments.

[0040] Figure 22 are the body weights (A) and blood glucose levels (B) of SD rats on days 0, 3, and 7 after STZ injection.

[0041] Figure 23 is the modeling process of periodontitis in the second maxillary left molar of SD rats; (A) Before silk ligation (B) Silk ligation (C) Removal of ligation wire.

[0042] Figure 24 are (A) Colony images of gingival crevicular fluid smear plates (B) Colony counting.

[0043] Figure 25 are (A) Micro-CT three-dimensional reconstruction, sagittal section, and x-ray images of the rat maxilla (B) Distance between the alveolar bone crest and the cementoenamel junction (ABC-CEJ) of the second maxillary molar of the rat Figure 26 is the bone volume fraction (BV / TV) and trabecular separation (Tb.Sp) of the alveolar bone between the first and second maxillary molars of the rat maxilla.

[0044] Figure 27 is the HE staining of the periodontal tissue of D rats.

[0045] Figure 28 is the Masson staining of the periodontal tissue of the rat.

[0046] Figure 29 is the HE staining of the heart, liver, spleen, lungs, and kidneys of the rat. Specific embodiments

[0047] 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0048] Example 1 Preparation and characterization of CuHP-loaded hydrogel Preparation of CuHP and CuHP-loaded hydrogel CuHP was provided by the Shanghai Institute of Ceramics, Chinese Academy of Sciences, and the preparation process is as follows. First, 0.768 g of copper sulfate powder was dissolved in 10 mL of double-distilled water, and then this solution was added to 500 mL of PBS (0.02 M) solution. Then, the reaction was shaken for 6 hours to ensure uniform mixing and complete reaction. After the reaction, the supernatant was separated by centrifugation (10,000 rpm / min, 5 minutes). Finally, after three washes and freeze-drying, CuHP was obtained.

[0049] When preparing the SA / CuHP composite hydrogel, first, SA was added to double-distilled water at 1.5% (wt%), and magnetically stirred until completely dissolved, sterilized at high temperature and high pressure (121 °C, 15 minutes), and cooled to obtain the SA solution; then, 1 g of calcium chloride and 100 mL of deionized water were weighed and stirred to prepare a 1% calcium chloride solution for standby; then, using a double-tube and a syringe, CuHP (0.25%, 0.5%, 1%, and 2% wt%) and the SA sol were mixed evenly; finally, the mixed solution was dropped into the CaCl 2 solution to form the SA / CuHP hydrogel, which was named SA / CuHP (0.25%), SA / CuHP (0.5%), SA / CuHP (1%), and SA / CuHP (2%), respectively. The SA solution was directly dropped into the CaCl 2 solution using a 1 mL syringe to form the SA hydrogel. Since the minimum dispensing accuracy of a 1 mL syringe is 10 μL, and a 10 μL volume can basically fill the periodontal pocket of a rat when applying the hydrogel in vivo, 10 μL hydrogel balls were prepared for in vitro cell and bacteria experiments.

[0050] Characterization of CuHP-loaded hydrogels The microtopography of SA / CuHP hydrogels with different concentrations (0.25%, 0.5%, 1%, and 2% wt%) was observed using SEM. The injectability of the hydrogel was evaluated by gross observation.

[0051] POD-like enzyme activity: SA, SA / CuHP (0.25%), SA / CuHP (0.5%), SA / CuHP (1%), and SA / CuHP (2%) hydrogels were added to 0.2 mL of 0.015% methylene blue (MB) solution. After the resulting mixture was stirred for 20 minutes, 0.8 mL of 3% H 2 O 2The reaction was carried out at room temperature for 10 minutes, and then the absorbance change at 664 nm was measured by UV-vis-NIR spectrophotometry. To further evaluate the POD enzyme activity of SA / CuHP at different pH values ​​(5, 6, and 7.4), SA / CuHP (1%) hydrogel was added to 0.2 mL of 0.015% MB solution (pH = 5, 6, and 7.4), respectively, and reacted at room temperature for 10 minutes, and then the absorbance change at 664 nm was measured by UV-vis-NIR spectrophotometry.

[0052] CAT enzyme activity: SA and SA / CuHP hydrogels were immersed in 10% H2O at pH 5, 6, and 7.4, respectively. 2 O 2 Solution, incubate at 37℃ for 5 minutes and then observe and record O 2 In addition, the CAT detection kit was used to evaluate the CAT-like enzyme activity of SA / CuHP hydrogels under different pH conditions (5, 6, and 7.4). According to the detection principle of the kit, CAT decomposes H 2 O 2 The reaction can be quickly terminated by adding ammonium molybdate, and the remaining H 2 O 2 It reacts with ammonium molybdate to produce a light yellow complex. The absorbance change at 405 nm is detected using an enzyme reader, and the activity of CAT can be calculated.

[0053] Experimental Results Basic characteristics of CuHP like Figure 2 As shown in A, the synthesized particles exhibit a micro-flower-like structure, which is composed of numerous petal-like nanosheets. Figure 2 As shown in B, copper, phosphorus and oxygen elements are evenly distributed ( Figure 2 B). Figure 2 C shows that the X-ray diffraction peak of the sample is consistent with Cu 4 H(PO 4 ) 3 ·3H 2 O (PDF No. 31-0458). These results prove that the CuHP microflowers were successfully synthesized in this study.

[0054] Basic characteristics, injectability and enzyme-like activity of CuHP-loaded hydrogels like Figure 3 As shown in A, pure SA hydrogel is colorless, and the blue color of SA / CuHP hydrogel gradually deepens with the increase of CuHP incorporation. Figure 3 As shown in B, the SA hydrogel has a porous structure, and CuHP with different concentrations is evenly distributed on the surface of the SA hydrogel without destroying its porous framework structure.

[0055] As shown Figure 4 in Figure A, when the SA / CuHP hydrogel comes into contact with the CaCl 2 solution, the hydrogel transforms from a sol state to a gel state. As shown Figure 4 in Figure B, the SA / CuHP hydrogel exhibits excellent injectability and can form different shapes. As shown Figure 4 in Figure C, the SA / CuHP hydrogel can form a gel in situ after being injected into the gingival sulcus of the second molar in rats.

[0056] Next, the POD-like enzyme activity of the SA / CuHP hydrogel was investigated. As shown Figure 5 in Figure A, as the concentration of the SA / CuHP hydrogel increases, the absorbance at 664 nm gradually decreases, and the blue solution gradually fades, indicating that the blue reduced MB is oxidized to the colorless oxidized state. This shows that the POD-like enzyme activity of the SA / CuHP hydrogel is positively correlated with its concentration, that is, the higher the concentration, the stronger the ability to catalyze the decomposition of H 2 O 2 to generate ·OH, and thus more effectively oxidize MB. Subsequently, the POD-like enzyme activity of the SA / CuHP (1%) hydrogel was evaluated under different pH conditions (pH = 5, 6, 7.4). As shown Figure 5 in Figure B, the absorbance at 664 nm decreases with the decrease of pH, indicating that the POD-like enzyme activity of the SA / CuHP hydrogel is higher under acidic conditions.

[0057] Furthermore, the effect of different pH on the CAT-like enzyme activity of the SA / CuHP hydrogel was investigated. As shown Figure 6 in Figure A, no obvious O 2 O 2 bubbles were observed in the H 2 solution with the added SA hydrogel; while after the SA / CuHP hydrogel was incubated for 5 minutes, H 2 O 2 decomposed: only a trace amount of O 2 bubbles were visible around the hydrogel at pH 5, the number of O 2 bubbles in the solution increased significantly at pH 6, and dense O 2 bubbles appeared in the solution when pH rose to 7.4. As shown Figure 6 in Figure B, the H 2 O 2 scavenging rate showed an increasing trend with the increase of pH (pH 7.4>6>5), and the scavenging efficiency under neutral conditions was higher than that under acidic conditions. The above results indicate that the SA / CuHP hydrogel has pH-sensitive CAT-like enzyme activity, and its catalytic decomposition of H 2 O 2 into H 2 O and O 2Its efficacy is stronger in a neutral environment than in an acidic environment.

[0058] Combined with the characteristic that the periodontal microenvironment is weakly acidic due to bacterial infection in diabetes-associated periodontitis, the present invention designs a material with various types of enzyme activities under different pH conditions, SA / CuHP hydrogel. The micron flower morphology of CuHP provides a large specific surface area, which is conducive to the interaction with surrounding substances and enhances the catalytic reaction. SA / CuHP hydrogel exhibits pH-sensitive various types of enzyme activities. Under weak acid conditions, it mainly exhibits peroxidase (POD)-like enzyme activity and catalyzes H 2 O 2 to produce more toxic ·OH. Under neutral conditions, it mainly exhibits catalase (CAT)-like enzyme activity and converts H 2 O 2 into non-toxic O 2 and H 2 O. Compared with single-functional nanozymes, this multifunctional enzyme-like system better meets the treatment needs of the complex pathological process of diabetes-associated periodontitis.

[0059] Example 2: Evaluation of in vitro biocompatibility and antioxidant properties of CuHP-loaded hydrogel Morphology of rat jaw bone BMSCs Rat jaw bone BMSCs were isolated and cultured by the tissue explant adherence method. It could be observed under an optical microscope that BMSCs (P2) adhered and grew, showing a swirling arrangement. The cell morphology was spindle-shaped, with a slender cell body and an oval nucleus in the center, and the chromatin was evenly distributed ( Figure 7 ). This spindle-shaped morphology is conducive to the cells connecting with each other in tissues and provides a structural basis for them to perform various biological functions such as proliferation, migration, and osteogenic differentiation.

[0060] In vitro biocompatibility analysis As Figure 8 shown, low concentrations of SA / CuHP hydrogel (0.25%, 0.5%, 1% wt%) promoted cell proliferation, while high concentrations of SA / CuHP hydrogel (2% wt%) showed cytotoxicity and inhibited cell proliferation. Among the groups, the SA / CuHP (1%) group had the highest absorbance at a wavelength of 450 nm, indicating that the proliferation of BMSCs was more effectively promoted in this group. Therefore, SA / CuHP hydrogel with a concentration of 1% was selected for subsequent experiments and abbreviated as SA / CuHP hydrogel.

[0061] Construction of in vitro oxidative stress model As Figure 9 shown, compared with the Control group, after stimulating cells with 300 µM H 2 O 2 the cell viability increased, while after stimulating cells with 500 µM H2 O 2 After stimulating the cells for 3 hours, the cell viability decreased significantly (p<0.05). Therefore, in this study, 500 µM H 2 O 2 was used to stimulate the cells for 3 hours to establish an oxidative stress model of BMSCs and simulate the pathological microenvironment of diabetic periodontitis.

[0062] Analysis of in vitro antioxidant performance Analysis of intracellular ROS level The intracellular ROS levels of each group of cells were detected using the DCFH-DA fluorescent probe. As Figure 10 shown in A and B, compared with the Control group, the H 2 O 2 group and the H 2 O 2 +SA group showed significantly enhanced green fluorescence signals (p<0.05), indicating that the intracellular ROS levels were significantly higher than those of the control group. It is worth noting that after the intervention with SA / CuHP hydrogel, the fluorescence intensity of the cells was significantly lower than that of the H 2 O 2 group (p<0.05), indicating that SA / CuHP hydrogel could effectively scavenge excessive ROS.

[0063] Analysis of cell viability The cell viability after different treatments was detected by the CCK8 assay. As Figure 11 shown, the stimulation with H 2 O 2 led to a decrease in cell viability (p<0.05); compared with the H 2 O 2 group and the H 2 O 2 +SA group, the cell viability of the H 2 O 2 +SA / CuHP group was significantly enhanced (p<0.05). This result indicates that SA / CuHP hydrogel can reduce the cytotoxicity caused by oxidative stress.

[0064] Analysis of cell survival rate The effects of different treatments on cell viability were further verified by live / dead cell staining. As Figure 12 shown, a large number of live cells stained green fluorescence were detected in the Control group; in the H 2 O 2 group and the H 2 O 2A decrease in green fluorescence was observed in the H₂O₂+SA group, while an increase in red fluorescence signal was observed in dead cells (p<0.05). After intervention with the SA / CuHP hydrogel, this cell death phenomenon was effectively inhibited (p<0.05). The results of semi-quantitative analysis were consistent with the trend of the live / dead cell staining results ( Figure 12 B). These results indicate that the SA / CuHP hydrogel can reduce the damage to cell viability caused by H 2 O 2 .

[0065] Analysis of cell migration ability In addition, a cell scratch assay was used to evaluate cell migration ability. As shown in Figure 13 A and B, at 24 h, the cell migration rate in the control group almost reached 100%, while the cell migration rates in the H 2 O 2 ₂ group and the H 2 O 2 ₂+SA group decreased (p<0.05). After intervention with SA / CuHP, the cell migration rate increased significantly (p<0.05). These results demonstrate that under oxidative stress conditions, the SA / CuHP hydrogel can promote cell migration.

[0066] The SA / CuHP hydrogel prepared in this invention has excellent antioxidant properties. Under oxidative stress conditions, the SA / CuHP hydrogel can reduce the production of intracellular reactive oxygen species and alleviate the inhibitory effects of oxidative stress on cell proliferation, cell viability, and cell migration.

[0067] Example 3: Evaluation of in vitro osteogenic ability of CuHP hydrogel ALP activity analysis ALP staining was used to analyze the ALP activity of cells after different treatments. As shown in Figure 14 A, dense dark blue-purple ALP-positive regions were observed in the cytoplasm of cells in the Control group, indicating high early osteogenic differentiation activity. In the other three H 2 O 2 ₂-induced groups, the stained area decreased and the color intensity weakened, suggesting that H 2 O 2 ₂ can inhibit the ALP activity of BMSCs. Compared with the H 2 O 2 ₂ group and the H 2 O 2 ₂+SA group, the stained area in the H 2 O 2 ₂+SA / CuHP group increased and the color depth recovered, demonstrating that SA / CuHP can effectively antagonize the inhibition of ALP activity induced by H 2 O 2 ₂. The quantitative results of ALP are shown in Figure 4. As shown in Figure 1B, the ALP activity of the H 2 O 2 group was significantly weakened (P<0.05). Compared with the H 2 O 2 group, there was no significant change in the ALP activity of the H 2 O 2 +SA group (P>0.05), and the ALP activity was significantly enhanced after the intervention of SA / CuHP hydrogel (P<0.05). The above results confirmed from the enzyme activity level that SA / CuHP could improve the inhibition of the early osteogenic differentiation function of BMSCs by H 2 O 2 .

[0068] Analysis of calcium nodule formation The formation of calcium nodules after different treatments was detected by ARS staining. As Figure 15 shown in Figure A, the Control group formed dense orange-red calcium nodules, showing typical characteristics of mineralized matrix deposition, indicating normal late osteogenic differentiation function of BMSCs. In the remaining three H 2 O 2 induction groups, the number of calcium nodules was significantly reduced and the distribution was sparse, suggesting that the oxidative stress environment severely damaged the mineralization ability of BMSCs. Compared with the H 2 O 2 +SA / CuHP treatment group, the calcium nodule deposition area increased in the H 2 O 2 group and the H 2 O 2 +SA group, indicating that SA / CuHP could effectively restore the late osteogenic differentiation function of BMSCs under oxidative stress conditions. The quantitative results of ARS are shown in Figure 15 Figure B. The relative calcium nodules in the H 2 O 2 group were significantly reduced (P<0.05); there was no statistical difference between the H 2 O 2 +SA group and the H 2 O 2 group (P>0.05); compared with the H 2 O 2 group, the relative calcium nodules in the H 2 O 2 +SA / CuHP group were significantly increased (P<0.05). The above results showed that SA / CuHP could significantly improve the late mineralization disorder induced by H 2 O 2 .

[0069] Analysis of RUNX2 expression The expression of cell RUNX2 was analyzed by immunofluorescence staining. As Figure 16As shown in A and B, strong green fluorescence signals were presented in the nuclei of the Control group, indicating that RUNX2 protein was effectively activated in the early stage of osteogenic differentiation. H 2 O 2 Stimulation led to a significant decrease in the nuclear fluorescence intensity of RUNX2 compared with the Control group, H 2 O 2 The RUNX2 fluorescence in the H 2 O 2 +SA group was enhanced to a certain extent, but there was no significant difference compared with the H 2 O 2 group (P>0.05). However, the green fluorescence was significantly stronger in the H 2 O 2 group after intervention with H

[0070] Analysis of the expression of osteogenesis-related genes (ALP, OCN, OSX) The expression levels of osteogenesis-related genes ALP, OCN, and OSX were detected by qPT-PCR. As Figure 17 shown, under the oxidative stress conditions induced by H 2 O 2 the expression of osteogenesis-related genes ALP, OCN, and OSX was significantly decreased (P<0.05); compared with the H 2 O 2 group, there was no significant change in the expression of ALP, OCN, and OSX in the H 2 O 2 +SA group (P>0.05), while the expression of ALP, OCN, and OSX in the H 2 O 2 +SA / CuHP group was significantly increased (P<0.05).

[0071] Analysis of the expression of osteogenesis-related proteins (RUNX2, OSX) The expression levels of osteogenesis-related proteins (RUNX2, OSX) were detected by WB. As Figure 18 shown, compared with the Control group, the expression of osteogenesis-related proteins RUNX2 and OSX in the H 2 O 2 group was significantly down-regulated (P<0.05); compared with the H 2 O 2 group, there was no obvious change in the expression of RUNX2 and OSX in the H 2 O 2 +SA group (P>0.05), while the expression of RUNX2 and OSX in the H 2 O 2 +SA / CuHP group was significantly up-regulated (P<0.05).

[0072] Conclusion An important clinical manifestation of diabetic periodontitis is alveolar bone resorption, and its core mechanism involves impaired osteogenic differentiation ability of BMSCs in the oxidative stress microenvironment. This study focused on this key pathological link of oxidative stress and systematically evaluated the protective effect of SA / CuHP hydrogel on the osteogenic function of BMSCs. In the osteogenic differentiation regulatory network, RUNX2 is the master transcription factor of osteogenic differentiation. By directly binding to the promoter regions of bone-specific protein-coding genes such as ALP and OCN, it precisely regulates their transcriptional activities, thereby leading the initiation and promotion of the osteogenic differentiation program. ALP is a core marker of early osteogenic differentiation. It can catalyze the release of inorganic phosphate, promote the deposition of hydroxyapatite crystals, and initiate bone matrix mineralization. OSX is a key effector factor downstream of RUNX2, mediating the terminal differentiation of osteoprogenitor cells into functional osteoblasts and being a marker of mid-stage osteogenic differentiation. OCN is a specific marker of late osteogenic differentiation, mainly secreted by mature osteoblasts. It specifically binds to Ca²⁺ through γ-carboxylated glutamate residues and mediates the directional arrangement of hydroxyapatite crystals in the bone matrix. Therefore, this invention explored and evaluated the effects of SA / CuHP on the expressions of ALP, RUN2, OSX, and OCN under oxidative stress conditions through qRT-PCR, immunofluorescence staining, and WB experiments. The results consistently showed that oxidative stress led to a downregulation of the protein expressions of osteogenesis-related genes in BMSCs, while the SA / CuHP hydrogel could promote the expressions of osteogenesis-related genes and proteins, reversing the osteogenic inhibitory effect of oxidative stress on BMSCs. In addition, the ALP staining results showed an increase in the ALP activity of cells after treatment with the SA / CuHP hydrogel, and the ARS staining results indicated an increase in the formation of calcified nodules after treatment with the SA / CuHP hydrogel, further demonstrating the osteogenic promoting performance of the SA / CuHP hydrogel under oxidative stress conditions.

[0073] The osteogenic protective effect of the SA / CuHP hydrogel may stem from the effective synergy of its CAT-like enzyme activity and the inherent osteogenic promoting property of copper. In the oxidative stress microenvironment, the CAT-like enzyme activity of the material significantly improved the osteogenic differentiation microenvironment of BMSCs by targeting the scavenging of H 2 O 2 This key oxidative mediator. Research has shown that the excessive accumulation of H 2 O 2 can directly inhibit the mineralization ability of osteoprogenitor cells and induce apoptosis, while local H 2 O 2Effective clearance creates the necessary redox homeostasis for osteogenic differentiation. Meanwhile, the inherent biological properties of copper play unique advantages in bone repair. Copper has been proven to promote the secretion of bone matrix proteins and calcium salt deposition by activating the expression profile of osteogenesis-related genes, and its osteogenic effect has been widely verified in the field of bone defect repair. In this study, SA / CuHP reduced the oxidative damage threshold through mimetic CAT enzyme activity in a neutral microenvironment, while copper directly enhanced the osteogenic activity of BMSCs. The synergistic effect of the two increased the protection of osteogenesis under oxidative stress conditions.

[0074] In an oxidative stress environment, the SA / CuHP hydrogel can significantly relieve the inhibitory effect of oxidative stress on osteogenesis, improve cell ALP activity, enhance osteogenic mineralization, and promote the expression of osteogenesis-related genes and proteins.

[0075] Example 4: Evaluation of the in vitro antibacterial properties of CuHP-loaded hydrogels Bacterial proliferation analysis The bacterial proliferation after different treatments was explored by bacterial spotting and colony counting. As Figure 19 shown in A and B, there was no significant difference in the number of colonies between the SA group and the control group (p>0.05), indicating that SA lacks antibacterial activity. The groups with the addition of H 2 O 2 or SA / CuHP alone and the H 2 O 2 +SA group only showed a weak antibacterial effect (p<0.05), suggesting that only H 2 O 2 and SA / CuHP were difficult to effectively inhibit A.a proliferation. The number of colonies in the H 2 O 2 +SA / CuHP group was significantly lower than that in the H 2 O 2 or SA / CuHP single treatment group (p<0.05). The above results indicate that H 2 O 2 +SA / CuHP showed a significant effect of inhibiting bacterial proliferation.

[0076] Bacterial viability analysis The bacterial viability after different treatments was analyzed by bacterial live / dead staining. As Figure 20 shown in A, almost all of the control group were green fluorescence (live bacteria); in the SA group, green fluorescence was dominant, and only sporadic red fluorescence (dead bacteria) was visible, indicating that SA itself did not show obvious antibacterial function; the H 2 O 2 group and the H 2 O 2In the +SA group, the red fluorescence signal increased slightly compared with the control group, but the green fluorescence was still dominant overall, indicating that low-concentration H 2 O 2 had limited bactericidal effect on bacteria; in the SA / CuHP group, the proportion of red fluorescence increased significantly; H 2 O 2 In the +SA / CuHP group, the intensity and coverage of the red fluorescence signal far exceeded those of other groups. The above results indicate that SA / CuHP has enhanced bactericidal effect in the weak acid environment where H 2 O 2 exists.

[0077] Observation of bacterial morphology The effects of different treatments on cell morphology were observed by SEM. As Figure 21 shown: The surfaces of bacteria in the control group and the SA group were smooth and intact, presenting short rod shapes; H 2 O 2 group and H 2 O 2 In the +SA group, slight shrinkage and deformation appeared on the bacterial surface, indicating partial damage to the cell membrane integrity; SA / CuHP treatment led to irregular bacterial morphology and damaged membrane structure, while H 2 O 2 In the +SA / CuHP group, the bacteria showed more severe shrinkage. The above results indicate that H 2 O 2 and SA / CuHP synergistically aggravated the damage of bacterial cell membrane structure and enhanced the antibacterial effect.

[0078] The experimental results show that the SA hydrogel did not show antibacterial activity; alone, H 2 O 2 and the SA / CuHP hydrogel also showed weak antibacterial effects; while in the H 2 O 2 +SA / CuHP group showed significant antibacterial effect, significantly stronger than that of the H 2 O 2 group or the SA / CuHP group. The antibacterial mechanism of the SA / CuHP hydrogel may be mainly attributed to its POD-like enzyme activity. It was proved that the POD-like enzyme activity was dominant in the SA / CuHP hydrogel in the weak acid environment, catalyzing H 2 O 2Generate more oxidizing ·OH. Previous studies have shown that the antibacterial mechanism of •OH mainly includes: (1) ·OH oxidizes the membrane lipids of the cell membrane, destroys the membrane integrity, resulting in the leakage of cell contents and osmotic imbalance. (2) Damages proteins and enzymes, leading to enzyme inactivation, structural denaturation or loss of function. (3) Damages DNA, hinders replication and transcription, and ultimately triggers bacterial apoptosis. In addition, the copper ions released by SA / CuHP play a synergistic antibacterial role. Previous studies have shown that the antibacterial mechanism of copper ions mainly includes: (1) Positively charged Cu²⁺ binds to the negatively charged bacterial cell membrane, destroys the membrane potential and increases permeability, resulting in ion imbalance and cytoplasmic leakage. (2) Competitively binds to the metal ions at the active center of the enzyme, leading to the inactivation of key enzymes such as respiratory chain enzymes. (3) Binds to the components of the electron transport chain on the mitochondria or cell membrane, hinders ATP synthesis, and inhibits energy metabolism. Therefore, the SA / CuHP hydrogel jointly destroys the membrane structure, key biomolecules (proteins, DNA) and metabolic pathways through the synergistic action of POD-like enzyme activity and Cu ions, enhancing the antibacterial effect.

[0079] Example 5: In vivo therapeutic effect and safety performance evaluation of CuHP-loaded hydrogel Establishment of type 2 diabetic rat model As Figure 22 Shown in A and B, the body weight of the rats in the Control group increased steadily, and the random blood glucose was lower than 16.7 mmol / L; after 4 weeks of high-fat diet feeding and injection of STZ in the experimental group rats (PBS group, SA group and SA / CuHP group) to induce diabetes, the body weight decreased, and at the same time, the random blood glucose value increased significantly and remained continuously above the diabetes diagnosis threshold of 16.7 mmol / L. The experimental group rats showed typical diabetes symptoms of "polyphagia, polydipsia, polyuria and weight loss": the food intake and water intake were significantly higher than those of the healthy control group; the phenomenon of polyuria led to an increase in the humidity and odor of the bedding, and it needed to be changed daily; the body weight continued to decrease. The observation of body posture and behavior further showed that the experimental group rats had manifestations such as withered and dull hair, reduced frequency of spontaneous activities and slow reaction. The changes in blood glucose, body weight and physiological state of the rats preliminarily confirmed the successful establishment of a type 2 diabetic rat model.

[0080] Establishment of periodontitis model associated with diabetes On the basis of the type 2 diabetic rat model, a periodontitis model was established by silk ligation. As Figure 23 Shown, the healthy periodontal tissue before silk ligation showed that the gingiva was pink, firm and tough, the gingival margin was closely attached to the tooth surface, and the probe was not easy to bleed; three weeks after silk ligation, after removing the ligature wire, a large amount of food residue accumulated in the interdental space of the second molar, the gingiva was red and swollen and retracted, the gingival margin was round and blunt and could not fit the tooth surface, and the probe was easy to bleed, which preliminarily proved the successful establishment of the periodontitis model.

[0081] Results of colony counting of gingival crevicular fluid smear The plate count method was used to quantitatively analyze the number of bacteria in the periodontal pockets of rats. As Figure 24 shown, the number of colonies in the periodontal pockets of the Control group was relatively small, while significant bacterial proliferation was observed in the PBS group (P<0.05), with colonies being dense and widely distributed on the plate, indicating severe bacterial infection in the diabetic periodontitis model. The number of colonies in the SA / CuHP group was significantly reduced compared to the PBS group (P<0.05), demonstrating excellent antibacterial efficacy. The pure SA hydrogel group also showed certain antibacterial effects (P<0.05).

[0082] Micro-CT analysis As Figure 25 shown in A and B, the Micro-CT three-dimensional reconstruction, sagittal cross-section, and x-ray images visually displayed the alveolar bone conditions of each group. In the Control group, the anatomic position relationship between ABC and CEJ was close, and the bone contour was intact. In the PBS group, the alveolar bone showed absorption and destruction characteristics: the distance between CEJ-ABC increased significantly (P<0.05), and the bone loss in the furcation area was exposed, further confirming the successful establishment of the diabetic periodontitis model. Although there was a decreasing trend in the CEJ-ABC distance in the SA intervention group, there was no significant difference compared to the PBS group (P>0.05). The SA / CuHP group showed a significant effect in improving alveolar bone destruction, with the CEJ-ABC distance being significantly shorter than that in the PBS group (P<0.05).

[0083] The alveolar bone between the first molar and the second molar was selected for analysis of bone microstructural parameters. As Figure 26 shown in A and B, the BV / TV in the PBS group was significantly lower than that in the Control group, and the trabecular separation (Tb.Sp) was significantly increased (P<0.05). Although the BV / TV and Tb.Sp parameters in the SA group were slightly improved compared to the PBS group, the difference was not statistically significant (P>0.05). The SA / CuHP group showed significant bone microstructural regulation ability: compared with the PBS group, BV / TV was significantly increased, and Tb.Sp was significantly decreased (p<0.05). The above results indicate that the SA / CuHP hydrogel effectively alleviated the alveolar bone destruction caused by diabetic periodontitis.

[0084] HE staining analysis As Figure 27As shown, the gingival papillae between the molars in the Control group were morphologically intact, the gingival epithelium was continuous without defects, the junctional epithelium attachment was located at the cementoenamel junction and was tightly attached to the root surface, and the alveolar crest height was consistent with the physiological bone contour. Different degrees of attachment loss were presented in the silk ligation intervention areas, manifested as the apical migration of the junctional epithelium. In the PBS group and the SA group, the junctional epithelium was loosely attached to the root surface, and the alveolar bone crest height was significantly reduced. The periodontal pathological features in the SA / CuHP group were significantly improved: the connections between epithelial cells were closer and stably attached to the cementum surface, and the alveolar bone height was relatively maintained intact.

[0085] Masson staining analysis As Figure 28 shown, the periodontal ligament fiber bundles in the Control group were regularly arranged. In the PBS group and the SA group, the collagen fiber bundles were disorderly arranged, showing fracture and dissolution. Although there was local disorder in the arrangement of the periodontal ligament fibers in the SA / CuHP group, the overall continuity was better than that in the PBS group and the SA group.

[0086] In vivo safety analysis As Figure 29 shown, after 4 weeks of treatment, through the histopathological analysis of the main organs of rats, it was found that there were no significant inflammatory infiltrations, necrosis or abnormal changes in the tissue structure in the heart, liver, spleen, lungs and kidneys of rats in each group, and there were no obvious pathological differences compared with the control group. This result indicates that the SA / CuHP hydrogel shows good biocompatibility in in vivo experiments, suggesting its potential clinical application safety.

[0087] Summary (1) By adopting the strategy of inducing with high-fat diet feeding combined with STZ intervention, a type 2 diabetic rat model was constructed, and by observing the rat status, body weight and blood glucose changes, it was confirmed that the type 2 diabetic rat model was successfully established.

[0088] (2) On the basis of type 2 diabetic rats, regional periodontitis of the left maxillary second molar was induced by the silk ligation method, effectively simulating the pathological process of clinical periodontitis with diabetes. The model successfully reproduced typical pathological features such as periodontal clinical attachment loss, bleeding on probing and alveolar bone resorption.

[0089] (3) In the treatment of the rat model of periodontitis with diabetes, the SA / CuHP hydrogel showed excellent antibacterial properties, significantly reduced the destruction and resorption of alveolar bone, and had in vivo safety.

[0090] All technicians should note that although the present invention has been illustrated by the above specific embodiments, the inventive concept of the present invention is not limited thereto. Any improvement or variation based on the inventive concept of the present invention falls within the protection scope of the patent right of the present invention.

[0091] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments and experimental examples. Any technical solution that follows the concept of the present invention shall be included in the protection scope of the present invention. It should be emphasized that for those of ordinary skill in the art, any modification or equivalent replacement shall be regarded as part of the protection scope of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A copper hydrogen phosphate-loaded hydrogel having pH-responsive multi-type enzyme activity, characterized in that: The copper hydrogen phosphate-loaded hydrogel is constructed by loading copper hydrogen phosphate (CuHP) on injectable sodium alginate hydrogel (SA), the copper hydrogen phosphate is copper hydrogen phosphate microflowers composed of nanosheets, and the mass fraction of copper hydrogen phosphate in the copper hydrogen phosphate-loaded hydrogel is 0.5-1wt%.

2. The copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-type enzyme activity according to claim 1, characterized in that: The mass fraction of copper hydrogen phosphate in the copper hydrogen phosphate-loaded hydrogel is 1wt%.

3. A method for preparing the copper hydrogen phosphate-loaded hydrogel having pH-responsive multi-type enzyme activity according to claim 1, characterized in that: The method comprises the following steps: (1) Preparation of CuHP microflowers: Dissolve copper sulfate powder in 10 mL of double distilled water, then add the solution to 500 mL of PBS. Dissolve copper sulfate (CuSO4) powder in double distilled water, then add the solution to phosphate buffer (PBS), and oscillate the reaction to ensure uniform mixing and complete reaction. After the reaction is completed, separate the supernatant by centrifugation, and finally, wash and freeze-dry to obtain CuHP microflowers. (2) Preparation of SA / CuHP composite hydrogel: SA was added to double distilled water, magnetically stirred until completely dissolved, sterilized at high temperature and high pressure, and cooled to obtain SA solution; calcium chloride and deionized water were then weighed and stirred to prepare a 1% calcium chloride solution for later use; CuHP and SA sol were then mixed evenly using a two-way tube and a syringe; the mixed solution was finally added dropwise to the CaCl2 solution using a syringe to form SA / CuHP hydrogel.

4. The preparation method according to claim 3, characterized in that: The concentration of SA in step (2) is 1.5 wt %.

5. The preparation method according to claim 3, characterized in that: The sterilization conditions for high temperature and high pressure sterilization in step (2) are sterilization at 121° C. for 15 minutes.

6. The preparation method according to claim 3, characterized in that: The mass fraction of CuHP in the step (2) is 0.5-1wt%.

7. Use of the copper hydrogen phosphate-loaded hydrogel with pH-responsive multi-type enzyme activity as claimed in claim 1 in preparing bone repair materials for diabetic periodontitis.

8. The use according to claim 7, characterized in that: The pH response conditions of the diabetic periodontitis bone repair material are: under weak acid conditions, it exhibits POD enzyme activity to catalyze the generation of ROS; under neutral conditions, it exhibits CAT enzyme activity to remove ROS.

9. The use according to claim 8, characterized in that: The weak acid condition is pH=5, and the neutral condition is pH=7.4.

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